glucose sensing in the intestinal epithelium

Glucose sensing in the intestinal epithelium

Jane Dyer1,*, Steven Vayro1,*, Timothy P. King2 and Soraya P. Shirazi-Beechey1

1Epithelial Function and Development Group, Department of Veterinary Preclinical Sciences, University of Liverpool, England, UK;2Rowett Research Institute, Aberdeen, Scotland, UK

Dietary sugars regulate expression of the intestinal Na+/glucose cotransporter, SGLT1, inmany species.Using sheepintestine as a model, we showed that lumenal monosaccha-rides, both metabolisable and nonmetabolisable, regulateSGLT1 expression. This regulation occurs not only at thelevel of transcription, but also at the post-transcriptionallevel. Introduction of D-glucose and some D-glucose ana-logues into ruminant sheep intestine resulted in >50-foldenhancement of SGLT1 expression.We aimed to determineif transport of sugar into the enterocytes is required forSGLT1 induction, and delineate the signal-transductionpathways involved.Amembrane impermeable D-glucose analogue, di(glucos-

6-yl)poly(ethylene glycol) 600, was synthesized and infusedinto the intestines of ruminant sheep. SGLT1 expressionwasdetermined using transport studies, Northern and Westernblotting, and immunohistochemistry. An intestinal cell line,STC-1, was used to investigate the signalling pathways.Intestinal infusion with di(glucos-6-yl)poly(ethylene gly-

col) 600 led to induction of functional SGLT1, but the

compound did not inhibit Na+/glucose transport intointestinal brush-border membrane vesicles. Studies usingcells showed that increased medium glucose up-regulatedSGLT1 abundance and SGLT1 promoter activity, andincreased intracellular cAMP levels. Glucose-induced acti-vation of theSGLT1 promoter wasmimicked by the proteinkinase A (PKA) agonist, 8Br-cAMP, and was inhibitedby H-89, a PKA inhibitor. Pertussis toxin, a G-protein(Gi)-specific inhibitor, enhanced SGLT1 protein abundanceto levels observed in response to glucose or 8Br-cAMP.We conclude that lumenal glucose is sensed by a glucose

sensor, distinct fromSGLT1, residing on the external face ofthe lumenal membrane. The glucose sensor initiates a sig-nalling pathway, involving a G-protein-coupled receptorlinked to a cAMP–PKA pathway resulting in enhancementof SGLT1 expression.

Keywords: intestine; Na+/glucose cotransport; nutrienttransport; sugar sensing.

The dietary monosaccharides, D-glucose and D-galactose,are transported across the brush-border membrane ofintestinal absorptive cells (enterocytes) by the Na+/glucosecotransporter, SGLT1. It has been demonstrated thatlumenal glucose enhances the number of functional SGLT1molecules in the intestinal brush-border membrane, andthat the metabolism of glucose is not required for theinduction [1–5].We have used sheep intestine, which is an excellent model

system, for the study of monosaccharide regulation ofintestinal sugar transport [3,6]. We have shown that dietarymonosaccharides regulate the expression of intestinal brush-border membrane Na+/glucose cotransporter at both thetranscriptional and post-transcriptional levels [3,7,8].

In preruminant lambs (birth to 3 weeks), milk sugar lactoseis hydrolysed by the intestinal lactase into D-glucose andD-galactose, and these sugars are transported by SGLT1.Lambs are normally weaned at 3–10 weeks of age and, asthe diet changes from milk to grass, the rumen develops.Dietary carbohydrates are fermented by rumen microflorato short chain fatty acids, and under these conditionsnegligible levels of monosaccharides reach the small intes-tine [9,10]. Associated with the decline in lumenal sugars,there is a decrease of over 50-fold in the levels of SGLT1protein and mRNA [8]. Introduction of either D-glucoseor nonmetabolisable analogues of D-glucose, via duodenalcannulae, into the intestinal lumenal contents of ruminantsheep enhances the levels of functional SGLT1 protein andmRNA to those detected in the preruminant state [4,8,11].Intestinal infusions of D-glucose induced SGLT1 expressionin the brush-border membrane of enterocytes just below thecrypt–villus junction, with SGLT1 expression spreading tothe villus tip, with cell migration along the crypt–villus axis[4,12]. We cloned and characterized the ovine SGLT1promoter [13], and using intestinal STC-1 cells as a suitablein vitro model [8], we identified (a) the basal SGLT1promoter, (b) a glucose-responsive element within thepromoter, and (c) a sugar-induced transcription factorinvolved in the transcriptional regulation of SGLT1 [8].In this study, we set out to assess if the transport of sugar

across the brush-border membrane into the enterocyte isrequired for enhancement in the expression of intestinal

Correspondence to S. P. Shirazi-Beechey, Epithelial Function and

Development Group, Department of Veterinary Preclinical Sciences,

University of Liverpool, Brownlow Hill, Liverpool L69 7ZJ, UK.

Fax: +44 (0) 151 794 4244, Tel.: +44 (0) 151 794 4255,

E-mail: [email protected]

Abbreviations: GPCR, G-protein coupled receptor; H-89, N-[2-

(p-bromocinnamylamino)-ethyl]-5-isoquinolinesulfonamide; PKA,

protein kinase A; SGLT1, Na+/glucose cotransporter; BBMV,

brush-border membrane vesicle; PEG, poly(ethylene glycol).

*Note: These two authors contributed equally to this work.

Note: A web site is available at http://www.liv.ac.uk/efdg

(Received 11 April 2003, accepted 16 June 2003)

Eur. J. Biochem. 270, 3377–3388 (2003) � FEBS 2003 doi:10.1046/j.1432-1033.2003.03721.x

SGLT1. To this end, we synthesized a membrane imper-meable glucose analogue, di(glucos-6-yl)poly(ethylene gly-col) 600 [di(glucos-6-yl)PEG600]. Introduction of thiscompound into the lumenal content of the ruminant sheepled to an increase in the expression of intestinalSGLT1. This glucose analogue did not, however, inhibitNa+-dependent glucose transport activity into ovine brush-border membrane vesicles. We conclude that the monosac-charide in the lumen of the intestine is sensed by a sugarsensor, which is located on the lumenal surface of theintestinal epithelial cell membrane, and is distinct fromSGLT1. To delineate the signal-transduction pathway bywhich the glucose sensor might operate, we investigated therole of some modulators of cAMP levels in induction ofSGLT1 protein expression and SGLT1 promoter activity.Using STC-1 cells, we report that 8-bromo-cAMP

(8Br-cAMP), a protein kinase A (PKA) agonist, mimi-cked the glucose-induced activation of the SGLT1promoter. 8Br-cAMP also increased the levels of SGLT1expressed endogenously in the cell line. The glucose-induced SGLT1 promoter activity was inhibited, in adose-dependent manner, by the PKA antagonist H-89.There was a 47% increase in the level of intracellularcAMP when cells were exposed to increased mediumD-glucose concentration; this paralleled the enhancementin SGLT1 abundance. The potential role of G-proteins inthe pathway was investigated. Addition of pertussis toxin,a G-protein (Gi)-specific inhibitor, to the intestinal cellline grown in low-glucose conditions enhanced theSGLT1 abundance to that observed with high-glucoseor with 8Br-cAMP.We propose that the intestinal epithelial cells have a

glucose sensor that resides on the external face of thelumenal membrane. Glucose binds to the sensor andgenerates an intracellular signal leading to enhancement ofthe expression of SGLT1. It is evident that the generatedsignal is independent of glucose metabolism and appears towork via a G-protein-coupled receptor and cAMP/PKAsignalling cascade.

