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Caveolae structure and importance in insulin action
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2004. , 53 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 875
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-24065Local ID: 3624ISBN: 91-7373-851-4 (print)OAI: oai:DiVA.org:liu-24065DiVA: diva2:244381
Public defence
2004-12-10, Berzeliussalen, Hälsouniversitetet, Linköping, 09:00
Opponent
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-10-25Bibliographically approved
List of papers
1. Insulin induces translocation of glucose transporter GLUT4 to plasma membrane caveolae in adipocytes
Open this publication in new window or tab >>Insulin induces translocation of glucose transporter GLUT4 to plasma membrane caveolae in adipocytes
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2002 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 16, no 2, 249-251 p.Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25037 (URN)10.1096/fj.01-0646fje (DOI)9461 (Local ID)9461 (Archive number)9461 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
2. Cell surface orifices of caveolae and localization of caveolin to the necks of caveolae in adipocytes
Open this publication in new window or tab >>Cell surface orifices of caveolae and localization of caveolin to the necks of caveolae in adipocytes
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2003 (English)In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 14, no 10, 3967-3976 p.Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14539 (URN)10.1091/mbc.E03-01-0050 (DOI)
Available from: 2007-06-01 Created: 2007-06-01 Last updated: 2017-12-13
3. Colocalization of insulin receptor and insulin receptor substrate-1 to caveolae in primary human adipocytes
Open this publication in new window or tab >>Colocalization of insulin receptor and insulin receptor substrate-1 to caveolae in primary human adipocytes
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2004 (English)In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 271, no 12, 2471-2479 p.Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-22408 (URN)10.1111/j.1432-1033.2004.04177.x (DOI)1620 (Local ID)1620 (Archive number)1620 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
4. Human, but not rat, IRS1 targets to the plasma membrane in both human and rat primary adipocytes
Open this publication in new window or tab >>Human, but not rat, IRS1 targets to the plasma membrane in both human and rat primary adipocytes
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2007 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 363, no 3, 840-845 p.Article in journal (Refereed) Published
Abstract [en]

Adipocytes are primary targets for insulin control of metabolism. The activated insulin receptor phosphorylates insulin receptor substrate-1 (IRS1), which acts as a docking protein for downstream signal mediators. In the absence of insulin stimulation, IRS1 in rat adipocytes is intracellular but in human adipocytes IRS1 is constitutively targeted to the plasma membrane. Stimulation of adipocytes with insulin increased the amount of IRS1 at the plasma membrane 2-fold in human adipocytes, but >10-fold in rat adipocytes, with the same final amount of IRS1 at the plasma membrane in cells from both species. Cross-transfection of rat adipocytes with human IRS1, or human adipocytes with rat IRS1, demonstrated that the species difference was due to the IRS1 protein and not the cellular milieus or posttranslational modifications. Chimeric IRS1, consisting of the conserved N-terminus of rat IRS1 with the variable C-terminal of human IRS1, did not target the plasma membrane, indicating that subtle sequence differences direct human IRS1 to the plasma membrane.

Keyword
Insulin receptor substrate; Human; Rat; Insulin; Plasma membrane; Signaling; Transfection; Caveolae
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14540 (URN)10.1016/j.bbrc.2007.09.065 (DOI)
Available from: 2007-06-01 Created: 2007-06-01 Last updated: 2017-12-13

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