Rapid insulin-dependent endocytosis of the insulin receptor by caveolae in primary adipocytes.

BACKGROUND: The insulin receptor is localized in caveolae and is dependent on caveolae or cholesterol for signaling in adipocytes. When stimulated with insulin, the receptor is internalized. METHODOLOGY/PRINCIPAL FINDINGS: We examined primary rat adipocytes by subcellular fractionation to examine if the insulin receptor was internalized in a caveolae-mediated process. Insulin induced a rapid, t(1/2)<3 min, endocytosis of the insulin receptor in parallel with receptor tyrosine autophosphorylation. Concomitantly, caveolin-1 was phosphorylated at tyrosine(14) and endocytosed. Vanadate increased the phosphorylation of caveolin-1 without affecting insulin receptor phosphorylation or endocytosis. Immunocapture of endosomal vesicles with antibodies against the insulin receptor co-captured caveolin-1 and immunocapture with antibodies against tyrosine(14)-phosphorylated caveolin-1 co-captured the insulin receptor, demonstrating that the insulin receptor was endocytosed together with tyrosine(14)-phosphorylated caveolin-1. By immunogold electron microscopy the insulin receptor and caveolin-1 were colocalized in endosome vesicles that resembled caveosomes. Clathrin was not endocytosed with the insulin receptor and the inhibitor of clathrin-coated pit-mediated endocytosis, chlorpromazine, did not inhibit internalization of the insulin receptor, while transferrin receptor internalization was inhibited. CONCLUSION: It is concluded that in response to insulin stimulation the autophosphorylated insulin receptor in primary adipocytes is rapidly endocytosed in a caveolae-mediated process, involving tyrosine phosphorylation of caveolin-1.

A new role for caveolae as metabolic platforms.

The plasma membrane of cells functions as a barrier to the environment. Caveolae are minute invaginations of the membrane that selectively carry out the exchange of information and materials with the environment, by functioning as organizers of signal transduction and through endocytosis. Recent findings of uptake of different metabolites and of lipid metabolism occurring in caveolae, point to a new general function of caveolae. As gateways for the uptake of nutrients across the plasma membrane, and as platforms for the metabolic conversion of nutrients, especially in adipocytes, caveolae are now emerging as active centers for many aspects of intermediary metabolism, with implications for our understanding of obesity, diabetes and other metabolic disorders.

Association and insulin regulated translocation of hormone-sensitive lipase with PTRF.

Polymerase I and transcript release factor (PTRF) is in human adipocytes mainly localized at the plasma membrane. This localization was under control of insulin, which translocated PTRF to the cytosol and nucleus, indicating a novel role for PTRF in insulin transcriptional control. In the plasma membrane PTRF was specifically bound to a triacylglycerol-metabolizing subclass of caveolae containing hormone-sensitive lipase (HSL). In response to insulin PTRF was translocated to the cytosol in parallel with HSL. PTRF and HSL were quantitatively immunoprecipitated from the cytosol by antibodies against either PTRF or HSL. The findings indicate also a novel extranuclear function for PTRF in the control of lipolysis.

Separation and characterization of caveolae subclasses in the plasma membrane of primary adipocytes; segregation of specific proteins and functions.

Caveolae are nearly ubiquitous plasma membrane domains that in adipocytes vary in size between 25 and 150 nm. They constitute sites of entry into the cell as well as platforms for cell signalling. We have previously reported that plasma membrane-associated caveolae that lack cell surface access can be identified by electron microscopy. We now report the identification, after density gradient ultracentrifugation, of a subclass of very high-density apparently closed caveolae that were not labelled by cell surface protein labelling of intact cells. These caveolae contained caveolin-1 and caveolin-2. Another class of high-density caveolae contained caveolin-1, caveolin-2 and specifically fatty acid transport protein-1, fatty acid transport protein-4, fatty acyl-CoA synthetase, hormone-sensitive lipase, perilipin, and insulin-regulated glucose transporter-4. This class of caveolae was specialized in fatty acid uptake and conversion to triacylglycerol. A third class of low-density caveolae contained the insulin receptor, class B scavenger receptor-1, and insulin-regulated glucose transporter-4. Small amounts of these proteins were also detected in the high-density caveolae. In response to insulin, the insulin receptor autophosphorylation and the amount of insulin-regulated glucose transporter-4 increased in these caveolae. The molar ratio of cholesterol to phospholipid in the three caveolae classes varied considerably, from 0.4 in very high-density caveolae to 0.9 in low-density caveolae. There was no correlation between the caveolar contents of caveolin and cholesterol. The low-density caveolae, with the highest cholesterol concentration, were particularly enriched with the cholesterol-rich lipoprotein receptor class B scavenger receptor-1, which mediated cholesteryl ester uptake from high-density lipoprotein and generation of free cholesterol in these caveolae, suggesting a specific role in cholesterol uptake/metabolism. These findings demonstrate a segregation of functions in caveolae subclasses.

Triacylglycerol is synthesized in a specific subclass of caveolae in primary adipocytes.

A principal metabolic function of adipocytes is to synthesize triacylglycerol (TG) from exogenous fatty acids. The level of fatty acids has to be tightly controlled in the adipocyte, as they can act as detergents that rapidly dissolve the plasma membrane, causing cell lysis if allowed to accumulate. Fatty acids therefore have to be efficiently converted to TG and stored in the central lipid droplet. We report that in intact primary adipocytes exogenous oleic acid was taken up and directly converted to TG in the plasma membrane, in a novel subclass of caveolae that specifically contains the protein perilipin. Isolated caveolae catalyzed de novo TG synthesis from oleic acid and glycerol 3-phosphate. Electron microscopy revealed the presence of caveolin and perilipin in caveolae and in lipid-laden bulbs in the plasma membrane, and fluorescence microscopy demonstrated colocalization of fatty acids/TG with caveolin and perilipin at the plasma membrane. A second caveolae fraction was isolated, which lacked perilipin and the triacylglycerol synthesizing enzymes. Both caveolae fractions contained caveolin-1 and the insulin receptor. The findings demonstrate that specific subclasses of caveolae carry out specific functions in cell metabolism. In particular, triacylglycerol is synthesized at the site of fatty acid entry in one of these caveolae classes.

Lipids and glycosphingolipids in caveolae and surrounding plasma membrane of primary rat adipocytes.

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. Carbonate-isolated 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 x 10(3) molecules of cholesterol, 7.5 x 10(3) of sphingomyelin and 23 x 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.

Cell surface orifices of caveolae and localization of caveolin to the necks of caveolae in adipocytes.

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.