THE KINETICS OF HEXOSE TRANSPORT AND THE INSULIN RESPONSE IN ADIPOCYTES Lucy Pauline Eperon. The facilitated diffusion of glucose in the rat adipocyte was examined. Kinetic parameters were determined for cells in both the insulin-stimulated and basal states. Transport was studied in both isolated intact cells and partially purified membrane sacs or "ghosts". Comparisons between the transport in adipocytes and human erythrocytes were made, particularly with respect to inhibition by xanthine derivatives. The external and internal kinetic parameters were determined by the use of zero trans. infinite cis and equilibrium exchange experiments. Initial rates were determined by direct measurement over short (one to two second) time intervals, critical for accurate estimates, and verified by integrated rate analyses of longer time courses for the sugar transport. Transport was measured using radiolabeled glucose analogues; the reaction was terminated with specific inhibitors followed by rapid separations of the cells from the extracellular medium. The transport of 3-0-methyl-D-glucose in isolated intact rat adipocytes was shown to be symmetrical in entry and exit. The results were compared to previous studies by a determination of the equilibrium exchange parameters. The procedure used to isolate adipocytes was such that the cells were highly responsive to insulin. 3-0-methyl-D-glucose transport in the insulin-treated adipocytes was also symmetrical. From the comprehensive kinetic determinations it was concluded that insulin stimulated hexose transport in adipocytes by increasing the maximum rate parameters equally for both influx and efflux, whilst both the external and internal affinity constants were unchanged. The significance of this finding for models of the insulin-mediated activation of transport was discussed. Some possible mechanisms for the activation were tested. In ghosts prepared from insulin-treated cells, the stimulation of transport was variable and at most a four-fold increase in initial transport rates was observed. Estimates of the kinetics parameters suggested that the transport symmetry of whole cells may not have been lost in ghosts. It was also shown that pink human erythrocye ghosts (from which 93% of the haemoglobin had been removed) retained the asymmetrical D-glucose transport found in the intact erythrocyte. The implications of these results for models of hexose transport were considered. The specific inhibition by xanthine derivatives of transport in adipocytes and the insulin-insensitive human erythrocyte ghosts was compared. There were differences in the kinetics of inhibition by 3-isobutyl-l-methylxanthine. In adipocytes the inhibition of zero trans entry and equilibrium exchange transport was competitive, whereas in human erythrocyte ghosts zero trans entry parameters were unaffected by 3-isobutyl-l-methylxanthine, although the efflux of D-glucose into sugar-free solutions was diminished. These differences were discussed.
|Date of Award||1983|