Tài liệu Báo cáo khoa học: Glucose sensing in the intestinal epithelium pdf

Glucose sensing in the intestinal epithelium Jane Dyer 1, *, Steven Vayro 1, *, Timothy P. King 2 and Soraya P. Shirazi-Beechey 1 1 Epithelial Function and Development Group, Department of Veterinary Preclinical Sciences, University of Liverpool, England, UK; 2 Rowett Research Institute, Aberdeen, Scotland, UK Dietary sugars regulate expression of the intestinal Na + / glucose cotransporter, SGLT1, in many species. Using sheep intestine as a model, we showed that lumenal monosaccha- rides, both metabolisable and nonmetabolisable, regulate SGLT1 expression. This regulation occurs not only at the level of transcription, but also at the post-transcriptional level. Introduction of D -glucose and some D -glucose ana- logues into ruminant sheep intestine resulted in > 50-fold enhancement of SGLT1 expression. We aimed to determine if transport of sugar into the enterocytes is required for SGLT1 induction, and delineate the signal-transduction pathways involved. A membrane impermeable D -glucose analogue, di(glucos- 6-yl)poly(ethylene glycol) 600, was synthesized and infused into the intestines of ruminant sheep. SGLT1 expression was determined using transport studies, Northern and Western blotting, 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 into intestinal brush-border membrane vesicles. Studies using cells showed that increased medium glucose up-regulated SGLT1 abundance and SGLT1 promoter activity, and increased intracellular cAMP levels. Glucose-induced acti- vation of the SGLT1 promoter was mimicked by the protein kinase A (PKA) agonist, 8Br-cAMP, and was inhibited by H-89, a PKA inhibitor. Pertussis toxin, a G-protein (G i )-specific inhibitor, enhanced SGLT1 protein abundance to levels observed in response to glucose or 8Br-cAMP. We conclude that lumenal glucose is sensed by a glucose sensor, distinct from SGLT1, residing on the external face of the lumenal membrane. The glucose sensor initiates a sig- nalling pathway, involving a G-protein-coupled receptor linked to a cAMP–PKA pathway resulting in enhancement of SGLT1 expression. Keywords: intestine; Na + /glucose cotransport; nutrient transport; sugar sensing. The dietary monosaccharides, D -glucose and D -galactose, are transported across the brush-border membrane of intestinal absorptive cells (enterocytes) by the Na + /glucose cotransporter, SGLT1. It has been demonstrated that lumenal glucose enhances the number of functional SGLT1 molecules in the intestinal brush-border membrane, and that the metabolism of glucose is not required for the induction [1–5]. We have used sheep intestine, which is an excellent model system, for the study of monosaccharide regulation of intestinal sugar transport [3,6]. We have shown that dietary monosaccharides regulate the expression of intestinal brush- border membrane Na + /glucose cotransporter at both the transcriptional and post-transcriptional levels [3,7,8]. In preruminant lambs (birth to 3 weeks), milk sugar lactose is hydrolysed by the intestinal lactase into D -glucose and D -galactose, and these sugars are transported by SGLT1. Lambs are normally weaned at 3–10 weeks of age and, as the diet changes from milk to grass, the rumen develops. Dietary carbohydrates are fermented by rumen microflora to short chain fatty acids, and under these conditions negligible 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 SGLT1 protein and mRNA [8]. Introduction of either D -glucose or nonmetabolisable analogues of D -glucose, via duodenal cannulae, into the intestinal lumenal contents of ruminant sheep enhances the levels of functional SGLT1 protein and mRNA to those detected in the preruminant state [4,8,11]. Intestinal infusions of D -glucose induced SGLT1 expression in the brush-border membrane of enterocytes just below the crypt–villus junction, with SGLT1 expression spreading to the villus tip, with cell migration along the crypt–villus axis [4,12]. We cloned and characterized the ovine SGLT1 promoter [13], and using intestinal STC-1 cells as a suitable in vitro model [8], we identified (a) the basal SGLT1 promoter, (b) a glucose-responsive element within the promoter, and (c) a sugar-induced transcription factor involved 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 is required 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: spsb@liv.ac.uk 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) Ó 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)PEG 600 ]. Introduction of this compound into the lumenal content of the ruminant sheep led to an increase in the expression of intestinal SGLT1. This glucose analogue did not, however, inhibit Na + -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 sugar sensor, which is located on the lumenal surface of the intestinal epithelial cell membrane, and is distinct from SGLT1. To delineate the signal-transduction pathway by which the glucose sensor might operate, we investigated the role of some modulators of cAMP levels in induction of SGLT1 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 SGLT1 promoter. 