Expression of Na+-D-glucose cotransporter SGLT2 in rodents is kidney-specific and exhibits sex and species differences

With a novel antibody against the rat Na(+)-D-glucose cotransporter SGLT2 (rSGLT2-Ab), which does not cross-react with rSGLT1 or rSGLT3, the ～75-kDa rSGLT2 protein was localized to the brush-border membrane (BBM) of the renal proximal tubule S1 and S2 segments (S1 > S2) with female-dominant expression in adult rats, whereas rSglt2 mRNA expression was similar in both sexes. Castration of adult males increased the abundance of rSGLT2 protein; this increase was further enhanced by estradiol and prevented by testosterone treatment. In the renal BBM vesicles, the rSGLT1-independent uptake of [(14)C]-α-methyl-D-glucopyranoside was similar in females and males, suggesting functional contribution of another Na(+)-D-glucose cotransporter to glucose reabsorption. Since immunoreactivity of rSGLT2-Ab could not be detected with certainty in rat extrarenal organs, the SGLT2 protein was immunocharacterized with the same antibody in wild-type (WT) mice, with SGLT2-deficient (Sglt2 knockout) mice as negative control. In WT mice, renal localization of mSGLT2 protein was similar to that in rats, whereas in extrarenal organs neither mSGLT2 protein nor mSglt2 mRNA expression was detected. At variance to the findings in rats, the abundance of mSGLT2 protein in the mouse kidneys was male dominant, whereas the expression of mSglt2 mRNA was female dominant. Our results indicate that in rodents the expression of SGLT2 is kidney-specific and point to distinct sex and species differences in SGLT2 protein expression that cannot be explained by differences in mRNA.     

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.     

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%.     

Inhibition of renal Na(+)-Pi cotransporter by mercuric chloride: role of sulfhydryl groups.

We studied the role of sulfhydryl groups in Na(+)-Pi cotransport across the renal brush border membrane (BBM), using HgCl2, an agent which penetrates membranes freely. HgCl2 inhibited the initial Na(+)-dependent 32Pi transport in a dose-dependent manner (IC50 = 54 microM). Na(+)-independent transport was not affected. The inhibitory effect persisted under Na+ equilibrium-exchange conditions. Additionally, HgCl2 had no effect on the diffusional uptake of 22Na up to 1 min incubation. Exposure to HgCl2 had no effect on vesicle integrity as determined by osmotic shrinking experiments. BBM vesicle (BBMV) volume, determined by D-glucose equilibrium uptake, was not affected at low HgCl2 concentrations, but decreased at higher concentrations (greater than 100 microM). Vesicle volumes, determined by flow cytometry, were not changed after exposure to HgCl2. Kinetic studies showed a reduction in the apparent Vmax for Pi transport from 1.40 +/- 0.13 to 0.75 +/- 0.19 nmoles/mg protein/5 sec, without a significant change in the apparent Km. In protection studies, dithiothreitol (DTT) completely protected against inhibition, but Pi, phosphonoformic acid (PFA), and Na+ gave no protection. The data suggest that sulfhydryl groups are essential for the function of Na(+)-Pi cotransporter of renal BBM.     

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.     

Sugar binding residue affects apparent Na+ affinity and transport stoichiometry in mouse sodium/glucose cotransporter type 3B

SGLT1 is a sodium/glucose cotransporter that moves two Na(+) ions with each glucose molecule per cycle. SGLT3 proteins belong to the same family and are described as glucose sensors rather than glucose transporters. Thus, human SGLT3 (hSGLT3) does not transport sugar, but extracellular glucose depolarizes the cell in which it is expressed. Mouse SGLT3b (mSGLT3b), although it transports sugar, has low apparent sugar affinity and partially uncoupled stoichiometry compared with SGLT1, suggesting that mSGLT3b is also a sugar sensor. The crystal structure of the Vibrio parahaemolyticus SGLT showed that residue Gln(428) interacts directly with the sugar. The corresponding amino acid in mammalian proteins, 457, is conserved in all SGLT1 proteins as glutamine. In SGLT3 proteins, glutamate is the most common residue at this position, although it is a glycine in mSGLT3b and a serine in rat SGLT3b. To test the contribution of this residue to the function of SGLT3 proteins, we constructed SGLT3b mutants that recapitulate residue 457 in SGLT1 and hSGLT3, glutamine and glutamate, respectively. The presence of glutamine at residue 457 increased the apparent Na(+) and sugar affinities, whereas glutamate decreased the apparent Na(+) affinity. Moreover, glutamate transported more cations per sugar molecule than the wild type protein. We propose a model where cations are released intracellularly without the release of sugar from an intermediate state. This model explains the uncoupled charge:sugar transport phenotype observed in wild type and G457E-mSGLT3b compared with SGLT1 and the sugar-activated cation transport without sugar transport that occurs in hSGLT3.     

