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Mechanisms of insulin signaling and the role of caveolae
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Insulin regulates metabolic as well as mitogenic processes in target cells, involving a large number of mediators of signal transduction. In its role as a growth factor, insulin stimulates cell growth, in a process we demonstrate requires the participation of the Raf-1 kinase.

Caveolae are invaginations of the plasma membrane, involved in signal transduction and intracellular transport of cholesterol. Caveolae are enriched in cholesterol, sphingolipids and the constituent protein caveolin. Herein we report that the insulin receptor is located in caveolae of plasma membrane from adipocytes. By confocal and electron microscopy we show co-localization of caveolin and the insulin receptor. Additionally, the insulin receptor independently of insulin stimulation is enriched in caveolae isolated by cell fractionation.

Cholesterol depletion has been shown to flatten caveolae and affect processes which occur in these domains. We show that depletion of cholesterol in adipocytes destroys caveolae and inhibits insulin-stimulated tyrosine phosphorylation of the insulin receptor substrate-1 (IRS-1 ), without affecting insulin receptor ligand binding or its autophosphorylation. Cholesterol-depleted adipocytes showed a decreased insulin-stimulated glucose uptake and phosphorylation of A TP citrate-lyase. Cholesterol depletion did not affect insulin's effect on the MAPK kinases ERK 1/2. She, which has been described to mediate an alternative pathway to that mediated by IRS-1 for insulin mitogenic regulation, was not involved in the regulation of the MAP kinases by insulin in adipocytes. We conclude that some other mediators which are not dependent on caveolae integrity must exist for regulation of this pathway.

The effects of cholesterol depletion on caveolae and insulin signaling prompted us to study caveolae in models of insulin resistance. We show that adipocytes from the obese and insulin resistant Zucker fa/fa rats have reduced amounts of cholesterol in caveolae compared with their lean littermates. Adipocytes of Zucker fa/fa rats have been shown to express high levels of TNF-α. We demonstrate that TNF-α treatment lowers the amount of cholesterol in caveolae in adipocytes from normal rats.

The results presented in this thesis demonstrate that insulin signaling originates in caveolae invaginations of the plasma membrane where the insulin receptor is located. Caveolae are required for certain metabolic effects of insulin but not for activation of the MAP kinase pathway, a scenario similar to what is found in cases of insulin resistance and type 2 diabetes. Moreover, alteration of the amount of cholesterol or caveolae leads to insulin resistance, suggesting that caveolae play a central role in insulin resistance and diabetes.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2001. , 45 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 684
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-25698Local ID: 10074ISBN: 91-7219-974-1 (print)OAI: oai:DiVA.org:liu-25698DiVA: diva2:246246
Public defence
2001-06-08, Berzeliussalen, Universitetssjukhuset, Linköping, 09:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-09-10Bibliographically approved
List of papers
1. Inhibition of Raf-1 kinase expression abolishes insulin stimulation of DNA synthesis in H4IIE hepatoma cells
Open this publication in new window or tab >>Inhibition of Raf-1 kinase expression abolishes insulin stimulation of DNA synthesis in H4IIE hepatoma cells
1994 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 269, no 19, 13919-13921 p.Article in journal (Refereed) Published
Abstract [en]

