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-beta-cyclodextrin, did not permeabilize the cells or interfere with clathrin-dependent internalization. However, at this concentration, methyl-beta-cyclodextrin perturbed CMD of hepatocyte PM and inhibited insulin-induced 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.
Highlights
Binding of insulin to its receptor is a high affinity specific process, the signaling cascades generated by the activated insulin receptor kinase (IRK)1 are shared by a number of other growth factors [1,2,3]
Since liver is an important insulin target tissue we investigated the significance of caveolin enriched membrane domains (CMD) in mediating insulin signaling in rat liver
Caveolin-1 was found in the Triton X-100 insoluble pellet and at the 10-30% sucrose interface (Fractions 4 & 5 in Fig.2a, left panel), which constituted detergent resistant membranes (DRM) with a buoyant density characteristic of lipid rafts (DRM/rafts)
Summary
Binding of insulin to its receptor is a high affinity specific process, the signaling cascades generated by the activated insulin receptor kinase (IRK) are shared by a number of other growth factors [1,2,3]. The 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, which are known as lipid rafts, can recruit or exclude signaling proteins. It has been shown that caveolae negatively affect EGF and Src signaling [13] In contrast to these inhibitory effects, a number of studies, mostly in adipocytes, have suggested that intact caveolae are necessary for insulin signaling. In 3T3-L1 adipocytes, IRK was reported to be concentrated in caveolae [14], and to interact with caveolin-1 to modulate insulin signaling [15,16]. Rafts/caveolae have been implicated in insulin-stimulated glucose transport in
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