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.

Insulin controls target cells by binding to its cell surface receptor. The further intracellular transmission of the insulin signal involves phosphorylation of the receptor as well as other proteins, in particular the insulin receptor substrate (IRS), 1 on specific tyrosine residues. After tyrosine phosphorylation IRS is recognized by Src homology 2 domain-containing proteins for metabolic and glucose transport control, or activation of the mitogen-activated protein kinase (MAP kinase) pathway and mitogenic control (1-4). In type 2 diabetes target cells of the hormone are not fully responsive, which is compensated temporarily by enhanced insulin secretion. The pathogenic mechanisms for this insulin resistance are not known, but an important common feature appears to be a reduced activation/ tyrosine phosphorylation of IRS-1 (5).The insulin receptors are sequestered in the caveolae microdomains of the plasma membrane in adipocytes, and caveolae appear to be critical for insulin control (6). By thin-section electron microscopy, caveolae appear as omega-shaped invaginations of 50 -100 nm diameter in the plasma membrane (7). Caveolae invaginations are found in the plasma membrane of many cell types, but are particularly abundant in adipocytes where they increase in number in conjunction with the differentiation of 3T3-L1 fibroblasts to mature adipocytes (8 -10). Caveolae are involved in receptor-mediated uptake of solutes into the cytosol (11) and in transcytosis (12). A number of proteins, in addition to the insulin receptor, involved in signal transduction are localized to caveolae, which suggests that they may be involved in cellular signaling and control (reviewed in Refs. 13-16).Caveolae are rich in cholesterol and sphingolipids. Caveolae may indeed form from cholesterol-and sphingolipid-rich rafts in the membrane in a process requiring the caveolae-specific structural protein caveolin. Caveolin is found in the plasma membrane and intracellularly, but in the plasma membrane is confined to caveolae; it is therefore used as a marker for these structures. The function of caveolae is dependent on a sufficient level of cholesterol in the plasma membrane and caveolae (12,17). We have also demonstrated a critical dependence of the insulin receptor signal transduction on cholesterol; depletion of cholesterol from the plasma membrane of rat adipocytes reversibly inhibited insulin stimulation of glucose transport and metabolic protein phosphorylation control (6). The importance of caveolae for insulin receptor signaling is further indicated by a consensus binding site for interaction with caveolin (18), and coprecipitation of the receptor with caveolin (4) indicates that the interaction may be physiological. Moreover, the insulin receptor appears to phosphorylate caveolin (19), whereas caveolin was shown to activate the isolated receptor, although the physiological relevance of this is not known (20).Herein we examine in detail the dependence of the insulin receptor on caveolae for signal transduction: the effects of cho...

Caveolae are noncoated invaginations of the plasma membrane that form in the presence of the protein caveolin. Caveolae are found in most cells, but are especially abundant in adipocytes. By high-resolution electron microscopy of plasma membrane sheets the detailed structure of individual caveolae of primary rat adipocytes was examined. Caveolin-1 and -2 binding was restricted to the membrane proximal region, such as the ducts or necks attaching the caveolar bulb to the membrane. This was confirmed by transfection with myc-tagged caveolin-1 and -2. Essentially the same results were obtained with human fibroblasts. Hence caveolin does not form the caveolar bulb in these cells, but rather the neck and may thus act to retain the caveolar constituents, indicating how caveolin participates in the formation of caveolae. Caveolae, randomly distributed over the plasma membrane, were very heterogeneous, varying in size between 25 and 150 nm. There was about one million caveolae in an adipocyte, which increased the surface area of the plasma membrane by 50%. Half of the caveolae, those larger than 50 nm, had access to the outside of the cell via ducts and 20-nm orifices at the cell surface. The rest of the caveolae, those smaller than 50 nm, were not open to the cell exterior. Cholesterol depletion destroyed both caveolae and the cell surface orifices.

Turbulent flow, characterized by velocity fluctuations, accompanies many forms of cardiovascular disease and may contribute to their progression and hemodynamic consequences. Several studies have investigated the effects of turbulence on the magnetic resonance imaging (MRI) signal. Quantitative MRI turbulence measurements have recently been shown to have great potential for application both in human cardiovascular flow and in engineering flow. In this article, potential pitfalls and sources of error in MRI turbulence measurements are theoretically and numerically investigated. Data acquisition strategies suitable for turbulence quantification are outlined. The results show that the sensitivity of MRI turbulence measurements to intravoxel mean velocity variations is negligible, but that noise may degrade the estimates if the turbulence encoding parameter is set improperly. Different approaches for utilizing a given amount of scan time were shown to influence the dynamic range and the uncertainty in the turbulence estimates due to noise. The findings reported in this work may be valuable for both in vitro and in vivo studies employing MRI methods for turbulence quantification.

We have made a comprehensive and quantitative analysis of the lipid composition of caveolae from primary rat fat cells and compared the composition of plasma membrane inside and outside caveolae. We isolated caveolae from purified plasma membranes using ultrasonication in carbonate buffer to disrupt the membrane, or extraction with nonionic detergent, followed by density gradient ultracentrifugation. The carbonate-isolated caveolae fraction was further immunopurified using caveolin antibodies. Carbonateisolated caveolae were enriched in cholesterol and sphingomyelin, and the concentration was three-and twofold higher, respectively, in caveolae compared to the surrounding plasma membrane. The concentration of glycerophospholipids was similar suggesting that glycerophospholipids constitute a constant core throughout the plasma membrane. The composition of detergent-insoluble fractions of the plasma membrane was very variable between preparations, but strongly enriched in sphingomyelin and depleted of glycerophospholipids compared to carbonate-isolated caveolae; indicating that detergent extraction is not a suitable technique for caveolae preparation. An average adipocyte caveola contained about 22 · 10 3 molecules of cholesterol, 7.5 · 10 3 of sphingomyelin and 23 · 10 3 of glycerophospholipid. The glycosphingolipid GD3 was highly enriched in caveolae, whereas GM3, GM1 and GD1a were present inside as well as outside the caveolae membrane. GD1b, GT1b, GM2, GQ1b, sulfatide and lactosylceramide sulfate were not detected in caveolae.