Characterization of insulin-stimulated sugar transport in the rat adipocyte.

Abstract

The effects of temperature, ions and sugar interaction on insulin-sensitive 3-0-methyl-D-glucose transport in rat adipocytes have been studied. Temperature affects the transport of sugar into fat cells. A protocol was devised whereby the effect of sub-physiological temperature on the activation and deactivation of sugar transport could be studied. Incubation of adipocytes at 20°C with a concentration of insulin which was sub-maximally stimulating at 37°C resulted in full expression of transport activity. The time-dependency of this was measured. No effect of sub-physiological temperature was noted in fully insulin-stimulated cells. The time-dependency of 125I-insulin association with adipocytes at low temperature was found to closely parallel the low-temperature stimulation of transport. Degradation of 125I-insulin was inhibited at 20°C. Deactivation of insulin-stimulated sugar transport was inhibited progressively at temperatures below 37°C, and completely at 20°C. Loss of cell-associated 125I-insulin closely matched the return to baseline transport. Adipocytes depleted of potassium or chloride by successive washing in buffers free of these ions showed accelerated basal transport at 37°C. Fully insulin-stimulated transport was unaffected under these conditions. The effect was reversible and time-dependent on replacement of the missing ion. The potassium ionophore valinomycin was able to lower insulin-stimulated transport to near-basal levels, but only in the presence of potassium. Valinomycin alone had little or no effect on transport stimulated by K+-free buffer. Cyanide inhibited the potassium-induced return to basal levels of transport stimulated in K+-free buffer. Inclusion of valinomycin overcame this inhibition. The interactions of the side-specific sugar transport inhibitors 4,6-O-ethylidene-D-glucoside and n'-propyl- beta-D-glucoside with the transport inhibitor cytochalasin B on sugar transport were studied. 4,6-O-ethylidene-D-glucoside is an inhibitor at the external site, while n'-propyl-beta-D-glucoside is inwardly directed. The results indicate competitive interactions between inhibitors at low concentration, apparently shifting to a non-competitive nature at higher concentration. The side-specificity of cytochalasin B inhibition was tested kinetically, an attempt being made to show side-specificity directly. The results show that cytochalasin B is not as specific for the inside inhibition site in fat cells as it is in the human erythrocyte.

Date of Award	1983
Original language	English
Awarding Institution	University of Bath