Mechanism of glucose sensing in the small intestine.

Sensing nutrients is a fundamental task for all living cells. For most eukaryotic cells glucose is a major source of energy, having significant and varied effects on cell function. Interest in identifying mechanisms by which cells sense and respond to variations in glucose concentration has increased recently. The epithelial cells lining the intestinal tract are exposed, from the luminal domain, to an environment with continuous and massive fluctuations in the levels of dietary monosaccharides. Enterocytes therefore have to sense and respond to the significant changes in the levels of luminal sugars, and regulate the expression of the intestinal glucose transporter (Na+/glucose co-transporter, SGLT1) accordingly. Our data, using a combination of in vivo and in vitro model systems, suggest that glucose in the lumen of the intestine is sensed by a glucose sensor residing on the external face of the enterocyte luminal membrane. Glucose binds to the sensor and generates an intracellular signal leading to enhancement in the expression of SGLT1. The generated signal is independent of glucose metabolism and is likely to operate via a G-protein-coupled receptor and cAMP/protein kinase A signalling cascade.

Transcriptional regulation of the ovine intestinal Na+/glucose cotransporter SGLT1 gene. Role of HNF-1 in glucose activation of promoter function.

Dietary sugars D-glucose and D-galactose are transported across the intestinal brush-border membrane by the Na+/glucose cotransporter, SGLT1. In various species studied, it has been shown that the activity, and expression, of intestinal SGLT1 is regulated by dietary sugars. We report in this paper that regulation of the intestinal SGLT1 gene by lumenal sugar is due, in part, to an increase in transcription. Using deletion analyses of the -66/+21-bp fragment, we have identified the minimal region of the ovine SGLT1 promoter able to support transcription. Site-directed mutagenesis of the hepatic nuclear factor-1 (HNF-1) consensus motif within this domain eliminates basal promoter function. In addition, we show direct evidence for glucose-induced activation of the -66/+21-bp promoter region. There is a co-ordinated decline in the abundance of ovine intestinal HNF-1 and SGLT1 transcripts during transition from preruminant to adult ruminant. This decline is recovered after glucose infusion of adult sheep intestine. Similarly, as shown using DNA mobility-shift assays, the intensity of the HNF-1-binding complex to the target promoter sequence decreases during maturation of the animal; this is restored after intestinal sugar infusion. These data indicate that HNF-1 plays an important role in the glucose responsiveness of the ovine SGLT1 gene. This is the first report of in vitro glucose-induced activation of the intestinal SGLT1 promoter and identification of a glucose-responsive region of the ovine SGLT1 promoter.

PKC regulates turnover rate of rabbit intestinal Na+-glucose transporter expressed in COS-7 cells.

We have used the recombinant NH2-terminal myc-tagged rabbit Na+-glucose transporter (SGLT1) to study the regulation of this carrier expressed in COS-7 cells. Treatment of cells with a protein kinase C (PKC) agonist, phorbol 12-myristate 13-acetate (PMA), caused a significant decrease (38.03 +/- 0.05%) in methyl alpha-D-glucopyranoside transport activity that could not be emulated by 4alpha-phorbol 12,13-didecanoate. The decrease in sugar uptake stimulated by PMA was reversed by the PKC inhibitor bisindolylmaleimide I. The maximal rate of Na+-glucose cotransport activity (Vmax) was decreased from 1.29 +/- 0.09 to 0.85 +/- 0.04 nmol. min-1. mg protein-1 after PMA exposure. However, measurement of high-affinity Na+-dependent phloridzin binding revealed that there was no difference in the number of cell surface transporters after PMA treatment; maximal binding capacities were 1.54 +/- 0.34 and 1.64 +/- 0.21 pmol/mg protein for untreated and treated cells, respectively. The apparent sugar binding affinity (Michaelis-Menten constant) and phloridzin binding affinity (dissociation constant) were not affected by PMA. Because PKC reduced Vmax without affecting the number of cell surface SGLT1 transporters, we conclude that PKC has a direct effect on the carrier, resulting in a lowering of the transporter turnover rate by a factor of two.