Materials and methods

Synthesis and characterization of di(glucos-6-yl)PEG600

Di(glucos-6-yl)PEG600 was synthesized by the route shownin Fig. 1.

Synthesis of dibromoPEG. Triphenylphosphine, finalconcentration 2 M, was added to a magnetically stirredsolution of 0.67 M PEG600 and 1.67 M tetrabromometh-ane in 15 mL dry dichloromethane at 40 �C. Thereaction mixture was refluxed, in the dark, for 4 days,by which time the reaction was complete, as indicated byTLC using fluorescent aluminium-backed silica plates(Merck type 5556) using methyl ethyl ketone/methanol/water/ 27% (w/w) concentrated ammonia (65 : 20 : 5 :10, v/v/v/v) as irrigant. TLC plates were developedinitially using iodine vapour and, after evaporation of theiodine, visualization was with methyl red spray, whichgave a bright red colour with the product [14]. Themixture was filtered and the filtrate washed three timeswith deionized water (15 mL) to remove triphenylphos-

phine oxide. The organic layer was concentrated and theresidue swirled with 20 mL deionized water for 6 h. Themixture was filtered, the residue washed with deionizedwater (10 mL), and the aqueous portions freeze-dried,redissolved in 5 mL water, and separated by gel filtrationon a Sephadex G15 gel column (75 · 2 cm) and elutedwith deionized water. An initial 25 mL was collected, andthen aliquots of 5 mL were taken. The halogenated PEG(Fig. 1) was isolated from fractions 1–5 as a clear,viscous, oil, which, when freeze-dried, gave an azure bluefollowed by a green colour in a flame test [15].

Reaction of methyl-a,D-glucopyranoside with halogenatedPEG in aqueous KOH. Methyl-a,D-glucopyranoside wasadded to amixture of 2 MKOH in 2 mL dimethyl sulfoxideto a final concentration of 1 M, followed immediately byan equimolar amount of the halogenated PEG derivative(Fig. 1). The reaction mixture was stirred in the dark for� 4 days until there was no remaining starting material, asassessed by TLC using a p-anisaldehyde spray. The sugarderivatives gave blue spots on a pink background. Thereaction mixture was then purified by gel filtration, asdescribed above, and the pure product was isolated fromfractions 1–3.Oxidation of the product with periodate indicated the

presence of two 6-O-glucosyl units per unit of PEG. Infraredspectroscopy and 1H NMR confirmed the presence of amethylglucose unit on either end of the PEG600 backbone.

Hydrolysis of di(methylglucos-6-yl)PEG600 using H2 SO4.To remove the methyl groups di(methylglucos-6-yl) PEG600

was dissolved in 10 mL 0.5 M H2SO4 to a concentration of0.5 mM and heated under reflux to produce the targetcompound. At all stages of the synthesis, reaction productswere analysed by TLC, infrared spectroscopy, and 1HNMR. The di(glucos-6-yl)PEG600 was tested for anypotential free glucose using a commercial glucose testingkit (Boehringer-Mannheim).

Fig. 1. Synthesis of di(glucos-6-yl)PEG600. D-Glucose was linked by

ether bonds to PEG600 by the synthesis pathway outlined.

3378 J. Dyer et al. (Eur. J. Biochem. 270) � FEBS 2003

Biological stability of di(glucos-6-yl)PEG600. To deter-mine if di(glucos-6-yl)PEG600 was stable, and resistant tohydrolysis when introduced into the intestinal contents,the following experiments were undertaken. Di(glucos-6-yl)PEG600 was mixed with 10 mL ovine intestinal digesta toa final concentration of 30 mM and incubated at 39 �C(sheep body temperature). Samples were removed atintervals of time up to 24 h and assayed for free glucoseusing a commercial kit (Boehringer-Mannheim), accordingto the manufacturer’s instructions. In addition, ovineintestinal crude cellular homogenate or purified brush-border membrane vesicles (1 mg protein) were incubated at39 �C in 0.1 mL of a solution containing 300 mMmannitol,20 mM Hepes/Tris, pH 7.4, 0.1 mM MgSO4 and 30 mM

di(glucos-6-yl)PEG600. Samples (10 lL) were removed at1 h intervals for up to 8 h and assayed for glucose using aglucose assay kit (Boehringer-Mannheim) as above.

Animals and intestinal infusions

Scottish Blackface ewes, all >1-year-old, were used.Animals were maintained on a conventional roughage diet,and fed grass pellets (1 kg a day) throughout the experi-ment, as described previously [6]. Perspex T-shaped cannu-lae were fitted into the duodenum 6 cm distal to the pylorus[6]. Animals were infused, through the duodenal cannulae,for 3 h with 30 mM solutions of D-glucose, PEG600, ordi(glucos-6-yl)PEG600 at a rate of 62.5 mLÆh

)1, as described[6,12]. They were killed with sodium pentobarbitone(Euthatal) [4,12], and sections of intestine were removed,flushed with ice-cold 0.9% (w/v) NaCl, and everted.Intestinal sections were rinsed clean, blotted with papertowels to remove mucous, and then wrapped in aluminiumfoil before immediate freezing in liquid nitrogen. Additionalsamples were frozen in isopentane cooled in liquid nitrogenfor immunohistochemical studies. Tissue was subsequentlystored at )80 �C until use.All procedures were carried out under an approved UK

Home Office project licence.

Cell culture

Intestinal cells, STC-1 [8,16] (passages 40–90) were grown inDulbeccos’ modified Eagle’s medium (Invitrogen) supple-mented with 10% (v/v) fetal bovine serum or 10% (v/v)dialysed fetal bovine serum (containing < 200 lMD-glucose), 50 UÆmL)1 antibiotic solution containing peni-cillin and streptomycin, and either 25 mM (high) or 5 mM

(low) D-glucose, as described [8]. Cells weremaintained at alltimes at 37 �C in 5% CO2. Stock cultures were grown in75-cm2 flasks (Corning, High Wycombe, Bucks, UK) andwere fed every 3–4 days. Subsequently cells were washedtwice with 5 mL Hanks’ balanced salt solution and thentrypsinized (1 min at 37 �C, 5% CO2) in 1 mL solutioncontaining Versene 1 : 5000 (Invitrogen) and 0.25% (w/v)trypsin (> 225 UÆmg)1; Invitrogen). Culture medium(10 mL) was added and the cells dispersed using a syringefitted with a Venflon 2 (Southern Syringe Services, Man-chester, UK). The cells were seeded into 12-well plates(22 mm; Corning) containing 2 mL of the medium at adensity of � 0.5 · 106 cells, and returned to 37 �C, untilthey were 60–70% confluent.

Preparation of brush-border membrane vesicles (BBMVs)

Brush-border membrane vesicles (BBMVs) were preparedfrom frozen intestinal sections using a combination ofcation precipitation and differential centrifugation as des-cribed previously [17]. The final purified BBMVs weresuspended in buffer containing 300 mM mannitol, 20 mM

Hepes/Tris, pH 7.4, and 0.1 mM MgSO4, and stored inliquid nitrogen until use.The protein concentration in the BBMVs was estimated

by its ability to bind Coomassie blue according to the Bio-Rad assay technique. Bovine c-globulin was used as thestandard [18]. The plasma membrane origin of the BBMVswas assessed by determination of the enrichment of theactivity and the abundance of the marker proteins of thebrush-border membrane. BBMV purity was determined byassessing the levels of marker proteins characteristic ofbasolateral and organelle membranes [6,19].

