research article synthesis, characterization, and preclinical...
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Research ArticleSynthesis Characterization and Preclinical Evaluation ofNew Thiazolidin-4-ones Substituted with p-ChlorophenoxyAcetic Acid and Clofibric Acid against Insulin Resistance andMetabolic Disorder
Vasantharaju S Gowdra1 Jayesh Mudgal2 Punit Bansal2 Pawan G Nayak2
Seethappa A Manohara Reddy2 Gautham G Shenoy3 Manna Valiathan4
Mallikarjuna R Chamallamudi2 and Gopalan K Nampurath2
1 Department of Pharmaceutical Quality Assurance Manipal College of Pharmaceutical Sciences Manipal University ManipalKarnataka 576104 India
2Department of Pharmacology Manipal College of Pharmaceutical Sciences Manipal University Manipal Karnataka 576104 India3 Department of Pharmaceutical Chemistry Manipal College of Pharmaceutical Sciences Manipal University ManipalKarnataka 576104 India
4Department of Pathology Kasturba Medical College Manipal University Manipal Karnataka 576104 India
Correspondence should be addressed to Gopalan K Nampurath ngkuttymanipaledu
Received 28 February 2014 Revised 3 May 2014 Accepted 6 May 2014 Published 3 June 2014
Academic Editor Konstantinos Kantartzis
Copyright copy 2014 Vasantharaju S Gowdra et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited
We synthesized twenty thiazolidin-4-one derivatives which were then characterized by standard chromatographic and spectro-scopic methods From the in vitro glucose uptake assay two compounds behaved as insulin sensitizers where they enhancedglucose uptake in isolated rat diaphragm In high-carbohydrate diet-induced insulin resistant mice these two thiazolidin-4-onesattenuated hyperglycemia hyperinsulinemia hypertriglyceridemia hypercholesterolemia and glucose intoleranceThey raised theplasma leptin but did not reverse the diabetes-induced hypoadiponectinemia Additionally compound 3a reduced adiposity Thetest compounds were also able to reverse the disturbed liver antioxidant milieu To conclude these two novel thiazolidin-4-onesmodulatedmultiplemechanisms involved inmetabolic disorders reversing insulin resistance and thus preventing the developmentof type-2 diabetes
1 Introduction
Type-2 diabetes is a major health problem worldwide Thismultifactorial disease and its complications like diabeticretinopathy neuropathy and nephropathy result in consider-able morbidity and mortality Diabetics are also at a greaterrisk to develop atherosclerosis and coronary heart disease(CHD) Metabolic syndrome characterized by dyslipidemiahyperglycemia insulin resistance hypertension and obesityalso is a predisposing condition for atherosclerosis and CHD[1] A number of drugs are routinely used to treat the variousconditions of metabolic syndromeThis often leads to patientnon-compliance besides raising the overall cost of treatment
[2 3] Further the drugs employed in treating dyslipidemiaand hyperglycemia have their limitations mainly adverseeffects like weight gain and increased cardiac toxicity There-fore there is a need for the discovery and development ofdrugs with multiple actions and lower toxicity profile Thiswill improve the treatment outcome reduce the number ofdrugs required reduce treatment cost and consequently leadto greater patient compliance
Further recent understanding of the pathomechanism ofmetabolic disorder has revealed the central role of inflam-mation [4 5] Downregulation of inflammatory milieu by ahypolipidemic agent such as clofibrate through modulationof PPAR120572 provides insights into adopting the approach for
Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 620434 14 pageshttpdxdoiorg1011552014620434
2 BioMed Research International
synthesizing the molecules having an impact on inflamma-tory and metabolic machinery [6]
Thiazolidin-4-one derivatives have been extensively stud-ied for their varied biological activities Panetta et al reportedthe anti-inflammatory effect of several 4-thiazolidinones [7]A number of thiazolidinones have been shown to possessantioxidant and calcium overload inhibiting activities [8]Suitably substituted thiazolidinones have been reported toreduce serum cholesterol triglyceride and glucose levels inour laboratory [9 10]Three thiazolidin-4-oneswere reportedto exhibit hypolipidemic activity in mice given high-fat dietand fructose [11] Another study fromour laboratory reportedthe antidiabetic effect of two thiazolidin-4-ones through isletcell protection in streptozotocin-induced diabetes [12]Thesemolecules carried a substituted benzene ring at C2 of thethiazolidine ring and nicotinoylamino moiety at the 3rdposition (N) of the ring
p-Chlorophenoxyacetic acid is structurally related to thehypolipidemic drug clofibrateThe latter molecule is a ligandfor the peroxisome proliferator activated receptor (PPAR120572)The present work was carried out on the presumption that aclofibrate or structurally related p-chlorophenoxyacetic acidlinked through an amide to the N of the thiazolidine ringwould enhance the hypolipidemic and antidiabetic effect ofthe molecule
In our earlier studies a few p-chlorophenoxyacetic acidderivatives were found to possess remedial effect on cellu-lar signalling of inflammation [13] These molecules sup-pressed the inflammatory signalling via inhibition of proin-flammatory mediators such as interleukin-6 (IL-6) tumornecrosis factor-120572 (TNF-120572) and prostaglandin-E
2(PGE2)
In this aspect the proposed study was aimed to synthesizeand evaluate a series of thiazolidin-4-ones from the samescaffold and to study their effects on multiple etiologicalfactors of metabolic disorder like glucose intolerance insulinresistance hyperglycemia hypertriglyceridemia and hyper-cholesterolemia
2 Materials and Methods
21 Chemical and Instruments All the chemicals used inthe synthesis of compounds were of analytical grade Com-pounds were characterized by using various analytical instru-ments such asmelting point apparatus thin layer chromatog-raphy UVVIS and IR spectrometer LC-MS and RP-HPLCwith the specification as reported previously [13]
22 General Synthetic Procedure for p-ChlorophenoxyaceticAcid Derivatives p-Chlorophenoxyacetic acid derivatives(Table 1) were synthesized as described previously [13] whichis schematically described in Figure 1
221 2-(4-Chlorophenoxy)-N-(2-(furan-2-yl)-4-oxothiazoli-din-3-yl)acetamide (Compound 2a) 1HNMR (CDCl
3) 120575
380 (s 2H CH2) 455 (s 2H CO-CH
2-O) 597 (s 1H CH)
685 (m 3H Furan) 742 (m 4H Ar) 813 (br s 1H NH) IR(KBr) cmminus1 3429 3053 2978 1708 1668 1589 1236 1068
933 827 744 669 UV 120582max (MeoH) 279 nm LC-MS (ESI)119898119911 353 (Calcd for C
15H13Cl N2O4S 35279)
222 2-(4-Chlorophenoxy)-N-(2-(4-chlorophenyl)-4- oxothia-zolidin-3-yl)acetamide (Compound 2b) 1HNMR (CDCl
3) 120575
370 (s 2H CH2) 447 (s 2H CO-CH
2-O) 590 (s 1H CH)
690 (m 4HAr) 736 (m 4HAr) 80 (br s 1HNH) IR (KBr)cmminus1 3450 3059 2989 1710 1678 1591 1236 1172 1060 962833 763 671 UV 120582max (MeoH) 280 nm LC-MS (ESI) 119898119911397 (Calcd for C
17H14Cl2N2O3S 39728)
223 2-(4-Chlorophenoxy)-N-(2-(35-di-tert-butyl-4-hydrox-yphenyl)-4-oxothiazolidin-3-yl)acetamide (Compound 2c)1HNMR (CDCl
3) 120575 149 (m 18H (CH
3)6) 375 (s 2H CH
2)
450 (s 2H CO-CH2-O) 540 (s 1H -OH) 590 (s 1H CH)
690 (m 2H Ar) 72 (m 4H Ar) 79 (br s 1H NH) IR (KBr)cmminus1 3620 3327 3064 2056 1726 1685 1595 1246 11551058 1008 889 833 675 UV 120582max (MeoHl) 278 nm LC-MS(ESI)119898119911 491 (Calcd C
25H31Cl N2O4S 49104)
224 2-(4-Chlorophenoxy)-N-(2-(2-nitrophenyl)-4-oxothia-zolidin-3-yl)acetamide (Compound 2d) 1HNMR (CDCl
3)
120575 377 (s 2H CH2) 453 (s 2H CO-CH
2-O) 64 (s 1H
CH) 719 (m 4H Ar) 80 (m 4H Ar) 818 (br s 1H NH)IR (KBr) cmminus1 3427 3072 2914 1722 1680 1597 1521 13861242 1170 1063 825 790 667 UV 120582max (MeOH) 258 nmLC-MS (ESI)119898119911 408 (Calcd for C
17H14Cl N3O3S 40783)
225 2-(4-Chlorophenoxy)-N-(2-(4-methoxyphenyl)-4-oxot-hiazolidin-3-yl)acetamide (Compound 2e) 1HNMR (CDCl
3)
120575 385 (t 3H CH3) 388 (s 2H CH
2) 465 (s 2H CO-CH
2-
O) 590 (s 1H CH) 70 (m 4H Ar) 750 (m 4H Ar) 86 (brs 1H NH) IR (KBr) cmminus1 3446 3030 2916 1703 1662 15951280 1238 1170 1074 950 823 763 669 UV 120582max (MeOH)280 nm LC-MS (ESI)119898119911 393 (Calcd for C
18H17Cl N2O4S
39286)
226 2-(4-Chlorophenoxy)-N-(2-(2-chlorophenyl)-4-oxothia-zolidin-3-yl)acetamide (Compound 2f) 1HNMR (CDCl
3) 120575
372 (s 2H CH2) 450 (s 2H CO-CH
2-O) 640 (s 1H CH)
690 (m 4H Ar) 747 (m 4H Ar) 803 (br s 1H NH) IR(KBr) cmminus1 3244 3064 2987 1719 1684 1589 1240 10951055 958 825 758 669 UV 120582max (MeOH) 279 nm LC-MS(ESI)119898119911 397 (Calcd for C
17H14Cl2N2O3S 39728)
227 2-(4-chlorophenoxy)-N-(4-oxo-2-(thiophen-2-yl)thia-zolidin-3-yl)acetamide (Compound 2g) 1HNMR (CDCl
3) 120575
330 (d 2H CH2) 465 (s 2H COCH
2-O) 690 (s 1H CH)
716 (m 3HThiophen) 746 (m 4H Ar) 875 (br s 1H NH)IR (KBr) cmminus1 3437 3030 2908 1716 1678 1604 1236 11821047 945 823 765 665 UV 120582max (MeOH) 327 nm LC-MS(ESI)119898119911 369 (Calcd for C
15H13Cl N2O3S2 36886)
228 2-(4-Chlorophenoxy)-N-(2-(4-fluorophenyl)-4-oxothia-zolidin-3-yl)acetamide (Compound 2h) 1HNMR (CDCl
3) 120575
370 (s 2H CH2) 460 (s 2H CO-CH
2-O) 590 (s 1H CH)
695 (m 8H Ar) 795 (br s 1H NH) IR (KBr) cmminus1 3209
BioMed Research International 3
Table 1 List of the twenty synthesized thiazolidin-4-ones
S no Compound code R
1 2aO
2-Furyl
2 2b Cl 4-Chlorophenyl
3 2c OH35-di-t-Butyl-4-hydroxyphenyl
4 2d 2-Nitrophenyl
O2N
5 2e 4-MethoxyphenylOCH3
6 2f
Cl
2-Chlorophenyl
7 2gS
2-Thiophenyl
8 2h F 4-Fluorophenyl
9 2iFF
F4-Trifluoromethylphenyl
10 2j 4-MethylphenylCH3
11 2k34-Dimethoxyphenyl
OCH3
OCH3
12 2l Br4-Bromophenyl
13 2mCl
Cl
34-Dichlorophenyl
14 3a 4-MethoxyphenylOCH3
15 3b F4-Fluorophenyl
4 BioMed Research International
Table 1 Continued
S no Compound code R
16 3c OH35-di-t-Butyl-4-hydroxyphenyl
17 4aCl
Cl
34-Dichlorophenyl
18 4b Cl 4-Chlorophenyl
19 4cN 2-Pyridinyl
20 4d4-Methoxyphenyl
OCH3
2922 1710 1678 1599 1230 1163 1070 1008 829 752 671UV 120582max (MeOH) 281 nm LC-MS (ESI) 119898119911 381 (Calcd forC17H14Cl F N
2O3S 38082)
229 2-(4-Chlorophenoxy)-N-(4-oxo-2-(4-(trifluoromethyl)phenyl)thiazolidin-3-yl)acetamide (Compound 2i) 1HNMR(CDCl
3) 120575 389 (s 2H CH
2) 465 (s 2H CO-CH
2-O) 595
(s 1H CH) 786 (m 8H Ar) 800 (br s 1H NH) IR (KBr)cmminus1 3275 3090 2937 1735 1716 1595 1228 1163 1055 960850 666UV 120582max (MeOH) 283 nm LC-MS (ESI) 119898119911 431(Calcd for C
18H14Cl F3N2O3S 43083)
2210 2-(4-Chlorophenoxy)-N-(4-oxo-2-p-tolyl thiazolidin-3-yl)acetamide (Compound 2j) 1HNMR (CDCl
3) 120575 290 (s
3H CH3) 370 (s 2H CH
2) 442 (s 2H CO-CH
2-O) 590
(s 1H CH) 695 (m 7H Ar) 805 (br s 1H NH) IR (KBr)cmminus1 3217 2989 1722 1676 1589 1292 1238 1220 1170 1060962 821 740 667 UV 120582max (MeOH) 279 nm LC-MS (ESI)119898119911 377 (Calcd for C
18H17Cl N2O3S 37686)
2211 2-(4-Chlorophenoxy)-N-(2-(34-dimethoxyphenyl)-4-oxothiazolidin-3-yl)acetamide (Compound 2k) 1HNMR(CDCl
3) 120575 385 (s 2H CH
2) 388 t 6H [(OCH
3)2] 420
(s 2H CO-CH2-O) 459 (s 2H CH
2) 589 (s 1H CH)
720 (m 7H Ar) 800 (br s 1H NH) IR (KBr) cmminus1 34483026 2974 1703 1662 1593 1236 1072 950 821 796 669UV 120582max (MeOH) 279 nm LC-MS (ESI)119898119911 423 (Calcd forC19H19Cl N2O5S 42288)
2212 N-(2-(4-Bromophenyl)-4-oxothiazolidin-3-yl)-2-(4-chlorophenoxy)acetamide (Compound 2l) 1HNMR (CDCl
3)
120575 305 (s 2H CH2) 378 (s 2H CO-CH
2-O) 564 (s 1H
CH) 672 (m 4H Ar) 715 (m 4H Ar) 784 (br s 1H NH)IR (KBr) cmminus1 3448 3051 2982 1714 1674 1587 1230 1176
1058 964 829 amp 673 UV 120582max (MeOH) 278 nm LC-MS(ESI)119898119911 442 (Calcd for C
17H14Br Cl N
2O3S 44173)
2213 2-(4-Chlorophenoxy)-N-(2-(34-dichlorophenyl)-4-ox-othiazolidin-3-yl)acetamide (Compound 2m) 1HNMR(CDCl
3) 120575 329 (s 2H -CH
2) 388 (s 2H -CH
2-CO) 564
(s 1H CH) 682 (m 3H Ar) 733 (m 4H Ar) 852 (br s1H NH) IR (KBr) cmminus1 3228 3022 2914 1730 1687 15851481 1369 1219 1130 1060 960 823 758 667 UV 120582max(MeOH) 279 nm LC-MS (ESI) 119898119911 432 (Calcd for C
17H13
Cl3N2O3S 43172)
2214 2-(4-Chlorophenoxy)-N-(2-(34-dichlorophenyl)-5-methyl-4-oxothiazolidin-3-yl)acetamide (Compound 4a)1HNMR (CDCl
3) 120575 153 (s 3H -CH
3) 367 (1H -CH-CH
3)
390 (s 2H -CH2-) 568 (s 1H -CH) 695 (m 3H Ar) 725
(m 4H Ar1015840) 836 (br s 1H -NH) IR (KBr) cmminus1 3277 30502980 1726 1695 1585 1490 1378 1217 1126 1060 966 821738 638 UV 120582max (MeOH) 278 nm LC-MS (ESI) 119898119911 448(Calcd for C
18H15Cl3N2O3S 44575)
2215 2-(4-Chlorophenoxy)-N-(2-(4-chlorophenyl)-5-methyl-4-oxothiazolidin-3-yl)acetamide (Compound 4b) 1HNMR(CDCl
3) 120575 155 (s 3H -CH
3) 340 (1H -CH-CH
3) 392 (s
2H -CH2-) 569 (s 1H -CH) 677 (m 4H Ar) 703 (m 4H
Ar1015840) 766 (br s 1H -NH) IR (KBr) cmminus1 3279 3057 29801726 1693 1585 1383 1205 1168 1070 962 773 636 UV 120582max(MeOH) 279 nm LC-MS (ESI) 119898119911 411 (Calcd for C
18H16
Cl2N2O3S 41130)
2216 2-(4-Chlorophenoxy)-N-(5-methyl-4-oxo-2-(pyridin-2-yl)thiazolidin-3-yl)acetamide (Compound 4c) 1HNMR(CDCl
3) 120575 145 (s 3H -CH
3) 355 (1H -CH-CH
3) 389 (s
2H-CH2) 610 (s 1H -CH) 652 (m 4H Pyridine) 690
BioMed Research International 5
Cl O C
Cl O C-CONH
a b ca b c
d e
N CH
R
N
S
O
H R
a b c
Cl O C-CONH N CH
R
d f
Cl O N
S
O
H R
Cl O C-CONH
Schiff rsquos bases
Schiff rsquos bases
Schiff rsquos bases
N CH
R
c d
Cl O N
S
O
H R
H2
Cl O C-CONHH2
H2
C-CONHH2
R1
R2
CONHNH2
R1 = R2 = H or CH3
CH3
CH3
CH3
C-CONH
CH3
CH3R = 2-furyl (2a)= 4-chlorophenyl (2b)= 35 di-t-butyl-4-hydroxyphenyl (2c)= 2-nitrophenyl (2d)= 4-methoxyphenyl (2e)= 2-chlorophenyl (2f)= 2-thiophenyl (2g)= 4-flurophenyl (2h)= 4-trifluromethylphenyl (2i)= 4-methylphenyl (2j)= 34-dimethoxyphenyl (2k)= 4-bromophenyl (2l)= 34-dichlorophenyl (2m)
R = 34-dichlorophenyl (4a)= 4-chlorophenyl (4b)= 2-pyridinyl (4c)= 4-methoxyphenyl (4d)
R = 4-methoxyphenyl (3a)= 4-flurophenyl (3b)= 35 di-t-butyl-4-hydroxyphenyl (3c)
Figure 1 Scheme for the synthesis of thiazolidin-4-ones Reagents and conditions (a) aromatic aldehydes (b) methanol (c) glacial aceticacid reflux for 15ndash45 minuts (d) dry benzenetoluene (e) thioglycolic acid reflux for 24ndash48 hrs or microwave irradiation at power setting of80 with 3 minutescycle for 16 minutes and (f) thiolactic acid
(m 4H Ar) 845 (br s 1H -NH) IR (KBr) cmminus1 3159 29661732 1691 1587 1496 1342 1238 1180 1060 997 829 754 661UV 120582max (MeOH) 263 nm LC-MS (ESI)119898119911 378 (Calcd forC17H16Cl N3O3S 37785)
2217 2-(4-Chlorophenoxy)-N-(2-(4-methoxyphenyl)-5-meth-yl-4-oxothiazolidin-3-yl)acetamide(Compound 4d) 1HNMR(CDCl
3) 120575 324 (s 3H -CH
3) 397 (s 3H -CH-CH
3) 594 (s
1H -CH) 666 (m 4H Ar) 715 (m 4H Ar1015840) 815 (br s 1H-NH) IR (KBr) cmminus1 3281 2935 1735 1683 1587 1383 12441165 1064 964 821 744 636 UV 120582max (MeOH) 280 nm LC-MS (ESI)119898119911 407 (Calcd for C
19H19Cl N2O4S 40688)
23 General Synthetic Procedure for 2-(4-Chlorophenoxy)-2-Methylpropanoic Acid Derivatives [14] Ethyl-2-(4-chlo-rophenoxy)-2-methylpropanoate was synthesized byesterifying 2-(4-chlorophenoxy)-2-methylpropanoic acid
(clofibric acid 006mol) with ethanol The ester wasconverted into hydrazide by reacting ethyl 2-(4-chloro-phenoxy)-2-methylpropanoate (01mol) with hydrazine hyd-rate (01mol) Schiff rsquos bases were prepared by reacting 2-(4-chlorophenoxy)-2-methylpropanehydrazide (01mol) withAr aldehydes (01mol) like anisaldehyde p-fluorobenzal-dehyde and 35-di-tert-butyl-4-hydroxybenzaldehyde inpresence of 4-5 drops of glacial acetic acid
Schiff rsquos base (001mol) and thioglycolic acid (002mol)were refluxed in dry benzene The water formed duringcyclization was removed azeotropically The completion ofthe reaction was checked chromatographically The periodof reflux varied from compound to compound After thecompletion of the reaction the solvent was distilled off underreducedpressureThe reactionmixturewas poured intowatercontaining sodium bicarbonate solution The solid separatedwas filtered dried and recrystallized from methanol asdescribed schematically in Figure 1
6 BioMed Research International
231 2-(4-Chlorophenoxy)-N-(2-(4-methoxyphenyl)-4-oxot-hiazolidin-3-yl)-2-methylpropanamide (Compound 3a)1HNMR (CDCl
3) 120575 = 137 (t 3H -CH
3) 147 (t 3H - CH
3)
370 (t 3H OCH3) 384 (s 2H CH
2) 590 (s 1H CH) 735
(m 8H Ar) 817 (br s 1H -NH) ppm IR (KBr) 3281 (-NH)3080 (Ar C-H Str) 2892 1710 amp 1680 (-C=O of -CH
2-C=O
amp -CONH) 1612 (C=C Str) 1251 (C-O Str) 1143 1095(C-Cl) 848 817 709 amp 663 (C-S-C) cmminus1 respectively MS(ESI)119898119911 = 421
232 2-(4-Chlorophenoxy)-N-(2-(4-fluorophenyl)-4-oxot-hiazolidin-3-yl)-2-methylpropanamide (Compound 3b)1HNMR (CDCl
3) 120575 = 137 (t 3H -CH
3) 147 (t 3H -CH
3)
385 (s 2H CH2) 590 (s 1H CH) 743 (m 8H Ar) 810 (br
s 1H -NH) ppm IR (KBr) 3250 (-NH) 3043 (Ar C-H Str)2985 1726 amp 1674 (-C=O of -CH
2-C=O amp -CONH) 1599
(C=C Str) 1228 (C-O Str) 1157 (C-F) 1085 (C-Cl) 937 850amp 648 (C-S-C) cmminus1 respectively MS (ESI)119898119911 = 409
233 2-(4-Chlorophenoxy)-N-(2-(35-di-tert-butyl-4-hydrox-yphenyl)-4-oxothiazolidin-3-yl)-2-methylpropanamide (Com-pound 3c) 1HNMR(CDCl
3) 120575= 128 (s 6H (CH
3)2) 142 (s
18H (CH3)6) 344 (s 2H -CH
2-CO-) 454 (s 1H -OH) 517
(s 2H Ar) 623 (s 1H CH) 757 (m 4H Ar) ppm IR (KBr)3636 (-OH) 3543 (-NH) 3001 (Ar C-H Str) 2955 1740 amp1697 (-C=O of -CH
2-C=O amp -CONH) 1600 (C=C Str) 1236
(C-O Str) 1209 1024 (C-Cl) 887 773 amp 626 (C-S-C) cmminus1respectively MS (ESI)119898119911 = 518
24 Experimental Subjects Male Swiss albino mice (22ndash25 g) and Wistar rats (220ndash250 g) (inbred in Central AnimalResearch Facility Manipal University Manipal KarnatakaIndia) were used At a temperature of 25 plusmn 05∘C animalswere housed in plastic cages with 12 h lightdark cycle andhumidity 50 plusmn 5 RH Animals were given standard foodpellet and water ad libitum The experimental protocolswere approved by the Institutional Animal Ethics Committee(no IAECKMC252009-2010) and the experiments werecarried out in accordance with the guidelines provided bythe Committee for the Purpose of Control and Supervision ofExperiments on Animals (CPCSEA) Government of India
25 In Vitro Glucose Uptake Assay [15] Overnight fastedmaleWistar rats (220ndash250 g) were sacrificed and diaphragmswere isolated avoiding trauma and divided into two hemidi-aphragms After isolation blood clots were removed by rins-ing the hemidiaphragms in cold Tyrodersquos solution (withoutglucose) and transferred to the mammalian organ bath con-taining Tyrodersquos solution with 05wv glucose withwithoutthe teststandard drug (1mM) followed by incubation for45min at 37 plusmn 1∘C in presence of aeration After theincubation the glucose content of the incubated organ bathwas measured spectrophotometrically using colorimetrickits (Aspen Laboratories Pvt Ltd New Delhi India) Thedifference between the initial and final glucose amount wasconsidered as amount of glucose uptake (mgg of tissueweight45min)
Table 2 Composition of high carbohydrate diet (HCD)
Sl no Contents Percent (ww)1 Cholesterol 22 Sucrose 553 Lard 34 Cellulose 3505 L-cysteine 0256 Choline bitartrate 0507 DL-methionine 0258 Vitamin and mineral mixture 0109 Normal pellet diet 3540
The following groups were made for screening the com-pounds Group 1 5mLof Tyrodersquos solutionwith 05wv glu-cose (glucose control in absence of insulin) Group 2 5mL ofTyrodersquos solution with 05wv glucose and regular insulin(Novo Nordisk India Pvt Ltd Bangalore India 40 IUmL)25 120583L containing 05 units of insulin (glucose control inpresence of insulin) Groups 3 to 23 5mL of Tyrodersquos solutionwith 05wv glucose and 1mM test compound (total num-bers = 20) Group 24 5mL of Tyrodersquos solution with 05wvglucose + 1mMpioglitazone (standard) Group 25 to 45 5mLof Tyrodersquos solution with 05wv glucose + regular insulin25 120583L containing 05 units of insulin + 1mM test drug (totalnumbers = 20) Group 46 5mL of Tyrodersquos solution with05wv glucose + regular insulin 25120583L containing 05 unitsof insulin + 1mM pioglitazone (standard)
26 Acute Oral Toxicity Study An acute toxicity study wasperformed on Swiss albino mice according to OECD 420guidelines according to the methods described earlier [13]All the newly synthesized test compounds were administeredat the dose of 2000mgkg po to the animals and observedfor any sign of toxicity as described earlier [13]
27 High Carbohydrate Diet- (HCD-) Induced Metabolic Dis-order in Mice [16] Four-week-old male Swiss albino micewere placed on the feeding of high sucrose diet (HCD)The detailed composition of diet where 55 energy source(kilocalories) was from sucrose is given in Table 2
After 24 weeks of HCD feeding mice displayed hyper-glycemia Animals with similar degrees of hyperglycemiawere randomly divided into six groups (119899 = 6) Thenormal pellet diet (NPD) fed mice were used as nondiabeticcontrols The diabetic control (HCD) and the normal control(NPD) groups received the vehicle (025CMC 10mLkg)while the treatment groups were given pioglitazone (5mgkgpo) and compound 2e and compound 3a (100mgkg po)respectively suspensions in 025wv CMC (10mLkg) Allthe treatments were given for 30 days Animalsrsquo body weightand cumulative food intake were recorded periodicallyFood efficiency ratio (FER) was calculated according to thereported method [17] using the following formula FER =change in body weight (day 0ndashday 30)cumulative foodconsumed in 30 days per animal Final body weight on day30 and FER were reported Plasma glucose triglycerides and
BioMed Research International 7
total cholesterol were monitored on day 14 and day 30 afterthe treatment using colorimetric kits (Aspen LaboratoriesPvt Ltd New Delhi India) Plasma insulin (Linco ResearchInc St Charles MO USA) leptin (BioVendor LLC CandlerNC USA) and adiponectin (Adipogen Corporation SanDiego CA USA) were estimated on day 30 using ELISAkits according to manufacturerrsquos instructions OGTT wasperformed on day 32 as described below On day 32 animalswere sacrificed and liver pancreas and white adipose tissues(from epididymal fat WAT) were isolated for histopatholog-ical investigation
271 Oral Glucose Tolerance Test (OGTT) in DiabeticMice [18] OGTT was performed according to the methoddescribed previously [18] In brief animals were fastedovernight and distilled water or glucose load of 2 gkgpo was administered and blood samples were collectedby retroorbital plexus puncture at 0 30 60 and 120minafter glucose challenge Plasma glucose was measured spec-trophotometrically using commercially available colorimet-ric kits (Aspen Laboratories Pvt Ltd New Delhi India) Thepercentage reduction in glucose excursion (AUC
0minus120 min)produced by test compounds was calculated from the areaunder the curve (AUC
0minus120 min) The results are expressedin time-dependent plasma glucose (mgdL) levels plasmaglucose (AUC
0minus120 min) and reduction in glucose excursion(AUC
0minus120 min)
272 Endogenous Liver Antioxidant Enzymes EstimationAnimals were sacrificed by cervical dislocation on the 32ndday after treatment Transcardial and whole liver perfusionwere performed using ice-cold saline [19] Liver was isolatedand 10wv homogenate was prepared using Teflon-glasshomogenizer (RQ-127AD REMI Group Mumbai India)with ice-cold saline-EDTAThe homogenate was centrifugedat 10000 rpm for 10min supernatant was collected andcentrifuged again at 20000 rpm for 1 h at 4∘C The super-natant obtained was used for the estimation of glutathione(GSH) glutathione-S-transferase (GST) catalase superoxidedismutase (SOD) and malondialdehyde (MDA) All the datapresented as mean plusmn SEM (119899 = 6)
(i) Glutathione (GSH) Assay [20] From the liver homogenateproteins were precipitated by 10 tri-carboxylic acid (TCA)and then centrifuged to collect the supernatant One mLsupernatant was mixed with 6mL 02MpH 80 and 1mL06mM 551015840-dithiobis-(2-nitrobenzoic acid) (DTNB) andincubated for 10min at room temperature The absorbancewas recorded against the blank at 412 nm in aUV-visible spec-trophotometer (model UV-1650PC Shimadzu Co KyotoJapan) and the GSH concentration was calculated from thestandard curve
(ii) Superoxide Dismutase Assay [21] The entire 1mL super-natant was added to 01M carbonate buffer (pH 102) andthe increase in absorbance after addition of epinephrine was
measured at 480 nm using a UV-visible spectrophotome-ter (model UV-1650PC Shimadzu Co Kyoto Japan) Theenzyme activity was expressed as Umg protein
(iii) Catalase Assay [22] The catalase activity was deter-mined spectrophotometrically according to the protocol ofClaiborne [22] The reaction was started by adding 005mLsupernatant to the reaction mixture (195mL 10mMH
2O2in
60mM phosphate buffer pH 70) Absorbance was recordedfor 3min at 240 nm Phosphate buffer (60mM pH 70) waskept as a reference To determine the specific activity ofcatalase extinction coefficient of 004mMminus1 cmminus1 was used
(iv) Glutathione-S-Transferase (GST) Assay [23] 01mL 1-chloro-24-dinitrobenzene (CDNB) was added to 06mLsupernatant of liver homogenate and 22mL phosphate bufferpH 65 incubated at 37∘C for 5min and added 01mL 30mMGSH Absorbance was recorded at 340 nm at intervals of12345min Blank was carried out in the same mannerwithout homogenate
(v) Malondialdehyde Assay [24]OnemL of liver homogenatewas combined with 2mL of reaction mixture [15wvtrichloroacetic acid (TCA) and 0375wv thiobarbituricacid (TBA) in 025N hydrochloric acid (HCl)] and mixedthoroughly The solution was heated for 20min on boilingwater bath Samples were cooled and centrifuged at 1000 rpmfor 10min to remove the flocculent precipitate Super-natant was collected and the absorbance of read at 532 nmagainst a blank which contained all the reagents exceptthe liver homogenate The extinction coefficient of 156 times105Mminus1 cmminus1 was used to calculate the malondialdehydeconcentration
273 Histopathological Investigation For histological exam-inations liver pancreas and white adipose tissue (WAT)from three animals per group were isolated on day 32 aftertreatment of compounds The tissue samples were fixed informalin solution (10) for one week at room temperaturedehydrated by graded ethanol cleared using graded xyleneand embedded in paraffin wax 5 120583m thick sections werecut using rotary microtome (RM2245 Leica MicrosystemsGmbHWetzlar Germany) fixed on glass slides stained witheosin and hematoxylin and observed using a microscope(Model BX41 Olympus Corporation Tokyo Japan) ForLangerhans cells the average areas of six islets per specimenwere measured using ImageJ software (version 148 NationalInstitute of Health MD USA) Area was expressed as 120583m2The quantification of adipocytes were performed as reported[25] using adipocyte quantification tool where area wasmeasured in 120583m2
28 Statistical Analysis Statistical analysis was performedby comparing the responses of the treatment groups torespective saline control and vehicle treated groups for allexperiments and the significance was determined by one-way ANOVA followed by post hoc Dunnettrsquos test Values
8 BioMed Research International
Table 3 Effect of thiazolidin-4-ones on in vitro glucose uptake in absence and presence of insulin
S no GroupsGlucose uptake (mgg45min)
In absence of insulin In presence of insulinMean plusmn SEM (119899 = 3) Mean plusmn SEM (119899 = 3)
1 Glucose control 809 plusmn 051 1211 plusmn 1342 Compound 2a 189 plusmn 145 1033 plusmn 1213 Compound 2b 619 plusmn 031 736 plusmn 0254 Compound 2c 1012 plusmn 052 1488 plusmn 0865 Compound 2d 984 plusmn 061 816 plusmn 0116 Compound 2e 1900 plusmn 127a 2342 plusmn 032b
7 Compound 2f 603 plusmn 033 833 plusmn 0158 Compound 2g 645 plusmn 013 708 plusmn 0199 Compound 2h 761 plusmn 018 728 plusmn 03810 Compound 2i 555 plusmn 014 644 plusmn 11911 Compound 2j 954 plusmn 021 368 plusmn 17912 Compound 2k 582 plusmn 018 721 plusmn 01513 Compound 2l 643 plusmn 010 999 plusmn 01014 Compound 2m 812 plusmn 082 1287 plusmn 01515 Compound 3a 2044 plusmn 017a 2501 plusmn 029b
16 Compound 3b 777 plusmn 029 1151 plusmn 00217 Compound 3c 1224 plusmn 010 1050 plusmn 08118 Compound 4a 753 plusmn 110 798 plusmn 08319 Compound 4b 610 plusmn 083 772 plusmn 02120 Compound 4c 097 plusmn 059 482 plusmn 05521 Compound 4d 377 plusmn 047 554 plusmn 04922 Pioglitazone 1337 plusmn 026a 2523 plusmn 034b
a and b represent 119875 lt 005 as compared to glucose control in absencepresence of insulin respectively
Table 4 Effect of thiazolidin-4-ones on body weight and food efficiency ratio in mice
NPD HCD Pioglitazone Compound 2e Compound 3aFinal body weight (g) 380 plusmn 21 412 plusmn 22 382 plusmn 14 376 plusmn 12 382 plusmn 21Food efficiency ratio 00025 plusmn 0005 00083 plusmn 0006 00020 plusmn 0005 00043 plusmn 0011 0071 plusmn 0010abaRepresents 119875 lt 005 as compared to NPD group (normal pellet diet)bRepresents 119875 lt 005 as compared to HCD group (high carbohydrate diet)
were expressed as mean plusmn SEM 119875 lt 005 was consideredsignificant
3 Results
31 In Vitro Biological Study
311 Glucose Uptake Assay Of the twenty thiazolidin-4-ones tested compounds 2e and 3a stimulated glucose uptake(119875 lt 005) when compared with control Uptake wasstimulated both in the absence and presence of externalinsulin The standard drug pioglitazone also increased theglucose uptake (Table 3)
32 In Vivo Biological Studies
321 Acute Oral Toxicity Study Acute toxicity studies werecarried out on Swiss albino mice as per the OECD guidelines
for test compounds 2e and 3a Both were found to be safe upto 2000mgkg which was the maximum dose tested
322 High Carbohydrate Diet- (HCD-) Induced MetabolicDisorder in Mice Animals put on HCD for 6 months hadsignificant hyperglycemia hypertriglyceridemia and hyperc-holesterolemiaThey were randomized to different treatmentgroups based on plasma glucose level and then treatment wasgiven for 30 days Firstly treatment did not have any signif-icant effect on body weight of animals (Table 4) Secondlyenergy expenditure was assessed indirectly using FER Noneof the treated groups except compound 3a-treated group hadany significant effect on FER The calculated FER was foundto be significantly higher in animal treated with compound3a compared to normal control and HCD group High FERvalue indicates the increased energy expenditure in animalstreated with compound 3a
BioMed Research International 9
0
50
100
150
200
250
14 day 30 day
NPDHCD
lowast
lowast
lowastlowast
lowastlowast
lowastlowast lowastlowastlowastlowast
lowastlowast
Pioglitazone (5mgkg)
Plas
ma g
luco
se (m
gdL
)(M
eanplusmn
SE
Mn=6
)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
60
120
180
240
300
lowast
lowastlowastlowastlowast
lowastlowast
Plas
ma T
G (m
gdL
)(M
eanplusmn
SE
Mn=6
)
14 day 30 day
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
60
120
180
240
300
lowast
lowast
lowastlowast
lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowast
Plas
ma c
hole
stero
l (m
gdL
)(M
eanplusmn
SE
Mn=6
)
14 day 30 day
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 2 Effect of thiazolidin-4-ones on plasma (a) glucose (b) triglyceride (TG) (c) cholesterol inHCD fedmice Data presented asmean plusmnSEM (119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 as compared to HCD group
(i) Effect of Thiazolidin-4-Ones on Plasma Glucose Triglyc-eride and Cholesterol Level Biochemical estimation ofmetabolic markers such as plasma glucose (PG) triglycerides(TG) and total cholesterol (TC)was performed on day 14 andday 30 after the drug treatment and it was observed that HCDcontrol group consistently had significant increase in PG andTC levels on days 14 and 30 compared to respective dayrsquosnormal control group However TG levels were significantlyincreased only on day 30 compared to respective dayrsquos normalcontrol group Pioglitazone and test compounds (2e and3a) significantly reversed hyperglycemia and elevated plasmacholesterol compared to HCD control group on both day14 and day 30 of drug treatment (Figures 2(a) and 2(c))while elevated TG levels were reduced on day 30 by the testcompounds and pioglitazone (Figure 2(b))
(ii) Effect of Thiazolidin-4-Ones on Plasma Insulin Leptinand Adiponectin Level Development of insulin resistance
in mouse was confirmed by estimating the plasma insulinlevels on day 30 after drug treatment HCD control group incontrast to NPD group showed two times increase in plasmainsulin levels (119875 lt 005) Hyperinsulinemia associated withhyperglycaemia and hypertriglyceridemia is considered tobe the sign of development of insulin resistance Thus highsucrose feeding for 6 months led to the development ofinsulin resistance in mice Treatment with pioglitazone andtest drugs attenuated the insulin resistance (Figure 3(a))
HCD feeding to animals did not elicit any significanteffect on plasma leptin levels as there was no significantdifference between HCD control and NPD group Howevertreatment with pioglitazone and test compounds significantly(119875 lt 005) raised the circulating leptin level in micecomparedwithHCDcontrol group (Figure 3(b)) Compound3a caused a fourfold elevation in leptin levels compared withHCD group HCD feeding to animals resulted in significant
10 BioMed Research International
0
1
2
3
4
5
NPDHCDPioglitazone (5mgkg)
Plas
ma i
nsul
in (n
gm
L)(M
eanplusmn
SE
Mn=6
)
lowastlowast
lowast
lowastlowastlowastlowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
1200
2400
3600
4800
6000
NPDHCDPioglitazone (5mgkg)
Plas
ma l
eptin
(pg
mL)
(Mea
nplusmn
SE
Mn=6
)
lowast lowast
lowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
12000
24000
36000
48000
60000
NPDHCDPioglitazone (5mgkg)
Plas
ma a
dipo
nect
in (n
gm
L)(M
eanplusmn
SE
Mn=6
)
lowast
lowastlowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 3 Effect of thiazolidin-4-ones on plasma (a) insulin (b) leptin (c) adiponectin in HCD fed mice Data presented as mean plusmn SEM(119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 as compared to HCD group
(119875 lt 005) hypoadiponectinemia comparedwithNPD Treat-ment with pioglitazone significantly (119875 lt 005) corrected thehypoadiponectinemia However both test compounds failedto correct hypoadiponectinemia (Figure 3(c))
(iii) Effect of Thiazolidin-4-Ones on Oral Glucose ToleranceTest In oral glucose tolerance test HCD group animalshowed significant (119875 lt 005) glucose intolerance (Figure 4)Pioglitazone and test compounds compound 2e and com-pound 3a corrected the glucose intolerance shown as sig-nificant (119875 lt 005) percent reductions in glucose excursion(AUC
0minus120min) by 1300 plusmn 33 1546 plusmn 554 and 1560 plusmn 349respectively compared with HCD group (Figures 4(a) and4(b))
(iv) Effect of Thiazolidin-4-Ones on Liver Enzymes Oxidativestress is the hallmark of metabolic disorder where disturbedhomeostasis between oxidative and antioxidative mechanismoccurs HCD feeding to mice resulted in an oxidative stress
observed as reduction in liver antioxidant enzymes such asglutathione (GSH) catalase (CAT) superoxide dismutase(SOD) and glutathione-S-transferase (GST) Treatment withpioglitazone and test compounds ameliorated the oxidativestress They also reversed the elevation in liver malondialde-hyde (MDA) levels in HCD-fed mice (Table 5)
(v) Histopathological ExaminationHistology of liver showednormal lobular architecture with normal hepatocytes inall groups (Figure 5(a)) Pancreas showed mild to mod-erate hyperplasia of islets of Langerhans (Figure 5(a)) inpioglitazone and compound 3a groups Pioglitazone and3a treatment significantly raised the area of Langerhansislets compared with HCD group (Figure 5(b)) Exocrineportion of the pancreas and vascularity appeared normalin all the groups Histopathological investigation of whiteadipose tissue (WAT) from epidydimal showed an increasein the size of adipocytes (Figure 5(c)) in HCD group whichwas reversed by pioglitazone and compound 3a treatment
BioMed Research International 11
Table 5 Effect of thiazolidin-4-ones on liver antioxidant enzyme and malondialdehyde level
Liver biomarkers NPD HCD Pioglitazone Compound 2e Compound 3aGSH (nmolemg of protein) 558 plusmn 21 464 plusmn 21a 632 plusmn 15b 683 plusmn 20b 678 plusmn 43b
CAT (Umg of protein) 892 plusmn 57 554 plusmn 31a 938 plusmn 10b 819 plusmn 24b 875 plusmn 74b
SOD (Umg of protein) 1868 plusmn 232 1081 plusmn 112a 1748 plusmn 144b 2022 plusmn 47b 2080 plusmn 154b
GST (Umg of protein) 043 plusmn 005 037 plusmn 001a 073 plusmn 002b 070 plusmn 001b 072 plusmn 004b
MDA (nmolemg of protein) 057 plusmn 006 10 plusmn 033a 046 plusmn 001b 040 plusmn 001b 041 plusmn 002baRepresents 119875 lt 005 as compared to NPD group (normal pellet diet)bRepresents 119875 lt 005 as compared to HCD group (high carbohydrate diet)
0 30 60 90 120100
130
160
190
220
Plas
ma g
luco
se (m
gdL
)(M
eanplusmn
SE
Mn=6
)
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
5000
10000
15000
20000
25000
(Mea
nplusmn
SE
Mn=6
)
lowast
lowastlowast lowastlowast lowastlowast
NPDHCDPioglitazone (5mgkg)
Plas
ma g
luco
se (A
UC 0
ndash120
min)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
Figure 4 Effect of thiazolidin-4-ones on (a) plasma glucose (mgdL) and (b) area under the curve (AUC0minus120 min) against oral glucose
tolerance test (OGTT) in HCD fed mice Data presented as mean plusmn SEM (119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD groupand lowastlowastrepresents 119875 lt 005 as compared to HCD group
However compound 2e did not show any significant effecton increased adipocyte size
4 Discussion and Conclusion
The present work was planned as a sequel to earlier studiesin our laboratory using thiazolidin-4-ones as antidiabetichypolipidemic and antiinflammatory molecules The substi-tutions in the thiazolidine ring were made at C2 and N3Attached to the latter position was a nicotinamide moietyand the substitution at C2 was either p-methoxyphenyl or2 5-di-tert-butyl-4-hydroxyphenyl group The compoundsshowed significant antidiabetic and hypolipidemic activities[9ndash11 26]
In the present study the substitution at N3 was changedto p-chloro-phenoxyacetylamino a group that is similar toclofibrate with a methylene bridge instead of gem-dimethylsubstitution
Four compounds out of 20 (ie compounds 4a 4b4c and 4d) had a methyl group attached at the C5 ofthiazolidine ring with cyclisation being made with thiolacticacid instead of thioglycolic acid In three other compounds(ie compounds 3a 3b and 3c) clofibrate was used to make
themoiety for substitution atN3Thiswas donewith a view toexamine the effect of the gem-dimethyl on the overall activityof the resulting molecule
All the 20 synthesised thiazolidin-4-ones were evaluatedfor glucose uptake in an in vitro system using the isolatedrat diaphragm In this experiment compounds 2e and 3asignificantly raised the amount of glucose uptake by thetissue both in the absence and presence of external insulinThis indicated the potential of the theses compounds insensitizing the tissues for the external insulin Hence thesetwo compounds were chosen to study their effect on a diet-induced model of insulin resistance Both compounds havethe same p-methoxyphenyl moiety attached to the C2 of thethiazolidine ring Compound 3a has a gem-dimethyl groupinstead of methylene in the substituent at N3
Apart from multiple risk factors diet-induced metabolicabnormalities contribute to the development of insulinresistance and 120573-cell failure in type-2 diabetes [16] Earlydetection and appropriate treatment are considered beneficialfor correcting the abnormality Among the various animalmodels induction of diabetes through diet provides moreresemblance to human type-2 diabetes Chronic intake ofdiet with high sucrose content has been reported to favourthe development of insulin resistance [16] Similarly in our
12 BioMed Research International
NPD HCD Pioglitazone Compound 3a Compound 2e
Liver
WAT
Green and red arrows indicate hyperplasia and increased size of adipocytes respectively
Pancreas
(a)
0
50000
100000
150000
200000
NPDHCDPioglitazone (5mgkg)
lowastlowastlowastlowast
(Mea
nplusmn
SE
Mn=3
)A
rea o
f lan
gerh
ans i
stle
ts (120583
m2)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
750
1500
2250
3000
NPDHCDPioglitazone (5mgkg)
lowastlowastlowastlowast
lowast
(Mea
nplusmn
SE
Mn=3
)A
rea o
f adi
pocy
tes (120583
m2)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 5 (a) Histological sections of mouse liver pancreas and white adipose tissue (WAT) in HCD model (10x) Green and red arrowsindicate hyperplasia of islets of Langerhans and increase in the size of adipocytes respectively Effect of thiazolidin-4-ones on (b) area ofLangerhans islets (120583m2) and (c) area of adipocytes (120583m2) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 ascompared to HCD group
study mice fed a high-sucrose diet for six months devel-oped metabolic abnormalities like hyperglycemia hyperin-sulinemia hypertriglyceridemia hypercholesterolemia andhypoadiponectinemia Hyperglycemia and hyperinsulinemiasuggest the inability of insulin to sensitize the tissue forglucose uptake allowing glucose to be diverted toward lipo-genesisThis led to hyperlipidemia and finally to insulin resis-tance In addition adiponectin and leptin the adipokinessecreted from white adipose tissue (WAT) are reported tobe involved in the metabolism of glucose and lipid [27]In our study we observed that HCD caused a reductionin plasma adiponectin level and an increase in the sizeof adipocytes However it did not cause any change inplasma leptin levels Thus we found a correlation between
the adiponectin level and size of adipocytes in HCD modelFurther OGTT results from HCD-fed mice correlated wellwith glucose intolerance hyperinsulinemia and hypoad-iponectinemia The authenticity of the model was validatedby the effect of pioglitazone which was able to correct themetabolic abnormalities Neither test compounds (2e or 3a)attenuated hypoadiponectinemia However they correctedthe impaired glucose tolerance and insulin resistance inmice Compounds 2e and 3a by their ability to enhanceglucose uptake and to sensitize the tissue for available insulinreduced hyperinsulinemia and raised the leptin levels Thiswould have resulted in better glucose utilization by periph-eral tissue Thus these compounds reduced the metabolicabnormalities like hyperglycemia hypertriglyceridemia and
BioMed Research International 13
hypercholesterolemia Prospective studies involving directassessment of insulin sensitivity in OGTT are requiredto analyze glucose stimulated insulin secretion (GSIS) inpresence of compounds to establish the mechanistic role ofthe test compounds on insulin signalling
Histological investigation showed no change in liverarchitecture in the various treatment groups In HCD controlmice there was an increase in the size of adipocytes in whiteadipose tissue (WAT) which was reversed by compound3a and pioglitazone treatment However only pioglitazone-treated animals showed positive correlation between reducedsize of the cells and correction of hypoadiponectinemiaApart from correction of hypoadiponectinemia pioglitazoneraised the peripheral leptin levels which was also true in caseof compounds 2e and 3a treatment Among the tested com-pounds 3a raised endogenous leptin levels four times morethan HCD group This finding points to the link betweenraised leptin levels and reduced adipocyte size caused bycompound 3a The pancreatic islets showed hyperplasia inpioglitazone and other treatment groups Pioglitazone is aPPAR-120574 agonist This could have been responsible for theproliferation of beta cells of pancreas The test compoundsare thiazolidin-4-ones with some similarity to thiazolidine-diones It is possible that hyperplasia observed in compound3a-treated animals might have been due to some agonisticactivity on PPAR-120574 receptors This needs to be investigatedthrough relevant assay
Oxidative stress has been implicated in the occurrence ofdiabetes and compounds reducing the oxidative stress havebeneficial role in correcting glucose intolerance and insulinresistance in diabetes [18 28]The test compounds and piogli-tazone reversed the depletion of endogenous antioxidantenzymes such as GSH CAT SOD and GST Further theyreduced malondialdehyde levels This suggests the inhibitoryeffect of these compounds on oxidative stress
Leptin serves as an insulin-sensitizing factor in the wholebody [29] However hyperleptinemia in the obese mouseand human is a sign of leptin resistance where increasedleptin levels are caused by disturbed homeostasis arisingfrom leptin receptor mutation ageing or obesity [29] Thushyperleptinemia further worsens impaired insulin actionin pathological condition In these conditions exogenouslyadministered leptin does not improve glucose tolerance andinsulin sensitivity In our study diseased animal (HCD fedmouse) per se did not develop hyperleptinemia which mightbe the sign of early stage of metabolic abnormality associatedwith insulin resistance At this state test compounds aswell as pioglitazone reversed the elevated glucose TG TCand insulin levels while facilitating leptin profile along withinsulin sensitivity Subsequently compound 3a treatmentresulted in increased energy expenditure demonstrated byelevated food efficiency ratio (FER) However reductionin body weight among the treated groups was not foundproportionate to the circulating leptin levels which suggeststhat increased leptin level in peripheral blood is not suffi-cient to induce a proportionate reduction on body weightA few questions remain unanswered such as (i) whetherthe compounds directly raised the leptin levels or are theresults due to indirect impact on metabolic signaling (ii) In
metabolic disorder combined with hyperleptinemia how dothese compounds affect leptin signaling Future studies areneeded to address these issues
No single mechanism would suffice to explain the ben-eficial effects of the test compounds They do not seem toact through insulinotropic activity unlike the sulfonylureasThey have no significant effect on adiponectin levels rulingout any involvement of this mechanism The increase in thelevel of serum leptin might point to the involvement of leptinin the antihyperlipidemic and antidiabetic potentials of thesemolecules
In conclusion thiazolidin-4-one derivatives act throughmultiple mechanisms to correct the metabolic abnormalitiesin type-2 diabetes In the present work compounds 2e and3a were found to be the most effective test compoundsto ameliorate insulin resistance and development of type-2diabetes
Conflict of Interests
The authors declare that they do not have any conflict ofinterests
Acknowledgment
The authors wish to thank All India Council for TechnicalEducation (AICTE) New Delhi India for providing thefinancial support and Manipal University Manipal Kar-nataka India for providing the facilities to carry out thepresent work
References
[1] S M Grundy I J Benjamin G L Burke et al ldquoDiabetes andcardiovascular disease a statement for healthcare professionalsfrom the american heart associationrdquo Circulation vol 100 no10 pp 1134ndash1146 1999
[2] K E Thorpe and D H Howard ldquoThe rise in spending amongMedicare beneficiaries the role of chronic disease prevalenceand changes in treatment intensity Increasing numbers ofbeneficiaries being treated for five or more conditions a year aredriving Medicare spending upwardrdquo Health Affairs vol 25 no5 pp w378ndashw388 2006
[3] S M Grundy ldquoDrug therapy of the metabolic syndrome min-imizing the emerging crisis in polypharmacyrdquo Nature ReviewsDrug Discovery vol 5 no 4 pp 295ndash309 2006
[4] M Navab N Gharavi and A D Watson ldquoInflammation andmetabolic disordersrdquo Current Opinion in Clinical Nutrition andMetabolic Care vol 11 no 4 pp 459ndash464 2008
[5] G S Hotamisligil ldquoInflammation and metabolic disordersrdquoNature vol 444 no 7121 pp 860ndash867 2006
[6] M Schmuth V Moosbrugger-Martinz S Blunder and SDubrac ldquoRole of PPAR LXR and PXR in epidermal homeosta-sis and inflammationrdquo Biochimica et Biophysica Acta vol 1841no 3 pp 463ndash473 2014
[7] J A Panetta J K Shadle M L Phillips D N Benslayand P P K Ho ldquo4-thiazolidinones potent antioxidants asantiinflammatory agentsrdquo Annals of the New York Academy ofSciences vol 696 pp 415ndash416 1993
14 BioMed Research International
[8] T Kato T Ozaki K Tamura Y Suzuki M Akima and NOhi ldquoNovel calcium antagonists with both calcium overloadinhibition and antioxidant activity 1 2-(35-Di-tert-butyl-4-hydroxyphenyl)-3- (aminopropyl)thiazolidinonesrdquo Journal ofMedicinal Chemistry vol 41 no 22 pp 4309ndash4316 1998
[9] JM Joy N Jacob andGN Kutty ldquoEvaluation of hypoglycemiceffects of 4-thiazolidinonesrdquo Indian Drugs vol 42 no 1 pp 17ndash21 2005
[10] N Jacob and G N Kutty ldquoSynthesis and hypolipidemic activityof a thiazolidinone derivativerdquo Indian Drugs vol 41 no 2 pp76ndash79 2004
[11] G K Nampurath S P Mathew V Khanna R T Zachariah SKanji andMRChamallamudi ldquoAssessment of hypolipidaemicactivity of three thiazolidin-4-ones in mice given high-fat dietand fructoserdquoChemico-Biological Interactions vol 171 no 3 pp363ndash368 2008
[12] A Kishore G K Nampurath S P Mathew et al ldquoAntidiabeticeffect through islet cell protection in streptozotocin diabetesa preliminary assessment of two thiazolidin-4-ones in Swissalbino micerdquo Chemico-Biological Interactions vol 177 no 3 pp242ndash246 2009
[13] J Mudgal V S Gowdra G Mathew et al ldquoRemedial effects ofnovel 2 3-disubstituted thiazolidin-4-ones in chemical medi-ated inflammationrdquo Chemico-Biological Interactions vol 210pp 34ndash42 2014
[14] A R Surrey ldquo4-Thiazolidones IV the preparation of some3-alkylaminoalkyl-2-aryl derivativesrdquo Journal of the AmericanChemical Society vol 71 no 10 pp 3354ndash3356 1949
[15] B R P Kumar and M J Nanjan ldquoNovel glitazones designsynthesis glucose uptake and structure-activity relationshipsrdquoBioorganic and Medicinal Chemistry Letters vol 20 no 6 pp1953ndash1956 2010
[16] M Sumiyoshi M Sakanaka and Y Kimura ldquoChronic intakeof high-fat and high-sucrose diets differentially affects glucoseintolerance inmicerdquo Journal of Nutrition vol 136 no 3 pp 582ndash587 2006
[17] S H Kim S H Hyun and S Y Choung ldquoAnti-diabetic effectof cinnamon extract on blood glucose in dbdb micerdquo Journalof Ethnopharmacology vol 104 no 1-2 pp 119ndash123 2006
[18] S A Manohara Reddy J Mudgal P Bansal et al ldquoAntioxidantanti-inflammatory and anti-hyperglycaemic activities of hete-rocyclic homoprostanoid derivativesrdquoBioorganic andMedicinalChemistry vol 19 no 1 pp 384ndash392 2011
[19] S T Shukla V H Kulkarni P V Habbu K S Jagadeesh BS Patil and D M Smita ldquoHepatoprotective and antioxidantactivities of crude fractions of endophytic fungi of Ocimumsanctum Linn in ratsrdquo Oriental Pharmacy and ExperimentalMedicine vol 12 no 2 pp 81ndash91 2012
[20] B Mannervik ldquoGlutathione peroxidaserdquo Methods in Enzymol-ogy vol 113 pp 490ndash495 1985
[21] H PMisra and I Fridovich ldquoThe role of superoxide anion in theautoxidation of epinephrine and a simple assay for superoxidedismutaserdquo Journal of Biological Chemistry vol 247 no 10 pp3170ndash3175 1972
[22] A Claiborne ldquoCatalase activityrdquo in Handbook of Methods forOxygen Radical Research R A Greenwald Ed pp 283ndash284CRC Press Boca Raton Fla USA 1985
