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Photolabelling of the liver-type glucose-transporter isoform GLUT2 with an azitrifluoroethylbenzoyl-substituted bis-D-mannose.

ABSTRACT: The bis-D-mannose photolabel ATB-BMPA (2-N-[4-(1-azi-2,2,2- trifluoroethyl)benzoyl]-1,3-bis-(D-mannos-4-yloxy) propyl-2-amine) has been used to radiolabel the glucose transporter present in liver plasma membranes. The labelling was inhibited by 4,6-O-ethylidene-D-glucose. Approx. 7% of the liver plasma-membrane protein that was photolabelled in a 4,6-O-ethylidene-D-glucose-inhibitable manner was specifically immunoprecipitated by either an anti-(GLUT2 C-terminal peptide) antibody or by an anti-(GLUT2 exofacial-loop peptide) antibody. After correction for non-specific labelling and precipitation, the ratio of immunoprecipitable GLUT2 to GLUT1 was approximately 5:1, suggesting that GLUT1 was not a major component of liver plasma membranes. The low levels of immunoprecipitation of the photolabelled transporter may be due to low antibody affinity for GLUT2 or may indicate that the photolabelling reagent has labelled another glucose-transporter-like protein. The hexose-transport inhibitors phloretin, cytochalasin B and 4,6-O-ethylidene-D-glucose all inhibited the photolabelling by ATB-BMPA of immunoprecipitable GLUT2. D-Glucose inhibited approx. 57% of the ATB-BMPA labelling of GLUT2. D-Fructose also inhibited the GLUT2 labelling confirming that it is a substrate for GLUT2 [Gould, Thomas, Jess & Bell (1991) Biochemistry 30, 5139-5145]. From photolabel displacement by a range of concentrations of non-labelled ATB-BMPA, the affinity constant (Kd) of ATB-BMPA was found to be 250 +/- 78 microM, whereas the Bmax. (total number of binding sites) value was 2.1 +/- 0.29 pmol of GLUT2/mg of membrane protein. Since GLUT1, GLUT4 and GLUT2 have approximately equal affinities for the external ligand ATB-BMPA, but have widely varying affinities for equilibrated and transported substrates, it is suggested that the isoforms may differ in their ability to bind hexoses at the internal site.

SUBMITTER: Jordan NJ

PROVIDER: S-EPMC1132949 | biostudies-other | 1992 Sep

REPOSITORIES: biostudies-other

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Project description:The liver-specific expression of the GLUT2 glucose transporter gene is suppressed in cultured hepatoma cell lines as well as in hepatocytes in primary culture. To understand the underlying mechanism involved in this process, we analysed the rat GLUT2 promoter region. A DNase I footprinting assay with rat liver nuclear extract revealed eight protected regions within a -500 bp region of the GLUT2 promoter (sites A to H). Three of these sites (B, F and H) were occupied by transcription factors that are considerably enriched in liver cells compared with spleen or kidney. The proteins binding to these sites were investigated by a combination of DNase I footprinting assay and electrophoretic mobility-shift assay with the addition of specific oligonucleotide competitors and specific antibody against known transcription factors. As a result it was revealed that hepatocyte nuclear factor 3 binds to site B (-120 to -70), and CCAAT/enhancer binding protein alpha (C/EBPalpha) and C/EBPbeta bind to site F (-375 to -356) and site H (-500 to -471). The binding of C/EBP to sites F and H was markedly decreased within 4 h when liver cells were subjected to primary culture, suggesting that C/EBP might be responsible for the decreased expression of GLUT2 in this process. In contrast, Western blot analysis revealed that C/EBPalpha began to decrease after 1 h of hepatocyte culture, and C/EBPbeta was not changed significantly throughout the culture period, suggesting that C/EBP could be regulated at the transcriptional level as well as the post-translational level when hepatocytes were put in culture. To confirm the role of C/EBP in the regulation of GLUT2 promoter activity, sites F and H were ligated to a chloramphenicol acetyltransferase (CAT) reporter gene and co-transfected with a C/EBP expression vector into HepG2 cells. The co-expression of C/EBPalpha and C/EBPbeta resulted in 9.1-fold and 3. 8-fold increases of CAT activities in the site F-CAT and site H-CAT constructs respectively. These results indicate that C/EBPalpha and C/EBPbeta regulate the promoter activity of the GLUT2 gene and might be responsible for the down-regulation of the GLUT2 gene when hepatocytes are subjected to primary culture.

Project description:Insulin stimulates glucose transport in fat and skeletal muscle cells primarily by inducing the translocation of GLUT4 (glucose transporter isoform 4) to the PM (plasma membrane) from specialized GSVs (GLUT4 storage vesicles). Glycosphingolipids are components of membrane microdomains and are involved in insulin-regulated glucose transport. Cellular glycosphingolipids decrease during adipocyte differentiation and have been suggested to be involved in adipocyte function. In the present study, we investigated the role of glycosphingolipids in regulating GLUT4 translocation. We decreased glycosphingolipids in 3T3-L1 adipocytes using glycosphingolipid synthesis inhibitors and investigated the effects on GLUT4 translocation using immunocytochemistry, preparation of PM sheets, isolation of GSVs and FRAP (fluorescence recovery after photobleaching) of GLUT4-GFP (green fluorescent protein) in intracellular structures. Glycosphingolipids were located in endosomal vesicles in pre-adipocytes and redistributed to the PM with decreased expression at day 2 after initiation of differentiation. In fully differentiated adipocytes, depletion of glycosphingolipids dramatically accelerated insulin-stimulated GLUT4 translocation. Although insulin-induced phosphorylation of IRS (insulin receptor substrate) and Akt remained intact in glycosphingolipid-depleted cells, both in vitro budding of GLUT4 vesicles and FRAP of GLUT4-GFP on GSVs were stimulated. Glycosphingolipid depletion also enhanced the insulin-induced translocation of VAMP2 (vesicle-associated membrane protein 2), but not the transferrin receptor or cellubrevin, indicating that the effect of glycosphingolipids was specific to VAMP2-positive GSVs. Our results strongly suggest that decreasing glycosphingolipid levels promotes the formation of GSVs and, thus, GLUT4 translocation. These studies provide a mechanistic basis for recent studies showing that inhibition of glycosphingolipid synthesis improves glycaemic control and enhances insulin sensitivity in animal models of Type 2 diabetes.

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Photolabelling of the liver-type glucose-transporter isoform GLUT2 with an azitrifluoroethylbenzoyl-substituted bis-D-mannose.

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