Measurement of monosaccharide transport activity

To assess the activity of SGLT1, the initial rate of 0.1 mM

D-glucose transport in BBMVs was measured at 39 �C inthe presence of NaSCN and KSCN, using the rapidfiltration stop technique, as described before [17,19]. Allinitial rate measurements were taken after a 3 s incubationperiod, as transport was determined to be linear up to 4 s[17]. Uptakes were measured in duplicate or triplicate.To assess the activity of any potential facilitative glucose

transporter, the initial rate of uptake of 1 mM 2-deoxy-D-glucopyranoside, a specific substrate of Na+-independentD-glucose transporter isoforms, was determined at 39 �C inincubation medium consisting of 300 mM mannitol, 20 mM

Hepes/Tris, pH 7.4, 0.1 mM MgSO4, and 0.02% (w/v)NaN3 in the presence and absence of 50 lM cytochalasin B,as described [20].Competition studies were carried out by determining the

initial rate of uptake of 0.1 mMD-glucose in the presence of1 mM competitor, using a standard technique as describedpreviously [21].

Immunodetection of SGLT1

Quantitative Western blotting. The abundance of SGLT1protein was measured by quantitative Western blotting asdescribed previously [22]. The BBMV protein contents wereseparated on an 8% polyacrylamide gel containing 0.1%(w/v) SDS and were electrotransferred to nitrocellulosemembrane (TransBlot; Bio-Rad).A standard calibration curve was constructed by slot-

blotting the synthetic peptide (amino acids 402–420 of theovine SGLT1 sequence, to which the antibody was raised)on to nitrocellulose membrane, and this was probedconcurrently with the BBMV samples. The specific immu-noreactive band was blocked when antibodies were pre-incubated with the immunizing peptide. The membraneswere developed using the ECL system (Amersham-Pharmacia, Little Chalfont, Bucks., UK), and exposed tofilm (XOMAT-LS; Kodak).The intensity of the immunoreactive bands detected in the

BBMVs and the peptide standard samples was quantifiedusing scanning densitometry (Phoretix 1D; Non-linear

� FEBS 2003 Intestinal glucose sensing (Eur. J. Biochem. 270) 3379

DynamicsLtd,Newcastle uponTyne,Tyne andWear,UK),and the abundance of SGLT1 protein per mg of BBMVprotein was calculated from the peptide standard curve.Immunodetection of SGLT1 in STC-1 cells was carried

out as described previously [8]. Cells were washed with ice-cold NaCl/Pi and then lysed in 300 lL buffer containing150 mM NaCl, 1% (w/v) SDS, 10 mM EDTA and 10 mM

Hepes/Tris, pH 7.4, with protease inhibitor cocktail (Boeh-ringer-Mannheim) and 0.2 mM phenylmethanesulfonylfluoride. Cells were scraped from the dish with a �rubberpoliceman� and homogenized by 10 passages through asyringe fitted with a 21-gauge needle. Protein (15 lg perlane) was separated on 8% (w/v) polyacrylamide gelscontaining 0.1% (w/v) SDS. After electrotransfer topoly(vinylidene difluoride) (0.2 lm), the membrane wasblocked for 30 min in buffer containing 150 mM NaCl,10 mMTris/HCl, pH 7.4, 0.05% (v/v) Tween 20, 0.5% (w/v)skimmed milk powder. Primary and secondary antibodyincubations were 1 h at room temperature; subsequentlymembranes were washed three times with buffer for 10 min.Detection and quantification were as described above.

Immunohistochemistry. Tissue sections (5 lm thick) werecut using a cryostat at)20 �C, air-dried on to gelatin-coatedmicroscope slides, and fixed in methanol for 15 min at)15 �C. Sections were washed in NaCl/Pi/Tween (10 mM

phosphate buffer, 150 mMNaCl, 0.05% Tween 20, pH 7.4)and incubated for 30 min at 37 �C in NaCl/Pi SuperBlock(Pierce and Warriner, Chester, UK). Sections were washedin six changes of NaCl/Pi/Tween over 20 min at roomtemperature and incubated for 60 min at 37 �C in5 lgÆmL)1 SGLT1 antiserum in NaCl/Pi/Tween containing0.1% acetylated BSA (Aurion, Wageningen, the Nether-lands). Sections were washed in six changes of NaCl/Pi/Tween over 20 min and incubated for 30 min at 37 �C witha mouse monoclonal anti-rabbit IgG (clone RG-96; Sigma)at a dilution of 1 : 400 in NaCl/Pi/Tween containing 0.1%acetylated BSA. Sections were washed in six changes ofNaCl/Pi/Tween over 20 min and incubated for 30 min at37 �C in 5 lgÆmL)1 Oregon Green goat anti-mouse IgG(Molecular Probes, Cambridge Bioscience, Cambridge,UK) in NaCl/Pi/Tween containing 0.1% acetylated BSA.Sections were washed in six changes of NaCl/Pi/Tween, andthen mounted in Vectorshield antifading mountant (VectorLaboratories) before examination by incident light fluores-cence microscopy on a Zeiss Axioscope microscope.Control sections were subjected to the same protocol exceptthat 0.1 lgÆmL)1 peptide (amino acids 402–420 of the ovineSGLT1) was added to the SGLT1 antibody, and thepeptide/antibody mixture was preincubated for 60 min at37 �C before use.

Immunodetection of G-proteins

The presence of G-proteins (Ga subunits) on the intestinalbrush-border membrane of preruminant lambs, ruminantsheep and age-matched glucose-infused ruminant sheep, aswell as in STC-1 cells, was determined by Westernblotting with a broad-range affinity-purified rabbit Ga

polyclonal antibody raised against the conserved GTP-binding domain (Sigma) at 1 : 1000 dilution, as describedabove for SGLT1.

Analysis of SGLT1 promoter function

The ovine SGLT1 promoter fragment used in these studieswas generated, and assayed, as described previously [8,13].STC-1 cells were seeded into 12-well plates (0.5 · 106 cells)containing 1 mL medium, and incubated for 24 h at 37 �C,5% CO2. Cells were then transiently transfected using thecationic lipid reagent Transfast (Promega) at a DNA/lipidratio of 1 : 1. A second plasmid, pRL-SV40 (Promega), wascotransfected (0.019 pmol) as an internal control. The cellswere incubated for 1 h at 37 �C, 5% CO2, and then 1 mLcomplete medium was added. After a further 48 h, the cellswere recovered and assayed for luciferase activity using theDual-Luciferase Reporter assay system (Promega) on aLumat LB9501 luminometer (Perkin–Elmer). Values arepresented as a ratio of the firefly luciferase to Renillaluciferase activity.

Intracellular cAMP determination

The cAMP levels in cellular homogenates were measuredusing a commercially available RIA kit (Amersham-Phar-macia), according to themanufacturer’s instructions. STC-1cells (1 · 106 per well) were harvested and homogenized,using a Polytron at setting 5 for 30 s, in 0.4 mL buffercontaining 100 mM mannitol, 2 mM Hepes/Tris, pH 7.4,5 mM EDTA, 0.2 mM phenylmethanesulfonyl fluoride,protease inhibitor cocktail (Boehringer-Mannheim) and1 mM 3-isobutyl-1-methylxanthine, a phosphodiesteraseinhibitor. The homogenates were then deproteinated byheating in a boiling water bath for 10 min, followed bycentrifugation at 15 000 g for 20 min at 4 �C to sedimentdenatured proteins. Supernatants were transferred to fresh1.5 mL tubes, placed on ice, and assayed for cAMPfollowing the instructions of the manufacturer. All proce-dures were carried out at 4 �C.