Normal molecular size of the Na(+)-phosphate cotransporter and normal Na(+)-dependent binding of phosphonoformic acid in renal brush border membranes of X-linked Hyp mice

X-linked Hyp mice have a specific defect in Na(+)-dependent phosphate (Pi) transport at the renal brush border membrane (BBM). In the present study we examined the effect of the Hyp mutation on the molecular size of the Pi transporting unit and on Na(+)-dependent 14C-phosphonoformic (PFA) binding in renal BBM vesicles. By radiation inactivation analysis, we demonstrated that the molecular size of the Na(+)-Pi cotransporter is similar in normal (242 +/- 16 kDa) and Hyp mice (227 +/- 39 kDa). Moreover, while BBM Na(+)-dependent Pi transport is significantly reduced in Hyp mice (249 +/- 54 vs 465 +/- 82 pmol/mg protein/6s), genotype differences in (1) Na(+)-dependent PFA binding (1020 +/- 115 vs 1009 +/- 97 pmol/mg protein/30 min), (2) Pi-displaceable Na(+)-dependent PFA binding (605 +/- 82 vs 624 +/- 65 pmol/mg protein/6s), and (3) phosphate uptake at Na(+)-equilibrium (67 +/- 10 vs 54 +/- 7 pmol/mg protein/6s) are not apparent. The present data demonstrate that the molecular size of the renal BBM Na(+)-Pi cotransporter is normal in Hyp mice and suggest that the number of Na(+)-Pi cotransporters may not be reduced in the mutant strain.     

Phosphatidylinositol 3-kinase mediates inhibitory effect of angiotensin II on sodium/glucose cotransporter in renal epithelial cells

Effects of angiotensin II (ANGII) on regulation of sodium/glucose cotransporter (SGLT1) activity were investigated in LLC-PK(1) cells, renal proximal epithelial cell line. ANGII inhibited alpha-[14C] methyl-D-glucopyranoside (AMG) uptake into LLC-PK(1) cells in a dose-dependent manner. This inhibition was based on a decrease in maximal transport rate (Vmax) of AMG from 2.20 nmol/mg protein/15 min to 1.19 nmol/mg protein/15 min, although apparent affinity constant (Km) did not alter. In western blot analysis, protein level of SGLT1 in brush border membrane (BBM) was decreased by ANGII, although total SGLT1 was not altered. In the aspect of intracellular signal transduction, ANGII blocked the formation of cAMP. Pertussis toxin, an inactivator of Gi protein that control intracellular cAMP level, completely prevented the decrease of AMG uptake caused by ANGII. 8-Br-cAMP, a cell membrane permeable cAMP analogue, increased AMG uptake and protein level of SGLT1 in BBM. Both wortmannin and LY294002 that are phosphatidylinositol (PI) 3-kinase inhibitors, inhibited the SGLT1 activity, and also attenuated the effect of 8-Br-cAMP on SGLT1 activity. Those inhibitors prevented the 8-Br-cAMP-induced expression of SGLT1 in plasma membrane. We conclude that ANGII plays an important role in post-translational regulation in SGLT1. Inhibition of SGLT1 translocation is suggested to be caused by inactivation of protein kinase A and decrease of PI 3-kinase activity.     

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.     

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.     

Functional studies of a chimeric protein containing portions of the Na(+)/glucose and Na(+)/myo-inositol cotransporters

We obtained cDNA chimeras between Na/glucose cotransporter (SGLT1) and the homologous Na(+)/myo-inositol cotransporter (SMIT) by creating random chimeras in plasmids. Of 12 chimeras, two were functional when expressed in Xenopus laevis oocytes but, upon sequencing, only one of them (C1) produced an actual chimeric protein. In C1, the first 69 amino acids of SGLT1 were replaced by the corresponding 50 amino acids of SMIT. C1 transports the same sugars as does SGLT1. C1's affinity for all sugar substrates was systematically increased by a factor of 3.3+/-0.4 but the V(max) was diminished by a factor of 15-40. In contrast, the cotransport affinity for Na(+) was unchanged. The surface expression of C1 was one seventh that of SGLT1, which explains part of the reduced V(max) and implies a significant reduction in turnover rate. N-terminal truncated constructs of SGLT1 cDNA showed that deleting amino acids 2-14 does not affect cotransporter activity, but that the pentapeptide T(14)RPVET(19) is important for normal levels of SGLT1 current. The main result of a kinetic analysis of the systematic increase in apparent affinity for sugars, together with the intact Na apparent affinity, suggests enhanced access to the sugar binding site in C1.     

