Insulin stimulates the transport of glucose into fat and muscle cells. Although the precise molecular mechanisms involved in this process remain uncertain, insulin initiates its actions by binding to its tyrosine kinase receptor, leading to the phosphorylation of intracellular substrates. One such substrate is the Cbl proto-oncogene product. Cbl is recruited to the insulin receptor by interaction with the adapter protein CAP, through one of three adjacent SH3 domains in the carboxy terminus of CAP. Upon phosphorylation of Cbl, the CAP-Cbl complex dissociates from the insulin receptor and moves to a caveolin-enriched, triton-insoluble membrane fraction. Here, to identify a molecular mechanism underlying this subcellular redistribution, we screened a yeast two-hybrid library using the amino-terminal region of CAP and identified the caveolar protein flotillin. Flotillin forms a ternary complex with CAP and Cbl, directing the localization of the CAP-Cbl complex to a lipid raft subdomain of the plasma membrane. Expression of the N-terminal domain of CAP in 3T3-L1 adipocytes blocks the stimulation of glucose transport by insulin, without affecting signalling events that depend on phosphatidylinositol-3-OH kinase. Thus, localization of the Cbl-CAP complex to lipid rafts generates a pathway that is crucial in the regulation of glucose uptake.

Recent studies indicate that insulin stimulation of glucose transporter (GLUT)4 translocation requires at least two distinct insulin receptor–mediated signals: one leading to the activation of phosphatidylinositol 3 (PI-3) kinase and the other to the activation of the small GTP binding protein TC10. We now demonstrate that TC10 is processed through the secretory membrane trafficking system and localizes to caveolin-enriched lipid raft microdomains. Although insulin activated the wild-type TC10 protein and a TC10/H-Ras chimera that were targeted to lipid raft microdomains, it was unable to activate a TC10/K-Ras chimera that was directed to the nonlipid raft domains. Similarly, only the lipid raft–localized TC10/ H-Ras chimera inhibited GLUT4 translocation, whereas the TC10/K-Ras chimera showed no significant inhibitory activity. Furthermore, disruption of lipid raft microdomains by expression of a dominant-interfering caveolin 3 mutant (Cav3/DGV) inhibited the insulin stimulation of GLUT4 translocation and TC10 lipid raft localization and activation without affecting PI-3 kinase signaling. These data demonstrate that the insulin stimulation of GLUT4 translocation in adipocytes requires the spatial separation and distinct compartmentalization of the PI-3 kinase and TC10 signaling pathways.

It is well established that insulin stimulation of glucose uptake requires the translocation of intracellular localized GLUT4 protein to the cell surface membrane. This plasma membrane-redistributed GLUT4 protein was partially co-localized with caveolin as determined by confocal fluorescent microscopy but was fully excluded from lipid rafts based upon Triton X-100 extractability. Cholesterol depletion with methyl-␤-cyclodextrin, filipin, or cholesterol oxidase resulted in an insulin-independent increase in the amount of plasma membrane-localized GLUT4 that was fully reversible by cholesterol replenishment. This basal accumulation of cell surface GLUT4 occurred due to an inhibition of GLUT4 endocytosis. However, this effect was not specific since cholesterol extraction also resulted in a dramatic inhibition of clathrin-mediated endocytosis as assessed by transferrin receptor internalization. To functionally distinguish between caveolin-and clathrindependent endocytic processes, we took advantage of a dominant-interfering caveolin 1 mutant (Cav1/S80E) that specifically disrupts caveolae organization. Expression of Cav1/S80E, but not the wild type (Cav1/WT) or Cav1/S80A mutant, inhibited cholera toxin B internalization without any significant effect on transferrin receptor endocytosis. In parallel, Cav1/S80E expression increased the amount of plasma membrane-localized GLUT4 protein in an insulin-independent manner. Although Cav1/S80E also decreased GLUT4 endocytosis, the extent of GLUT4 internalization was only partially reduced (ϳ40%). In addition, expression of Cav1/WT and Cav1/S80A enhanced GLUT4 endocytosis by ϳ20%. Together, these data indicate that the endocytosis of GLUT4 requires clathrin-mediated endocytosis but that the higher order structural organization of plasma membrane caveolin has a significant influence on this process.One of the major acute actions of insulin is enhanced glucose uptake in striated muscle and adipose tissue (1-3). This results from the rapid translocation of the intracellular-sequestered GLUT4 1 glucose transporter isoform to the plasma membrane (4, 5). The increase in plasma membrane GLUT4 occurs due to a large increase in the rate of GLUT4 exocytosis coupled with a smaller decrease in the rate of GLUT4 endocytosis (6, 7). Recent data suggest that two independent signal transduction pathways are necessary for the full extent of insulin-stimulated GLUT4 translocation. In one case, the insulin receptor tyrosine phosphorylates insulin receptor substrate-family proteins, resulting in the activation of phosphatidylinositol 3-kinase and the generation of phosphatidylinositol 3,4,5-triphosphate. Although less well defined, the serine/threonine kinases phosphoinositide-dependent protein kinase 1 and protein kinase B/Akt as well as protein kinase C/ have been implicated in signaling events functioning downstream of phosphatidylinositol 3-kinase (8 -10). This pathway appears to be spatially segregated from a parallel insulin receptor-signaling pathway that results in the tyrosine phosphory...