Statistical analysis

Data are expressed as mean ± SEM. Statistical compari-sons are made using Student’s t test, and results areconsidered significant if P<0.05.

Results

Synthesis of di(glucos-6-yl)PEG600

In this molecule we chose to link the glucose and PEG byether bonds, which made it improbable that enzymatichydrolysis would occur in vivo. On the basis of our previousinvestigations on the stereoselectivity of the glucose sensor[4,12], we chose the ether linkage to O6 of glucose; being aprimary alcohol, the 6-OH is the most reactive once theanomeric position is blocked.Bromination of primary alcohols using tetrabromometh-

ane/triphenylphosphine usually gives good yields of alkylbromides [23], but with PEG600 as the alcohol, brominationwas accompanied by chlorination. However, the dihalogen-ated products were all eluted in the same fraction from aSephadex G-15 column, and the mixture reacted fully withmethyl-a,D-glucopyranoside in dimethyl sulfoxide to givegood yields of the glucoside, provided that KOH was used


as base. The presence of two 6-O-glucosyl units per unit ofPEG was demonstrated by oxidation with periodate;� 4 mol was consumed per mol di(methylglucosyl)PEG600,with production of formic acid. Hydrolysis of the glucosidewith 0.5 M H2SO4 was used to remove the methyl groups inquantitative yield, giving the target compound (Fig. 1).

Stability of di(glucos-6-yl)PEG600

The synthesized di(glucos-6-yl)PEG600 was assayed for anypotential free glucose using a commercial kit (Boehringer-Mannheim). The results indicated total absence of freeglucose, and this was confirmed by TLC. To determinebiological stability, di(glucos-6-yl)PEG600 (30 mM) wasincubated with ovine intestinal digesta (10 mL) at 39 �C for24 h, and fractions were removed periodically and assayedfor free glucose. Glucose was not detected in the samples upto8 h, indicating that the compoundwasnothydrolysedandwould remain intact over the infusion period. Similarlyglucose was not detected when di(glucos-6-yl)PEG600 wasincubated with either the ovine intestinal mucosal homogen-ate or purified BBMVs. Results indicate that free glucosewouldnotbepresentduringthe infusionperiod,orduring thepassage of the compound through the small intestine. Thiseliminated the possibility that the induction of SGLT1expression may be due to the glucose released in the smallintestine as a result of the breakdown of the compound.

Induction of SGLT1 by intestinal infusion

Western blot analysis. The protein component of BBMVsisolated from the jejunum of control ruminant sheep andruminant sheep the intestines of which were infused withPEG600, D-glucose or di(glucos-6-yl)PEG600 were separatedby SDS/PAGE and electrotransferred to nitrocellulose.Samples were immunoblotted to determine the presence ofSGLT1 protein using an affinity-purified polyclonal peptideantibody, as described previously [22]. The results arepresented in Fig. 2. The antibody recognizes a single proteinwith an apparent molecular mass of 75 kDa in the BBMVsisolated from the D-glucose and di(glucos-6-yl)PEG600-infused animals, but not the PEG600-infused animals orcontrols, indicating that the presence of D-glucose or thenontransportable, membrane-impermeable, di(glucos-6-yl)-PEG600 in the lumen of the intestine induces expression ofSGLT1. The abundance of SGLT1 protein in the BBMVsisolated from the intestine of sheep infused with D-glucoseor with di(glucos-6-yl)PEG600 (Fig. 2) are 12.6 ± 1.3 and13.1 ± 1.2 pmolÆ(mg protein))1, respectively.

Transport of D-glucose. To determine if the sugar-inducedSGLT1 is capable of transport, the ability of the BBMVs totransport glucose in aNa+-dependentmannerwas assessed.The initial rates of 0.1 mM D-glucose uptake into BBMVsisolated from the intestine of control and infused ruminantsheep are presented in Fig. 3. The initial rates of Na+-dependent D-glucose transport were 108.8 ± 10.5 and111 ± 6.4 pmolÆs)1Æ(mg protein))1 in BBMVs isolatedfrom the jejuna for D-glucose and di(glucos-6-yl)PEG600,respectively. The initial rate of uptake in vesicles isolatedfrom PEG600-infused sheep was 3.6 ± 1.2 pmolÆs)1Æ(mgprotein))1, a rate identical with that measured in adult

ruminant control BBMVs [11,22]. The results indicate thatthe SGLT1 protein that is expressed in response to lumenalinfusion is functional. There was no cytochalasin B-sensitive2-deoxy-D-glucose transport detected in any of the BBMVsamples (data not shown), indicating the absence ofGLUT2from the BBMVs and therefore any basolateral membranecontamination.To investigate any interaction between the di(glucos-6-

yl)PEG600 and SGLT1 function, the ability of this com-pound to inhibit Na+-dependent D-glucose transport intoBBMVs was investigated. Concurrently the effect onSGLT1 activity of other glucose analogues was alsodetermined. The results are presented in Fig. 4. The initialrate of uptake of 0.1 mM D-glucose into lamb jejunalBBMVs was reduced in the presence of 1 mM concentra-tions of inducers of SGLT1 expression such as D-glucose,

Fig. 2. Abundance of SGLT1 protein in the intestinal brush-border

membrane of ruminant sheep after intestinal infusion with various solutes.

Ruminant sheep (3 years old) had their intestines infused with 30 mM

solutions of PEG600, D-glucose, or di(glucos-6-yl)PEG600 through

duodenal cannulae. BBMVs were prepared from the intestine of these

animals, and brush-border membrane proteins (20 lg per lane) wereseparated on 8%polyacrylamide gels containing 0.1%SDS. Separated

proteins were electrotransferred to nitrocellulose membranes and

blotted for the presence of SGLT1, as described previously [8,22]. The

abundance of SGLT1 protein in the brush-border membrane samples

was quantified using the peptide antigen as a standard [22]. N.D., Not

detected.

Fig. 3. Initial rate of Na+-dependent glucose uptake in ovine intestine

BBMVs after intestinal infusion. The initial (3 s) rate of 0.1 mM

D-glucose uptake into BBMVs (0.1 mg protein) wasmeasured at 39 �Cin the presence of 100 mM NaSCN, as described in Materials and

methods. Results are presented as mean ± SEM (n ¼ 3).


D-galactose (natural SGLT1 substrates), a-methylglucose or3-O-methylglucose (nonmetabolisable SGLT1 substrates).However, D-glucose uptakewas unchangedwhen D-fructoseor di(glucos-6-yl)PEG600 (not transported by SGLT1 butinduce SGLT1 expression), L-glucose or PEG600 (nottransported by SGLT1 and do not induce SGLT1 expres-sion) were included in the incubation medium. The resultssuggest that there is no interaction between di(glucos-6-yl)PEG600 and SGLT1 protein that affects SGLT1function.

Immunofluorescence localization of SGLT1. The distribu-tion of SGLT1 protein along the crypt–villus axes of theintestine of the infused ruminant sheep was also determinedby immunohistochemistry. Typical results, presented inFig. 5, indicate that infusion of the intestine of ruminantsheep with either D-glucose or di(glucos-6-yl)PEG600 resultsin induction of SGLT1 protein expression. Infusion of theintestine with PEG600 has no effect (Fig. 5A). Immuno-fluorescence localization of SGLT1 protein along the crypt–villus axes of the ruminant sheep, with the intestinal infusionof either di(glucos-6-yl)PEG600 or D-glucose, shows labellingon theentirebrush-border surface, including the lower regionof the villus (Fig. 5B,C). The distribution of SGLT1 proteinin infusedruminantsheep(Fig. 5B,C) is similar tothatseen inthe intestineof thepreruminant lamb(Fig. 5D).The labellingis specific, as it was blocked when the primary antibody waspreincubated with the peptide antigen (Fig. 5E).

