| 1. |
- Gustavsson, Johanna, 1956-, et al.
(author)
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Localization of the insulin receptor in caveolae of adipocyte plasma membrane
- 1999
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In: The FASEB Journal. - 0892-6638 .- 1530-6860. ; 13:14, s. 1961-1971
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Journal article (peer-reviewed)abstract
- 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 β-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. β-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.
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| 2. |
- Karlsson, Margareta, 1942-, et al.
(author)
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Colocalization of insulin receptor and insulin receptor substrate-1 to caveolae in primary human adipocytes
- 2004
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In: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 271:12, s. 2471-2479
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Journal article (peer-reviewed)abstract
- Caveolae are plasma membrane invaginations with several functions, one of which appears to be to organize receptor mediated signalling. Here we report that in primary human subcutaneous adipocytes the insulin receptor was localized to caveolae by electron microscopy/immunogold detection and by isolating caveolae from plasma membranes. Part of insulin receptor substrate 1 (IRS1), the immediate downstream signal mediator, was colocalized with the insulin receptor in the plasma membrane and caveolae, as demonstrated by immunofluorescence microscopy, immunogold electron microscopy, and immunogold electron microscopy of transfected recombinant HA-IRS1. In contrast, rat epididymal adipocytes lacked IRS1 at the plasma membrane. Depletion of cholesterol from the cells using β-cyclodextrin blocked insulin stimulation of glucose uptake, insulin inhibition of perilipin phosphorylation in response to isoproterenol, and insulin stimulation of protein kinase B and Map-kinases extracellular signal-related kinase (ERK)1/2 phosphorylation. Insulin-stimulated phosphorylation of the insulin receptor and IRS1 was not affected, indicating that caveolae integrity is required downstream of IRS1. In conclusion we show that insulin receptor and IRS1 are both caveolar proteins and that caveolae are required for both metabolic and mitogenic control in human adipocytes. Our results establish caveolae as foci of insulin action and stress the importance of examining human cells in addition to animal cells and cell lines.
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| 3. |
- Karlsson, Margareta, 1942-, et al.
(author)
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Insulin induces translocation of glucose transporter GLUT4 to plasma membrane caveolae in adipocytes
- 2002
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In: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 16:2, s. 249-251
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Journal article (peer-reviewed)abstract
- Insulin-stimulated glucose uptake in muscle and adipose tissue is the result of translocation of insulin-regulated glucose transporters (GLUT4) from intracellular vesicles to the plasma membrane. Here we report that GLUT4 in the plasma membrane of 3T3-L1 adipocytes were located predominantly in caveolae invaginations: by immunogold electron microscopy of plasma membranes, 88% of GLUT4 were localized to caveolae structures and this distribution within the plasma membrane was not affected by insulin. By immunofluorescence microscopy, a major part of GLUT 4 was colocalized with caveolin. The total amount of GLUT4 in the plasma membrane increased 2.2-fold in response to insulin as determined by immunogold electron or immunofluorescence microscopy. GLUT4 were enriched in caveolae fractions isolated without detergents from plasma membranes of rat adipocytes. In these fractions, GLUT4 were largely confined to caveolin-containing membranes of the caveolae preparation isolated from insulin-stimulated cells, determined by electron microscopy. Insulin increased the amount of GLUT4 2.7-fold in this caveolae fraction. Caveolae were purified further by immunoisolation with antibodies against caveolin. The amount of GLUT4 increased to the same extent in the immunopurified caveolae as in the cruder caveolae fractions from insulin-stimulated cells. We conclude that insulin induces translocation of GLUT4 to caveolae.
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| 4. |
- Thorn, Hans, 1967-
(author)
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Caveolae structure and importance in insulin action
- 2004
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Doctoral thesis (other academic/artistic)abstract
- Type II diabetes is a disease characterized by chronic hyperglycaemia and abnormalities in lipid metabolism that affects approximately 5% of the population in the Western World. Caveolae are invaginations of the plasma membrane, described as 25-150 nm omega shaped structures, which are enriched in cholesterol, sphingolipids and the constituent protein caveolin. Caveolae have been shown to be involved in signal transduction, uptake over the plasma membrane and intracellular transport. By electron microscopy studies of cell membranes and biochemical analyses of isolated caveolae, we report that in rat adipocytes glucose transporter GLUT4 was translocated to caveolae in response to insulin. Insulin stimulation increased the amount of GLUT4 in the plasma membrane, but the ratio between GLUT4 in the planar and caveolae membrane remained constant. These findings indicate that caveolae are the locales for glucose uptake in the cell. We also report that the insulin receptor, independently of insulin stimulation, was localised in caveolae in human adipocytes. In these cells depletion of cholesterol destroyed the caveolae structure and the adipocytes became insulin resistant. Cholesterol depletion did not affect the insulinstimulated autophosphorylation of the insulin receptor nor the phosphorylation of the downstream IRS1. Further signalling to metabolic control or mitogenic control was inhibited, however. With transmission electron-, scanning electron- and fluorescence-microscopic techniques, we studied the ultrastructure and distribution of caveolae in the rat adipocyte. We found that caveolae can be divided into two subpopulations, small (<50 nm) and large (50-150 nm). The large caveolae are connected to the extracellular space via narrow necks and the orifices of caveolae were herein shown in primary adipocytes for the first time. Caveolin is located in the membrane proximal part of the small caveolae and to the neck in the large caveolae. The insulin receptor substrate IRS 1 was shown to be localized to caveolae in human adipocytes and to colocalize with the insulin receptor. In rat adipocytes, however, IRS1 was not localized to the plasma membrane in the absence of insulin stimulation. By transfection of rat adipocytes with human IRS1 we found that human IRS1 bound to the plasma membrane in the rat adipocyte, whereas the endogenous rat IRS1 did not. Taken together, caveolae seem to be closely involved in regulation of insulin action in the adipocyte.
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