Insulin control of glucose transport in caveolae microdomains of the plasma membrane

Sammanfattning: Caveolae are invaginated, dynamic microdomains in the plasma membrane and believed to be involved in receptor-mediated uptake of small molecules (potocytosis) and in signal transduction. A phosphatidylinositol glycan, a precursor of potential insulin second messengers, has been found to be enriched in the caveolae-fraction of adipocyte plasma membranes (Parpal et al., 1995, J Cell Biol 131:125-135). We now demonstrate that the insulin receptor is localized to caveolae microdomains. This was investigated in i) 3T3-Ll adipocyte plasma membranes by a morphological method (double immunofluorescence labeling and contbcal microscopy) and in ii) caveolae isolated by a biochemical, detergent-free method. The insulin receptor was enriched in caveolae and, in response to insulin, phosphorylated on tyrosine which indicated that the insulin receptor was active.Insulin stimulates the translocation of glUcose transporter protcins from intracellular stores to the plasma membrane which leads to an increased glucose uptake. Long-chain 1 ,2-diacylgiycerol, one of two potential second messengers for insulin, has been found to stimulate glucose uptake in rat adipocytes (Stn1fors, 1988, Nature, 335:554-556). Here, we report that long-chain 1,2-diacylglycerol, emulsified in taurodeoxycholate, stimulates the translocation of GLUT4 to the plasma membrane. Moreover, physiological long-chain 1,2~diacylglycerols are taken up by different cell types in amounts sufficient to have biological eftects, equally well in the absence or presence of taurodeoxycholate.We also report that a rapid translocation of GLUT4 to the plasma membrane was followed by a slower transition of GLUT4 into caveolae. Accumulation of GLUT4 in caveolae coincided with the insulin-stimulated increase in glucose uptake. This offers a mechanistic explanation for the observed discrepancy between the appearance of GLUT4 in the plasma membrane and the delayed increase in glucose uptake.Non-hydrolyzable GTP-analogs stimulate the translocation of GLUT4 and increase glucose uptake in permeabilized cells. The small GTP·binding protein RaM is suggested to be involved in these processes since Rab4 has been localized to GLUT4-containing vesicles and is redistributed in response to insulin. We found that Rab4 is enriched in caveolae and that the amount of Rab4 increased in caveolae, in the same extent a<> GLUT4 did, in re!.J)Onse to insulin.Caveolae are characterized by high levels of sphingolipids and cholesterol. Depletion of cholesterol, which disrupts the integrity of caveolae, abolished insulin-stimulated glucose uptake reversibly. Insulin's control of protein pho.<:phorylation was also abolished while j3-adrenergic signaling was unaffected.The results suggest that caveolae are crucial Jor insulin-signuling in adipocytes and a disruption of these structures may have consequences for the development of insulin re.~istance and diabetes mellitus.