Functional studies of the rabbit intestinal Na+/glucose carrier (SGLT1) expressed in COS-7 cells: evaluation of the mutant A166C indicates this region is important for Na+-activation of the carrier.

We have exploited two mutants of the rabbit intestinal Na+/glucose carrier SGLT1 to explore the structure/function relationship of this Na+/glucose transporter in COS-7 cells. A functional N-terminal myc-epitope-tagged SGLT1 protein was constructed and used to determine the plasma-membrane localization of SGLT1. The kinetic and specificity characteristics of the myc-tagged SGLT1 mutant were identical with those of wild-type SGLT1. Immunogold labelling and electron microscopy confirmed the topology of the N-terminal region to be extracellular. Expression of the SGLT1 A166C mutant in these cells showed diminished levels of Na+-dependent alpha-methyl-d-glucopyranoside transport activity compared with wild-type SGLT1. For SGLT1 A166C, Vmax was 0.92+/-0.08 nmol/min per mg of protein and Km was 0.98+/-0.13 mM; for wild-type SGLT1, Vmax was 1.98+/-0.47 nmol/min per mg of protein and Km was 0.36+/-0.16 mM. Significantly, phlorrhizin (phloridzin) binding experiments confirmed equal expression of Na+-dependent high-affinity phlorrhizin binding to COS-7 cells expressing SGLT1 A166C or wild-type SGLT1 (Bmax 1.55+/-0.18 and 1.69+/-0.57 pmol/mg of protein respectively); Kd values were 0.46+/-0.15 and 0.51+/-0.11 microM for SGLT1 A166C and wild-type SGLT1 respectively. The specificity of sugar interaction was unchanged by the A166C mutation. We conclude that the replacement of an alanine residue by cysteine at position 166 has a profound effect on transporter function, resulting in a decrease in transporter turnover rate by a factor of 2. Taken as a whole the functional changes observed by SGLT1 A166C are most consistent with the mutation having caused an altered Na+ interaction with the transporter.

An effect of Ca2+ on the Intrinsic Cl(-)-conductance of rat kidney cortex brush border membrane vesicles.

Brush-border membrane vesicles (BBMV) were prepared from superficial rat renal cortex by a divalent(2+)-precipitation technique using either CaCl2 or MgCl2. The dependence of the initial [14C]-D-glucose (or [3H]-L-proline) uptake rate and the extent of the overshoot of D-glucose or L-proline uphill accumulation from solutions containing 100 mM Na+ salt, was found to be dependent upon the precipitating divalent cation. With Mg2+ precipitation the initial uptake and overshoot accumulation of either D-glucose or L-proline were enhanced compared to BBMV prepared by Ca2+ precipitation. When the anion composition of the media was varied (uptake in Cl- media in comparison to gluconate(-)-containing media) it was found that the Cl(-)-dependent component of the initial uptake was markedly depressed with Ca(2+)-prepared BBMV (104.99 +/- 33.31 vs. 13.83 +/- 1.44 pmoles/sec/mg protein for Mg2+ and Ca2+ prepared vesicles respectively). When Ca2+ was loaded into Mg2+ prepared BBMV using a freeze-thaw technique, it was found that the magnitude and Cl- enhancement of D-glucose transport was reduced in a dose-dependent manner. Neomycin, an inhibitor of phospholipase C, had no effect on the reduction of D-glucose uptake by Ca2+ in Mg2+ prepared vesicles. In contrast, phosphatase inhibitors such as vanadate and fluoride were able to partially reverse the Ca2+ inhibition of D-glucose uptake and restore the enhancement due to Cl- media. In addition, inhibitors of protein phosphatase 2B, deltamethrin (50 nM) and trifluoperazine (10 microM), caused partial reversal of Ca2(+)-dependent inhibition of D-glucose uptake. Direct measurement of changes in the bi-ionic (Cl-vs. gluconate-) transmembrane electrical potential differences using the cyanine dye, 3,3'-dipropylthiodicarbocyanine iodide DiSC3-(5) confirmed that Cl- conductance was reduced in Ca(2+)-prepared vesicles. We conclude that a Cl- conductance coexists with Na+ cotransport in rat renal BBMV and this may be subject to negative regulation by Ca2+ via stimulation of protein phosphatase (PP2B).

