Segmental diversity of electrogenic glucose transport characteristics in the small intestines of weaned pigs

It has been shown in several species that the intestinal Na(+)-dependent glucose co-transporter 1 (SGLT1) is more abundant in the jejunum than in ileum. In contrast, the efficiency of intestinal glucose uptake rates in suckling piglets or weaned pigs is not clearly fitting with this segmental distribution. The aim of this study was to evaluate SGLT1 mediated glucose absorption in the jejunum and ileum of growing pigs (Sus scrofa) in more detail. In Ussing chambers, basal short-circuit currents were significantly more positive in the jejunum. It could be demonstrated that the electrogenic ileal glucose transport was significantly more pronounced in different breeds and occurred at 5 mmol?L(-1) glucose 7 times faster in the ileum, although slightly higher jejunal expression of glycosylated SGLT1 was detected by Western blotting. This expression pattern was connected to significantly lower phlorizin sensitivity in the jejunum. As the more efficient ileal glucose absorption was also observable with glucose uptake studies into isolated brush-border membrane vesicles without differences in abundance and activity of the Na(+)/K(+)-ATPase in both segments, we conclude that the segmental differences in porcine glucose transport characteristics may be based on direct or indirect modulations of SGLT1 activity.     

Measuring ion transport activities in Xenopus oocytes using the ion-trap technique

The ion-trap technique is an experimental approach allowing measurement of changes in ionic concentrations within a restricted space (the trap) comprised of a large-diameter ion-selective electrode apposed to a voltage-clamped Xenopus laevis oocyte. The technique is demonstrated with oocytes expressing the Na(+)/glucose cotransporter (SGLT1) using Na(+)- and H(+)-selective electrodes and with the electroneutral H(+)/monocarboxylate transporter (MCT1). In SGLT1-expressing oocytes, bath substrate diffused into the trap within 20 s, stimulating Na(+)/glucose influx, which generated a measurable decrease in the trap Na(+) concentration ([Na(+)](T)) by 0.080 +/- 0.009 mM. Membrane hyperpolarization produced a further decrease in [Na(+)](T), which was proportional to the increased cotransport current. In a Na(+)-free, weakly buffered solution (pH 5.5), H(+) drives glucose transport through SGLT1, and this was monitored with a H(+)-selective electrode. Proton movements can also be clearly detected on adding lactate to an oocyte expressing MCT1 (pH 6.5). For SGLT1, time-dependent changes in [Na(+)](T) or [H(+)](T) were also detected during a membrane potential pulse (150 ms) in the presence of substrate. In the absence of substrate, hyperpolarization triggered rapid reorientation of SGLT1 cation binding sites, accompanied by cation capture from the trap. The resulting change in [Na(+)](T) or [H(+)](T) is proportional to the pre-steady-state charge movement. The ion-trap technique can thus be used to measure steady-state and pre-steady-state transport activities and provides new opportunities for studying electrogenic and electroneutral ion transport mechanisms.     

Molecular evidence for two renal Na+/glucose cotransporters.

Previous studies have shown that two kinetically and genetically distinct Na+/glucose cotransporters exist in mammalian kidney. We have recently cloned and sequenced one of the rabbit renal Na+/glucose cotransporters (SGLT1) and have found that it is identical in sequence to the intestinal Na+/glucose cotransporter. Northern blots showed that SGLT1 mRNA was found predominantly in the outer medulla of rabbit kidney. Injection of mRNA from outer medulla and outer cortex into Xenopus oocytes resulted in equal expression of Na(+)-dependent sugar uptake, indicating that the outer cortex sample contained mRNA encoding both SGLT1 and a second Na+/glucose cotransporter. Western blots using antipeptide antibodies against SGLT1 showed that the SGLT1 protein is more abundant in outer medulla than outer cortex. However, brush border membrane vesicles prepared from outer cortex had a greater capacity for Na(+)-dependent glucose transport, indicating the presence of a second transporter in the vesicles from outer cortex. It appears that the cloned renal Na+/glucose cotransporter, SGLT1, is the 'high affinity, low capacity' transporter found predominantly in outer medulla. There is evidence that a second transporter, the 'low affinity, high capacity' transporter, is in outer cortex. Finally, the cDNA and protein sequences of the two renal Na+/glucose cotransporters are predicted to differ by more than 20%.     

