More than apical: distribution of SGLT1 in Caco-2 cells

Abstract

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 employing 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 approximately 2:1. Further immunocytochemical investigation of SGLT1 distribution in fixed Caco-2 cells by epifluorescence and confocal microscopy revealed that the intracellular compartments containing SGLT1 were associated with microtubules. 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 days 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 the regulation of SGLT1 abundance at the apical cell surface.

SGLT-1-mediated glucose uptake protects human intestinal epithelial cells against Giardia duodenalis-induced apoptosis

“…Stainings of SGLT-1 on the cell surface as well as in cytoplasmic pools were observed in control cells. This observation is consistent with previously described intracellular pools containing SGLT-1 in enterocytes ( [Chung et al, 2002a], [Chung et al, 2002b] and [Kipp et al, 2003]). Our findings indicate that the increased SGLT-1-specific sugar uptake triggered by exposure to G. duodenalis resulted from an augmentation of V max of the cotransporter, without changes in K m. These data indicate that G. duodenalis increased the maximal rate of SGLT-1 sugar transport (increased surface expression of transporter), whereas the affinity between the transporter and ligand was not altered.…”

“…Additional Western blot results showed that the total amount of SGLT-1 protein in epithelial cells was not increased postchallenge, suggesting that novel synthesis of the protein may not be involved in this phenomenon. Further evidence indicated that the increased expression of SGLT-1 on apical membranes of enterocytes requires intact cytoskeletal proteins and microtubules, in agreement with previous reports suggesting that intracellular compartments containing SGLT-1 are attached to microtubules (Kipp et al, 2003). In contrast, as cytochalasin D had no effect on SGLT-1 activation, actin filaments do not appear to be involved in this process.…”

“…The media was supplemented with 10% FBS, 15 mM Hepes, 100 U/ml Penicillin, 0.1 mg/ml Streptomycin (Sigma, St. Louis, MO) and 0.25 mg/ml Geneticin (Life technologies, Inc.) (Turner et al, 1996). None of the polarized intestinal epithelial cell lines available today express detectable levels of transepithelial Na + -dependent glucose transport, which has been attributed to a deficiency of surface expression of SGLT-1 ( [Turner et al, 1996] and [Kipp et al, 2003]). In these transfected cells, the expression of SGLT-1 protein on the apical membrane is associated with functional Na + -dependent uptake of glucose, and physiological transduction pathways in response to SGLT-1 activation ( [Turner et al, 1996], [Shiue et al, 2005] and [Hu et al, 2006]).…”

Three Surface Subdomains Form the Vestibule of the Na+/Glucose Cotransporter SGLT1

“…We demonstrated that antibodies against these regions coupled separately to AFM tips could interact specifically with the antigenic-binding epitopes of SGLT1. These results confirm the extracellular orientations of the regions of the subdomain I (aa 243-272), II (aa 339 -356), and III (aa 606 -630), which support the assumptions derived from previous studies (5-8, 33) and also from our immunohistochemistry labeling the surface of SGLT1-expressing cells (26). Furthermore, the binding probabilities of the three antibodies to SGLT1 are quite similar.…”

“…Immunopurified polyclonal antibodies QIS30 (subdomain I), PAN2-2 (subdomain II), and PAN3-2 (subdomain III) were raised against oligopeptides from the amino acid sequences aa 243-272 (loop 6 -7, i.e. the loop between the transmembrane helices 6 and 7), aa 339 -356 (loop 8 -9), and aa 606 -630 (loop 13-14) of rbSGLT1 (rabbit isoform), respectively, as described before (6,25,26). The specificity of the interaction of the antibodies with the supposed epitopes has been shown in independent previous immunohistochemistry studies where surface labeling of the cells could be prevented in the presence of the peptides used for their generation.…”