Proton/solute cotransport in rat kidney brush-border membrane vesicles: relative importance to both D-glucose and peptide transport.

We have determined the relative importance of the transmembrane proton electrochemical gradient to the transport of D-[14C]glucose and [14C]glycylsarcosine (gly-sar) in rat kidney brush-border membrane vesicles (BBMV) from superficial renal cortex. Electrogenic [14C]gly-sar transport was first optimised by imposing a pH gradient (pHo = 5.7, pHi = 8.4) and an interior negative p.d. (using outwardly directed K+ gradient plus valinomycin). Under identical conditions (pHo = 5.7, pHi = 8.4), an acceleration of initial D-[14C]glucose (at 100 microM) transport by 2.0 +/- 0.7-fold was observed compared to no proton gradient (pHo = 8.4, pHi = 8.4). This increase was due primarily to an effect of external protons, since acidic conditions (pHo = pHi = 5.7) also resulted in acceleration of D-glucose influx (2-fold). The increase in D-glucose transport in the presence of external acidity was reduced by the uncoupler FCCP, even in the absence of a proton gradient. Furthermore, the increased D-glucose transport with external acidity in the presence of a proton gradient was insensitive to a K+ gradient-driven diffusion potential in the presence of valinomycin. In no instance was an overshoot accumulation of D-[14C]glucose observed in H+ gradient conditions. H(+)-stimulated D-[14C]glucose transport showed a linear dependence on D-glucose concentration up to 20 mM D-glucose, unlike electrogenic Na(+)-dependent D-glucose transport, whose Km was 1.77 +/- 0.35 mM. In contrast, the initial rate of [14C]gly-sar (100 microM) transport by the renal H+/di-tripeptide transporter was accelerated 15.7 +/- 3.3-fold and stimulated a marked overshoot of 5.1 +/- 0.4-fold over equilibrium values. Conversely, the electrogenic, Na+/glucose transporter could be readily demonstrated, whilst [14C]gly-sar transport could not be energised by an inward Na+ gradient. The absence of electrogenic D-glucose transport in H+ gradient conditions is clear evidence against H+/glucose cotransport in Na(+)-free conditions mediated by SGLT2 (sodium-glucose transporter, renal cortex). Furthermore, since a proton gradient does not increase brush-border membrane D-glucose uptake in Na(+)-rich media, it is unlikely that in vivo renal D-glucose transport mediated via SGLT2 may be energised by the transmembrane proton gradient.

Preparation and characterization of basolateral plasma-membrane vesicles from sheep parotid glands. Mechanisms of phosphate and D-glucose transport.

A procedure is described for the preparation of basolateral membrane vesicles from the acinar cells of the sheep parotid gland. The ouabain-sensitive K(+)-activated phosphatase activity was enriched 30-fold over the tissue homogenate; 45% of this activity was recovered in the final membrane fraction. The presence of membranes from other organelles was negligible. Evidence is presented for the location of Na(+)-dependent symporters for phosphate and D-glucose on the basolateral membrane.

Mechanisms of phosphate transport in sheep intestine and parotid gland: response to variation in dietary phosphate supply.

The transport of phosphate in intestinal brush-border membrane and parotid basolateral membrane vesicles isolated from sheep maintained on high and low phosphate diets have been studied. The mechanism of the transport of phosphate in the intestine is via a proton symporter whilst in the parotid gland it is effected by a Na+ coupled transporter. In sheep fed a low-P diet there is no change in the capacity of the parotid basolateral membrane to transport phosphate into the parotid end piece cells. This is in marked contrast to the response of the enterocyte brush-border membrane, where there is a significant enhancement of the capacity of the membrane to transport phosphate. We conclude that in sheep the gut appears to play a major role in response to phosphate deprivation, by increasing the capacity to transport phosphate. This enhancement is not achieved by increases in the levels of circulating 1,25-dihydroxycholecalciferol.