The overall aim of the project is to dissect the mechanism by which insulin increase the transport of glucose into cultured 3T3-F442A fat cells. This will be achieved by modifying certain structural features of the predominant insulin-sensitive glucose carrier in these cells (IRGT) and assessing effects on its functional mechanism and regulation. The transport and regulatory features unique to the IRGT will be determined by comparison to another type of glucose carrier also present in these cells, but which predominates in insulin-insensitive cell types, such as erythrocytes and brain. Initial studies will involve comparison of transport kinetics between the two carrier subtypes under conditions in which one subtype is alternatively absent or blocked. Selected structural modifications will then be made, and the resulting transport, biochemical behavior and responsiveness to insulin of the two subtypes contrasted. Methods for biochemically distinguishing between subtypes will include cell-surface labeling, Northern blot analysis with carrier-specific cDNA probes, and immunoprecipitation and immunoblotting with carrier-specific antibodies. The planned structural dissection of the IRGT will include 1) analysis of its transmembrane loop structure by susceptibility to proteolytic cleavage and by accessibility to membrane-impermeant labeling reagents; 2) investigation of whether the IRGT, like the erythrocyte carrier protein, is linked directly to the plasma membrane through a fatty acyl-thiol ester and if so, whether loss of such an anchoring mechanism affects its function or regulation by insulin; 3) determination of whether the IRGT-selective effects of the sulfhydryl reagent phenylarsine oxide are due to direct reaction with the IRGT, or to indirect effects on insulin-stimulated recruitment of carriers to the plasma membrane; and 4) assessment of whether and how phosphorylation of the IRGT or associated proteins affects insulin-stimulated transport. The specific components of insulin-stimulated transport to be clarified by this approach include: the relative contributions of intrinsic activation and translocation of the IRGT from internal sites to the plasma membrane in response to insulin, and whether unique structural features of the IRGT contribute substantially to its sensitivity to regulation by insulin and other agents. By increasing the understanding of how the structure of the IRGT contributes to its function and regulation, these results may also be relevant to its study in clinical situations including exercise, and insulin resistant and insulin-deficient states.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK038794-05
Application #
3238297
Study Section
Metabolism Study Section (MET)
Project Start
1986-09-01
Project End
1994-06-30
Budget Start
1992-07-01
Budget End
1993-06-30
Support Year
5
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Whitesell, R R; Ward, M; McCall, A L et al. (1995) Coupled glucose transport and metabolism in cultured neuronal cells: determination of the rate-limiting step. J Cereb Blood Flow Metab 15:814-26
Due, A D; Qu, Z C; Thomas, J M et al. (1995) Role of the C-terminal tail of the GLUT1 glucose transporter in its expression and function in Xenopus laevis oocytes. Biochemistry 34:5462-71
Due, A D; Cook, J A; Fletcher, S J et al. (1995) A ""cysteineless"" GLUT1 glucose transporter has normal function when expressed in Xenopus oocytes. Biochem Biophys Res Commun 208:590-6
Morita, H; Yano, Y; Niswender, K D et al. (1994) Coexpression of glucose transporters and glucokinase in Xenopus oocytes indicates that both glucose transport and phosphorylation determine glucose utilization. J Clin Invest 94:1373-82
May, J M; Qu, Z C; Beechem, J M (1993) Tryptic digestion of the human erythrocyte glucose transporter: effects on ligand binding and tryptophan fluorescence. Biochemistry 32:9524-31
Yano, Y; May, J M (1993) Ligand-induced conformational changes modify proteolytic cleavage of the adipocyte insulin-sensitive glucose transporter. Biochem J 295 ( Pt 1):183-8
May, J M; Beechem, J M (1993) Monitoring conformational change in the human erythrocyte glucose carrier: use of a fluorescent probe attached to an exofacial carrier sulfhydryl. Biochemistry 32:2907-15
May, J M (1991) The one-site model of human erythrocyte glucose transport: testing its predictions using network thermodynamic computer simulations. Biochim Biophys Acta 1064:1-6
May, J M; Buchs, A; Carter-Su, C (1990) Localization of a reactive exofacial sulfhydryl on the glucose carrier of human erythrocytes. Biochemistry 29:10393-8
May, J M (1989) Inhibition of hexose transport in the human erythrocyte by 5, 5'-dithiobis(2-nitrobenzoic acid): role of an exofacial carrier sulfhydryl group. J Membr Biol 108:227-33

Showing the most recent 10 out of 16 publications