The PI's long term objectives are: 1) To determine the role of GLUT1 in mediating IGF1, TGFbeta1, PDGF, and D-glucose-induced extracellular matrix (ECM) synthesis in mesangial cells; and 2) To identify the specific growth factor (GF)- and glucose response elements in the GLUT1 gene which may mediate its transcription. The unifying hypothesis is that mesangial cell GLUT1 expression level is the determinant of ECM synthesis, and factors which regulate GLUT1 expression regulate ECM synthesis. If the hypothesis is confirmed, treatments targeting GLUT1 can be designed to prevent glomerulosclerosis.
Specific Aim 1 : To test the hypothesis that the growth factors IGF1, TGFbeta1, and PDGF stimulate 3HS-DOG uptake into normal rat mesangial cells, and that this correlates with ECM synthesis. The PI has demonstrated IGF1 stimulation of 3HS-DOG uptake but this has not yet been correlated with EMC synthesis.
Specific Aim 2 : To determine the mechanism(s) by which GLUT1 is recruited to transport glucose into the normal mesangial cell by the 3 growth factors, IGF1, TGFbeta1, and PDGF, and by D-glucose. GLUT1 translocation, activity, and expression in mesangial cells will be examined in response to each of these agents, using growth factor doses which give maximal glucose uptake rates in the experiments described above.
Specific Aim 3 : To test the hypothesis that overexpression and underexpression of GLUT1 will enhance and suppress respectively, GF- induced- and D-glucose-induced 3H2-DOG uptake and ECM and MCGT1AS GLUT1- underexpressing cells will be used here. These experiments have the potential to dissociate growth factor exposure from ECM synthesis if a change in GLUT1 is required for their effects.
Specific Aim 4 : To determine the mechanism(s) by which the 3 growth factors and D-glucose induce GLUT1 expression in mesangial cells, the PI will assess GLUT1 genes transcription, transcript stability, and peptide half-life in control and GLUT1-overexpressing mesangial cells.
Specific Aim 5 : To identify the growth factor- and glucose-responsive (G1RE) elements in the 5' flanking region and enhancers of the GLUT1 gene. Based upon the results obtained in Aim 4, the PI plans to analyze the GLUT1 gene for these cis-acting elements. They will be identified by transient transfection of control and GLUT1-overexpressing cells with the appropriate GLUT1-CAT reporter constructs and an internal standard beta- galactosidase reporter gene construct. Deletion mutation analysis of activated reporter constructs will locate the limiting sequences of these cis-acting elements.