Our previous studies revealed that insulin stimulates the tyrosine phosphorylation of caveolin in 3T3L1 adipocytes. To explore the mechanisms involved in this event, we evaluated the association of the insulin receptor with caveolin. The receptor was detected in a Tritoninsoluble low density fraction, co-sedimenting with caveolin and flotillin on sucrose density gradients. We also detected the receptor in caveolin-enriched rosette structures by immunohistochemical analysis of plasma membrane sheets from 3T3L1 adipocytes. Insulin stimulated the phosphorylation of caveolin-1 on Tyr 14 . This effect of the hormone was not blocked by overexpression of mutant forms of the Cbl-associated protein that block the translocation of phospho-Cbl to the caveolinenriched, lipid raft microdomains. Moreover, this phosphorylation event was also unaffected by inhibitors of the MAPK and phosphatidylinositol 3-kinase pathways. Although previous studies demonstrated that the Src family kinase Fyn was highly enriched in caveolae, an inhibitor of this kinase had no effect on insulin-stimulated caveolin phosphorylation. Interestingly, overexpression of a mutant form of caveolin that failed to interact with the insulin receptor did not undergo phosphorylation. Taken together, these data indicate that the insulin receptor directly catalyzes the tyrosine phosphorylation of caveolin.The insulin receptor is a tyrosine kinase that undergoes ligand-stimulated autophosphorylation and activation of its intrinsic substrate kinase activity (1). Once activated, the receptor phosphorylates intracellular substrates on tyrosine, including members of the insulin receptor substrate family (IRS1/2/3/4), 1 Shc, SIRP, Gab-1, Cbl, and APS (2-4). Tyrosine phosphorylation of these proteins creates recognition sites for effector molecules containing Src homology 2 (SH2) and phosphotyrosine binding domains. These include the small adapter proteins Grb2, CrkII, and Nck, the SHP2 protein-tyrosine phosphatase and the regulatory subunit of the type 1A phosphatidylinositol 3-kinase (PI3K) (4). The insulin receptor-dependent tyrosine phosphorylation of both IRS1 and IRS2 are critical in maintaining proper glucose homeostasis through its interaction with the PI3K (5-9). This interaction appears to serve a dual function by stimulating PI3K activity and targeting the enzyme to a critical intracellular site (10). Studies using various pharmacological inhibitors, microinjection of blocking antibodies, expression of dominant-interfering, and constitutively active mutants have consistently demonstrated a necessary role for PI3K activity in most of the metabolic effects of insulin (11,12). Furthermore, inhibition of PI(3,4,5)P 3 formation (a product of PI3K) by expression of the 3Ј-phosphatase phosphatase and tensin homolog or the 5Ј-phosphatase SH2 inositol 5-phosphatase prevents insulin-stimulated glucose uptake and Glut4 translocation (13,14).Despite the general agreement that PI3K activity is necessary for insulin action, several studies have demonstrated the requirement...

The effects of a high concentration of glucose on the insulin receptor-down signaling were investigated in human hepatoma (HepG2) cells in vitro to delineate the molecular mechanism of insulin resistance under glucose toxicity. Treatment of the cells with high concentrations of glucose (15-33 mM) caused phosphorylation of serine residues of the insulin receptor substrate 1 (IRS-1), leading to reduced electrophoretic mobility of it. The phosphorylation of IRS-1 with high glucose treatment was blocked only by protein kinase C (PKC) inhibitors. The high glucose treatment attenuated insulininduced association of IRS-1 and phosphatidylinositol 3-kinase and insulin-stimulated phosphorylation of Akt. A metabolic effect of insulin, stimulation of glycogen synthesis, was also inhibited by the treatment. In contrast, insulin-induced association of Shc and Grb2 was not inhibited. Treatment of the cells with high glucose promoted the translocation of PKC⑀ and PKC␦ from the cytosol to the plasma membrane but not that of other PKC isoforms. Finally, PKC⑀ and PKC␦ directly phosphorylated IRS-1 under cell-free conditions. We conclude that a high concentration of glucose causes phosphorylation of IRS-1, leading to selective attenuation of metabolic signaling of insulin. PKC⑀ and PKC␦ are involved in the down-regulation of insulin signaling, and they may lie in a pathway regulating the phosphorylation of IRS-1.