[23] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974
[24] W G Niehaus Jr and B Samuelsson ldquoFormation of malonalde-hyde from phospholipid arachidonate during microsomal lipidperoxidationrdquoEuropean Journal of Biochemistry vol 6 no 1 pp126ndash130 1968
[25] O S Osman J L Selway M A Kępczynska et al ldquoA novelautomated image analysis method for accurate adipocyte quan-tificationrdquo Adipocyte vol 2 no 3 pp 160ndash164 2013
[26] R Kalia C M Rao and N Gopalan Kutty ldquoSynthesisand evaluation of the anti-inflammatory activity of N-[2-(35-di-tert-butyl-4-hydroxyphenyl)-4-oxothiazolidin-3-yl]-nicotinamiderdquo Drug Research vol 57 no 9 pp 616ndash6222007
[27] N Maeda M Takahashi T Funahashi et al ldquoPPAR120574 ligandsincrease expression and plasma concentrations of adiponectinan adipose-derived proteinrdquo Diabetes vol 50 no 9 pp 2094ndash2099 2001
[28] P Bansal P Paul J Mudgal et al ldquoAntidiabetic antihyperlipi-demic and antioxidant effects of the flavonoid rich fraction ofPilea microphylla (L) in high fat dietstreptozotocin-induceddiabetes in micerdquo Experimental and Toxicologic Pathology vol64 no 6 pp 651ndash658 2012
[29] C Perez C Fernandez-Galaz T Fernandez-Agullo et alldquoLeptin impairs insulin signaling in rat adipocytesrdquo Diabetesvol 53 no 2 pp 347ndash353 2004
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MEDIATORSINFLAMMATION
of
2 BioMed Research International
synthesizing the molecules having an impact on inflamma-tory and metabolic machinery [6]
Thiazolidin-4-one derivatives have been extensively stud-ied for their varied biological activities Panetta et al reportedthe anti-inflammatory effect of several 4-thiazolidinones [7]A number of thiazolidinones have been shown to possessantioxidant and calcium overload inhibiting activities [8]Suitably substituted thiazolidinones have been reported toreduce serum cholesterol triglyceride and glucose levels inour laboratory [9 10]Three thiazolidin-4-oneswere reportedto exhibit hypolipidemic activity in mice given high-fat dietand fructose [11] Another study fromour laboratory reportedthe antidiabetic effect of two thiazolidin-4-ones through isletcell protection in streptozotocin-induced diabetes [12]Thesemolecules carried a substituted benzene ring at C2 of thethiazolidine ring and nicotinoylamino moiety at the 3rdposition (N) of the ring
p-Chlorophenoxyacetic acid is structurally related to thehypolipidemic drug clofibrateThe latter molecule is a ligandfor the peroxisome proliferator activated receptor (PPAR120572)The present work was carried out on the presumption that aclofibrate or structurally related p-chlorophenoxyacetic acidlinked through an amide to the N of the thiazolidine ringwould enhance the hypolipidemic and antidiabetic effect ofthe molecule
In our earlier studies a few p-chlorophenoxyacetic acidderivatives were found to possess remedial effect on cellu-lar signalling of inflammation [13] These molecules sup-pressed the inflammatory signalling via inhibition of proin-flammatory mediators such as interleukin-6 (IL-6) tumornecrosis factor-120572 (TNF-120572) and prostaglandin-E
2(PGE2)
In this aspect the proposed study was aimed to synthesizeand evaluate a series of thiazolidin-4-ones from the samescaffold and to study their effects on multiple etiologicalfactors of metabolic disorder like glucose intolerance insulinresistance hyperglycemia hypertriglyceridemia and hyper-cholesterolemia
2 Materials and Methods
21 Chemical and Instruments All the chemicals used inthe synthesis of compounds were of analytical grade Com-pounds were characterized by using various analytical instru-ments such asmelting point apparatus thin layer chromatog-raphy UVVIS and IR spectrometer LC-MS and RP-HPLCwith the specification as reported previously [13]
22 General Synthetic Procedure for p-ChlorophenoxyaceticAcid Derivatives p-Chlorophenoxyacetic acid derivatives(Table 1) were synthesized as described previously [13] whichis schematically described in Figure 1
221 2-(4-Chlorophenoxy)-N-(2-(furan-2-yl)-4-oxothiazoli-din-3-yl)acetamide (Compound 2a) 1HNMR (CDCl
3) 120575
380 (s 2H CH2) 455 (s 2H CO-CH
2-O) 597 (s 1H CH)
685 (m 3H Furan) 742 (m 4H Ar) 813 (br s 1H NH) IR(KBr) cmminus1 3429 3053 2978 1708 1668 1589 1236 1068
933 827 744 669 UV 120582max (MeoH) 279 nm LC-MS (ESI)119898119911 353 (Calcd for C
15H13Cl N2O4S 35279)
222 2-(4-Chlorophenoxy)-N-(2-(4-chlorophenyl)-4- oxothia-zolidin-3-yl)acetamide (Compound 2b) 1HNMR (CDCl
3) 120575
370 (s 2H CH2) 447 (s 2H CO-CH
2-O) 590 (s 1H CH)
690 (m 4HAr) 736 (m 4HAr) 80 (br s 1HNH) IR (KBr)cmminus1 3450 3059 2989 1710 1678 1591 1236 1172 1060 962833 763 671 UV 120582max (MeoH) 280 nm LC-MS (ESI) 119898119911397 (Calcd for C
17H14Cl2N2O3S 39728)
223 2-(4-Chlorophenoxy)-N-(2-(35-di-tert-butyl-4-hydrox-yphenyl)-4-oxothiazolidin-3-yl)acetamide (Compound 2c)1HNMR (CDCl
3) 120575 149 (m 18H (CH
3)6) 375 (s 2H CH
2)
450 (s 2H CO-CH2-O) 540 (s 1H -OH) 590 (s 1H CH)
690 (m 2H Ar) 72 (m 4H Ar) 79 (br s 1H NH) IR (KBr)cmminus1 3620 3327 3064 2056 1726 1685 1595 1246 11551058 1008 889 833 675 UV 120582max (MeoHl) 278 nm LC-MS(ESI)119898119911 491 (Calcd C
25H31Cl N2O4S 49104)
224 2-(4-Chlorophenoxy)-N-(2-(2-nitrophenyl)-4-oxothia-zolidin-3-yl)acetamide (Compound 2d) 1HNMR (CDCl
3)
120575 377 (s 2H CH2) 453 (s 2H CO-CH
2-O) 64 (s 1H
CH) 719 (m 4H Ar) 80 (m 4H Ar) 818 (br s 1H NH)IR (KBr) cmminus1 3427 3072 2914 1722 1680 1597 1521 13861242 1170 1063 825 790 667 UV 120582max (MeOH) 258 nmLC-MS (ESI)119898119911 408 (Calcd for C
17H14Cl N3O3S 40783)
225 2-(4-Chlorophenoxy)-N-(2-(4-methoxyphenyl)-4-oxot-hiazolidin-3-yl)acetamide (Compound 2e) 1HNMR (CDCl
3)
120575 385 (t 3H CH3) 388 (s 2H CH
2) 465 (s 2H CO-CH
2-
O) 590 (s 1H CH) 70 (m 4H Ar) 750 (m 4H Ar) 86 (brs 1H NH) IR (KBr) cmminus1 3446 3030 2916 1703 1662 15951280 1238 1170 1074 950 823 763 669 UV 120582max (MeOH)280 nm LC-MS (ESI)119898119911 393 (Calcd for C
18H17Cl N2O4S
39286)
226 2-(4-Chlorophenoxy)-N-(2-(2-chlorophenyl)-4-oxothia-zolidin-3-yl)acetamide (Compound 2f) 1HNMR (CDCl
3) 120575
372 (s 2H CH2) 450 (s 2H CO-CH
2-O) 640 (s 1H CH)
690 (m 4H Ar) 747 (m 4H Ar) 803 (br s 1H NH) IR(KBr) cmminus1 3244 3064 2987 1719 1684 1589 1240 10951055 958 825 758 669 UV 120582max (MeOH) 279 nm LC-MS(ESI)119898119911 397 (Calcd for C
17H14Cl2N2O3S 39728)
227 2-(4-chlorophenoxy)-N-(4-oxo-2-(thiophen-2-yl)thia-zolidin-3-yl)acetamide (Compound 2g) 1HNMR (CDCl
3) 120575
330 (d 2H CH2) 465 (s 2H COCH
2-O) 690 (s 1H CH)
716 (m 3HThiophen) 746 (m 4H Ar) 875 (br s 1H NH)IR (KBr) cmminus1 3437 3030 2908 1716 1678 1604 1236 11821047 945 823 765 665 UV 120582max (MeOH) 327 nm LC-MS(ESI)119898119911 369 (Calcd for C
15H13Cl N2O3S2 36886)
228 2-(4-Chlorophenoxy)-N-(2-(4-fluorophenyl)-4-oxothia-zolidin-3-yl)acetamide (Compound 2h) 1HNMR (CDCl
3) 120575
370 (s 2H CH2) 460 (s 2H CO-CH
2-O) 590 (s 1H CH)
695 (m 8H Ar) 795 (br s 1H NH) IR (KBr) cmminus1 3209
BioMed Research International 3
Table 1 List of the twenty synthesized thiazolidin-4-ones
S no Compound code R
1 2aO
2-Furyl
2 2b Cl 4-Chlorophenyl
3 2c OH35-di-t-Butyl-4-hydroxyphenyl
4 2d 2-Nitrophenyl
O2N
5 2e 4-MethoxyphenylOCH3
6 2f
Cl
2-Chlorophenyl
7 2gS
2-Thiophenyl
8 2h F 4-Fluorophenyl
9 2iFF
F4-Trifluoromethylphenyl
10 2j 4-MethylphenylCH3
11 2k34-Dimethoxyphenyl
OCH3
OCH3
12 2l Br4-Bromophenyl
13 2mCl
Cl
34-Dichlorophenyl
14 3a 4-MethoxyphenylOCH3
15 3b F4-Fluorophenyl
4 BioMed Research International
Table 1 Continued
S no Compound code R
16 3c OH35-di-t-Butyl-4-hydroxyphenyl
17 4aCl
Cl
34-Dichlorophenyl
18 4b Cl 4-Chlorophenyl
19 4cN 2-Pyridinyl
20 4d4-Methoxyphenyl
OCH3
2922 1710 1678 1599 1230 1163 1070 1008 829 752 671UV 120582max (MeOH) 281 nm LC-MS (ESI) 119898119911 381 (Calcd forC17H14Cl F N
2O3S 38082)
229 2-(4-Chlorophenoxy)-N-(4-oxo-2-(4-(trifluoromethyl)phenyl)thiazolidin-3-yl)acetamide (Compound 2i) 1HNMR(CDCl
3) 120575 389 (s 2H CH
2) 465 (s 2H CO-CH
2-O) 595
(s 1H CH) 786 (m 8H Ar) 800 (br s 1H NH) IR (KBr)cmminus1 3275 3090 2937 1735 1716 1595 1228 1163 1055 960850 666UV 120582max (MeOH) 283 nm LC-MS (ESI) 119898119911 431(Calcd for C
18H14Cl F3N2O3S 43083)
2210 2-(4-Chlorophenoxy)-N-(4-oxo-2-p-tolyl thiazolidin-3-yl)acetamide (Compound 2j) 1HNMR (CDCl
3) 120575 290 (s
3H CH3) 370 (s 2H CH
2) 442 (s 2H CO-CH
2-O) 590
(s 1H CH) 695 (m 7H Ar) 805 (br s 1H NH) IR (KBr)cmminus1 3217 2989 1722 1676 1589 1292 1238 1220 1170 1060962 821 740 667 UV 120582max (MeOH) 279 nm LC-MS (ESI)119898119911 377 (Calcd for C
18H17Cl N2O3S 37686)
2211 2-(4-Chlorophenoxy)-N-(2-(34-dimethoxyphenyl)-4-oxothiazolidin-3-yl)acetamide (Compound 2k) 1HNMR(CDCl
3) 120575 385 (s 2H CH
2) 388 t 6H [(OCH
3)2] 420
(s 2H CO-CH2-O) 459 (s 2H CH
2) 589 (s 1H CH)
720 (m 7H Ar) 800 (br s 1H NH) IR (KBr) cmminus1 34483026 2974 1703 1662 1593 1236 1072 950 821 796 669UV 120582max (MeOH) 279 nm LC-MS (ESI)119898119911 423 (Calcd forC19H19Cl N2O5S 42288)
2212 N-(2-(4-Bromophenyl)-4-oxothiazolidin-3-yl)-2-(4-chlorophenoxy)acetamide (Compound 2l) 1HNMR (CDCl
3)
120575 305 (s 2H CH2) 378 (s 2H CO-CH
2-O) 564 (s 1H
CH) 672 (m 4H Ar) 715 (m 4H Ar) 784 (br s 1H NH)IR (KBr) cmminus1 3448 3051 2982 1714 1674 1587 1230 1176
1058 964 829 amp 673 UV 120582max (MeOH) 278 nm LC-MS(ESI)119898119911 442 (Calcd for C
17H14Br Cl N
2O3S 44173)
2213 2-(4-Chlorophenoxy)-N-(2-(34-dichlorophenyl)-4-ox-othiazolidin-3-yl)acetamide (Compound 2m) 1HNMR(CDCl
3) 120575 329 (s 2H -CH
2) 388 (s 2H -CH
2-CO) 564
(s 1H CH) 682 (m 3H Ar) 733 (m 4H Ar) 852 (br s1H NH) IR (KBr) cmminus1 3228 3022 2914 1730 1687 15851481 1369 1219 1130 1060 960 823 758 667 UV 120582max(MeOH) 279 nm LC-MS (ESI) 119898119911 432 (Calcd for C
17H13
Cl3N2O3S 43172)
2214 2-(4-Chlorophenoxy)-N-(2-(34-dichlorophenyl)-5-methyl-4-oxothiazolidin-3-yl)acetamide (Compound 4a)1HNMR (CDCl
3) 120575 153 (s 3H -CH
3) 367 (1H -CH-CH
3)
390 (s 2H -CH2-) 568 (s 1H -CH) 695 (m 3H Ar) 725
(m 4H Ar1015840) 836 (br s 1H -NH) IR (KBr) cmminus1 3277 30502980 1726 1695 1585 1490 1378 1217 1126 1060 966 821738 638 UV 120582max (MeOH) 278 nm LC-MS (ESI) 119898119911 448(Calcd for C
18H15Cl3N2O3S 44575)
2215 2-(4-Chlorophenoxy)-N-(2-(4-chlorophenyl)-5-methyl-4-oxothiazolidin-3-yl)acetamide (Compound 4b) 1HNMR(CDCl
3) 120575 155 (s 3H -CH
3) 340 (1H -CH-CH
3) 392 (s
2H -CH2-) 569 (s 1H -CH) 677 (m 4H Ar) 703 (m 4H
Ar1015840) 766 (br s 1H -NH) IR (KBr) cmminus1 3279 3057 29801726 1693 1585 1383 1205 1168 1070 962 773 636 UV 120582max(MeOH) 279 nm LC-MS (ESI) 119898119911 411 (Calcd for C
18H16
Cl2N2O3S 41130)
2216 2-(4-Chlorophenoxy)-N-(5-methyl-4-oxo-2-(pyridin-2-yl)thiazolidin-3-yl)acetamide (Compound 4c) 1HNMR(CDCl
3) 120575 145 (s 3H -CH
3) 355 (1H -CH-CH
3) 389 (s
2H-CH2) 610 (s 1H -CH) 652 (m 4H Pyridine) 690
BioMed Research International 5
Cl O C
Cl O C-CONH
a b ca b c
d e
N CH
R
N
S
O
H R
a b c
Cl O C-CONH N CH
R
d f
Cl O N
S
O
H R
Cl O C-CONH
Schiff rsquos bases
Schiff rsquos bases
Schiff rsquos bases
N CH
R
c d
Cl O N
S
O
H R
H2
Cl O C-CONHH2
H2
C-CONHH2
R1
R2
CONHNH2
R1 = R2 = H or CH3
CH3
CH3
CH3
C-CONH
CH3
CH3R = 2-furyl (2a)= 4-chlorophenyl (2b)= 35 di-t-butyl-4-hydroxyphenyl (2c)= 2-nitrophenyl (2d)= 4-methoxyphenyl (2e)= 2-chlorophenyl (2f)= 2-thiophenyl (2g)= 4-flurophenyl (2h)= 4-trifluromethylphenyl (2i)= 4-methylphenyl (2j)= 34-dimethoxyphenyl (2k)= 4-bromophenyl (2l)= 34-dichlorophenyl (2m)
R = 34-dichlorophenyl (4a)= 4-chlorophenyl (4b)= 2-pyridinyl (4c)= 4-methoxyphenyl (4d)
R = 4-methoxyphenyl (3a)= 4-flurophenyl (3b)= 35 di-t-butyl-4-hydroxyphenyl (3c)
Figure 1 Scheme for the synthesis of thiazolidin-4-ones Reagents and conditions (a) aromatic aldehydes (b) methanol (c) glacial aceticacid reflux for 15ndash45 minuts (d) dry benzenetoluene (e) thioglycolic acid reflux for 24ndash48 hrs or microwave irradiation at power setting of80 with 3 minutescycle for 16 minutes and (f) thiolactic acid
(m 4H Ar) 845 (br s 1H -NH) IR (KBr) cmminus1 3159 29661732 1691 1587 1496 1342 1238 1180 1060 997 829 754 661UV 120582max (MeOH) 263 nm LC-MS (ESI)119898119911 378 (Calcd forC17H16Cl N3O3S 37785)
2217 2-(4-Chlorophenoxy)-N-(2-(4-methoxyphenyl)-5-meth-yl-4-oxothiazolidin-3-yl)acetamide(Compound 4d) 1HNMR(CDCl
3) 120575 324 (s 3H -CH
3) 397 (s 3H -CH-CH
3) 594 (s
1H -CH) 666 (m 4H Ar) 715 (m 4H Ar1015840) 815 (br s 1H-NH) IR (KBr) cmminus1 3281 2935 1735 1683 1587 1383 12441165 1064 964 821 744 636 UV 120582max (MeOH) 280 nm LC-MS (ESI)119898119911 407 (Calcd for C
19H19Cl N2O4S 40688)
23 General Synthetic Procedure for 2-(4-Chlorophenoxy)-2-Methylpropanoic Acid Derivatives [14] Ethyl-2-(4-chlo-rophenoxy)-2-methylpropanoate was synthesized byesterifying 2-(4-chlorophenoxy)-2-methylpropanoic acid
(clofibric acid 006mol) with ethanol The ester wasconverted into hydrazide by reacting ethyl 2-(4-chloro-phenoxy)-2-methylpropanoate (01mol) with hydrazine hyd-rate (01mol) Schiff rsquos bases were prepared by reacting 2-(4-chlorophenoxy)-2-methylpropanehydrazide (01mol) withAr aldehydes (01mol) like anisaldehyde p-fluorobenzal-dehyde and 35-di-tert-butyl-4-hydroxybenzaldehyde inpresence of 4-5 drops of glacial acetic acid
Schiff rsquos base (001mol) and thioglycolic acid (002mol)were refluxed in dry benzene The water formed duringcyclization was removed azeotropically The completion ofthe reaction was checked chromatographically The periodof reflux varied from compound to compound After thecompletion of the reaction the solvent was distilled off underreducedpressureThe reactionmixturewas poured intowatercontaining sodium bicarbonate solution The solid separatedwas filtered dried and recrystallized from methanol asdescribed schematically in Figure 1
6 BioMed Research International
231 2-(4-Chlorophenoxy)-N-(2-(4-methoxyphenyl)-4-oxot-hiazolidin-3-yl)-2-methylpropanamide (Compound 3a)1HNMR (CDCl
3) 120575 = 137 (t 3H -CH
3) 147 (t 3H - CH
3)
370 (t 3H OCH3) 384 (s 2H CH
2) 590 (s 1H CH) 735
(m 8H Ar) 817 (br s 1H -NH) ppm IR (KBr) 3281 (-NH)3080 (Ar C-H Str) 2892 1710 amp 1680 (-C=O of -CH
2-C=O
amp -CONH) 1612 (C=C Str) 1251 (C-O Str) 1143 1095(C-Cl) 848 817 709 amp 663 (C-S-C) cmminus1 respectively MS(ESI)119898119911 = 421
232 2-(4-Chlorophenoxy)-N-(2-(4-fluorophenyl)-4-oxot-hiazolidin-3-yl)-2-methylpropanamide (Compound 3b)1HNMR (CDCl
3) 120575 = 137 (t 3H -CH
3) 147 (t 3H -CH
3)
385 (s 2H CH2) 590 (s 1H CH) 743 (m 8H Ar) 810 (br
s 1H -NH) ppm IR (KBr) 3250 (-NH) 3043 (Ar C-H Str)2985 1726 amp 1674 (-C=O of -CH
2-C=O amp -CONH) 1599
(C=C Str) 1228 (C-O Str) 1157 (C-F) 1085 (C-Cl) 937 850amp 648 (C-S-C) cmminus1 respectively MS (ESI)119898119911 = 409
233 2-(4-Chlorophenoxy)-N-(2-(35-di-tert-butyl-4-hydrox-yphenyl)-4-oxothiazolidin-3-yl)-2-methylpropanamide (Com-pound 3c) 1HNMR(CDCl
3) 120575= 128 (s 6H (CH
3)2) 142 (s
18H (CH3)6) 344 (s 2H -CH
2-CO-) 454 (s 1H -OH) 517
(s 2H Ar) 623 (s 1H CH) 757 (m 4H Ar) ppm IR (KBr)3636 (-OH) 3543 (-NH) 3001 (Ar C-H Str) 2955 1740 amp1697 (-C=O of -CH
2-C=O amp -CONH) 1600 (C=C Str) 1236
(C-O Str) 1209 1024 (C-Cl) 887 773 amp 626 (C-S-C) cmminus1respectively MS (ESI)119898119911 = 518
24 Experimental Subjects Male Swiss albino mice (22ndash25 g) and Wistar rats (220ndash250 g) (inbred in Central AnimalResearch Facility Manipal University Manipal KarnatakaIndia) were used At a temperature of 25 plusmn 05∘C animalswere housed in plastic cages with 12 h lightdark cycle andhumidity 50 plusmn 5 RH Animals were given standard foodpellet and water ad libitum The experimental protocolswere approved by the Institutional Animal Ethics Committee(no IAECKMC252009-2010) and the experiments werecarried out in accordance with the guidelines provided bythe Committee for the Purpose of Control and Supervision ofExperiments on Animals (CPCSEA) Government of India
25 In Vitro Glucose Uptake Assay [15] Overnight fastedmaleWistar rats (220ndash250 g) were sacrificed and diaphragmswere isolated avoiding trauma and divided into two hemidi-aphragms After isolation blood clots were removed by rins-ing the hemidiaphragms in cold Tyrodersquos solution (withoutglucose) and transferred to the mammalian organ bath con-taining Tyrodersquos solution with 05wv glucose withwithoutthe teststandard drug (1mM) followed by incubation for45min at 37 plusmn 1∘C in presence of aeration After theincubation the glucose content of the incubated organ bathwas measured spectrophotometrically using colorimetrickits (Aspen Laboratories Pvt Ltd New Delhi India) Thedifference between the initial and final glucose amount wasconsidered as amount of glucose uptake (mgg of tissueweight45min)
Table 2 Composition of high carbohydrate diet (HCD)
Sl no Contents Percent (ww)1 Cholesterol 22 Sucrose 553 Lard 34 Cellulose 3505 L-cysteine 0256 Choline bitartrate 0507 DL-methionine 0258 Vitamin and mineral mixture 0109 Normal pellet diet 3540
The following groups were made for screening the com-pounds Group 1 5mLof Tyrodersquos solutionwith 05wv glu-cose (glucose control in absence of insulin) Group 2 5mL ofTyrodersquos solution with 05wv glucose and regular insulin(Novo Nordisk India Pvt Ltd Bangalore India 40 IUmL)25 120583L containing 05 units of insulin (glucose control inpresence of insulin) Groups 3 to 23 5mL of Tyrodersquos solutionwith 05wv glucose and 1mM test compound (total num-bers = 20) Group 24 5mL of Tyrodersquos solution with 05wvglucose + 1mMpioglitazone (standard) Group 25 to 45 5mLof Tyrodersquos solution with 05wv glucose + regular insulin25 120583L containing 05 units of insulin + 1mM test drug (totalnumbers = 20) Group 46 5mL of Tyrodersquos solution with05wv glucose + regular insulin 25120583L containing 05 unitsof insulin + 1mM pioglitazone (standard)
26 Acute Oral Toxicity Study An acute toxicity study wasperformed on Swiss albino mice according to OECD 420guidelines according to the methods described earlier [13]All the newly synthesized test compounds were administeredat the dose of 2000mgkg po to the animals and observedfor any sign of toxicity as described earlier [13]
27 High Carbohydrate Diet- (HCD-) Induced Metabolic Dis-order in Mice [16] Four-week-old male Swiss albino micewere placed on the feeding of high sucrose diet (HCD)The detailed composition of diet where 55 energy source(kilocalories) was from sucrose is given in Table 2
After 24 weeks of HCD feeding mice displayed hyper-glycemia Animals with similar degrees of hyperglycemiawere randomly divided into six groups (119899 = 6) Thenormal pellet diet (NPD) fed mice were used as nondiabeticcontrols The diabetic control (HCD) and the normal control(NPD) groups received the vehicle (025CMC 10mLkg)while the treatment groups were given pioglitazone (5mgkgpo) and compound 2e and compound 3a (100mgkg po)respectively suspensions in 025wv CMC (10mLkg) Allthe treatments were given for 30 days Animalsrsquo body weightand cumulative food intake were recorded periodicallyFood efficiency ratio (FER) was calculated according to thereported method [17] using the following formula FER =change in body weight (day 0ndashday 30)cumulative foodconsumed in 30 days per animal Final body weight on day30 and FER were reported Plasma glucose triglycerides and
BioMed Research International 7
total cholesterol were monitored on day 14 and day 30 afterthe treatment using colorimetric kits (Aspen LaboratoriesPvt Ltd New Delhi India) Plasma insulin (Linco ResearchInc St Charles MO USA) leptin (BioVendor LLC CandlerNC USA) and adiponectin (Adipogen Corporation SanDiego CA USA) were estimated on day 30 using ELISAkits according to manufacturerrsquos instructions OGTT wasperformed on day 32 as described below On day 32 animalswere sacrificed and liver pancreas and white adipose tissues(from epididymal fat WAT) were isolated for histopatholog-ical investigation
271 Oral Glucose Tolerance Test (OGTT) in DiabeticMice [18] OGTT was performed according to the methoddescribed previously [18] In brief animals were fastedovernight and distilled water or glucose load of 2 gkgpo was administered and blood samples were collectedby retroorbital plexus puncture at 0 30 60 and 120minafter glucose challenge Plasma glucose was measured spec-trophotometrically using commercially available colorimet-ric kits (Aspen Laboratories Pvt Ltd New Delhi India) Thepercentage reduction in glucose excursion (AUC
0minus120 min)produced by test compounds was calculated from the areaunder the curve (AUC
0minus120 min) The results are expressedin time-dependent plasma glucose (mgdL) levels plasmaglucose (AUC
0minus120 min) and reduction in glucose excursion(AUC
0minus120 min)
272 Endogenous Liver Antioxidant Enzymes EstimationAnimals were sacrificed by cervical dislocation on the 32ndday after treatment Transcardial and whole liver perfusionwere performed using ice-cold saline [19] Liver was isolatedand 10wv homogenate was prepared using Teflon-glasshomogenizer (RQ-127AD REMI Group Mumbai India)with ice-cold saline-EDTAThe homogenate was centrifugedat 10000 rpm for 10min supernatant was collected andcentrifuged again at 20000 rpm for 1 h at 4∘C The super-natant obtained was used for the estimation of glutathione(GSH) glutathione-S-transferase (GST) catalase superoxidedismutase (SOD) and malondialdehyde (MDA) All the datapresented as mean plusmn SEM (119899 = 6)
(i) Glutathione (GSH) Assay [20] From the liver homogenateproteins were precipitated by 10 tri-carboxylic acid (TCA)and then centrifuged to collect the supernatant One mLsupernatant was mixed with 6mL 02MpH 80 and 1mL06mM 551015840-dithiobis-(2-nitrobenzoic acid) (DTNB) andincubated for 10min at room temperature The absorbancewas recorded against the blank at 412 nm in aUV-visible spec-trophotometer (model UV-1650PC Shimadzu Co KyotoJapan) and the GSH concentration was calculated from thestandard curve
(ii) Superoxide Dismutase Assay [21] The entire 1mL super-natant was added to 01M carbonate buffer (pH 102) andthe increase in absorbance after addition of epinephrine was
measured at 480 nm using a UV-visible spectrophotome-ter (model UV-1650PC Shimadzu Co Kyoto Japan) Theenzyme activity was expressed as Umg protein
(iii) Catalase Assay [22] The catalase activity was deter-mined spectrophotometrically according to the protocol ofClaiborne [22] The reaction was started by adding 005mLsupernatant to the reaction mixture (195mL 10mMH
2O2in
60mM phosphate buffer pH 70) Absorbance was recordedfor 3min at 240 nm Phosphate buffer (60mM pH 70) waskept as a reference To determine the specific activity ofcatalase extinction coefficient of 004mMminus1 cmminus1 was used
(iv) Glutathione-S-Transferase (GST) Assay [23] 01mL 1-chloro-24-dinitrobenzene (CDNB) was added to 06mLsupernatant of liver homogenate and 22mL phosphate bufferpH 65 incubated at 37∘C for 5min and added 01mL 30mMGSH Absorbance was recorded at 340 nm at intervals of12345min Blank was carried out in the same mannerwithout homogenate
(v) Malondialdehyde Assay [24]OnemL of liver homogenatewas combined with 2mL of reaction mixture [15wvtrichloroacetic acid (TCA) and 0375wv thiobarbituricacid (TBA) in 025N hydrochloric acid (HCl)] and mixedthoroughly The solution was heated for 20min on boilingwater bath Samples were cooled and centrifuged at 1000 rpmfor 10min to remove the flocculent precipitate Super-natant was collected and the absorbance of read at 532 nmagainst a blank which contained all the reagents exceptthe liver homogenate The extinction coefficient of 156 times105Mminus1 cmminus1 was used to calculate the malondialdehydeconcentration
273 Histopathological Investigation For histological exam-inations liver pancreas and white adipose tissue (WAT)from three animals per group were isolated on day 32 aftertreatment of compounds The tissue samples were fixed informalin solution (10) for one week at room temperaturedehydrated by graded ethanol cleared using graded xyleneand embedded in paraffin wax 5 120583m thick sections werecut using rotary microtome (RM2245 Leica MicrosystemsGmbHWetzlar Germany) fixed on glass slides stained witheosin and hematoxylin and observed using a microscope(Model BX41 Olympus Corporation Tokyo Japan) ForLangerhans cells the average areas of six islets per specimenwere measured using ImageJ software (version 148 NationalInstitute of Health MD USA) Area was expressed as 120583m2The quantification of adipocytes were performed as reported[25] using adipocyte quantification tool where area wasmeasured in 120583m2
28 Statistical Analysis Statistical analysis was performedby comparing the responses of the treatment groups torespective saline control and vehicle treated groups for allexperiments and the significance was determined by one-way ANOVA followed by post hoc Dunnettrsquos test Values
8 BioMed Research International
Table 3 Effect of thiazolidin-4-ones on in vitro glucose uptake in absence and presence of insulin
S no GroupsGlucose uptake (mgg45min)
In absence of insulin In presence of insulinMean plusmn SEM (119899 = 3) Mean plusmn SEM (119899 = 3)
1 Glucose control 809 plusmn 051 1211 plusmn 1342 Compound 2a 189 plusmn 145 1033 plusmn 1213 Compound 2b 619 plusmn 031 736 plusmn 0254 Compound 2c 1012 plusmn 052 1488 plusmn 0865 Compound 2d 984 plusmn 061 816 plusmn 0116 Compound 2e 1900 plusmn 127a 2342 plusmn 032b
7 Compound 2f 603 plusmn 033 833 plusmn 0158 Compound 2g 645 plusmn 013 708 plusmn 0199 Compound 2h 761 plusmn 018 728 plusmn 03810 Compound 2i 555 plusmn 014 644 plusmn 11911 Compound 2j 954 plusmn 021 368 plusmn 17912 Compound 2k 582 plusmn 018 721 plusmn 01513 Compound 2l 643 plusmn 010 999 plusmn 01014 Compound 2m 812 plusmn 082 1287 plusmn 01515 Compound 3a 2044 plusmn 017a 2501 plusmn 029b
16 Compound 3b 777 plusmn 029 1151 plusmn 00217 Compound 3c 1224 plusmn 010 1050 plusmn 08118 Compound 4a 753 plusmn 110 798 plusmn 08319 Compound 4b 610 plusmn 083 772 plusmn 02120 Compound 4c 097 plusmn 059 482 plusmn 05521 Compound 4d 377 plusmn 047 554 plusmn 04922 Pioglitazone 1337 plusmn 026a 2523 plusmn 034b
a and b represent 119875 lt 005 as compared to glucose control in absencepresence of insulin respectively
Table 4 Effect of thiazolidin-4-ones on body weight and food efficiency ratio in mice
NPD HCD Pioglitazone Compound 2e Compound 3aFinal body weight (g) 380 plusmn 21 412 plusmn 22 382 plusmn 14 376 plusmn 12 382 plusmn 21Food efficiency ratio 00025 plusmn 0005 00083 plusmn 0006 00020 plusmn 0005 00043 plusmn 0011 0071 plusmn 0010abaRepresents 119875 lt 005 as compared to NPD group (normal pellet diet)bRepresents 119875 lt 005 as compared to HCD group (high carbohydrate diet)
were expressed as mean plusmn SEM 119875 lt 005 was consideredsignificant
3 Results
31 In Vitro Biological Study
311 Glucose Uptake Assay Of the twenty thiazolidin-4-ones tested compounds 2e and 3a stimulated glucose uptake(119875 lt 005) when compared with control Uptake wasstimulated both in the absence and presence of externalinsulin The standard drug pioglitazone also increased theglucose uptake (Table 3)
32 In Vivo Biological Studies
321 Acute Oral Toxicity Study Acute toxicity studies werecarried out on Swiss albino mice as per the OECD guidelines
for test compounds 2e and 3a Both were found to be safe upto 2000mgkg which was the maximum dose tested
322 High Carbohydrate Diet- (HCD-) Induced MetabolicDisorder in Mice Animals put on HCD for 6 months hadsignificant hyperglycemia hypertriglyceridemia and hyperc-holesterolemiaThey were randomized to different treatmentgroups based on plasma glucose level and then treatment wasgiven for 30 days Firstly treatment did not have any signif-icant effect on body weight of animals (Table 4) Secondlyenergy expenditure was assessed indirectly using FER Noneof the treated groups except compound 3a-treated group hadany significant effect on FER The calculated FER was foundto be significantly higher in animal treated with compound3a compared to normal control and HCD group High FERvalue indicates the increased energy expenditure in animalstreated with compound 3a
BioMed Research International 9
0
50
100
150
200
250
14 day 30 day
NPDHCD
lowast
lowast
lowastlowast
lowastlowast
lowastlowast lowastlowastlowastlowast
lowastlowast
Pioglitazone (5mgkg)
Plas
ma g
luco
se (m
gdL
)(M
eanplusmn
SE
Mn=6
)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
60
120
180
240
300
lowast
lowastlowastlowastlowast
lowastlowast
Plas
ma T
G (m
gdL
)(M
eanplusmn
SE
Mn=6
)
14 day 30 day
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
60
120
180
240
300
lowast
lowast
lowastlowast
lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowast
Plas
ma c
hole
stero
l (m
gdL
)(M
eanplusmn
SE
Mn=6
)
14 day 30 day
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 2 Effect of thiazolidin-4-ones on plasma (a) glucose (b) triglyceride (TG) (c) cholesterol inHCD fedmice Data presented asmean plusmnSEM (119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 as compared to HCD group
(i) Effect of Thiazolidin-4-Ones on Plasma Glucose Triglyc-eride and Cholesterol Level Biochemical estimation ofmetabolic markers such as plasma glucose (PG) triglycerides(TG) and total cholesterol (TC)was performed on day 14 andday 30 after the drug treatment and it was observed that HCDcontrol group consistently had significant increase in PG andTC levels on days 14 and 30 compared to respective dayrsquosnormal control group However TG levels were significantlyincreased only on day 30 compared to respective dayrsquos normalcontrol group Pioglitazone and test compounds (2e and3a) significantly reversed hyperglycemia and elevated plasmacholesterol compared to HCD control group on both day14 and day 30 of drug treatment (Figures 2(a) and 2(c))while elevated TG levels were reduced on day 30 by the testcompounds and pioglitazone (Figure 2(b))
(ii) Effect of Thiazolidin-4-Ones on Plasma Insulin Leptinand Adiponectin Level Development of insulin resistance
in mouse was confirmed by estimating the plasma insulinlevels on day 30 after drug treatment HCD control group incontrast to NPD group showed two times increase in plasmainsulin levels (119875 lt 005) Hyperinsulinemia associated withhyperglycaemia and hypertriglyceridemia is considered tobe the sign of development of insulin resistance Thus highsucrose feeding for 6 months led to the development ofinsulin resistance in mice Treatment with pioglitazone andtest drugs attenuated the insulin resistance (Figure 3(a))
HCD feeding to animals did not elicit any significanteffect on plasma leptin levels as there was no significantdifference between HCD control and NPD group Howevertreatment with pioglitazone and test compounds significantly(119875 lt 005) raised the circulating leptin level in micecomparedwithHCDcontrol group (Figure 3(b)) Compound3a caused a fourfold elevation in leptin levels compared withHCD group HCD feeding to animals resulted in significant
10 BioMed Research International
0
1
2
3
4
5
NPDHCDPioglitazone (5mgkg)
Plas
ma i
nsul
in (n
gm
L)(M
eanplusmn
SE
Mn=6
)
lowastlowast
lowast
lowastlowastlowastlowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
1200
2400
3600
4800
6000
NPDHCDPioglitazone (5mgkg)
Plas
ma l
eptin
(pg
mL)
(Mea
nplusmn
SE
Mn=6
)
lowast lowast
lowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
12000
24000
36000
48000
60000
NPDHCDPioglitazone (5mgkg)
Plas
ma a
dipo
nect
in (n
gm
L)(M
eanplusmn
SE
Mn=6
)
lowast
lowastlowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 3 Effect of thiazolidin-4-ones on plasma (a) insulin (b) leptin (c) adiponectin in HCD fed mice Data presented as mean plusmn SEM(119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 as compared to HCD group
(119875 lt 005) hypoadiponectinemia comparedwithNPD Treat-ment with pioglitazone significantly (119875 lt 005) corrected thehypoadiponectinemia However both test compounds failedto correct hypoadiponectinemia (Figure 3(c))
(iii) Effect of Thiazolidin-4-Ones on Oral Glucose ToleranceTest In oral glucose tolerance test HCD group animalshowed significant (119875 lt 005) glucose intolerance (Figure 4)Pioglitazone and test compounds compound 2e and com-pound 3a corrected the glucose intolerance shown as sig-nificant (119875 lt 005) percent reductions in glucose excursion(AUC
0minus120min) by 1300 plusmn 33 1546 plusmn 554 and 1560 plusmn 349respectively compared with HCD group (Figures 4(a) and4(b))
(iv) Effect of Thiazolidin-4-Ones on Liver Enzymes Oxidativestress is the hallmark of metabolic disorder where disturbedhomeostasis between oxidative and antioxidative mechanismoccurs HCD feeding to mice resulted in an oxidative stress
observed as reduction in liver antioxidant enzymes such asglutathione (GSH) catalase (CAT) superoxide dismutase(SOD) and glutathione-S-transferase (GST) Treatment withpioglitazone and test compounds ameliorated the oxidativestress They also reversed the elevation in liver malondialde-hyde (MDA) levels in HCD-fed mice (Table 5)
(v) Histopathological ExaminationHistology of liver showednormal lobular architecture with normal hepatocytes inall groups (Figure 5(a)) Pancreas showed mild to mod-erate hyperplasia of islets of Langerhans (Figure 5(a)) inpioglitazone and compound 3a groups Pioglitazone and3a treatment significantly raised the area of Langerhansislets compared with HCD group (Figure 5(b)) Exocrineportion of the pancreas and vascularity appeared normalin all the groups Histopathological investigation of whiteadipose tissue (WAT) from epidydimal showed an increasein the size of adipocytes (Figure 5(c)) in HCD group whichwas reversed by pioglitazone and compound 3a treatment
BioMed Research International 11
Table 5 Effect of thiazolidin-4-ones on liver antioxidant enzyme and malondialdehyde level
Liver biomarkers NPD HCD Pioglitazone Compound 2e Compound 3aGSH (nmolemg of protein) 558 plusmn 21 464 plusmn 21a 632 plusmn 15b 683 plusmn 20b 678 plusmn 43b
CAT (Umg of protein) 892 plusmn 57 554 plusmn 31a 938 plusmn 10b 819 plusmn 24b 875 plusmn 74b
SOD (Umg of protein) 1868 plusmn 232 1081 plusmn 112a 1748 plusmn 144b 2022 plusmn 47b 2080 plusmn 154b
GST (Umg of protein) 043 plusmn 005 037 plusmn 001a 073 plusmn 002b 070 plusmn 001b 072 plusmn 004b
MDA (nmolemg of protein) 057 plusmn 006 10 plusmn 033a 046 plusmn 001b 040 plusmn 001b 041 plusmn 002baRepresents 119875 lt 005 as compared to NPD group (normal pellet diet)bRepresents 119875 lt 005 as compared to HCD group (high carbohydrate diet)
0 30 60 90 120100
130
160
190
220
Plas
ma g
luco
se (m
gdL
)(M
eanplusmn
SE
Mn=6
)
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
5000
10000
15000
20000
25000
(Mea
nplusmn
SE
Mn=6
)
lowast
lowastlowast lowastlowast lowastlowast
NPDHCDPioglitazone (5mgkg)
Plas
ma g
luco
se (A
UC 0
ndash120
min)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
Figure 4 Effect of thiazolidin-4-ones on (a) plasma glucose (mgdL) and (b) area under the curve (AUC0minus120 min) against oral glucose
tolerance test (OGTT) in HCD fed mice Data presented as mean plusmn SEM (119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD groupand lowastlowastrepresents 119875 lt 005 as compared to HCD group
However compound 2e did not show any significant effecton increased adipocyte size
4 Discussion and Conclusion
The present work was planned as a sequel to earlier studiesin our laboratory using thiazolidin-4-ones as antidiabetichypolipidemic and antiinflammatory molecules The substi-tutions in the thiazolidine ring were made at C2 and N3Attached to the latter position was a nicotinamide moietyand the substitution at C2 was either p-methoxyphenyl or2 5-di-tert-butyl-4-hydroxyphenyl group The compoundsshowed significant antidiabetic and hypolipidemic activities[9ndash11 26]
In the present study the substitution at N3 was changedto p-chloro-phenoxyacetylamino a group that is similar toclofibrate with a methylene bridge instead of gem-dimethylsubstitution
Four compounds out of 20 (ie compounds 4a 4b4c and 4d) had a methyl group attached at the C5 ofthiazolidine ring with cyclisation being made with thiolacticacid instead of thioglycolic acid In three other compounds(ie compounds 3a 3b and 3c) clofibrate was used to make
themoiety for substitution atN3Thiswas donewith a view toexamine the effect of the gem-dimethyl on the overall activityof the resulting molecule
All the 20 synthesised thiazolidin-4-ones were evaluatedfor glucose uptake in an in vitro system using the isolatedrat diaphragm In this experiment compounds 2e and 3asignificantly raised the amount of glucose uptake by thetissue both in the absence and presence of external insulinThis indicated the potential of the theses compounds insensitizing the tissues for the external insulin Hence thesetwo compounds were chosen to study their effect on a diet-induced model of insulin resistance Both compounds havethe same p-methoxyphenyl moiety attached to the C2 of thethiazolidine ring Compound 3a has a gem-dimethyl groupinstead of methylene in the substituent at N3
Apart from multiple risk factors diet-induced metabolicabnormalities contribute to the development of insulinresistance and 120573-cell failure in type-2 diabetes [16] Earlydetection and appropriate treatment are considered beneficialfor correcting the abnormality Among the various animalmodels induction of diabetes through diet provides moreresemblance to human type-2 diabetes Chronic intake ofdiet with high sucrose content has been reported to favourthe development of insulin resistance [16] Similarly in our
12 BioMed Research International
NPD HCD Pioglitazone Compound 3a Compound 2e
Liver
WAT
Green and red arrows indicate hyperplasia and increased size of adipocytes respectively
Pancreas
(a)
0
50000
100000
150000
200000
NPDHCDPioglitazone (5mgkg)
lowastlowastlowastlowast
(Mea
nplusmn
SE
Mn=3
)A
rea o
f lan
gerh
ans i
stle
ts (120583
m2)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
750
1500
2250
3000
NPDHCDPioglitazone (5mgkg)
lowastlowastlowastlowast
lowast
(Mea
nplusmn
SE
Mn=3
)A
rea o
f adi
pocy
tes (120583
m2)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 5 (a) Histological sections of mouse liver pancreas and white adipose tissue (WAT) in HCD model (10x) Green and red arrowsindicate hyperplasia of islets of Langerhans and increase in the size of adipocytes respectively Effect of thiazolidin-4-ones on (b) area ofLangerhans islets (120583m2) and (c) area of adipocytes (120583m2) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 ascompared to HCD group
study mice fed a high-sucrose diet for six months devel-oped metabolic abnormalities like hyperglycemia hyperin-sulinemia hypertriglyceridemia hypercholesterolemia andhypoadiponectinemia Hyperglycemia and hyperinsulinemiasuggest the inability of insulin to sensitize the tissue forglucose uptake allowing glucose to be diverted toward lipo-genesisThis led to hyperlipidemia and finally to insulin resis-tance In addition adiponectin and leptin the adipokinessecreted from white adipose tissue (WAT) are reported tobe involved in the metabolism of glucose and lipid [27]In our study we observed that HCD caused a reductionin plasma adiponectin level and an increase in the sizeof adipocytes However it did not cause any change inplasma leptin levels Thus we found a correlation between
the adiponectin level and size of adipocytes in HCD modelFurther OGTT results from HCD-fed mice correlated wellwith glucose intolerance hyperinsulinemia and hypoad-iponectinemia The authenticity of the model was validatedby the effect of pioglitazone which was able to correct themetabolic abnormalities Neither test compounds (2e or 3a)attenuated hypoadiponectinemia However they correctedthe impaired glucose tolerance and insulin resistance inmice Compounds 2e and 3a by their ability to enhanceglucose uptake and to sensitize the tissue for available insulinreduced hyperinsulinemia and raised the leptin levels Thiswould have resulted in better glucose utilization by periph-eral tissue Thus these compounds reduced the metabolicabnormalities like hyperglycemia hypertriglyceridemia and
BioMed Research International 13
hypercholesterolemia Prospective studies involving directassessment of insulin sensitivity in OGTT are requiredto analyze glucose stimulated insulin secretion (GSIS) inpresence of compounds to establish the mechanistic role ofthe test compounds on insulin signalling
Histological investigation showed no change in liverarchitecture in the various treatment groups In HCD controlmice there was an increase in the size of adipocytes in whiteadipose tissue (WAT) which was reversed by compound3a and pioglitazone treatment However only pioglitazone-treated animals showed positive correlation between reducedsize of the cells and correction of hypoadiponectinemiaApart from correction of hypoadiponectinemia pioglitazoneraised the peripheral leptin levels which was also true in caseof compounds 2e and 3a treatment Among the tested com-pounds 3a raised endogenous leptin levels four times morethan HCD group This finding points to the link betweenraised leptin levels and reduced adipocyte size caused bycompound 3a The pancreatic islets showed hyperplasia inpioglitazone and other treatment groups Pioglitazone is aPPAR-120574 agonist This could have been responsible for theproliferation of beta cells of pancreas The test compoundsare thiazolidin-4-ones with some similarity to thiazolidine-diones It is possible that hyperplasia observed in compound3a-treated animals might have been due to some agonisticactivity on PPAR-120574 receptors This needs to be investigatedthrough relevant assay
Oxidative stress has been implicated in the occurrence ofdiabetes and compounds reducing the oxidative stress havebeneficial role in correcting glucose intolerance and insulinresistance in diabetes [18 28]The test compounds and piogli-tazone reversed the depletion of endogenous antioxidantenzymes such as GSH CAT SOD and GST Further theyreduced malondialdehyde levels This suggests the inhibitoryeffect of these compounds on oxidative stress
Leptin serves as an insulin-sensitizing factor in the wholebody [29] However hyperleptinemia in the obese mouseand human is a sign of leptin resistance where increasedleptin levels are caused by disturbed homeostasis arisingfrom leptin receptor mutation ageing or obesity [29] Thushyperleptinemia further worsens impaired insulin actionin pathological condition In these conditions exogenouslyadministered leptin does not improve glucose tolerance andinsulin sensitivity In our study diseased animal (HCD fedmouse) per se did not develop hyperleptinemia which mightbe the sign of early stage of metabolic abnormality associatedwith insulin resistance At this state test compounds aswell as pioglitazone reversed the elevated glucose TG TCand insulin levels while facilitating leptin profile along withinsulin sensitivity Subsequently compound 3a treatmentresulted in increased energy expenditure demonstrated byelevated food efficiency ratio (FER) However reductionin body weight among the treated groups was not foundproportionate to the circulating leptin levels which suggeststhat increased leptin level in peripheral blood is not suffi-cient to induce a proportionate reduction on body weightA few questions remain unanswered such as (i) whetherthe compounds directly raised the leptin levels or are theresults due to indirect impact on metabolic signaling (ii) In
metabolic disorder combined with hyperleptinemia how dothese compounds affect leptin signaling Future studies areneeded to address these issues
No single mechanism would suffice to explain the ben-eficial effects of the test compounds They do not seem toact through insulinotropic activity unlike the sulfonylureasThey have no significant effect on adiponectin levels rulingout any involvement of this mechanism The increase in thelevel of serum leptin might point to the involvement of leptinin the antihyperlipidemic and antidiabetic potentials of thesemolecules
In conclusion thiazolidin-4-one derivatives act throughmultiple mechanisms to correct the metabolic abnormalitiesin type-2 diabetes In the present work compounds 2e and3a were found to be the most effective test compoundsto ameliorate insulin resistance and development of type-2diabetes
Conflict of Interests
The authors declare that they do not have any conflict ofinterests
Acknowledgment
The authors wish to thank All India Council for TechnicalEducation (AICTE) New Delhi India for providing thefinancial support and Manipal University Manipal Kar-nataka India for providing the facilities to carry out thepresent work
References
[1] S M Grundy I J Benjamin G L Burke et al ldquoDiabetes andcardiovascular disease a statement for healthcare professionalsfrom the american heart associationrdquo Circulation vol 100 no10 pp 1134ndash1146 1999
[2] K E Thorpe and D H Howard ldquoThe rise in spending amongMedicare beneficiaries the role of chronic disease prevalenceand changes in treatment intensity Increasing numbers ofbeneficiaries being treated for five or more conditions a year aredriving Medicare spending upwardrdquo Health Affairs vol 25 no5 pp w378ndashw388 2006
[3] S M Grundy ldquoDrug therapy of the metabolic syndrome min-imizing the emerging crisis in polypharmacyrdquo Nature ReviewsDrug Discovery vol 5 no 4 pp 295ndash309 2006
[4] M Navab N Gharavi and A D Watson ldquoInflammation andmetabolic disordersrdquo Current Opinion in Clinical Nutrition andMetabolic Care vol 11 no 4 pp 459ndash464 2008
[5] G S Hotamisligil ldquoInflammation and metabolic disordersrdquoNature vol 444 no 7121 pp 860ndash867 2006
[6] M Schmuth V Moosbrugger-Martinz S Blunder and SDubrac ldquoRole of PPAR LXR and PXR in epidermal homeosta-sis and inflammationrdquo Biochimica et Biophysica Acta vol 1841no 3 pp 463ndash473 2014
[7] J A Panetta J K Shadle M L Phillips D N Benslayand P P K Ho ldquo4-thiazolidinones potent antioxidants asantiinflammatory agentsrdquo Annals of the New York Academy ofSciences vol 696 pp 415ndash416 1993
14 BioMed Research International
[8] T Kato T Ozaki K Tamura Y Suzuki M Akima and NOhi ldquoNovel calcium antagonists with both calcium overloadinhibition and antioxidant activity 1 2-(35-Di-tert-butyl-4-hydroxyphenyl)-3- (aminopropyl)thiazolidinonesrdquo Journal ofMedicinal Chemistry vol 41 no 22 pp 4309ndash4316 1998
[9] JM Joy N Jacob andGN Kutty ldquoEvaluation of hypoglycemiceffects of 4-thiazolidinonesrdquo Indian Drugs vol 42 no 1 pp 17ndash21 2005
[10] N Jacob and G N Kutty ldquoSynthesis and hypolipidemic activityof a thiazolidinone derivativerdquo Indian Drugs vol 41 no 2 pp76ndash79 2004
[11] G K Nampurath S P Mathew V Khanna R T Zachariah SKanji andMRChamallamudi ldquoAssessment of hypolipidaemicactivity of three thiazolidin-4-ones in mice given high-fat dietand fructoserdquoChemico-Biological Interactions vol 171 no 3 pp363ndash368 2008
[12] A Kishore G K Nampurath S P Mathew et al ldquoAntidiabeticeffect through islet cell protection in streptozotocin diabetesa preliminary assessment of two thiazolidin-4-ones in Swissalbino micerdquo Chemico-Biological Interactions vol 177 no 3 pp242ndash246 2009
[13] J Mudgal V S Gowdra G Mathew et al ldquoRemedial effects ofnovel 2 3-disubstituted thiazolidin-4-ones in chemical medi-ated inflammationrdquo Chemico-Biological Interactions vol 210pp 34ndash42 2014
[14] A R Surrey ldquo4-Thiazolidones IV the preparation of some3-alkylaminoalkyl-2-aryl derivativesrdquo Journal of the AmericanChemical Society vol 71 no 10 pp 3354ndash3356 1949
[15] B R P Kumar and M J Nanjan ldquoNovel glitazones designsynthesis glucose uptake and structure-activity relationshipsrdquoBioorganic and Medicinal Chemistry Letters vol 20 no 6 pp1953ndash1956 2010
[16] M Sumiyoshi M Sakanaka and Y Kimura ldquoChronic intakeof high-fat and high-sucrose diets differentially affects glucoseintolerance inmicerdquo Journal of Nutrition vol 136 no 3 pp 582ndash587 2006
[17] S H Kim S H Hyun and S Y Choung ldquoAnti-diabetic effectof cinnamon extract on blood glucose in dbdb micerdquo Journalof Ethnopharmacology vol 104 no 1-2 pp 119ndash123 2006
[18] S A Manohara Reddy J Mudgal P Bansal et al ldquoAntioxidantanti-inflammatory and anti-hyperglycaemic activities of hete-rocyclic homoprostanoid derivativesrdquoBioorganic andMedicinalChemistry vol 19 no 1 pp 384ndash392 2011
[19] S T Shukla V H Kulkarni P V Habbu K S Jagadeesh BS Patil and D M Smita ldquoHepatoprotective and antioxidantactivities of crude fractions of endophytic fungi of Ocimumsanctum Linn in ratsrdquo Oriental Pharmacy and ExperimentalMedicine vol 12 no 2 pp 81ndash91 2012
[20] B Mannervik ldquoGlutathione peroxidaserdquo Methods in Enzymol-ogy vol 113 pp 490ndash495 1985
[21] H PMisra and I Fridovich ldquoThe role of superoxide anion in theautoxidation of epinephrine and a simple assay for superoxidedismutaserdquo Journal of Biological Chemistry vol 247 no 10 pp3170ndash3175 1972
[22] A Claiborne ldquoCatalase activityrdquo in Handbook of Methods forOxygen Radical Research R A Greenwald Ed pp 283ndash284CRC Press Boca Raton Fla USA 1985
[23] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974
[24] W G Niehaus Jr and B Samuelsson ldquoFormation of malonalde-hyde from phospholipid arachidonate during microsomal lipidperoxidationrdquoEuropean Journal of Biochemistry vol 6 no 1 pp126ndash130 1968
[25] O S Osman J L Selway M A Kępczynska et al ldquoA novelautomated image analysis method for accurate adipocyte quan-tificationrdquo Adipocyte vol 2 no 3 pp 160ndash164 2013
[26] R Kalia C M Rao and N Gopalan Kutty ldquoSynthesisand evaluation of the anti-inflammatory activity of N-[2-(35-di-tert-butyl-4-hydroxyphenyl)-4-oxothiazolidin-3-yl]-nicotinamiderdquo Drug Research vol 57 no 9 pp 616ndash6222007
[27] N Maeda M Takahashi T Funahashi et al ldquoPPAR120574 ligandsincrease expression and plasma concentrations of adiponectinan adipose-derived proteinrdquo Diabetes vol 50 no 9 pp 2094ndash2099 2001
[28] P Bansal P Paul J Mudgal et al ldquoAntidiabetic antihyperlipi-demic and antioxidant effects of the flavonoid rich fraction ofPilea microphylla (L) in high fat dietstreptozotocin-induceddiabetes in micerdquo Experimental and Toxicologic Pathology vol64 no 6 pp 651ndash658 2012
[29] C Perez C Fernandez-Galaz T Fernandez-Agullo et alldquoLeptin impairs insulin signaling in rat adipocytesrdquo Diabetesvol 53 no 2 pp 347ndash353 2004
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BioMed Research International 3
Table 1 List of the twenty synthesized thiazolidin-4-ones
S no Compound code R
1 2aO
2-Furyl
2 2b Cl 4-Chlorophenyl
3 2c OH35-di-t-Butyl-4-hydroxyphenyl
4 2d 2-Nitrophenyl
O2N
5 2e 4-MethoxyphenylOCH3
6 2f
Cl
2-Chlorophenyl
7 2gS
2-Thiophenyl
8 2h F 4-Fluorophenyl
9 2iFF
F4-Trifluoromethylphenyl
10 2j 4-MethylphenylCH3
11 2k34-Dimethoxyphenyl
OCH3
OCH3
12 2l Br4-Bromophenyl
13 2mCl
Cl
34-Dichlorophenyl
14 3a 4-MethoxyphenylOCH3
15 3b F4-Fluorophenyl
4 BioMed Research International
Table 1 Continued
S no Compound code R
16 3c OH35-di-t-Butyl-4-hydroxyphenyl
17 4aCl
Cl
34-Dichlorophenyl
18 4b Cl 4-Chlorophenyl
19 4cN 2-Pyridinyl
20 4d4-Methoxyphenyl
OCH3
2922 1710 1678 1599 1230 1163 1070 1008 829 752 671UV 120582max (MeOH) 281 nm LC-MS (ESI) 119898119911 381 (Calcd forC17H14Cl F N