8Br-cAMP also increased the levels of SGLT1 expressed endogenously in the cell line. The glucose- induced SGLT1 promoter activity was inhibited, in a dose-dependent manner, by the PKA antagonist H-89. There was a 47% increase in the level of intracellular cAMP when cells were exposed to increased medium D -glucose concentration; this paralleled the enhancement in SGLT1 abundance. The potential role of G-proteins in the pathway was investigated. Addition of pertussis toxin, a G-protein (G i )-specific inhibitor, to the intestinal cell line grown in low-glucose conditions enhanced the SGLT1 abundance to that observed with high-glucose or with 8Br-cAMP. We propose that the intestinal epithelial cells have a glucose sensor that resides on the external face of the lumenal membrane. Glucose binds to the sensor and generates an intracellular signal leading to enhancement of the expression of SGLT1. It is evident that the generated signal is independent of glucose metabolism and appears to work via a G-protein-coupled receptor and cAMP/PKA signalling cascade. Materials and methods Synthesis and characterization of di(glucos-6-yl)PEG 600 Di(glucos-6-yl)PEG 600 was synthesized by the route shown in Fig. 1. Synthesis of dibromoPEG. Triphenylphosphine, final concentration 2 M , was added to a magnetically stirred solution of 0.67 M PEG 600 and 1.67 M tetrabromometh- ane in 15 mL dry dichloromethane at 40 °C. The reaction mixture was refluxed, in the dark, for 4 days, by which time the reaction was complete, as indicated by TLC 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 developed initially using iodine vapour and, after evaporation of the iodine, visualization was with methyl red spray, which gave a bright red colour with the product [14]. The mixture was filtered and the filtrate washed three times with deionized water (15 mL) to remove triphenylphos- phine oxide. The organic layer was concentrated and the residue swirled with 20 mL deionized water for 6 h. The mixture was filtered, the residue washed with deionized water (10 mL), and the aqueous portions freeze-dried, redissolved in 5 mL water, and separated by gel filtration on a Sephadex G15 gel column (75 · 2cm) and eluted with deionized water. An initial 25 mL was collected, and then 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 blue followed by a green colour in a flame test [15]. Reaction of methyl-a, D -glucopyranoside with halogenated PEG in aqueous KOH. Methyl-a, D -glucopyranoside was added to a mixture of 2 M KOH in 2 mL dimethyl sulfoxide to a final concentration of 1 M , followed immediately by an 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, as assessed by TLC using a p-anisaldehyde spray. The sugar derivatives gave blue spots on a pink background. The reaction mixture was then purified by gel filtration, as described above, and the pure product was isolated from fractions 1–3. Oxidation of the product with periodate indicated the presence of two 6-O-glucosyl units per unit of PEG. Infrared spectroscopy and 1 H NMR confirmed the presence of a methylglucose unit on either end of the PEG 600 backbone. Hydrolysis of di(methylglucos-6-yl)PEG 600 using H 2 SO 4 . To remove the methyl groups di(methylglucos-6-yl) PEG 600 was dissolved in 10 mL 0.5 M H 2 SO 4 to a concentration of 0.5 m M and heated under reflux to produce the target compound. At all stages of the synthesis, reaction products were analysed by TLC, infrared spectroscopy, and 1 H NMR. The di(glucos-6-yl)PEG 600 was tested for any potential free glucose using a commercial glucose testing kit (Boehringer-Mannheim). Fig. 1. Synthesis of di(glucos-6-yl)PEG 600 . D -Glucose was linked by ether bonds to PEG 600 by the synthesis pathway outlined. 3378 J. Dyer et al.(Eur. J. Biochem. 270) Ó FEBS 2003 Biological stability of di(glucos-6-yl)PEG 600 . To deter- mine if di(glucos-6-yl)PEG 600 was stable, and resistant to hydrolysis when introduced into the intestinal contents, the following experiments were undertaken. Di(glucos-6- yl)PEG 600 was mixed with 10 mL ovine intestinal digesta to a final concentration of 30 m M and incubated at 39 °C (sheep body temperature). Samples were removed at intervals of time up to 24 h and assayed for free glucose using a commercial kit (Boehringer-Mannheim), according to the manufacturer’s instructions. In addition, ovine intestinal crude cellular homogenate or purified brush- border membrane vesicles (1 mg protein) were incubated at 39 °C in 0.1 mL of a solution containing 300 m M mannitol, 20 m M Hepes/Tris, pH 7.4, 0.1 m M MgSO 4 and 30 m M di(glucos-6-yl)PEG 600 .Samples(10lL) were removed at 1 h intervals for up to 8 h and assayed for glucose using a glucose 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 m M solutions of D -glucose, PEG 600 ,or di(glucos-6-yl)PEG 600 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 paper towels to remove mucous, and then wrapped in aluminium foil before immediate freezing in liquid nitrogen. Additional samples were frozen in isopentane cooled in liquid nitrogen for immunohistochemical studies. Tissue was subsequently stored 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 in Dulbeccos’ modified Eagle’s medium (Invitrogen) supple- mented with 10% (v/v) fetal bovine serum or 10% (v/v) dialysed fetal bovine serum (containing < 200 l M D -glucose), 50 UÆmL )1 antibiotic solution containing peni- cillin and streptomycin, and either 25 m M (high) or 5 m M (low) D -glucose, as described [8]. Cells were maintained at all times at 37 °Cin5%CO 2 . Stock cultures were grown in 75-cm 2 flasks (Corning, High Wycombe, Bucks, UK) and were fed every 3–4 days. Subsequently cells were