brian covello: diabetes mellitus type 2 research poster
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1,25-Dihydroxyvitamin D3 and Retinoic Acid in vitro Modulation of PPARγand Insulin Resistance in Diabetes Mellitus II
Brian Covello, Emily BradshawFlorida Southern College
Abstract1,25-Dihydroxyvitamin D3 (D3) and Retinoic Acid (RA) have previ-
ously been reported to down-regulate the master controller of adipoge-nesis, nuclear receptor PPARγ (Yoshifumi, 1998). This receptor playsa significant role in understanding diabetes mellitus II, and a class ofanti-diabetic drugs has been found to agonistically bind to PPARγ. Forthe first time, this study seeks to combine the aforementioned metabo-lites together in one treatment scheme. This is a crucial component forcreating a direct relation to in vivo studies, as only concentrations ofthese metabolites are found in human serum. We hypothesized that acombined treatment of D3 and RA will have a synergistic effect uponPPARγ, causing novel protein changes when compared to individualtreatments and leading to a greater down-regulation than previouslyreported (Yoshifumi, 1998). Visualization of protein location throughimmunofluorescence microscopy revealed localization of PPARγ to thenucleus, and densitometric analysis immunoblotted proteins indicatedsignificant downregulation with µM D3 treatments. The combinedtreatments failed to downregulate PPARγ more intensely than solitaryµM D3, thus, no synergistic mechanism was found and the hypothe-sis was not supported. Surprisingly, human serum concentrations ofmixed vitamin metabolites upregulated PPARγ epxression. These re-sults seem to suggest that diabetics may decrease their resistance to in-sulin and improve their condition by maintaining appropriate nanomo-lar serum levels of these two vitamins. Further research is warranted tobetter translate these findings into accurate medical recommendations.
IntroductionTwenty five million people within the United States are currently
inflicted by Diabetes Mellitus 2, a disease whose progression may becharacterized by insulin resistance (Powers, 2005). The nuclear re-ceptor PPARγ effects both insulin resistance and adipogenesis (Di-radourian, 2005). A class of anti-diabetic drugs known as TZDs workagonistically on PPARγ (Rangwala, 2003).
Upon ligand binding, PPARγ heterodimerizes with retinoid X re-ceptors (RXR). RXR supply within a cell is limited, and a series of otherreceptors such as vitamin D receptor (VDR) and retinoid A receptor(RAR) compete with PPARγ for the common heterodimeric partner,RXR (Grimaldi, 2007). As such, ligands of RAR and VDR such as1,25-dihydroxyvitamin D3 and retinoic acid have been implemented instudies of PPARγ.
This research aims to mimic normal human physiological condi-tions, providing a bridge towards translational research. By examiningthe combined interactions of these vitamins, doctors may one day beable to prescribe a more detailed and analytical supplemental regimenfor diabetics based upon their various anti-diabetic medications.
MethodsCHO and 3T3-L1 cells were treated with different treatments (see
table 1) for 24 hours. Protein lysates were quantified and equal pro-tein loads resolved through SDS-PAGE. Proteins were transferred to aPVDF membrane, and immunoblotted for PPARγ and β-actin.
For immunofluorescent staining, the treated cells were fixed with3 percent paraformaldehyde.The cells were then blocked and perme-abilized. Primary antibodies were utilized in a 1:25 dilution, and cysecondary antibodies were utilized for visualization. Bright light mi-croscopy was taken with 40X objective, while fluorescent pictures weretaken under oil immersion with a 100X objective.
Concentration Curve
Figure 1: Cells are elongated with distinct nuclei after treat-ment, indicating survival. Protein changes are not caused bydeath of the cells. N=5.
Densitometric Analysis
Figure 2: Densitometric analysis carried out through imagejsoftware provided by NIH approximates protein quantifica-tion within the PVDF membranes. N=2.
Western Blot
Figure 3: 3T3-L1 cells were treated as labeled for 24 hours.β-actin indicates equal protein loads. N=1.
Immunofluorescence
Figure 4: Only the positive control causes translocation ofPPARγ out of the nucleus and into the cytosol. All othertreatments fail to produce this result. Thus, any proteinchanges that occur through immunoblot are not caused bymovement of PPARγ, but expression alone. N=3.
Results
Treatments IF Western Blot+ Control + 14- Control - —µM D3 - 29nM D3 - 88µM RA - 111nM RA - 64µM Combined - 48nM Combined - 105
Table 1: + indicates PPARγ translocation, while - indicates localiza-tion to nucleus. Western blot data represented as percent changes inprotein expressions as compared to the negative control
Conclusion* Treatment of CHO cells with vitamin A and vitamin D metabolitesfails to translocate PPARγ from the nucleus
* CHO and 3T3-L1 cells maintained an elongated form with distinctnuclei upon all treatments, indicating no cellular death
*Western blot data with densitometric analysis indicates thestrongest downregulation of PPARγ with µM D3 treatment,followed by treatment with µM combined.
* Combined micromolar treatments of RA and D3 downregulatePPARγ, while nanomolar combined treatments of RA and D3upregulate PPARγ
*Human physiological nanomolar concentrations ofmixed Vitamin A and Vitamin D may help decreaseinsulin resistance and serve to help diabetics byupregulating PPARγ
* Future work will concentration on 1. Differentiation ofpre-adipocytes into adipocytes 2. Oil Red O staining for lipidaccumulation visualization 3. Aiming results towards concretemedical recommendations for diabetics and 4. Confirming datathrough trials and statistical verification
References1. Desvergne B, Wahli W. Peroxisome proliferator-activated receptors: nuclear control of metabolism.Endocr Rev 1999;20:649-882. Diradourian C, Girard J, Pegorier JP. Phosphorylation of PPARS: from molecular characterizationto physiological relevance. Biochimie 2005;87:33-83. Kong J, Li YC. Molecular mechanism of 1,25-dihydroxyvitamin D3 inhibition of adipogenesis in3T3-L1 cells. Am J Physiol Endocrinol Metab 2006:290:E916-9244. Ishida, Y. (1988). Possible involvement of 1,25-dihydroxyvitamine d3 in proliferation and differen-tiation of 3t3-l1 cells. Biochemical and Biophysical Research Communications,151(3), 1122-1127.5. Yoshifumi. (1998). Counteraction of retinoic acid and 1,25-dihydroxyvitamin d3 on up-regulationof adipocyte differentiation with ppar ligand, an antidiabetic thiazolidinedione, in 3t3-l1 cells. Phar-macology Letters, 62(14), 205-211.
AcknowledgementsThank you Florida Southern College for providing the funding and laboratory spacethat made this project possible. Dr. Jun Liu from Mayo Clinic for the 3T3-L1 Cells,Dr. Emily Bradshaw for material acquisition and cell culture maintenance along withmuch needed guidance and direction, and fellow researchers and colleagues for theirsupport.
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