2O3S 38082)
229 2-(4-Chlorophenoxy)-N-(4-oxo-2-(4-(trifluoromethyl)phenyl)thiazolidin-3-yl)acetamide (Compound 2i) 1HNMR(CDCl
3) 120575 389 (s 2H CH
2) 465 (s 2H CO-CH
2-O) 595
(s 1H CH) 786 (m 8H Ar) 800 (br s 1H NH) IR (KBr)cmminus1 3275 3090 2937 1735 1716 1595 1228 1163 1055 960850 666UV 120582max (MeOH) 283 nm LC-MS (ESI) 119898119911 431(Calcd for C
18H14Cl F3N2O3S 43083)
2210 2-(4-Chlorophenoxy)-N-(4-oxo-2-p-tolyl thiazolidin-3-yl)acetamide (Compound 2j) 1HNMR (CDCl
3) 120575 290 (s
3H CH3) 370 (s 2H CH
2) 442 (s 2H CO-CH
2-O) 590
(s 1H CH) 695 (m 7H Ar) 805 (br s 1H NH) IR (KBr)cmminus1 3217 2989 1722 1676 1589 1292 1238 1220 1170 1060962 821 740 667 UV 120582max (MeOH) 279 nm LC-MS (ESI)119898119911 377 (Calcd for C
18H17Cl N2O3S 37686)
2211 2-(4-Chlorophenoxy)-N-(2-(34-dimethoxyphenyl)-4-oxothiazolidin-3-yl)acetamide (Compound 2k) 1HNMR(CDCl
3) 120575 385 (s 2H CH
2) 388 t 6H [(OCH
3)2] 420
(s 2H CO-CH2-O) 459 (s 2H CH
2) 589 (s 1H CH)
720 (m 7H Ar) 800 (br s 1H NH) IR (KBr) cmminus1 34483026 2974 1703 1662 1593 1236 1072 950 821 796 669UV 120582max (MeOH) 279 nm LC-MS (ESI)119898119911 423 (Calcd forC19H19Cl N2O5S 42288)
2212 N-(2-(4-Bromophenyl)-4-oxothiazolidin-3-yl)-2-(4-chlorophenoxy)acetamide (Compound 2l) 1HNMR (CDCl
3)
120575 305 (s 2H CH2) 378 (s 2H CO-CH
2-O) 564 (s 1H
CH) 672 (m 4H Ar) 715 (m 4H Ar) 784 (br s 1H NH)IR (KBr) cmminus1 3448 3051 2982 1714 1674 1587 1230 1176
1058 964 829 amp 673 UV 120582max (MeOH) 278 nm LC-MS(ESI)119898119911 442 (Calcd for C
17H14Br Cl N
2O3S 44173)
2213 2-(4-Chlorophenoxy)-N-(2-(34-dichlorophenyl)-4-ox-othiazolidin-3-yl)acetamide (Compound 2m) 1HNMR(CDCl
3) 120575 329 (s 2H -CH
2) 388 (s 2H -CH
2-CO) 564
(s 1H CH) 682 (m 3H Ar) 733 (m 4H Ar) 852 (br s1H NH) IR (KBr) cmminus1 3228 3022 2914 1730 1687 15851481 1369 1219 1130 1060 960 823 758 667 UV 120582max(MeOH) 279 nm LC-MS (ESI) 119898119911 432 (Calcd for C
17H13
Cl3N2O3S 43172)
2214 2-(4-Chlorophenoxy)-N-(2-(34-dichlorophenyl)-5-methyl-4-oxothiazolidin-3-yl)acetamide (Compound 4a)1HNMR (CDCl
3) 120575 153 (s 3H -CH
3) 367 (1H -CH-CH
3)
390 (s 2H -CH2-) 568 (s 1H -CH) 695 (m 3H Ar) 725
(m 4H Ar1015840) 836 (br s 1H -NH) IR (KBr) cmminus1 3277 30502980 1726 1695 1585 1490 1378 1217 1126 1060 966 821738 638 UV 120582max (MeOH) 278 nm LC-MS (ESI) 119898119911 448(Calcd for C
18H15Cl3N2O3S 44575)
2215 2-(4-Chlorophenoxy)-N-(2-(4-chlorophenyl)-5-methyl-4-oxothiazolidin-3-yl)acetamide (Compound 4b) 1HNMR(CDCl
3) 120575 155 (s 3H -CH
3) 340 (1H -CH-CH
3) 392 (s
2H -CH2-) 569 (s 1H -CH) 677 (m 4H Ar) 703 (m 4H
Ar1015840) 766 (br s 1H -NH) IR (KBr) cmminus1 3279 3057 29801726 1693 1585 1383 1205 1168 1070 962 773 636 UV 120582max(MeOH) 279 nm LC-MS (ESI) 119898119911 411 (Calcd for C
18H16
Cl2N2O3S 41130)
2216 2-(4-Chlorophenoxy)-N-(5-methyl-4-oxo-2-(pyridin-2-yl)thiazolidin-3-yl)acetamide (Compound 4c) 1HNMR(CDCl
3) 120575 145 (s 3H -CH
3) 355 (1H -CH-CH
3) 389 (s
2H-CH2) 610 (s 1H -CH) 652 (m 4H Pyridine) 690
BioMed Research International 5
Cl O C
Cl O C-CONH
a b ca b c
d e
N CH
R
N
S
O
H R
a b c
Cl O C-CONH N CH
R
d f
Cl O N
S
O
H R
Cl O C-CONH
Schiff rsquos bases
Schiff rsquos bases
Schiff rsquos bases
N CH
R
c d
Cl O N
S
O
H R
H2
Cl O C-CONHH2
H2
C-CONHH2
R1
R2
CONHNH2
R1 = R2 = H or CH3
CH3
CH3
CH3
C-CONH
CH3
CH3R = 2-furyl (2a)= 4-chlorophenyl (2b)= 35 di-t-butyl-4-hydroxyphenyl (2c)= 2-nitrophenyl (2d)= 4-methoxyphenyl (2e)= 2-chlorophenyl (2f)= 2-thiophenyl (2g)= 4-flurophenyl (2h)= 4-trifluromethylphenyl (2i)= 4-methylphenyl (2j)= 34-dimethoxyphenyl (2k)= 4-bromophenyl (2l)= 34-dichlorophenyl (2m)
R = 34-dichlorophenyl (4a)= 4-chlorophenyl (4b)= 2-pyridinyl (4c)= 4-methoxyphenyl (4d)
R = 4-methoxyphenyl (3a)= 4-flurophenyl (3b)= 35 di-t-butyl-4-hydroxyphenyl (3c)
Figure 1 Scheme for the synthesis of thiazolidin-4-ones Reagents and conditions (a) aromatic aldehydes (b) methanol (c) glacial aceticacid reflux for 15ndash45 minuts (d) dry benzenetoluene (e) thioglycolic acid reflux for 24ndash48 hrs or microwave irradiation at power setting of80 with 3 minutescycle for 16 minutes and (f) thiolactic acid
(m 4H Ar) 845 (br s 1H -NH) IR (KBr) cmminus1 3159 29661732 1691 1587 1496 1342 1238 1180 1060 997 829 754 661UV 120582max (MeOH) 263 nm LC-MS (ESI)119898119911 378 (Calcd forC17H16Cl N3O3S 37785)
2217 2-(4-Chlorophenoxy)-N-(2-(4-methoxyphenyl)-5-meth-yl-4-oxothiazolidin-3-yl)acetamide(Compound 4d) 1HNMR(CDCl
3) 120575 324 (s 3H -CH
3) 397 (s 3H -CH-CH
3) 594 (s
1H -CH) 666 (m 4H Ar) 715 (m 4H Ar1015840) 815 (br s 1H-NH) IR (KBr) cmminus1 3281 2935 1735 1683 1587 1383 12441165 1064 964 821 744 636 UV 120582max (MeOH) 280 nm LC-MS (ESI)119898119911 407 (Calcd for C
19H19Cl N2O4S 40688)
23 General Synthetic Procedure for 2-(4-Chlorophenoxy)-2-Methylpropanoic Acid Derivatives [14] Ethyl-2-(4-chlo-rophenoxy)-2-methylpropanoate was synthesized byesterifying 2-(4-chlorophenoxy)-2-methylpropanoic acid
(clofibric acid 006mol) with ethanol The ester wasconverted into hydrazide by reacting ethyl 2-(4-chloro-phenoxy)-2-methylpropanoate (01mol) with hydrazine hyd-rate (01mol) Schiff rsquos bases were prepared by reacting 2-(4-chlorophenoxy)-2-methylpropanehydrazide (01mol) withAr aldehydes (01mol) like anisaldehyde p-fluorobenzal-dehyde and 35-di-tert-butyl-4-hydroxybenzaldehyde inpresence of 4-5 drops of glacial acetic acid
Schiff rsquos base (001mol) and thioglycolic acid (002mol)were refluxed in dry benzene The water formed duringcyclization was removed azeotropically The completion ofthe reaction was checked chromatographically The periodof reflux varied from compound to compound After thecompletion of the reaction the solvent was distilled off underreducedpressureThe reactionmixturewas poured intowatercontaining sodium bicarbonate solution The solid separatedwas filtered dried and recrystallized from methanol asdescribed schematically in Figure 1
6 BioMed Research International
231 2-(4-Chlorophenoxy)-N-(2-(4-methoxyphenyl)-4-oxot-hiazolidin-3-yl)-2-methylpropanamide (Compound 3a)1HNMR (CDCl
3) 120575 = 137 (t 3H -CH
3) 147 (t 3H - CH
3)
370 (t 3H OCH3) 384 (s 2H CH
2) 590 (s 1H CH) 735
(m 8H Ar) 817 (br s 1H -NH) ppm IR (KBr) 3281 (-NH)3080 (Ar C-H Str) 2892 1710 amp 1680 (-C=O of -CH
2-C=O
amp -CONH) 1612 (C=C Str) 1251 (C-O Str) 1143 1095(C-Cl) 848 817 709 amp 663 (C-S-C) cmminus1 respectively MS(ESI)119898119911 = 421
232 2-(4-Chlorophenoxy)-N-(2-(4-fluorophenyl)-4-oxot-hiazolidin-3-yl)-2-methylpropanamide (Compound 3b)1HNMR (CDCl
3) 120575 = 137 (t 3H -CH
3) 147 (t 3H -CH
3)
385 (s 2H CH2) 590 (s 1H CH) 743 (m 8H Ar) 810 (br
s 1H -NH) ppm IR (KBr) 3250 (-NH) 3043 (Ar C-H Str)2985 1726 amp 1674 (-C=O of -CH
2-C=O amp -CONH) 1599
(C=C Str) 1228 (C-O Str) 1157 (C-F) 1085 (C-Cl) 937 850amp 648 (C-S-C) cmminus1 respectively MS (ESI)119898119911 = 409
233 2-(4-Chlorophenoxy)-N-(2-(35-di-tert-butyl-4-hydrox-yphenyl)-4-oxothiazolidin-3-yl)-2-methylpropanamide (Com-pound 3c) 1HNMR(CDCl
3) 120575= 128 (s 6H (CH
3)2) 142 (s
18H (CH3)6) 344 (s 2H -CH
2-CO-) 454 (s 1H -OH) 517
(s 2H Ar) 623 (s 1H CH) 757 (m 4H Ar) ppm IR (KBr)3636 (-OH) 3543 (-NH) 3001 (Ar C-H Str) 2955 1740 amp1697 (-C=O of -CH
2-C=O amp -CONH) 1600 (C=C Str) 1236
(C-O Str) 1209 1024 (C-Cl) 887 773 amp 626 (C-S-C) cmminus1respectively MS (ESI)119898119911 = 518
24 Experimental Subjects Male Swiss albino mice (22ndash25 g) and Wistar rats (220ndash250 g) (inbred in Central AnimalResearch Facility Manipal University Manipal KarnatakaIndia) were used At a temperature of 25 plusmn 05∘C animalswere housed in plastic cages with 12 h lightdark cycle andhumidity 50 plusmn 5 RH Animals were given standard foodpellet and water ad libitum The experimental protocolswere approved by the Institutional Animal Ethics Committee(no IAECKMC252009-2010) and the experiments werecarried out in accordance with the guidelines provided bythe Committee for the Purpose of Control and Supervision ofExperiments on Animals (CPCSEA) Government of India
25 In Vitro Glucose Uptake Assay [15] Overnight fastedmaleWistar rats (220ndash250 g) were sacrificed and diaphragmswere isolated avoiding trauma and divided into two hemidi-aphragms After isolation blood clots were removed by rins-ing the hemidiaphragms in cold Tyrodersquos solution (withoutglucose) and transferred to the mammalian organ bath con-taining Tyrodersquos solution with 05wv glucose withwithoutthe teststandard drug (1mM) followed by incubation for45min at 37 plusmn 1∘C in presence of aeration After theincubation the glucose content of the incubated organ bathwas measured spectrophotometrically using colorimetrickits (Aspen Laboratories Pvt Ltd New Delhi India) Thedifference between the initial and final glucose amount wasconsidered as amount of glucose uptake (mgg of tissueweight45min)
Table 2 Composition of high carbohydrate diet (HCD)
Sl no Contents Percent (ww)1 Cholesterol 22 Sucrose 553 Lard 34 Cellulose 3505 L-cysteine 0256 Choline bitartrate 0507 DL-methionine 0258 Vitamin and mineral mixture 0109 Normal pellet diet 3540
The following groups were made for screening the com-pounds Group 1 5mLof Tyrodersquos solutionwith 05wv glu-cose (glucose control in absence of insulin) Group 2 5mL ofTyrodersquos solution with 05wv glucose and regular insulin(Novo Nordisk India Pvt Ltd Bangalore India 40 IUmL)25 120583L containing 05 units of insulin (glucose control inpresence of insulin) Groups 3 to 23 5mL of Tyrodersquos solutionwith 05wv glucose and 1mM test compound (total num-bers = 20) Group 24 5mL of Tyrodersquos solution with 05wvglucose + 1mMpioglitazone (standard) Group 25 to 45 5mLof Tyrodersquos solution with 05wv glucose + regular insulin25 120583L containing 05 units of insulin + 1mM test drug (totalnumbers = 20) Group 46 5mL of Tyrodersquos solution with05wv glucose + regular insulin 25120583L containing 05 unitsof insulin + 1mM pioglitazone (standard)
26 Acute Oral Toxicity Study An acute toxicity study wasperformed on Swiss albino mice according to OECD 420guidelines according to the methods described earlier [13]All the newly synthesized test compounds were administeredat the dose of 2000mgkg po to the animals and observedfor any sign of toxicity as described earlier [13]
27 High Carbohydrate Diet- (HCD-) Induced Metabolic Dis-order in Mice [16] Four-week-old male Swiss albino micewere placed on the feeding of high sucrose diet (HCD)The detailed composition of diet where 55 energy source(kilocalories) was from sucrose is given in Table 2
After 24 weeks of HCD feeding mice displayed hyper-glycemia Animals with similar degrees of hyperglycemiawere randomly divided into six groups (119899 = 6) Thenormal pellet diet (NPD) fed mice were used as nondiabeticcontrols The diabetic control (HCD) and the normal control(NPD) groups received the vehicle (025CMC 10mLkg)while the treatment groups were given pioglitazone (5mgkgpo) and compound 2e and compound 3a (100mgkg po)respectively suspensions in 025wv CMC (10mLkg) Allthe treatments were given for 30 days Animalsrsquo body weightand cumulative food intake were recorded periodicallyFood efficiency ratio (FER) was calculated according to thereported method [17] using the following formula FER =change in body weight (day 0ndashday 30)cumulative foodconsumed in 30 days per animal Final body weight on day30 and FER were reported Plasma glucose triglycerides and
BioMed Research International 7
total cholesterol were monitored on day 14 and day 30 afterthe treatment using colorimetric kits (Aspen LaboratoriesPvt Ltd New Delhi India) Plasma insulin (Linco ResearchInc St Charles MO USA) leptin (BioVendor LLC CandlerNC USA) and adiponectin (Adipogen Corporation SanDiego CA USA) were estimated on day 30 using ELISAkits according to manufacturerrsquos instructions OGTT wasperformed on day 32 as described below On day 32 animalswere sacrificed and liver pancreas and white adipose tissues(from epididymal fat WAT) were isolated for histopatholog-ical investigation
271 Oral Glucose Tolerance Test (OGTT) in DiabeticMice [18] OGTT was performed according to the methoddescribed previously [18] In brief animals were fastedovernight and distilled water or glucose load of 2 gkgpo was administered and blood samples were collectedby retroorbital plexus puncture at 0 30 60 and 120minafter glucose challenge Plasma glucose was measured spec-trophotometrically using commercially available colorimet-ric kits (Aspen Laboratories Pvt Ltd New Delhi India) Thepercentage reduction in glucose excursion (AUC
0minus120 min)produced by test compounds was calculated from the areaunder the curve (AUC
0minus120 min) The results are expressedin time-dependent plasma glucose (mgdL) levels plasmaglucose (AUC
0minus120 min) and reduction in glucose excursion(AUC
0minus120 min)
272 Endogenous Liver Antioxidant Enzymes EstimationAnimals were sacrificed by cervical dislocation on the 32ndday after treatment Transcardial and whole liver perfusionwere performed using ice-cold saline [19] Liver was isolatedand 10wv homogenate was prepared using Teflon-glasshomogenizer (RQ-127AD REMI Group Mumbai India)with ice-cold saline-EDTAThe homogenate was centrifugedat 10000 rpm for 10min supernatant was collected andcentrifuged again at 20000 rpm for 1 h at 4∘C The super-natant obtained was used for the estimation of glutathione(GSH) glutathione-S-transferase (GST) catalase superoxidedismutase (SOD) and malondialdehyde (MDA) All the datapresented as mean plusmn SEM (119899 = 6)
(i) Glutathione (GSH) Assay [20] From the liver homogenateproteins were precipitated by 10 tri-carboxylic acid (TCA)and then centrifuged to collect the supernatant One mLsupernatant was mixed with 6mL 02MpH 80 and 1mL06mM 551015840-dithiobis-(2-nitrobenzoic acid) (DTNB) andincubated for 10min at room temperature The absorbancewas recorded against the blank at 412 nm in aUV-visible spec-trophotometer (model UV-1650PC Shimadzu Co KyotoJapan) and the GSH concentration was calculated from thestandard curve
(ii) Superoxide Dismutase Assay [21] The entire 1mL super-natant was added to 01M carbonate buffer (pH 102) andthe increase in absorbance after addition of epinephrine was
measured at 480 nm using a UV-visible spectrophotome-ter (model UV-1650PC Shimadzu Co Kyoto Japan) Theenzyme activity was expressed as Umg protein
(iii) Catalase Assay [22] The catalase activity was deter-mined spectrophotometrically according to the protocol ofClaiborne [22] The reaction was started by adding 005mLsupernatant to the reaction mixture (195mL 10mMH
2O2in
60mM phosphate buffer pH 70) Absorbance was recordedfor 3min at 240 nm Phosphate buffer (60mM pH 70) waskept as a reference To determine the specific activity ofcatalase extinction coefficient of 004mMminus1 cmminus1 was used
(iv) Glutathione-S-Transferase (GST) Assay [23] 01mL 1-chloro-24-dinitrobenzene (CDNB) was added to 06mLsupernatant of liver homogenate and 22mL phosphate bufferpH 65 incubated at 37∘C for 5min and added 01mL 30mMGSH Absorbance was recorded at 340 nm at intervals of12345min Blank was carried out in the same mannerwithout homogenate
(v) Malondialdehyde Assay [24]OnemL of liver homogenatewas combined with 2mL of reaction mixture [15wvtrichloroacetic acid (TCA) and 0375wv thiobarbituricacid (TBA) in 025N hydrochloric acid (HCl)] and mixedthoroughly The solution was heated for 20min on boilingwater bath Samples were cooled and centrifuged at 1000 rpmfor 10min to remove the flocculent precipitate Super-natant was collected and the absorbance of read at 532 nmagainst a blank which contained all the reagents exceptthe liver homogenate The extinction coefficient of 156 times105Mminus1 cmminus1 was used to calculate the malondialdehydeconcentration
273 Histopathological Investigation For histological exam-inations liver pancreas and white adipose tissue (WAT)from three animals per group were isolated on day 32 aftertreatment of compounds The tissue samples were fixed informalin solution (10) for one week at room temperaturedehydrated by graded ethanol cleared using graded xyleneand embedded in paraffin wax 5 120583m thick sections werecut using rotary microtome (RM2245 Leica MicrosystemsGmbHWetzlar Germany) fixed on glass slides stained witheosin and hematoxylin and observed using a microscope(Model BX41 Olympus Corporation Tokyo Japan) ForLangerhans cells the average areas of six islets per specimenwere measured using ImageJ software (version 148 NationalInstitute of Health MD USA) Area was expressed as 120583m2The quantification of adipocytes were performed as reported[25] using adipocyte quantification tool where area wasmeasured in 120583m2
28 Statistical Analysis Statistical analysis was performedby comparing the responses of the treatment groups torespective saline control and vehicle treated groups for allexperiments and the significance was determined by one-way ANOVA followed by post hoc Dunnettrsquos test Values
8 BioMed Research International
Table 3 Effect of thiazolidin-4-ones on in vitro glucose uptake in absence and presence of insulin
S no GroupsGlucose uptake (mgg45min)
In absence of insulin In presence of insulinMean plusmn SEM (119899 = 3) Mean plusmn SEM (119899 = 3)
1 Glucose control 809 plusmn 051 1211 plusmn 1342 Compound 2a 189 plusmn 145 1033 plusmn 1213 Compound 2b 619 plusmn 031 736 plusmn 0254 Compound 2c 1012 plusmn 052 1488 plusmn 0865 Compound 2d 984 plusmn 061 816 plusmn 0116 Compound 2e 1900 plusmn 127a 2342 plusmn 032b
7 Compound 2f 603 plusmn 033 833 plusmn 0158 Compound 2g 645 plusmn 013 708 plusmn 0199 Compound 2h 761 plusmn 018 728 plusmn 03810 Compound 2i 555 plusmn 014 644 plusmn 11911 Compound 2j 954 plusmn 021 368 plusmn 17912 Compound 2k 582 plusmn 018 721 plusmn 01513 Compound 2l 643 plusmn 010 999 plusmn 01014 Compound 2m 812 plusmn 082 1287 plusmn 01515 Compound 3a 2044 plusmn 017a 2501 plusmn 029b
16 Compound 3b 777 plusmn 029 1151 plusmn 00217 Compound 3c 1224 plusmn 010 1050 plusmn 08118 Compound 4a 753 plusmn 110 798 plusmn 08319 Compound 4b 610 plusmn 083 772 plusmn 02120 Compound 4c 097 plusmn 059 482 plusmn 05521 Compound 4d 377 plusmn 047 554 plusmn 04922 Pioglitazone 1337 plusmn 026a 2523 plusmn 034b
a and b represent 119875 lt 005 as compared to glucose control in absencepresence of insulin respectively
Table 4 Effect of thiazolidin-4-ones on body weight and food efficiency ratio in mice
NPD HCD Pioglitazone Compound 2e Compound 3aFinal body weight (g) 380 plusmn 21 412 plusmn 22 382 plusmn 14 376 plusmn 12 382 plusmn 21Food efficiency ratio 00025 plusmn 0005 00083 plusmn 0006 00020 plusmn 0005 00043 plusmn 0011 0071 plusmn 0010abaRepresents 119875 lt 005 as compared to NPD group (normal pellet diet)bRepresents 119875 lt 005 as compared to HCD group (high carbohydrate diet)
were expressed as mean plusmn SEM 119875 lt 005 was consideredsignificant
3 Results
31 In Vitro Biological Study
311 Glucose Uptake Assay Of the twenty thiazolidin-4-ones tested compounds 2e and 3a stimulated glucose uptake(119875 lt 005) when compared with control Uptake wasstimulated both in the absence and presence of externalinsulin The standard drug pioglitazone also increased theglucose uptake (Table 3)
32 In Vivo Biological Studies
321 Acute Oral Toxicity Study Acute toxicity studies werecarried out on Swiss albino mice as per the OECD guidelines
for test compounds 2e and 3a Both were found to be safe upto 2000mgkg which was the maximum dose tested
322 High Carbohydrate Diet- (HCD-) Induced MetabolicDisorder in Mice Animals put on HCD for 6 months hadsignificant hyperglycemia hypertriglyceridemia and hyperc-holesterolemiaThey were randomized to different treatmentgroups based on plasma glucose level and then treatment wasgiven for 30 days Firstly treatment did not have any signif-icant effect on body weight of animals (Table 4) Secondlyenergy expenditure was assessed indirectly using FER Noneof the treated groups except compound 3a-treated group hadany significant effect on FER The calculated FER was foundto be significantly higher in animal treated with compound3a compared to normal control and HCD group High FERvalue indicates the increased energy expenditure in animalstreated with compound 3a
BioMed Research International 9
0
50
100
150
200
250
14 day 30 day
NPDHCD
lowast
lowast
lowastlowast
lowastlowast
lowastlowast lowastlowastlowastlowast
lowastlowast
Pioglitazone (5mgkg)
Plas
ma g
luco
se (m
gdL
)(M
eanplusmn
SE
Mn=6
)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
60
120
180
240
300
lowast
lowastlowastlowastlowast
lowastlowast
Plas
ma T
G (m
gdL
)(M
eanplusmn
SE
Mn=6
)
14 day 30 day
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
60
120
180
240
300
lowast
lowast
lowastlowast
lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowast
Plas
ma c
hole
stero
l (m
gdL
)(M
eanplusmn
SE
Mn=6
)
14 day 30 day
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 2 Effect of thiazolidin-4-ones on plasma (a) glucose (b) triglyceride (TG) (c) cholesterol inHCD fedmice Data presented asmean plusmnSEM (119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 as compared to HCD group
(i) Effect of Thiazolidin-4-Ones on Plasma Glucose Triglyc-eride and Cholesterol Level Biochemical estimation ofmetabolic markers such as plasma glucose (PG) triglycerides(TG) and total cholesterol (TC)was performed on day 14 andday 30 after the drug treatment and it was observed that HCDcontrol group consistently had significant increase in PG andTC levels on days 14 and 30 compared to respective dayrsquosnormal control group However TG levels were significantlyincreased only on day 30 compared to respective dayrsquos normalcontrol group Pioglitazone and test compounds (2e and3a) significantly reversed hyperglycemia and elevated plasmacholesterol compared to HCD control group on both day14 and day 30 of drug treatment (Figures 2(a) and 2(c))while elevated TG levels were reduced on day 30 by the testcompounds and pioglitazone (Figure 2(b))
(ii) Effect of Thiazolidin-4-Ones on Plasma Insulin Leptinand Adiponectin Level Development of insulin resistance
in mouse was confirmed by estimating the plasma insulinlevels on day 30 after drug treatment HCD control group incontrast to NPD group showed two times increase in plasmainsulin levels (119875 lt 005) Hyperinsulinemia associated withhyperglycaemia and hypertriglyceridemia is considered tobe the sign of development of insulin resistance Thus highsucrose feeding for 6 months led to the development ofinsulin resistance in mice Treatment with pioglitazone andtest drugs attenuated the insulin resistance (Figure 3(a))
HCD feeding to animals did not elicit any significanteffect on plasma leptin levels as there was no significantdifference between HCD control and NPD group Howevertreatment with pioglitazone and test compounds significantly(119875 lt 005) raised the circulating leptin level in micecomparedwithHCDcontrol group (Figure 3(b)) Compound3a caused a fourfold elevation in leptin levels compared withHCD group HCD feeding to animals resulted in significant
10 BioMed Research International
0
1
2
3
4
5
NPDHCDPioglitazone (5mgkg)
Plas
ma i
nsul
in (n
gm
L)(M
eanplusmn
SE
Mn=6
)
lowastlowast
lowast
lowastlowastlowastlowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
1200
2400
3600
4800
6000
NPDHCDPioglitazone (5mgkg)
Plas
ma l
eptin
(pg
mL)
(Mea
nplusmn
SE
Mn=6
)
lowast lowast
lowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
12000
24000
36000
48000
60000
NPDHCDPioglitazone (5mgkg)
Plas
ma a
dipo
nect
in (n
gm
L)(M
eanplusmn
SE
Mn=6
)
lowast
lowastlowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 3 Effect of thiazolidin-4-ones on plasma (a) insulin (b) leptin (c) adiponectin in HCD fed mice Data presented as mean plusmn SEM(119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 as compared to HCD group
(119875 lt 005) hypoadiponectinemia comparedwithNPD Treat-ment with pioglitazone significantly (119875 lt 005) corrected thehypoadiponectinemia However both test compounds failedto correct hypoadiponectinemia (Figure 3(c))
(iii) Effect of Thiazolidin-4-Ones on Oral Glucose ToleranceTest In oral glucose tolerance test HCD group animalshowed significant (119875 lt 005) glucose intolerance (Figure 4)Pioglitazone and test compounds compound 2e and com-pound 3a corrected the glucose intolerance shown as sig-nificant (119875 lt 005) percent reductions in glucose excursion(AUC
0minus120min) by 1300 plusmn 33 1546 plusmn 554 and 1560 plusmn 349respectively compared with HCD group (Figures 4(a) and4(b))
(iv) Effect of Thiazolidin-4-Ones on Liver Enzymes Oxidativestress is the hallmark of metabolic disorder where disturbedhomeostasis between oxidative and antioxidative mechanismoccurs HCD feeding to mice resulted in an oxidative stress
observed as reduction in liver antioxidant enzymes such asglutathione (GSH) catalase (CAT) superoxide dismutase(SOD) and glutathione-S-transferase (GST) Treatment withpioglitazone and test compounds ameliorated the oxidativestress They also reversed the elevation in liver malondialde-hyde (MDA) levels in HCD-fed mice (Table 5)
(v) Histopathological ExaminationHistology of liver showednormal lobular architecture with normal hepatocytes inall groups (Figure 5(a)) Pancreas showed mild to mod-erate hyperplasia of islets of Langerhans (Figure 5(a)) inpioglitazone and compound 3a groups Pioglitazone and3a treatment significantly raised the area of Langerhansislets compared with HCD group (Figure 5(b)) Exocrineportion of the pancreas and vascularity appeared normalin all the groups Histopathological investigation of whiteadipose tissue (WAT) from epidydimal showed an increasein the size of adipocytes (Figure 5(c)) in HCD group whichwas reversed by pioglitazone and compound 3a treatment
BioMed Research International 11
Table 5 Effect of thiazolidin-4-ones on liver antioxidant enzyme and malondialdehyde level
Liver biomarkers NPD HCD Pioglitazone Compound 2e Compound 3aGSH (nmolemg of protein) 558 plusmn 21 464 plusmn 21a 632 plusmn 15b 683 plusmn 20b 678 plusmn 43b
CAT (Umg of protein) 892 plusmn 57 554 plusmn 31a 938 plusmn 10b 819 plusmn 24b 875 plusmn 74b
SOD (Umg of protein) 1868 plusmn 232 1081 plusmn 112a 1748 plusmn 144b 2022 plusmn 47b 2080 plusmn 154b
GST (Umg of protein) 043 plusmn 005 037 plusmn 001a 073 plusmn 002b 070 plusmn 001b 072 plusmn 004b
MDA (nmolemg of protein) 057 plusmn 006 10 plusmn 033a 046 plusmn 001b 040 plusmn 001b 041 plusmn 002baRepresents 119875 lt 005 as compared to NPD group (normal pellet diet)bRepresents 119875 lt 005 as compared to HCD group (high carbohydrate diet)
0 30 60 90 120100
130
160
190
220
Plas
ma g
luco
se (m
gdL
)(M
eanplusmn
SE
Mn=6
)
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
5000
10000
15000
20000
25000
(Mea
nplusmn
SE
Mn=6
)
lowast
lowastlowast lowastlowast lowastlowast
NPDHCDPioglitazone (5mgkg)
Plas
ma g
luco
se (A
UC 0
ndash120
min)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
Figure 4 Effect of thiazolidin-4-ones on (a) plasma glucose (mgdL) and (b) area under the curve (AUC0minus120 min) against oral glucose
tolerance test (OGTT) in HCD fed mice Data presented as mean plusmn SEM (119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD groupand lowastlowastrepresents 119875 lt 005 as compared to HCD group
However compound 2e did not show any significant effecton increased adipocyte size
4 Discussion and Conclusion
The present work was planned as a sequel to earlier studiesin our laboratory using thiazolidin-4-ones as antidiabetichypolipidemic and antiinflammatory molecules The substi-tutions in the thiazolidine ring were made at C2 and N3Attached to the latter position was a nicotinamide moietyand the substitution at C2 was either p-methoxyphenyl or2 5-di-tert-butyl-4-hydroxyphenyl group The compoundsshowed significant antidiabetic and hypolipidemic activities[9ndash11 26]
In the present study the substitution at N3 was changedto p-chloro-phenoxyacetylamino a group that is similar toclofibrate with a methylene bridge instead of gem-dimethylsubstitution
Four compounds out of 20 (ie compounds 4a 4b4c and 4d) had a methyl group attached at the C5 ofthiazolidine ring with cyclisation being made with thiolacticacid instead of thioglycolic acid In three other compounds(ie compounds 3a 3b and 3c) clofibrate was used to make
themoiety for substitution atN3Thiswas donewith a view toexamine the effect of the gem-dimethyl on the overall activityof the resulting molecule
All the 20 synthesised thiazolidin-4-ones were evaluatedfor glucose uptake in an in vitro system using the isolatedrat diaphragm In this experiment compounds 2e and 3asignificantly raised the amount of glucose uptake by thetissue both in the absence and presence of external insulinThis indicated the potential of the theses compounds insensitizing the tissues for the external insulin Hence thesetwo compounds were chosen to study their effect on a diet-induced model of insulin resistance Both compounds havethe same p-methoxyphenyl moiety attached to the C2 of thethiazolidine ring Compound 3a has a gem-dimethyl groupinstead of methylene in the substituent at N3
Apart from multiple risk factors diet-induced metabolicabnormalities contribute to the development of insulinresistance and 120573-cell failure in type-2 diabetes [16] Earlydetection and appropriate treatment are considered beneficialfor correcting the abnormality Among the various animalmodels induction of diabetes through diet provides moreresemblance to human type-2 diabetes Chronic intake ofdiet with high sucrose content has been reported to favourthe development of insulin resistance [16] Similarly in our
12 BioMed Research International
NPD HCD Pioglitazone Compound 3a Compound 2e
Liver
WAT
Green and red arrows indicate hyperplasia and increased size of adipocytes respectively
Pancreas
(a)
0
50000
100000
150000
200000
NPDHCDPioglitazone (5mgkg)
lowastlowastlowastlowast
(Mea
nplusmn
SE
Mn=3
)A
rea o
f lan
gerh
ans i
stle
ts (120583
m2)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
750
1500
2250
3000
NPDHCDPioglitazone (5mgkg)
lowastlowastlowastlowast
lowast
(Mea
nplusmn
SE
Mn=3
)A
rea o
f adi
pocy
tes (120583
m2)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 5 (a) Histological sections of mouse liver pancreas and white adipose tissue (WAT) in HCD model (10x) Green and red arrowsindicate hyperplasia of islets of Langerhans and increase in the size of adipocytes respectively Effect of thiazolidin-4-ones on (b) area ofLangerhans islets (120583m2) and (c) area of adipocytes (120583m2) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 ascompared to HCD group
study mice fed a high-sucrose diet for six months devel-oped metabolic abnormalities like hyperglycemia hyperin-sulinemia hypertriglyceridemia hypercholesterolemia andhypoadiponectinemia Hyperglycemia and hyperinsulinemiasuggest the inability of insulin to sensitize the tissue forglucose uptake allowing glucose to be diverted toward lipo-genesisThis led to hyperlipidemia and finally to insulin resis-tance In addition adiponectin and leptin the adipokinessecreted from white adipose tissue (WAT) are reported tobe involved in the metabolism of glucose and lipid [27]In our study we observed that HCD caused a reductionin plasma adiponectin level and an increase in the sizeof adipocytes However it did not cause any change inplasma leptin levels Thus we found a correlation between
the adiponectin level and size of adipocytes in HCD modelFurther OGTT results from HCD-fed mice correlated wellwith glucose intolerance hyperinsulinemia and hypoad-iponectinemia The authenticity of the model was validatedby the effect of pioglitazone which was able to correct themetabolic abnormalities Neither test compounds (2e or 3a)attenuated hypoadiponectinemia However they correctedthe impaired glucose tolerance and insulin resistance inmice Compounds 2e and 3a by their ability to enhanceglucose uptake and to sensitize the tissue for available insulinreduced hyperinsulinemia and raised the leptin levels Thiswould have resulted in better glucose utilization by periph-eral tissue Thus these compounds reduced the metabolicabnormalities like hyperglycemia hypertriglyceridemia and
BioMed Research International 13
hypercholesterolemia Prospective studies involving directassessment of insulin sensitivity in OGTT are requiredto analyze glucose stimulated insulin secretion (GSIS) inpresence of compounds to establish the mechanistic role ofthe test compounds on insulin signalling
Histological investigation showed no change in liverarchitecture in the various treatment groups In HCD controlmice there was an increase in the size of adipocytes in whiteadipose tissue (WAT) which was reversed by compound3a and pioglitazone treatment However only pioglitazone-treated animals showed positive correlation between reducedsize of the cells and correction of hypoadiponectinemiaApart from correction of hypoadiponectinemia pioglitazoneraised the peripheral leptin levels which was also true in caseof compounds 2e and 3a treatment Among the tested com-pounds 3a raised endogenous leptin levels four times morethan HCD group This finding points to the link betweenraised leptin levels and reduced adipocyte size caused bycompound 3a The pancreatic islets showed hyperplasia inpioglitazone and other treatment groups Pioglitazone is aPPAR-120574 agonist This could have been responsible for theproliferation of beta cells of pancreas The test compoundsare thiazolidin-4-ones with some similarity to thiazolidine-diones It is possible that hyperplasia observed in compound3a-treated animals might have been due to some agonisticactivity on PPAR-120574 receptors This needs to be investigatedthrough relevant assay
Oxidative stress has been implicated in the occurrence ofdiabetes and compounds reducing the oxidative stress havebeneficial role in correcting glucose intolerance and insulinresistance in diabetes [18 28]The test compounds and piogli-tazone reversed the depletion of endogenous antioxidantenzymes such as GSH CAT SOD and GST Further theyreduced malondialdehyde levels This suggests the inhibitoryeffect of these compounds on oxidative stress
Leptin serves as an insulin-sensitizing factor in the wholebody [29] However hyperleptinemia in the obese mouseand human is a sign of leptin resistance where increasedleptin levels are caused by disturbed homeostasis arisingfrom leptin receptor mutation ageing or obesity [29] Thushyperleptinemia further worsens impaired insulin actionin pathological condition In these conditions exogenouslyadministered leptin does not improve glucose tolerance andinsulin sensitivity In our study diseased animal (HCD fedmouse) per se did not develop hyperleptinemia which mightbe the sign of early stage of metabolic abnormality associatedwith insulin resistance At this state test compounds aswell as pioglitazone reversed the elevated glucose TG TCand insulin levels while facilitating leptin profile along withinsulin sensitivity Subsequently compound 3a treatmentresulted in increased energy expenditure demonstrated byelevated food efficiency ratio (FER) However reductionin body weight among the treated groups was not foundproportionate to the circulating leptin levels which suggeststhat increased leptin level in peripheral blood is not suffi-cient to induce a proportionate reduction on body weightA few questions remain unanswered such as (i) whetherthe compounds directly raised the leptin levels or are theresults due to indirect impact on metabolic signaling (ii) In
metabolic disorder combined with hyperleptinemia how dothese compounds affect leptin signaling Future studies areneeded to address these issues
No single mechanism would suffice to explain the ben-eficial effects of the test compounds They do not seem toact through insulinotropic activity unlike the sulfonylureasThey have no significant effect on adiponectin levels rulingout any involvement of this mechanism The increase in thelevel of serum leptin might point to the involvement of leptinin the antihyperlipidemic and antidiabetic potentials of thesemolecules
In conclusion thiazolidin-4-one derivatives act throughmultiple mechanisms to correct the metabolic abnormalitiesin type-2 diabetes In the present work compounds 2e and3a were found to be the most effective test compoundsto ameliorate insulin resistance and development of type-2diabetes
Conflict of Interests
The authors declare that they do not have any conflict ofinterests
Acknowledgment
The authors wish to thank All India Council for TechnicalEducation (AICTE) New Delhi India for providing thefinancial support and Manipal University Manipal Kar-nataka India for providing the facilities to carry out thepresent work
References
[1] S M Grundy I J Benjamin G L Burke et al ldquoDiabetes andcardiovascular disease a statement for healthcare professionalsfrom the american heart associationrdquo Circulation vol 100 no10 pp 1134ndash1146 1999
[2] K E Thorpe and D H Howard ldquoThe rise in spending amongMedicare beneficiaries the role of chronic disease prevalenceand changes in treatment intensity Increasing numbers ofbeneficiaries being treated for five or more conditions a year aredriving Medicare spending upwardrdquo Health Affairs vol 25 no5 pp w378ndashw388 2006
[3] S M Grundy ldquoDrug therapy of the metabolic syndrome min-imizing the emerging crisis in polypharmacyrdquo Nature ReviewsDrug Discovery vol 5 no 4 pp 295ndash309 2006
[4] M Navab N Gharavi and A D Watson ldquoInflammation andmetabolic disordersrdquo Current Opinion in Clinical Nutrition andMetabolic Care vol 11 no 4 pp 459ndash464 2008
[5] G S Hotamisligil ldquoInflammation and metabolic disordersrdquoNature vol 444 no 7121 pp 860ndash867 2006
[6] M Schmuth V Moosbrugger-Martinz S Blunder and SDubrac ldquoRole of PPAR LXR and PXR in epidermal homeosta-sis and inflammationrdquo Biochimica et Biophysica Acta vol 1841no 3 pp 463ndash473 2014
[7] J A Panetta J K Shadle M L Phillips D N Benslayand P P K Ho ldquo4-thiazolidinones potent antioxidants asantiinflammatory agentsrdquo Annals of the New York Academy ofSciences vol 696 pp 415ndash416 1993
14 BioMed Research International
[8] T Kato T Ozaki K Tamura Y Suzuki M Akima and NOhi ldquoNovel calcium antagonists with both calcium overloadinhibition and antioxidant activity 1 2-(35-Di-tert-butyl-4-hydroxyphenyl)-3- (aminopropyl)thiazolidinonesrdquo Journal ofMedicinal Chemistry vol 41 no 22 pp 4309ndash4316 1998
[9] JM Joy N Jacob andGN Kutty ldquoEvaluation of hypoglycemiceffects of 4-thiazolidinonesrdquo Indian Drugs vol 42 no 1 pp 17ndash21 2005
[10] N Jacob and G N Kutty ldquoSynthesis and hypolipidemic activityof a thiazolidinone derivativerdquo Indian Drugs vol 41 no 2 pp76ndash79 2004
[11] G K Nampurath S P Mathew V Khanna R T Zachariah SKanji andMRChamallamudi ldquoAssessment of hypolipidaemicactivity of three thiazolidin-4-ones in mice given high-fat dietand fructoserdquoChemico-Biological Interactions vol 171 no 3 pp363ndash368 2008
[12] A Kishore G K Nampurath S P Mathew et al ldquoAntidiabeticeffect through islet cell protection in streptozotocin diabetesa preliminary assessment of two thiazolidin-4-ones in Swissalbino micerdquo Chemico-Biological Interactions vol 177 no 3 pp242ndash246 2009
[13] J Mudgal V S Gowdra G Mathew et al ldquoRemedial effects ofnovel 2 3-disubstituted thiazolidin-4-ones in chemical medi-ated inflammationrdquo Chemico-Biological Interactions vol 210pp 34ndash42 2014
[14] A R Surrey ldquo4-Thiazolidones IV the preparation of some3-alkylaminoalkyl-2-aryl derivativesrdquo Journal of the AmericanChemical Society vol 71 no 10 pp 3354ndash3356 1949
[15] B R P Kumar and M J Nanjan ldquoNovel glitazones designsynthesis glucose uptake and structure-activity relationshipsrdquoBioorganic and Medicinal Chemistry Letters vol 20 no 6 pp1953ndash1956 2010
[16] M Sumiyoshi M Sakanaka and Y Kimura ldquoChronic intakeof high-fat and high-sucrose diets differentially affects glucoseintolerance inmicerdquo Journal of Nutrition vol 136 no 3 pp 582ndash587 2006
[17] S H Kim S H Hyun and S Y Choung ldquoAnti-diabetic effectof cinnamon extract on blood glucose in dbdb micerdquo Journalof Ethnopharmacology vol 104 no 1-2 pp 119ndash123 2006
[18] S A Manohara Reddy J Mudgal P Bansal et al ldquoAntioxidantanti-inflammatory and anti-hyperglycaemic activities of hete-rocyclic homoprostanoid derivativesrdquoBioorganic andMedicinalChemistry vol 19 no 1 pp 384ndash392 2011
[19] S T Shukla V H Kulkarni P V Habbu K S Jagadeesh BS Patil and D M Smita ldquoHepatoprotective and antioxidantactivities of crude fractions of endophytic fungi of Ocimumsanctum Linn in ratsrdquo Oriental Pharmacy and ExperimentalMedicine vol 12 no 2 pp 81ndash91 2012
[20] B Mannervik ldquoGlutathione peroxidaserdquo Methods in Enzymol-ogy vol 113 pp 490ndash495 1985
[21] H PMisra and I Fridovich ldquoThe role of superoxide anion in theautoxidation of epinephrine and a simple assay for superoxidedismutaserdquo Journal of Biological Chemistry vol 247 no 10 pp3170ndash3175 1972
[22] A Claiborne ldquoCatalase activityrdquo in Handbook of Methods forOxygen Radical Research R A Greenwald Ed pp 283ndash284CRC Press Boca Raton Fla USA 1985
[23] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974
[24] W G Niehaus Jr and B Samuelsson ldquoFormation of malonalde-hyde from phospholipid arachidonate during microsomal lipidperoxidationrdquoEuropean Journal of Biochemistry vol 6 no 1 pp126ndash130 1968
[25] O S Osman J L Selway M A Kępczynska et al ldquoA novelautomated image analysis method for accurate adipocyte quan-tificationrdquo Adipocyte vol 2 no 3 pp 160ndash164 2013
[26] R Kalia C M Rao and N Gopalan Kutty ldquoSynthesisand evaluation of the anti-inflammatory activity of N-[2-(35-di-tert-butyl-4-hydroxyphenyl)-4-oxothiazolidin-3-yl]-nicotinamiderdquo Drug Research vol 57 no 9 pp 616ndash6222007
[27] N Maeda M Takahashi T Funahashi et al ldquoPPAR120574 ligandsincrease expression and plasma concentrations of adiponectinan adipose-derived proteinrdquo Diabetes vol 50 no 9 pp 2094ndash2099 2001
[28] P Bansal P Paul J Mudgal et al ldquoAntidiabetic antihyperlipi-demic and antioxidant effects of the flavonoid rich fraction ofPilea microphylla (L) in high fat dietstreptozotocin-induceddiabetes in micerdquo Experimental and Toxicologic Pathology vol64 no 6 pp 651ndash658 2012
[29] C Perez C Fernandez-Galaz T Fernandez-Agullo et alldquoLeptin impairs insulin signaling in rat adipocytesrdquo Diabetesvol 53 no 2 pp 347ndash353 2004
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MEDIATORSINFLAMMATION
of
4 BioMed Research International
Table 1 Continued
S no Compound code R
16 3c OH35-di-t-Butyl-4-hydroxyphenyl
17 4aCl
Cl
34-Dichlorophenyl
18 4b Cl 4-Chlorophenyl
19 4cN 2-Pyridinyl
20 4d4-Methoxyphenyl
OCH3
2922 1710 1678 1599 1230 1163 1070 1008 829 752 671UV 120582max (MeOH) 281 nm LC-MS (ESI) 119898119911 381 (Calcd forC17H14Cl F N
2O3S 38082)
229 2-(4-Chlorophenoxy)-N-(4-oxo-2-(4-(trifluoromethyl)phenyl)thiazolidin-3-yl)acetamide (Compound 2i) 1HNMR(CDCl
3) 120575 389 (s 2H CH
2) 465 (s 2H CO-CH
2-O) 595
(s 1H CH) 786 (m 8H Ar) 800 (br s 1H NH) IR (KBr)cmminus1 3275 3090 2937 1735 1716 1595 1228 1163 1055 960850 666UV 120582max (MeOH) 283 nm LC-MS (ESI) 119898119911 431(Calcd for C
18H14Cl F3N2O3S 43083)
2210 2-(4-Chlorophenoxy)-N-(4-oxo-2-p-tolyl thiazolidin-3-yl)acetamide (Compound 2j) 1HNMR (CDCl
3) 120575 290 (s
3H CH3) 370 (s 2H CH
2) 442 (s 2H CO-CH
2-O) 590
(s 1H CH) 695 (m 7H Ar) 805 (br s 1H NH) IR (KBr)cmminus1 3217 2989 1722 1676 1589 1292 1238 1220 1170 1060962 821 740 667 UV 120582max (MeOH) 279 nm LC-MS (ESI)119898119911 377 (Calcd for C
18H17Cl N2O3S 37686)
2211 2-(4-Chlorophenoxy)-N-(2-(34-dimethoxyphenyl)-4-oxothiazolidin-3-yl)acetamide (Compound 2k) 1HNMR(CDCl
3) 120575 385 (s 2H CH
2) 388 t 6H [(OCH
3)2] 420
(s 2H CO-CH2-O) 459 (s 2H CH
2) 589 (s 1H CH)
720 (m 7H Ar) 800 (br s 1H NH) IR (KBr) cmminus1 34483026 2974 1703 1662 1593 1236 1072 950 821 796 669UV 120582max (MeOH) 279 nm LC-MS (ESI)119898119911 423 (Calcd forC19H19Cl N2O5S 42288)
2212 N-(2-(4-Bromophenyl)-4-oxothiazolidin-3-yl)-2-(4-chlorophenoxy)acetamide (Compound 2l) 1HNMR (CDCl
3)
120575 305 (s 2H CH2) 378 (s 2H CO-CH
2-O) 564 (s 1H
CH) 672 (m 4H Ar) 715 (m 4H Ar) 784 (br s 1H NH)IR (KBr) cmminus1 3448 3051 2982 1714 1674 1587 1230 1176
1058 964 829 amp 673 UV 120582max (MeOH) 278 nm LC-MS(ESI)119898119911 442 (Calcd for C
17H14Br Cl N
2O3S 44173)
2213 2-(4-Chlorophenoxy)-N-(2-(34-dichlorophenyl)-4-ox-othiazolidin-3-yl)acetamide (Compound 2m) 1HNMR(CDCl
3) 120575 329 (s 2H -CH
2) 388 (s 2H -CH
2-CO) 564
(s 1H CH) 682 (m 3H Ar) 733 (m 4H Ar) 852 (br s1H NH) IR (KBr) cmminus1 3228 3022 2914 1730 1687 15851481 1369 1219 1130 1060 960 823 758 667 UV 120582max(MeOH) 279 nm LC-MS (ESI) 119898119911 432 (Calcd for C
17H13
Cl3N2O3S 43172)
2214 2-(4-Chlorophenoxy)-N-(2-(34-dichlorophenyl)-5-methyl-4-oxothiazolidin-3-yl)acetamide (Compound 4a)1HNMR (CDCl
3) 120575 153 (s 3H -CH
3) 367 (1H -CH-CH
3)
390 (s 2H -CH2-) 568 (s 1H -CH) 695 (m 3H Ar) 725
(m 4H Ar1015840) 836 (br s 1H -NH) IR (KBr) cmminus1 3277 30502980 1726 1695 1585 1490 1378 1217 1126 1060 966 821738 638 UV 120582max (MeOH) 278 nm LC-MS (ESI) 119898119911 448(Calcd for C
18H15Cl3N2O3S 44575)
2215 2-(4-Chlorophenoxy)-N-(2-(4-chlorophenyl)-5-methyl-4-oxothiazolidin-3-yl)acetamide (Compound 4b) 1HNMR(CDCl
3) 120575 155 (s 3H -CH
3) 340 (1H -CH-CH
3) 392 (s
2H -CH2-) 569 (s 1H -CH) 677 (m 4H Ar) 703 (m 4H
Ar1015840) 766 (br s 1H -NH) IR (KBr) cmminus1 3279 3057 29801726 1693 1585 1383 1205 1168 1070 962 773 636 UV 120582max(MeOH) 279 nm LC-MS (ESI) 119898119911 411 (Calcd for C
18H16
Cl2N2O3S 41130)
2216 2-(4-Chlorophenoxy)-N-(5-methyl-4-oxo-2-(pyridin-2-yl)thiazolidin-3-yl)acetamide (Compound 4c) 1HNMR(CDCl
3) 120575 145 (s 3H -CH
3) 355 (1H -CH-CH
3) 389 (s
2H-CH2) 610 (s 1H -CH) 652 (m 4H Pyridine) 690
BioMed Research International 5
Cl O C
Cl O C-CONH
a b ca b c
d e
N CH
R
N
S
O
H R
a b c
Cl O C-CONH N CH
R
d f
Cl O N
S
O
H R
Cl O C-CONH
Schiff rsquos bases
Schiff rsquos bases
Schiff rsquos bases
N CH
R
c d
Cl O N
S
O
H R
H2
Cl O C-CONHH2
H2
C-CONHH2
R1
R2
CONHNH2
R1 = R2 = H or CH3
CH3
CH3
CH3
C-CONH
CH3
CH3R = 2-furyl (2a)= 4-chlorophenyl (2b)= 35 di-t-butyl-4-hydroxyphenyl (2c)= 2-nitrophenyl (2d)= 4-methoxyphenyl (2e)= 2-chlorophenyl (2f)= 2-thiophenyl (2g)= 4-flurophenyl (2h)= 4-trifluromethylphenyl (2i)= 4-methylphenyl (2j)= 34-dimethoxyphenyl (2k)= 4-bromophenyl (2l)= 34-dichlorophenyl (2m)
R = 34-dichlorophenyl (4a)= 4-chlorophenyl (4b)= 2-pyridinyl (4c)= 4-methoxyphenyl (4d)
R = 4-methoxyphenyl (3a)= 4-flurophenyl (3b)= 35 di-t-butyl-4-hydroxyphenyl (3c)
Figure 1 Scheme for the synthesis of thiazolidin-4-ones Reagents and conditions (a) aromatic aldehydes (b) methanol (c) glacial aceticacid reflux for 15ndash45 minuts (d) dry benzenetoluene (e) thioglycolic acid reflux for 24ndash48 hrs or microwave irradiation at power setting of80 with 3 minutescycle for 16 minutes and (f) thiolactic acid
(m 4H Ar) 845 (br s 1H -NH) IR (KBr) cmminus1 3159 29661732 1691 1587 1496 1342 1238 1180 1060 997 829 754 661UV 120582max (MeOH) 263 nm LC-MS (ESI)119898119911 378 (Calcd forC17H16Cl N3O3S 37785)
2217 2-(4-Chlorophenoxy)-N-(2-(4-methoxyphenyl)-5-meth-yl-4-oxothiazolidin-3-yl)acetamide(Compound 4d) 1HNMR(CDCl
3) 120575 324 (s 3H -CH
3) 397 (s 3H -CH-CH
3) 594 (s
1H -CH) 666 (m 4H Ar) 715 (m 4H Ar1015840) 815 (br s 1H-NH) IR (KBr) cmminus1 3281 2935 1735 1683 1587 1383 12441165 1064 964 821 744 636 UV 120582max (MeOH) 280 nm LC-MS (ESI)119898119911 407 (Calcd for C
19H19Cl N2O4S 40688)
23 General Synthetic Procedure for 2-(4-Chlorophenoxy)-2-Methylpropanoic Acid Derivatives [14] Ethyl-2-(4-chlo-rophenoxy)-2-methylpropanoate was synthesized byesterifying 2-(4-chlorophenoxy)-2-methylpropanoic acid
(clofibric acid 006mol) with ethanol The ester wasconverted into hydrazide by reacting ethyl 2-(4-chloro-phenoxy)-2-methylpropanoate (01mol) with hydrazine hyd-rate (01mol) Schiff rsquos bases were prepared by reacting 2-(4-chlorophenoxy)-2-methylpropanehydrazide (01mol) withAr aldehydes (01mol) like anisaldehyde p-fluorobenzal-dehyde and 35-di-tert-butyl-4-hydroxybenzaldehyde inpresence of 4-5 drops of glacial acetic acid
Schiff rsquos base (001mol) and thioglycolic acid (002mol)were refluxed in dry benzene The water formed duringcyclization was removed azeotropically The completion ofthe reaction was checked chromatographically The periodof reflux varied from compound to compound After thecompletion of the reaction the solvent was distilled off underreducedpressureThe reactionmixturewas poured intowatercontaining sodium bicarbonate solution The solid separatedwas filtered dried and recrystallized from methanol asdescribed schematically in Figure 1
6 BioMed Research International
231 2-(4-Chlorophenoxy)-N-(2-(4-methoxyphenyl)-4-oxot-hiazolidin-3-yl)-2-methylpropanamide (Compound 3a)1HNMR (CDCl
3) 120575 = 137 (t 3H -CH
3) 147 (t 3H - CH
3)
370 (t 3H OCH3) 384 (s 2H CH
2) 590 (s 1H CH) 735
(m 8H Ar) 817 (br s 1H -NH) ppm IR (KBr) 3281 (-NH)3080 (Ar C-H Str) 2892 1710 amp 1680 (-C=O of -CH
2-C=O
amp -CONH) 1612 (C=C Str) 1251 (C-O Str) 1143 1095(C-Cl) 848 817 709 amp 663 (C-S-C) cmminus1 respectively MS(ESI)119898119911 = 421
232 2-(4-Chlorophenoxy)-N-(2-(4-fluorophenyl)-4-oxot-hiazolidin-3-yl)-2-methylpropanamide (Compound 3b)1HNMR (CDCl
3) 120575 = 137 (t 3H -CH
3) 147 (t 3H -CH
3)
385 (s 2H CH2) 590 (s 1H CH) 743 (m 8H Ar) 810 (br
s 1H -NH) ppm IR (KBr) 3250 (-NH) 3043 (Ar C-H Str)2985 1726 amp 1674 (-C=O of -CH
2-C=O amp -CONH) 1599
(C=C Str) 1228 (C-O Str) 1157 (C-F) 1085 (C-Cl) 937 850amp 648 (C-S-C) cmminus1 respectively MS (ESI)119898119911 = 409
233 2-(4-Chlorophenoxy)-N-(2-(35-di-tert-butyl-4-hydrox-yphenyl)-4-oxothiazolidin-3-yl)-2-methylpropanamide (Com-pound 3c) 1HNMR(CDCl
3) 120575= 128 (s 6H (CH
3)2) 142 (s
18H (CH3)6) 344 (s 2H -CH
2-CO-) 454 (s 1H -OH) 517
(s 2H Ar) 623 (s 1H CH) 757 (m 4H Ar) ppm IR (KBr)3636 (-OH) 3543 (-NH) 3001 (Ar C-H Str) 2955 1740 amp1697 (-C=O of -CH
2-C=O amp -CONH) 1600 (C=C Str) 1236
(C-O Str) 1209 1024 (C-Cl) 887 773 amp 626 (C-S-C) cmminus1respectively MS (ESI)119898119911 = 518
24 Experimental Subjects Male Swiss albino mice (22ndash25 g) and Wistar rats (220ndash250 g) (inbred in Central AnimalResearch Facility Manipal University Manipal KarnatakaIndia) were used At a temperature of 25 plusmn 05∘C animalswere housed in plastic cages with 12 h lightdark cycle andhumidity 50 plusmn 5 RH Animals were given standard foodpellet and water ad libitum The experimental protocolswere approved by the Institutional Animal Ethics Committee(no IAECKMC252009-2010) and the experiments werecarried out in accordance with the guidelines provided bythe Committee for the Purpose of Control and Supervision ofExperiments on Animals (CPCSEA) Government of India