Effect of cAMP and PKA on ovine SGLT1 promoteractivity and SGLT1 expression

We have demonstrated that di(glucos-6-yl)PEG600, a mem-brane-impermeable glucose analogue, enhances the level ofSGLT1. Furthermore, we have determined that SGLT1function is not inhibited in the presence of D-fructose,2-deoxy-D-glucose and di(glucos-6-yl)PEG600, compoundsthat induce the expression of functional SGLT1. Weconclude therefore that a glucose sensor, with differentsugar specificity from SGLT1, is located on the external faceof the intestinal brush-border membrane. The sensor woulddetect changes in the lumenal sugar concentration andinitiate signalling pathways, leading to modulations in theexpression of functional SGLT1. Using the intestinal cell

line, STC-1, as an in vitro expression system [8], we assessedthe potential role of cAMP/PKA in the transcriptionalregulation of the )66/+21-bp SGLT1 glucose-responsivepromoter fragment [8]. To this end, we used (a) 8Br-cAMP,a membrane-permeable cAMP analogue and a PKAagonist, and (b) H-89, a PKA antagonist.Cells were cultured in medium containing 5 mM glucose,

transfected with the ovine SGLT1 promoter fragment, andthen eithermaintained in the samemedium or transferred toone containing 25 mM glucose, in the presence or absence of(a) 0.5 mM 8Br-cAMP or (b) H-89. The results are shown inFig. 6 and Fig. 7, respectively. Figure 6 shows that SGLT1promoter activity increased twofold, after the addition of25 mM glucose, in agreement with our previous data [8].Cells maintained in low-glucose (5 mM) medium, buttreated with 0.5 mM 8Br-cAMP, also showed a significantincrease in promoter activity compared with controls. 8Br-cAMP also augmented the increase in promoter activityobserved in response to high-glucose medium by a further30%. When the reporter gene construct was placed in thereverse orientation, neither glucose nor 8Br-cAMP had anyeffect. We conclude that an increase in intracellular cAMPresults in the activation of SGLT1 promoter function.Glucose-induced SGLT1 promoter activity was inhibited,

in a dose-dependent manner, in response to increasingconcentrations of H-89 (0.1, 0.5, 1.0 lM; Fig. 7). In cellsswitched to 25 mM glucose, in the presence of 1 lM H-89,SGLT1 promoter activity was reduced to the level detectedin cells maintained in 5 mM glucose. Promoter function wasnot inhibited by H-89 in cells maintained in low glucose, orin cells transfected with the reporter gene construct in thereverse orientation (not shown). Therefore, the inhibitoryaction of H-89 appears to be specific to the glucose-inducedSGLT1 promoter activity. These data suggest that PKA hasan important role in the transcriptional activation of theovine SGLT1 promoter.We also examined the effects of both 8Br-cAMP and

H-89 on the level of endogenous SGLT1 protein expressedin the STC-1 cells (Fig. 8A,B). Cells transferred from 5 mM

to 25 mM glucose showed a (2.88 ± 0.22)-fold increase inthe abundance of SGLT1 (Fig. 8A, lanes 1 and 3, andFig. 8B, lanes 1 and 2), consistent with our previous findings[8]. In cells cultured in 5 mM glucose in the presence of0.5 mM 8Br-cAMP there was a (4.49 ± 0.56)-fold enhance-ment in SGLT1 abundance, compared with controls(Fig. 8A, lanes 1 and 2). Cells switched to mediumcontaining 25 mM glucose and 8Br-cAMP showed a further(1.81 ± 0.71)-fold enhancement in SGLT1 protein abun-dance (Fig. 8A, lane 4). Cells maintained throughout in25 mM glucose medium did not respond to 8Br-cAMP (notshown). 8-Br-cAMP had no effect on the levels of b-actin(Fig. 8A). Treatment of STC-1 cells with 0.1 lM H-89resulted in 34.1 ± 3.3% reduction in glucose-inducedSGLT1 protein abundance (Fig. 8B, lane 3), and increasingthe concentration of H-89 to 1 lM had no further effect(Fig. 8B, lanes 4 and 5), suggesting a role for PKA in theregulatory process.Interestingly, cAMP concentrations measured in depro-

teinated homogenates of cells cultured in 5 mM D-glucosewere 47% lower than cAMP levels detected in cellstransferred to 25 mM glucose [0.19 ± 0.04 vs. 0.28 ±0.03 pmolÆ(mg protein))1; mean ± SEM, n ¼ 3], implying

Fig. 4. Competition studies. The initial rate of the Na+-dependent

uptake of 0.1 mM D-glucose into preruminant lamb jejunal BBMVs

was measured at 39 �C in the presence of of the indicated competitor

(1 mM). Results are expressed as percentage of control and are

means ± SEM (n ¼ 3).


Fig. 5. Immunofluorescence localization of SGLT1 protein along the crypt–villus axes of ruminant sheep after intestinal infusion with various solutes.

Typical immunofluorescence images are presented showing localization of SGLT1 protein on the jejunal villi of 3-year-old sheep after intestinal

infusion of 30 mM solutions of (A) PEG600, (B) di(glucos-6-yl)PEG600 and (C) D-glucose. SGLT1 localization in preruminant lamb jejunum is also

shown (D), with signals blocked by preincubation of the antibody with the immunizing peptide antigen (E). Labelling over the entire villus (V)

brush-border surface is shown. Scale bar represents 100 lm.


that there is an increase in intracellular cAMP levels inresponse to increasing levels of medium D-glucose.

Involvement of a GPCR in intestinal glucose sensing

Onemechanism for sensing glucose in yeast (Saccharomycescerevisiae) is through the plasma membrane GPRC, Gpr1,which initiates a cAMP signal-transduction cascade cul-minating in the expression of hexose transporter genes [24–29]. To investigate the potential role of a G-protein inglucose sensing, we used the G-protein (Gi)-specific inhi-bitor, pertussis toxin.In STC-1 cells cultured in 5 mM glucose, the abundance

of SGLT1 protein increased in a dose-dependent manner(1.83 ± 0.42-fold, 1.70 ± 0.20-fold and 2.47 ± 0.84-fold)

in response to increasing concentrations of pertussis toxin(100, 250 and 500 ngÆmL)1; Fig. 9 lanes 1, 2, 3 and 4). Incells transferred from 5 mM to 25 mM glucose, SGLT1expression was up-regulated twofold, as expected (lanes 1and 5), and pertussis toxin had no effect on this response(lanes 6, 7 and 8). Pertussis toxin had no effect on the levelsof b-actin.The presence ofG-proteins (Ga subunits) on the intestinal

brush-border membrane of preruminant lambs, ruminantsheep and age-matched glucose-infused ruminant sheep, aswell as in STC-1 cells, was determined by Western blottingwith a broad-range Ga subunit antibody. The abundance ofSGLT1 and b-actin were also determined in the samesamples (Fig. 10A,B). Ga subunits were detected, as animmunoreactive band of � 41 kDa, in the intestinalbrush-border membrane of lambs, ruminant sheep, and

Fig. 6. Effect of 8Br-cAMP on SGLT1 promoter activity. STC-1 cells

were cultured in medium containing 5 mM D-glucose and transfected

with the )66/+21-bp ovine SGLT1 promoter construct, as described

in the Methods section. After transfection, the cells were incubated in

medium containing either 5 mM or 25 mM D-glucose with (j) or

without (h) 0.5 mM 8Br-cAMP, for a further 48 h, before assaying.

