We have examined the expression and function of a previously undescribed human member (SGLT3͞SLC5A4) of the sodium͞glu-cose cotransporter gene family (SLC5) that was first identified by the chromosome 22 genome project. The cDNA was cloned and sequenced, confirming that the gene coded for a 659-residue protein with 70% amino acid identity to the human SGLT1. RT-PCR and Western blotting showed that the gene was transcribed and mRNA was translated in human skeletal muscle and small intestine. Immunofluorescence microscopy indicated that in the small intestine the protein was expressed in cholinergic neurons in the submucosal and myenteric plexuses, but not in enterocytes. In skeletal muscle SGLT3 immunoreactivity colocalized with the nicotinic acetylcholine receptor. Functional studies using the Xenopus laevis oocyte expression system showed that hSGLT3 was incapable of sugar transport, even though SGLT3 was efficiently inserted into the plasma membrane. Electrophysiological assays revealed that glucose caused a specific, phlorizin-sensitive, Na ؉ -dependent depolarization of the membrane potential. Uptake assays under voltage clamp showed that the glucose-induced inward currents were not accompanied by glucose transport. We suggest that SGLT3 is not a Na ؉ ͞glucose cotransporter but instead a glucose sensor in the plasma membrane of cholinergic neurons, skeletal muscle, and other tissues. This points to an unexpected role of glucose and SLC5 proteins in physiology, and highlights the importance of determining the tissue expression and function of new members of gene families.Na͞sugar cotransporter ͉ human SGLT3 ͉ muscle

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The product of the intronless single copy gene RSC1A1, named RS1, is an intracellular 617-amino-acid protein that is involved in the regulation of the Na ؉ -D-glucose cotransporter SGLT1. We generated and characterized RS1 knockout (RS1 ؊/؊ ) mice. In the small intestines of RS1 ؊/؊ mice, the SGLT1 protein was up-regulated sevenfold compared to that of wild-type mice but was not changed in the kidneys. The upregulation of SGLT1 was posttranscriptional. Small intestinal D-glucose uptake measured in jointly perfused small bowel and liver was increased twofold compared to that of the wild-type, with increased peak concentrations of D-glucose in the portal vein. At birth, the weights of RS1 ؊/؊ and wild-type mice were similar. At the age of 3 months, male RS1 ؊/؊ mice had 5% higher weights and 15% higher food intakes, whereas their energy expenditures and serum leptin concentrations were similar to those of wild-type mice. At the age of 5 months, male and female RS1 ؊/؊ mice were obese, with 30% increased body weight, 80% increased total fat, and 30% increased serum cholesterol. At this age, serum leptin was increased, whereas food intake was the same as for wild-type mice. The data suggest that the removal of RS1 leads to leptin-independent up-regulation of food intake, which causes obesity.Glucose absorption in the small intestine is essential for energy supply through carbohydrates. It is mediated by two transporters in the enterocytes, the sodium-dependent D-glucose cotransporter SGLT1 in the brush border membrane and the sodium-independent glucose transporter GLUT2 in the basolateral membrane (11). Na ϩ -D-glucose cotransporter expression and activity in the small intestine exhibits circadian periodicity and is increased following a carbohydrate-rich diet (5, 27). The regulation of SGLT1 can be mediated by adrenergic innervation, insulin, glucagon 37, glucagon-like peptide 2, and cholecystokinin (2,13,14,29,30). SGLT1 may be regulated by changes in transcription (23, 35), mRNA stability (21), endocytosis (12), and transport activity within the plasma membrane (34).Previously, several related 67-kDa polypeptides from humans, pigs, and rabbits, termed RS1, which show about 70% amino acid identity and are involved in the regulation of SGLT1, were cloned (17,18,26,36). The RS1 polypeptides are encoded by intronless single copy genes (RSC1A1 on chromosome 1p36.1 in humans). These genes are expressed in many cell types, including small intestinal enterocytes and renal proximal tubular cells (18,26,36). RS1 contains consensus sequences for protein kinase C and casein kinase II and a ubiquitin-associated domain that is conserved between different species (33). The RS1 protein is localized intracellularly and associated with the plasma membrane (33).Coexpression experiments with Xenopus laevis oocytes showed that human RS1 (hRS1) is involved in posttranscriptional down-regulation of hSGLT1 (18,26,36,37). The downregulation of hSGLT1 by hRS1 was dynamin dependent and increased by activation of protein kinase C (PKC) (37)...

Previously we cloned membrane associated (M(r) 62000-67000) polypeptides from pig (pRS1), rabbit (rbRS1) and man (hRS1) which modified transport activities that were expressed in Xenopus laevis oocytes by the Na(+)-D-glucose cotransporter SGLT1 and/or the organic cation transporter OCT2. These effects were dependent on the species of RS1 and on the target transporters. hRS1 and rbRS1 were shown to be intronless single copy genes which are expressed in various tissues and cell types. Earlier immunohistochemical data with a monoclonal IgM antibody suggested an extracellular membrane association of RS1. In the present paper antibodies against recombinant pRS1 were raised and the distribution and membrane localization of RS1 reevaluated. After subcellular fractionation of renal cortex RS1 was found associated with brush border membranes and an about 1:200 relation between RS1 and SGLT1 protein was estimated. Also after overexpression in X. laevis oocytes RS1 was associated with the plasma membrane, however, at variance to the kidney it was also observed in the cytosol. Labeling experiments with covalently binding lipid-permeable and lipid-impermeable biotin analogues showed that RS1 is localized at the inner side of the plasma membrane. Western blots with plasma membranes from Xenopus oocytes revealed that SGLT1 protein in the plasma membrane was reduced when hRS1 was coexpressed with human SGLT1 which leads to a reduction in V(max) of expressed glucose transport. Measurements of membrane capacitance and electron microscopic inspection showed that the expression of hRS1 leads to a reduction of the oocyte plasma membrane surface. The data suggest that RS1 is an intracellular regulatory protein that associates with the plasma membrane. Overexpression of RS1 may effect the incorporation and/or retrieval of transporters into the plasma membrane.