25 In Vitro Glucose Uptake Assay [15] Overnight fastedmaleWistar rats (220ndash250 g) were sacrificed and diaphragmswere isolated avoiding trauma and divided into two hemidi-aphragms After isolation blood clots were removed by rins-ing the hemidiaphragms in cold Tyrodersquos solution (withoutglucose) and transferred to the mammalian organ bath con-taining Tyrodersquos solution with 05wv glucose withwithoutthe teststandard drug (1mM) followed by incubation for45min at 37 plusmn 1∘C in presence of aeration After theincubation the glucose content of the incubated organ bathwas measured spectrophotometrically using colorimetrickits (Aspen Laboratories Pvt Ltd New Delhi India) Thedifference between the initial and final glucose amount wasconsidered as amount of glucose uptake (mgg of tissueweight45min)
Table 2 Composition of high carbohydrate diet (HCD)
Sl no Contents Percent (ww)1 Cholesterol 22 Sucrose 553 Lard 34 Cellulose 3505 L-cysteine 0256 Choline bitartrate 0507 DL-methionine 0258 Vitamin and mineral mixture 0109 Normal pellet diet 3540
The following groups were made for screening the com-pounds Group 1 5mLof Tyrodersquos solutionwith 05wv glu-cose (glucose control in absence of insulin) Group 2 5mL ofTyrodersquos solution with 05wv glucose and regular insulin(Novo Nordisk India Pvt Ltd Bangalore India 40 IUmL)25 120583L containing 05 units of insulin (glucose control inpresence of insulin) Groups 3 to 23 5mL of Tyrodersquos solutionwith 05wv glucose and 1mM test compound (total num-bers = 20) Group 24 5mL of Tyrodersquos solution with 05wvglucose + 1mMpioglitazone (standard) Group 25 to 45 5mLof Tyrodersquos solution with 05wv glucose + regular insulin25 120583L containing 05 units of insulin + 1mM test drug (totalnumbers = 20) Group 46 5mL of Tyrodersquos solution with05wv glucose + regular insulin 25120583L containing 05 unitsof insulin + 1mM pioglitazone (standard)
26 Acute Oral Toxicity Study An acute toxicity study wasperformed on Swiss albino mice according to OECD 420guidelines according to the methods described earlier [13]All the newly synthesized test compounds were administeredat the dose of 2000mgkg po to the animals and observedfor any sign of toxicity as described earlier [13]
27 High Carbohydrate Diet- (HCD-) Induced Metabolic Dis-order in Mice [16] Four-week-old male Swiss albino micewere placed on the feeding of high sucrose diet (HCD)The detailed composition of diet where 55 energy source(kilocalories) was from sucrose is given in Table 2
After 24 weeks of HCD feeding mice displayed hyper-glycemia Animals with similar degrees of hyperglycemiawere randomly divided into six groups (119899 = 6) Thenormal pellet diet (NPD) fed mice were used as nondiabeticcontrols The diabetic control (HCD) and the normal control(NPD) groups received the vehicle (025CMC 10mLkg)while the treatment groups were given pioglitazone (5mgkgpo) and compound 2e and compound 3a (100mgkg po)respectively suspensions in 025wv CMC (10mLkg) Allthe treatments were given for 30 days Animalsrsquo body weightand cumulative food intake were recorded periodicallyFood efficiency ratio (FER) was calculated according to thereported method [17] using the following formula FER =change in body weight (day 0ndashday 30)cumulative foodconsumed in 30 days per animal Final body weight on day30 and FER were reported Plasma glucose triglycerides and
BioMed Research International 7
total cholesterol were monitored on day 14 and day 30 afterthe treatment using colorimetric kits (Aspen LaboratoriesPvt Ltd New Delhi India) Plasma insulin (Linco ResearchInc St Charles MO USA) leptin (BioVendor LLC CandlerNC USA) and adiponectin (Adipogen Corporation SanDiego CA USA) were estimated on day 30 using ELISAkits according to manufacturerrsquos instructions OGTT wasperformed on day 32 as described below On day 32 animalswere sacrificed and liver pancreas and white adipose tissues(from epididymal fat WAT) were isolated for histopatholog-ical investigation
271 Oral Glucose Tolerance Test (OGTT) in DiabeticMice [18] OGTT was performed according to the methoddescribed previously [18] In brief animals were fastedovernight and distilled water or glucose load of 2 gkgpo was administered and blood samples were collectedby retroorbital plexus puncture at 0 30 60 and 120minafter glucose challenge Plasma glucose was measured spec-trophotometrically using commercially available colorimet-ric kits (Aspen Laboratories Pvt Ltd New Delhi India) Thepercentage reduction in glucose excursion (AUC
0minus120 min)produced by test compounds was calculated from the areaunder the curve (AUC
0minus120 min) The results are expressedin time-dependent plasma glucose (mgdL) levels plasmaglucose (AUC
0minus120 min) and reduction in glucose excursion(AUC
0minus120 min)
272 Endogenous Liver Antioxidant Enzymes EstimationAnimals were sacrificed by cervical dislocation on the 32ndday after treatment Transcardial and whole liver perfusionwere performed using ice-cold saline [19] Liver was isolatedand 10wv homogenate was prepared using Teflon-glasshomogenizer (RQ-127AD REMI Group Mumbai India)with ice-cold saline-EDTAThe homogenate was centrifugedat 10000 rpm for 10min supernatant was collected andcentrifuged again at 20000 rpm for 1 h at 4∘C The super-natant obtained was used for the estimation of glutathione(GSH) glutathione-S-transferase (GST) catalase superoxidedismutase (SOD) and malondialdehyde (MDA) All the datapresented as mean plusmn SEM (119899 = 6)
(i) Glutathione (GSH) Assay [20] From the liver homogenateproteins were precipitated by 10 tri-carboxylic acid (TCA)and then centrifuged to collect the supernatant One mLsupernatant was mixed with 6mL 02MpH 80 and 1mL06mM 551015840-dithiobis-(2-nitrobenzoic acid) (DTNB) andincubated for 10min at room temperature The absorbancewas recorded against the blank at 412 nm in aUV-visible spec-trophotometer (model UV-1650PC Shimadzu Co KyotoJapan) and the GSH concentration was calculated from thestandard curve
(ii) Superoxide Dismutase Assay [21] The entire 1mL super-natant was added to 01M carbonate buffer (pH 102) andthe increase in absorbance after addition of epinephrine was
measured at 480 nm using a UV-visible spectrophotome-ter (model UV-1650PC Shimadzu Co Kyoto Japan) Theenzyme activity was expressed as Umg protein
(iii) Catalase Assay [22] The catalase activity was deter-mined spectrophotometrically according to the protocol ofClaiborne [22] The reaction was started by adding 005mLsupernatant to the reaction mixture (195mL 10mMH
2O2in
60mM phosphate buffer pH 70) Absorbance was recordedfor 3min at 240 nm Phosphate buffer (60mM pH 70) waskept as a reference To determine the specific activity ofcatalase extinction coefficient of 004mMminus1 cmminus1 was used
(iv) Glutathione-S-Transferase (GST) Assay [23] 01mL 1-chloro-24-dinitrobenzene (CDNB) was added to 06mLsupernatant of liver homogenate and 22mL phosphate bufferpH 65 incubated at 37∘C for 5min and added 01mL 30mMGSH Absorbance was recorded at 340 nm at intervals of12345min Blank was carried out in the same mannerwithout homogenate
(v) Malondialdehyde Assay [24]OnemL of liver homogenatewas combined with 2mL of reaction mixture [15wvtrichloroacetic acid (TCA) and 0375wv thiobarbituricacid (TBA) in 025N hydrochloric acid (HCl)] and mixedthoroughly The solution was heated for 20min on boilingwater bath Samples were cooled and centrifuged at 1000 rpmfor 10min to remove the flocculent precipitate Super-natant was collected and the absorbance of read at 532 nmagainst a blank which contained all the reagents exceptthe liver homogenate The extinction coefficient of 156 times105Mminus1 cmminus1 was used to calculate the malondialdehydeconcentration
273 Histopathological Investigation For histological exam-inations liver pancreas and white adipose tissue (WAT)from three animals per group were isolated on day 32 aftertreatment of compounds The tissue samples were fixed informalin solution (10) for one week at room temperaturedehydrated by graded ethanol cleared using graded xyleneand embedded in paraffin wax 5 120583m thick sections werecut using rotary microtome (RM2245 Leica MicrosystemsGmbHWetzlar Germany) fixed on glass slides stained witheosin and hematoxylin and observed using a microscope(Model BX41 Olympus Corporation Tokyo Japan) ForLangerhans cells the average areas of six islets per specimenwere measured using ImageJ software (version 148 NationalInstitute of Health MD USA) Area was expressed as 120583m2The quantification of adipocytes were performed as reported[25] using adipocyte quantification tool where area wasmeasured in 120583m2
28 Statistical Analysis Statistical analysis was performedby comparing the responses of the treatment groups torespective saline control and vehicle treated groups for allexperiments and the significance was determined by one-way ANOVA followed by post hoc Dunnettrsquos test Values
8 BioMed Research International
Table 3 Effect of thiazolidin-4-ones on in vitro glucose uptake in absence and presence of insulin
S no GroupsGlucose uptake (mgg45min)
In absence of insulin In presence of insulinMean plusmn SEM (119899 = 3) Mean plusmn SEM (119899 = 3)
1 Glucose control 809 plusmn 051 1211 plusmn 1342 Compound 2a 189 plusmn 145 1033 plusmn 1213 Compound 2b 619 plusmn 031 736 plusmn 0254 Compound 2c 1012 plusmn 052 1488 plusmn 0865 Compound 2d 984 plusmn 061 816 plusmn 0116 Compound 2e 1900 plusmn 127a 2342 plusmn 032b
7 Compound 2f 603 plusmn 033 833 plusmn 0158 Compound 2g 645 plusmn 013 708 plusmn 0199 Compound 2h 761 plusmn 018 728 plusmn 03810 Compound 2i 555 plusmn 014 644 plusmn 11911 Compound 2j 954 plusmn 021 368 plusmn 17912 Compound 2k 582 plusmn 018 721 plusmn 01513 Compound 2l 643 plusmn 010 999 plusmn 01014 Compound 2m 812 plusmn 082 1287 plusmn 01515 Compound 3a 2044 plusmn 017a 2501 plusmn 029b
16 Compound 3b 777 plusmn 029 1151 plusmn 00217 Compound 3c 1224 plusmn 010 1050 plusmn 08118 Compound 4a 753 plusmn 110 798 plusmn 08319 Compound 4b 610 plusmn 083 772 plusmn 02120 Compound 4c 097 plusmn 059 482 plusmn 05521 Compound 4d 377 plusmn 047 554 plusmn 04922 Pioglitazone 1337 plusmn 026a 2523 plusmn 034b
a and b represent 119875 lt 005 as compared to glucose control in absencepresence of insulin respectively
Table 4 Effect of thiazolidin-4-ones on body weight and food efficiency ratio in mice
NPD HCD Pioglitazone Compound 2e Compound 3aFinal body weight (g) 380 plusmn 21 412 plusmn 22 382 plusmn 14 376 plusmn 12 382 plusmn 21Food efficiency ratio 00025 plusmn 0005 00083 plusmn 0006 00020 plusmn 0005 00043 plusmn 0011 0071 plusmn 0010abaRepresents 119875 lt 005 as compared to NPD group (normal pellet diet)bRepresents 119875 lt 005 as compared to HCD group (high carbohydrate diet)
were expressed as mean plusmn SEM 119875 lt 005 was consideredsignificant
3 Results
31 In Vitro Biological Study
311 Glucose Uptake Assay Of the twenty thiazolidin-4-ones tested compounds 2e and 3a stimulated glucose uptake(119875 lt 005) when compared with control Uptake wasstimulated both in the absence and presence of externalinsulin The standard drug pioglitazone also increased theglucose uptake (Table 3)
32 In Vivo Biological Studies
321 Acute Oral Toxicity Study Acute toxicity studies werecarried out on Swiss albino mice as per the OECD guidelines
for test compounds 2e and 3a Both were found to be safe upto 2000mgkg which was the maximum dose tested
322 High Carbohydrate Diet- (HCD-) Induced MetabolicDisorder in Mice Animals put on HCD for 6 months hadsignificant hyperglycemia hypertriglyceridemia and hyperc-holesterolemiaThey were randomized to different treatmentgroups based on plasma glucose level and then treatment wasgiven for 30 days Firstly treatment did not have any signif-icant effect on body weight of animals (Table 4) Secondlyenergy expenditure was assessed indirectly using FER Noneof the treated groups except compound 3a-treated group hadany significant effect on FER The calculated FER was foundto be significantly higher in animal treated with compound3a compared to normal control and HCD group High FERvalue indicates the increased energy expenditure in animalstreated with compound 3a
BioMed Research International 9
0
50
100
150
200
250
14 day 30 day
NPDHCD
lowast
lowast
lowastlowast
lowastlowast
lowastlowast lowastlowastlowastlowast
lowastlowast
Pioglitazone (5mgkg)
Plas
ma g
luco
se (m
gdL
)(M
eanplusmn
SE
Mn=6
)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
60
120
180
240
300
lowast
lowastlowastlowastlowast
lowastlowast
Plas
ma T
G (m
gdL
)(M
eanplusmn
SE
Mn=6
)
14 day 30 day
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
60
120
180
240
300
lowast
lowast
lowastlowast
lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowast
Plas
ma c
hole
stero
l (m
gdL
)(M
eanplusmn
SE
Mn=6
)
14 day 30 day
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 2 Effect of thiazolidin-4-ones on plasma (a) glucose (b) triglyceride (TG) (c) cholesterol inHCD fedmice Data presented asmean plusmnSEM (119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 as compared to HCD group
(i) Effect of Thiazolidin-4-Ones on Plasma Glucose Triglyc-eride and Cholesterol Level Biochemical estimation ofmetabolic markers such as plasma glucose (PG) triglycerides(TG) and total cholesterol (TC)was performed on day 14 andday 30 after the drug treatment and it was observed that HCDcontrol group consistently had significant increase in PG andTC levels on days 14 and 30 compared to respective dayrsquosnormal control group However TG levels were significantlyincreased only on day 30 compared to respective dayrsquos normalcontrol group Pioglitazone and test compounds (2e and3a) significantly reversed hyperglycemia and elevated plasmacholesterol compared to HCD control group on both day14 and day 30 of drug treatment (Figures 2(a) and 2(c))while elevated TG levels were reduced on day 30 by the testcompounds and pioglitazone (Figure 2(b))
(ii) Effect of Thiazolidin-4-Ones on Plasma Insulin Leptinand Adiponectin Level Development of insulin resistance
in mouse was confirmed by estimating the plasma insulinlevels on day 30 after drug treatment HCD control group incontrast to NPD group showed two times increase in plasmainsulin levels (119875 lt 005) Hyperinsulinemia associated withhyperglycaemia and hypertriglyceridemia is considered tobe the sign of development of insulin resistance Thus highsucrose feeding for 6 months led to the development ofinsulin resistance in mice Treatment with pioglitazone andtest drugs attenuated the insulin resistance (Figure 3(a))
HCD feeding to animals did not elicit any significanteffect on plasma leptin levels as there was no significantdifference between HCD control and NPD group Howevertreatment with pioglitazone and test compounds significantly(119875 lt 005) raised the circulating leptin level in micecomparedwithHCDcontrol group (Figure 3(b)) Compound3a caused a fourfold elevation in leptin levels compared withHCD group HCD feeding to animals resulted in significant
10 BioMed Research International
0
1
2
3
4
5
NPDHCDPioglitazone (5mgkg)
Plas
ma i
nsul
in (n
gm
L)(M
eanplusmn
SE
Mn=6
)
lowastlowast
lowast
lowastlowastlowastlowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
1200
2400
3600
4800
6000
NPDHCDPioglitazone (5mgkg)
Plas
ma l
eptin
(pg
mL)
(Mea
nplusmn
SE
Mn=6
)
lowast lowast
lowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
12000
24000
36000
48000
60000
NPDHCDPioglitazone (5mgkg)
Plas
ma a
dipo
nect
in (n
gm
L)(M
eanplusmn
SE
Mn=6
)
lowast
lowastlowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 3 Effect of thiazolidin-4-ones on plasma (a) insulin (b) leptin (c) adiponectin in HCD fed mice Data presented as mean plusmn SEM(119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 as compared to HCD group
(119875 lt 005) hypoadiponectinemia comparedwithNPD Treat-ment with pioglitazone significantly (119875 lt 005) corrected thehypoadiponectinemia However both test compounds failedto correct hypoadiponectinemia (Figure 3(c))
(iii) Effect of Thiazolidin-4-Ones on Oral Glucose ToleranceTest In oral glucose tolerance test HCD group animalshowed significant (119875 lt 005) glucose intolerance (Figure 4)Pioglitazone and test compounds compound 2e and com-pound 3a corrected the glucose intolerance shown as sig-nificant (119875 lt 005) percent reductions in glucose excursion(AUC
0minus120min) by 1300 plusmn 33 1546 plusmn 554 and 1560 plusmn 349respectively compared with HCD group (Figures 4(a) and4(b))
(iv) Effect of Thiazolidin-4-Ones on Liver Enzymes Oxidativestress is the hallmark of metabolic disorder where disturbedhomeostasis between oxidative and antioxidative mechanismoccurs HCD feeding to mice resulted in an oxidative stress
observed as reduction in liver antioxidant enzymes such asglutathione (GSH) catalase (CAT) superoxide dismutase(SOD) and glutathione-S-transferase (GST) Treatment withpioglitazone and test compounds ameliorated the oxidativestress They also reversed the elevation in liver malondialde-hyde (MDA) levels in HCD-fed mice (Table 5)
(v) Histopathological ExaminationHistology of liver showednormal lobular architecture with normal hepatocytes inall groups (Figure 5(a)) Pancreas showed mild to mod-erate hyperplasia of islets of Langerhans (Figure 5(a)) inpioglitazone and compound 3a groups Pioglitazone and3a treatment significantly raised the area of Langerhansislets compared with HCD group (Figure 5(b)) Exocrineportion of the pancreas and vascularity appeared normalin all the groups Histopathological investigation of whiteadipose tissue (WAT) from epidydimal showed an increasein the size of adipocytes (Figure 5(c)) in HCD group whichwas reversed by pioglitazone and compound 3a treatment
BioMed Research International 11
Table 5 Effect of thiazolidin-4-ones on liver antioxidant enzyme and malondialdehyde level
Liver biomarkers NPD HCD Pioglitazone Compound 2e Compound 3aGSH (nmolemg of protein) 558 plusmn 21 464 plusmn 21a 632 plusmn 15b 683 plusmn 20b 678 plusmn 43b
CAT (Umg of protein) 892 plusmn 57 554 plusmn 31a 938 plusmn 10b 819 plusmn 24b 875 plusmn 74b
SOD (Umg of protein) 1868 plusmn 232 1081 plusmn 112a 1748 plusmn 144b 2022 plusmn 47b 2080 plusmn 154b
GST (Umg of protein) 043 plusmn 005 037 plusmn 001a 073 plusmn 002b 070 plusmn 001b 072 plusmn 004b
MDA (nmolemg of protein) 057 plusmn 006 10 plusmn 033a 046 plusmn 001b 040 plusmn 001b 041 plusmn 002baRepresents 119875 lt 005 as compared to NPD group (normal pellet diet)bRepresents 119875 lt 005 as compared to HCD group (high carbohydrate diet)
0 30 60 90 120100
130
160
190
220
Plas
ma g
luco
se (m
gdL
)(M
eanplusmn
SE
Mn=6
)
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
5000
10000
15000
20000
25000
(Mea
nplusmn
SE
Mn=6
)
lowast
lowastlowast lowastlowast lowastlowast
NPDHCDPioglitazone (5mgkg)
Plas
ma g
luco
se (A
UC 0
ndash120
min)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
Figure 4 Effect of thiazolidin-4-ones on (a) plasma glucose (mgdL) and (b) area under the curve (AUC0minus120 min) against oral glucose
tolerance test (OGTT) in HCD fed mice Data presented as mean plusmn SEM (119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD groupand lowastlowastrepresents 119875 lt 005 as compared to HCD group
However compound 2e did not show any significant effecton increased adipocyte size
4 Discussion and Conclusion
The present work was planned as a sequel to earlier studiesin our laboratory using thiazolidin-4-ones as antidiabetichypolipidemic and antiinflammatory molecules The substi-tutions in the thiazolidine ring were made at C2 and N3Attached to the latter position was a nicotinamide moietyand the substitution at C2 was either p-methoxyphenyl or2 5-di-tert-butyl-4-hydroxyphenyl group The compoundsshowed significant antidiabetic and hypolipidemic activities[9ndash11 26]
In the present study the substitution at N3 was changedto p-chloro-phenoxyacetylamino a group that is similar toclofibrate with a methylene bridge instead of gem-dimethylsubstitution
Four compounds out of 20 (ie compounds 4a 4b4c and 4d) had a methyl group attached at the C5 ofthiazolidine ring with cyclisation being made with thiolacticacid instead of thioglycolic acid In three other compounds(ie compounds 3a 3b and 3c) clofibrate was used to make
themoiety for substitution atN3Thiswas donewith a view toexamine the effect of the gem-dimethyl on the overall activityof the resulting molecule
All the 20 synthesised thiazolidin-4-ones were evaluatedfor glucose uptake in an in vitro system using the isolatedrat diaphragm In this experiment compounds 2e and 3asignificantly raised the amount of glucose uptake by thetissue both in the absence and presence of external insulinThis indicated the potential of the theses compounds insensitizing the tissues for the external insulin Hence thesetwo compounds were chosen to study their effect on a diet-induced model of insulin resistance Both compounds havethe same p-methoxyphenyl moiety attached to the C2 of thethiazolidine ring Compound 3a has a gem-dimethyl groupinstead of methylene in the substituent at N3
Apart from multiple risk factors diet-induced metabolicabnormalities contribute to the development of insulinresistance and 120573-cell failure in type-2 diabetes [16] Earlydetection and appropriate treatment are considered beneficialfor correcting the abnormality Among the various animalmodels induction of diabetes through diet provides moreresemblance to human type-2 diabetes Chronic intake ofdiet with high sucrose content has been reported to favourthe development of insulin resistance [16] Similarly in our
12 BioMed Research International
NPD HCD Pioglitazone Compound 3a Compound 2e
Liver
WAT
Green and red arrows indicate hyperplasia and increased size of adipocytes respectively
Pancreas
(a)
0
50000
100000
150000
200000
NPDHCDPioglitazone (5mgkg)
lowastlowastlowastlowast
(Mea
nplusmn
SE
Mn=3
)A
rea o
f lan
gerh
ans i
stle
ts (120583
m2)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
750
1500
2250
3000
NPDHCDPioglitazone (5mgkg)
lowastlowastlowastlowast
lowast
(Mea
nplusmn
SE
Mn=3
)A
rea o
f adi
pocy
tes (120583
m2)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 5 (a) Histological sections of mouse liver pancreas and white adipose tissue (WAT) in HCD model (10x) Green and red arrowsindicate hyperplasia of islets of Langerhans and increase in the size of adipocytes respectively Effect of thiazolidin-4-ones on (b) area ofLangerhans islets (120583m2) and (c) area of adipocytes (120583m2) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 ascompared to HCD group
study mice fed a high-sucrose diet for six months devel-oped metabolic abnormalities like hyperglycemia hyperin-sulinemia hypertriglyceridemia hypercholesterolemia andhypoadiponectinemia Hyperglycemia and hyperinsulinemiasuggest the inability of insulin to sensitize the tissue forglucose uptake allowing glucose to be diverted toward lipo-genesisThis led to hyperlipidemia and finally to insulin resis-tance In addition adiponectin and leptin the adipokinessecreted from white adipose tissue (WAT) are reported tobe involved in the metabolism of glucose and lipid [27]In our study we observed that HCD caused a reductionin plasma adiponectin level and an increase in the sizeof adipocytes However it did not cause any change inplasma leptin levels Thus we found a correlation between
the adiponectin level and size of adipocytes in HCD modelFurther OGTT results from HCD-fed mice correlated wellwith glucose intolerance hyperinsulinemia and hypoad-iponectinemia The authenticity of the model was validatedby the effect of pioglitazone which was able to correct themetabolic abnormalities Neither test compounds (2e or 3a)attenuated hypoadiponectinemia However they correctedthe impaired glucose tolerance and insulin resistance inmice Compounds 2e and 3a by their ability to enhanceglucose uptake and to sensitize the tissue for available insulinreduced hyperinsulinemia and raised the leptin levels Thiswould have resulted in better glucose utilization by periph-eral tissue Thus these compounds reduced the metabolicabnormalities like hyperglycemia hypertriglyceridemia and
BioMed Research International 13
hypercholesterolemia Prospective studies involving directassessment of insulin sensitivity in OGTT are requiredto analyze glucose stimulated insulin secretion (GSIS) inpresence of compounds to establish the mechanistic role ofthe test compounds on insulin signalling
Histological investigation showed no change in liverarchitecture in the various treatment groups In HCD controlmice there was an increase in the size of adipocytes in whiteadipose tissue (WAT) which was reversed by compound3a and pioglitazone treatment However only pioglitazone-treated animals showed positive correlation between reducedsize of the cells and correction of hypoadiponectinemiaApart from correction of hypoadiponectinemia pioglitazoneraised the peripheral leptin levels which was also true in caseof compounds 2e and 3a treatment Among the tested com-pounds 3a raised endogenous leptin levels four times morethan HCD group This finding points to the link betweenraised leptin levels and reduced adipocyte size caused bycompound 3a The pancreatic islets showed hyperplasia inpioglitazone and other treatment groups Pioglitazone is aPPAR-120574 agonist This could have been responsible for theproliferation of beta cells of pancreas The test compoundsare thiazolidin-4-ones with some similarity to thiazolidine-diones It is possible that hyperplasia observed in compound3a-treated animals might have been due to some agonisticactivity on PPAR-120574 receptors This needs to be investigatedthrough relevant assay
Oxidative stress has been implicated in the occurrence ofdiabetes and compounds reducing the oxidative stress havebeneficial role in correcting glucose intolerance and insulinresistance in diabetes [18 28]The test compounds and piogli-tazone reversed the depletion of endogenous antioxidantenzymes such as GSH CAT SOD and GST Further theyreduced malondialdehyde levels This suggests the inhibitoryeffect of these compounds on oxidative stress
Leptin serves as an insulin-sensitizing factor in the wholebody [29] However hyperleptinemia in the obese mouseand human is a sign of leptin resistance where increasedleptin levels are caused by disturbed homeostasis arisingfrom leptin receptor mutation ageing or obesity [29] Thushyperleptinemia further worsens impaired insulin actionin pathological condition In these conditions exogenouslyadministered leptin does not improve glucose tolerance andinsulin sensitivity In our study diseased animal (HCD fedmouse) per se did not develop hyperleptinemia which mightbe the sign of early stage of metabolic abnormality associatedwith insulin resistance At this state test compounds aswell as pioglitazone reversed the elevated glucose TG TCand insulin levels while facilitating leptin profile along withinsulin sensitivity Subsequently compound 3a treatmentresulted in increased energy expenditure demonstrated byelevated food efficiency ratio (FER) However reductionin body weight among the treated groups was not foundproportionate to the circulating leptin levels which suggeststhat increased leptin level in peripheral blood is not suffi-cient to induce a proportionate reduction on body weightA few questions remain unanswered such as (i) whetherthe compounds directly raised the leptin levels or are theresults due to indirect impact on metabolic signaling (ii) In
metabolic disorder combined with hyperleptinemia how dothese compounds affect leptin signaling Future studies areneeded to address these issues
No single mechanism would suffice to explain the ben-eficial effects of the test compounds They do not seem toact through insulinotropic activity unlike the sulfonylureasThey have no significant effect on adiponectin levels rulingout any involvement of this mechanism The increase in thelevel of serum leptin might point to the involvement of leptinin the antihyperlipidemic and antidiabetic potentials of thesemolecules
In conclusion thiazolidin-4-one derivatives act throughmultiple mechanisms to correct the metabolic abnormalitiesin type-2 diabetes In the present work compounds 2e and3a were found to be the most effective test compoundsto ameliorate insulin resistance and development of type-2diabetes
Conflict of Interests
The authors declare that they do not have any conflict ofinterests
Acknowledgment
The authors wish to thank All India Council for TechnicalEducation (AICTE) New Delhi India for providing thefinancial support and Manipal University Manipal Kar-nataka India for providing the facilities to carry out thepresent work
References
[1] S M Grundy I J Benjamin G L Burke et al ldquoDiabetes andcardiovascular disease a statement for healthcare professionalsfrom the american heart associationrdquo Circulation vol 100 no10 pp 1134ndash1146 1999
[2] K E Thorpe and D H Howard ldquoThe rise in spending amongMedicare beneficiaries the role of chronic disease prevalenceand changes in treatment intensity Increasing numbers ofbeneficiaries being treated for five or more conditions a year aredriving Medicare spending upwardrdquo Health Affairs vol 25 no5 pp w378ndashw388 2006
[3] S M Grundy ldquoDrug therapy of the metabolic syndrome min-imizing the emerging crisis in polypharmacyrdquo Nature ReviewsDrug Discovery vol 5 no 4 pp 295ndash309 2006
[4] M Navab N Gharavi and A D Watson ldquoInflammation andmetabolic disordersrdquo Current Opinion in Clinical Nutrition andMetabolic Care vol 11 no 4 pp 459ndash464 2008
[5] G S Hotamisligil ldquoInflammation and metabolic disordersrdquoNature vol 444 no 7121 pp 860ndash867 2006
[6] M Schmuth V Moosbrugger-Martinz S Blunder and SDubrac ldquoRole of PPAR LXR and PXR in epidermal homeosta-sis and inflammationrdquo Biochimica et Biophysica Acta vol 1841no 3 pp 463ndash473 2014
[7] J A Panetta J K Shadle M L Phillips D N Benslayand P P K Ho ldquo4-thiazolidinones potent antioxidants asantiinflammatory agentsrdquo Annals of the New York Academy ofSciences vol 696 pp 415ndash416 1993
14 BioMed Research International
[8] T Kato T Ozaki K Tamura Y Suzuki M Akima and NOhi ldquoNovel calcium antagonists with both calcium overloadinhibition and antioxidant activity 1 2-(35-Di-tert-butyl-4-hydroxyphenyl)-3- (aminopropyl)thiazolidinonesrdquo Journal ofMedicinal Chemistry vol 41 no 22 pp 4309ndash4316 1998
[9] JM Joy N Jacob andGN Kutty ldquoEvaluation of hypoglycemiceffects of 4-thiazolidinonesrdquo Indian Drugs vol 42 no 1 pp 17ndash21 2005
[10] N Jacob and G N Kutty ldquoSynthesis and hypolipidemic activityof a thiazolidinone derivativerdquo Indian Drugs vol 41 no 2 pp76ndash79 2004
[11] G K Nampurath S P Mathew V Khanna R T Zachariah SKanji andMRChamallamudi ldquoAssessment of hypolipidaemicactivity of three thiazolidin-4-ones in mice given high-fat dietand fructoserdquoChemico-Biological Interactions vol 171 no 3 pp363ndash368 2008
[12] A Kishore G K Nampurath S P Mathew et al ldquoAntidiabeticeffect through islet cell protection in streptozotocin diabetesa preliminary assessment of two thiazolidin-4-ones in Swissalbino micerdquo Chemico-Biological Interactions vol 177 no 3 pp242ndash246 2009
[13] J Mudgal V S Gowdra G Mathew et al ldquoRemedial effects ofnovel 2 3-disubstituted thiazolidin-4-ones in chemical medi-ated inflammationrdquo Chemico-Biological Interactions vol 210pp 34ndash42 2014
[14] A R Surrey ldquo4-Thiazolidones IV the preparation of some3-alkylaminoalkyl-2-aryl derivativesrdquo Journal of the AmericanChemical Society vol 71 no 10 pp 3354ndash3356 1949
[15] B R P Kumar and M J Nanjan ldquoNovel glitazones designsynthesis glucose uptake and structure-activity relationshipsrdquoBioorganic and Medicinal Chemistry Letters vol 20 no 6 pp1953ndash1956 2010
[16] M Sumiyoshi M Sakanaka and Y Kimura ldquoChronic intakeof high-fat and high-sucrose diets differentially affects glucoseintolerance inmicerdquo Journal of Nutrition vol 136 no 3 pp 582ndash587 2006
[17] S H Kim S H Hyun and S Y Choung ldquoAnti-diabetic effectof cinnamon extract on blood glucose in dbdb micerdquo Journalof Ethnopharmacology vol 104 no 1-2 pp 119ndash123 2006
[18] S A Manohara Reddy J Mudgal P Bansal et al ldquoAntioxidantanti-inflammatory and anti-hyperglycaemic activities of hete-rocyclic homoprostanoid derivativesrdquoBioorganic andMedicinalChemistry vol 19 no 1 pp 384ndash392 2011
[19] S T Shukla V H Kulkarni P V Habbu K S Jagadeesh BS Patil and D M Smita ldquoHepatoprotective and antioxidantactivities of crude fractions of endophytic fungi of Ocimumsanctum Linn in ratsrdquo Oriental Pharmacy and ExperimentalMedicine vol 12 no 2 pp 81ndash91 2012
[20] B Mannervik ldquoGlutathione peroxidaserdquo Methods in Enzymol-ogy vol 113 pp 490ndash495 1985
[21] H PMisra and I Fridovich ldquoThe role of superoxide anion in theautoxidation of epinephrine and a simple assay for superoxidedismutaserdquo Journal of Biological Chemistry vol 247 no 10 pp3170ndash3175 1972
[22] A Claiborne ldquoCatalase activityrdquo in Handbook of Methods forOxygen Radical Research R A Greenwald Ed pp 283ndash284CRC Press Boca Raton Fla USA 1985
[23] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974
[24] W G Niehaus Jr and B Samuelsson ldquoFormation of malonalde-hyde from phospholipid arachidonate during microsomal lipidperoxidationrdquoEuropean Journal of Biochemistry vol 6 no 1 pp126ndash130 1968
[25] O S Osman J L Selway M A Kępczynska et al ldquoA novelautomated image analysis method for accurate adipocyte quan-tificationrdquo Adipocyte vol 2 no 3 pp 160ndash164 2013
[26] R Kalia C M Rao and N Gopalan Kutty ldquoSynthesisand evaluation of the anti-inflammatory activity of N-[2-(35-di-tert-butyl-4-hydroxyphenyl)-4-oxothiazolidin-3-yl]-nicotinamiderdquo Drug Research vol 57 no 9 pp 616ndash6222007
[27] N Maeda M Takahashi T Funahashi et al ldquoPPAR120574 ligandsincrease expression and plasma concentrations of adiponectinan adipose-derived proteinrdquo Diabetes vol 50 no 9 pp 2094ndash2099 2001
[28] P Bansal P Paul J Mudgal et al ldquoAntidiabetic antihyperlipi-demic and antioxidant effects of the flavonoid rich fraction ofPilea microphylla (L) in high fat dietstreptozotocin-induceddiabetes in micerdquo Experimental and Toxicologic Pathology vol64 no 6 pp 651ndash658 2012
[29] C Perez C Fernandez-Galaz T Fernandez-Agullo et alldquoLeptin impairs insulin signaling in rat adipocytesrdquo Diabetesvol 53 no 2 pp 347ndash353 2004
Submit your manuscripts athttpwwwhindawicom
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Tropical MedicineJournal of
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Medicinal ChemistryInternational Journal of
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AddictionJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Autoimmune Diseases
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Pharmaceutics
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MEDIATORSINFLAMMATION
of
BioMed Research International 5
Cl O C
Cl O C-CONH
a b ca b c
d e
N CH
R
N
S
O
H R
a b c
Cl O C-CONH N CH
R
d f
Cl O N
S
O
H R
Cl O C-CONH
Schiff rsquos bases
Schiff rsquos bases
Schiff rsquos bases
N CH
R
c d
Cl O N
S
O
H R
H2
Cl O C-CONHH2
H2
C-CONHH2
R1
R2
CONHNH2
R1 = R2 = H or CH3
CH3
CH3
CH3
C-CONH
CH3
CH3R = 2-furyl (2a)= 4-chlorophenyl (2b)= 35 di-t-butyl-4-hydroxyphenyl (2c)= 2-nitrophenyl (2d)= 4-methoxyphenyl (2e)= 2-chlorophenyl (2f)= 2-thiophenyl (2g)= 4-flurophenyl (2h)= 4-trifluromethylphenyl (2i)= 4-methylphenyl (2j)= 34-dimethoxyphenyl (2k)= 4-bromophenyl (2l)= 34-dichlorophenyl (2m)
R = 34-dichlorophenyl (4a)= 4-chlorophenyl (4b)= 2-pyridinyl (4c)= 4-methoxyphenyl (4d)
R = 4-methoxyphenyl (3a)= 4-flurophenyl (3b)= 35 di-t-butyl-4-hydroxyphenyl (3c)
Figure 1 Scheme for the synthesis of thiazolidin-4-ones Reagents and conditions (a) aromatic aldehydes (b) methanol (c) glacial aceticacid reflux for 15ndash45 minuts (d) dry benzenetoluene (e) thioglycolic acid reflux for 24ndash48 hrs or microwave irradiation at power setting of80 with 3 minutescycle for 16 minutes and (f) thiolactic acid
(m 4H Ar) 845 (br s 1H -NH) IR (KBr) cmminus1 3159 29661732 1691 1587 1496 1342 1238 1180 1060 997 829 754 661UV 120582max (MeOH) 263 nm LC-MS (ESI)119898119911 378 (Calcd forC17H16Cl N3O3S 37785)
2217 2-(4-Chlorophenoxy)-N-(2-(4-methoxyphenyl)-5-meth-yl-4-oxothiazolidin-3-yl)acetamide(Compound 4d) 1HNMR(CDCl
3) 120575 324 (s 3H -CH
3) 397 (s 3H -CH-CH
3) 594 (s
1H -CH) 666 (m 4H Ar) 715 (m 4H Ar1015840) 815 (br s 1H-NH) IR (KBr) cmminus1 3281 2935 1735 1683 1587 1383 12441165 1064 964 821 744 636 UV 120582max (MeOH) 280 nm LC-MS (ESI)119898119911 407 (Calcd for C
19H19Cl N2O4S 40688)
23 General Synthetic Procedure for 2-(4-Chlorophenoxy)-2-Methylpropanoic Acid Derivatives [14] Ethyl-2-(4-chlo-rophenoxy)-2-methylpropanoate was synthesized byesterifying 2-(4-chlorophenoxy)-2-methylpropanoic acid
(clofibric acid 006mol) with ethanol The ester wasconverted into hydrazide by reacting ethyl 2-(4-chloro-phenoxy)-2-methylpropanoate (01mol) with hydrazine hyd-rate (01mol) Schiff rsquos bases were prepared by reacting 2-(4-chlorophenoxy)-2-methylpropanehydrazide (01mol) withAr aldehydes (01mol) like anisaldehyde p-fluorobenzal-dehyde and 35-di-tert-butyl-4-hydroxybenzaldehyde inpresence of 4-5 drops of glacial acetic acid
Schiff rsquos base (001mol) and thioglycolic acid (002mol)were refluxed in dry benzene The water formed duringcyclization was removed azeotropically The completion ofthe reaction was checked chromatographically The periodof reflux varied from compound to compound After thecompletion of the reaction the solvent was distilled off underreducedpressureThe reactionmixturewas poured intowatercontaining sodium bicarbonate solution The solid separatedwas filtered dried and recrystallized from methanol asdescribed schematically in Figure 1
6 BioMed Research International
231 2-(4-Chlorophenoxy)-N-(2-(4-methoxyphenyl)-4-oxot-hiazolidin-3-yl)-2-methylpropanamide (Compound 3a)1HNMR (CDCl
3) 120575 = 137 (t 3H -CH
3) 147 (t 3H - CH
3)
370 (t 3H OCH3) 384 (s 2H CH
2) 590 (s 1H CH) 735
(m 8H Ar) 817 (br s 1H -NH) ppm IR (KBr) 3281 (-NH)3080 (Ar C-H Str) 2892 1710 amp 1680 (-C=O of -CH
2-C=O
amp -CONH) 1612 (C=C Str) 1251 (C-O Str) 1143 1095(C-Cl) 848 817 709 amp 663 (C-S-C) cmminus1 respectively MS(ESI)119898119911 = 421
232 2-(4-Chlorophenoxy)-N-(2-(4-fluorophenyl)-4-oxot-hiazolidin-3-yl)-2-methylpropanamide (Compound 3b)1HNMR (CDCl
3) 120575 = 137 (t 3H -CH
3) 147 (t 3H -CH
3)
385 (s 2H CH2) 590 (s 1H CH) 743 (m 8H Ar) 810 (br
s 1H -NH) ppm IR (KBr) 3250 (-NH) 3043 (Ar C-H Str)2985 1726 amp 1674 (-C=O of -CH
2-C=O amp -CONH) 1599
(C=C Str) 1228 (C-O Str) 1157 (C-F) 1085 (C-Cl) 937 850amp 648 (C-S-C) cmminus1 respectively MS (ESI)119898119911 = 409
233 2-(4-Chlorophenoxy)-N-(2-(35-di-tert-butyl-4-hydrox-yphenyl)-4-oxothiazolidin-3-yl)-2-methylpropanamide (Com-pound 3c) 1HNMR(CDCl
3) 120575= 128 (s 6H (CH
3)2) 142 (s
18H (CH3)6) 344 (s 2H -CH
2-CO-) 454 (s 1H -OH) 517
(s 2H Ar) 623 (s 1H CH) 757 (m 4H Ar) ppm IR (KBr)3636 (-OH) 3543 (-NH) 3001 (Ar C-H Str) 2955 1740 amp1697 (-C=O of -CH
2-C=O amp -CONH) 1600 (C=C Str) 1236
(C-O Str) 1209 1024 (C-Cl) 887 773 amp 626 (C-S-C) cmminus1respectively MS (ESI)119898119911 = 518
24 Experimental Subjects Male Swiss albino mice (22ndash25 g) and Wistar rats (220ndash250 g) (inbred in Central AnimalResearch Facility Manipal University Manipal KarnatakaIndia) were used At a temperature of 25 plusmn 05∘C animalswere housed in plastic cages with 12 h lightdark cycle andhumidity 50 plusmn 5 RH Animals were given standard foodpellet and water ad libitum The experimental protocolswere approved by the Institutional Animal Ethics Committee(no IAECKMC252009-2010) and the experiments werecarried out in accordance with the guidelines provided bythe Committee for the Purpose of Control and Supervision ofExperiments on Animals (CPCSEA) Government of India
25 In Vitro Glucose Uptake Assay [15] Overnight fastedmaleWistar rats (220ndash250 g) were sacrificed and diaphragmswere isolated avoiding trauma and divided into two hemidi-aphragms After isolation blood clots were removed by rins-ing the hemidiaphragms in cold Tyrodersquos solution (withoutglucose) and transferred to the mammalian organ bath con-taining Tyrodersquos solution with 05wv glucose withwithoutthe teststandard drug (1mM) followed by incubation for45min at 37 plusmn 1∘C in presence of aeration After theincubation the glucose content of the incubated organ bathwas measured spectrophotometrically using colorimetrickits (Aspen Laboratories Pvt Ltd New Delhi India) Thedifference between the initial and final glucose amount wasconsidered as amount of glucose uptake (mgg of tissueweight45min)
Table 2 Composition of high carbohydrate diet (HCD)
Sl no Contents Percent (ww)1 Cholesterol 22 Sucrose 553 Lard 34 Cellulose 3505 L-cysteine 0256 Choline bitartrate 0507 DL-methionine 0258 Vitamin and mineral mixture 0109 Normal pellet diet 3540
The following groups were made for screening the com-pounds Group 1 5mLof Tyrodersquos solutionwith 05wv glu-cose (glucose control in absence of insulin) Group 2 5mL ofTyrodersquos solution with 05wv glucose and regular insulin(Novo Nordisk India Pvt Ltd Bangalore India 40 IUmL)25 120583L containing 05 units of insulin (glucose control inpresence of insulin) Groups 3 to 23 5mL of Tyrodersquos solutionwith 05wv glucose and 1mM test compound (total num-bers = 20) Group 24 5mL of Tyrodersquos solution with 05wvglucose + 1mMpioglitazone (standard) Group 25 to 45 5mLof Tyrodersquos solution with 05wv glucose + regular insulin25 120583L containing 05 units of insulin + 1mM test drug (totalnumbers = 20) Group 46 5mL of Tyrodersquos solution with05wv glucose + regular insulin 25120583L containing 05 unitsof insulin + 1mM pioglitazone (standard)
26 Acute Oral Toxicity Study An acute toxicity study wasperformed on Swiss albino mice according to OECD 420guidelines according to the methods described earlier [13]All the newly synthesized test compounds were administeredat the dose of 2000mgkg po to the animals and observedfor any sign of toxicity as described earlier [13]
27 High Carbohydrate Diet- (HCD-) Induced Metabolic Dis-order in Mice [16] Four-week-old male Swiss albino micewere placed on the feeding of high sucrose diet (HCD)The detailed composition of diet where 55 energy source(kilocalories) was from sucrose is given in Table 2
After 24 weeks of HCD feeding mice displayed hyper-glycemia Animals with similar degrees of hyperglycemiawere randomly divided into six groups (119899 = 6) Thenormal pellet diet (NPD) fed mice were used as nondiabeticcontrols The diabetic control (HCD) and the normal control(NPD) groups received the vehicle (025CMC 10mLkg)while the treatment groups were given pioglitazone (5mgkgpo) and compound 2e and compound 3a (100mgkg po)respectively suspensions in 025wv CMC (10mLkg) Allthe treatments were given for 30 days Animalsrsquo body weightand cumulative food intake were recorded periodicallyFood efficiency ratio (FER) was calculated according to thereported method [17] using the following formula FER =change in body weight (day 0ndashday 30)cumulative foodconsumed in 30 days per animal Final body weight on day30 and FER were reported Plasma glucose triglycerides and
BioMed Research International 7
total cholesterol were monitored on day 14 and day 30 afterthe treatment using colorimetric kits (Aspen LaboratoriesPvt Ltd New Delhi India) Plasma insulin (Linco ResearchInc St Charles MO USA) leptin (BioVendor LLC CandlerNC USA) and adiponectin (Adipogen Corporation SanDiego CA USA) were estimated on day 30 using ELISAkits according to manufacturerrsquos instructions OGTT wasperformed on day 32 as described below On day 32 animalswere sacrificed and liver pancreas and white adipose tissues(from epididymal fat WAT) were isolated for histopatholog-ical investigation
271 Oral Glucose Tolerance Test (OGTT) in DiabeticMice [18] OGTT was performed according to the methoddescribed previously [18] In brief animals were fastedovernight and distilled water or glucose load of 2 gkgpo was administered and blood samples were collectedby retroorbital plexus puncture at 0 30 60 and 120minafter glucose challenge Plasma glucose was measured spec-trophotometrically using commercially available colorimet-ric kits (Aspen Laboratories Pvt Ltd New Delhi India) Thepercentage reduction in glucose excursion (AUC
0minus120 min)produced by test compounds was calculated from the areaunder the curve (AUC
0minus120 min) The results are expressedin time-dependent plasma glucose (mgdL) levels plasmaglucose (AUC
0minus120 min) and reduction in glucose excursion(AUC
0minus120 min)
272 Endogenous Liver Antioxidant Enzymes EstimationAnimals were sacrificed by cervical dislocation on the 32ndday after treatment Transcardial and whole liver perfusionwere performed using ice-cold saline [19] Liver was isolatedand 10wv homogenate was prepared using Teflon-glasshomogenizer (RQ-127AD REMI Group Mumbai India)with ice-cold saline-EDTAThe homogenate was centrifugedat 10000 rpm for 10min supernatant was collected andcentrifuged again at 20000 rpm for 1 h at 4∘C The super-natant obtained was used for the estimation of glutathione(GSH) glutathione-S-transferase (GST) catalase superoxidedismutase (SOD) and malondialdehyde (MDA) All the datapresented as mean plusmn SEM (119899 = 6)
(i) Glutathione (GSH) Assay [20] From the liver homogenateproteins were precipitated by 10 tri-carboxylic acid (TCA)and then centrifuged to collect the supernatant One mLsupernatant was mixed with 6mL 02MpH 80 and 1mL06mM 551015840-dithiobis-(2-nitrobenzoic acid) (DTNB) andincubated for 10min at room temperature The absorbancewas recorded against the blank at 412 nm in aUV-visible spec-trophotometer (model UV-1650PC Shimadzu Co KyotoJapan) and the GSH concentration was calculated from thestandard curve
(ii) Superoxide Dismutase Assay [21] The entire 1mL super-natant was added to 01M carbonate buffer (pH 102) andthe increase in absorbance after addition of epinephrine was
measured at 480 nm using a UV-visible spectrophotome-ter (model UV-1650PC Shimadzu Co Kyoto Japan) Theenzyme activity was expressed as Umg protein
(iii) Catalase Assay [22] The catalase activity was deter-mined spectrophotometrically according to the protocol ofClaiborne [22] The reaction was started by adding 005mLsupernatant to the reaction mixture (195mL 10mMH
2O2in
60mM phosphate buffer pH 70) Absorbance was recordedfor 3min at 240 nm Phosphate buffer (60mM pH 70) waskept as a reference To determine the specific activity ofcatalase extinction coefficient of 004mMminus1 cmminus1 was used
(iv) Glutathione-S-Transferase (GST) Assay [23] 01mL 1-chloro-24-dinitrobenzene (CDNB) was added to 06mLsupernatant of liver homogenate and 22mL phosphate bufferpH 65 incubated at 37∘C for 5min and added 01mL 30mMGSH Absorbance was recorded at 340 nm at intervals of12345min Blank was carried out in the same mannerwithout homogenate
(v) Malondialdehyde Assay [24]OnemL of liver homogenatewas combined with 2mL of reaction mixture [15wvtrichloroacetic acid (TCA) and 0375wv thiobarbituricacid (TBA) in 025N hydrochloric acid (HCl)] and mixedthoroughly The solution was heated for 20min on boilingwater bath Samples were cooled and centrifuged at 1000 rpmfor 10min to remove the flocculent precipitate Super-natant was collected and the absorbance of read at 532 nmagainst a blank which contained all the reagents exceptthe liver homogenate The extinction coefficient of 156 times105Mminus1 cmminus1 was used to calculate the malondialdehydeconcentration
273 Histopathological Investigation For histological exam-inations liver pancreas and white adipose tissue (WAT)from three animals per group were isolated on day 32 aftertreatment of compounds The tissue samples were fixed informalin solution (10) for one week at room temperaturedehydrated by graded ethanol cleared using graded xyleneand embedded in paraffin wax 5 120583m thick sections werecut using rotary microtome (RM2245 Leica MicrosystemsGmbHWetzlar Germany) fixed on glass slides stained witheosin and hematoxylin and observed using a microscope(Model BX41 Olympus Corporation Tokyo Japan) ForLangerhans cells the average areas of six islets per specimenwere measured using ImageJ software (version 148 NationalInstitute of Health MD USA) Area was expressed as 120583m2The quantification of adipocytes were performed as reported[25] using adipocyte quantification tool where area wasmeasured in 120583m2
28 Statistical Analysis Statistical analysis was performedby comparing the responses of the treatment groups torespective saline control and vehicle treated groups for allexperiments and the significance was determined by one-way ANOVA followed by post hoc Dunnettrsquos test Values
8 BioMed Research International
Table 3 Effect of thiazolidin-4-ones on in vitro glucose uptake in absence and presence of insulin
S no GroupsGlucose uptake (mgg45min)
In absence of insulin In presence of insulinMean plusmn SEM (119899 = 3) Mean plusmn SEM (119899 = 3)
1 Glucose control 809 plusmn 051 1211 plusmn 1342 Compound 2a 189 plusmn 145 1033 plusmn 1213 Compound 2b 619 plusmn 031 736 plusmn 0254 Compound 2c 1012 plusmn 052 1488 plusmn 0865 Compound 2d 984 plusmn 061 816 plusmn 0116 Compound 2e 1900 plusmn 127a 2342 plusmn 032b
7 Compound 2f 603 plusmn 033 833 plusmn 0158 Compound 2g 645 plusmn 013 708 plusmn 0199 Compound 2h 761 plusmn 018 728 plusmn 03810 Compound 2i 555 plusmn 014 644 plusmn 11911 Compound 2j 954 plusmn 021 368 plusmn 17912 Compound 2k 582 plusmn 018 721 plusmn 01513 Compound 2l 643 plusmn 010 999 plusmn 01014 Compound 2m 812 plusmn 082 1287 plusmn 01515 Compound 3a 2044 plusmn 017a 2501 plusmn 029b
16 Compound 3b 777 plusmn 029 1151 plusmn 00217 Compound 3c 1224 plusmn 010 1050 plusmn 08118 Compound 4a 753 plusmn 110 798 plusmn 08319 Compound 4b 610 plusmn 083 772 plusmn 02120 Compound 4c 097 plusmn 059 482 plusmn 05521 Compound 4d 377 plusmn 047 554 plusmn 04922 Pioglitazone 1337 plusmn 026a 2523 plusmn 034b
a and b represent 119875 lt 005 as compared to glucose control in absencepresence of insulin respectively
Table 4 Effect of thiazolidin-4-ones on body weight and food efficiency ratio in mice
NPD HCD Pioglitazone Compound 2e Compound 3aFinal body weight (g) 380 plusmn 21 412 plusmn 22 382 plusmn 14 376 plusmn 12 382 plusmn 21Food efficiency ratio 00025 plusmn 0005 00083 plusmn 0006 00020 plusmn 0005 00043 plusmn 0011 0071 plusmn 0010abaRepresents 119875 lt 005 as compared to NPD group (normal pellet diet)bRepresents 119875 lt 005 as compared to HCD group (high carbohydrate diet)
were expressed as mean plusmn SEM 119875 lt 005 was consideredsignificant
3 Results
31 In Vitro Biological Study
311 Glucose Uptake Assay Of the twenty thiazolidin-4-ones tested compounds 2e and 3a stimulated glucose uptake(119875 lt 005) when compared with control Uptake wasstimulated both in the absence and presence of externalinsulin The standard drug pioglitazone also increased theglucose uptake (Table 3)
32 In Vivo Biological Studies
321 Acute Oral Toxicity Study Acute toxicity studies werecarried out on Swiss albino mice as per the OECD guidelines