Values are the means ± SEM from four determinations.

Fig. 7. Effect of PKA inhibitor, H-89, on SGLT1 promoter activity.

STC-1 cells were treated as described in the legend to Fig. 6. After

transfection with the promoter construct, the cells were incubated in

medium containing either 5 mM or 25 mM D-glucose with increasing

concentrations of H-89 (0.1, 0.5, 1.0 mM), for a further 48 h, before

assay. Values are the means ± SEM from three to seven determina-

tions.

Fig. 8. Effect of 8Br-cAMP andH-89 on the levels of SGLT1 expressed

endogenously in STC-1 cells. STC-1 cells were cultured in 5 mM

D-glucose medium and then exposed to medium containing either

5 mM or 25 mMD-glucose, in the presence of (A) 0.5 mM 8-Br-cAMP,

or (B) increasing concentrations of H-89 (0.1, 0.5 or 1.0 lM) for afurther 48 h. Cell lysates were then prepared for Western blotting.

Equal amounts of protein (15 lg) were loaded per lane. Immuno-

detection of SGLT1 and b-actin were carried out using the SGLT1

antibody (1 : 5000 dilution) and amouse monoclonal b-actin antibody(1 : 10 000 dilution), respectively. Data shown are representative of

three experiments.


age-matched glucose-infused animals, as well as in lysatesfrom STC-1 cells cultured under low or high glucoseconditions. The abundance of SGLT1 protein in BBMVsisolated from the intestinal tissues and the STC-1 cell lysateswas a good representation of the glucose induction ofSGLT1 [22]. b-Actin abundancewas constant in all samples.These observations confirm the presence of Ga subunits inthe ovine intestinal lumenal membrane and support thepotential involvement of a GPCR in the signalling pathwayfor glucose regulation of SGLT1 expression.

Discussion

Glucose is amajor source of energy formost eukaryotic cells,and has significant and varied effects on cell function.Consequently maintenance of glucose homoeostasis is ofgreat importance to many organisms. Interest in identifyingmechanismsbywhich cells sense and respond tovariations inglucose concentration has increased recently, and promisingadvances have been made [30]. It has been shown thatdifferent eukaryotic cells use specificmechanisms to sense thepresence of glucose, and that the physiological roles of thesemechanisms are dependent on the particular cell type [27].

Yeast cells, S. cerevisiae, have a remarkable preferencefor glucose as a carbon source [27] and have evolvedmechanisms for sensing and responding to wildly fluctu-ating levels of extracellular glucose. These mechanismsinvolve a large family of hexose transporters (HXTproteins), and the glucose transporter homologues Snf3and Rgt2. Snf3 and Rgt2 are plasma membrane glucose-sensing proteins with no detectable transport activity. They

Fig. 9. Effect of pertussis toxin on SGLT1 protein abundance. STC-1

cells were treated as described in the legend to Fig. 8. They were then

transferred to medium containing either 5 mM or 25 mMD-glucose, in

the presence of 100, 250 or 500 ngÆmL)1 pertussis toxin for a further

48 h. Cell lysates were then prepared for Western blotting, and equal

amounts of protein (15 lg) were loaded per lane. Immunodetection ofSGLT1 and b-actin were carried out using the SGLT1 antibody

(1 : 5000 dilution) and a mouse monoclonal b-actin antibody

(1 : 10 000 dilution), respectively. Data shown are representative of

three experiments.

Fig. 10. Immunodetection of Ga subunits in ovine BBMV and STC-1

cells. BBMVs were prepared from the jejunal mucosal scrapings of

preruminant lambs, 3-year-old-adult sheep, and age-matched sheep

after glucose infusion. STC-1 cells were cultured in the presence of

medium containing either 5 mM or 25 mMD-glucose for 48 h and then

cell lysates prepared. Equal amounts of protein (15 lg), from (A)

intestinal BBMVs or (B) STC-1 cell lysates were loaded per lane.

Immunodetection of SGLT1 was carried out using the SGLT1 anti-

body (1 : 5000 dilution). Immunodetection of Ga subunits was per-

formed using an affinity-purified rabbit Ga polyclonal antibody raised

against the conserved GTP-binding domain at 1 : 1000 dilution.


sense the extracellular glucose and generate an intracellularglucose signal that triggers the induction of HXT geneexpression [31]. Using Snf3/Rgt2 double mutants it wasshown that, in yeast, glucose-sensing and signalling arereceptor-mediated processes and are independent of glucosemetabolism [31]. In addition, a novel GPCR, Gpr1, whichsenses external medium glucose, has also been identified.Gpr1 acts via theG-protein, Gpa2, to initiate a cAMP/PKAsignalling cascade [25,26,29]. Gpr1 is activated by glucoseand transmits a signal, via Gpa2, to adenylate cyclase.Glucose activation of cAMP synthesis requires active sugarphosphorylation but no further metabolism of sugar. Theglucose-sensing Gpr1–Gpa2 system for activation of thecAMP pathway in S. cerevisiae appears to be the firstexample of a nutrient-sensing GPCR system. If nutrientsensing GPCRs were common to eukaryotic cells, theywould provide a means of regulating major signal-trans-duction pathways by the nutrient status of the cellularenvironment. The latter is supported by a recent reportshowing that aGPCR,GPR40, abundantly expressed in thepancreas, functions as a receptor for long-chain free fattyacids. The latter amplify glucose-stimulated insulin secretionfrom pancreatic b-cells by activating GPR40 [32].Intestinal epithelial cells are exposed from the lumenal

domain to an environment with continuous and massivefluctuations in the level of dietary monosaccharides. This isin contrast with other mammalian cells, which are exposedto a relatively constant blood glucose concentration regu-lated by endocrine hormones. Enterocytes therefore have tosense and respond to the significant fluctuations in lumenalsugars and regulate their function accordingly. Dietarysugars have been shown to regulate the expression of theintestinal lumenal membrane glucose transporter, the Na+/glucose cotransporter (SGLT1), in a wide range of species[1,2,4,5]. Using the sheep intestine as a model system, wehave shown that lumenal sugars regulate the expression ofSGLT1 at both transcriptional and post-transcriptionallevel [3,8]. It was demonstrated, using nuclear run-on assays,that the transcriptional activity of the ovine SGLT1 geneincreased 2–3-fold, in response to lumenal sugar. Thisincrease did not account entirely for the overall enhance-ment in steady-state levels of SGLT1mRNAdetermined byNorthern blot analysis [8,11].Rumen development in sheep is a natural and efficient

way of ensuring a virtual block in the delivery of monosac-charides to the small intestine. Nutrients, such as peptides,amino acids and fats, enter the intestinal lumen, butmonosaccharides are selectively excluded [10]. Associatedwith the decline in the levels of monosaccharides, there is a>50-fold decline in the levels of functional SGLT1 proteinand mRNA [8,11,22]. Introduction of monosaccharides,D-glucose, D-galactose, a-methyl-D-glucose, 3-O-methyl-D-glucose, D-fructose and 2-deoxy-D-glucose into the lumenalcontents of ruminant sheep intestine, through duodenalcannulae, resulted in increased expression of SGLT1 to thelevels detected in the preruminant lamb. We concluded thatinduction of SGLT1 by lumenal sugar is independent ofglucose metabolism and that the inducing sugar need not bea substrate of SGLT1 [4,6,22].To determine if transport of sugar into the enterocyte is