washed twice with 5 mL Hanks’ balanced salt solution and then trypsinized (1 min at 37 °C, 5% CO 2 )in1mLsolution containing 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 syringe fitted 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 a density of  0.5 · 10 6 cells, and returned to 37 °C, until they were 60–70% confluent. Preparation of brush-border membrane vesicles (BBMVs) Brush-border membrane vesicles (BBMVs) were prepared from frozen intestinal sections using a combination of cation precipitation and differential centrifugation as des- cribed previously [17]. The final purified BBMVs were suspended in buffer containing 300 m M mannitol, 20 m M Hepes/Tris, pH 7.4, and 0.1 m M MgSO 4 ,andstoredin liquid 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 the standard [18]. The plasma membrane origin of the BBMVs was assessed by determination of the enrichment of the activity and the abundance of the marker proteins of the brush-border membrane. BBMV purity was determined by assessing the levels of marker proteins characteristic of basolateral and organelle membranes [6,19]. Measurement of monosaccharide transport activity To assess the activity of SGLT1, the initial rate of 0.1 m M D -glucose transport in BBMVs was measured at 39 °Cin the presence of NaSCN and KSCN, using the rapid filtration stop technique, as described before [17,19]. All initial rate measurements were taken after a 3 s incubation period, 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 m M 2-deoxy- D - glucopyranoside, a specific substrate of Na + -independent D -glucose transporter isoforms, was determined at 39 °Cin incubation medium consisting of 300 m M mannitol, 20 m M Hepes/Tris, pH 7.4, 0.1 m M MgSO 4 , and 0.02% (w/v) NaN 3 in the presence and absence of 50 l M cytochalasin B, as described [20]. Competition studies were carried out by determining the initial rate of uptake of 0.1 m MD -glucose in the presence of 1m M competitor, using a standard technique as described previously [21]. Immunodetection of SGLT1 Quantitative Western blotting. The abundance of SGLT1 protein was measured by quantitative Western blotting as described previously [22]. The BBMV protein contents were separated on an 8% polyacrylamide gel containing 0.1% (w/v) SDS and were electrotransferred to nitrocellulose membrane (TransBlot; Bio-Rad). A standard calibration curve was constructed by slot- blotting the synthetic peptide (amino acids 402–420 of the ovine SGLT1 sequence, to which the antibody was raised) on to nitrocellulose membrane, and this was probed concurrently with the BBMV samples. The specific immu- noreactive band was blocked when antibodies were pre- incubated with the immunizing peptide. The membranes were developed using the ECL system (Amersham- Pharmacia, Little Chalfont, Bucks., UK), and exposed to film (XOMAT-LS; Kodak). The intensity of the immunoreactive bands detected in the BBMVs and the peptide standard samples was quantified using scanning densitometry (Phoretix 1D; Non-linear Ó FEBS 2003 Intestinal glucose sensing (Eur. J. Biochem. 270) 3379 Dynamics Ltd, Newcastle upon Tyne, Tyne and Wear, UK), and the abundance of SGLT1 protein per mg of BBMV protein 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/P i and then lysed in 300 lL buffer containing 150 m M NaCl, 1% (w/v) SDS, 10 m M EDTA and 10 m M Hepes/Tris, pH 7.4, with protease inhibitor cocktail (Boeh- ringer-Mannheim) and 0.2 m M phenylmethanesulfonyl fluoride. Cells were scraped from the dish with a Ôrubber policemanÕ and homogenized by 10 passages through a syringe fitted with a 21-gauge needle. Protein (15 lgper lane) was separated on 8% (w/v) polyacrylamide gels containing 0.1% (w/v) SDS. After electrotransfer to poly(vinylidene difluoride) (0.2 lm), the membrane was blocked for 30 min in buffer containing 150 m M NaCl, 10 m M Tris/HCl, pH 7.4, 0.05% (v/v) Tween 20, 0.5% (w/v) skimmed milk powder. Primary and secondary antibody incubations were 1 h at room temperature; subsequently membranes were washed three times with buffer for 10 min. Detection and quantification were as described above. Immunohistochemistry. Tissue sections (5 lm thick) were cut using a cryostat at )20 °C, air-dried on to gelatin-coated microscope slides, and fixed in methanol for 15 min at )15 °C. Sections were washed in NaCl/P i /Tween (10 m M phosphate buffer, 150 m M NaCl, 0.05% Tween 20, pH 7.4) and incubated for 30 min at 37 °CinNaCl/P i SuperBlock (Pierce and Warriner, Chester, UK). Sections were washed in six changes of NaCl/P i /Tween over 20 min at room temperature and incubated for 60 min at 37 °Cin 5 lgÆmL )1 SGLT1 antiserum in NaCl/P i /Tween containing 0.1% acetylated BSA (Aurion, Wageningen, the Nether- lands). Sections were washed in six changes of NaCl/P i / Tween over 20 min and incubated for 30 min at 37 °Cwith a mouse monoclonal anti-rabbit IgG (clone RG-96; Sigma) at a dilution of 1 : 400 in NaCl/P i /Tween containing 0.1% acetylated BSA. Sections were washed in six changes of NaCl/P i /Tween over 20 min and incubated for 30 min at 37 °Cin5lgÆmL )1 Oregon Green goat anti-mouse IgG (Molecular Probes, Cambridge Bioscience, Cambridge, UK) in NaCl/P i /Tween containing 0.1% acetylated BSA. Sections were washed in six changes of NaCl/P i /Tween, and then mounted in Vectorshield antifading mountant (Vector Laboratories) before examination by incident light fluores- cence