Effect of Insulin on Caveolin-enriched Membrane Domains in Rat Liver

Abstract

Compartmentalization of signaling molecules may explain, at least in part, how insulin or growth factors achieve specificity. Caveolae/rafts are specialized lipid compartments that have been implicated in insulin signaling. In the present study, we investigated the role of caveolin-enriched membrane domains (CMD) in mediating insulin signaling in rat liver. We report the existence of at least two different populations of CMD in rat liver plasma membranes (PM). One population is soluble in Triton X-100 and seems to be constitutively associated with cytoskeletal elements. The other population of CMD is located in a membrane compartment insoluble in Triton X-100 with light buoyant density and is hence designated CMD/rafts. We found evidence of rapid actin reorganization in rat liver PM in response to insulin, along with the association of CMD/rafts and insulin signaling molecules with a cell fraction enriched in cytoskeletal elements. The presence of CMD in liver parenchyma cells was confirmed by the presence of caveolin-1 in primary rat hepatocyte cultures. Cholesterol depletion, effected by incubating hepatocytes with 2 mM methyl-␤-cyclodextrin, did not permeabilize the cells or interfere with clathrin-dependent internalization. However, at this concentration, methyl-␤-cyclodextrin perturbed CMD of hepatocyte PM and inhibited insulininduced Akt activation and glycogen synthesis but did not affect insulin-induced insulin receptor kinase tyrosine phosphorylation. These events, together with the presence of a functional insulin receptor in CMD of rat liver PM, suggest that insulin signaling is influenced by the interaction of caveolae with cytoskeletal elements in liver.Although binding of insulin to its receptor is a process specific to high affinity, the signaling cascades generated by the activated insulin receptor kinase (IRK) 1 are shared by a number of other growth factors (1-3). Nevertheless, the metabolic actions of insulin cannot be reproduced in intensity and quality by other hormones or growth factors. The mechanism by which insulin achieves its specificity of action remains to be satisfactorily determined. Compartmentalization of signaling molecules in plasma membranes and endosomes may play an important role in determining the specificity of signal transduction (4 -7).The recent isolation of detergent-insoluble, low-density membrane fragments from cells suggests that sphingolipid and cholesterol rich domains could exist as a liquid-ordered phase in the membrane (8). These lipid domains, known as lipid rafts, can recruit or exclude signaling proteins. Thus, lipid rafts have been implicated in the regulation of hormone and growth factor signaling (8, 9). Caveolae, which constitute a subset of lipid rafts, are invaginated cell surface microdomains that are enriched in caveolin oligomers, the major protein constituent of these structures (10, 11). Three caveolins (caveolin-1, -2, and -3) have been discovered. Caveolin-1 and caveolin-2 are found most abundantly in adipocyte and endothelial cells, whereas c...

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

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The Plasma Membrane Ca2+-ATPase Isoform 4 Is Localized in Lipid Rafts of Cerebellum Synaptic Plasma Membranes

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