title: Insulin regulation of hexose transport in mouse 3T3-L1 cells expressing the human HepG2 glucose transporter.
abstract: Complementary DNA encoding a HepG2 cell-facilitated glucose transporter (GLUT1) was subcloned into a metal-inducible, mammalian expression vector, pLEN. Mouse 3T3-L1 fibroblasts transfected with this new construct, pLENGT, exhibited zinc-inducible expression of human glucose transporter mRNA, protein, and glucose transport activity, before and after differentiation into adipocytes. Both mouse host GLUT1 and expressed human GLUT1 proteins distributed about equally between 3T3-L1 adipocyte plasma membranes and low density microsomal membranes, while host skeletal muscle/adipocyte-type glucose transporter (GLUT4) was concentrated in the latter fraction. Mouse GLUT1 and GLUT4 proteins and the constitutively expressed human GLUT1 protein in pLENGT adipocytes were all redistributed from low density microsomal membrane to plasma membrane fractions in response to insulin. Insulin stimulated 2-deoxyglucose uptake in untransfected fibroblasts about 2-fold, while untransfected adipocytes displayed a 14-fold increase in deoxyglucose uptake in response to insulin. Both the expression of human GLUT1 protein and basal 2-deoxyglucose uptake by 75 microM zinc-treated pLENGT fibroblasts and adipocytes were increased approximately 3-fold over untransfected cells. In such pLENGT fibroblasts expressing human GLUT1 protein, however, the absolute values for insulin-stimulated increases in sugar uptake were no different than in control fibroblasts. As was observed in pLENGT fibroblasts, the increased basal sugar uptake by pLENGT adipocytes was additive with the insulin-stimulated increase in the rate of sugar uptake and, therefore, the -fold stimulation by insulin was markedly reduced. These data indicate that: 1) the membrane distributions of a glucose transporter protein, which is not responsive to insulin in HepG2 cells, and both mouse GLUT1 and GLUT4 glucose transporter isoforms are regulated by insulin in mouse 3T3-L1 adipocytes, and 2) the expressed human GLUT1 appears to contribute significantly to the rate of basal uptake but not to the insulin-stimulated increase in 2-deoxyglucose uptake by 3T3-L1 fibroblasts and adipocytes.
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester 01605.
authors.forename: S A
authors.initials: SA
authors.lastname: Harrison
authors.forename: J M
authors.initials: JM
authors.lastname: Buxton
authors.forename: B M
authors.initials: BM
authors.lastname: Clancy
authors.forename: M P
authors.initials: MP
authors.lastname: Czech
mesh_terms: D000273:Adipose Tissue / Q000378:metabolism; D000818:Animals; D006528:Carcinoma, Hepatocellular; D002454:Cell Differentiation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003847:Deoxyglucose / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D008113:Liver Neoplasms; D051379:Mice; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection; D015032:Zinc / Q000494:pharmacology
chemical_list: D007328:Insulin; D009004:Monosaccharide Transport Proteins; D012333:RNA, Messenger; D003847:Deoxyglucose; D015032:Zinc