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.
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.
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.
Background (1): In the wake of COVID-19, elderly people have been labelled a risk group. As the pandemic is a new crisis in Sweden, we have no knowledge on how this group perceives the information and recommendations being provided. Complying with these recommendations entails physical distancing and, for some, isolation at home. Methods (2): From 16 April to 15 May 2020, we conducted an online survey targeting people aged 70 and older in Sweden (n = 1854). Results (3): A vast majority of the participants find the information and recommendations clear and reliable. Half of the participants report staying at home all the time, and up to half report decreased mental health in terms of, e.g., feeling depressed, having sleeping problems and that isolation makes them feel bad. However, elderly people are not a homogenous group, and there are gender and demographic differences. (4) Conclusion: At this point, we do not know the full extent of the ongoing pandemic, either in terms of duration or in terms of losses. The Swedish model for action on COVID-19 has not included a lock down. However, elderly people seem to comply with recommendations and practice social distancing to a high degree. This might lead to decreased mental health and long-term effects.
Insulin controls cell metabolism via metabolic signal transduction pathways and cell proliferation via mitogenic signal pathways. Metabolic signalling occurs through receptor-activated phosphorylation of insulin receptor substrate (IRS) proteins that subsequently activate phosphatidylinositide 3-kinase (PI3-kinase) to generate second messengers that produce increased phosphorylation and activation of protein kinase B ⁄ Akt (PKB). PKB appears to be central to downstream control of both glucose uptake and glycogen synthesis by insulin [1,2]. Although adipocytes are terminally differentiated cells that do not divide further, insulin has the potential for genomic control via a mitogenic signalling pathway. This may also be mediated by IRS; insulin activation of the G-protein Ras leads to phosphorylation and activation of mitogenactivated protein (MAP) kinases -extracellular signal-related kinase (ERK) 1 and 2 [3], and p38 [4,5] Insulin resistance is a cardinal feature of type 2 diabetes and also a consequence of trauma such as surgery. Directly after surgery and cell isolation, adipocytes were insulin resistant, but this was reversed after overnight incubation in 10% CO 2 at 37°C 2 . Tyrosine phosphorylation of the insulin receptor and insulin receptor substrate (IRS)1 was insulin sensitive, but protein kinase B (PKB) and downstream metabolic effects exhibited insulin resistance that was reversed by overnight incubation. MAP-kinases ERK1 ⁄ 2 and p38 were strongly phosphorylated after surgery, but was dephosphorylated during reversal of insulin resistance. Phosphorylation of MAP-kinase was not caused by collagenase treatment during cell isolation and was present also in tissue pieces that were not subjected to cell isolation procedures. The insulin resistance directly after surgery and cell isolation was different from insulin resistance of type 2 diabetes; adipocytes from patients with type 2 diabetes remained insulin resistant after overnight incubation. IRS1, PKB, and downstream metabolic effects, but not insulinstimulated tyrosine phosphorylation of insulin receptor, exhibited insulin resistance. These findings suggest a new approach in the study of surgeryinduced insulin resistance and indicate that human adipocytes should recover after surgical procedures for analysis of insulin signalling. Moreover, we pinpoint the signalling dysregulation in type 2 diabetes to be the insulin-stimulated phosphorylation of IRS1 in human adipocytes.Abbreviations ERK, extracellular signal-related kinase; GLUT4, insulin-sensitive glucose transporter-4; IRS, insulin receptor substrate; MAP, mitogenactivated protein; PKB, protein kinase B; PI3-kinase, phosphatidylinositide 3-kinase.
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