microscopy on a Zeiss Axioscope microscope. Control sections were subjected to the same protocol except that 0.1 lgÆmL )1 peptide (amino acids 402–420 of the ovine SGLT1) was added to the SGLT1 antibody, and the peptide/antibody mixture was preincubated for 60 min at 37 °Cbeforeuse. Immunodetection of G-proteins The presence of G-proteins (G a subunits) on the intestinal brush-border membrane of preruminant lambs, ruminant sheep and age-matched glucose-infused ruminant sheep, as well as in STC-1 cells, was determined by Western blotting with a broad-range affinity-purified rabbit G a polyclonal antibody raised against the conserved GTP- binding domain (Sigma) at 1 : 1000 dilution, as described above for SGLT1. Analysis of SGLT1 promoter function The ovine SGLT1 promoter fragment used in these studies was generated, and assayed, as described previously [8,13]. STC-1 cells were seeded into 12-well plates (0.5 · 10 6 cells) containing 1 mL medium, and incubated for 24 h at 37 °C, 5% CO 2 . Cells were then transiently transfected using the cationic lipid reagent Transfast (Promega) at a DNA/lipid ratioof1 :1.Asecondplasmid,pRL-SV40(Promega),was cotransfected (0.019 pmol) as an internal control. The cells were incubated for 1 h at 37 °C, 5% CO 2 ,andthen1mL complete medium was added. After a further 48 h, the cells were recovered and assayed for luciferase activity using the Dual-Luciferase Reporter assay system (Promega) on a Lumat LB9501 luminometer (Perkin–Elmer). Values are presented as a ratio of the firefly luciferase to Renilla luciferase activity. Intracellular cAMP determination The cAMP levels in cellular homogenates were measured using a commercially available RIA kit (Amersham-Phar- macia), according to the manufacturer’s instructions. STC-1 cells (1 · 10 6 per well) were harvested and homogenized, using a Polytron at setting 5 for 30 s, in 0.4 mL buffer containing 100 m M mannitol, 2 m M Hepes/Tris, pH 7.4, 5m M EDTA, 0.2 m M phenylmethanesulfonyl fluoride, protease inhibitor cocktail (Boehringer-Mannheim) and 1m M 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor. The homogenates were then deproteinated by heating in a boiling water bath for 10 min, followed by centrifugation at 15 000 g for 20 min at 4 °C to sediment denatured proteins. Supernatants were transferred to fresh 1.5 mL tubes, placed on ice, and assayed for cAMP following 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 are considered significant if P <0.05. Results Synthesis of di(glucos-6-yl)PEG 600 In this molecule we chose to link the glucose and PEG by ether bonds, which made it improbable that enzymatic hydrolysis would occur in vivo. On the basis of our previous investigations on the stereoselectivity of the glucose sensor [4,12], we chose the ether linkage to O6 of glucose; being a primary alcohol, the 6-OH is the most reactive once the anomeric position is blocked. Bromination of primary alcohols using tetrabromometh- ane/triphenylphosphine usually gives good yields of alkyl bromides [23], but with PEG 600 as the alcohol, bromination was accompanied by chlorination. However, the dihalogen- ated products were all eluted in the same fraction from a Sephadex G-15 column, and the mixture reacted fully with methyl-a, D -glucopyranoside in dimethyl sulfoxide to give good yields of the glucoside, provided that KOH was used 3380 J. Dyer et al.(Eur. J. Biochem. 270) Ó FEBS 2003 as base. The presence of two 6-O-glucosyl units per unit of PEG was demonstrated by oxidation with periodate;  4 mol was consumed per mol di(methylglucosyl)PEG 600 , with production of formic acid. Hydrolysis of the glucoside with 0.5 M H 2 SO 4 was used to remove the methyl groups in quantitative yield, giving the target compound (Fig. 1). Stability of di(glucos-6-yl)PEG 600 The synthesized di(glucos-6-yl)PEG 600 was assayed for any potential free glucose using a commercial kit (Boehringer- Mannheim). The results indicated total absence of free glucose, and this was confirmed by TLC. To determine biological stability, di(glucos-6-yl)PEG 600 (30 m M )was incubated with ovine intestinal digesta (10 mL) at 39 °Cfor 24 h, and fractions were removed periodically and assayed for free glucose. Glucose was not detected in the samples up to 8 h, indicating that the compound was not hydrolysed and would remain intact over the infusion period. Similarly glucose was not detected when di(glucos-6-yl)PEG 600 was incubated with either the ovine intestinal mucosal homogen- ate or purified BBMVs. Results indicate that free glucose would not be present during the infusion period, or during the passage of the compound through the small intestine. This eliminated the possibility that the induction of SGLT1 expression may be due to the glucose released in the small intestine as a result of the breakdown of the compound. Induction of SGLT1 by intestinal infusion Western blot analysis. The protein component of BBMVs isolated from the jejunum of control ruminant sheep and ruminant sheep the intestines of which were infused with PEG 600 , D -glucose or di(glucos-6-yl)PEG 600 were separated by SDS/PAGE and electrotransferred to nitrocellulose. Samples were immunoblotted to determine the presence of SGLT1 protein using an affinity-purified polyclonal peptide antibody, as described previously [22]. The results are presented in Fig. 2. The antibody recognizes a single protein with an