for test compounds 2e and 3a Both were found to be safe upto 2000mgkg which was the maximum dose tested
322 High Carbohydrate Diet- (HCD-) Induced MetabolicDisorder in Mice Animals put on HCD for 6 months hadsignificant hyperglycemia hypertriglyceridemia and hyperc-holesterolemiaThey were randomized to different treatmentgroups based on plasma glucose level and then treatment wasgiven for 30 days Firstly treatment did not have any signif-icant effect on body weight of animals (Table 4) Secondlyenergy expenditure was assessed indirectly using FER Noneof the treated groups except compound 3a-treated group hadany significant effect on FER The calculated FER was foundto be significantly higher in animal treated with compound3a compared to normal control and HCD group High FERvalue indicates the increased energy expenditure in animalstreated with compound 3a
BioMed Research International 9
0
50
100
150
200
250
14 day 30 day
NPDHCD
lowast
lowast
lowastlowast
lowastlowast
lowastlowast lowastlowastlowastlowast
lowastlowast
Pioglitazone (5mgkg)
Plas
ma g
luco
se (m
gdL
)(M
eanplusmn
SE
Mn=6
)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
60
120
180
240
300
lowast
lowastlowastlowastlowast
lowastlowast
Plas
ma T
G (m
gdL
)(M
eanplusmn
SE
Mn=6
)
14 day 30 day
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
60
120
180
240
300
lowast
lowast
lowastlowast
lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowast
Plas
ma c
hole
stero
l (m
gdL
)(M
eanplusmn
SE
Mn=6
)
14 day 30 day
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 2 Effect of thiazolidin-4-ones on plasma (a) glucose (b) triglyceride (TG) (c) cholesterol inHCD fedmice Data presented asmean plusmnSEM (119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 as compared to HCD group
(i) Effect of Thiazolidin-4-Ones on Plasma Glucose Triglyc-eride and Cholesterol Level Biochemical estimation ofmetabolic markers such as plasma glucose (PG) triglycerides(TG) and total cholesterol (TC)was performed on day 14 andday 30 after the drug treatment and it was observed that HCDcontrol group consistently had significant increase in PG andTC levels on days 14 and 30 compared to respective dayrsquosnormal control group However TG levels were significantlyincreased only on day 30 compared to respective dayrsquos normalcontrol group Pioglitazone and test compounds (2e and3a) significantly reversed hyperglycemia and elevated plasmacholesterol compared to HCD control group on both day14 and day 30 of drug treatment (Figures 2(a) and 2(c))while elevated TG levels were reduced on day 30 by the testcompounds and pioglitazone (Figure 2(b))
(ii) Effect of Thiazolidin-4-Ones on Plasma Insulin Leptinand Adiponectin Level Development of insulin resistance
in mouse was confirmed by estimating the plasma insulinlevels on day 30 after drug treatment HCD control group incontrast to NPD group showed two times increase in plasmainsulin levels (119875 lt 005) Hyperinsulinemia associated withhyperglycaemia and hypertriglyceridemia is considered tobe the sign of development of insulin resistance Thus highsucrose feeding for 6 months led to the development ofinsulin resistance in mice Treatment with pioglitazone andtest drugs attenuated the insulin resistance (Figure 3(a))
HCD feeding to animals did not elicit any significanteffect on plasma leptin levels as there was no significantdifference between HCD control and NPD group Howevertreatment with pioglitazone and test compounds significantly(119875 lt 005) raised the circulating leptin level in micecomparedwithHCDcontrol group (Figure 3(b)) Compound3a caused a fourfold elevation in leptin levels compared withHCD group HCD feeding to animals resulted in significant
10 BioMed Research International
0
1
2
3
4
5
NPDHCDPioglitazone (5mgkg)
Plas
ma i
nsul
in (n
gm
L)(M
eanplusmn
SE
Mn=6
)
lowastlowast
lowast
lowastlowastlowastlowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
1200
2400
3600
4800
6000
NPDHCDPioglitazone (5mgkg)
Plas
ma l
eptin
(pg
mL)
(Mea
nplusmn
SE
Mn=6
)
lowast lowast
lowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
12000
24000
36000
48000
60000
NPDHCDPioglitazone (5mgkg)
Plas
ma a
dipo
nect
in (n
gm
L)(M
eanplusmn
SE
Mn=6
)
lowast
lowastlowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 3 Effect of thiazolidin-4-ones on plasma (a) insulin (b) leptin (c) adiponectin in HCD fed mice Data presented as mean plusmn SEM(119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 as compared to HCD group
(119875 lt 005) hypoadiponectinemia comparedwithNPD Treat-ment with pioglitazone significantly (119875 lt 005) corrected thehypoadiponectinemia However both test compounds failedto correct hypoadiponectinemia (Figure 3(c))
(iii) Effect of Thiazolidin-4-Ones on Oral Glucose ToleranceTest In oral glucose tolerance test HCD group animalshowed significant (119875 lt 005) glucose intolerance (Figure 4)Pioglitazone and test compounds compound 2e and com-pound 3a corrected the glucose intolerance shown as sig-nificant (119875 lt 005) percent reductions in glucose excursion(AUC
0minus120min) by 1300 plusmn 33 1546 plusmn 554 and 1560 plusmn 349respectively compared with HCD group (Figures 4(a) and4(b))
(iv) Effect of Thiazolidin-4-Ones on Liver Enzymes Oxidativestress is the hallmark of metabolic disorder where disturbedhomeostasis between oxidative and antioxidative mechanismoccurs HCD feeding to mice resulted in an oxidative stress
observed as reduction in liver antioxidant enzymes such asglutathione (GSH) catalase (CAT) superoxide dismutase(SOD) and glutathione-S-transferase (GST) Treatment withpioglitazone and test compounds ameliorated the oxidativestress They also reversed the elevation in liver malondialde-hyde (MDA) levels in HCD-fed mice (Table 5)
(v) Histopathological ExaminationHistology of liver showednormal lobular architecture with normal hepatocytes inall groups (Figure 5(a)) Pancreas showed mild to mod-erate hyperplasia of islets of Langerhans (Figure 5(a)) inpioglitazone and compound 3a groups Pioglitazone and3a treatment significantly raised the area of Langerhansislets compared with HCD group (Figure 5(b)) Exocrineportion of the pancreas and vascularity appeared normalin all the groups Histopathological investigation of whiteadipose tissue (WAT) from epidydimal showed an increasein the size of adipocytes (Figure 5(c)) in HCD group whichwas reversed by pioglitazone and compound 3a treatment
BioMed Research International 11
Table 5 Effect of thiazolidin-4-ones on liver antioxidant enzyme and malondialdehyde level
Liver biomarkers NPD HCD Pioglitazone Compound 2e Compound 3aGSH (nmolemg of protein) 558 plusmn 21 464 plusmn 21a 632 plusmn 15b 683 plusmn 20b 678 plusmn 43b
CAT (Umg of protein) 892 plusmn 57 554 plusmn 31a 938 plusmn 10b 819 plusmn 24b 875 plusmn 74b
SOD (Umg of protein) 1868 plusmn 232 1081 plusmn 112a 1748 plusmn 144b 2022 plusmn 47b 2080 plusmn 154b
GST (Umg of protein) 043 plusmn 005 037 plusmn 001a 073 plusmn 002b 070 plusmn 001b 072 plusmn 004b
MDA (nmolemg of protein) 057 plusmn 006 10 plusmn 033a 046 plusmn 001b 040 plusmn 001b 041 plusmn 002baRepresents 119875 lt 005 as compared to NPD group (normal pellet diet)bRepresents 119875 lt 005 as compared to HCD group (high carbohydrate diet)
0 30 60 90 120100
130
160
190
220
Plas
ma g
luco
se (m
gdL
)(M
eanplusmn
SE
Mn=6
)
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
5000
10000
15000
20000
25000
(Mea
nplusmn
SE
Mn=6
)
lowast
lowastlowast lowastlowast lowastlowast
NPDHCDPioglitazone (5mgkg)
Plas
ma g
luco
se (A
UC 0
ndash120
min)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
Figure 4 Effect of thiazolidin-4-ones on (a) plasma glucose (mgdL) and (b) area under the curve (AUC0minus120 min) against oral glucose
tolerance test (OGTT) in HCD fed mice Data presented as mean plusmn SEM (119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD groupand lowastlowastrepresents 119875 lt 005 as compared to HCD group
However compound 2e did not show any significant effecton increased adipocyte size
4 Discussion and Conclusion
The present work was planned as a sequel to earlier studiesin our laboratory using thiazolidin-4-ones as antidiabetichypolipidemic and antiinflammatory molecules The substi-tutions in the thiazolidine ring were made at C2 and N3Attached to the latter position was a nicotinamide moietyand the substitution at C2 was either p-methoxyphenyl or2 5-di-tert-butyl-4-hydroxyphenyl group The compoundsshowed significant antidiabetic and hypolipidemic activities[9ndash11 26]
In the present study the substitution at N3 was changedto p-chloro-phenoxyacetylamino a group that is similar toclofibrate with a methylene bridge instead of gem-dimethylsubstitution
Four compounds out of 20 (ie compounds 4a 4b4c and 4d) had a methyl group attached at the C5 ofthiazolidine ring with cyclisation being made with thiolacticacid instead of thioglycolic acid In three other compounds(ie compounds 3a 3b and 3c) clofibrate was used to make
themoiety for substitution atN3Thiswas donewith a view toexamine the effect of the gem-dimethyl on the overall activityof the resulting molecule
All the 20 synthesised thiazolidin-4-ones were evaluatedfor glucose uptake in an in vitro system using the isolatedrat diaphragm In this experiment compounds 2e and 3asignificantly raised the amount of glucose uptake by thetissue both in the absence and presence of external insulinThis indicated the potential of the theses compounds insensitizing the tissues for the external insulin Hence thesetwo compounds were chosen to study their effect on a diet-induced model of insulin resistance Both compounds havethe same p-methoxyphenyl moiety attached to the C2 of thethiazolidine ring Compound 3a has a gem-dimethyl groupinstead of methylene in the substituent at N3
Apart from multiple risk factors diet-induced metabolicabnormalities contribute to the development of insulinresistance and 120573-cell failure in type-2 diabetes [16] Earlydetection and appropriate treatment are considered beneficialfor correcting the abnormality Among the various animalmodels induction of diabetes through diet provides moreresemblance to human type-2 diabetes Chronic intake ofdiet with high sucrose content has been reported to favourthe development of insulin resistance [16] Similarly in our
12 BioMed Research International
NPD HCD Pioglitazone Compound 3a Compound 2e
Liver
WAT
Green and red arrows indicate hyperplasia and increased size of adipocytes respectively
Pancreas
(a)
0
50000
100000
150000
200000
NPDHCDPioglitazone (5mgkg)
lowastlowastlowastlowast
(Mea
nplusmn
SE
Mn=3
)A
rea o
f lan
gerh
ans i
stle
ts (120583
m2)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
750
1500
2250
3000
NPDHCDPioglitazone (5mgkg)
lowastlowastlowastlowast
lowast
(Mea
nplusmn
SE
Mn=3
)A
rea o
f adi
pocy
tes (120583
m2)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 5 (a) Histological sections of mouse liver pancreas and white adipose tissue (WAT) in HCD model (10x) Green and red arrowsindicate hyperplasia of islets of Langerhans and increase in the size of adipocytes respectively Effect of thiazolidin-4-ones on (b) area ofLangerhans islets (120583m2) and (c) area of adipocytes (120583m2) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 ascompared to HCD group
study mice fed a high-sucrose diet for six months devel-oped metabolic abnormalities like hyperglycemia hyperin-sulinemia hypertriglyceridemia hypercholesterolemia andhypoadiponectinemia Hyperglycemia and hyperinsulinemiasuggest the inability of insulin to sensitize the tissue forglucose uptake allowing glucose to be diverted toward lipo-genesisThis led to hyperlipidemia and finally to insulin resis-tance In addition adiponectin and leptin the adipokinessecreted from white adipose tissue (WAT) are reported tobe involved in the metabolism of glucose and lipid [27]In our study we observed that HCD caused a reductionin plasma adiponectin level and an increase in the sizeof adipocytes However it did not cause any change inplasma leptin levels Thus we found a correlation between
the adiponectin level and size of adipocytes in HCD modelFurther OGTT results from HCD-fed mice correlated wellwith glucose intolerance hyperinsulinemia and hypoad-iponectinemia The authenticity of the model was validatedby the effect of pioglitazone which was able to correct themetabolic abnormalities Neither test compounds (2e or 3a)attenuated hypoadiponectinemia However they correctedthe impaired glucose tolerance and insulin resistance inmice Compounds 2e and 3a by their ability to enhanceglucose uptake and to sensitize the tissue for available insulinreduced hyperinsulinemia and raised the leptin levels Thiswould have resulted in better glucose utilization by periph-eral tissue Thus these compounds reduced the metabolicabnormalities like hyperglycemia hypertriglyceridemia and
BioMed Research International 13
hypercholesterolemia Prospective studies involving directassessment of insulin sensitivity in OGTT are requiredto analyze glucose stimulated insulin secretion (GSIS) inpresence of compounds to establish the mechanistic role ofthe test compounds on insulin signalling
Histological investigation showed no change in liverarchitecture in the various treatment groups In HCD controlmice there was an increase in the size of adipocytes in whiteadipose tissue (WAT) which was reversed by compound3a and pioglitazone treatment However only pioglitazone-treated animals showed positive correlation between reducedsize of the cells and correction of hypoadiponectinemiaApart from correction of hypoadiponectinemia pioglitazoneraised the peripheral leptin levels which was also true in caseof compounds 2e and 3a treatment Among the tested com-pounds 3a raised endogenous leptin levels four times morethan HCD group This finding points to the link betweenraised leptin levels and reduced adipocyte size caused bycompound 3a The pancreatic islets showed hyperplasia inpioglitazone and other treatment groups Pioglitazone is aPPAR-120574 agonist This could have been responsible for theproliferation of beta cells of pancreas The test compoundsare thiazolidin-4-ones with some similarity to thiazolidine-diones It is possible that hyperplasia observed in compound3a-treated animals might have been due to some agonisticactivity on PPAR-120574 receptors This needs to be investigatedthrough relevant assay
Oxidative stress has been implicated in the occurrence ofdiabetes and compounds reducing the oxidative stress havebeneficial role in correcting glucose intolerance and insulinresistance in diabetes [18 28]The test compounds and piogli-tazone reversed the depletion of endogenous antioxidantenzymes such as GSH CAT SOD and GST Further theyreduced malondialdehyde levels This suggests the inhibitoryeffect of these compounds on oxidative stress
Leptin serves as an insulin-sensitizing factor in the wholebody [29] However hyperleptinemia in the obese mouseand human is a sign of leptin resistance where increasedleptin levels are caused by disturbed homeostasis arisingfrom leptin receptor mutation ageing or obesity [29] Thushyperleptinemia further worsens impaired insulin actionin pathological condition In these conditions exogenouslyadministered leptin does not improve glucose tolerance andinsulin sensitivity In our study diseased animal (HCD fedmouse) per se did not develop hyperleptinemia which mightbe the sign of early stage of metabolic abnormality associatedwith insulin resistance At this state test compounds aswell as pioglitazone reversed the elevated glucose TG TCand insulin levels while facilitating leptin profile along withinsulin sensitivity Subsequently compound 3a treatmentresulted in increased energy expenditure demonstrated byelevated food efficiency ratio (FER) However reductionin body weight among the treated groups was not foundproportionate to the circulating leptin levels which suggeststhat increased leptin level in peripheral blood is not suffi-cient to induce a proportionate reduction on body weightA few questions remain unanswered such as (i) whetherthe compounds directly raised the leptin levels or are theresults due to indirect impact on metabolic signaling (ii) In
metabolic disorder combined with hyperleptinemia how dothese compounds affect leptin signaling Future studies areneeded to address these issues
No single mechanism would suffice to explain the ben-eficial effects of the test compounds They do not seem toact through insulinotropic activity unlike the sulfonylureasThey have no significant effect on adiponectin levels rulingout any involvement of this mechanism The increase in thelevel of serum leptin might point to the involvement of leptinin the antihyperlipidemic and antidiabetic potentials of thesemolecules
In conclusion thiazolidin-4-one derivatives act throughmultiple mechanisms to correct the metabolic abnormalitiesin type-2 diabetes In the present work compounds 2e and3a were found to be the most effective test compoundsto ameliorate insulin resistance and development of type-2diabetes
Conflict of Interests
The authors declare that they do not have any conflict ofinterests
Acknowledgment
The authors wish to thank All India Council for TechnicalEducation (AICTE) New Delhi India for providing thefinancial support and Manipal University Manipal Kar-nataka India for providing the facilities to carry out thepresent work
References
[1] S M Grundy I J Benjamin G L Burke et al ldquoDiabetes andcardiovascular disease a statement for healthcare professionalsfrom the american heart associationrdquo Circulation vol 100 no10 pp 1134ndash1146 1999
[2] K E Thorpe and D H Howard ldquoThe rise in spending amongMedicare beneficiaries the role of chronic disease prevalenceand changes in treatment intensity Increasing numbers ofbeneficiaries being treated for five or more conditions a year aredriving Medicare spending upwardrdquo Health Affairs vol 25 no5 pp w378ndashw388 2006
[3] S M Grundy ldquoDrug therapy of the metabolic syndrome min-imizing the emerging crisis in polypharmacyrdquo Nature ReviewsDrug Discovery vol 5 no 4 pp 295ndash309 2006
[4] M Navab N Gharavi and A D Watson ldquoInflammation andmetabolic disordersrdquo Current Opinion in Clinical Nutrition andMetabolic Care vol 11 no 4 pp 459ndash464 2008
[5] G S Hotamisligil ldquoInflammation and metabolic disordersrdquoNature vol 444 no 7121 pp 860ndash867 2006
[6] M Schmuth V Moosbrugger-Martinz S Blunder and SDubrac ldquoRole of PPAR LXR and PXR in epidermal homeosta-sis and inflammationrdquo Biochimica et Biophysica Acta vol 1841no 3 pp 463ndash473 2014
[7] J A Panetta J K Shadle M L Phillips D N Benslayand P P K Ho ldquo4-thiazolidinones potent antioxidants asantiinflammatory agentsrdquo Annals of the New York Academy ofSciences vol 696 pp 415ndash416 1993
14 BioMed Research International
[8] T Kato T Ozaki K Tamura Y Suzuki M Akima and NOhi ldquoNovel calcium antagonists with both calcium overloadinhibition and antioxidant activity 1 2-(35-Di-tert-butyl-4-hydroxyphenyl)-3- (aminopropyl)thiazolidinonesrdquo Journal ofMedicinal Chemistry vol 41 no 22 pp 4309ndash4316 1998
[9] JM Joy N Jacob andGN Kutty ldquoEvaluation of hypoglycemiceffects of 4-thiazolidinonesrdquo Indian Drugs vol 42 no 1 pp 17ndash21 2005
[10] N Jacob and G N Kutty ldquoSynthesis and hypolipidemic activityof a thiazolidinone derivativerdquo Indian Drugs vol 41 no 2 pp76ndash79 2004
[11] G K Nampurath S P Mathew V Khanna R T Zachariah SKanji andMRChamallamudi ldquoAssessment of hypolipidaemicactivity of three thiazolidin-4-ones in mice given high-fat dietand fructoserdquoChemico-Biological Interactions vol 171 no 3 pp363ndash368 2008
[12] A Kishore G K Nampurath S P Mathew et al ldquoAntidiabeticeffect through islet cell protection in streptozotocin diabetesa preliminary assessment of two thiazolidin-4-ones in Swissalbino micerdquo Chemico-Biological Interactions vol 177 no 3 pp242ndash246 2009
[13] J Mudgal V S Gowdra G Mathew et al ldquoRemedial effects ofnovel 2 3-disubstituted thiazolidin-4-ones in chemical medi-ated inflammationrdquo Chemico-Biological Interactions vol 210pp 34ndash42 2014
[14] A R Surrey ldquo4-Thiazolidones IV the preparation of some3-alkylaminoalkyl-2-aryl derivativesrdquo Journal of the AmericanChemical Society vol 71 no 10 pp 3354ndash3356 1949
[15] B R P Kumar and M J Nanjan ldquoNovel glitazones designsynthesis glucose uptake and structure-activity relationshipsrdquoBioorganic and Medicinal Chemistry Letters vol 20 no 6 pp1953ndash1956 2010
[16] M Sumiyoshi M Sakanaka and Y Kimura ldquoChronic intakeof high-fat and high-sucrose diets differentially affects glucoseintolerance inmicerdquo Journal of Nutrition vol 136 no 3 pp 582ndash587 2006
[17] S H Kim S H Hyun and S Y Choung ldquoAnti-diabetic effectof cinnamon extract on blood glucose in dbdb micerdquo Journalof Ethnopharmacology vol 104 no 1-2 pp 119ndash123 2006
[18] S A Manohara Reddy J Mudgal P Bansal et al ldquoAntioxidantanti-inflammatory and anti-hyperglycaemic activities of hete-rocyclic homoprostanoid derivativesrdquoBioorganic andMedicinalChemistry vol 19 no 1 pp 384ndash392 2011
[19] S T Shukla V H Kulkarni P V Habbu K S Jagadeesh BS Patil and D M Smita ldquoHepatoprotective and antioxidantactivities of crude fractions of endophytic fungi of Ocimumsanctum Linn in ratsrdquo Oriental Pharmacy and ExperimentalMedicine vol 12 no 2 pp 81ndash91 2012
[20] B Mannervik ldquoGlutathione peroxidaserdquo Methods in Enzymol-ogy vol 113 pp 490ndash495 1985
[21] H PMisra and I Fridovich ldquoThe role of superoxide anion in theautoxidation of epinephrine and a simple assay for superoxidedismutaserdquo Journal of Biological Chemistry vol 247 no 10 pp3170ndash3175 1972
[22] A Claiborne ldquoCatalase activityrdquo in Handbook of Methods forOxygen Radical Research R A Greenwald Ed pp 283ndash284CRC Press Boca Raton Fla USA 1985
[23] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974
[24] W G Niehaus Jr and B Samuelsson ldquoFormation of malonalde-hyde from phospholipid arachidonate during microsomal lipidperoxidationrdquoEuropean Journal of Biochemistry vol 6 no 1 pp126ndash130 1968
[25] O S Osman J L Selway M A Kępczynska et al ldquoA novelautomated image analysis method for accurate adipocyte quan-tificationrdquo Adipocyte vol 2 no 3 pp 160ndash164 2013
[26] R Kalia C M Rao and N Gopalan Kutty ldquoSynthesisand evaluation of the anti-inflammatory activity of N-[2-(35-di-tert-butyl-4-hydroxyphenyl)-4-oxothiazolidin-3-yl]-nicotinamiderdquo Drug Research vol 57 no 9 pp 616ndash6222007
[27] N Maeda M Takahashi T Funahashi et al ldquoPPAR120574 ligandsincrease expression and plasma concentrations of adiponectinan adipose-derived proteinrdquo Diabetes vol 50 no 9 pp 2094ndash2099 2001
[28] P Bansal P Paul J Mudgal et al ldquoAntidiabetic antihyperlipi-demic and antioxidant effects of the flavonoid rich fraction ofPilea microphylla (L) in high fat dietstreptozotocin-induceddiabetes in micerdquo Experimental and Toxicologic Pathology vol64 no 6 pp 651ndash658 2012
[29] C Perez C Fernandez-Galaz T Fernandez-Agullo et alldquoLeptin impairs insulin signaling in rat adipocytesrdquo Diabetesvol 53 no 2 pp 347ndash353 2004
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MEDIATORSINFLAMMATION
of
6 BioMed Research International
231 2-(4-Chlorophenoxy)-N-(2-(4-methoxyphenyl)-4-oxot-hiazolidin-3-yl)-2-methylpropanamide (Compound 3a)1HNMR (CDCl
3) 120575 = 137 (t 3H -CH
3) 147 (t 3H - CH
3)
370 (t 3H OCH3) 384 (s 2H CH
2) 590 (s 1H CH) 735
(m 8H Ar) 817 (br s 1H -NH) ppm IR (KBr) 3281 (-NH)3080 (Ar C-H Str) 2892 1710 amp 1680 (-C=O of -CH
2-C=O
amp -CONH) 1612 (C=C Str) 1251 (C-O Str) 1143 1095(C-Cl) 848 817 709 amp 663 (C-S-C) cmminus1 respectively MS(ESI)119898119911 = 421
232 2-(4-Chlorophenoxy)-N-(2-(4-fluorophenyl)-4-oxot-hiazolidin-3-yl)-2-methylpropanamide (Compound 3b)1HNMR (CDCl
3) 120575 = 137 (t 3H -CH
3) 147 (t 3H -CH
3)
385 (s 2H CH2) 590 (s 1H CH) 743 (m 8H Ar) 810 (br
s 1H -NH) ppm IR (KBr) 3250 (-NH) 3043 (Ar C-H Str)2985 1726 amp 1674 (-C=O of -CH
2-C=O amp -CONH) 1599
(C=C Str) 1228 (C-O Str) 1157 (C-F) 1085 (C-Cl) 937 850amp 648 (C-S-C) cmminus1 respectively MS (ESI)119898119911 = 409
233 2-(4-Chlorophenoxy)-N-(2-(35-di-tert-butyl-4-hydrox-yphenyl)-4-oxothiazolidin-3-yl)-2-methylpropanamide (Com-pound 3c) 1HNMR(CDCl
3) 120575= 128 (s 6H (CH
3)2) 142 (s
18H (CH3)6) 344 (s 2H -CH
2-CO-) 454 (s 1H -OH) 517
(s 2H Ar) 623 (s 1H CH) 757 (m 4H Ar) ppm IR (KBr)3636 (-OH) 3543 (-NH) 3001 (Ar C-H Str) 2955 1740 amp1697 (-C=O of -CH
2-C=O amp -CONH) 1600 (C=C Str) 1236
(C-O Str) 1209 1024 (C-Cl) 887 773 amp 626 (C-S-C) cmminus1respectively MS (ESI)119898119911 = 518
24 Experimental Subjects Male Swiss albino mice (22ndash25 g) and Wistar rats (220ndash250 g) (inbred in Central AnimalResearch Facility Manipal University Manipal KarnatakaIndia) were used At a temperature of 25 plusmn 05∘C animalswere housed in plastic cages with 12 h lightdark cycle andhumidity 50 plusmn 5 RH Animals were given standard foodpellet and water ad libitum The experimental protocolswere approved by the Institutional Animal Ethics Committee(no IAECKMC252009-2010) and the experiments werecarried out in accordance with the guidelines provided bythe Committee for the Purpose of Control and Supervision ofExperiments on Animals (CPCSEA) Government of India
25 In Vitro Glucose Uptake Assay [15] Overnight fastedmaleWistar rats (220ndash250 g) were sacrificed and diaphragmswere isolated avoiding trauma and divided into two hemidi-aphragms After isolation blood clots were removed by rins-ing the hemidiaphragms in cold Tyrodersquos solution (withoutglucose) and transferred to the mammalian organ bath con-taining Tyrodersquos solution with 05wv glucose withwithoutthe teststandard drug (1mM) followed by incubation for45min at 37 plusmn 1∘C in presence of aeration After theincubation the glucose content of the incubated organ bathwas measured spectrophotometrically using colorimetrickits (Aspen Laboratories Pvt Ltd New Delhi India) Thedifference between the initial and final glucose amount wasconsidered as amount of glucose uptake (mgg of tissueweight45min)
Table 2 Composition of high carbohydrate diet (HCD)
Sl no Contents Percent (ww)1 Cholesterol 22 Sucrose 553 Lard 34 Cellulose 3505 L-cysteine 0256 Choline bitartrate 0507 DL-methionine 0258 Vitamin and mineral mixture 0109 Normal pellet diet 3540
The following groups were made for screening the com-pounds Group 1 5mLof Tyrodersquos solutionwith 05wv glu-cose (glucose control in absence of insulin) Group 2 5mL ofTyrodersquos solution with 05wv glucose and regular insulin(Novo Nordisk India Pvt Ltd Bangalore India 40 IUmL)25 120583L containing 05 units of insulin (glucose control inpresence of insulin) Groups 3 to 23 5mL of Tyrodersquos solutionwith 05wv glucose and 1mM test compound (total num-bers = 20) Group 24 5mL of Tyrodersquos solution with 05wvglucose + 1mMpioglitazone (standard) Group 25 to 45 5mLof Tyrodersquos solution with 05wv glucose + regular insulin25 120583L containing 05 units of insulin + 1mM test drug (totalnumbers = 20) Group 46 5mL of Tyrodersquos solution with05wv glucose + regular insulin 25120583L containing 05 unitsof insulin + 1mM pioglitazone (standard)
26 Acute Oral Toxicity Study An acute toxicity study wasperformed on Swiss albino mice according to OECD 420guidelines according to the methods described earlier [13]All the newly synthesized test compounds were administeredat the dose of 2000mgkg po to the animals and observedfor any sign of toxicity as described earlier [13]
27 High Carbohydrate Diet- (HCD-) Induced Metabolic Dis-order in Mice [16] Four-week-old male Swiss albino micewere placed on the feeding of high sucrose diet (HCD)The detailed composition of diet where 55 energy source(kilocalories) was from sucrose is given in Table 2
After 24 weeks of HCD feeding mice displayed hyper-glycemia Animals with similar degrees of hyperglycemiawere randomly divided into six groups (119899 = 6) Thenormal pellet diet (NPD) fed mice were used as nondiabeticcontrols The diabetic control (HCD) and the normal control(NPD) groups received the vehicle (025CMC 10mLkg)while the treatment groups were given pioglitazone (5mgkgpo) and compound 2e and compound 3a (100mgkg po)respectively suspensions in 025wv CMC (10mLkg) Allthe treatments were given for 30 days Animalsrsquo body weightand cumulative food intake were recorded periodicallyFood efficiency ratio (FER) was calculated according to thereported method [17] using the following formula FER =change in body weight (day 0ndashday 30)cumulative foodconsumed in 30 days per animal Final body weight on day30 and FER were reported Plasma glucose triglycerides and
BioMed Research International 7
total cholesterol were monitored on day 14 and day 30 afterthe treatment using colorimetric kits (Aspen LaboratoriesPvt Ltd New Delhi India) Plasma insulin (Linco ResearchInc St Charles MO USA) leptin (BioVendor LLC CandlerNC USA) and adiponectin (Adipogen Corporation SanDiego CA USA) were estimated on day 30 using ELISAkits according to manufacturerrsquos instructions OGTT wasperformed on day 32 as described below On day 32 animalswere sacrificed and liver pancreas and white adipose tissues(from epididymal fat WAT) were isolated for histopatholog-ical investigation
271 Oral Glucose Tolerance Test (OGTT) in DiabeticMice [18] OGTT was performed according to the methoddescribed previously [18] In brief animals were fastedovernight and distilled water or glucose load of 2 gkgpo was administered and blood samples were collectedby retroorbital plexus puncture at 0 30 60 and 120minafter glucose challenge Plasma glucose was measured spec-trophotometrically using commercially available colorimet-ric kits (Aspen Laboratories Pvt Ltd New Delhi India) Thepercentage reduction in glucose excursion (AUC
0minus120 min)produced by test compounds was calculated from the areaunder the curve (AUC
0minus120 min) The results are expressedin time-dependent plasma glucose (mgdL) levels plasmaglucose (AUC
0minus120 min) and reduction in glucose excursion(AUC
0minus120 min)
272 Endogenous Liver Antioxidant Enzymes EstimationAnimals were sacrificed by cervical dislocation on the 32ndday after treatment Transcardial and whole liver perfusionwere performed using ice-cold saline [19] Liver was isolatedand 10wv homogenate was prepared using Teflon-glasshomogenizer (RQ-127AD REMI Group Mumbai India)with ice-cold saline-EDTAThe homogenate was centrifugedat 10000 rpm for 10min supernatant was collected andcentrifuged again at 20000 rpm for 1 h at 4∘C The super-natant obtained was used for the estimation of glutathione(GSH) glutathione-S-transferase (GST) catalase superoxidedismutase (SOD) and malondialdehyde (MDA) All the datapresented as mean plusmn SEM (119899 = 6)
(i) Glutathione (GSH) Assay [20] From the liver homogenateproteins were precipitated by 10 tri-carboxylic acid (TCA)and then centrifuged to collect the supernatant One mLsupernatant was mixed with 6mL 02MpH 80 and 1mL06mM 551015840-dithiobis-(2-nitrobenzoic acid) (DTNB) andincubated for 10min at room temperature The absorbancewas recorded against the blank at 412 nm in aUV-visible spec-trophotometer (model UV-1650PC Shimadzu Co KyotoJapan) and the GSH concentration was calculated from thestandard curve
(ii) Superoxide Dismutase Assay [21] The entire 1mL super-natant was added to 01M carbonate buffer (pH 102) andthe increase in absorbance after addition of epinephrine was
measured at 480 nm using a UV-visible spectrophotome-ter (model UV-1650PC Shimadzu Co Kyoto Japan) Theenzyme activity was expressed as Umg protein
(iii) Catalase Assay [22] The catalase activity was deter-mined spectrophotometrically according to the protocol ofClaiborne [22] The reaction was started by adding 005mLsupernatant to the reaction mixture (195mL 10mMH
2O2in
60mM phosphate buffer pH 70) Absorbance was recordedfor 3min at 240 nm Phosphate buffer (60mM pH 70) waskept as a reference To determine the specific activity ofcatalase extinction coefficient of 004mMminus1 cmminus1 was used
(iv) Glutathione-S-Transferase (GST) Assay [23] 01mL 1-chloro-24-dinitrobenzene (CDNB) was added to 06mLsupernatant of liver homogenate and 22mL phosphate bufferpH 65 incubated at 37∘C for 5min and added 01mL 30mMGSH Absorbance was recorded at 340 nm at intervals of12345min Blank was carried out in the same mannerwithout homogenate
(v) Malondialdehyde Assay [24]OnemL of liver homogenatewas combined with 2mL of reaction mixture [15wvtrichloroacetic acid (TCA) and 0375wv thiobarbituricacid (TBA) in 025N hydrochloric acid (HCl)] and mixedthoroughly The solution was heated for 20min on boilingwater bath Samples were cooled and centrifuged at 1000 rpmfor 10min to remove the flocculent precipitate Super-natant was collected and the absorbance of read at 532 nmagainst a blank which contained all the reagents exceptthe liver homogenate The extinction coefficient of 156 times105Mminus1 cmminus1 was used to calculate the malondialdehydeconcentration
273 Histopathological Investigation For histological exam-inations liver pancreas and white adipose tissue (WAT)from three animals per group were isolated on day 32 aftertreatment of compounds The tissue samples were fixed informalin solution (10) for one week at room temperaturedehydrated by graded ethanol cleared using graded xyleneand embedded in paraffin wax 5 120583m thick sections werecut using rotary microtome (RM2245 Leica MicrosystemsGmbHWetzlar Germany) fixed on glass slides stained witheosin and hematoxylin and observed using a microscope(Model BX41 Olympus Corporation Tokyo Japan) ForLangerhans cells the average areas of six islets per specimenwere measured using ImageJ software (version 148 NationalInstitute of Health MD USA) Area was expressed as 120583m2The quantification of adipocytes were performed as reported[25] using adipocyte quantification tool where area wasmeasured in 120583m2
28 Statistical Analysis Statistical analysis was performedby comparing the responses of the treatment groups torespective saline control and vehicle treated groups for allexperiments and the significance was determined by one-way ANOVA followed by post hoc Dunnettrsquos test Values
8 BioMed Research International
Table 3 Effect of thiazolidin-4-ones on in vitro glucose uptake in absence and presence of insulin
S no GroupsGlucose uptake (mgg45min)
In absence of insulin In presence of insulinMean plusmn SEM (119899 = 3) Mean plusmn SEM (119899 = 3)
1 Glucose control 809 plusmn 051 1211 plusmn 1342 Compound 2a 189 plusmn 145 1033 plusmn 1213 Compound 2b 619 plusmn 031 736 plusmn 0254 Compound 2c 1012 plusmn 052 1488 plusmn 0865 Compound 2d 984 plusmn 061 816 plusmn 0116 Compound 2e 1900 plusmn 127a 2342 plusmn 032b
7 Compound 2f 603 plusmn 033 833 plusmn 0158 Compound 2g 645 plusmn 013 708 plusmn 0199 Compound 2h 761 plusmn 018 728 plusmn 03810 Compound 2i 555 plusmn 014 644 plusmn 11911 Compound 2j 954 plusmn 021 368 plusmn 17912 Compound 2k 582 plusmn 018 721 plusmn 01513 Compound 2l 643 plusmn 010 999 plusmn 01014 Compound 2m 812 plusmn 082 1287 plusmn 01515 Compound 3a 2044 plusmn 017a 2501 plusmn 029b
16 Compound 3b 777 plusmn 029 1151 plusmn 00217 Compound 3c 1224 plusmn 010 1050 plusmn 08118 Compound 4a 753 plusmn 110 798 plusmn 08319 Compound 4b 610 plusmn 083 772 plusmn 02120 Compound 4c 097 plusmn 059 482 plusmn 05521 Compound 4d 377 plusmn 047 554 plusmn 04922 Pioglitazone 1337 plusmn 026a 2523 plusmn 034b
a and b represent 119875 lt 005 as compared to glucose control in absencepresence of insulin respectively
Table 4 Effect of thiazolidin-4-ones on body weight and food efficiency ratio in mice
NPD HCD Pioglitazone Compound 2e Compound 3aFinal body weight (g) 380 plusmn 21 412 plusmn 22 382 plusmn 14 376 plusmn 12 382 plusmn 21Food efficiency ratio 00025 plusmn 0005 00083 plusmn 0006 00020 plusmn 0005 00043 plusmn 0011 0071 plusmn 0010abaRepresents 119875 lt 005 as compared to NPD group (normal pellet diet)bRepresents 119875 lt 005 as compared to HCD group (high carbohydrate diet)
were expressed as mean plusmn SEM 119875 lt 005 was consideredsignificant
3 Results
31 In Vitro Biological Study
311 Glucose Uptake Assay Of the twenty thiazolidin-4-ones tested compounds 2e and 3a stimulated glucose uptake(119875 lt 005) when compared with control Uptake wasstimulated both in the absence and presence of externalinsulin The standard drug pioglitazone also increased theglucose uptake (Table 3)
32 In Vivo Biological Studies
321 Acute Oral Toxicity Study Acute toxicity studies werecarried out on Swiss albino mice as per the OECD guidelines
for test compounds 2e and 3a Both were found to be safe upto 2000mgkg which was the maximum dose tested
322 High Carbohydrate Diet- (HCD-) Induced MetabolicDisorder in Mice Animals put on HCD for 6 months hadsignificant hyperglycemia hypertriglyceridemia and hyperc-holesterolemiaThey were randomized to different treatmentgroups based on plasma glucose level and then treatment wasgiven for 30 days Firstly treatment did not have any signif-icant effect on body weight of animals (Table 4) Secondlyenergy expenditure was assessed indirectly using FER Noneof the treated groups except compound 3a-treated group hadany significant effect on FER The calculated FER was foundto be significantly higher in animal treated with compound3a compared to normal control and HCD group High FERvalue indicates the increased energy expenditure in animalstreated with compound 3a
BioMed Research International 9
0
50
100
150
200
250
14 day 30 day
NPDHCD
lowast
lowast
lowastlowast
lowastlowast
lowastlowast lowastlowastlowastlowast
lowastlowast
Pioglitazone (5mgkg)
Plas
ma g
luco
se (m
gdL
)(M
eanplusmn
SE
Mn=6
)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
60
120
180
240
300
lowast
lowastlowastlowastlowast
lowastlowast
Plas
ma T
G (m
gdL
)(M
eanplusmn
SE
Mn=6
)
14 day 30 day
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
60
120
180
240
300
lowast
lowast
lowastlowast
lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowast
Plas
ma c
hole
stero
l (m
gdL
)(M
eanplusmn
SE
Mn=6
)
14 day 30 day
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 2 Effect of thiazolidin-4-ones on plasma (a) glucose (b) triglyceride (TG) (c) cholesterol inHCD fedmice Data presented asmean plusmnSEM (119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 as compared to HCD group
(i) Effect of Thiazolidin-4-Ones on Plasma Glucose Triglyc-eride and Cholesterol Level Biochemical estimation ofmetabolic markers such as plasma glucose (PG) triglycerides(TG) and total cholesterol (TC)was performed on day 14 andday 30 after the drug treatment and it was observed that HCDcontrol group consistently had significant increase in PG andTC levels on days 14 and 30 compared to respective dayrsquosnormal control group However TG levels were significantlyincreased only on day 30 compared to respective dayrsquos normalcontrol group Pioglitazone and test compounds (2e and3a) significantly reversed hyperglycemia and elevated plasmacholesterol compared to HCD control group on both day14 and day 30 of drug treatment (Figures 2(a) and 2(c))while elevated TG levels were reduced on day 30 by the testcompounds and pioglitazone (Figure 2(b))
(ii) Effect of Thiazolidin-4-Ones on Plasma Insulin Leptinand Adiponectin Level Development of insulin resistance
in mouse was confirmed by estimating the plasma insulinlevels on day 30 after drug treatment HCD control group incontrast to NPD group showed two times increase in plasmainsulin levels (119875 lt 005) Hyperinsulinemia associated withhyperglycaemia and hypertriglyceridemia is considered tobe the sign of development of insulin resistance Thus highsucrose feeding for 6 months led to the development ofinsulin resistance in mice Treatment with pioglitazone andtest drugs attenuated the insulin resistance (Figure 3(a))
HCD feeding to animals did not elicit any significanteffect on plasma leptin levels as there was no significantdifference between HCD control and NPD group Howevertreatment with pioglitazone and test compounds significantly(119875 lt 005) raised the circulating leptin level in micecomparedwithHCDcontrol group (Figure 3(b)) Compound3a caused a fourfold elevation in leptin levels compared withHCD group HCD feeding to animals resulted in significant
10 BioMed Research International
0
1
2
3
4
5
NPDHCDPioglitazone (5mgkg)
Plas
ma i
nsul
in (n
gm
L)(M
eanplusmn
SE
Mn=6
)
lowastlowast
lowast
lowastlowastlowastlowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
1200
2400
3600
4800
6000
NPDHCDPioglitazone (5mgkg)
Plas
ma l
eptin
(pg
mL)
(Mea
nplusmn
SE
Mn=6
)
lowast lowast
lowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
12000
24000
36000
48000
60000
NPDHCDPioglitazone (5mgkg)
Plas
ma a
dipo
nect
in (n
gm
L)(M
eanplusmn
SE
Mn=6
)
lowast
lowastlowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 3 Effect of thiazolidin-4-ones on plasma (a) insulin (b) leptin (c) adiponectin in HCD fed mice Data presented as mean plusmn SEM(119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 as compared to HCD group
(119875 lt 005) hypoadiponectinemia comparedwithNPD Treat-ment with pioglitazone significantly (119875 lt 005) corrected thehypoadiponectinemia However both test compounds failedto correct hypoadiponectinemia (Figure 3(c))
(iii) Effect of Thiazolidin-4-Ones on Oral Glucose ToleranceTest In oral glucose tolerance test HCD group animalshowed significant (119875 lt 005) glucose intolerance (Figure 4)Pioglitazone and test compounds compound 2e and com-pound 3a corrected the glucose intolerance shown as sig-nificant (119875 lt 005) percent reductions in glucose excursion(AUC
0minus120min) by 1300 plusmn 33 1546 plusmn 554 and 1560 plusmn 349respectively compared with HCD group (Figures 4(a) and4(b))
(iv) Effect of Thiazolidin-4-Ones on Liver Enzymes Oxidativestress is the hallmark of metabolic disorder where disturbedhomeostasis between oxidative and antioxidative mechanismoccurs HCD feeding to mice resulted in an oxidative stress
observed as reduction in liver antioxidant enzymes such asglutathione (GSH) catalase (CAT) superoxide dismutase(SOD) and glutathione-S-transferase (GST) Treatment withpioglitazone and test compounds ameliorated the oxidativestress They also reversed the elevation in liver malondialde-hyde (MDA) levels in HCD-fed mice (Table 5)
(v) Histopathological ExaminationHistology of liver showednormal lobular architecture with normal hepatocytes inall groups (Figure 5(a)) Pancreas showed mild to mod-erate hyperplasia of islets of Langerhans (Figure 5(a)) inpioglitazone and compound 3a groups Pioglitazone and3a treatment significantly raised the area of Langerhansislets compared with HCD group (Figure 5(b)) Exocrineportion of the pancreas and vascularity appeared normalin all the groups Histopathological investigation of whiteadipose tissue (WAT) from epidydimal showed an increasein the size of adipocytes (Figure 5(c)) in HCD group whichwas reversed by pioglitazone and compound 3a treatment
BioMed Research International 11
Table 5 Effect of thiazolidin-4-ones on liver antioxidant enzyme and malondialdehyde level
Liver biomarkers NPD HCD Pioglitazone Compound 2e Compound 3aGSH (nmolemg of protein) 558 plusmn 21 464 plusmn 21a 632 plusmn 15b 683 plusmn 20b 678 plusmn 43b
CAT (Umg of protein) 892 plusmn 57 554 plusmn 31a 938 plusmn 10b 819 plusmn 24b 875 plusmn 74b
SOD (Umg of protein) 1868 plusmn 232 1081 plusmn 112a 1748 plusmn 144b 2022 plusmn 47b 2080 plusmn 154b
GST (Umg of protein) 043 plusmn 005 037 plusmn 001a 073 plusmn 002b 070 plusmn 001b 072 plusmn 004b
MDA (nmolemg of protein) 057 plusmn 006 10 plusmn 033a 046 plusmn 001b 040 plusmn 001b 041 plusmn 002baRepresents 119875 lt 005 as compared to NPD group (normal pellet diet)bRepresents 119875 lt 005 as compared to HCD group (high carbohydrate diet)
0 30 60 90 120100
130
160
190
220
Plas
ma g
luco
se (m
gdL
)(M
eanplusmn
SE
Mn=6
)
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
5000
10000
15000
20000
25000
(Mea
nplusmn
SE
Mn=6
)
lowast
lowastlowast lowastlowast lowastlowast
NPDHCDPioglitazone (5mgkg)
Plas
ma g
luco
se (A
UC 0
ndash120
min)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
Figure 4 Effect of thiazolidin-4-ones on (a) plasma glucose (mgdL) and (b) area under the curve (AUC0minus120 min) against oral glucose
tolerance test (OGTT) in HCD fed mice Data presented as mean plusmn SEM (119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD groupand lowastlowastrepresents 119875 lt 005 as compared to HCD group
However compound 2e did not show any significant effecton increased adipocyte size
4 Discussion and Conclusion
The present work was planned as a sequel to earlier studiesin our laboratory using thiazolidin-4-ones as antidiabetichypolipidemic and antiinflammatory molecules The substi-tutions in the thiazolidine ring were made at C2 and N3Attached to the latter position was a nicotinamide moietyand the substitution at C2 was either p-methoxyphenyl or2 5-di-tert-butyl-4-hydroxyphenyl group The compoundsshowed significant antidiabetic and hypolipidemic activities[9ndash11 26]
In the present study the substitution at N3 was changedto p-chloro-phenoxyacetylamino a group that is similar toclofibrate with a methylene bridge instead of gem-dimethylsubstitution
Four compounds out of 20 (ie compounds 4a 4b4c and 4d) had a methyl group attached at the C5 ofthiazolidine ring with cyclisation being made with thiolacticacid instead of thioglycolic acid In three other compounds(ie compounds 3a 3b and 3c) clofibrate was used to make
themoiety for substitution atN3Thiswas donewith a view toexamine the effect of the gem-dimethyl on the overall activityof the resulting molecule
All the 20 synthesised thiazolidin-4-ones were evaluatedfor glucose uptake in an in vitro system using the isolatedrat diaphragm In this experiment compounds 2e and 3asignificantly raised the amount of glucose uptake by thetissue both in the absence and presence of external insulinThis indicated the potential of the theses compounds insensitizing the tissues for the external insulin Hence thesetwo compounds were chosen to study their effect on a diet-induced model of insulin resistance Both compounds havethe same p-methoxyphenyl moiety attached to the C2 of thethiazolidine ring Compound 3a has a gem-dimethyl groupinstead of methylene in the substituent at N3
Apart from multiple risk factors diet-induced metabolicabnormalities contribute to the development of insulinresistance and 120573-cell failure in type-2 diabetes [16] Earlydetection and appropriate treatment are considered beneficialfor correcting the abnormality Among the various animalmodels induction of diabetes through diet provides moreresemblance to human type-2 diabetes Chronic intake ofdiet with high sucrose content has been reported to favourthe development of insulin resistance [16] Similarly in our
12 BioMed Research International
NPD HCD Pioglitazone Compound 3a Compound 2e
Liver
WAT
Green and red arrows indicate hyperplasia and increased size of adipocytes respectively
Pancreas
(a)
0
50000
100000
150000
200000
NPDHCDPioglitazone (5mgkg)
lowastlowastlowastlowast
(Mea
nplusmn
SE
Mn=3
)A
rea o
f lan
gerh
ans i
stle
ts (120583
m2)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
750
1500
2250
3000
NPDHCDPioglitazone (5mgkg)
lowastlowastlowastlowast
lowast
(Mea
nplusmn
SE
Mn=3
)A
rea o
f adi
pocy
tes (120583
m2)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 5 (a) Histological sections of mouse liver pancreas and white adipose tissue (WAT) in HCD model (10x) Green and red arrowsindicate hyperplasia of islets of Langerhans and increase in the size of adipocytes respectively Effect of thiazolidin-4-ones on (b) area ofLangerhans islets (120583m2) and (c) area of adipocytes (120583m2) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 ascompared to HCD group
study mice fed a high-sucrose diet for six months devel-oped metabolic abnormalities like hyperglycemia hyperin-sulinemia hypertriglyceridemia hypercholesterolemia andhypoadiponectinemia Hyperglycemia and hyperinsulinemiasuggest the inability of insulin to sensitize the tissue forglucose uptake allowing glucose to be diverted toward lipo-genesisThis led to hyperlipidemia and finally to insulin resis-tance In addition adiponectin and leptin the adipokinessecreted from white adipose tissue (WAT) are reported tobe involved in the metabolism of glucose and lipid [27]In our study we observed that HCD caused a reductionin plasma adiponectin level and an increase in the sizeof adipocytes However it did not cause any change inplasma leptin levels Thus we found a correlation between
the adiponectin level and size of adipocytes in HCD modelFurther OGTT results from HCD-fed mice correlated wellwith glucose intolerance hyperinsulinemia and hypoad-iponectinemia The authenticity of the model was validatedby the effect of pioglitazone which was able to correct themetabolic abnormalities Neither test compounds (2e or 3a)attenuated hypoadiponectinemia However they correctedthe impaired glucose tolerance and insulin resistance inmice Compounds 2e and 3a by their ability to enhanceglucose uptake and to sensitize the tissue for available insulinreduced hyperinsulinemia and raised the leptin levels Thiswould have resulted in better glucose utilization by periph-eral tissue Thus these compounds reduced the metabolicabnormalities like hyperglycemia hypertriglyceridemia and
BioMed Research International 13
hypercholesterolemia Prospective studies involving directassessment of insulin sensitivity in OGTT are requiredto analyze glucose stimulated insulin secretion (GSIS) inpresence of compounds to establish the mechanistic role ofthe test compounds on insulin signalling
Histological investigation showed no change in liverarchitecture in the various treatment groups In HCD controlmice there was an increase in the size of adipocytes in whiteadipose tissue (WAT) which was reversed by compound3a and pioglitazone treatment However only pioglitazone-treated animals showed positive correlation between reducedsize of the cells and correction of hypoadiponectinemiaApart from correction of hypoadiponectinemia pioglitazoneraised the peripheral leptin levels which was also true in caseof compounds 2e and 3a treatment Among the tested com-pounds 3a raised endogenous leptin levels four times morethan HCD group This finding points to the link betweenraised leptin levels and reduced adipocyte size caused bycompound 3a The pancreatic islets showed hyperplasia inpioglitazone and other treatment groups Pioglitazone is aPPAR-120574 agonist This could have been responsible for theproliferation of beta cells of pancreas The test compoundsare thiazolidin-4-ones with some similarity to thiazolidine-diones It is possible that hyperplasia observed in compound3a-treated animals might have been due to some agonisticactivity on PPAR-120574 receptors This needs to be investigatedthrough relevant assay
