Na(+)-D-glucose cotransporter SGLT1 is pivotal for intestinal glucose absorption and glucose-dependent incretin secretion.

To clarify the physiological role of Na(+)-D-glucose cotransporter SGLT1 in small intestine and kidney, Sglt1(-/-) mice were generated and characterized phenotypically. After gavage of d-glucose, small intestinal glucose absorption across the brush-border membrane (BBM) via SGLT1 and GLUT2 were analyzed. Glucose-induced secretion of insulinotropic hormone (GIP) and glucagon-like peptide 1 (GLP-1) in wild-type and Sglt1(-/-) mice were compared. The impact of SGLT1 on renal glucose handling was investigated by micropuncture studies. It was observed that Sglt1(-/-) mice developed a glucose-galactose malabsorption syndrome but thrive normally when fed a glucose-galactose-free diet. In wild-type mice, passage of D-glucose across the intestinal BBM was predominantly mediated by SGLT1, independent the glucose load. High glucose concentrations increased the amounts of SGLT1 and GLUT2 in the BBM, and SGLT1 was required for upregulation of GLUT2. SGLT1 was located in luminal membranes of cells immunopositive for GIP and GLP-1, and Sglt1(-/-) mice exhibited reduced glucose-triggered GIP and GLP-1 levels. In the kidney, SGLT1 reabsorbed ∼3% of the filtered glucose under normoglycemic conditions. The data indicate that SGLT1 is 1) pivotal for intestinal mass absorption of d-glucose, 2) triggers the glucose-induced secretion of GIP and GLP-1, and 3) triggers the upregulation of GLUT2

Trafficking of canalicular ABC transporters in hepatocytes

ATP-binding cassette (ABC) transporters located in the hepatocyte canalicular membrane of mammalian liver are critical players in bile formation and detoxification. Although ABC transporters have been well characterized functionally, only recently have several canalicular ABC transporters been cloned and their molecular nature revealed. Subsequently, development of specific antibodies has permitted a detailed investigation of ABC transporter intrahepatic distribution under varying physiological conditions. It is now apparent that there is a complex array of ABC transporters in hepatocytes. ABC transporter molecules reside in intrahepatic compartments and are delivered to the canalicular domain following increased physiological demand to secrete bile. Insufficient amounts of ABC transporters in the bile canalicular membrane result in cholestasis (i.e., bile secretory failure). Therefore, elucidation of the intrahepatic pathways and regulation of ABC transporters may help to understand the cause of cholestasis at a molecular level and provide clues for novel therapies

Dynamics of GFP-SGLT1 in COS-7 Cells

We recently showed that in Caco-2 cells the high affinity Na+-/Dglucose cotransporter (SGLTl) resides in intracellular endosomes which are attached to microtubules and therefore proposed that the activity of SGLTl is regulated by intracellular trafficking (Kipp et al., 2003). To visualize the dynamics of the transporter, we expressed a GFP-SGLT1 fusion in COS-7 cells. Using fluorescence microscopy, we discovered the GFP-SGLT1 protein in vesicular stmctures resembling those of endogenous SGLTl in Caco-2 cells. We were able to show the SGLT1-containing vesicles moving rapidly within COS-7 cells transiently expressing GFP-SGLT1 by means of time-lapse fluorescence microscopy. This movement was completely abolished by the disruption of the microtubule network with nocodazole. Next, we compared the features of the SGLTl vesicles and vesicles containing the transferrin receptor (TR). Therefore we incubated COS-7 cells expressing GFP-SGLT1 with Alexa Fluor 546-transferrin to compare the localization and the trafficking of SGLTl and the TR. Fluorescence microscopy of these specimens revealed a localization of the TR in vesicular structures that do not colocalize with SGLT1-vesicles and move with a higher speed. Our presented results confirm a vesicular localization of SGLTl and display the microtubule-dependent transport of these vesicles in hing cells. The mechanism of SGLTl endo-/exocytosis is clearly distinguishable from the transport of the transferrin recepto

In Caco-2 cells, most of the "Apical" SGLT1 resides in intracellular, microtubuli-associated vesicles

We investigated the distribution of the endogenous sodium/D-glucose cotransporter (SGLT1) in polarized Caco-2 cells, a model for enterocytes. A cellular organelle fraction was separated by free flow electrophoresis and subjected to the analysis of endogenous and exogenous marker enzymes for various membrane vesicle components. Furthermore, the presence of SGLT1 was tested by an ELISA assay using newly developed epitope-specific antibodies. Thereby it was found that the major amount of SGLT1 resided in intracellular compartments and only a minor amount in apical plasma membranes. The distribution ratio between intracellular SGLT1 and apical membrane-associated SGLT1 was ~2:1. Further immunohistochemical investigation of SGLT1 distribution in fixed Caco-2 cells by epifluorescence and confocal microscopy revealed that the intracellular compartments containing SGLT1 were associated with microtubuli. Elimination of SGLT1 synthesis by incubation of cells with cycloheximide did not significantly reduce the size of the intracellular SGLT1 pool. Furthermore, the half-life of SGLT1 in Caco-2 cells was determined to be 2.5 d by metabolic labeling followed by immunoprecipitation. Our data suggest that most of the intracellular SGLT1 are not transporters en route from biosynthesis to their cellular destination, but represent an intracellular reserve pool. We therefore propose that intracellular compartments containing SGLT1 are involved in an endo-/exocytosis process, which regulates SGLT1 abundance at the apical cell surface