apparent molecular mass of 75 kDa in the BBMVs isolated from the D -glucose and di(glucos-6-yl)PEG 600 - infused animals, but not the PEG 600 -infused animals or controls, indicating that the presence of D -glucose or the nontransportable, membrane-impermeable, di(glucos-6-yl)- PEG 600 in the lumen of the intestine induces expression of SGLT1. The abundance of SGLT1 protein in the BBMVs isolated from the intestine of sheep infused with D -glucose or with di(glucos-6-yl)PEG 600 (Fig. 2) are 12.6 ± 1.3 and 13.1 ± 1.2 pmolÆ(mg protein) )1 , respectively. Transport of D -glucose. To determine if the sugar-induced SGLT1 is capable of transport, the ability of the BBMVs to transport glucose in a Na + -dependent manner was assessed. The initial rates of 0.1 m MD -glucose uptake into BBMVs isolated from the intestine of control and infused ruminant sheep are presented in Fig. 3. The initial rates of Na + - dependent D -glucose transport were 108.8 ± 10.5 and 111 ± 6.4 pmolÆs )1 Æ(mg protein) )1 in BBMVs isolated from the jejuna for D -glucose and di(glucos-6-yl)PEG 600 , respectively. The initial rate of uptake in vesicles isolated from PEG 600 -infused sheep was 3.6 ± 1.2 pmolÆs )1 Æ(mg protein) )1 , a rate identical with that measured in adult ruminant control BBMVs [11,22]. The results indicate that the SGLT1 protein that is expressed in response to lumenal infusion is functional. There was no cytochalasin B-sensitive 2-deoxy- D -glucose transport detected in any of the BBMV samples (data not shown), indicating the absence of GLUT2 from the BBMVs and therefore any basolateral membrane contamination. To investigate any interaction between the di(glucos-6- yl)PEG 600 and SGLT1 function, the ability of this com- pound to inhibit Na + -dependent D -glucose transport into BBMVs was investigated. Concurrently the effect on SGLT1 activity of other glucose analogues was also determined. The results are presented in Fig. 4. The initial rate of uptake of 0.1 m MD -glucose into lamb jejunal BBMVs was reduced in the presence of 1 m M 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 m M solutions of PEG 600 , D -glucose, or di(glucos-6-yl)PEG 600 through duodenal cannulae. BBMVs were prepared from the intestine of these animals, and brush-border membrane proteins (20 lg per lane) were separated 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 m M D -glucose uptake into BBMVs (0.1 mg protein) was measured at 39 °C inthepresenceof100m M NaSCN, as described in Materials and methods. Results are presented as mean ± SEM (n ¼ 3). Ó FEBS 2003 Intestinal glucose sensing (Eur. J. Biochem. 270) 3381 D -galactose (natural SGLT1 substrates), a-methylglucose or 3-O-methylglucose (nonmetabolisable SGLT1 substrates). However, D -glucose uptake was unchanged when D -fructose or di(glucos-6-yl)PEG 600 (not transported by SGLT1 but induce SGLT1 expression), L -glucose or PEG 600 (not transported by SGLT1 and do not induce SGLT1 expres- sion) were included in the incubation medium. The results suggest that there is no interaction between di(glucos- 6-yl)PEG 600 and SGLT1 protein that affects SGLT1 function. Immunofluorescence localization of SGLT1. The distribu- tion of SGLT1 protein along the crypt–villus axes of the intestine of the infused ruminant sheep was also determined by immunohistochemistry. Typical results, presented in Fig. 5, indicate that infusion of the intestine of ruminant sheep with either D -glucose or di(glucos-6-yl)PEG 600 results in induction of SGLT1 protein expression. Infusion of the intestine with PEG 600 has no effect (Fig. 5A). Immuno- fluorescence localization of SGLT1 protein along the crypt– villus axes of the ruminant sheep, with the intestinal infusion of either di(glucos-6-yl)PEG 600 or D -glucose, shows labelling on the entire brush-border surface, including the lower region of the villus (Fig. 5B,C). The distribution of SGLT1 protein in infused ruminant sheep (Fig. 5B,C)is similar to that seen in the intestine of thepreruminant lamb (Fig. 5D). The labelling is specific, as it was blocked when the primary antibody was preincubated with the peptide antigen (Fig. 5E). Effect of cAMP and PKA on ovine SGLT1 promoter activity and SGLT1 expression We have demonstrated that di(glucos-6-yl)PEG 600 , a mem- brane-impermeable glucose analogue, enhances the level of SGLT1. Furthermore, we have determined that SGLT1 function is not inhibited in the presence of D -fructose, 2-deoxy- D -glucose and di(glucos-6-yl)PEG 600 , compounds that induce the expression of functional SGLT1. We conclude therefore that a glucose sensor, with different sugar specificity from SGLT1, is located on the external face of the intestinal brush-border membrane. The sensor would detect changes in the lumenal sugar concentration and initiate signalling pathways, leading to modulations in the expression of functional SGLT1. Using the intestinal cell line, STC-1, as an in vitro expression system [8], we assessed the potential role of cAMP/PKA in the transcriptional regulation of the )66/+21-bp SGLT1 glucose-responsive promoter fragment [8]. To this end, we used (a) 8Br-cAMP, a membrane-permeable cAMP analogue and a PKA agonist, and (b) H-89, a PKA antagonist. Cells were cultured in medium containing 5 m M glucose, transfected with the ovine SGLT1 promoter fragment, and then either maintained in the same medium or transferred to one containing 25 m M glucose, in the presence or absence of (a) 0.5 m M 8Br-cAMP or (b) H-89. The results are shown in Fig. 6 and Fig. 7, respectively. Figure 6 shows that SGLT1 promoter activity increased twofold, after the addition of 25 m M glucose, in agreement with our previous data [8]. Cells maintained in low-glucose (5 m M ) medium, but treated with 0.5 m M 8Br-cAMP, also showed a significant increase in promoter activity compared with controls. 