Oxidative stress has been implicated in the occurrence ofdiabetes and compounds reducing the oxidative stress havebeneficial role in correcting glucose intolerance and insulinresistance in diabetes [18 28]The test compounds and piogli-tazone reversed the depletion of endogenous antioxidantenzymes such as GSH CAT SOD and GST Further theyreduced malondialdehyde levels This suggests the inhibitoryeffect of these compounds on oxidative stress
Leptin serves as an insulin-sensitizing factor in the wholebody [29] However hyperleptinemia in the obese mouseand human is a sign of leptin resistance where increasedleptin levels are caused by disturbed homeostasis arisingfrom leptin receptor mutation ageing or obesity [29] Thushyperleptinemia further worsens impaired insulin actionin pathological condition In these conditions exogenouslyadministered leptin does not improve glucose tolerance andinsulin sensitivity In our study diseased animal (HCD fedmouse) per se did not develop hyperleptinemia which mightbe the sign of early stage of metabolic abnormality associatedwith insulin resistance At this state test compounds aswell as pioglitazone reversed the elevated glucose TG TCand insulin levels while facilitating leptin profile along withinsulin sensitivity Subsequently compound 3a treatmentresulted in increased energy expenditure demonstrated byelevated food efficiency ratio (FER) However reductionin body weight among the treated groups was not foundproportionate to the circulating leptin levels which suggeststhat increased leptin level in peripheral blood is not suffi-cient to induce a proportionate reduction on body weightA few questions remain unanswered such as (i) whetherthe compounds directly raised the leptin levels or are theresults due to indirect impact on metabolic signaling (ii) In
metabolic disorder combined with hyperleptinemia how dothese compounds affect leptin signaling Future studies areneeded to address these issues
No single mechanism would suffice to explain the ben-eficial effects of the test compounds They do not seem toact through insulinotropic activity unlike the sulfonylureasThey have no significant effect on adiponectin levels rulingout any involvement of this mechanism The increase in thelevel of serum leptin might point to the involvement of leptinin the antihyperlipidemic and antidiabetic potentials of thesemolecules
In conclusion thiazolidin-4-one derivatives act throughmultiple mechanisms to correct the metabolic abnormalitiesin type-2 diabetes In the present work compounds 2e and3a were found to be the most effective test compoundsto ameliorate insulin resistance and development of type-2diabetes
Conflict of Interests
The authors declare that they do not have any conflict ofinterests
Acknowledgment
The authors wish to thank All India Council for TechnicalEducation (AICTE) New Delhi India for providing thefinancial support and Manipal University Manipal Kar-nataka India for providing the facilities to carry out thepresent work
References
[1] S M Grundy I J Benjamin G L Burke et al ldquoDiabetes andcardiovascular disease a statement for healthcare professionalsfrom the american heart associationrdquo Circulation vol 100 no10 pp 1134ndash1146 1999
[2] K E Thorpe and D H Howard ldquoThe rise in spending amongMedicare beneficiaries the role of chronic disease prevalenceand changes in treatment intensity Increasing numbers ofbeneficiaries being treated for five or more conditions a year aredriving Medicare spending upwardrdquo Health Affairs vol 25 no5 pp w378ndashw388 2006
[3] S M Grundy ldquoDrug therapy of the metabolic syndrome min-imizing the emerging crisis in polypharmacyrdquo Nature ReviewsDrug Discovery vol 5 no 4 pp 295ndash309 2006
[4] M Navab N Gharavi and A D Watson ldquoInflammation andmetabolic disordersrdquo Current Opinion in Clinical Nutrition andMetabolic Care vol 11 no 4 pp 459ndash464 2008
[5] G S Hotamisligil ldquoInflammation and metabolic disordersrdquoNature vol 444 no 7121 pp 860ndash867 2006
[6] M Schmuth V Moosbrugger-Martinz S Blunder and SDubrac ldquoRole of PPAR LXR and PXR in epidermal homeosta-sis and inflammationrdquo Biochimica et Biophysica Acta vol 1841no 3 pp 463ndash473 2014
[7] J A Panetta J K Shadle M L Phillips D N Benslayand P P K Ho ldquo4-thiazolidinones potent antioxidants asantiinflammatory agentsrdquo Annals of the New York Academy ofSciences vol 696 pp 415ndash416 1993
14 BioMed Research International
[8] T Kato T Ozaki K Tamura Y Suzuki M Akima and NOhi ldquoNovel calcium antagonists with both calcium overloadinhibition and antioxidant activity 1 2-(35-Di-tert-butyl-4-hydroxyphenyl)-3- (aminopropyl)thiazolidinonesrdquo Journal ofMedicinal Chemistry vol 41 no 22 pp 4309ndash4316 1998
[9] JM Joy N Jacob andGN Kutty ldquoEvaluation of hypoglycemiceffects of 4-thiazolidinonesrdquo Indian Drugs vol 42 no 1 pp 17ndash21 2005
[10] N Jacob and G N Kutty ldquoSynthesis and hypolipidemic activityof a thiazolidinone derivativerdquo Indian Drugs vol 41 no 2 pp76ndash79 2004
[11] G K Nampurath S P Mathew V Khanna R T Zachariah SKanji andMRChamallamudi ldquoAssessment of hypolipidaemicactivity of three thiazolidin-4-ones in mice given high-fat dietand fructoserdquoChemico-Biological Interactions vol 171 no 3 pp363ndash368 2008
[12] A Kishore G K Nampurath S P Mathew et al ldquoAntidiabeticeffect through islet cell protection in streptozotocin diabetesa preliminary assessment of two thiazolidin-4-ones in Swissalbino micerdquo Chemico-Biological Interactions vol 177 no 3 pp242ndash246 2009
[13] J Mudgal V S Gowdra G Mathew et al ldquoRemedial effects ofnovel 2 3-disubstituted thiazolidin-4-ones in chemical medi-ated inflammationrdquo Chemico-Biological Interactions vol 210pp 34ndash42 2014
[14] A R Surrey ldquo4-Thiazolidones IV the preparation of some3-alkylaminoalkyl-2-aryl derivativesrdquo Journal of the AmericanChemical Society vol 71 no 10 pp 3354ndash3356 1949
[15] B R P Kumar and M J Nanjan ldquoNovel glitazones designsynthesis glucose uptake and structure-activity relationshipsrdquoBioorganic and Medicinal Chemistry Letters vol 20 no 6 pp1953ndash1956 2010
[16] M Sumiyoshi M Sakanaka and Y Kimura ldquoChronic intakeof high-fat and high-sucrose diets differentially affects glucoseintolerance inmicerdquo Journal of Nutrition vol 136 no 3 pp 582ndash587 2006
[17] S H Kim S H Hyun and S Y Choung ldquoAnti-diabetic effectof cinnamon extract on blood glucose in dbdb micerdquo Journalof Ethnopharmacology vol 104 no 1-2 pp 119ndash123 2006
[18] S A Manohara Reddy J Mudgal P Bansal et al ldquoAntioxidantanti-inflammatory and anti-hyperglycaemic activities of hete-rocyclic homoprostanoid derivativesrdquoBioorganic andMedicinalChemistry vol 19 no 1 pp 384ndash392 2011
[19] S T Shukla V H Kulkarni P V Habbu K S Jagadeesh BS Patil and D M Smita ldquoHepatoprotective and antioxidantactivities of crude fractions of endophytic fungi of Ocimumsanctum Linn in ratsrdquo Oriental Pharmacy and ExperimentalMedicine vol 12 no 2 pp 81ndash91 2012
[20] B Mannervik ldquoGlutathione peroxidaserdquo Methods in Enzymol-ogy vol 113 pp 490ndash495 1985
[21] H PMisra and I Fridovich ldquoThe role of superoxide anion in theautoxidation of epinephrine and a simple assay for superoxidedismutaserdquo Journal of Biological Chemistry vol 247 no 10 pp3170ndash3175 1972
[22] A Claiborne ldquoCatalase activityrdquo in Handbook of Methods forOxygen Radical Research R A Greenwald Ed pp 283ndash284CRC Press Boca Raton Fla USA 1985
[23] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974
[24] W G Niehaus Jr and B Samuelsson ldquoFormation of malonalde-hyde from phospholipid arachidonate during microsomal lipidperoxidationrdquoEuropean Journal of Biochemistry vol 6 no 1 pp126ndash130 1968
[25] O S Osman J L Selway M A Kępczynska et al ldquoA novelautomated image analysis method for accurate adipocyte quan-tificationrdquo Adipocyte vol 2 no 3 pp 160ndash164 2013
[26] R Kalia C M Rao and N Gopalan Kutty ldquoSynthesisand evaluation of the anti-inflammatory activity of N-[2-(35-di-tert-butyl-4-hydroxyphenyl)-4-oxothiazolidin-3-yl]-nicotinamiderdquo Drug Research vol 57 no 9 pp 616ndash6222007
[27] N Maeda M Takahashi T Funahashi et al ldquoPPAR120574 ligandsincrease expression and plasma concentrations of adiponectinan adipose-derived proteinrdquo Diabetes vol 50 no 9 pp 2094ndash2099 2001
[28] P Bansal P Paul J Mudgal et al ldquoAntidiabetic antihyperlipi-demic and antioxidant effects of the flavonoid rich fraction ofPilea microphylla (L) in high fat dietstreptozotocin-induceddiabetes in micerdquo Experimental and Toxicologic Pathology vol64 no 6 pp 651ndash658 2012
[29] C Perez C Fernandez-Galaz T Fernandez-Agullo et alldquoLeptin impairs insulin signaling in rat adipocytesrdquo Diabetesvol 53 no 2 pp 347ndash353 2004
Submit your manuscripts athttpwwwhindawicom
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StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Tropical MedicineJournal of
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Medicinal ChemistryInternational Journal of
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AddictionJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Autoimmune Diseases
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Pharmaceutics
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MEDIATORSINFLAMMATION
of
BioMed Research International 7
total cholesterol were monitored on day 14 and day 30 afterthe treatment using colorimetric kits (Aspen LaboratoriesPvt Ltd New Delhi India) Plasma insulin (Linco ResearchInc St Charles MO USA) leptin (BioVendor LLC CandlerNC USA) and adiponectin (Adipogen Corporation SanDiego CA USA) were estimated on day 30 using ELISAkits according to manufacturerrsquos instructions OGTT wasperformed on day 32 as described below On day 32 animalswere sacrificed and liver pancreas and white adipose tissues(from epididymal fat WAT) were isolated for histopatholog-ical investigation
271 Oral Glucose Tolerance Test (OGTT) in DiabeticMice [18] OGTT was performed according to the methoddescribed previously [18] In brief animals were fastedovernight and distilled water or glucose load of 2 gkgpo was administered and blood samples were collectedby retroorbital plexus puncture at 0 30 60 and 120minafter glucose challenge Plasma glucose was measured spec-trophotometrically using commercially available colorimet-ric kits (Aspen Laboratories Pvt Ltd New Delhi India) Thepercentage reduction in glucose excursion (AUC
0minus120 min)produced by test compounds was calculated from the areaunder the curve (AUC
0minus120 min) The results are expressedin time-dependent plasma glucose (mgdL) levels plasmaglucose (AUC
0minus120 min) and reduction in glucose excursion(AUC
0minus120 min)
272 Endogenous Liver Antioxidant Enzymes EstimationAnimals were sacrificed by cervical dislocation on the 32ndday after treatment Transcardial and whole liver perfusionwere performed using ice-cold saline [19] Liver was isolatedand 10wv homogenate was prepared using Teflon-glasshomogenizer (RQ-127AD REMI Group Mumbai India)with ice-cold saline-EDTAThe homogenate was centrifugedat 10000 rpm for 10min supernatant was collected andcentrifuged again at 20000 rpm for 1 h at 4∘C The super-natant obtained was used for the estimation of glutathione(GSH) glutathione-S-transferase (GST) catalase superoxidedismutase (SOD) and malondialdehyde (MDA) All the datapresented as mean plusmn SEM (119899 = 6)
(i) Glutathione (GSH) Assay [20] From the liver homogenateproteins were precipitated by 10 tri-carboxylic acid (TCA)and then centrifuged to collect the supernatant One mLsupernatant was mixed with 6mL 02MpH 80 and 1mL06mM 551015840-dithiobis-(2-nitrobenzoic acid) (DTNB) andincubated for 10min at room temperature The absorbancewas recorded against the blank at 412 nm in aUV-visible spec-trophotometer (model UV-1650PC Shimadzu Co KyotoJapan) and the GSH concentration was calculated from thestandard curve
(ii) Superoxide Dismutase Assay [21] The entire 1mL super-natant was added to 01M carbonate buffer (pH 102) andthe increase in absorbance after addition of epinephrine was
measured at 480 nm using a UV-visible spectrophotome-ter (model UV-1650PC Shimadzu Co Kyoto Japan) Theenzyme activity was expressed as Umg protein
(iii) Catalase Assay [22] The catalase activity was deter-mined spectrophotometrically according to the protocol ofClaiborne [22] The reaction was started by adding 005mLsupernatant to the reaction mixture (195mL 10mMH
2O2in
60mM phosphate buffer pH 70) Absorbance was recordedfor 3min at 240 nm Phosphate buffer (60mM pH 70) waskept as a reference To determine the specific activity ofcatalase extinction coefficient of 004mMminus1 cmminus1 was used
(iv) Glutathione-S-Transferase (GST) Assay [23] 01mL 1-chloro-24-dinitrobenzene (CDNB) was added to 06mLsupernatant of liver homogenate and 22mL phosphate bufferpH 65 incubated at 37∘C for 5min and added 01mL 30mMGSH Absorbance was recorded at 340 nm at intervals of12345min Blank was carried out in the same mannerwithout homogenate
(v) Malondialdehyde Assay [24]OnemL of liver homogenatewas combined with 2mL of reaction mixture [15wvtrichloroacetic acid (TCA) and 0375wv thiobarbituricacid (TBA) in 025N hydrochloric acid (HCl)] and mixedthoroughly The solution was heated for 20min on boilingwater bath Samples were cooled and centrifuged at 1000 rpmfor 10min to remove the flocculent precipitate Super-natant was collected and the absorbance of read at 532 nmagainst a blank which contained all the reagents exceptthe liver homogenate The extinction coefficient of 156 times105Mminus1 cmminus1 was used to calculate the malondialdehydeconcentration
273 Histopathological Investigation For histological exam-inations liver pancreas and white adipose tissue (WAT)from three animals per group were isolated on day 32 aftertreatment of compounds The tissue samples were fixed informalin solution (10) for one week at room temperaturedehydrated by graded ethanol cleared using graded xyleneand embedded in paraffin wax 5 120583m thick sections werecut using rotary microtome (RM2245 Leica MicrosystemsGmbHWetzlar Germany) fixed on glass slides stained witheosin and hematoxylin and observed using a microscope(Model BX41 Olympus Corporation Tokyo Japan) ForLangerhans cells the average areas of six islets per specimenwere measured using ImageJ software (version 148 NationalInstitute of Health MD USA) Area was expressed as 120583m2The quantification of adipocytes were performed as reported[25] using adipocyte quantification tool where area wasmeasured in 120583m2
28 Statistical Analysis Statistical analysis was performedby comparing the responses of the treatment groups torespective saline control and vehicle treated groups for allexperiments and the significance was determined by one-way ANOVA followed by post hoc Dunnettrsquos test Values
8 BioMed Research International
Table 3 Effect of thiazolidin-4-ones on in vitro glucose uptake in absence and presence of insulin
S no GroupsGlucose uptake (mgg45min)
In absence of insulin In presence of insulinMean plusmn SEM (119899 = 3) Mean plusmn SEM (119899 = 3)
1 Glucose control 809 plusmn 051 1211 plusmn 1342 Compound 2a 189 plusmn 145 1033 plusmn 1213 Compound 2b 619 plusmn 031 736 plusmn 0254 Compound 2c 1012 plusmn 052 1488 plusmn 0865 Compound 2d 984 plusmn 061 816 plusmn 0116 Compound 2e 1900 plusmn 127a 2342 plusmn 032b
7 Compound 2f 603 plusmn 033 833 plusmn 0158 Compound 2g 645 plusmn 013 708 plusmn 0199 Compound 2h 761 plusmn 018 728 plusmn 03810 Compound 2i 555 plusmn 014 644 plusmn 11911 Compound 2j 954 plusmn 021 368 plusmn 17912 Compound 2k 582 plusmn 018 721 plusmn 01513 Compound 2l 643 plusmn 010 999 plusmn 01014 Compound 2m 812 plusmn 082 1287 plusmn 01515 Compound 3a 2044 plusmn 017a 2501 plusmn 029b
16 Compound 3b 777 plusmn 029 1151 plusmn 00217 Compound 3c 1224 plusmn 010 1050 plusmn 08118 Compound 4a 753 plusmn 110 798 plusmn 08319 Compound 4b 610 plusmn 083 772 plusmn 02120 Compound 4c 097 plusmn 059 482 plusmn 05521 Compound 4d 377 plusmn 047 554 plusmn 04922 Pioglitazone 1337 plusmn 026a 2523 plusmn 034b
a and b represent 119875 lt 005 as compared to glucose control in absencepresence of insulin respectively
Table 4 Effect of thiazolidin-4-ones on body weight and food efficiency ratio in mice
NPD HCD Pioglitazone Compound 2e Compound 3aFinal body weight (g) 380 plusmn 21 412 plusmn 22 382 plusmn 14 376 plusmn 12 382 plusmn 21Food efficiency ratio 00025 plusmn 0005 00083 plusmn 0006 00020 plusmn 0005 00043 plusmn 0011 0071 plusmn 0010abaRepresents 119875 lt 005 as compared to NPD group (normal pellet diet)bRepresents 119875 lt 005 as compared to HCD group (high carbohydrate diet)
were expressed as mean plusmn SEM 119875 lt 005 was consideredsignificant
3 Results
31 In Vitro Biological Study
311 Glucose Uptake Assay Of the twenty thiazolidin-4-ones tested compounds 2e and 3a stimulated glucose uptake(119875 lt 005) when compared with control Uptake wasstimulated both in the absence and presence of externalinsulin The standard drug pioglitazone also increased theglucose uptake (Table 3)
32 In Vivo Biological Studies
321 Acute Oral Toxicity Study Acute toxicity studies werecarried out on Swiss albino mice as per the OECD guidelines
for test compounds 2e and 3a Both were found to be safe upto 2000mgkg which was the maximum dose tested
322 High Carbohydrate Diet- (HCD-) Induced MetabolicDisorder in Mice Animals put on HCD for 6 months hadsignificant hyperglycemia hypertriglyceridemia and hyperc-holesterolemiaThey were randomized to different treatmentgroups based on plasma glucose level and then treatment wasgiven for 30 days Firstly treatment did not have any signif-icant effect on body weight of animals (Table 4) Secondlyenergy expenditure was assessed indirectly using FER Noneof the treated groups except compound 3a-treated group hadany significant effect on FER The calculated FER was foundto be significantly higher in animal treated with compound3a compared to normal control and HCD group High FERvalue indicates the increased energy expenditure in animalstreated with compound 3a
BioMed Research International 9
0
50
100
150
200
250
14 day 30 day
NPDHCD
lowast
lowast
lowastlowast
lowastlowast
lowastlowast lowastlowastlowastlowast
lowastlowast
Pioglitazone (5mgkg)
Plas
ma g
luco
se (m
gdL
)(M
eanplusmn
SE
Mn=6
)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
60
120
180
240
300
lowast
lowastlowastlowastlowast
lowastlowast
Plas
ma T
G (m
gdL
)(M
eanplusmn
SE
Mn=6
)
14 day 30 day
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
60
120
180
240
300
lowast
lowast
lowastlowast
lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowast
Plas
ma c
hole
stero
l (m
gdL
)(M
eanplusmn
SE
Mn=6
)
14 day 30 day
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 2 Effect of thiazolidin-4-ones on plasma (a) glucose (b) triglyceride (TG) (c) cholesterol inHCD fedmice Data presented asmean plusmnSEM (119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 as compared to HCD group
(i) Effect of Thiazolidin-4-Ones on Plasma Glucose Triglyc-eride and Cholesterol Level Biochemical estimation ofmetabolic markers such as plasma glucose (PG) triglycerides(TG) and total cholesterol (TC)was performed on day 14 andday 30 after the drug treatment and it was observed that HCDcontrol group consistently had significant increase in PG andTC levels on days 14 and 30 compared to respective dayrsquosnormal control group However TG levels were significantlyincreased only on day 30 compared to respective dayrsquos normalcontrol group Pioglitazone and test compounds (2e and3a) significantly reversed hyperglycemia and elevated plasmacholesterol compared to HCD control group on both day14 and day 30 of drug treatment (Figures 2(a) and 2(c))while elevated TG levels were reduced on day 30 by the testcompounds and pioglitazone (Figure 2(b))
(ii) Effect of Thiazolidin-4-Ones on Plasma Insulin Leptinand Adiponectin Level Development of insulin resistance
in mouse was confirmed by estimating the plasma insulinlevels on day 30 after drug treatment HCD control group incontrast to NPD group showed two times increase in plasmainsulin levels (119875 lt 005) Hyperinsulinemia associated withhyperglycaemia and hypertriglyceridemia is considered tobe the sign of development of insulin resistance Thus highsucrose feeding for 6 months led to the development ofinsulin resistance in mice Treatment with pioglitazone andtest drugs attenuated the insulin resistance (Figure 3(a))
HCD feeding to animals did not elicit any significanteffect on plasma leptin levels as there was no significantdifference between HCD control and NPD group Howevertreatment with pioglitazone and test compounds significantly(119875 lt 005) raised the circulating leptin level in micecomparedwithHCDcontrol group (Figure 3(b)) Compound3a caused a fourfold elevation in leptin levels compared withHCD group HCD feeding to animals resulted in significant
10 BioMed Research International
0
1
2
3
4
5
NPDHCDPioglitazone (5mgkg)
Plas
ma i
nsul
in (n
gm
L)(M
eanplusmn
SE
Mn=6
)
lowastlowast
lowast
lowastlowastlowastlowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
1200
2400
3600
4800
6000
NPDHCDPioglitazone (5mgkg)
Plas
ma l
eptin
(pg
mL)
(Mea
nplusmn
SE
Mn=6
)
lowast lowast
lowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
12000
24000
36000
48000
60000
NPDHCDPioglitazone (5mgkg)
Plas
ma a
dipo
nect
in (n
gm
L)(M
eanplusmn
SE
Mn=6
)
lowast
lowastlowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 3 Effect of thiazolidin-4-ones on plasma (a) insulin (b) leptin (c) adiponectin in HCD fed mice Data presented as mean plusmn SEM(119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 as compared to HCD group
(119875 lt 005) hypoadiponectinemia comparedwithNPD Treat-ment with pioglitazone significantly (119875 lt 005) corrected thehypoadiponectinemia However both test compounds failedto correct hypoadiponectinemia (Figure 3(c))
(iii) Effect of Thiazolidin-4-Ones on Oral Glucose ToleranceTest In oral glucose tolerance test HCD group animalshowed significant (119875 lt 005) glucose intolerance (Figure 4)Pioglitazone and test compounds compound 2e and com-pound 3a corrected the glucose intolerance shown as sig-nificant (119875 lt 005) percent reductions in glucose excursion(AUC
0minus120min) by 1300 plusmn 33 1546 plusmn 554 and 1560 plusmn 349respectively compared with HCD group (Figures 4(a) and4(b))
(iv) Effect of Thiazolidin-4-Ones on Liver Enzymes Oxidativestress is the hallmark of metabolic disorder where disturbedhomeostasis between oxidative and antioxidative mechanismoccurs HCD feeding to mice resulted in an oxidative stress
observed as reduction in liver antioxidant enzymes such asglutathione (GSH) catalase (CAT) superoxide dismutase(SOD) and glutathione-S-transferase (GST) Treatment withpioglitazone and test compounds ameliorated the oxidativestress They also reversed the elevation in liver malondialde-hyde (MDA) levels in HCD-fed mice (Table 5)
(v) Histopathological ExaminationHistology of liver showednormal lobular architecture with normal hepatocytes inall groups (Figure 5(a)) Pancreas showed mild to mod-erate hyperplasia of islets of Langerhans (Figure 5(a)) inpioglitazone and compound 3a groups Pioglitazone and3a treatment significantly raised the area of Langerhansislets compared with HCD group (Figure 5(b)) Exocrineportion of the pancreas and vascularity appeared normalin all the groups Histopathological investigation of whiteadipose tissue (WAT) from epidydimal showed an increasein the size of adipocytes (Figure 5(c)) in HCD group whichwas reversed by pioglitazone and compound 3a treatment
BioMed Research International 11
Table 5 Effect of thiazolidin-4-ones on liver antioxidant enzyme and malondialdehyde level
Liver biomarkers NPD HCD Pioglitazone Compound 2e Compound 3aGSH (nmolemg of protein) 558 plusmn 21 464 plusmn 21a 632 plusmn 15b 683 plusmn 20b 678 plusmn 43b
CAT (Umg of protein) 892 plusmn 57 554 plusmn 31a 938 plusmn 10b 819 plusmn 24b 875 plusmn 74b
SOD (Umg of protein) 1868 plusmn 232 1081 plusmn 112a 1748 plusmn 144b 2022 plusmn 47b 2080 plusmn 154b
GST (Umg of protein) 043 plusmn 005 037 plusmn 001a 073 plusmn 002b 070 plusmn 001b 072 plusmn 004b
MDA (nmolemg of protein) 057 plusmn 006 10 plusmn 033a 046 plusmn 001b 040 plusmn 001b 041 plusmn 002baRepresents 119875 lt 005 as compared to NPD group (normal pellet diet)bRepresents 119875 lt 005 as compared to HCD group (high carbohydrate diet)
0 30 60 90 120100
130
160
190
220
Plas
ma g
luco
se (m
gdL
)(M
eanplusmn
SE
Mn=6
)
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
5000
10000
15000
20000
25000
(Mea
nplusmn
SE
Mn=6
)
lowast
lowastlowast lowastlowast lowastlowast
NPDHCDPioglitazone (5mgkg)
Plas
ma g
luco
se (A
UC 0
ndash120
min)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
Figure 4 Effect of thiazolidin-4-ones on (a) plasma glucose (mgdL) and (b) area under the curve (AUC0minus120 min) against oral glucose
tolerance test (OGTT) in HCD fed mice Data presented as mean plusmn SEM (119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD groupand lowastlowastrepresents 119875 lt 005 as compared to HCD group
However compound 2e did not show any significant effecton increased adipocyte size
4 Discussion and Conclusion
The present work was planned as a sequel to earlier studiesin our laboratory using thiazolidin-4-ones as antidiabetichypolipidemic and antiinflammatory molecules The substi-tutions in the thiazolidine ring were made at C2 and N3Attached to the latter position was a nicotinamide moietyand the substitution at C2 was either p-methoxyphenyl or2 5-di-tert-butyl-4-hydroxyphenyl group The compoundsshowed significant antidiabetic and hypolipidemic activities[9ndash11 26]
In the present study the substitution at N3 was changedto p-chloro-phenoxyacetylamino a group that is similar toclofibrate with a methylene bridge instead of gem-dimethylsubstitution
Four compounds out of 20 (ie compounds 4a 4b4c and 4d) had a methyl group attached at the C5 ofthiazolidine ring with cyclisation being made with thiolacticacid instead of thioglycolic acid In three other compounds(ie compounds 3a 3b and 3c) clofibrate was used to make
themoiety for substitution atN3Thiswas donewith a view toexamine the effect of the gem-dimethyl on the overall activityof the resulting molecule
All the 20 synthesised thiazolidin-4-ones were evaluatedfor glucose uptake in an in vitro system using the isolatedrat diaphragm In this experiment compounds 2e and 3asignificantly raised the amount of glucose uptake by thetissue both in the absence and presence of external insulinThis indicated the potential of the theses compounds insensitizing the tissues for the external insulin Hence thesetwo compounds were chosen to study their effect on a diet-induced model of insulin resistance Both compounds havethe same p-methoxyphenyl moiety attached to the C2 of thethiazolidine ring Compound 3a has a gem-dimethyl groupinstead of methylene in the substituent at N3
Apart from multiple risk factors diet-induced metabolicabnormalities contribute to the development of insulinresistance and 120573-cell failure in type-2 diabetes [16] Earlydetection and appropriate treatment are considered beneficialfor correcting the abnormality Among the various animalmodels induction of diabetes through diet provides moreresemblance to human type-2 diabetes Chronic intake ofdiet with high sucrose content has been reported to favourthe development of insulin resistance [16] Similarly in our
12 BioMed Research International
NPD HCD Pioglitazone Compound 3a Compound 2e
Liver
WAT
Green and red arrows indicate hyperplasia and increased size of adipocytes respectively
Pancreas
(a)
0
50000
100000
150000
200000
NPDHCDPioglitazone (5mgkg)
lowastlowastlowastlowast
(Mea
nplusmn
SE
Mn=3
)A
rea o
f lan
gerh
ans i
stle
ts (120583
m2)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
750
1500
2250
3000
NPDHCDPioglitazone (5mgkg)
lowastlowastlowastlowast
lowast
(Mea
nplusmn
SE
Mn=3
)A
rea o
f adi
pocy
tes (120583
m2)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 5 (a) Histological sections of mouse liver pancreas and white adipose tissue (WAT) in HCD model (10x) Green and red arrowsindicate hyperplasia of islets of Langerhans and increase in the size of adipocytes respectively Effect of thiazolidin-4-ones on (b) area ofLangerhans islets (120583m2) and (c) area of adipocytes (120583m2) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 ascompared to HCD group
study mice fed a high-sucrose diet for six months devel-oped metabolic abnormalities like hyperglycemia hyperin-sulinemia hypertriglyceridemia hypercholesterolemia andhypoadiponectinemia Hyperglycemia and hyperinsulinemiasuggest the inability of insulin to sensitize the tissue forglucose uptake allowing glucose to be diverted toward lipo-genesisThis led to hyperlipidemia and finally to insulin resis-tance In addition adiponectin and leptin the adipokinessecreted from white adipose tissue (WAT) are reported tobe involved in the metabolism of glucose and lipid [27]In our study we observed that HCD caused a reductionin plasma adiponectin level and an increase in the sizeof adipocytes However it did not cause any change inplasma leptin levels Thus we found a correlation between
the adiponectin level and size of adipocytes in HCD modelFurther OGTT results from HCD-fed mice correlated wellwith glucose intolerance hyperinsulinemia and hypoad-iponectinemia The authenticity of the model was validatedby the effect of pioglitazone which was able to correct themetabolic abnormalities Neither test compounds (2e or 3a)attenuated hypoadiponectinemia However they correctedthe impaired glucose tolerance and insulin resistance inmice Compounds 2e and 3a by their ability to enhanceglucose uptake and to sensitize the tissue for available insulinreduced hyperinsulinemia and raised the leptin levels Thiswould have resulted in better glucose utilization by periph-eral tissue Thus these compounds reduced the metabolicabnormalities like hyperglycemia hypertriglyceridemia and
BioMed Research International 13
hypercholesterolemia Prospective studies involving directassessment of insulin sensitivity in OGTT are requiredto analyze glucose stimulated insulin secretion (GSIS) inpresence of compounds to establish the mechanistic role ofthe test compounds on insulin signalling
Histological investigation showed no change in liverarchitecture in the various treatment groups In HCD controlmice there was an increase in the size of adipocytes in whiteadipose tissue (WAT) which was reversed by compound3a and pioglitazone treatment However only pioglitazone-treated animals showed positive correlation between reducedsize of the cells and correction of hypoadiponectinemiaApart from correction of hypoadiponectinemia pioglitazoneraised the peripheral leptin levels which was also true in caseof compounds 2e and 3a treatment Among the tested com-pounds 3a raised endogenous leptin levels four times morethan HCD group This finding points to the link betweenraised leptin levels and reduced adipocyte size caused bycompound 3a The pancreatic islets showed hyperplasia inpioglitazone and other treatment groups Pioglitazone is aPPAR-120574 agonist This could have been responsible for theproliferation of beta cells of pancreas The test compoundsare thiazolidin-4-ones with some similarity to thiazolidine-diones It is possible that hyperplasia observed in compound3a-treated animals might have been due to some agonisticactivity on PPAR-120574 receptors This needs to be investigatedthrough relevant assay
Oxidative stress has been implicated in the occurrence ofdiabetes and compounds reducing the oxidative stress havebeneficial role in correcting glucose intolerance and insulinresistance in diabetes [18 28]The test compounds and piogli-tazone reversed the depletion of endogenous antioxidantenzymes such as GSH CAT SOD and GST Further theyreduced malondialdehyde levels This suggests the inhibitoryeffect of these compounds on oxidative stress
Leptin serves as an insulin-sensitizing factor in the wholebody [29] However hyperleptinemia in the obese mouseand human is a sign of leptin resistance where increasedleptin levels are caused by disturbed homeostasis arisingfrom leptin receptor mutation ageing or obesity [29] Thushyperleptinemia further worsens impaired insulin actionin pathological condition In these conditions exogenouslyadministered leptin does not improve glucose tolerance andinsulin sensitivity In our study diseased animal (HCD fedmouse) per se did not develop hyperleptinemia which mightbe the sign of early stage of metabolic abnormality associatedwith insulin resistance At this state test compounds aswell as pioglitazone reversed the elevated glucose TG TCand insulin levels while facilitating leptin profile along withinsulin sensitivity Subsequently compound 3a treatmentresulted in increased energy expenditure demonstrated byelevated food efficiency ratio (FER) However reductionin body weight among the treated groups was not foundproportionate to the circulating leptin levels which suggeststhat increased leptin level in peripheral blood is not suffi-cient to induce a proportionate reduction on body weightA few questions remain unanswered such as (i) whetherthe compounds directly raised the leptin levels or are theresults due to indirect impact on metabolic signaling (ii) In
metabolic disorder combined with hyperleptinemia how dothese compounds affect leptin signaling Future studies areneeded to address these issues
No single mechanism would suffice to explain the ben-eficial effects of the test compounds They do not seem toact through insulinotropic activity unlike the sulfonylureasThey have no significant effect on adiponectin levels rulingout any involvement of this mechanism The increase in thelevel of serum leptin might point to the involvement of leptinin the antihyperlipidemic and antidiabetic potentials of thesemolecules
In conclusion thiazolidin-4-one derivatives act throughmultiple mechanisms to correct the metabolic abnormalitiesin type-2 diabetes In the present work compounds 2e and3a were found to be the most effective test compoundsto ameliorate insulin resistance and development of type-2diabetes
Conflict of Interests
The authors declare that they do not have any conflict ofinterests
Acknowledgment
The authors wish to thank All India Council for TechnicalEducation (AICTE) New Delhi India for providing thefinancial support and Manipal University Manipal Kar-nataka India for providing the facilities to carry out thepresent work
References
[1] S M Grundy I J Benjamin G L Burke et al ldquoDiabetes andcardiovascular disease a statement for healthcare professionalsfrom the american heart associationrdquo Circulation vol 100 no10 pp 1134ndash1146 1999
[2] K E Thorpe and D H Howard ldquoThe rise in spending amongMedicare beneficiaries the role of chronic disease prevalenceand changes in treatment intensity Increasing numbers ofbeneficiaries being treated for five or more conditions a year aredriving Medicare spending upwardrdquo Health Affairs vol 25 no5 pp w378ndashw388 2006
[3] S M Grundy ldquoDrug therapy of the metabolic syndrome min-imizing the emerging crisis in polypharmacyrdquo Nature ReviewsDrug Discovery vol 5 no 4 pp 295ndash309 2006
[4] M Navab N Gharavi and A D Watson ldquoInflammation andmetabolic disordersrdquo Current Opinion in Clinical Nutrition andMetabolic Care vol 11 no 4 pp 459ndash464 2008
[5] G S Hotamisligil ldquoInflammation and metabolic disordersrdquoNature vol 444 no 7121 pp 860ndash867 2006
[6] M Schmuth V Moosbrugger-Martinz S Blunder and SDubrac ldquoRole of PPAR LXR and PXR in epidermal homeosta-sis and inflammationrdquo Biochimica et Biophysica Acta vol 1841no 3 pp 463ndash473 2014
[7] J A Panetta J K Shadle M L Phillips D N Benslayand P P K Ho ldquo4-thiazolidinones potent antioxidants asantiinflammatory agentsrdquo Annals of the New York Academy ofSciences vol 696 pp 415ndash416 1993
14 BioMed Research International
[8] T Kato T Ozaki K Tamura Y Suzuki M Akima and NOhi ldquoNovel calcium antagonists with both calcium overloadinhibition and antioxidant activity 1 2-(35-Di-tert-butyl-4-hydroxyphenyl)-3- (aminopropyl)thiazolidinonesrdquo Journal ofMedicinal Chemistry vol 41 no 22 pp 4309ndash4316 1998
[9] JM Joy N Jacob andGN Kutty ldquoEvaluation of hypoglycemiceffects of 4-thiazolidinonesrdquo Indian Drugs vol 42 no 1 pp 17ndash21 2005
[10] N Jacob and G N Kutty ldquoSynthesis and hypolipidemic activityof a thiazolidinone derivativerdquo Indian Drugs vol 41 no 2 pp76ndash79 2004
[11] G K Nampurath S P Mathew V Khanna R T Zachariah SKanji andMRChamallamudi ldquoAssessment of hypolipidaemicactivity of three thiazolidin-4-ones in mice given high-fat dietand fructoserdquoChemico-Biological Interactions vol 171 no 3 pp363ndash368 2008
[12] A Kishore G K Nampurath S P Mathew et al ldquoAntidiabeticeffect through islet cell protection in streptozotocin diabetesa preliminary assessment of two thiazolidin-4-ones in Swissalbino micerdquo Chemico-Biological Interactions vol 177 no 3 pp242ndash246 2009
[13] J Mudgal V S Gowdra G Mathew et al ldquoRemedial effects ofnovel 2 3-disubstituted thiazolidin-4-ones in chemical medi-ated inflammationrdquo Chemico-Biological Interactions vol 210pp 34ndash42 2014
[14] A R Surrey ldquo4-Thiazolidones IV the preparation of some3-alkylaminoalkyl-2-aryl derivativesrdquo Journal of the AmericanChemical Society vol 71 no 10 pp 3354ndash3356 1949
[15] B R P Kumar and M J Nanjan ldquoNovel glitazones designsynthesis glucose uptake and structure-activity relationshipsrdquoBioorganic and Medicinal Chemistry Letters vol 20 no 6 pp1953ndash1956 2010
[16] M Sumiyoshi M Sakanaka and Y Kimura ldquoChronic intakeof high-fat and high-sucrose diets differentially affects glucoseintolerance inmicerdquo Journal of Nutrition vol 136 no 3 pp 582ndash587 2006
[17] S H Kim S H Hyun and S Y Choung ldquoAnti-diabetic effectof cinnamon extract on blood glucose in dbdb micerdquo Journalof Ethnopharmacology vol 104 no 1-2 pp 119ndash123 2006
[18] S A Manohara Reddy J Mudgal P Bansal et al ldquoAntioxidantanti-inflammatory and anti-hyperglycaemic activities of hete-rocyclic homoprostanoid derivativesrdquoBioorganic andMedicinalChemistry vol 19 no 1 pp 384ndash392 2011
[19] S T Shukla V H Kulkarni P V Habbu K S Jagadeesh BS Patil and D M Smita ldquoHepatoprotective and antioxidantactivities of crude fractions of endophytic fungi of Ocimumsanctum Linn in ratsrdquo Oriental Pharmacy and ExperimentalMedicine vol 12 no 2 pp 81ndash91 2012
[20] B Mannervik ldquoGlutathione peroxidaserdquo Methods in Enzymol-ogy vol 113 pp 490ndash495 1985
[21] H PMisra and I Fridovich ldquoThe role of superoxide anion in theautoxidation of epinephrine and a simple assay for superoxidedismutaserdquo Journal of Biological Chemistry vol 247 no 10 pp3170ndash3175 1972
[22] A Claiborne ldquoCatalase activityrdquo in Handbook of Methods forOxygen Radical Research R A Greenwald Ed pp 283ndash284CRC Press Boca Raton Fla USA 1985
[23] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974
[24] W G Niehaus Jr and B Samuelsson ldquoFormation of malonalde-hyde from phospholipid arachidonate during microsomal lipidperoxidationrdquoEuropean Journal of Biochemistry vol 6 no 1 pp126ndash130 1968
[25] O S Osman J L Selway M A Kępczynska et al ldquoA novelautomated image analysis method for accurate adipocyte quan-tificationrdquo Adipocyte vol 2 no 3 pp 160ndash164 2013
[26] R Kalia C M Rao and N Gopalan Kutty ldquoSynthesisand evaluation of the anti-inflammatory activity of N-[2-(35-di-tert-butyl-4-hydroxyphenyl)-4-oxothiazolidin-3-yl]-nicotinamiderdquo Drug Research vol 57 no 9 pp 616ndash6222007
[27] N Maeda M Takahashi T Funahashi et al ldquoPPAR120574 ligandsincrease expression and plasma concentrations of adiponectinan adipose-derived proteinrdquo Diabetes vol 50 no 9 pp 2094ndash2099 2001
[28] P Bansal P Paul J Mudgal et al ldquoAntidiabetic antihyperlipi-demic and antioxidant effects of the flavonoid rich fraction ofPilea microphylla (L) in high fat dietstreptozotocin-induceddiabetes in micerdquo Experimental and Toxicologic Pathology vol64 no 6 pp 651ndash658 2012
[29] C Perez C Fernandez-Galaz T Fernandez-Agullo et alldquoLeptin impairs insulin signaling in rat adipocytesrdquo Diabetesvol 53 no 2 pp 347ndash353 2004
Submit your manuscripts athttpwwwhindawicom
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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BioMed Research International
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MEDIATORSINFLAMMATION
of
8 BioMed Research International
Table 3 Effect of thiazolidin-4-ones on in vitro glucose uptake in absence and presence of insulin
S no GroupsGlucose uptake (mgg45min)
In absence of insulin In presence of insulinMean plusmn SEM (119899 = 3) Mean plusmn SEM (119899 = 3)
1 Glucose control 809 plusmn 051 1211 plusmn 1342 Compound 2a 189 plusmn 145 1033 plusmn 1213 Compound 2b 619 plusmn 031 736 plusmn 0254 Compound 2c 1012 plusmn 052 1488 plusmn 0865 Compound 2d 984 plusmn 061 816 plusmn 0116 Compound 2e 1900 plusmn 127a 2342 plusmn 032b
7 Compound 2f 603 plusmn 033 833 plusmn 0158 Compound 2g 645 plusmn 013 708 plusmn 0199 Compound 2h 761 plusmn 018 728 plusmn 03810 Compound 2i 555 plusmn 014 644 plusmn 11911 Compound 2j 954 plusmn 021 368 plusmn 17912 Compound 2k 582 plusmn 018 721 plusmn 01513 Compound 2l 643 plusmn 010 999 plusmn 01014 Compound 2m 812 plusmn 082 1287 plusmn 01515 Compound 3a 2044 plusmn 017a 2501 plusmn 029b
16 Compound 3b 777 plusmn 029 1151 plusmn 00217 Compound 3c 1224 plusmn 010 1050 plusmn 08118 Compound 4a 753 plusmn 110 798 plusmn 08319 Compound 4b 610 plusmn 083 772 plusmn 02120 Compound 4c 097 plusmn 059 482 plusmn 05521 Compound 4d 377 plusmn 047 554 plusmn 04922 Pioglitazone 1337 plusmn 026a 2523 plusmn 034b
a and b represent 119875 lt 005 as compared to glucose control in absencepresence of insulin respectively
Table 4 Effect of thiazolidin-4-ones on body weight and food efficiency ratio in mice
NPD HCD Pioglitazone Compound 2e Compound 3aFinal body weight (g) 380 plusmn 21 412 plusmn 22 382 plusmn 14 376 plusmn 12 382 plusmn 21Food efficiency ratio 00025 plusmn 0005 00083 plusmn 0006 00020 plusmn 0005 00043 plusmn 0011 0071 plusmn 0010abaRepresents 119875 lt 005 as compared to NPD group (normal pellet diet)bRepresents 119875 lt 005 as compared to HCD group (high carbohydrate diet)
were expressed as mean plusmn SEM 119875 lt 005 was consideredsignificant
3 Results
31 In Vitro Biological Study
311 Glucose Uptake Assay Of the twenty thiazolidin-4-ones tested compounds 2e and 3a stimulated glucose uptake(119875 lt 005) when compared with control Uptake wasstimulated both in the absence and presence of externalinsulin The standard drug pioglitazone also increased theglucose uptake (Table 3)
32 In Vivo Biological Studies
321 Acute Oral Toxicity Study Acute toxicity studies werecarried out on Swiss albino mice as per the OECD guidelines
for test compounds 2e and 3a Both were found to be safe upto 2000mgkg which was the maximum dose tested
322 High Carbohydrate Diet- (HCD-) Induced MetabolicDisorder in Mice Animals put on HCD for 6 months hadsignificant hyperglycemia hypertriglyceridemia and hyperc-holesterolemiaThey were randomized to different treatmentgroups based on plasma glucose level and then treatment wasgiven for 30 days Firstly treatment did not have any signif-icant effect on body weight of animals (Table 4) Secondlyenergy expenditure was assessed indirectly using FER Noneof the treated groups except compound 3a-treated group hadany significant effect on FER The calculated FER was foundto be significantly higher in animal treated with compound3a compared to normal control and HCD group High FERvalue indicates the increased energy expenditure in animalstreated with compound 3a
BioMed Research International 9
0
50
100
150
200
250
14 day 30 day
NPDHCD
lowast
lowast
lowastlowast
lowastlowast
lowastlowast lowastlowastlowastlowast
lowastlowast
Pioglitazone (5mgkg)
Plas
ma g
luco
se (m
gdL
)(M
eanplusmn
SE
Mn=6
)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
60
120
180
240
300
lowast
lowastlowastlowastlowast
lowastlowast
Plas
ma T
G (m
gdL
)(M
eanplusmn
SE
Mn=6
)
14 day 30 day
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
60
120
180
240
300
lowast
lowast
lowastlowast
lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowast
Plas
ma c
hole
stero
l (m
gdL
)(M
eanplusmn
SE
Mn=6
)
14 day 30 day
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 2 Effect of thiazolidin-4-ones on plasma (a) glucose (b) triglyceride (TG) (c) cholesterol inHCD fedmice Data presented asmean plusmnSEM (119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 as compared to HCD group
(i) Effect of Thiazolidin-4-Ones on Plasma Glucose Triglyc-eride and Cholesterol Level Biochemical estimation ofmetabolic markers such as plasma glucose (PG) triglycerides(TG) and total cholesterol (TC)was performed on day 14 andday 30 after the drug treatment and it was observed that HCDcontrol group consistently had significant increase in PG andTC levels on days 14 and 30 compared to respective dayrsquosnormal control group However TG levels were significantlyincreased only on day 30 compared to respective dayrsquos normalcontrol group Pioglitazone and test compounds (2e and3a) significantly reversed hyperglycemia and elevated plasmacholesterol compared to HCD control group on both day14 and day 30 of drug treatment (Figures 2(a) and 2(c))while elevated TG levels were reduced on day 30 by the testcompounds and pioglitazone (Figure 2(b))
(ii) Effect of Thiazolidin-4-Ones on Plasma Insulin Leptinand Adiponectin Level Development of insulin resistance
in mouse was confirmed by estimating the plasma insulinlevels on day 30 after drug treatment HCD control group incontrast to NPD group showed two times increase in plasmainsulin levels (119875 lt 005) Hyperinsulinemia associated withhyperglycaemia and hypertriglyceridemia is considered tobe the sign of development of insulin resistance Thus highsucrose feeding for 6 months led to the development ofinsulin resistance in mice Treatment with pioglitazone andtest drugs attenuated the insulin resistance (Figure 3(a))
HCD feeding to animals did not elicit any significanteffect on plasma leptin levels as there was no significantdifference between HCD control and NPD group Howevertreatment with pioglitazone and test compounds significantly(119875 lt 005) raised the circulating leptin level in micecomparedwithHCDcontrol group (Figure 3(b)) Compound3a caused a fourfold elevation in leptin levels compared withHCD group HCD feeding to animals resulted in significant
10 BioMed Research International
0
1
2
3
4
5
NPDHCDPioglitazone (5mgkg)
Plas
ma i
nsul
in (n
gm
L)(M
eanplusmn
SE
Mn=6
)
lowastlowast
lowast
lowastlowastlowastlowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
1200
2400
3600
4800
6000
NPDHCDPioglitazone (5mgkg)
Plas
ma l
eptin
(pg
mL)
(Mea
nplusmn
SE
Mn=6
)
lowast lowast
lowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
12000
24000
36000
48000
60000
NPDHCDPioglitazone (5mgkg)
Plas
ma a
dipo
nect
in (n
gm
L)(M
eanplusmn
SE
Mn=6
)
lowast
lowastlowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 3 Effect of thiazolidin-4-ones on plasma (a) insulin (b) leptin (c) adiponectin in HCD fed mice Data presented as mean plusmn SEM(119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 as compared to HCD group
(119875 lt 005) hypoadiponectinemia comparedwithNPD Treat-ment with pioglitazone significantly (119875 lt 005) corrected thehypoadiponectinemia However both test compounds failedto correct hypoadiponectinemia (Figure 3(c))
(iii) Effect of Thiazolidin-4-Ones on Oral Glucose ToleranceTest In oral glucose tolerance test HCD group animalshowed significant (119875 lt 005) glucose intolerance (Figure 4)Pioglitazone and test compounds compound 2e and com-pound 3a corrected the glucose intolerance shown as sig-nificant (119875 lt 005) percent reductions in glucose excursion(AUC
0minus120min) by 1300 plusmn 33 1546 plusmn 554 and 1560 plusmn 349respectively compared with HCD group (Figures 4(a) and4(b))
(iv) Effect of Thiazolidin-4-Ones on Liver Enzymes Oxidativestress is the hallmark of metabolic disorder where disturbedhomeostasis between oxidative and antioxidative mechanismoccurs HCD feeding to mice resulted in an oxidative stress
observed as reduction in liver antioxidant enzymes such asglutathione (GSH) catalase (CAT) superoxide dismutase(SOD) and glutathione-S-transferase (GST) Treatment withpioglitazone and test compounds ameliorated the oxidativestress They also reversed the elevation in liver malondialde-hyde (MDA) levels in HCD-fed mice (Table 5)
(v) Histopathological ExaminationHistology of liver showednormal lobular architecture with normal hepatocytes inall groups (Figure 5(a)) Pancreas showed mild to mod-erate hyperplasia of islets of Langerhans (Figure 5(a)) inpioglitazone and compound 3a groups Pioglitazone and3a treatment significantly raised the area of Langerhansislets compared with HCD group (Figure 5(b)) Exocrineportion of the pancreas and vascularity appeared normalin all the groups Histopathological investigation of whiteadipose tissue (WAT) from epidydimal showed an increasein the size of adipocytes (Figure 5(c)) in HCD group whichwas reversed by pioglitazone and compound 3a treatment
BioMed Research International 11
Table 5 Effect of thiazolidin-4-ones on liver antioxidant enzyme and malondialdehyde level
Liver biomarkers NPD HCD Pioglitazone Compound 2e Compound 3aGSH (nmolemg of protein) 558 plusmn 21 464 plusmn 21a 632 plusmn 15b 683 plusmn 20b 678 plusmn 43b
CAT (Umg of protein) 892 plusmn 57 554 plusmn 31a 938 plusmn 10b 819 plusmn 24b 875 plusmn 74b
SOD (Umg of protein) 1868 plusmn 232 1081 plusmn 112a 1748 plusmn 144b 2022 plusmn 47b 2080 plusmn 154b
GST (Umg of protein) 043 plusmn 005 037 plusmn 001a 073 plusmn 002b 070 plusmn 001b 072 plusmn 004b
MDA (nmolemg of protein) 057 plusmn 006 10 plusmn 033a 046 plusmn 001b 040 plusmn 001b 041 plusmn 002baRepresents 119875 lt 005 as compared to NPD group (normal pellet diet)bRepresents 119875 lt 005 as compared to HCD group (high carbohydrate diet)
0 30 60 90 120100
130
160
190
220
Plas
ma g
luco
se (m
gdL
)(M
eanplusmn
SE
Mn=6
)
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
5000
10000
15000
20000
25000
(Mea
nplusmn
SE
Mn=6
)
lowast
lowastlowast lowastlowast lowastlowast
NPDHCDPioglitazone (5mgkg)
Plas
ma g
luco
se (A
UC 0
ndash120
min)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
Figure 4 Effect of thiazolidin-4-ones on (a) plasma glucose (mgdL) and (b) area under the curve (AUC0minus120 min) against oral glucose
tolerance test (OGTT) in HCD fed mice Data presented as mean plusmn SEM (119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD groupand lowastlowastrepresents 119875 lt 005 as compared to HCD group
However compound 2e did not show any significant effecton increased adipocyte size
