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- 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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| 2. |
- Parpal, Santiago, et al.
(author)
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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
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 276:13, s. 9670-9678
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Journal article (peer-reviewed)abstract
- 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.
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| 3. |
- Parpal, Santiago
(author)
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Mechanisms of insulin signaling and the role of caveolae
- 2001
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Doctoral thesis (other academic/artistic)abstract
- 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.
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