required for SGLT1 induction, we set out to synthesize awater-soluble, metabolically inert, membrane-impermeable

glucose analogue. Our overall objective was to join glucoseto a water-soluble polymer in such a way that the conjugatewould activate the glucose sensor, but would not liberatefree glucose by chemical or enzymatic reactions in the gut.We decided to join the water-soluble polymer PEG600,which is sufficiently large to be impermeable to the gutplasmamembrane [33], via an ether linkage to the 6-positionof glucose. The requirement for stability led us to reject aglycoside linkage to PEG and also any ester link. Weprevented linkage at the aromatic position by usingmethylglucose and anticipated that reaction with anelectrophilic derivative would occur largely at the primaryalcohol (O6) of glucose. Using spectroscopic, chromato-graphic, chemical, and enzymatic analyses, we confirmedthe structure and stability of the compound di(glucos-6-yl)PEG600. Competition studies indicated that this com-pound did not inhibit Na+/glucose transport into intestinalBBMVs, and the infusion of the intestine with di(glucos-6-yl)PEG600, but not PEG600, led to induction of functionalSGLT1.We conclude that the lumenal sugar is sensed by a glucose

sensor, with a sugar specificity different from that ofSGLT1, which is located on the external face of theintestinal lumenal membrane. This initiates a signallingpathway, independent of glucose metabolism, leading toenhanced SGLT1 expression.To identify the molecular components of the signalling

pathway, we used the intestinal cell line, STC-1, as an in vitrosystem. We have shown previously that these cells respondto medium glucose, and regulate SGLT1 expression, in amanner similar to that shown in the native intestinal tissue[8]. In STC-1 cells, SGLT1 abundance is up-regulated inresponse to increased medium glucose concentration [8](Fig. 8). Measurements of intracellular cAMP under thesame experimental conditions, indicated that the levels arealso increased when cells are exposed to high-glucosemedium. Inclusion of 8Br-cAMP in the culture mediumresulted in an increase in SGLT1 protein abundance similarto that observed in response to glucose. 8Br-cAMP alsoenhanced the ovine SGLT1 promoter activity. The PKA-specific inhibitor, H-89, completely abolished the glucose-induced SGLT1 promoter activity, and significantlyinhibited the induction of endogenous SGLT1 expressionin the STC-1 cells. Therefore, we conclude that changes inthe intracellular cAMP level, and activation of PKA couldbe mechanisms for glucose-responsive SGLT1 gene expres-sion. These data are consistent with other reports showingSGLT1 upregulation by elevated cAMP levels in theporcine kidney-derived cell line, LLC-PK1 [34]. Our previ-ous studies indicated that induction of SGLT1 expressionrequires de novo protein synthesis; there is no evidence for anintracellular pool of SGLT1, and therefore the increase inSGLT1 protein abundance is unlikely to be due to therecruitment of the protein from intracellular stores [8].Having shown that increases in intracellular cAMP

increased SGLT1 promoter activity, and SGLT1 expres-sion, we investigated the possibility that glucose sensingmaybe linked to a G-protein, analogous to Gpr1, the GPCR inyeast. The presence of Ga subunits was confirmed in theBBMVs of lambs, adult ruminant sheep, and glucose-infused ruminant sheep, as well as in STC-1 cells. Theaddition of pertussis toxin, an inhibitor of the inhibitory


G-protein (Gi), resulted in a twofold increase in SGLT1abundance, identical with the effect of adding 8Br-cAMP.Inhibition of Gi by pertussis toxin results in hyperstimula-tion of adenylate cyclase leading to increased cAMP levels.Adenylate cyclase is under negative regulation by Gi andpositive activation by Gas. It is tempting to propose that theG-protein, Gas, could be linked to the glucose-sensingmechanism in the intestine.We suggest that glucose-inducedSGLT1 gene activation may be initiated through a GPCR,via an adenylate cyclase–PKA pathway.In summary, our data indicate that lumenal glucose is

sensed by a sugar sensor, probably distinct from SGLT1,and located on the external face of the intestinal lumenalmembrane. The glucose sensor initiates a signalling path-way, involving a GPCR linked to a cAMP–PKA pathway,which eventually leads to enhancement of SGLT1 expres-sion, resulting in an increase in the number of functionalintestinal Na+-dependent sugar transporters.It would be intriguing to determine if enterocytes, which,

like S. cerevisiae, have to adjust their function to wildlyfluctuating levels of extracellular glucose, have developedsimilar mechanisms to sense glucose and regulate theirfunction, and furthermore, if the glucose-sensing GPCRsystem is a common, but unexplored, mechanism present inother eukaryotic cells. In any case, the data from thesestudies should allow a better understanding of the mech-anisms of glucose sensing and glucose-induced signalling inthe control of intestinal glucose absorption.

Acknowledgements

We thank Drs Richard Simmonds and Kishore Bagga for their help in

the chemical synthesis and Dr Dennis Scott for his assistance with the

ovine intestinal infusions. Financial support from theWellcome Trust is

gratefully acknowledged.

References

1. Solberg, D.H. & Diamond, J.M. (1987) Comparison of different

dietary sugars as inducers of intestinal sugar transporters. Am. J.

Physiol. 252, G574–G584.

2. Ferraris, R.P. & Diamond, J.M. (1989) Specific regulation of

intestinal nutrient transporters by their dietary substrates. Annu.

Rev. Physiol. 51, 125–141.

3. Lescale-Matys, L., Dyer, J., Scott, D., Freeman, T.C., Wright,

E.M. & Shirazi-Beechey, S.P. (1993) Regulation of the ovine

intestinal Na+/glucose co-transporter (SGLT1) is dissociated

from mRNA abundance. Biochem. J. 91, 435–440.

4. Shirazi-Beechey, S.P. (1996) Intestinal sodium-dependent D-glu-

cose co-transporter: dietary regulation. Proc. Nutr. Soc. 55,

167–178.

5. Dyer, J., Hosie, K.B. & Shirazi-Beechey, S.P. (1997) Nutrient

regulation of human intestinal sugar transporter (SGLT1)

expression. Gut 41, 56–59.

6. Shirazi-Beechey, S.P., Hirayama, B.A., Wang, Y., Scott, D.,

Smith, M.W. & Wright, E.M. (1991) Ontogenic development of

lamb intestinal sodium-glucose co-transporter is regulated by diet.

J. Physiol. (Lond.) 437, 699–708.

7. Freeman, T.C., Wood, I.S., Sirinathsinghji, D.J., Beechey, R.B.,

Dyer, J. & Shirazi-Beechey, S.P. (1993) The expression of the

Na+/glucose cotransporter (SGLT1) gene in lamb small intestine

during postnatal development. Biochim. Biophys. Acta. 1146,

203–212.

8. Vayro, S., Wood, I.S., Dyer, J. & Shirazi-Beechey, S.P. (2001)

Transcriptional regulation of the ovine intestinal Na+/glucose

co-transporter SGLT1 gene: the role of HNF-1 in glucose acti-

vation of promoter function. Eur. J. Biochem. 268, 5460–5470.

9. Wallace, R.J. (1995) Biochemistry andmicrobiology in the rumen.

In Physiological and Clinical Aspects of Short Chain Fatty Acids

(Cummings, J.H., Rombeau, J.L. & Sakarta, T., eds), pp. 57–71.

Cambridge University Press, Cambridge, UK.