The involvement of Raf-1 kinase in the insulin signal transduction chain leading to control of cell proliferation was studied in the H4IIE rat hepatoma cell line by inhibiting expression of the kinase with antisense oligodeoxyribonucleotide directed against Raf-1 mRNA. Antisense oligonucleotide was found to reduce (at 2 microM) or completely block (at 15 microM) the stimulation by insulin of DNA synthesis, measured as thymidine incorporation. The residual DNA synthesis seen in the absence of insulin stimulation was also inhibited by the Raf-1 kinase antisense oligonucleotide.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-81239 (URN)
Available from: 2012-09-10 Created: 2012-09-10 Last updated: 2017-12-07Bibliographically approved
2. Localization of the insulin receptor in caveolae of adipocyte plasma membrane
Open this publication in new window or tab >>Localization of the insulin receptor in caveolae of adipocyte plasma membrane
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1999 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 13, no 14, 1961-1971 p.Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25036 (URN)9460 (Local ID)9460 (Archive number)9460 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
3. Cholesterol Depletion Disrupts Caveolae and Insulin Receptor Signaling for Metabolic Control via Insulin Receptor Substrate-1, but Not for Mitogen-activated Protein Kinase Control
Open this publication in new window or tab >>Cholesterol Depletion Disrupts Caveolae and Insulin Receptor Signaling for Metabolic Control via Insulin Receptor Substrate-1, but Not for Mitogen-activated Protein Kinase Control
2001 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 13, 9670-9678 p.Article in journal (Refereed) Published
Abstract [en]

Insulin exerts its cellular control through receptor binding in caveolae in plasmalemma of target cells (Gustavsson, J., Parpal, S., Karlsson, M., Ramsing, C., Thorn, H., Borg, M., Lindroth, M., Peterson, K. H., Magnusson, K.-E., and Strålfors, P. (1999) FASEB. J. 13, 1961–1971). We now report that a progressive cholesterol depletion of 3T3-L1 adipocytes with β-cyclodextrin gradually destroyed caveolae structures and concomitantly attenuated insulin stimulation of glucose transport, in effect making cells insulin-resistant. Insulin access to or affinity for the insulin receptor on rat adipocytes was not affected as determined by 125I-insulin binding. By immunoblotting of plasma membranes, total amount of insulin receptor and of caveolin remained unchanged. Receptor autophosphorylation in response to insulin was not affected by cholesterol depletion. Insulin treatment of isolated caveolae preparations increased autophosphorylation of receptor before and following cholesterol depletion. Insulin-increased tyrosine phosphorylation of an immediate downstream signal transducer, insulin receptor substrate-1, and activation of the further downstream protein kinase B were inhibited. In contrast, insulin signaling to mitogenic control as determined by control of the extracellular signal-related kinases 1/2, mitogen-activated protein kinase pathway was not affected. Insulin did not control Shc phosphorylation, and Shc did not control extracellular signal-related kinases 1/2, whereas cholesterol depletion constitutively phosphorylated Shc. In conclusion, caveolae are critical for propagating the insulin receptor signal to downstream targets and have the potential for sorting signal transduction for metabolic and mitogenic effects.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-47429 (URN)10.1074/jbc.M007454200 (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13Bibliographically approved
4. Reduced content of cholesterol in caveolae of adipocytes from insulin resistant, obese, and diabetic Zucker fa/fa rats
Open this publication in new window or tab >>Reduced content of cholesterol in caveolae of adipocytes from insulin resistant, obese, and diabetic Zucker fa/fa rats
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The Zucker fa/fa rat has a mutation in the leptin receptor and therefore over-eats and becomes grossly obese. The animal develops insulin resistance and tumor necrosis factor a (TNFa) has been implicated in the pathogenesis of the insulin resistance that thls animal model of type 2 diabetes exhibits. We have shown that the insulin receptor is located in caveolae of the plasma membrane and that reduction of caveolar cholesterol content makes adipocytes insulin resistant (Parpal et a., J. Biol. Chem. 276 (2001)9670-9678). Here we show that adipocytes from the Zucker fa/fa rat are insulin resistant and that this can be explained by a 50 % lower content of cholesterol in the caveolae compared to lean rats. Moreover, TNFa treatment reduces the caveolar content of cholesterol in normal rat adipocytes. An enhanced production of TNFa may thus reduce caveolar cholesterol levels and make the Zucker fa/fa rat insulin resistant.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-81241 (URN)
Available from: 2012-09-10 Created: 2012-09-10 Last updated: 2012-09-10Bibliographically approved

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