Luminal glucose sensing in the rat intestine has characteristics of a sodium-glucose cotransporter

The presence of glucose in the intestinal lumen elicits a number of changes in gastrointestinal function, including inhibition of gastric emptying and food intake and stimulation of pancreatic and intestinal secretion. The present study tested the hypothesis that Na(+)-glucose cotransporter (SGLT)-3, a member of the SGLT family of transport proteins, is involved in detection of luminal glucose in the intestine. Gastric emptying, measured in awake rats, was significantly inhibited by perfusion of the intestine with glucose (60 and 90 mg); this effect was mimicked by alpha-methyl glucose (nonmetabolizable substrate of SGLT-1 and -3) but not 2-deoxy-d-glucose (substrate for GLUT-2) or isoosmotic mannitol. Gastric motility and intestinal fluid secretion, measured in anesthetised rats, were significantly inhibited and stimulated, respectively, by duodenal glucose but not galactose, which has a much lower affinity for SGLT-3 than glucose. Duodenal glucose but not galactose stimulated the release of 5-HT into mesenteric lymph and stimulated the discharge of duodenal vagal afferent fibers. mRNA for SGLT-3 was identified in the duodenal mucosa. Together these data suggest that detection of glucose in the intestine may involve SGLT-3, possibly expressed by enterochromaffin cells in the intestinal mucosa, and release of 5-HT.     

Regulation of the human Na⁺-dependent glucose cotransporter hSGLT2

The human Na(+)-glucose cotransporter SGLT2 is expressed mainly in the kidney proximal convoluted tubule where it is considered to be responsible for the bulk of glucose reabsorption. Phosphorylation profiling has revealed that SGLT2 exists in a phosphorylated state in the rat renal proximal tubule cortex, so we decided to investigate the regulation of human SGLT2 (hSGLT2) by protein kinases. hSGLT2 was expressed in human embryonic kidney (HEK) 293T cells, and the activity of the protein was measured using radiotracer and whole cell patch-clamp electrophysiology assays before and after activation of protein kinases. 8-Bromo-adenosine cAMP (8-Br-cAMP) was used to activate protein kinase A, and sn-1,2-dioctanoylglycerol (DOG) was used to activate protein kinase C (PKC). 8-Br-cAMP stimulated D-[α-methyl-(14)C]glucopyranoside ([(14)C]α-MDG) uptake and Na(+)-glucose currents by 200% and DOG increased [(14)C]α-MDG uptake and Na(+)-glucose currents by 50%. In both cases the increase in SGLT2 activity was marked by an increase in the maximum rate of transport with no change in glucose affinity. These effects were completely negated by mutation of serine 624 to alanine. Insulin induced a 250% increase in Na(+)-glucose transport by wild-type but not S624A SGLT2. Parallel studies confirmed that the activity of hSGLT1 was regulated by PKA and PKC due to changes in the number of transporters in the cell membrane. hSGLT1 was relatively insensitive to insulin. We conclude that hSGLT1 and hSGLT2 are regulated by different mechanisms and suggest that insulin is an SGLT2 agonist in vivo.     

SLC5A9/SGLT4, a new Na+-dependent glucose transporter, is an essential transporter for mannose, 1,5-anhydro-D-glucitol, and fructose

We isolated a cDNA clone of SLC5A9/SGLT4 from human small intestinal full-length cDNA libraries, and functionally characterized it in vitro. The messenger RNA encoding SGLT4 was mainly expressed in the small intestine and kidney, among the human tissues tested. COS-7 cells transiently expressing SGLT4 exhibited Na(+)-dependent alpha-methyl-D-glucopyranoside (AMG) transport activity with an apparent K(m) of 2.6 mM, suggesting that SGLT4 is a low affinity-type transporter. The rank order of naturally occurring sugar analogs for the inhibition of AMG transport was: D-mannose (Man) > D-glucose (Glc) > D-fructose (Fru) = 1,5-anhydro-D-glucitol (1,5AG) > D-galactose (Gal). Recognition of Man as a substrate was confirmed by direct uptake of Man into the cell. COS-7 cells expressing a putative murine SGLT4 ortholog showed similar Na(+)-dependent AMG transport activity and a similar deduced substrate specificity. These results suggest that SGLT4 would have unique physiological functions (i.e., absorption and/or reabsorption of Man, 1,5AG, and Fru, in addition to Glc).     