8Br- cAMP also augmented the increase in promoter activity observed in response to high-glucose medium by a further 30%. When the reporter gene construct was placed in the reverse orientation, neither glucose nor 8Br-cAMP had any effect. We conclude that an increase in intracellular cAMP results in the activation of SGLT1 promoter function. Glucose-induced SGLT1 promoter activity was inhibited, in a dose-dependent manner, in response to increasing concentrations of H-89 (0.1, 0.5, 1.0 l M ; Fig. 7). In cells switched to 25 m M glucose, in the presence of 1 l M H-89, SGLT1 promoter activity was reduced to the level detected in cells maintained in 5 m M glucose. Promoter function was not inhibited by H-89 in cells maintained in low glucose, or in cells transfected with the reporter gene construct in the reverse orientation (not shown). Therefore, the inhibitory action of H-89 appears to be specific to the glucose-induced SGLT1 promoter activity. These data suggest that PKA has an important role in the transcriptional activation of the ovine SGLT1 promoter. We also examined the effects of both 8Br-cAMP and H-89 on the level of endogenous SGLT1 protein expressed in the STC-1 cells (Fig. 8A,B). Cells transferred from 5 m M to 25 m M glucose showed a (2.88 ± 0.22)-fold increase in the abundance of SGLT1 (Fig. 8A, lanes 1 and 3, and Fig. 8B, lanes 1 and 2), consistent with our previous findings [8]. In cells cultured in 5 m M glucose in the presence of 0.5 m M 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 medium containing 25 m M 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 in 25 m M glucose medium did not respond to 8Br-cAMP (not shown). 8-Br-cAMP had no effect on the levels of b-actin (Fig. 8A). Treatment of STC-1 cells with 0.1 l M H-89 resulted in 34.1 ± 3.3% reduction in glucose-induced SGLT1 protein abundance (Fig. 8B, lane 3), and increasing the concentration of H-89 to 1 l M had no further effect (Fig. 8B, lanes 4 and 5), suggesting a role for PKA in the regulatory process. Interestingly, cAMP concentrations measured in depro- teinated homogenates of cells cultured in 5 m MD -glucose were 47% lower than cAMP levels detected in cells transferred to 25 m M 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 m MD -glucose into preruminant lamb jejunal BBMVs was measured at 39 °C in the presence of of the indicated competitor (1 m M ). Results are expressed as percentage of control and are means ± SEM (n ¼ 3). 3382 J. Dyer et al.(Eur. J. Biochem. 270) Ó FEBS 2003 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 m M solutions of (A) PEG 600 , (B) di(glucos-6-yl)PEG 600 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. Ó FEBS 2003 Intestinal glucose sensing (Eur. J. Biochem. 270) 3383 that there is an increase in intracellular cAMP levels in response to increasing levels of medium D -glucose. Involvement of a GPCR in intestinal glucose sensing One mechanism for sensing glucose in yeast (Saccharomyces cerevisiae) 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 in glucose sensing, we used the G-protein (G i )-specific inhi- bitor, pertussis toxin. In STC-1 cells cultured in 5 m M 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.9lanes1,2,3and4).In cells transferred from 5 m M to 25 m M glucose, SGLT1 expression was up-regulated twofold, as expected (lanes 1 and 5), and pertussis toxin had no effect on this response (lanes 6, 7 and 8). Pertussis toxin had no effect on the levels of b-actin. The presence of G-proteins (G a subunits) on the intestinal brush-border membrane of preruminant lambs, ruminant sheep and age-matched glucose-infused ruminant sheep, as well as in STC-1 cells, was determined by Western blotting with a broad-range G a subunit antibody. The abundance of SGLT1 and b-actin were also determined in the same samples (Fig. 10A,B). G a subunits were detected, as an immunoreactive band of  41 kDa, in the intestinal brush-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 m MD -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 m M or 25 m MD -glucose with (j)or without (h)0.5m M 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 m M or 25 m MD -glucose with increasing concentrations of H-89 (0.1, 0.5, 1.0 m M ), for a further 48 h, before assay. Values are the means ± SEM from three to seven determina- tions. Fig. 8. Effect of 8Br-cAMP and H-89 on the levels of SGLT1 expressed endogenously in STC-1 cells. STC-1 cells were cultured in 5 m M D -glucose medium and then exposed to medium containing either 5m M or 25 m MD -glucose, in the presence of (A) 0.5 m M 8-Br-cAMP, or (B) increasing concentrations of H-89 (0.1, 0.5 or 1.0 l M )fora further 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 a mouse monoclonal b-actin antibody (1 : 10 000 dilution), respectively. Data shown are representative of three experiments. 3384 J. Dyer et al.(Eur. J. Biochem. 