Localization of the insulin receptor in caveolae of adipocyte plasma membrane

Abstract

The insulin receptor is a transmembrane protein of the plasma membrane, where it recognizes extracellular insulin and transmits signals into the cellular signaling network. We report that insulin receptors are localized and signal in caveolae microdomains of adipocyte plasma membrane. Immunogold electron microscopy and immunofluorescence microscopy show that insulin receptors are restricted to caveolae and are colocalized with caveolin over the plasma membrane. Insulin receptor was enriched in a caveolae-enriched fraction of plasma membrane. By extraction with beta-cyclodextrin or destruction with cholesterol oxidase, cholesterol reduction attenuated insulin receptor signaling to protein phosphorylation or glucose transport. Insulin signaling was regained by spontaneous recovery or by exogenous replenishment of cholesterol. beta-Cyclodextrin treatment caused a nearly complete annihilation of caveolae invaginations as examined by electron microscopy. This suggests that the receptor is dependent on the caveolae environment for signaling. Insulin stimulation of cells prior to isolation of caveolae or insulin stimulation of the isolated caveolae fraction increased tyrosine phosphorylation of the insulin receptor in caveolae, demonstrating that insulin receptors in caveolae are functional. Our results indicate that insulin receptors are localized to caveolae in the plasma membrane of adipocytes, are signaling in caveolae, and are dependent on caveolae for signaling.

Obesity and related consequences to ageing

“…Although there is missing information on Mest and Sfrp5 gene expressions and their regulation in the process of ageing, caveolae are currently being intensively studied. Recent research has shown that adipocytes from animals in a process of ageing presented a decrease in mean expression of caveolar proteins per unit cell surface (Hulstrom et al 2013); in addition, Cav1 was reported to be relevant for insulin signaling in AT (Gustavsson et al 1999).…”

Selective Caveolin-1–dependent Endocytosis of Glycosphingolipids

“…For inhibition of clathrin-dependent endocytosis, samples were pretreated with 8 g/ml chlorpromazine (CPZ; Gustavsson et al, 1999;Okamoto et al, 2000) or were potassium depleted (Larkin et al, 1983;Hansen et al, 1993); for disruption of caveolar endocytosis, cells were pretreated with 25 g/ml nystatin (Rothberg et al, 1992) or 200 M genistein (Aoki et al, 1999;Chen and Norkin, 1999;Liu and Anderson, 1999 g/ml DNA using the manufacturer's protocol. After a 4 -6-h treatment, the cells were washed and subsequently cultured for 24 -48 h in DMEM containing 10% FBS before treatment with the fluorescent lipids or toxins as above.…”

Oxidation–reduction respiratory chains and ATP synthase complex are localized in detergent-resistant lipid rafts

“…These specific membrane compartments, also commonly referred to as the lipid rafts, are composed of cholesterol and glycolipids [1,2], and predominantly harbor a host of receptors and signaling molecules, allowing them to mediate a variety of cellular events, including cellular signaling, pathogenic invasion, immune responses, and cholesterol homeostasis [3][4][5][6]. Because the lipid rafts are loaded with many receptors for epidermal growth factor (EGF) [7], platelet-derived growth factor [8], nerve growth factor [9], insulin [10], insulin-like growth factor [11], transforming growth factor [12], and tumor necrosis factor [13], these rafts might act as platforms which initiate various cellular signal pathways.…”