Direct inhibitory effect of rotavirus NSP4(114-135) peptide on the Na(+)-D-glucose symporter of rabbit intestinal brush border membrane

The direct effect of a rotavirus nonstructural glycoprotein, NSP4, and certain related peptides on the sodium-coupled transport of D-glucose and of L-leucine was studied by using intestinal brush border membrane vesicles isolated from young rabbits. Kinetic analyses revealed that the NSP4(114-135) peptide, which causes diarrhea in young rodents, is a specific, fully noncompetitive inhibitor of the Na(+)-D-glucose symporter (SGLT1). This interaction involves three peptide-binding sites per carrier unit. In contrast, the Norwalk virus NV(464-483) and mNSP4(131K) peptides, neither of which causes diarrhea, both behave inertly. The NSP4(114-135) and NV(464-483) peptides inhibited Na(+)-L-leucine symport about equally and partially via a different transport mechanism, in that Na(+) behaves as a nonobligatory activator. The selective and strong inhibition caused by the NSP4(114-135) peptide on SGLT1 in vitro suggests that during rotavirus infection in vivo, NSP4 can be one effector directly causing SGLT1 inhibition. This effect, implying a concomitant inhibition of water reabsorption, is postulated to play a mechanistic role in the pathogenesis of rotavirus diarrhea.     

Immunohistochemical localization of Na+-dependent glucose transporter in the rat digestive tract

Summary Glucose is actively absorbed in the intestine by the action of the Na⁺-dependent glucose transporter. Using an antibody against the rabbit intestinal Na⁺-dependent glucose transporter (SGLT1), we examined the localization of SGLT1 immunohistochemically along the rat digestive tract (oesophagus, stomach, duodenum, jejunum, ileum, colon and rectum). SGLT1 was detected in the small intestine (duodenum, jejunum and ileum), but not in the oesophagus, stomach, colon or rectum. SGLT1 was localized at the brush border of the absorptive epithelium cells in the small intestine. Electron microscopical examination showed that SGLT1 was localized at the apical plasma membrane of the absorptive epithelial cells. SGLT1 was not detected at the basolateral plasma membrane. Along the crypt-villus axis, all the absorptive epithelial cells in the villus were positive for SGLT1, whose amount increased from the bottom of the villus to its tip. On the other hand, cells in the crypts exhibited little or no staining for SGLT1. Goblet cells scattered throughout the intestinal epithelium were negative for SGLT1. These observations show that SGLT1 is specific to the apical plasma membrane of differentiated absorptive epithelial cells in the small intestine, and suggest that active uptake of glucose occurs mainly in the absorptive epithelial cells in the small intestine.     

Oral polyamine administration modifies the ontogeny of hexose transporter gene expression in the postnatal rat intestine

Gastrointestinal mucosal polyamines influence enterocyte proliferation and differentiation during small intestinal maturation in the rat. Studies in postnatal rats have shown that ornithine decarboxylase (ODC) protein and mRNA peak before the maximal expression of brush-border membrane (BBM) sucrase-isomaltase (SI) and the sugar transporters sodium-dependent glucose transporter 1 (SGLT1) and glucose transporter 2 (GLUT2). This study was undertaken to test the hypothesis that the oral administration of spermidine in postnatal rats upregulates the expression of ODC, thereby enhancing the expression of SI and SGLT1 in the brush-border membrane as well as basolateral membrane-facilitative GLUT2 and Na(+)-K(+)-ATPase. Northern and Western blot analyses were performed with antibodies and cDNA probes specific for SI, SGLT1, GLUT2, alpha(1)- and beta(1)-subunits of Na(+)-K(+)-ATPase, and ODC. Postnatal rats fed 6 mumol spermidine daily for 3 days from days 7 to 9 were killed either on postnatal day 10 (Sp10) or day 13 following a 3-day washout period (Sp13). Sp10 rats showed a precocious increase in the abundance of mRNAs for SI, SGLT1, and GLUT2 and Na(+)-K(+)-ATPase activity and alpha(1)- and beta(1)-isoform gene expression compared with controls. ODC activity and protein and mRNA abundance were also increased in Sp10 animals. The increased expression of these genes was not sustained in Sp13 rats, suggesting that these effects were transient. Thus, 3 days of oral polyamine administration induces the precocious maturation of glucose transporters in the postnatal rat small intestine, which may be mediated by alterations in ODC expression.     

Baculovirus-mediated expression of the Na+/glucose cotransporter in Sf9 cells.