Apical Na+-D-glucose cotransporter 1 (SGLT1) activity and protein abundance are expressed along the jejunal crypt-villus axis in the neonatal pig

Gut apical Na(+)-glucose cotransporter 1 (SGLT1) activity is high at the birth and during suckling, thus contributing substantially to neonatal glucose homeostasis. We hypothesize that neonates possess high SGLT1 maximal activity by expressing apical SGLT1 protein along the intestinal crypt-villus axis via unique control mechanisms. Kinetics of SGLT1 activity in apical membrane vesicles, prepared from epithelial cells sequentially isolated along the jejunal crypt-villus axis from neonatal piglets by the distended intestinal sac method, were measured. High levels of maximal SGLT1 uptake activity were shown to exist along the jejunal crypt-villus axis in the piglets. Real-time RT-PCR analyses showed that SGLT1 mRNA abundance was lower (P < 0.05) by 30-35% in crypt cells than in villus cells. There were no significant differences in SGLT1 protein abundances on the jejunal apical membrane among upper villus, middle villus, and crypt cells, consistent with the immunohistochemical staining pattern. Higher abundances (P < 0.05) of total eukaryotic initiation factor 4E (eIF4E) protein and eIE4E-binding protein 1 γ-isoform in contrast to a lower (P < 0.05) abundance of phosphorylated (Pi) eukaryotic elongation factor 2 (eEF2) protein and the eEF2-Pi to total eEF2 abundance ratio suggest higher global protein translational efficiency in the crypt cells than in the upper villus cells. In conclusion, neonates have high intestinal apical SGLT1 uptake activity by abundantly expressing SGLT1 protein in the epithelia and on the apical membrane along the entire crypt-villus axis in association with enhanced protein translational control mechanisms in the crypt cells.     

Voltage and substrate dependence of the inverse transport mode of the rabbit Na(+)/glucose cotransporter (SGLT1)

Properties of the cytoplasmic binding sites of the rabbit Na(+)/glucose cotransporter, SGLT1, expressed in Xenopus oocytes were investigated using the giant excised patch clamp technique. Voltage and substrate dependence of the outward cotransport were studied using alpha-methyl D-glucopyranoside (alphaMDG) as a substrate. The apparent affinity for alphaMDG depends on the cytoplasmic Na(+) concentration and voltage. At 0 mV the K(M) for alphaMDG is 7 mM at 110 mM Na(+) and 31 mM at 10 mM Na(+). The apparent affinity for alphaMDG and Na(+) is voltage dependent and increases at positive potentials. At 0 mV holding potential the outward current is half-maximal at about 70 mM. The results show that SGLT1 can mediate sugar transport out of the cell under appropriate concentration and voltage conditions, but under physiological conditions this transport is highly improbable due to the low affinity for sugar.     

Differential regulation of three glucose transporter genes in a renal epithelial cell line

Three hexose transporter genes, the Na(+)/glucose cotransporters SGLT1 and SGLT3 (formerly SAAT1/pSGLT2) and the facilitative transporter GLUT1, are expressed in a renal epithelial cell line with proximal tubule characteristics. A number of studies have demonstrated that SGLT1 expression is coupled to the cellular differentiation state and is also negatively regulated by its substrate glucose. In the present study, we demonstrate that SGLT3 mRNA expression is relatively unaffected by conditions promoting dedifferentiation (reseeding to a subconfluent density, activation of protein kinase C) or differentiation (confluent cell density, activation of protein kinase A) nor was expression sensitive to hyperglycemic glucose levels in the medium. We further demonstrate that protein kinase A and protein kinase C exert opposing effects on GLUT1 and SGLT1 mRNA levels in polarized cell monolayers, indicating that GLUT1 mRNA is also highly regulated in polarized epithelial cells by agents affecting cell differentiation. The relatively constitutive expression of SGLT3 mRNA suggests a novel role for this low-affinity Na(+)/glucose cotransporter, to provide concentrative glucose uptake under hyperglycemic conditions where expression of high-affinity glucose cotransporter SGLT1 mRNA is significantly downregulated.