4 Discussion and Conclusion
The present work was planned as a sequel to earlier studiesin our laboratory using thiazolidin-4-ones as antidiabetichypolipidemic and antiinflammatory molecules The substi-tutions in the thiazolidine ring were made at C2 and N3Attached to the latter position was a nicotinamide moietyand the substitution at C2 was either p-methoxyphenyl or2 5-di-tert-butyl-4-hydroxyphenyl group The compoundsshowed significant antidiabetic and hypolipidemic activities[9ndash11 26]
In the present study the substitution at N3 was changedto p-chloro-phenoxyacetylamino a group that is similar toclofibrate with a methylene bridge instead of gem-dimethylsubstitution
Four compounds out of 20 (ie compounds 4a 4b4c and 4d) had a methyl group attached at the C5 ofthiazolidine ring with cyclisation being made with thiolacticacid instead of thioglycolic acid In three other compounds(ie compounds 3a 3b and 3c) clofibrate was used to make
themoiety for substitution atN3Thiswas donewith a view toexamine the effect of the gem-dimethyl on the overall activityof the resulting molecule
All the 20 synthesised thiazolidin-4-ones were evaluatedfor glucose uptake in an in vitro system using the isolatedrat diaphragm In this experiment compounds 2e and 3asignificantly raised the amount of glucose uptake by thetissue both in the absence and presence of external insulinThis indicated the potential of the theses compounds insensitizing the tissues for the external insulin Hence thesetwo compounds were chosen to study their effect on a diet-induced model of insulin resistance Both compounds havethe same p-methoxyphenyl moiety attached to the C2 of thethiazolidine ring Compound 3a has a gem-dimethyl groupinstead of methylene in the substituent at N3
Apart from multiple risk factors diet-induced metabolicabnormalities contribute to the development of insulinresistance and 120573-cell failure in type-2 diabetes [16] Earlydetection and appropriate treatment are considered beneficialfor correcting the abnormality Among the various animalmodels induction of diabetes through diet provides moreresemblance to human type-2 diabetes Chronic intake ofdiet with high sucrose content has been reported to favourthe development of insulin resistance [16] Similarly in our
12 BioMed Research International
NPD HCD Pioglitazone Compound 3a Compound 2e
Liver
WAT
Green and red arrows indicate hyperplasia and increased size of adipocytes respectively
Pancreas
(a)
0
50000
100000
150000
200000
NPDHCDPioglitazone (5mgkg)
lowastlowastlowastlowast
(Mea
nplusmn
SE
Mn=3
)A
rea o
f lan
gerh
ans i
stle
ts (120583
m2)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
750
1500
2250
3000
NPDHCDPioglitazone (5mgkg)
lowastlowastlowastlowast
lowast
(Mea
nplusmn
SE
Mn=3
)A
rea o
f adi
pocy
tes (120583
m2)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 5 (a) Histological sections of mouse liver pancreas and white adipose tissue (WAT) in HCD model (10x) Green and red arrowsindicate hyperplasia of islets of Langerhans and increase in the size of adipocytes respectively Effect of thiazolidin-4-ones on (b) area ofLangerhans islets (120583m2) and (c) area of adipocytes (120583m2) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 ascompared to HCD group
study mice fed a high-sucrose diet for six months devel-oped metabolic abnormalities like hyperglycemia hyperin-sulinemia hypertriglyceridemia hypercholesterolemia andhypoadiponectinemia Hyperglycemia and hyperinsulinemiasuggest the inability of insulin to sensitize the tissue forglucose uptake allowing glucose to be diverted toward lipo-genesisThis led to hyperlipidemia and finally to insulin resis-tance In addition adiponectin and leptin the adipokinessecreted from white adipose tissue (WAT) are reported tobe involved in the metabolism of glucose and lipid [27]In our study we observed that HCD caused a reductionin plasma adiponectin level and an increase in the sizeof adipocytes However it did not cause any change inplasma leptin levels Thus we found a correlation between
the adiponectin level and size of adipocytes in HCD modelFurther OGTT results from HCD-fed mice correlated wellwith glucose intolerance hyperinsulinemia and hypoad-iponectinemia The authenticity of the model was validatedby the effect of pioglitazone which was able to correct themetabolic abnormalities Neither test compounds (2e or 3a)attenuated hypoadiponectinemia However they correctedthe impaired glucose tolerance and insulin resistance inmice Compounds 2e and 3a by their ability to enhanceglucose uptake and to sensitize the tissue for available insulinreduced hyperinsulinemia and raised the leptin levels Thiswould have resulted in better glucose utilization by periph-eral tissue Thus these compounds reduced the metabolicabnormalities like hyperglycemia hypertriglyceridemia and
BioMed Research International 13
hypercholesterolemia Prospective studies involving directassessment of insulin sensitivity in OGTT are requiredto analyze glucose stimulated insulin secretion (GSIS) inpresence of compounds to establish the mechanistic role ofthe test compounds on insulin signalling
Histological investigation showed no change in liverarchitecture in the various treatment groups In HCD controlmice there was an increase in the size of adipocytes in whiteadipose tissue (WAT) which was reversed by compound3a and pioglitazone treatment However only pioglitazone-treated animals showed positive correlation between reducedsize of the cells and correction of hypoadiponectinemiaApart from correction of hypoadiponectinemia pioglitazoneraised the peripheral leptin levels which was also true in caseof compounds 2e and 3a treatment Among the tested com-pounds 3a raised endogenous leptin levels four times morethan HCD group This finding points to the link betweenraised leptin levels and reduced adipocyte size caused bycompound 3a The pancreatic islets showed hyperplasia inpioglitazone and other treatment groups Pioglitazone is aPPAR-120574 agonist This could have been responsible for theproliferation of beta cells of pancreas The test compoundsare thiazolidin-4-ones with some similarity to thiazolidine-diones It is possible that hyperplasia observed in compound3a-treated animals might have been due to some agonisticactivity on PPAR-120574 receptors This needs to be investigatedthrough relevant assay
Oxidative stress has been implicated in the occurrence ofdiabetes and compounds reducing the oxidative stress havebeneficial role in correcting glucose intolerance and insulinresistance in diabetes [18 28]The test compounds and piogli-tazone reversed the depletion of endogenous antioxidantenzymes such as GSH CAT SOD and GST Further theyreduced malondialdehyde levels This suggests the inhibitoryeffect of these compounds on oxidative stress
Leptin serves as an insulin-sensitizing factor in the wholebody [29] However hyperleptinemia in the obese mouseand human is a sign of leptin resistance where increasedleptin levels are caused by disturbed homeostasis arisingfrom leptin receptor mutation ageing or obesity [29] Thushyperleptinemia further worsens impaired insulin actionin pathological condition In these conditions exogenouslyadministered leptin does not improve glucose tolerance andinsulin sensitivity In our study diseased animal (HCD fedmouse) per se did not develop hyperleptinemia which mightbe the sign of early stage of metabolic abnormality associatedwith insulin resistance At this state test compounds aswell as pioglitazone reversed the elevated glucose TG TCand insulin levels while facilitating leptin profile along withinsulin sensitivity Subsequently compound 3a treatmentresulted in increased energy expenditure demonstrated byelevated food efficiency ratio (FER) However reductionin body weight among the treated groups was not foundproportionate to the circulating leptin levels which suggeststhat increased leptin level in peripheral blood is not suffi-cient to induce a proportionate reduction on body weightA few questions remain unanswered such as (i) whetherthe compounds directly raised the leptin levels or are theresults due to indirect impact on metabolic signaling (ii) In
metabolic disorder combined with hyperleptinemia how dothese compounds affect leptin signaling Future studies areneeded to address these issues
No single mechanism would suffice to explain the ben-eficial effects of the test compounds They do not seem toact through insulinotropic activity unlike the sulfonylureasThey have no significant effect on adiponectin levels rulingout any involvement of this mechanism The increase in thelevel of serum leptin might point to the involvement of leptinin the antihyperlipidemic and antidiabetic potentials of thesemolecules
In conclusion thiazolidin-4-one derivatives act throughmultiple mechanisms to correct the metabolic abnormalitiesin type-2 diabetes In the present work compounds 2e and3a were found to be the most effective test compoundsto ameliorate insulin resistance and development of type-2diabetes
Conflict of Interests
The authors declare that they do not have any conflict ofinterests
Acknowledgment
The authors wish to thank All India Council for TechnicalEducation (AICTE) New Delhi India for providing thefinancial support and Manipal University Manipal Kar-nataka India for providing the facilities to carry out thepresent work
References
[1] S M Grundy I J Benjamin G L Burke et al ldquoDiabetes andcardiovascular disease a statement for healthcare professionalsfrom the american heart associationrdquo Circulation vol 100 no10 pp 1134ndash1146 1999
[2] K E Thorpe and D H Howard ldquoThe rise in spending amongMedicare beneficiaries the role of chronic disease prevalenceand changes in treatment intensity Increasing numbers ofbeneficiaries being treated for five or more conditions a year aredriving Medicare spending upwardrdquo Health Affairs vol 25 no5 pp w378ndashw388 2006
[3] S M Grundy ldquoDrug therapy of the metabolic syndrome min-imizing the emerging crisis in polypharmacyrdquo Nature ReviewsDrug Discovery vol 5 no 4 pp 295ndash309 2006
[4] M Navab N Gharavi and A D Watson ldquoInflammation andmetabolic disordersrdquo Current Opinion in Clinical Nutrition andMetabolic Care vol 11 no 4 pp 459ndash464 2008
[5] G S Hotamisligil ldquoInflammation and metabolic disordersrdquoNature vol 444 no 7121 pp 860ndash867 2006
[6] M Schmuth V Moosbrugger-Martinz S Blunder and SDubrac ldquoRole of PPAR LXR and PXR in epidermal homeosta-sis and inflammationrdquo Biochimica et Biophysica Acta vol 1841no 3 pp 463ndash473 2014
[7] J A Panetta J K Shadle M L Phillips D N Benslayand P P K Ho ldquo4-thiazolidinones potent antioxidants asantiinflammatory agentsrdquo Annals of the New York Academy ofSciences vol 696 pp 415ndash416 1993
14 BioMed Research International
[8] T Kato T Ozaki K Tamura Y Suzuki M Akima and NOhi ldquoNovel calcium antagonists with both calcium overloadinhibition and antioxidant activity 1 2-(35-Di-tert-butyl-4-hydroxyphenyl)-3- (aminopropyl)thiazolidinonesrdquo Journal ofMedicinal Chemistry vol 41 no 22 pp 4309ndash4316 1998
[9] JM Joy N Jacob andGN Kutty ldquoEvaluation of hypoglycemiceffects of 4-thiazolidinonesrdquo Indian Drugs vol 42 no 1 pp 17ndash21 2005
[10] N Jacob and G N Kutty ldquoSynthesis and hypolipidemic activityof a thiazolidinone derivativerdquo Indian Drugs vol 41 no 2 pp76ndash79 2004
[11] G K Nampurath S P Mathew V Khanna R T Zachariah SKanji andMRChamallamudi ldquoAssessment of hypolipidaemicactivity of three thiazolidin-4-ones in mice given high-fat dietand fructoserdquoChemico-Biological Interactions vol 171 no 3 pp363ndash368 2008
[12] A Kishore G K Nampurath S P Mathew et al ldquoAntidiabeticeffect through islet cell protection in streptozotocin diabetesa preliminary assessment of two thiazolidin-4-ones in Swissalbino micerdquo Chemico-Biological Interactions vol 177 no 3 pp242ndash246 2009
[13] J Mudgal V S Gowdra G Mathew et al ldquoRemedial effects ofnovel 2 3-disubstituted thiazolidin-4-ones in chemical medi-ated inflammationrdquo Chemico-Biological Interactions vol 210pp 34ndash42 2014
[14] A R Surrey ldquo4-Thiazolidones IV the preparation of some3-alkylaminoalkyl-2-aryl derivativesrdquo Journal of the AmericanChemical Society vol 71 no 10 pp 3354ndash3356 1949
[15] B R P Kumar and M J Nanjan ldquoNovel glitazones designsynthesis glucose uptake and structure-activity relationshipsrdquoBioorganic and Medicinal Chemistry Letters vol 20 no 6 pp1953ndash1956 2010
[16] M Sumiyoshi M Sakanaka and Y Kimura ldquoChronic intakeof high-fat and high-sucrose diets differentially affects glucoseintolerance inmicerdquo Journal of Nutrition vol 136 no 3 pp 582ndash587 2006
[17] S H Kim S H Hyun and S Y Choung ldquoAnti-diabetic effectof cinnamon extract on blood glucose in dbdb micerdquo Journalof Ethnopharmacology vol 104 no 1-2 pp 119ndash123 2006
[18] S A Manohara Reddy J Mudgal P Bansal et al ldquoAntioxidantanti-inflammatory and anti-hyperglycaemic activities of hete-rocyclic homoprostanoid derivativesrdquoBioorganic andMedicinalChemistry vol 19 no 1 pp 384ndash392 2011
[19] S T Shukla V H Kulkarni P V Habbu K S Jagadeesh BS Patil and D M Smita ldquoHepatoprotective and antioxidantactivities of crude fractions of endophytic fungi of Ocimumsanctum Linn in ratsrdquo Oriental Pharmacy and ExperimentalMedicine vol 12 no 2 pp 81ndash91 2012
[20] B Mannervik ldquoGlutathione peroxidaserdquo Methods in Enzymol-ogy vol 113 pp 490ndash495 1985
[21] H PMisra and I Fridovich ldquoThe role of superoxide anion in theautoxidation of epinephrine and a simple assay for superoxidedismutaserdquo Journal of Biological Chemistry vol 247 no 10 pp3170ndash3175 1972
[22] A Claiborne ldquoCatalase activityrdquo in Handbook of Methods forOxygen Radical Research R A Greenwald Ed pp 283ndash284CRC Press Boca Raton Fla USA 1985
[23] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974
[24] W G Niehaus Jr and B Samuelsson ldquoFormation of malonalde-hyde from phospholipid arachidonate during microsomal lipidperoxidationrdquoEuropean Journal of Biochemistry vol 6 no 1 pp126ndash130 1968
[25] O S Osman J L Selway M A Kępczynska et al ldquoA novelautomated image analysis method for accurate adipocyte quan-tificationrdquo Adipocyte vol 2 no 3 pp 160ndash164 2013
[26] R Kalia C M Rao and N Gopalan Kutty ldquoSynthesisand evaluation of the anti-inflammatory activity of N-[2-(35-di-tert-butyl-4-hydroxyphenyl)-4-oxothiazolidin-3-yl]-nicotinamiderdquo Drug Research vol 57 no 9 pp 616ndash6222007
[27] N Maeda M Takahashi T Funahashi et al ldquoPPAR120574 ligandsincrease expression and plasma concentrations of adiponectinan adipose-derived proteinrdquo Diabetes vol 50 no 9 pp 2094ndash2099 2001
[28] P Bansal P Paul J Mudgal et al ldquoAntidiabetic antihyperlipi-demic and antioxidant effects of the flavonoid rich fraction ofPilea microphylla (L) in high fat dietstreptozotocin-induceddiabetes in micerdquo Experimental and Toxicologic Pathology vol64 no 6 pp 651ndash658 2012
[29] C Perez C Fernandez-Galaz T Fernandez-Agullo et alldquoLeptin impairs insulin signaling in rat adipocytesrdquo Diabetesvol 53 no 2 pp 347ndash353 2004
Submit your manuscripts athttpwwwhindawicom
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StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Autoimmune Diseases
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Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
BioMed Research International 9
0
50
100
150
200
250
14 day 30 day
NPDHCD
lowast
lowast
lowastlowast
lowastlowast
lowastlowast lowastlowastlowastlowast
lowastlowast
Pioglitazone (5mgkg)
Plas
ma g
luco
se (m
gdL
)(M
eanplusmn
SE
Mn=6
)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
60
120
180
240
300
lowast
lowastlowastlowastlowast
lowastlowast
Plas
ma T
G (m
gdL
)(M
eanplusmn
SE
Mn=6
)
14 day 30 day
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
60
120
180
240
300
lowast
lowast
lowastlowast
lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowast
Plas
ma c
hole
stero
l (m
gdL
)(M
eanplusmn
SE
Mn=6
)
14 day 30 day
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 2 Effect of thiazolidin-4-ones on plasma (a) glucose (b) triglyceride (TG) (c) cholesterol inHCD fedmice Data presented asmean plusmnSEM (119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 as compared to HCD group
(i) Effect of Thiazolidin-4-Ones on Plasma Glucose Triglyc-eride and Cholesterol Level Biochemical estimation ofmetabolic markers such as plasma glucose (PG) triglycerides(TG) and total cholesterol (TC)was performed on day 14 andday 30 after the drug treatment and it was observed that HCDcontrol group consistently had significant increase in PG andTC levels on days 14 and 30 compared to respective dayrsquosnormal control group However TG levels were significantlyincreased only on day 30 compared to respective dayrsquos normalcontrol group Pioglitazone and test compounds (2e and3a) significantly reversed hyperglycemia and elevated plasmacholesterol compared to HCD control group on both day14 and day 30 of drug treatment (Figures 2(a) and 2(c))while elevated TG levels were reduced on day 30 by the testcompounds and pioglitazone (Figure 2(b))
(ii) Effect of Thiazolidin-4-Ones on Plasma Insulin Leptinand Adiponectin Level Development of insulin resistance
in mouse was confirmed by estimating the plasma insulinlevels on day 30 after drug treatment HCD control group incontrast to NPD group showed two times increase in plasmainsulin levels (119875 lt 005) Hyperinsulinemia associated withhyperglycaemia and hypertriglyceridemia is considered tobe the sign of development of insulin resistance Thus highsucrose feeding for 6 months led to the development ofinsulin resistance in mice Treatment with pioglitazone andtest drugs attenuated the insulin resistance (Figure 3(a))
HCD feeding to animals did not elicit any significanteffect on plasma leptin levels as there was no significantdifference between HCD control and NPD group Howevertreatment with pioglitazone and test compounds significantly(119875 lt 005) raised the circulating leptin level in micecomparedwithHCDcontrol group (Figure 3(b)) Compound3a caused a fourfold elevation in leptin levels compared withHCD group HCD feeding to animals resulted in significant
10 BioMed Research International
0
1
2
3
4
5
NPDHCDPioglitazone (5mgkg)
Plas
ma i
nsul
in (n
gm
L)(M
eanplusmn
SE
Mn=6
)
lowastlowast
lowast
lowastlowastlowastlowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
1200
2400
3600
4800
6000
NPDHCDPioglitazone (5mgkg)
Plas
ma l
eptin
(pg
mL)
(Mea
nplusmn
SE
Mn=6
)
lowast lowast
lowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
12000
24000
36000
48000
60000
NPDHCDPioglitazone (5mgkg)
Plas
ma a
dipo
nect
in (n
gm
L)(M
eanplusmn
SE
Mn=6
)
lowast
lowastlowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 3 Effect of thiazolidin-4-ones on plasma (a) insulin (b) leptin (c) adiponectin in HCD fed mice Data presented as mean plusmn SEM(119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 as compared to HCD group
(119875 lt 005) hypoadiponectinemia comparedwithNPD Treat-ment with pioglitazone significantly (119875 lt 005) corrected thehypoadiponectinemia However both test compounds failedto correct hypoadiponectinemia (Figure 3(c))
(iii) Effect of Thiazolidin-4-Ones on Oral Glucose ToleranceTest In oral glucose tolerance test HCD group animalshowed significant (119875 lt 005) glucose intolerance (Figure 4)Pioglitazone and test compounds compound 2e and com-pound 3a corrected the glucose intolerance shown as sig-nificant (119875 lt 005) percent reductions in glucose excursion(AUC
0minus120min) by 1300 plusmn 33 1546 plusmn 554 and 1560 plusmn 349respectively compared with HCD group (Figures 4(a) and4(b))
(iv) Effect of Thiazolidin-4-Ones on Liver Enzymes Oxidativestress is the hallmark of metabolic disorder where disturbedhomeostasis between oxidative and antioxidative mechanismoccurs HCD feeding to mice resulted in an oxidative stress
observed as reduction in liver antioxidant enzymes such asglutathione (GSH) catalase (CAT) superoxide dismutase(SOD) and glutathione-S-transferase (GST) Treatment withpioglitazone and test compounds ameliorated the oxidativestress They also reversed the elevation in liver malondialde-hyde (MDA) levels in HCD-fed mice (Table 5)
(v) Histopathological ExaminationHistology of liver showednormal lobular architecture with normal hepatocytes inall groups (Figure 5(a)) Pancreas showed mild to mod-erate hyperplasia of islets of Langerhans (Figure 5(a)) inpioglitazone and compound 3a groups Pioglitazone and3a treatment significantly raised the area of Langerhansislets compared with HCD group (Figure 5(b)) Exocrineportion of the pancreas and vascularity appeared normalin all the groups Histopathological investigation of whiteadipose tissue (WAT) from epidydimal showed an increasein the size of adipocytes (Figure 5(c)) in HCD group whichwas reversed by pioglitazone and compound 3a treatment
BioMed Research International 11
Table 5 Effect of thiazolidin-4-ones on liver antioxidant enzyme and malondialdehyde level
Liver biomarkers NPD HCD Pioglitazone Compound 2e Compound 3aGSH (nmolemg of protein) 558 plusmn 21 464 plusmn 21a 632 plusmn 15b 683 plusmn 20b 678 plusmn 43b
CAT (Umg of protein) 892 plusmn 57 554 plusmn 31a 938 plusmn 10b 819 plusmn 24b 875 plusmn 74b
SOD (Umg of protein) 1868 plusmn 232 1081 plusmn 112a 1748 plusmn 144b 2022 plusmn 47b 2080 plusmn 154b
GST (Umg of protein) 043 plusmn 005 037 plusmn 001a 073 plusmn 002b 070 plusmn 001b 072 plusmn 004b
MDA (nmolemg of protein) 057 plusmn 006 10 plusmn 033a 046 plusmn 001b 040 plusmn 001b 041 plusmn 002baRepresents 119875 lt 005 as compared to NPD group (normal pellet diet)bRepresents 119875 lt 005 as compared to HCD group (high carbohydrate diet)
0 30 60 90 120100
130
160
190
220
Plas
ma g
luco
se (m
gdL
)(M
eanplusmn
SE
Mn=6
)
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
5000
10000
15000
20000
25000
(Mea
nplusmn
SE
Mn=6
)
lowast
lowastlowast lowastlowast lowastlowast
NPDHCDPioglitazone (5mgkg)
Plas
ma g
luco
se (A
UC 0
ndash120
min)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
Figure 4 Effect of thiazolidin-4-ones on (a) plasma glucose (mgdL) and (b) area under the curve (AUC0minus120 min) against oral glucose
tolerance test (OGTT) in HCD fed mice Data presented as mean plusmn SEM (119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD groupand lowastlowastrepresents 119875 lt 005 as compared to HCD group
However compound 2e did not show any significant effecton increased adipocyte size
4 Discussion and Conclusion
The present work was planned as a sequel to earlier studiesin our laboratory using thiazolidin-4-ones as antidiabetichypolipidemic and antiinflammatory molecules The substi-tutions in the thiazolidine ring were made at C2 and N3Attached to the latter position was a nicotinamide moietyand the substitution at C2 was either p-methoxyphenyl or2 5-di-tert-butyl-4-hydroxyphenyl group The compoundsshowed significant antidiabetic and hypolipidemic activities[9ndash11 26]
In the present study the substitution at N3 was changedto p-chloro-phenoxyacetylamino a group that is similar toclofibrate with a methylene bridge instead of gem-dimethylsubstitution
Four compounds out of 20 (ie compounds 4a 4b4c and 4d) had a methyl group attached at the C5 ofthiazolidine ring with cyclisation being made with thiolacticacid instead of thioglycolic acid In three other compounds(ie compounds 3a 3b and 3c) clofibrate was used to make
themoiety for substitution atN3Thiswas donewith a view toexamine the effect of the gem-dimethyl on the overall activityof the resulting molecule
All the 20 synthesised thiazolidin-4-ones were evaluatedfor glucose uptake in an in vitro system using the isolatedrat diaphragm In this experiment compounds 2e and 3asignificantly raised the amount of glucose uptake by thetissue both in the absence and presence of external insulinThis indicated the potential of the theses compounds insensitizing the tissues for the external insulin Hence thesetwo compounds were chosen to study their effect on a diet-induced model of insulin resistance Both compounds havethe same p-methoxyphenyl moiety attached to the C2 of thethiazolidine ring Compound 3a has a gem-dimethyl groupinstead of methylene in the substituent at N3
Apart from multiple risk factors diet-induced metabolicabnormalities contribute to the development of insulinresistance and 120573-cell failure in type-2 diabetes [16] Earlydetection and appropriate treatment are considered beneficialfor correcting the abnormality Among the various animalmodels induction of diabetes through diet provides moreresemblance to human type-2 diabetes Chronic intake ofdiet with high sucrose content has been reported to favourthe development of insulin resistance [16] Similarly in our
12 BioMed Research International
NPD HCD Pioglitazone Compound 3a Compound 2e
Liver
WAT
Green and red arrows indicate hyperplasia and increased size of adipocytes respectively
Pancreas
(a)
0
50000
100000
150000
200000
NPDHCDPioglitazone (5mgkg)
lowastlowastlowastlowast
(Mea
nplusmn
SE
Mn=3
)A
rea o
f lan
gerh
ans i
stle
ts (120583
m2)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
750
1500
2250
3000
NPDHCDPioglitazone (5mgkg)
lowastlowastlowastlowast
lowast
(Mea
nplusmn
SE
Mn=3
)A
rea o
f adi
pocy
tes (120583
m2)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 5 (a) Histological sections of mouse liver pancreas and white adipose tissue (WAT) in HCD model (10x) Green and red arrowsindicate hyperplasia of islets of Langerhans and increase in the size of adipocytes respectively Effect of thiazolidin-4-ones on (b) area ofLangerhans islets (120583m2) and (c) area of adipocytes (120583m2) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 ascompared to HCD group
study mice fed a high-sucrose diet for six months devel-oped metabolic abnormalities like hyperglycemia hyperin-sulinemia hypertriglyceridemia hypercholesterolemia andhypoadiponectinemia Hyperglycemia and hyperinsulinemiasuggest the inability of insulin to sensitize the tissue forglucose uptake allowing glucose to be diverted toward lipo-genesisThis led to hyperlipidemia and finally to insulin resis-tance In addition adiponectin and leptin the adipokinessecreted from white adipose tissue (WAT) are reported tobe involved in the metabolism of glucose and lipid [27]In our study we observed that HCD caused a reductionin plasma adiponectin level and an increase in the sizeof adipocytes However it did not cause any change inplasma leptin levels Thus we found a correlation between
the adiponectin level and size of adipocytes in HCD modelFurther OGTT results from HCD-fed mice correlated wellwith glucose intolerance hyperinsulinemia and hypoad-iponectinemia The authenticity of the model was validatedby the effect of pioglitazone which was able to correct themetabolic abnormalities Neither test compounds (2e or 3a)attenuated hypoadiponectinemia However they correctedthe impaired glucose tolerance and insulin resistance inmice Compounds 2e and 3a by their ability to enhanceglucose uptake and to sensitize the tissue for available insulinreduced hyperinsulinemia and raised the leptin levels Thiswould have resulted in better glucose utilization by periph-eral tissue Thus these compounds reduced the metabolicabnormalities like hyperglycemia hypertriglyceridemia and
BioMed Research International 13
hypercholesterolemia Prospective studies involving directassessment of insulin sensitivity in OGTT are requiredto analyze glucose stimulated insulin secretion (GSIS) inpresence of compounds to establish the mechanistic role ofthe test compounds on insulin signalling
Histological investigation showed no change in liverarchitecture in the various treatment groups In HCD controlmice there was an increase in the size of adipocytes in whiteadipose tissue (WAT) which was reversed by compound3a and pioglitazone treatment However only pioglitazone-treated animals showed positive correlation between reducedsize of the cells and correction of hypoadiponectinemiaApart from correction of hypoadiponectinemia pioglitazoneraised the peripheral leptin levels which was also true in caseof compounds 2e and 3a treatment Among the tested com-pounds 3a raised endogenous leptin levels four times morethan HCD group This finding points to the link betweenraised leptin levels and reduced adipocyte size caused bycompound 3a The pancreatic islets showed hyperplasia inpioglitazone and other treatment groups Pioglitazone is aPPAR-120574 agonist This could have been responsible for theproliferation of beta cells of pancreas The test compoundsare thiazolidin-4-ones with some similarity to thiazolidine-diones It is possible that hyperplasia observed in compound3a-treated animals might have been due to some agonisticactivity on PPAR-120574 receptors This needs to be investigatedthrough relevant assay
Oxidative stress has been implicated in the occurrence ofdiabetes and compounds reducing the oxidative stress havebeneficial role in correcting glucose intolerance and insulinresistance in diabetes [18 28]The test compounds and piogli-tazone reversed the depletion of endogenous antioxidantenzymes such as GSH CAT SOD and GST Further theyreduced malondialdehyde levels This suggests the inhibitoryeffect of these compounds on oxidative stress
Leptin serves as an insulin-sensitizing factor in the wholebody [29] However hyperleptinemia in the obese mouseand human is a sign of leptin resistance where increasedleptin levels are caused by disturbed homeostasis arisingfrom leptin receptor mutation ageing or obesity [29] Thushyperleptinemia further worsens impaired insulin actionin pathological condition In these conditions exogenouslyadministered leptin does not improve glucose tolerance andinsulin sensitivity In our study diseased animal (HCD fedmouse) per se did not develop hyperleptinemia which mightbe the sign of early stage of metabolic abnormality associatedwith insulin resistance At this state test compounds aswell as pioglitazone reversed the elevated glucose TG TCand insulin levels while facilitating leptin profile along withinsulin sensitivity Subsequently compound 3a treatmentresulted in increased energy expenditure demonstrated byelevated food efficiency ratio (FER) However reductionin body weight among the treated groups was not foundproportionate to the circulating leptin levels which suggeststhat increased leptin level in peripheral blood is not suffi-cient to induce a proportionate reduction on body weightA few questions remain unanswered such as (i) whetherthe compounds directly raised the leptin levels or are theresults due to indirect impact on metabolic signaling (ii) In
metabolic disorder combined with hyperleptinemia how dothese compounds affect leptin signaling Future studies areneeded to address these issues
No single mechanism would suffice to explain the ben-eficial effects of the test compounds They do not seem toact through insulinotropic activity unlike the sulfonylureasThey have no significant effect on adiponectin levels rulingout any involvement of this mechanism The increase in thelevel of serum leptin might point to the involvement of leptinin the antihyperlipidemic and antidiabetic potentials of thesemolecules
In conclusion thiazolidin-4-one derivatives act throughmultiple mechanisms to correct the metabolic abnormalitiesin type-2 diabetes In the present work compounds 2e and3a were found to be the most effective test compoundsto ameliorate insulin resistance and development of type-2diabetes
Conflict of Interests
The authors declare that they do not have any conflict ofinterests
Acknowledgment
The authors wish to thank All India Council for TechnicalEducation (AICTE) New Delhi India for providing thefinancial support and Manipal University Manipal Kar-nataka India for providing the facilities to carry out thepresent work
References
[1] S M Grundy I J Benjamin G L Burke et al ldquoDiabetes andcardiovascular disease a statement for healthcare professionalsfrom the american heart associationrdquo Circulation vol 100 no10 pp 1134ndash1146 1999
[2] K E Thorpe and D H Howard ldquoThe rise in spending amongMedicare beneficiaries the role of chronic disease prevalenceand changes in treatment intensity Increasing numbers ofbeneficiaries being treated for five or more conditions a year aredriving Medicare spending upwardrdquo Health Affairs vol 25 no5 pp w378ndashw388 2006
[3] S M Grundy ldquoDrug therapy of the metabolic syndrome min-imizing the emerging crisis in polypharmacyrdquo Nature ReviewsDrug Discovery vol 5 no 4 pp 295ndash309 2006
[4] M Navab N Gharavi and A D Watson ldquoInflammation andmetabolic disordersrdquo Current Opinion in Clinical Nutrition andMetabolic Care vol 11 no 4 pp 459ndash464 2008
[5] G S Hotamisligil ldquoInflammation and metabolic disordersrdquoNature vol 444 no 7121 pp 860ndash867 2006
[6] M Schmuth V Moosbrugger-Martinz S Blunder and SDubrac ldquoRole of PPAR LXR and PXR in epidermal homeosta-sis and inflammationrdquo Biochimica et Biophysica Acta vol 1841no 3 pp 463ndash473 2014
[7] J A Panetta J K Shadle M L Phillips D N Benslayand P P K Ho ldquo4-thiazolidinones potent antioxidants asantiinflammatory agentsrdquo Annals of the New York Academy ofSciences vol 696 pp 415ndash416 1993
14 BioMed Research International
[8] T Kato T Ozaki K Tamura Y Suzuki M Akima and NOhi ldquoNovel calcium antagonists with both calcium overloadinhibition and antioxidant activity 1 2-(35-Di-tert-butyl-4-hydroxyphenyl)-3- (aminopropyl)thiazolidinonesrdquo Journal ofMedicinal Chemistry vol 41 no 22 pp 4309ndash4316 1998
[9] JM Joy N Jacob andGN Kutty ldquoEvaluation of hypoglycemiceffects of 4-thiazolidinonesrdquo Indian Drugs vol 42 no 1 pp 17ndash21 2005
[10] N Jacob and G N Kutty ldquoSynthesis and hypolipidemic activityof a thiazolidinone derivativerdquo Indian Drugs vol 41 no 2 pp76ndash79 2004
[11] G K Nampurath S P Mathew V Khanna R T Zachariah SKanji andMRChamallamudi ldquoAssessment of hypolipidaemicactivity of three thiazolidin-4-ones in mice given high-fat dietand fructoserdquoChemico-Biological Interactions vol 171 no 3 pp363ndash368 2008
[12] A Kishore G K Nampurath S P Mathew et al ldquoAntidiabeticeffect through islet cell protection in streptozotocin diabetesa preliminary assessment of two thiazolidin-4-ones in Swissalbino micerdquo Chemico-Biological Interactions vol 177 no 3 pp242ndash246 2009
[13] J Mudgal V S Gowdra G Mathew et al ldquoRemedial effects ofnovel 2 3-disubstituted thiazolidin-4-ones in chemical medi-ated inflammationrdquo Chemico-Biological Interactions vol 210pp 34ndash42 2014
[14] A R Surrey ldquo4-Thiazolidones IV the preparation of some3-alkylaminoalkyl-2-aryl derivativesrdquo Journal of the AmericanChemical Society vol 71 no 10 pp 3354ndash3356 1949
[15] B R P Kumar and M J Nanjan ldquoNovel glitazones designsynthesis glucose uptake and structure-activity relationshipsrdquoBioorganic and Medicinal Chemistry Letters vol 20 no 6 pp1953ndash1956 2010
[16] M Sumiyoshi M Sakanaka and Y Kimura ldquoChronic intakeof high-fat and high-sucrose diets differentially affects glucoseintolerance inmicerdquo Journal of Nutrition vol 136 no 3 pp 582ndash587 2006
[17] S H Kim S H Hyun and S Y Choung ldquoAnti-diabetic effectof cinnamon extract on blood glucose in dbdb micerdquo Journalof Ethnopharmacology vol 104 no 1-2 pp 119ndash123 2006
[18] S A Manohara Reddy J Mudgal P Bansal et al ldquoAntioxidantanti-inflammatory and anti-hyperglycaemic activities of hete-rocyclic homoprostanoid derivativesrdquoBioorganic andMedicinalChemistry vol 19 no 1 pp 384ndash392 2011
[19] S T Shukla V H Kulkarni P V Habbu K S Jagadeesh BS Patil and D M Smita ldquoHepatoprotective and antioxidantactivities of crude fractions of endophytic fungi of Ocimumsanctum Linn in ratsrdquo Oriental Pharmacy and ExperimentalMedicine vol 12 no 2 pp 81ndash91 2012
[20] B Mannervik ldquoGlutathione peroxidaserdquo Methods in Enzymol-ogy vol 113 pp 490ndash495 1985
[21] H PMisra and I Fridovich ldquoThe role of superoxide anion in theautoxidation of epinephrine and a simple assay for superoxidedismutaserdquo Journal of Biological Chemistry vol 247 no 10 pp3170ndash3175 1972
[22] A Claiborne ldquoCatalase activityrdquo in Handbook of Methods forOxygen Radical Research R A Greenwald Ed pp 283ndash284CRC Press Boca Raton Fla USA 1985
[23] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974
[24] W G Niehaus Jr and B Samuelsson ldquoFormation of malonalde-hyde from phospholipid arachidonate during microsomal lipidperoxidationrdquoEuropean Journal of Biochemistry vol 6 no 1 pp126ndash130 1968
[25] O S Osman J L Selway M A Kępczynska et al ldquoA novelautomated image analysis method for accurate adipocyte quan-tificationrdquo Adipocyte vol 2 no 3 pp 160ndash164 2013
[26] R Kalia C M Rao and N Gopalan Kutty ldquoSynthesisand evaluation of the anti-inflammatory activity of N-[2-(35-di-tert-butyl-4-hydroxyphenyl)-4-oxothiazolidin-3-yl]-nicotinamiderdquo Drug Research vol 57 no 9 pp 616ndash6222007
[27] N Maeda M Takahashi T Funahashi et al ldquoPPAR120574 ligandsincrease expression and plasma concentrations of adiponectinan adipose-derived proteinrdquo Diabetes vol 50 no 9 pp 2094ndash2099 2001
[28] P Bansal P Paul J Mudgal et al ldquoAntidiabetic antihyperlipi-demic and antioxidant effects of the flavonoid rich fraction ofPilea microphylla (L) in high fat dietstreptozotocin-induceddiabetes in micerdquo Experimental and Toxicologic Pathology vol64 no 6 pp 651ndash658 2012
[29] C Perez C Fernandez-Galaz T Fernandez-Agullo et alldquoLeptin impairs insulin signaling in rat adipocytesrdquo Diabetesvol 53 no 2 pp 347ndash353 2004
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
10 BioMed Research International
0
1
2
3
4
5
NPDHCDPioglitazone (5mgkg)
Plas
ma i
nsul
in (n
gm
L)(M
eanplusmn
SE
Mn=6
)
lowastlowast
lowast
lowastlowastlowastlowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
1200
2400
3600
4800
6000
NPDHCDPioglitazone (5mgkg)
Plas
ma l
eptin
(pg
mL)
(Mea
nplusmn
SE
Mn=6
)
lowast lowast
lowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
12000
24000
36000
48000
60000
NPDHCDPioglitazone (5mgkg)
Plas
ma a
dipo
nect
in (n
gm
L)(M
eanplusmn
SE
Mn=6
)
lowast
lowastlowast
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 3 Effect of thiazolidin-4-ones on plasma (a) insulin (b) leptin (c) adiponectin in HCD fed mice Data presented as mean plusmn SEM(119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 as compared to HCD group
(119875 lt 005) hypoadiponectinemia comparedwithNPD Treat-ment with pioglitazone significantly (119875 lt 005) corrected thehypoadiponectinemia However both test compounds failedto correct hypoadiponectinemia (Figure 3(c))
(iii) Effect of Thiazolidin-4-Ones on Oral Glucose ToleranceTest In oral glucose tolerance test HCD group animalshowed significant (119875 lt 005) glucose intolerance (Figure 4)Pioglitazone and test compounds compound 2e and com-pound 3a corrected the glucose intolerance shown as sig-nificant (119875 lt 005) percent reductions in glucose excursion(AUC
0minus120min) by 1300 plusmn 33 1546 plusmn 554 and 1560 plusmn 349respectively compared with HCD group (Figures 4(a) and4(b))
(iv) Effect of Thiazolidin-4-Ones on Liver Enzymes Oxidativestress is the hallmark of metabolic disorder where disturbedhomeostasis between oxidative and antioxidative mechanismoccurs HCD feeding to mice resulted in an oxidative stress
observed as reduction in liver antioxidant enzymes such asglutathione (GSH) catalase (CAT) superoxide dismutase(SOD) and glutathione-S-transferase (GST) Treatment withpioglitazone and test compounds ameliorated the oxidativestress They also reversed the elevation in liver malondialde-hyde (MDA) levels in HCD-fed mice (Table 5)
(v) Histopathological ExaminationHistology of liver showednormal lobular architecture with normal hepatocytes inall groups (Figure 5(a)) Pancreas showed mild to mod-erate hyperplasia of islets of Langerhans (Figure 5(a)) inpioglitazone and compound 3a groups Pioglitazone and3a treatment significantly raised the area of Langerhansislets compared with HCD group (Figure 5(b)) Exocrineportion of the pancreas and vascularity appeared normalin all the groups Histopathological investigation of whiteadipose tissue (WAT) from epidydimal showed an increasein the size of adipocytes (Figure 5(c)) in HCD group whichwas reversed by pioglitazone and compound 3a treatment
BioMed Research International 11
Table 5 Effect of thiazolidin-4-ones on liver antioxidant enzyme and malondialdehyde level
Liver biomarkers NPD HCD Pioglitazone Compound 2e Compound 3aGSH (nmolemg of protein) 558 plusmn 21 464 plusmn 21a 632 plusmn 15b 683 plusmn 20b 678 plusmn 43b
CAT (Umg of protein) 892 plusmn 57 554 plusmn 31a 938 plusmn 10b 819 plusmn 24b 875 plusmn 74b
SOD (Umg of protein) 1868 plusmn 232 1081 plusmn 112a 1748 plusmn 144b 2022 plusmn 47b 2080 plusmn 154b
GST (Umg of protein) 043 plusmn 005 037 plusmn 001a 073 plusmn 002b 070 plusmn 001b 072 plusmn 004b
MDA (nmolemg of protein) 057 plusmn 006 10 plusmn 033a 046 plusmn 001b 040 plusmn 001b 041 plusmn 002baRepresents 119875 lt 005 as compared to NPD group (normal pellet diet)bRepresents 119875 lt 005 as compared to HCD group (high carbohydrate diet)
0 30 60 90 120100
130
160
190
220
Plas
ma g
luco
se (m
gdL
)(M
eanplusmn
SE
Mn=6
)
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
5000
10000
15000
20000
25000
(Mea
nplusmn
SE
Mn=6
)
lowast
lowastlowast lowastlowast lowastlowast
NPDHCDPioglitazone (5mgkg)
Plas
ma g
luco
se (A
UC 0
ndash120
min)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
Figure 4 Effect of thiazolidin-4-ones on (a) plasma glucose (mgdL) and (b) area under the curve (AUC0minus120 min) against oral glucose
tolerance test (OGTT) in HCD fed mice Data presented as mean plusmn SEM (119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD groupand lowastlowastrepresents 119875 lt 005 as compared to HCD group
However compound 2e did not show any significant effecton increased adipocyte size
4 Discussion and Conclusion
The present work was planned as a sequel to earlier studiesin our laboratory using thiazolidin-4-ones as antidiabetichypolipidemic and antiinflammatory molecules The substi-tutions in the thiazolidine ring were made at C2 and N3Attached to the latter position was a nicotinamide moietyand the substitution at C2 was either p-methoxyphenyl or2 5-di-tert-butyl-4-hydroxyphenyl group The compoundsshowed significant antidiabetic and hypolipidemic activities[9ndash11 26]
In the present study the substitution at N3 was changedto p-chloro-phenoxyacetylamino a group that is similar toclofibrate with a methylene bridge instead of gem-dimethylsubstitution
Four compounds out of 20 (ie compounds 4a 4b4c and 4d) had a methyl group attached at the C5 ofthiazolidine ring with cyclisation being made with thiolacticacid instead of thioglycolic acid In three other compounds(ie compounds 3a 3b and 3c) clofibrate was used to make
themoiety for substitution atN3Thiswas donewith a view toexamine the effect of the gem-dimethyl on the overall activityof the resulting molecule
All the 20 synthesised thiazolidin-4-ones were evaluatedfor glucose uptake in an in vitro system using the isolatedrat diaphragm In this experiment compounds 2e and 3asignificantly raised the amount of glucose uptake by thetissue both in the absence and presence of external insulinThis indicated the potential of the theses compounds insensitizing the tissues for the external insulin Hence thesetwo compounds were chosen to study their effect on a diet-induced model of insulin resistance Both compounds havethe same p-methoxyphenyl moiety attached to the C2 of thethiazolidine ring Compound 3a has a gem-dimethyl groupinstead of methylene in the substituent at N3
Apart from multiple risk factors diet-induced metabolicabnormalities contribute to the development of insulinresistance and 120573-cell failure in type-2 diabetes [16] Earlydetection and appropriate treatment are considered beneficialfor correcting the abnormality Among the various animalmodels induction of diabetes through diet provides moreresemblance to human type-2 diabetes Chronic intake ofdiet with high sucrose content has been reported to favourthe development of insulin resistance [16] Similarly in our
12 BioMed Research International
NPD HCD Pioglitazone Compound 3a Compound 2e
Liver
WAT
Green and red arrows indicate hyperplasia and increased size of adipocytes respectively
Pancreas
(a)
0
50000
100000
150000
200000
NPDHCDPioglitazone (5mgkg)
lowastlowastlowastlowast
(Mea
nplusmn
SE
Mn=3
)A
rea o
f lan
gerh
ans i
stle
ts (120583
m2)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
750
1500
2250
3000
NPDHCDPioglitazone (5mgkg)
lowastlowastlowastlowast
lowast
(Mea
nplusmn
SE
Mn=3
)A
rea o
f adi
pocy
tes (120583
m2)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 5 (a) Histological sections of mouse liver pancreas and white adipose tissue (WAT) in HCD model (10x) Green and red arrowsindicate hyperplasia of islets of Langerhans and increase in the size of adipocytes respectively Effect of thiazolidin-4-ones on (b) area ofLangerhans islets (120583m2) and (c) area of adipocytes (120583m2) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 ascompared to HCD group
study mice fed a high-sucrose diet for six months devel-oped metabolic abnormalities like hyperglycemia hyperin-sulinemia hypertriglyceridemia hypercholesterolemia andhypoadiponectinemia Hyperglycemia and hyperinsulinemiasuggest the inability of insulin to sensitize the tissue forglucose uptake allowing glucose to be diverted toward lipo-genesisThis led to hyperlipidemia and finally to insulin resis-tance In addition adiponectin and leptin the adipokinessecreted from white adipose tissue (WAT) are reported tobe involved in the metabolism of glucose and lipid [27]In our study we observed that HCD caused a reductionin plasma adiponectin level and an increase in the sizeof adipocytes However it did not cause any change inplasma leptin levels Thus we found a correlation between
the adiponectin level and size of adipocytes in HCD modelFurther OGTT results from HCD-fed mice correlated wellwith glucose intolerance hyperinsulinemia and hypoad-iponectinemia The authenticity of the model was validatedby the effect of pioglitazone which was able to correct themetabolic abnormalities Neither test compounds (2e or 3a)attenuated hypoadiponectinemia However they correctedthe impaired glucose tolerance and insulin resistance inmice Compounds 2e and 3a by their ability to enhanceglucose uptake and to sensitize the tissue for available insulinreduced hyperinsulinemia and raised the leptin levels Thiswould have resulted in better glucose utilization by periph-eral tissue Thus these compounds reduced the metabolicabnormalities like hyperglycemia hypertriglyceridemia and
BioMed Research International 13
hypercholesterolemia Prospective studies involving directassessment of insulin sensitivity in OGTT are requiredto analyze glucose stimulated insulin secretion (GSIS) inpresence of compounds to establish the mechanistic role ofthe test compounds on insulin signalling
Histological investigation showed no change in liverarchitecture in the various treatment groups In HCD controlmice there was an increase in the size of adipocytes in whiteadipose tissue (WAT) which was reversed by compound3a and pioglitazone treatment However only pioglitazone-treated animals showed positive correlation between reducedsize of the cells and correction of hypoadiponectinemiaApart from correction of hypoadiponectinemia pioglitazoneraised the peripheral leptin levels which was also true in caseof compounds 2e and 3a treatment Among the tested com-pounds 3a raised endogenous leptin levels four times morethan HCD group This finding points to the link betweenraised leptin levels and reduced adipocyte size caused bycompound 3a The pancreatic islets showed hyperplasia inpioglitazone and other treatment groups Pioglitazone is aPPAR-120574 agonist This could have been responsible for theproliferation of beta cells of pancreas The test compoundsare thiazolidin-4-ones with some similarity to thiazolidine-diones It is possible that hyperplasia observed in compound3a-treated animals might have been due to some agonisticactivity on PPAR-120574 receptors This needs to be investigatedthrough relevant assay
Oxidative stress has been implicated in the occurrence ofdiabetes and compounds reducing the oxidative stress havebeneficial role in correcting glucose intolerance and insulinresistance in diabetes [18 28]The test compounds and piogli-tazone reversed the depletion of endogenous antioxidantenzymes such as GSH CAT SOD and GST Further theyreduced malondialdehyde levels This suggests the inhibitoryeffect of these compounds on oxidative stress
Leptin serves as an insulin-sensitizing factor in the wholebody [29] However hyperleptinemia in the obese mouseand human is a sign of leptin resistance where increasedleptin levels are caused by disturbed homeostasis arisingfrom leptin receptor mutation ageing or obesity [29] Thushyperleptinemia further worsens impaired insulin actionin pathological condition In these conditions exogenouslyadministered leptin does not improve glucose tolerance andinsulin sensitivity In our study diseased animal (HCD fedmouse) per se did not develop hyperleptinemia which mightbe the sign of early stage of metabolic abnormality associatedwith insulin resistance At this state test compounds aswell as pioglitazone reversed the elevated glucose TG TCand insulin levels while facilitating leptin profile along withinsulin sensitivity Subsequently compound 3a treatmentresulted in increased energy expenditure demonstrated byelevated food efficiency ratio (FER) However reductionin body weight among the treated groups was not foundproportionate to the circulating leptin levels which suggeststhat increased leptin level in peripheral blood is not suffi-cient to induce a proportionate reduction on body weightA few questions remain unanswered such as (i) whetherthe compounds directly raised the leptin levels or are theresults due to indirect impact on metabolic signaling (ii) In
metabolic disorder combined with hyperleptinemia how dothese compounds affect leptin signaling Future studies areneeded to address these issues
No single mechanism would suffice to explain the ben-eficial effects of the test compounds They do not seem toact through insulinotropic activity unlike the sulfonylureasThey have no significant effect on adiponectin levels rulingout any involvement of this mechanism The increase in thelevel of serum leptin might point to the involvement of leptinin the antihyperlipidemic and antidiabetic potentials of thesemolecules
In conclusion thiazolidin-4-one derivatives act throughmultiple mechanisms to correct the metabolic abnormalitiesin type-2 diabetes In the present work compounds 2e and3a were found to be the most effective test compoundsto ameliorate insulin resistance and development of type-2diabetes
