title: Characterisation of glucose transport in rat aortic vascular smooth muscle cells
Creators: name: Wakefield, Jill Margaret
Publisher: name: University of Glasgow
Year: 1999

description: Cardiovascular disease is the principal cause of death in the Western world. Atherosclerosis is a major contributor to the progression of cardiovascular disease. Increased proliferation of vascular smooth muscle cells plays an important role in the development of atherosclerotic plaques and, as increased glucose transport is an early response common to all mitogens, it is reasonable to presume that increased proliferation of VSMCs in atherosclerotic plaques is accompanied by an increase in glucose transport in these cells. To date, however, very little is known about the regulation of glucose transport in VSMCs. The work presented in this thesis examines the regulation of glucose transport in VSMCs. Firstly the glucose transporters expressed in VSMCs were studied. It was determined that in VSMC freshly isolated from aorta GLUT-4 was present, however, as the cells were passaged in culture GLUT-4 was lost and GLUT-3 levels increased. GLUT-1 was present in both freshly isolated VSMCs and in cells that had been passaged in cell culture. The effects of PDGF, a growth factor believed to play an important role in mediating increased VSMC proliferation in atherosclerotic plaque development, on glucose transport was examined. PDGF could stimulate a 2- to 5-fold increase in glucose transport in cultured VSMCs, and this was dependent on both MAP kinase (p38 and p42/44) and PI3' kinase activity, as determined by the use of specific inhibitors of these kinases. Both cAMP and cGMP are known to be important regulators of VSMC function and as such the ability of cyclic nucleotides to regulate glucose transport was studied. Analogues of cAMP were capable of stimulating a 2-fold increase in glucose transport, and like PDGF-stimulated transport it was dependent on PI3' kinase and p38 MAP kinase activity, however there was no role for p42/44 MAP kinase. Agonists that stimulated adenyl cyclase activity in VSMCs were unable to stimulate an increase in glucose transport, but inhibitors of PDE 3 activity did stimulate a two-fold increase in glucose transport in VSMCs. The cGMP analogue (8-Br cGMP) had no effect on basal glucose transport rates, but was inhibitory with regard to PDGF-stimulated glucose transport. Finally, the mechanisms by which glucose transport is increased in response to mitogens were studied. To determine if translocation of GLUT-1 may be responsible for increased glucose transport, chimeras of GLUT-1 and green fluorescent protein were made to allow the movement of GLUT-1 within the cell to be followed. As well as translocation of GLUTs it is possible that altered GLUT activity, perhaps mediated by phosphorylation of the transporters, may be responsible for increased glucose transport. To address this possibility the phosphorylation state of both GLUT-1 and GLUT-3 from VSMCs was investigated under different conditions. The results from this work indicated that phosphorylation of the GLUTs is unlikely to be responsible for increased glucose transport in VSMCs.