10. Mills, J., France, J. & Dijkstra, J. (1999) A review of starch

digestion in the lactating dairy cow and proposal for a mechanistic

model. 1. Dietary starch characterization and ruminal starch

digestion. J. Anim. Feed. Sci. 8, 291–340.

11. Wood, I.S., Dyer, J., Hofmann, R.R. & Shirazi-Beechey, S.P.

(2000) Expression of the Na+/glucose co-transporter (SGLT1) in

the intestine of domestic and wild ruminants. Pflugers Arch. 441,

155–162.

12. Dyer, J., Scott, D., Beechey, R.B., Care, A.D., Abbas, S.K. &

Shirazi-Beechey, S.P. (1993) Dietary regulation of intestinal glu-

cose transport. InMammalian Brush-Border Membrane Proteins,

Part II (Lentze, M.D. & Grand, R., eds), pp. 65–72. Thieme

Verlag, Stuttgart and New York.

13. Wood, I.S., Allison, G.G. & Shirazi-Beechey, S.P. (1999) Isolation

and characterization of a genomic region upstream from the ovine

Na+/D-glucose cotransporter (SGLT1) cDNA. Biochem. Biophys.

Res. Commun. 257, 533–537.

14. Krzeminski, L.F. & Landmann, W.A. (1963) Methyl yellow as a

spray reagent in the paper chromatograpy of chlorinated hydro-

carbon pesticides. J. Chromatogr. 10, 515–516.

15. Dittrich, K. (1986) Analyses by emission, absorption, and fluor-

escence of small molecules in the visible and ultraviolet range in

gaseous-phase. CRC Crit. Rev. Anal. Chem. 16, 223–279.

16. Rindi, G., Grant, S.G.N., Yiangou, Y., Ghatei, M.A., Bloom,

S.R., Bautch, V.L., Solcia, E. & Polak, L.M. (1990) Development

of neuroendocrine tumors in the gastrointestinal tract of trans-

genic mice. Heterogeneity of hormone expression. Am. J. Pathol.

136, 13449–13463.

17. Shirazi-Beechey, S.P., Davies, A.G., Tebbutt, K., Dyer, J., Ellis,

A., Taylor, C.J., Fairclough, P. & Beechey, R.B. (1990) Prepara-

tion and properties of brush-border membrane vesicles from

human small intestine. Gastroenterology 98, 676–685.

18. Tarpey, P.S., Wood, I.S., Shirazi-Beechey, S.P. & Beechey, R.B.

(1995) Amino acid sequence and the cellular location of the Na+-

dependent D-glucose symporters (SGLT1) in the ovine enterocyte

and the parotid acinar cell. Biochem. J. 312, 293–300.

19. Shirazi-Beechey, S.P., Kemp, R.B., Dyer, J. & Beechey, R.B.

(1989) Changes in the functions of the intestinal brush border

membrane during the development of the ruminant habit in

lambs. Comp. Biochem. Physiol. B 94, 801–806.

20. Dyer, J., Fernandez-Castano Merediz, E., Salmon, K.S.H.,

Proudman, C.J., Edwards, G.B. & Shirazi-Beechey, S.P. (2002)

Molecular characterisation of carbohydrate digestion and

absorption in equine small intestine. Equine Vet. J. 34, 349–358.

21. Dyer, J., Wood, I.S., Palejwala, A., Ellis, A. & Shirazi-Beechey,

S.P. (2002) Expression of monosaccharide transporters in the

intestine of human diabetics. Am. J. Physiol. 282, G241–G248.

22. Dyer, J., Barker, P.J. & Shirazi-Beechey, S.P. (1997) Nutrient

regulation of the intestinal Na+/glucose co-transporter (SGLT1)

gene expression. Biochem. Biophys. Res. Commun. 230, 624–629.

23. Kocienski, P.J., Cemigliaro, G. & Feldstein, G. (1977) Pheromone

synthesis. 4. A synthesis of (+)-methyl n-tetradeca-trans-2,4,5-

trienoate, an allenic ester produced by the male dried bean beetle

Acanthoscelides obtectus (Say). J. Org. Chem. 42, 353–355.

24. Nakafuku, M., Obara, T., Kaibuchi, K., Miyajima, I., Miyajima,

A., Itoh, H., Nakamura, S., Arai, K.-I.,Matsumoto, K. &Kaziro,

Y. (1988) Isolation of a second yeast Saccharomyces cerevisiae


(GPA2) coding for guanine nucleotide-binding regulatory protein:

studies on its structure and possible functions. Proc. Natl Acad.

Sci. USA 85, 1374–1378.

25. Xue, Y., Batlle, M. &Hirsch, J.P. (1998) GPR1 encodes a putative

G-protein coupled receptor that associates with the Gpa2p Gasubunit and functions in a Ras-independent pathway. EMBO

J. 17, 1996–2007.

26. Kraakman, L., Lemarie, K., Pingsheng, M., Teunissen,

A.W.R.H., Donaton, M.C., Van Dijck, P., Winderickx, J., de

Winde, J.H. & Thevelein, J.M. (1999) A Saccharomyces cerevisiae

G-protein coupled receptor, Gpr1, is specifically required for

glucose activation of the cAMP pahway during the transition to

growth on glucose.Mol. Microbiol. 32, 1002–1012.

27. Rolland, F., Winderickx, J. & Thevelein, J.M. (2001) Glucose-

sensing mechanisms in eukaryotic cells. Trends Biochem. Sci. 26,

310–317.

28. Lorenz, M.C., Pan, X., Harashima, T., Cardenas, M., Xue, Y.,

Hirsch, J.P. & Heitman, J. (2000) The G-protein-coupled receptor

Gpr1 is a nutrient sensor that regulates pseudohyphal differ-

entiation in Saccharomyces cerevisiae. Genetics 154, 609–622.

29. Colombo, S., Pingsheng, M., Cauwenberg, L., Winderickx, J.,

Crauwels, M., Teunissen, A., Nauwelaers, D., de Winde, J.H.,

Gorwa, M.-F., Colavizza, D. & Thevelein, J. (1998) Involvement

of distinct G-proteins, Gpa2 and Ras, in glucose-and intracellular

acidification-induced cAMP signalling in the yeast Saccharomyces

cerevisiae. EMBO J. 17, 3326–3341.

30. Johnston, M. (1999) Feasting, fasting and fermenting, glucose

sensing in yeast and other cells. Trends Genet. 15, 29–33.

31. Ozcan, S., Dover, J. & Johnston, M. (1998) Glucose sensing and

signalling by two glucose receptors in the yeast Saccharomyces

cerevisiae. EMBO J. 17, 2566–2573.

32. Itoh, Y., Kawamata, Y., Harada, M., Kobayashi, M., Fujii, Y.,

Fukusumi, S., Ogi, K., Hosoya, M., Tanaka, Y., Uejima,

H., Tanaka, H., Maruyama, M., Satoh, R., Okubo, S., Kizawa,

H., Komatsu, H., Matsumura, F., Noguchi, Y., Shinohara, T.,

Hinuma, S., Fujisawa, Y. & Fujino, M. (2003) Free fatty acids

regulate insulin secretion from pancreatic b-cells through GPR40.Nature (London) 422, 173–176.

33. Lloyd, J.B. (1998) Intestinal permeability to polyethyleneglycol

and sugars: a re-evaluation. Clin. Sci. 95, 107–110.

34. Yet, S.F., Kong, C.T., Peng, H. &Lever, J.E. (1994) Regulation of

Na+/glucose cotransporter (SGLT1) mRNA in LLC-PK1 cells.

J. Cell Physiol. 158, 506–512.


glucose sensing in the intestinal epithelium

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