Conflict of Interests
The authors declare that they do not have any conflict ofinterests
Acknowledgment
The authors wish to thank All India Council for TechnicalEducation (AICTE) New Delhi India for providing thefinancial support and Manipal University Manipal Kar-nataka India for providing the facilities to carry out thepresent work
References
[1] S M Grundy I J Benjamin G L Burke et al ldquoDiabetes andcardiovascular disease a statement for healthcare professionalsfrom the american heart associationrdquo Circulation vol 100 no10 pp 1134ndash1146 1999
[2] K E Thorpe and D H Howard ldquoThe rise in spending amongMedicare beneficiaries the role of chronic disease prevalenceand changes in treatment intensity Increasing numbers ofbeneficiaries being treated for five or more conditions a year aredriving Medicare spending upwardrdquo Health Affairs vol 25 no5 pp w378ndashw388 2006
[3] S M Grundy ldquoDrug therapy of the metabolic syndrome min-imizing the emerging crisis in polypharmacyrdquo Nature ReviewsDrug Discovery vol 5 no 4 pp 295ndash309 2006
[4] M Navab N Gharavi and A D Watson ldquoInflammation andmetabolic disordersrdquo Current Opinion in Clinical Nutrition andMetabolic Care vol 11 no 4 pp 459ndash464 2008
[5] G S Hotamisligil ldquoInflammation and metabolic disordersrdquoNature vol 444 no 7121 pp 860ndash867 2006
[6] M Schmuth V Moosbrugger-Martinz S Blunder and SDubrac ldquoRole of PPAR LXR and PXR in epidermal homeosta-sis and inflammationrdquo Biochimica et Biophysica Acta vol 1841no 3 pp 463ndash473 2014
[7] J A Panetta J K Shadle M L Phillips D N Benslayand P P K Ho ldquo4-thiazolidinones potent antioxidants asantiinflammatory agentsrdquo Annals of the New York Academy ofSciences vol 696 pp 415ndash416 1993
14 BioMed Research International
[8] T Kato T Ozaki K Tamura Y Suzuki M Akima and NOhi ldquoNovel calcium antagonists with both calcium overloadinhibition and antioxidant activity 1 2-(35-Di-tert-butyl-4-hydroxyphenyl)-3- (aminopropyl)thiazolidinonesrdquo Journal ofMedicinal Chemistry vol 41 no 22 pp 4309ndash4316 1998
[9] JM Joy N Jacob andGN Kutty ldquoEvaluation of hypoglycemiceffects of 4-thiazolidinonesrdquo Indian Drugs vol 42 no 1 pp 17ndash21 2005
[10] N Jacob and G N Kutty ldquoSynthesis and hypolipidemic activityof a thiazolidinone derivativerdquo Indian Drugs vol 41 no 2 pp76ndash79 2004
[11] G K Nampurath S P Mathew V Khanna R T Zachariah SKanji andMRChamallamudi ldquoAssessment of hypolipidaemicactivity of three thiazolidin-4-ones in mice given high-fat dietand fructoserdquoChemico-Biological Interactions vol 171 no 3 pp363ndash368 2008
[12] A Kishore G K Nampurath S P Mathew et al ldquoAntidiabeticeffect through islet cell protection in streptozotocin diabetesa preliminary assessment of two thiazolidin-4-ones in Swissalbino micerdquo Chemico-Biological Interactions vol 177 no 3 pp242ndash246 2009
[13] J Mudgal V S Gowdra G Mathew et al ldquoRemedial effects ofnovel 2 3-disubstituted thiazolidin-4-ones in chemical medi-ated inflammationrdquo Chemico-Biological Interactions vol 210pp 34ndash42 2014
[14] A R Surrey ldquo4-Thiazolidones IV the preparation of some3-alkylaminoalkyl-2-aryl derivativesrdquo Journal of the AmericanChemical Society vol 71 no 10 pp 3354ndash3356 1949
[15] B R P Kumar and M J Nanjan ldquoNovel glitazones designsynthesis glucose uptake and structure-activity relationshipsrdquoBioorganic and Medicinal Chemistry Letters vol 20 no 6 pp1953ndash1956 2010
[16] M Sumiyoshi M Sakanaka and Y Kimura ldquoChronic intakeof high-fat and high-sucrose diets differentially affects glucoseintolerance inmicerdquo Journal of Nutrition vol 136 no 3 pp 582ndash587 2006
[17] S H Kim S H Hyun and S Y Choung ldquoAnti-diabetic effectof cinnamon extract on blood glucose in dbdb micerdquo Journalof Ethnopharmacology vol 104 no 1-2 pp 119ndash123 2006
[18] S A Manohara Reddy J Mudgal P Bansal et al ldquoAntioxidantanti-inflammatory and anti-hyperglycaemic activities of hete-rocyclic homoprostanoid derivativesrdquoBioorganic andMedicinalChemistry vol 19 no 1 pp 384ndash392 2011
[19] S T Shukla V H Kulkarni P V Habbu K S Jagadeesh BS Patil and D M Smita ldquoHepatoprotective and antioxidantactivities of crude fractions of endophytic fungi of Ocimumsanctum Linn in ratsrdquo Oriental Pharmacy and ExperimentalMedicine vol 12 no 2 pp 81ndash91 2012
[20] B Mannervik ldquoGlutathione peroxidaserdquo Methods in Enzymol-ogy vol 113 pp 490ndash495 1985
[21] H PMisra and I Fridovich ldquoThe role of superoxide anion in theautoxidation of epinephrine and a simple assay for superoxidedismutaserdquo Journal of Biological Chemistry vol 247 no 10 pp3170ndash3175 1972
[22] A Claiborne ldquoCatalase activityrdquo in Handbook of Methods forOxygen Radical Research R A Greenwald Ed pp 283ndash284CRC Press Boca Raton Fla USA 1985
[23] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974
[24] W G Niehaus Jr and B Samuelsson ldquoFormation of malonalde-hyde from phospholipid arachidonate during microsomal lipidperoxidationrdquoEuropean Journal of Biochemistry vol 6 no 1 pp126ndash130 1968
[25] O S Osman J L Selway M A Kępczynska et al ldquoA novelautomated image analysis method for accurate adipocyte quan-tificationrdquo Adipocyte vol 2 no 3 pp 160ndash164 2013
[26] R Kalia C M Rao and N Gopalan Kutty ldquoSynthesisand evaluation of the anti-inflammatory activity of N-[2-(35-di-tert-butyl-4-hydroxyphenyl)-4-oxothiazolidin-3-yl]-nicotinamiderdquo Drug Research vol 57 no 9 pp 616ndash6222007
[27] N Maeda M Takahashi T Funahashi et al ldquoPPAR120574 ligandsincrease expression and plasma concentrations of adiponectinan adipose-derived proteinrdquo Diabetes vol 50 no 9 pp 2094ndash2099 2001
[28] P Bansal P Paul J Mudgal et al ldquoAntidiabetic antihyperlipi-demic and antioxidant effects of the flavonoid rich fraction ofPilea microphylla (L) in high fat dietstreptozotocin-induceddiabetes in micerdquo Experimental and Toxicologic Pathology vol64 no 6 pp 651ndash658 2012
[29] C Perez C Fernandez-Galaz T Fernandez-Agullo et alldquoLeptin impairs insulin signaling in rat adipocytesrdquo Diabetesvol 53 no 2 pp 347ndash353 2004
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
BioMed Research International 11
Table 5 Effect of thiazolidin-4-ones on liver antioxidant enzyme and malondialdehyde level
Liver biomarkers NPD HCD Pioglitazone Compound 2e Compound 3aGSH (nmolemg of protein) 558 plusmn 21 464 plusmn 21a 632 plusmn 15b 683 plusmn 20b 678 plusmn 43b
CAT (Umg of protein) 892 plusmn 57 554 plusmn 31a 938 plusmn 10b 819 plusmn 24b 875 plusmn 74b
SOD (Umg of protein) 1868 plusmn 232 1081 plusmn 112a 1748 plusmn 144b 2022 plusmn 47b 2080 plusmn 154b
GST (Umg of protein) 043 plusmn 005 037 plusmn 001a 073 plusmn 002b 070 plusmn 001b 072 plusmn 004b
MDA (nmolemg of protein) 057 plusmn 006 10 plusmn 033a 046 plusmn 001b 040 plusmn 001b 041 plusmn 002baRepresents 119875 lt 005 as compared to NPD group (normal pellet diet)bRepresents 119875 lt 005 as compared to HCD group (high carbohydrate diet)
0 30 60 90 120100
130
160
190
220
Plas
ma g
luco
se (m
gdL
)(M
eanplusmn
SE
Mn=6
)
NPDHCDPioglitazone (5mgkg)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(a)
0
5000
10000
15000
20000
25000
(Mea
nplusmn
SE
Mn=6
)
lowast
lowastlowast lowastlowast lowastlowast
NPDHCDPioglitazone (5mgkg)
Plas
ma g
luco
se (A
UC 0
ndash120
min)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
Figure 4 Effect of thiazolidin-4-ones on (a) plasma glucose (mgdL) and (b) area under the curve (AUC0minus120 min) against oral glucose
tolerance test (OGTT) in HCD fed mice Data presented as mean plusmn SEM (119899 = 6) lowastrepresents 119875 lt 005 as compared to NPD groupand lowastlowastrepresents 119875 lt 005 as compared to HCD group
However compound 2e did not show any significant effecton increased adipocyte size
4 Discussion and Conclusion
The present work was planned as a sequel to earlier studiesin our laboratory using thiazolidin-4-ones as antidiabetichypolipidemic and antiinflammatory molecules The substi-tutions in the thiazolidine ring were made at C2 and N3Attached to the latter position was a nicotinamide moietyand the substitution at C2 was either p-methoxyphenyl or2 5-di-tert-butyl-4-hydroxyphenyl group The compoundsshowed significant antidiabetic and hypolipidemic activities[9ndash11 26]
In the present study the substitution at N3 was changedto p-chloro-phenoxyacetylamino a group that is similar toclofibrate with a methylene bridge instead of gem-dimethylsubstitution
Four compounds out of 20 (ie compounds 4a 4b4c and 4d) had a methyl group attached at the C5 ofthiazolidine ring with cyclisation being made with thiolacticacid instead of thioglycolic acid In three other compounds(ie compounds 3a 3b and 3c) clofibrate was used to make
themoiety for substitution atN3Thiswas donewith a view toexamine the effect of the gem-dimethyl on the overall activityof the resulting molecule
All the 20 synthesised thiazolidin-4-ones were evaluatedfor glucose uptake in an in vitro system using the isolatedrat diaphragm In this experiment compounds 2e and 3asignificantly raised the amount of glucose uptake by thetissue both in the absence and presence of external insulinThis indicated the potential of the theses compounds insensitizing the tissues for the external insulin Hence thesetwo compounds were chosen to study their effect on a diet-induced model of insulin resistance Both compounds havethe same p-methoxyphenyl moiety attached to the C2 of thethiazolidine ring Compound 3a has a gem-dimethyl groupinstead of methylene in the substituent at N3
Apart from multiple risk factors diet-induced metabolicabnormalities contribute to the development of insulinresistance and 120573-cell failure in type-2 diabetes [16] Earlydetection and appropriate treatment are considered beneficialfor correcting the abnormality Among the various animalmodels induction of diabetes through diet provides moreresemblance to human type-2 diabetes Chronic intake ofdiet with high sucrose content has been reported to favourthe development of insulin resistance [16] Similarly in our
12 BioMed Research International
NPD HCD Pioglitazone Compound 3a Compound 2e
Liver
WAT
Green and red arrows indicate hyperplasia and increased size of adipocytes respectively
Pancreas
(a)
0
50000
100000
150000
200000
NPDHCDPioglitazone (5mgkg)
lowastlowastlowastlowast
(Mea
nplusmn
SE
Mn=3
)A
rea o
f lan
gerh
ans i
stle
ts (120583
m2)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
750
1500
2250
3000
NPDHCDPioglitazone (5mgkg)
lowastlowastlowastlowast
lowast
(Mea
nplusmn
SE
Mn=3
)A
rea o
f adi
pocy
tes (120583
m2)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 5 (a) Histological sections of mouse liver pancreas and white adipose tissue (WAT) in HCD model (10x) Green and red arrowsindicate hyperplasia of islets of Langerhans and increase in the size of adipocytes respectively Effect of thiazolidin-4-ones on (b) area ofLangerhans islets (120583m2) and (c) area of adipocytes (120583m2) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 ascompared to HCD group
study mice fed a high-sucrose diet for six months devel-oped metabolic abnormalities like hyperglycemia hyperin-sulinemia hypertriglyceridemia hypercholesterolemia andhypoadiponectinemia Hyperglycemia and hyperinsulinemiasuggest the inability of insulin to sensitize the tissue forglucose uptake allowing glucose to be diverted toward lipo-genesisThis led to hyperlipidemia and finally to insulin resis-tance In addition adiponectin and leptin the adipokinessecreted from white adipose tissue (WAT) are reported tobe involved in the metabolism of glucose and lipid [27]In our study we observed that HCD caused a reductionin plasma adiponectin level and an increase in the sizeof adipocytes However it did not cause any change inplasma leptin levels Thus we found a correlation between
the adiponectin level and size of adipocytes in HCD modelFurther OGTT results from HCD-fed mice correlated wellwith glucose intolerance hyperinsulinemia and hypoad-iponectinemia The authenticity of the model was validatedby the effect of pioglitazone which was able to correct themetabolic abnormalities Neither test compounds (2e or 3a)attenuated hypoadiponectinemia However they correctedthe impaired glucose tolerance and insulin resistance inmice Compounds 2e and 3a by their ability to enhanceglucose uptake and to sensitize the tissue for available insulinreduced hyperinsulinemia and raised the leptin levels Thiswould have resulted in better glucose utilization by periph-eral tissue Thus these compounds reduced the metabolicabnormalities like hyperglycemia hypertriglyceridemia and
BioMed Research International 13
hypercholesterolemia Prospective studies involving directassessment of insulin sensitivity in OGTT are requiredto analyze glucose stimulated insulin secretion (GSIS) inpresence of compounds to establish the mechanistic role ofthe test compounds on insulin signalling
Histological investigation showed no change in liverarchitecture in the various treatment groups In HCD controlmice there was an increase in the size of adipocytes in whiteadipose tissue (WAT) which was reversed by compound3a and pioglitazone treatment However only pioglitazone-treated animals showed positive correlation between reducedsize of the cells and correction of hypoadiponectinemiaApart from correction of hypoadiponectinemia pioglitazoneraised the peripheral leptin levels which was also true in caseof compounds 2e and 3a treatment Among the tested com-pounds 3a raised endogenous leptin levels four times morethan HCD group This finding points to the link betweenraised leptin levels and reduced adipocyte size caused bycompound 3a The pancreatic islets showed hyperplasia inpioglitazone and other treatment groups Pioglitazone is aPPAR-120574 agonist This could have been responsible for theproliferation of beta cells of pancreas The test compoundsare thiazolidin-4-ones with some similarity to thiazolidine-diones It is possible that hyperplasia observed in compound3a-treated animals might have been due to some agonisticactivity on PPAR-120574 receptors This needs to be investigatedthrough relevant assay
Oxidative stress has been implicated in the occurrence ofdiabetes and compounds reducing the oxidative stress havebeneficial role in correcting glucose intolerance and insulinresistance in diabetes [18 28]The test compounds and piogli-tazone reversed the depletion of endogenous antioxidantenzymes such as GSH CAT SOD and GST Further theyreduced malondialdehyde levels This suggests the inhibitoryeffect of these compounds on oxidative stress
Leptin serves as an insulin-sensitizing factor in the wholebody [29] However hyperleptinemia in the obese mouseand human is a sign of leptin resistance where increasedleptin levels are caused by disturbed homeostasis arisingfrom leptin receptor mutation ageing or obesity [29] Thushyperleptinemia further worsens impaired insulin actionin pathological condition In these conditions exogenouslyadministered leptin does not improve glucose tolerance andinsulin sensitivity In our study diseased animal (HCD fedmouse) per se did not develop hyperleptinemia which mightbe the sign of early stage of metabolic abnormality associatedwith insulin resistance At this state test compounds aswell as pioglitazone reversed the elevated glucose TG TCand insulin levels while facilitating leptin profile along withinsulin sensitivity Subsequently compound 3a treatmentresulted in increased energy expenditure demonstrated byelevated food efficiency ratio (FER) However reductionin body weight among the treated groups was not foundproportionate to the circulating leptin levels which suggeststhat increased leptin level in peripheral blood is not suffi-cient to induce a proportionate reduction on body weightA few questions remain unanswered such as (i) whetherthe compounds directly raised the leptin levels or are theresults due to indirect impact on metabolic signaling (ii) In
metabolic disorder combined with hyperleptinemia how dothese compounds affect leptin signaling Future studies areneeded to address these issues
No single mechanism would suffice to explain the ben-eficial effects of the test compounds They do not seem toact through insulinotropic activity unlike the sulfonylureasThey have no significant effect on adiponectin levels rulingout any involvement of this mechanism The increase in thelevel of serum leptin might point to the involvement of leptinin the antihyperlipidemic and antidiabetic potentials of thesemolecules
In conclusion thiazolidin-4-one derivatives act throughmultiple mechanisms to correct the metabolic abnormalitiesin type-2 diabetes In the present work compounds 2e and3a were found to be the most effective test compoundsto ameliorate insulin resistance and development of type-2diabetes
Conflict of Interests
The authors declare that they do not have any conflict ofinterests
Acknowledgment
The authors wish to thank All India Council for TechnicalEducation (AICTE) New Delhi India for providing thefinancial support and Manipal University Manipal Kar-nataka India for providing the facilities to carry out thepresent work
References
[1] S M Grundy I J Benjamin G L Burke et al ldquoDiabetes andcardiovascular disease a statement for healthcare professionalsfrom the american heart associationrdquo Circulation vol 100 no10 pp 1134ndash1146 1999
[2] K E Thorpe and D H Howard ldquoThe rise in spending amongMedicare beneficiaries the role of chronic disease prevalenceand changes in treatment intensity Increasing numbers ofbeneficiaries being treated for five or more conditions a year aredriving Medicare spending upwardrdquo Health Affairs vol 25 no5 pp w378ndashw388 2006
[3] S M Grundy ldquoDrug therapy of the metabolic syndrome min-imizing the emerging crisis in polypharmacyrdquo Nature ReviewsDrug Discovery vol 5 no 4 pp 295ndash309 2006
[4] M Navab N Gharavi and A D Watson ldquoInflammation andmetabolic disordersrdquo Current Opinion in Clinical Nutrition andMetabolic Care vol 11 no 4 pp 459ndash464 2008
[5] G S Hotamisligil ldquoInflammation and metabolic disordersrdquoNature vol 444 no 7121 pp 860ndash867 2006
[6] M Schmuth V Moosbrugger-Martinz S Blunder and SDubrac ldquoRole of PPAR LXR and PXR in epidermal homeosta-sis and inflammationrdquo Biochimica et Biophysica Acta vol 1841no 3 pp 463ndash473 2014
[7] J A Panetta J K Shadle M L Phillips D N Benslayand P P K Ho ldquo4-thiazolidinones potent antioxidants asantiinflammatory agentsrdquo Annals of the New York Academy ofSciences vol 696 pp 415ndash416 1993
14 BioMed Research International
[8] T Kato T Ozaki K Tamura Y Suzuki M Akima and NOhi ldquoNovel calcium antagonists with both calcium overloadinhibition and antioxidant activity 1 2-(35-Di-tert-butyl-4-hydroxyphenyl)-3- (aminopropyl)thiazolidinonesrdquo Journal ofMedicinal Chemistry vol 41 no 22 pp 4309ndash4316 1998
[9] JM Joy N Jacob andGN Kutty ldquoEvaluation of hypoglycemiceffects of 4-thiazolidinonesrdquo Indian Drugs vol 42 no 1 pp 17ndash21 2005
[10] N Jacob and G N Kutty ldquoSynthesis and hypolipidemic activityof a thiazolidinone derivativerdquo Indian Drugs vol 41 no 2 pp76ndash79 2004
[11] G K Nampurath S P Mathew V Khanna R T Zachariah SKanji andMRChamallamudi ldquoAssessment of hypolipidaemicactivity of three thiazolidin-4-ones in mice given high-fat dietand fructoserdquoChemico-Biological Interactions vol 171 no 3 pp363ndash368 2008
[12] A Kishore G K Nampurath S P Mathew et al ldquoAntidiabeticeffect through islet cell protection in streptozotocin diabetesa preliminary assessment of two thiazolidin-4-ones in Swissalbino micerdquo Chemico-Biological Interactions vol 177 no 3 pp242ndash246 2009
[13] J Mudgal V S Gowdra G Mathew et al ldquoRemedial effects ofnovel 2 3-disubstituted thiazolidin-4-ones in chemical medi-ated inflammationrdquo Chemico-Biological Interactions vol 210pp 34ndash42 2014
[14] A R Surrey ldquo4-Thiazolidones IV the preparation of some3-alkylaminoalkyl-2-aryl derivativesrdquo Journal of the AmericanChemical Society vol 71 no 10 pp 3354ndash3356 1949
[15] B R P Kumar and M J Nanjan ldquoNovel glitazones designsynthesis glucose uptake and structure-activity relationshipsrdquoBioorganic and Medicinal Chemistry Letters vol 20 no 6 pp1953ndash1956 2010
[16] M Sumiyoshi M Sakanaka and Y Kimura ldquoChronic intakeof high-fat and high-sucrose diets differentially affects glucoseintolerance inmicerdquo Journal of Nutrition vol 136 no 3 pp 582ndash587 2006
[17] S H Kim S H Hyun and S Y Choung ldquoAnti-diabetic effectof cinnamon extract on blood glucose in dbdb micerdquo Journalof Ethnopharmacology vol 104 no 1-2 pp 119ndash123 2006
[18] S A Manohara Reddy J Mudgal P Bansal et al ldquoAntioxidantanti-inflammatory and anti-hyperglycaemic activities of hete-rocyclic homoprostanoid derivativesrdquoBioorganic andMedicinalChemistry vol 19 no 1 pp 384ndash392 2011
[19] S T Shukla V H Kulkarni P V Habbu K S Jagadeesh BS Patil and D M Smita ldquoHepatoprotective and antioxidantactivities of crude fractions of endophytic fungi of Ocimumsanctum Linn in ratsrdquo Oriental Pharmacy and ExperimentalMedicine vol 12 no 2 pp 81ndash91 2012
[20] B Mannervik ldquoGlutathione peroxidaserdquo Methods in Enzymol-ogy vol 113 pp 490ndash495 1985
[21] H PMisra and I Fridovich ldquoThe role of superoxide anion in theautoxidation of epinephrine and a simple assay for superoxidedismutaserdquo Journal of Biological Chemistry vol 247 no 10 pp3170ndash3175 1972
[22] A Claiborne ldquoCatalase activityrdquo in Handbook of Methods forOxygen Radical Research R A Greenwald Ed pp 283ndash284CRC Press Boca Raton Fla USA 1985
[23] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974
[24] W G Niehaus Jr and B Samuelsson ldquoFormation of malonalde-hyde from phospholipid arachidonate during microsomal lipidperoxidationrdquoEuropean Journal of Biochemistry vol 6 no 1 pp126ndash130 1968
[25] O S Osman J L Selway M A Kępczynska et al ldquoA novelautomated image analysis method for accurate adipocyte quan-tificationrdquo Adipocyte vol 2 no 3 pp 160ndash164 2013
[26] R Kalia C M Rao and N Gopalan Kutty ldquoSynthesisand evaluation of the anti-inflammatory activity of N-[2-(35-di-tert-butyl-4-hydroxyphenyl)-4-oxothiazolidin-3-yl]-nicotinamiderdquo Drug Research vol 57 no 9 pp 616ndash6222007
[27] N Maeda M Takahashi T Funahashi et al ldquoPPAR120574 ligandsincrease expression and plasma concentrations of adiponectinan adipose-derived proteinrdquo Diabetes vol 50 no 9 pp 2094ndash2099 2001
[28] P Bansal P Paul J Mudgal et al ldquoAntidiabetic antihyperlipi-demic and antioxidant effects of the flavonoid rich fraction ofPilea microphylla (L) in high fat dietstreptozotocin-induceddiabetes in micerdquo Experimental and Toxicologic Pathology vol64 no 6 pp 651ndash658 2012
[29] C Perez C Fernandez-Galaz T Fernandez-Agullo et alldquoLeptin impairs insulin signaling in rat adipocytesrdquo Diabetesvol 53 no 2 pp 347ndash353 2004
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
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StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
12 BioMed Research International
NPD HCD Pioglitazone Compound 3a Compound 2e
Liver
WAT
Green and red arrows indicate hyperplasia and increased size of adipocytes respectively
Pancreas
(a)
0
50000
100000
150000
200000
NPDHCDPioglitazone (5mgkg)
lowastlowastlowastlowast
(Mea
nplusmn
SE
Mn=3
)A
rea o
f lan
gerh
ans i
stle
ts (120583
m2)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(b)
0
750
1500
2250
3000
NPDHCDPioglitazone (5mgkg)
lowastlowastlowastlowast
lowast
(Mea
nplusmn
SE
Mn=3
)A
rea o
f adi
pocy
tes (120583
m2)
Compound 2e (100mgkg)Compound 3a (100mgkg)
(c)
Figure 5 (a) Histological sections of mouse liver pancreas and white adipose tissue (WAT) in HCD model (10x) Green and red arrowsindicate hyperplasia of islets of Langerhans and increase in the size of adipocytes respectively Effect of thiazolidin-4-ones on (b) area ofLangerhans islets (120583m2) and (c) area of adipocytes (120583m2) lowastrepresents 119875 lt 005 as compared to NPD group and lowastlowastrepresents 119875 lt 005 ascompared to HCD group
study mice fed a high-sucrose diet for six months devel-oped metabolic abnormalities like hyperglycemia hyperin-sulinemia hypertriglyceridemia hypercholesterolemia andhypoadiponectinemia Hyperglycemia and hyperinsulinemiasuggest the inability of insulin to sensitize the tissue forglucose uptake allowing glucose to be diverted toward lipo-genesisThis led to hyperlipidemia and finally to insulin resis-tance In addition adiponectin and leptin the adipokinessecreted from white adipose tissue (WAT) are reported tobe involved in the metabolism of glucose and lipid [27]In our study we observed that HCD caused a reductionin plasma adiponectin level and an increase in the sizeof adipocytes However it did not cause any change inplasma leptin levels Thus we found a correlation between
the adiponectin level and size of adipocytes in HCD modelFurther OGTT results from HCD-fed mice correlated wellwith glucose intolerance hyperinsulinemia and hypoad-iponectinemia The authenticity of the model was validatedby the effect of pioglitazone which was able to correct themetabolic abnormalities Neither test compounds (2e or 3a)attenuated hypoadiponectinemia However they correctedthe impaired glucose tolerance and insulin resistance inmice Compounds 2e and 3a by their ability to enhanceglucose uptake and to sensitize the tissue for available insulinreduced hyperinsulinemia and raised the leptin levels Thiswould have resulted in better glucose utilization by periph-eral tissue Thus these compounds reduced the metabolicabnormalities like hyperglycemia hypertriglyceridemia and
BioMed Research International 13
hypercholesterolemia Prospective studies involving directassessment of insulin sensitivity in OGTT are requiredto analyze glucose stimulated insulin secretion (GSIS) inpresence of compounds to establish the mechanistic role ofthe test compounds on insulin signalling
Histological investigation showed no change in liverarchitecture in the various treatment groups In HCD controlmice there was an increase in the size of adipocytes in whiteadipose tissue (WAT) which was reversed by compound3a and pioglitazone treatment However only pioglitazone-treated animals showed positive correlation between reducedsize of the cells and correction of hypoadiponectinemiaApart from correction of hypoadiponectinemia pioglitazoneraised the peripheral leptin levels which was also true in caseof compounds 2e and 3a treatment Among the tested com-pounds 3a raised endogenous leptin levels four times morethan HCD group This finding points to the link betweenraised leptin levels and reduced adipocyte size caused bycompound 3a The pancreatic islets showed hyperplasia inpioglitazone and other treatment groups Pioglitazone is aPPAR-120574 agonist This could have been responsible for theproliferation of beta cells of pancreas The test compoundsare thiazolidin-4-ones with some similarity to thiazolidine-diones It is possible that hyperplasia observed in compound3a-treated animals might have been due to some agonisticactivity on PPAR-120574 receptors This needs to be investigatedthrough relevant assay
Oxidative stress has been implicated in the occurrence ofdiabetes and compounds reducing the oxidative stress havebeneficial role in correcting glucose intolerance and insulinresistance in diabetes [18 28]The test compounds and piogli-tazone reversed the depletion of endogenous antioxidantenzymes such as GSH CAT SOD and GST Further theyreduced malondialdehyde levels This suggests the inhibitoryeffect of these compounds on oxidative stress
Leptin serves as an insulin-sensitizing factor in the wholebody [29] However hyperleptinemia in the obese mouseand human is a sign of leptin resistance where increasedleptin levels are caused by disturbed homeostasis arisingfrom leptin receptor mutation ageing or obesity [29] Thushyperleptinemia further worsens impaired insulin actionin pathological condition In these conditions exogenouslyadministered leptin does not improve glucose tolerance andinsulin sensitivity In our study diseased animal (HCD fedmouse) per se did not develop hyperleptinemia which mightbe the sign of early stage of metabolic abnormality associatedwith insulin resistance At this state test compounds aswell as pioglitazone reversed the elevated glucose TG TCand insulin levels while facilitating leptin profile along withinsulin sensitivity Subsequently compound 3a treatmentresulted in increased energy expenditure demonstrated byelevated food efficiency ratio (FER) However reductionin body weight among the treated groups was not foundproportionate to the circulating leptin levels which suggeststhat increased leptin level in peripheral blood is not suffi-cient to induce a proportionate reduction on body weightA few questions remain unanswered such as (i) whetherthe compounds directly raised the leptin levels or are theresults due to indirect impact on metabolic signaling (ii) In
metabolic disorder combined with hyperleptinemia how dothese compounds affect leptin signaling Future studies areneeded to address these issues
No single mechanism would suffice to explain the ben-eficial effects of the test compounds They do not seem toact through insulinotropic activity unlike the sulfonylureasThey have no significant effect on adiponectin levels rulingout any involvement of this mechanism The increase in thelevel of serum leptin might point to the involvement of leptinin the antihyperlipidemic and antidiabetic potentials of thesemolecules
In conclusion thiazolidin-4-one derivatives act throughmultiple mechanisms to correct the metabolic abnormalitiesin type-2 diabetes In the present work compounds 2e and3a were found to be the most effective test compoundsto ameliorate insulin resistance and development of type-2diabetes
Conflict of Interests
The authors declare that they do not have any conflict ofinterests
Acknowledgment
The authors wish to thank All India Council for TechnicalEducation (AICTE) New Delhi India for providing thefinancial support and Manipal University Manipal Kar-nataka India for providing the facilities to carry out thepresent work
References
[1] S M Grundy I J Benjamin G L Burke et al ldquoDiabetes andcardiovascular disease a statement for healthcare professionalsfrom the american heart associationrdquo Circulation vol 100 no10 pp 1134ndash1146 1999
[2] K E Thorpe and D H Howard ldquoThe rise in spending amongMedicare beneficiaries the role of chronic disease prevalenceand changes in treatment intensity Increasing numbers ofbeneficiaries being treated for five or more conditions a year aredriving Medicare spending upwardrdquo Health Affairs vol 25 no5 pp w378ndashw388 2006
[3] S M Grundy ldquoDrug therapy of the metabolic syndrome min-imizing the emerging crisis in polypharmacyrdquo Nature ReviewsDrug Discovery vol 5 no 4 pp 295ndash309 2006
[4] M Navab N Gharavi and A D Watson ldquoInflammation andmetabolic disordersrdquo Current Opinion in Clinical Nutrition andMetabolic Care vol 11 no 4 pp 459ndash464 2008
[5] G S Hotamisligil ldquoInflammation and metabolic disordersrdquoNature vol 444 no 7121 pp 860ndash867 2006
[6] M Schmuth V Moosbrugger-Martinz S Blunder and SDubrac ldquoRole of PPAR LXR and PXR in epidermal homeosta-sis and inflammationrdquo Biochimica et Biophysica Acta vol 1841no 3 pp 463ndash473 2014
[7] J A Panetta J K Shadle M L Phillips D N Benslayand P P K Ho ldquo4-thiazolidinones potent antioxidants asantiinflammatory agentsrdquo Annals of the New York Academy ofSciences vol 696 pp 415ndash416 1993
14 BioMed Research International
[8] T Kato T Ozaki K Tamura Y Suzuki M Akima and NOhi ldquoNovel calcium antagonists with both calcium overloadinhibition and antioxidant activity 1 2-(35-Di-tert-butyl-4-hydroxyphenyl)-3- (aminopropyl)thiazolidinonesrdquo Journal ofMedicinal Chemistry vol 41 no 22 pp 4309ndash4316 1998
[9] JM Joy N Jacob andGN Kutty ldquoEvaluation of hypoglycemiceffects of 4-thiazolidinonesrdquo Indian Drugs vol 42 no 1 pp 17ndash21 2005
[10] N Jacob and G N Kutty ldquoSynthesis and hypolipidemic activityof a thiazolidinone derivativerdquo Indian Drugs vol 41 no 2 pp76ndash79 2004
[11] G K Nampurath S P Mathew V Khanna R T Zachariah SKanji andMRChamallamudi ldquoAssessment of hypolipidaemicactivity of three thiazolidin-4-ones in mice given high-fat dietand fructoserdquoChemico-Biological Interactions vol 171 no 3 pp363ndash368 2008
[12] A Kishore G K Nampurath S P Mathew et al ldquoAntidiabeticeffect through islet cell protection in streptozotocin diabetesa preliminary assessment of two thiazolidin-4-ones in Swissalbino micerdquo Chemico-Biological Interactions vol 177 no 3 pp242ndash246 2009
[13] J Mudgal V S Gowdra G Mathew et al ldquoRemedial effects ofnovel 2 3-disubstituted thiazolidin-4-ones in chemical medi-ated inflammationrdquo Chemico-Biological Interactions vol 210pp 34ndash42 2014
[14] A R Surrey ldquo4-Thiazolidones IV the preparation of some3-alkylaminoalkyl-2-aryl derivativesrdquo Journal of the AmericanChemical Society vol 71 no 10 pp 3354ndash3356 1949
[15] B R P Kumar and M J Nanjan ldquoNovel glitazones designsynthesis glucose uptake and structure-activity relationshipsrdquoBioorganic and Medicinal Chemistry Letters vol 20 no 6 pp1953ndash1956 2010
[16] M Sumiyoshi M Sakanaka and Y Kimura ldquoChronic intakeof high-fat and high-sucrose diets differentially affects glucoseintolerance inmicerdquo Journal of Nutrition vol 136 no 3 pp 582ndash587 2006
[17] S H Kim S H Hyun and S Y Choung ldquoAnti-diabetic effectof cinnamon extract on blood glucose in dbdb micerdquo Journalof Ethnopharmacology vol 104 no 1-2 pp 119ndash123 2006
[18] S A Manohara Reddy J Mudgal P Bansal et al ldquoAntioxidantanti-inflammatory and anti-hyperglycaemic activities of hete-rocyclic homoprostanoid derivativesrdquoBioorganic andMedicinalChemistry vol 19 no 1 pp 384ndash392 2011
[19] S T Shukla V H Kulkarni P V Habbu K S Jagadeesh BS Patil and D M Smita ldquoHepatoprotective and antioxidantactivities of crude fractions of endophytic fungi of Ocimumsanctum Linn in ratsrdquo Oriental Pharmacy and ExperimentalMedicine vol 12 no 2 pp 81ndash91 2012
[20] B Mannervik ldquoGlutathione peroxidaserdquo Methods in Enzymol-ogy vol 113 pp 490ndash495 1985
[21] H PMisra and I Fridovich ldquoThe role of superoxide anion in theautoxidation of epinephrine and a simple assay for superoxidedismutaserdquo Journal of Biological Chemistry vol 247 no 10 pp3170ndash3175 1972
[22] A Claiborne ldquoCatalase activityrdquo in Handbook of Methods forOxygen Radical Research R A Greenwald Ed pp 283ndash284CRC Press Boca Raton Fla USA 1985
[23] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974
[24] W G Niehaus Jr and B Samuelsson ldquoFormation of malonalde-hyde from phospholipid arachidonate during microsomal lipidperoxidationrdquoEuropean Journal of Biochemistry vol 6 no 1 pp126ndash130 1968
[25] O S Osman J L Selway M A Kępczynska et al ldquoA novelautomated image analysis method for accurate adipocyte quan-tificationrdquo Adipocyte vol 2 no 3 pp 160ndash164 2013
[26] R Kalia C M Rao and N Gopalan Kutty ldquoSynthesisand evaluation of the anti-inflammatory activity of N-[2-(35-di-tert-butyl-4-hydroxyphenyl)-4-oxothiazolidin-3-yl]-nicotinamiderdquo Drug Research vol 57 no 9 pp 616ndash6222007
[27] N Maeda M Takahashi T Funahashi et al ldquoPPAR120574 ligandsincrease expression and plasma concentrations of adiponectinan adipose-derived proteinrdquo Diabetes vol 50 no 9 pp 2094ndash2099 2001
[28] P Bansal P Paul J Mudgal et al ldquoAntidiabetic antihyperlipi-demic and antioxidant effects of the flavonoid rich fraction ofPilea microphylla (L) in high fat dietstreptozotocin-induceddiabetes in micerdquo Experimental and Toxicologic Pathology vol64 no 6 pp 651ndash658 2012
[29] C Perez C Fernandez-Galaz T Fernandez-Agullo et alldquoLeptin impairs insulin signaling in rat adipocytesrdquo Diabetesvol 53 no 2 pp 347ndash353 2004
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
BioMed Research International 13
hypercholesterolemia Prospective studies involving directassessment of insulin sensitivity in OGTT are requiredto analyze glucose stimulated insulin secretion (GSIS) inpresence of compounds to establish the mechanistic role ofthe test compounds on insulin signalling
Histological investigation showed no change in liverarchitecture in the various treatment groups In HCD controlmice there was an increase in the size of adipocytes in whiteadipose tissue (WAT) which was reversed by compound3a and pioglitazone treatment However only pioglitazone-treated animals showed positive correlation between reducedsize of the cells and correction of hypoadiponectinemiaApart from correction of hypoadiponectinemia pioglitazoneraised the peripheral leptin levels which was also true in caseof compounds 2e and 3a treatment Among the tested com-pounds 3a raised endogenous leptin levels four times morethan HCD group This finding points to the link betweenraised leptin levels and reduced adipocyte size caused bycompound 3a The pancreatic islets showed hyperplasia inpioglitazone and other treatment groups Pioglitazone is aPPAR-120574 agonist This could have been responsible for theproliferation of beta cells of pancreas The test compoundsare thiazolidin-4-ones with some similarity to thiazolidine-diones It is possible that hyperplasia observed in compound3a-treated animals might have been due to some agonisticactivity on PPAR-120574 receptors This needs to be investigatedthrough relevant assay
Oxidative stress has been implicated in the occurrence ofdiabetes and compounds reducing the oxidative stress havebeneficial role in correcting glucose intolerance and insulinresistance in diabetes [18 28]The test compounds and piogli-tazone reversed the depletion of endogenous antioxidantenzymes such as GSH CAT SOD and GST Further theyreduced malondialdehyde levels This suggests the inhibitoryeffect of these compounds on oxidative stress
Leptin serves as an insulin-sensitizing factor in the wholebody [29] However hyperleptinemia in the obese mouseand human is a sign of leptin resistance where increasedleptin levels are caused by disturbed homeostasis arisingfrom leptin receptor mutation ageing or obesity [29] Thushyperleptinemia further worsens impaired insulin actionin pathological condition In these conditions exogenouslyadministered leptin does not improve glucose tolerance andinsulin sensitivity In our study diseased animal (HCD fedmouse) per se did not develop hyperleptinemia which mightbe the sign of early stage of metabolic abnormality associatedwith insulin resistance At this state test compounds aswell as pioglitazone reversed the elevated glucose TG TCand insulin levels while facilitating leptin profile along withinsulin sensitivity Subsequently compound 3a treatmentresulted in increased energy expenditure demonstrated byelevated food efficiency ratio (FER) However reductionin body weight among the treated groups was not foundproportionate to the circulating leptin levels which suggeststhat increased leptin level in peripheral blood is not suffi-cient to induce a proportionate reduction on body weightA few questions remain unanswered such as (i) whetherthe compounds directly raised the leptin levels or are theresults due to indirect impact on metabolic signaling (ii) In
metabolic disorder combined with hyperleptinemia how dothese compounds affect leptin signaling Future studies areneeded to address these issues
No single mechanism would suffice to explain the ben-eficial effects of the test compounds They do not seem toact through insulinotropic activity unlike the sulfonylureasThey have no significant effect on adiponectin levels rulingout any involvement of this mechanism The increase in thelevel of serum leptin might point to the involvement of leptinin the antihyperlipidemic and antidiabetic potentials of thesemolecules
In conclusion thiazolidin-4-one derivatives act throughmultiple mechanisms to correct the metabolic abnormalitiesin type-2 diabetes In the present work compounds 2e and3a were found to be the most effective test compoundsto ameliorate insulin resistance and development of type-2diabetes
Conflict of Interests
The authors declare that they do not have any conflict ofinterests
Acknowledgment
The authors wish to thank All India Council for TechnicalEducation (AICTE) New Delhi India for providing thefinancial support and Manipal University Manipal Kar-nataka India for providing the facilities to carry out thepresent work
References
[1] S M Grundy I J Benjamin G L Burke et al ldquoDiabetes andcardiovascular disease a statement for healthcare professionalsfrom the american heart associationrdquo Circulation vol 100 no10 pp 1134ndash1146 1999
[2] K E Thorpe and D H Howard ldquoThe rise in spending amongMedicare beneficiaries the role of chronic disease prevalenceand changes in treatment intensity Increasing numbers ofbeneficiaries being treated for five or more conditions a year aredriving Medicare spending upwardrdquo Health Affairs vol 25 no5 pp w378ndashw388 2006
[3] S M Grundy ldquoDrug therapy of the metabolic syndrome min-imizing the emerging crisis in polypharmacyrdquo Nature ReviewsDrug Discovery vol 5 no 4 pp 295ndash309 2006
[4] M Navab N Gharavi and A D Watson ldquoInflammation andmetabolic disordersrdquo Current Opinion in Clinical Nutrition andMetabolic Care vol 11 no 4 pp 459ndash464 2008
[5] G S Hotamisligil ldquoInflammation and metabolic disordersrdquoNature vol 444 no 7121 pp 860ndash867 2006
[6] M Schmuth V Moosbrugger-Martinz S Blunder and SDubrac ldquoRole of PPAR LXR and PXR in epidermal homeosta-sis and inflammationrdquo Biochimica et Biophysica Acta vol 1841no 3 pp 463ndash473 2014
[7] J A Panetta J K Shadle M L Phillips D N Benslayand P P K Ho ldquo4-thiazolidinones potent antioxidants asantiinflammatory agentsrdquo Annals of the New York Academy ofSciences vol 696 pp 415ndash416 1993
14 BioMed Research International
[8] T Kato T Ozaki K Tamura Y Suzuki M Akima and NOhi ldquoNovel calcium antagonists with both calcium overloadinhibition and antioxidant activity 1 2-(35-Di-tert-butyl-4-hydroxyphenyl)-3- (aminopropyl)thiazolidinonesrdquo Journal ofMedicinal Chemistry vol 41 no 22 pp 4309ndash4316 1998
[9] JM Joy N Jacob andGN Kutty ldquoEvaluation of hypoglycemiceffects of 4-thiazolidinonesrdquo Indian Drugs vol 42 no 1 pp 17ndash21 2005
[10] N Jacob and G N Kutty ldquoSynthesis and hypolipidemic activityof a thiazolidinone derivativerdquo Indian Drugs vol 41 no 2 pp76ndash79 2004
[11] G K Nampurath S P Mathew V Khanna R T Zachariah SKanji andMRChamallamudi ldquoAssessment of hypolipidaemicactivity of three thiazolidin-4-ones in mice given high-fat dietand fructoserdquoChemico-Biological Interactions vol 171 no 3 pp363ndash368 2008
[12] A Kishore G K Nampurath S P Mathew et al ldquoAntidiabeticeffect through islet cell protection in streptozotocin diabetesa preliminary assessment of two thiazolidin-4-ones in Swissalbino micerdquo Chemico-Biological Interactions vol 177 no 3 pp242ndash246 2009
[13] J Mudgal V S Gowdra G Mathew et al ldquoRemedial effects ofnovel 2 3-disubstituted thiazolidin-4-ones in chemical medi-ated inflammationrdquo Chemico-Biological Interactions vol 210pp 34ndash42 2014
[14] A R Surrey ldquo4-Thiazolidones IV the preparation of some3-alkylaminoalkyl-2-aryl derivativesrdquo Journal of the AmericanChemical Society vol 71 no 10 pp 3354ndash3356 1949
[15] B R P Kumar and M J Nanjan ldquoNovel glitazones designsynthesis glucose uptake and structure-activity relationshipsrdquoBioorganic and Medicinal Chemistry Letters vol 20 no 6 pp1953ndash1956 2010
[16] M Sumiyoshi M Sakanaka and Y Kimura ldquoChronic intakeof high-fat and high-sucrose diets differentially affects glucoseintolerance inmicerdquo Journal of Nutrition vol 136 no 3 pp 582ndash587 2006
[17] S H Kim S H Hyun and S Y Choung ldquoAnti-diabetic effectof cinnamon extract on blood glucose in dbdb micerdquo Journalof Ethnopharmacology vol 104 no 1-2 pp 119ndash123 2006
[18] S A Manohara Reddy J Mudgal P Bansal et al ldquoAntioxidantanti-inflammatory and anti-hyperglycaemic activities of hete-rocyclic homoprostanoid derivativesrdquoBioorganic andMedicinalChemistry vol 19 no 1 pp 384ndash392 2011
[19] S T Shukla V H Kulkarni P V Habbu K S Jagadeesh BS Patil and D M Smita ldquoHepatoprotective and antioxidantactivities of crude fractions of endophytic fungi of Ocimumsanctum Linn in ratsrdquo Oriental Pharmacy and ExperimentalMedicine vol 12 no 2 pp 81ndash91 2012
[20] B Mannervik ldquoGlutathione peroxidaserdquo Methods in Enzymol-ogy vol 113 pp 490ndash495 1985
[21] H PMisra and I Fridovich ldquoThe role of superoxide anion in theautoxidation of epinephrine and a simple assay for superoxidedismutaserdquo Journal of Biological Chemistry vol 247 no 10 pp3170ndash3175 1972
[22] A Claiborne ldquoCatalase activityrdquo in Handbook of Methods forOxygen Radical Research R A Greenwald Ed pp 283ndash284CRC Press Boca Raton Fla USA 1985
[23] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974
[24] W G Niehaus Jr and B Samuelsson ldquoFormation of malonalde-hyde from phospholipid arachidonate during microsomal lipidperoxidationrdquoEuropean Journal of Biochemistry vol 6 no 1 pp126ndash130 1968
[25] O S Osman J L Selway M A Kępczynska et al ldquoA novelautomated image analysis method for accurate adipocyte quan-tificationrdquo Adipocyte vol 2 no 3 pp 160ndash164 2013
[26] R Kalia C M Rao and N Gopalan Kutty ldquoSynthesisand evaluation of the anti-inflammatory activity of N-[2-(35-di-tert-butyl-4-hydroxyphenyl)-4-oxothiazolidin-3-yl]-nicotinamiderdquo Drug Research vol 57 no 9 pp 616ndash6222007
[27] N Maeda M Takahashi T Funahashi et al ldquoPPAR120574 ligandsincrease expression and plasma concentrations of adiponectinan adipose-derived proteinrdquo Diabetes vol 50 no 9 pp 2094ndash2099 2001
[28] P Bansal P Paul J Mudgal et al ldquoAntidiabetic antihyperlipi-demic and antioxidant effects of the flavonoid rich fraction ofPilea microphylla (L) in high fat dietstreptozotocin-induceddiabetes in micerdquo Experimental and Toxicologic Pathology vol64 no 6 pp 651ndash658 2012
[29] C Perez C Fernandez-Galaz T Fernandez-Agullo et alldquoLeptin impairs insulin signaling in rat adipocytesrdquo Diabetesvol 53 no 2 pp 347ndash353 2004
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
14 BioMed Research International
[8] T Kato T Ozaki K Tamura Y Suzuki M Akima and NOhi ldquoNovel calcium antagonists with both calcium overloadinhibition and antioxidant activity 1 2-(35-Di-tert-butyl-4-hydroxyphenyl)-3- (aminopropyl)thiazolidinonesrdquo Journal ofMedicinal Chemistry vol 41 no 22 pp 4309ndash4316 1998
[9] JM Joy N Jacob andGN Kutty ldquoEvaluation of hypoglycemiceffects of 4-thiazolidinonesrdquo Indian Drugs vol 42 no 1 pp 17ndash21 2005
[10] N Jacob and G N Kutty ldquoSynthesis and hypolipidemic activityof a thiazolidinone derivativerdquo Indian Drugs vol 41 no 2 pp76ndash79 2004
[11] G K Nampurath S P Mathew V Khanna R T Zachariah SKanji andMRChamallamudi ldquoAssessment of hypolipidaemicactivity of three thiazolidin-4-ones in mice given high-fat dietand fructoserdquoChemico-Biological Interactions vol 171 no 3 pp363ndash368 2008
[12] A Kishore G K Nampurath S P Mathew et al ldquoAntidiabeticeffect through islet cell protection in streptozotocin diabetesa preliminary assessment of two thiazolidin-4-ones in Swissalbino micerdquo Chemico-Biological Interactions vol 177 no 3 pp242ndash246 2009
[13] J Mudgal V S Gowdra G Mathew et al ldquoRemedial effects ofnovel 2 3-disubstituted thiazolidin-4-ones in chemical medi-ated inflammationrdquo Chemico-Biological Interactions vol 210pp 34ndash42 2014
[14] A R Surrey ldquo4-Thiazolidones IV the preparation of some3-alkylaminoalkyl-2-aryl derivativesrdquo Journal of the AmericanChemical Society vol 71 no 10 pp 3354ndash3356 1949
[15] B R P Kumar and M J Nanjan ldquoNovel glitazones designsynthesis glucose uptake and structure-activity relationshipsrdquoBioorganic and Medicinal Chemistry Letters vol 20 no 6 pp1953ndash1956 2010
[16] M Sumiyoshi M Sakanaka and Y Kimura ldquoChronic intakeof high-fat and high-sucrose diets differentially affects glucoseintolerance inmicerdquo Journal of Nutrition vol 136 no 3 pp 582ndash587 2006
[17] S H Kim S H Hyun and S Y Choung ldquoAnti-diabetic effectof cinnamon extract on blood glucose in dbdb micerdquo Journalof Ethnopharmacology vol 104 no 1-2 pp 119ndash123 2006
[18] S A Manohara Reddy J Mudgal P Bansal et al ldquoAntioxidantanti-inflammatory and anti-hyperglycaemic activities of hete-rocyclic homoprostanoid derivativesrdquoBioorganic andMedicinalChemistry vol 19 no 1 pp 384ndash392 2011
[19] S T Shukla V H Kulkarni P V Habbu K S Jagadeesh BS Patil and D M Smita ldquoHepatoprotective and antioxidantactivities of crude fractions of endophytic fungi of Ocimumsanctum Linn in ratsrdquo Oriental Pharmacy and ExperimentalMedicine vol 12 no 2 pp 81ndash91 2012
[20] B Mannervik ldquoGlutathione peroxidaserdquo Methods in Enzymol-ogy vol 113 pp 490ndash495 1985
[21] H PMisra and I Fridovich ldquoThe role of superoxide anion in theautoxidation of epinephrine and a simple assay for superoxidedismutaserdquo Journal of Biological Chemistry vol 247 no 10 pp3170ndash3175 1972
[22] A Claiborne ldquoCatalase activityrdquo in Handbook of Methods forOxygen Radical Research R A Greenwald Ed pp 283ndash284CRC Press Boca Raton Fla USA 1985
[23] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974
[24] W G Niehaus Jr and B Samuelsson ldquoFormation of malonalde-hyde from phospholipid arachidonate during microsomal lipidperoxidationrdquoEuropean Journal of Biochemistry vol 6 no 1 pp126ndash130 1968
[25] O S Osman J L Selway M A Kępczynska et al ldquoA novelautomated image analysis method for accurate adipocyte quan-tificationrdquo Adipocyte vol 2 no 3 pp 160ndash164 2013
[26] R Kalia C M Rao and N Gopalan Kutty ldquoSynthesisand evaluation of the anti-inflammatory activity of N-[2-(35-di-tert-butyl-4-hydroxyphenyl)-4-oxothiazolidin-3-yl]-nicotinamiderdquo Drug Research vol 57 no 9 pp 616ndash6222007
[27] N Maeda M Takahashi T Funahashi et al ldquoPPAR120574 ligandsincrease expression and plasma concentrations of adiponectinan adipose-derived proteinrdquo Diabetes vol 50 no 9 pp 2094ndash2099 2001
[28] P Bansal P Paul J Mudgal et al ldquoAntidiabetic antihyperlipi-demic and antioxidant effects of the flavonoid rich fraction ofPilea microphylla (L) in high fat dietstreptozotocin-induceddiabetes in micerdquo Experimental and Toxicologic Pathology vol64 no 6 pp 651ndash658 2012
[29] C Perez C Fernandez-Galaz T Fernandez-Agullo et alldquoLeptin impairs insulin signaling in rat adipocytesrdquo Diabetesvol 53 no 2 pp 347ndash353 2004
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of