270) Ó FEBS 2003 age-matched glucose-infused animals, as well as in lysates from STC-1 cells cultured under low or high glucose conditions. The abundance of SGLT1 protein in BBMVs isolated from the intestinal tissues and the STC-1 cell lysates was a good representation of the glucose induction of SGLT1 [22]. b-Actin abundance was constant in all samples. These observations confirm the presence of G a subunits in the ovine intestinal lumenal membrane and support the potential involvement of a GPCR in the signalling pathway for glucose regulation of SGLT1 expression. Discussion Glucose is a major source of energy for most eukaryotic cells, and has significant and varied effects on cell function. Consequently maintenance of glucose homoeostasis is of great importance to many organisms. Interest in identifying mechanisms by which cells sense and respond to variations in glucose concentration has increased recently, and promising advances have been made [30]. It has been shown that different eukaryotic cells use specific mechanisms to sense the presence of glucose, and that the physiological roles of these mechanisms are dependent on the particular cell type [27]. Yeast cells, S. cerevisiae, have a remarkable preference for glucose as a carbon source [27] and have evolved mechanisms for sensing and responding to wildly fluctu- ating levels of extracellular glucose. These mechanisms involve a large family of hexose transporters (HXT proteins), and the glucose transporter homologues Snf3 and 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 m M or 25 m MD -glucose, in thepresenceof100,250or500ngÆmL )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 of SGLT1 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 G a 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 m M or 25 m MD -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 G a subunits was per- formed using an affinity-purified rabbit G a polyclonal antibody raised against the conserved GTP-binding domain at 1 : 1000 dilution. Ó FEBS 2003 Intestinal glucose sensing (Eur. J. Biochem. 270) 3385 sense the extracellular glucose and generate an intracellular glucose signal that triggers the induction of HXT gene expression [31]. Using Snf3/Rgt2 double mutants it was shown that, in yeast, glucose-sensing and signalling are receptor-mediated processes and are independent of glucose metabolism [31]. In addition, a novel GPCR, Gpr1, which senses external medium glucose, has also been identified. Gpr1 acts via the G-protein, Gpa2, to initiate a cAMP/PKA signalling cascade [25,26,29]. Gpr1 is activated by glucose and transmits a signal, via Gpa2, to adenylate cyclase. Glucose activation of cAMP synthesis requires active sugar phosphorylation but no further metabolism of sugar. The glucose-sensing Gpr1–Gpa2 system for activation of the cAMP pathway in S. cerevisiae appears to be the first example of a nutrient-sensing GPCR system. If nutrient sensing GPCRs were common to eukaryotic cells, they would provide a means of regulating major signal-trans- duction pathways by the nutrient status of the cellular environment. The latter is supported by a recent report showing that a GPCR, GPR40, abundantly expressed in the pancreas, functions as a receptor for long-chain free fatty acids. The latter amplify glucose-stimulated insulin secretion from pancreatic b-cells by activating GPR40 [32]. Intestinal epithelial cells are exposed from the lumenal domain to an environment with continuous and massive fluctuations in the level of dietary monosaccharides. This is in contrast with other mammalian cells, which are exposed to a relatively constant blood glucose concentration regu- lated by endocrine hormones. Enterocytes therefore have to sense and respond to the significant fluctuations in lumenal sugars and regulate their function accordingly. Dietary sugars have been shown to regulate the expression of the intestinal 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, we have shown that lumenal sugars regulate the expression of SGLT1 at both transcriptional and post-transcriptional level [3,8]. It was demonstrated, using nuclear run-on assays, that the transcriptional activity of the ovine SGLT1 gene increased 2–3-fold, in response to lumenal sugar. This increase did not account entirely for the overall enhance- ment in steady-state levels of SGLT1 mRNA determined by Northern 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, but monosaccharides are selectively excluded [10]. Associated with the decline in the levels of monosaccharides, there is a > 50-fold decline in the levels of functional SGLT1 protein and 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 lumenal contents of ruminant sheep intestine, through duodenal cannulae, resulted in increased expression of SGLT1 to the levels detected in the preruminant lamb. We concluded that induction of SGLT1 by lumenal sugar is independent of glucose metabolism and that the inducing sugar need not be a 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 a water-soluble, metabolically inert, membrane-impermeable glucose analogue. Our overall objective was to join glucose to a water-soluble polymer in such a way that the conjugate would activate the glucose sensor, but would not liberate free glucose by chemical or enzymatic reactions in the gut. We decided to join the water-soluble polymer PEG 600 , which is sufficiently large to be impermeable to the gut plasma membrane [33], via an ether linkage to