We have used baculovirus (AcNPV) to express the Na+/glucose cotransporter protein in cultured Sf9 cells. We constructed a baculovirus transfer vector containing the cDNA for the rabbit intestinal Na+/glucose cotransporter (SGLT1) under the control of the polyhedrin gene promoter. Recombinant baculovirus was obtained by cotransfection of SF9 cells with wild-type AcNPV DNA and the transfer vector. Recombinant virus was identified by Southern blotting and then purified. Recombinant infected Sf9 cells expressed a protein which was recognized by anti-peptide antibodies raised to sequences of the cloned Na+/glucose cotransporter. This protein migrated with a molecular mass of 55 kD by SDS-PAGE, similar to the in vitro translation product of SGLT1. An identical protein was metabolically labeled with [35S]methionine. Cells which synthesized the transport protein showed Na(+)-dependent alpha MeGlc transport. Micromolar phlorizin inhibited transport. Uninfected and wild-type virus infected Sf9 cells did not have Na(+)-dependent glucose transport. All transport protein migrated at 45% sucrose (w/w) by density gradient sedimentation, suggesting that the expressed transporter is membrane associated. We conclude that we have functionally expressed the rabbit intestinal Na+/glucose cotransporter in Sf9 cells. The transporter is not heavily glycosylated, and this is consistent with previous work showing that glycosylation is not necessary for function. We are poised to purify and characterize this protein from a structure-function perspective.     

Role of Cl- in electrogenic Na+-coupled cotransporters GAT1 and SGLT1

We have investigated the functional role of Cl(-) in the human Na(+)/Cl(-)/gamma-aminobutyric acid (GABA) and Na(+)/glucose cotransporters (GAT1 and SGLT1, respectively) expressed in Xenopus laevis oocytes. Substrate-evoked steady-state inward currents were examined in the presence and absence of external Cl(-). Replacement of Cl(-) by gluconate or 2-(N-morpholino)ethanesulfonic acid decreased the apparent affinity of GAT1 and SGLT1 for Na(+) and the organic substrate. In the absence of substrate, GAT1 and SGLT1 exhibited charge movements that manifested as pre-steady-state current transients. Removal of Cl(-) shifted the voltage dependence of charge movements to more negative potentials, with apparent affinity constants (K(0.5)) for Cl(-) of 21 and 115 mm for SGLT1 and GAT1, respectively. The maximum charge moved and the apparent valence were not altered. GAT1 stoichiometry was determined by measuring GABA-evoked currents and the unidirectional influx of (36)Cl(-), (22)Na(+), or [(3)H]GABA. Uptake of each GABA molecule was accompanied by inward movement of 2 positive charges, which was entirely accounted for by the influx of Na(+) in the presence or absence of Cl(-). Thus, the GAT1 stoichiometry was 2Na(+):1GABA. However, Cl(-) was transported by GAT1 because the inward movement of 2 positive charges was accompanied by the influx of one Cl(-) ion, suggesting unidirectional influx of 2Na(+):1Cl(-):1GABA per transport cycle. Activation of forward Na(+)/Cl(-)/GABA transport evoked (36)Cl(-) efflux and was blocked by the inhibitor SKF 89976A. These data suggest a Cl(-)/Cl(-) exchange mechanism during the GAT1 transport cycle. In contrast, Cl(-) was not transported by SGLT1. Thus, in both GAT1 and SGLT1, Cl(-) modulates the kinetics of cotransport by altering Na(+) affinity, but does not contribute to net charge transported per transport cycle. We conclude that Cl(-) dependence per se is not a useful criterion to classify Na(+) cotransporters.     

cAMP-mediated regulation of murine intestinal/pancreatic Na+/HCO3- cotransporter subtype pNBC1

Basolateral Na(+)-HCO(3)(-) cotransport is essential for intestinal anion secretion, and indirect evidence suggests that it may be stimulated by a rise of intracellular cAMP. We therefore investigated the expression, activity, and regulation by cAMP of the Na(+)-HCO(3)(-) cotransporter isoforms NBC1 and NBCn1 in isolated murine colonic crypts. Na(+)-HCO(3)(-) transport rates were measured fluorometrically in BCECF-loaded crypts, and mRNA expression levels and localization were determined by semiquantitative PCR and in situ hybridization. Acid-activated Na(+)-HCO(3)(-) cotransport rates were 5.07 +/- 0.7 mM/min and increased by 62% after forskolin stimulation. NBC1 mRNA was more abundant in colonic crypts than in surface cells, and crypts expressed far more NBC1 than NBCn1. To investigate whether the cAMP-induced Na(+)-HCO(3)(-) cotransport activation was secondary to secretion-associated changes in HCO(3)(-) or cell volume, we measured potential forskolin-induced changes in intracellular pH and assessed Na(+)-HCO(3)(-) transport activity in CFTR -/- crypts (in which no forskolin-induced cell shrinkage occurs). We found 30% reduced Na(+)-HCO(3)(-) transport rates in CFTR -/- compared with CFTR +/+ crypts but similar Na(+)-HCO(3)(-) cotransport activation by forskolin. These studies establish the existence of an intracellular HCO(3)(-) concentration- and cell volume-independent activation of colonic NBC by an increase in intracellular cAMP.