Lysosomal Degradation of Receptor-bound 125I-labeled Insulin by Rat Adipocytes: Its Characterization and Dissociation from the Short-term Biologic Effects of Insulin

Abstract

In this study we used chloroquine to characterize the interalization and lysosomal degradation of receptor-bound 125I-insulin by rat adipocytes and to determine the role of lysosomal processing of insulin in the short-term biologic effects of the hormone. Chloroquine inhibited the degradation of 125I-insulin bound to adipocytes by both association and disslociation experiments. In the former experiments, chloroquine caused a time- and concentration-dependent increase in specifically bound insulin owing to an increase in intact insulin and a decrease in degradation products, as determined by trichloroacetic acid precipitability and gel chromatographic analysis of material extracted from the cells. In the dissociation experiments, 50 microM chloroquine decreased the rate of degradation by two third, as reflected in the release of degradation to or degraded by isolated plasma membranes, on the degradation of 125I-insulin by proteases in the incubation medium, or on the endocytotic uptake of receptor-bound insulin. Quantitative electron miroscopy, using monomeric ferritin-insulin, showed 50 microM chloroquine doubled the number of lysosomal structures containing ferritin. These findings are consistent with an inhibition by chloroquine of lysosomal degradation of internalized receptor-bound insulin. Chloroquine, at these same concentrations, had no effect on the ability of insulin to stimulate glucose transport and oxidation or to inhibit epinephrine-stimulated lipolysis. In these studies, we show that lysosomal degradation of internalized receptor-bound insulin is not necessary for insulin to cause short-term biologic effects in the adipocyte.

Receptor-mediated insulin degradation and insulin-stimulated glycogenesis in cultured foetal hepatocytes

“…The functional relationship between the degradation and short-term biological effects of insulin in adipocytes had been examined in the presence of the lysosomotropic agent chloroquine. These experiments indicated that insulin degradation was not a prerequisite for biological action (Suzuki & Kono, 1979;Hammons & Jarett, 1980;Marshall & Olefsky, 1980). Cultured foetal hepatocytes have been shown to bind insulin specifically and to respond to the presence of the hormone by an enhanced ability to synthesize glycogen (Plas et aL, 1979; Menuelle & Plas, 1981).…”

Binding, Internalization, and Degradation of Insulin in Vascular Endothelial Cells

“…4 ' 15 ' 17 - 18 The ability of chloroquine to lower the level of low-mol-wt degradation products released into the medium may indicate that at least part of the degradation is in the lysosomes. 24 Pre-exposure of the endothelial cells to insulin led to downregulation of its cell surface receptor sites, as described for other cell systems. 1i17 ' 21 It should be emphasized that all our studies were performed with cells attached to culture dishes, in which only the apical cell surface is fully available for hormone binding.…”

Binding and Degradation of [125I]Human Growth Hormone in Rat Adipocytes*

“…Failure of ammonium chloride, leupeptin, and colchicine to block or enhance insulin-like responses is not surprising in view of the rapidity in onset of some insulin-like responses (15)(16)(17)(18)(19)(20)(21)(22) and the failure of some of these agents to interfere with similar actions of insulin in adipocytes (23). Even under conditions in which hormone degradation was severely impeded by agents that act at various loci, we could detect no impairment or enhancement of the three biological responses tested.…”