the 6-position of glucose. The requirement for stability led us to reject a glycoside linkage to PEG and also any ester link. We prevented linkage at the aromatic position by using methylglucose and anticipated that reaction with an electrophilic derivative would occur largely at the primary alcohol (O6) of glucose. Using spectroscopic, chromato- graphic, chemical, and enzymatic analyses, we confirmed the structure and stability of the compound di(glucos-6- yl)PEG 600 . Competition studies indicated that this com- pound did not inhibit Na + /glucose transport into intestinal BBMVs, and the infusion of the intestine with di(glucos-6- yl)PEG 600 , but not PEG 600 , led to induction of functional SGLT1. We conclude that the lumenal sugar is sensed by a glucose sensor, with a sugar specificity different from that of SGLT1, which is located on the external face of the intestinal lumenal membrane. This initiates a signalling pathway, independent of glucose metabolism, leading to enhanced SGLT1 expression. To identify the molecular components of the signalling pathway, we used the intestinal cell line, STC-1, as an in vitro system. We have shown previously that these cells respond to medium glucose, and regulate SGLT1 expression, in a manner similar to that shown in the native intestinal tissue [8]. In STC-1 cells, SGLT1 abundance is up-regulated in response to increased medium glucose concentration [8] (Fig. 8). Measurements of intracellular cAMP under the same experimental conditions, indicated that the levels are also increased when cells are exposed to high-glucose medium. Inclusion of 8Br-cAMP in the culture medium resulted in an increase in SGLT1 protein abundance similar to that observed in response to glucose. 8Br-cAMP also enhanced the ovine SGLT1 promoter activity. The PKA- specific inhibitor, H-89, completely abolished the glucose- induced SGLT1 promoter activity, and significantly inhibited the induction of endogenous SGLT1 expression in the STC-1 cells. Therefore, we conclude that changes in the intracellular cAMP level, and activation of PKA could be mechanisms for glucose-responsive SGLT1 gene expres- sion. These data are consistent with other reports showing SGLT1 upregulation by elevated cAMP levels in the porcine kidney-derived cell line, LLC-PK 1 [34]. Our previ- ous studies indicated that induction of SGLT1 expression requires de novo protein synthesis; there is no evidence for an intracellular pool of SGLT1, and therefore the increase in SGLT1 protein abundance is unlikely to be due to the recruitment 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 sensing may be linked to a G-protein, analogous to Gpr1, the GPCR in yeast. The presence of G a subunits was confirmed in the BBMVs of lambs, adult ruminant sheep, and glucose- infused ruminant sheep, as well as in STC-1 cells. The addition of pertussis toxin, an inhibitor of the inhibitory 3386 J. Dyer et al.(Eur. J. Biochem. 270) Ó FEBS 2003 [...]... present in other eukaryotic cells In any case, the data from these studies should allow a better understanding of the mechanisms of glucose sensing and glucose- induced signalling in the 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... mechanism in the intestine We suggest that glucose- induced SGLT1 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 lumenal membrane The glucose sensor initiates a signalling pathway, involving a GPCR linked to... expression, resulting in an increase in the number of functional intestinal Na+-dependent sugar transporters It would be intriguing to determine if enterocytes, which, like S cerevisiae, have to adjust their function to wildly fluctuating levels of extracellular glucose, have developed similar mechanisms to sense glucose and regulate their function, and furthermore, if the glucose- sensing GPCR system... 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G-protein (Gi), resulted in a twofold increase in SGLT1 abundance, identical with the effect of adding 8Br-cAMP Inhibition of Gi by pertussis toxin results in hyperstimulation of adenylate cyclase leading to increased cAMP levels Adenylate cyclase is under negative regulation by Gi and positive activation by Gas It is tempting to propose that the G-protein, Gas, could be linked to the glucose- sensing. .. and Ras, in glucose- and intracellular acidification-induced cAMP signalling in the yeast Saccharomyces cerevisiae EMBO J 17, 3326–3341 Johnston, M (1999) Feasting, fasting and fermenting, glucose sensing in yeast and other cells Trends Genet 15, 29–33 ¨ 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... 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The G-protein-coupled receptor Gpr1 is a nutrient sensor that regulates pseudohyphal differentiation in Saccharomyces cerevisiae Genetics 154, 609–622 Colombo, S., Pingsheng, M., Cauwenberg, L., Winderickx, J., Crauwels, M., Teunissen, A., Nauwelaers, D., de Winde, J.H., 30 31 32 33 34 Gorwa, M.-F., Colavizza, D & Thevelein, J (1998) Involvement of distinct G-proteins, Gpa2 and Ras, in glucose- and intracellular . 3383 that there is an increase in intracellular cAMP levels in response to increasing levels of medium D -glucose. Involvement of a GPCR in intestinal glucose sensing One. Na + -dependent glucose uptake in ovine intestine BBMVs after intestinal infusion. The initial (3 s) rate of 0.1 m M D -glucose uptake into BBMVs (0.1 mg protein)