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Rapid insulin-dependent endocytosis of the insulin receptor by caveolae in primary adipocytes
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
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2009 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 4, no 6, e5985- p.Article in journal (Refereed) Published
Abstract [en]

Background: The insulin receptor is localized in caveolae and is dependent on caveolae or cholesterol for signaling in adipocytes. When stimulated with insulin, the receptor is internalized. Methodology/Principal Findings: We examined primary rat adipocytes by subcellular fractionation to examine if the insulin receptor was internalized in a caveolae-mediated process. Insulin induced a rapid, t1/2 less than3 min, endocytosis of the insulin receptor in parallel with receptor tyrosine autophosphorylation. Concomitantly, caveolin-1 was phosphorylated at tyrosine(14) and endocytosed. Vanadate increased the phosphorylation of caveolin-1 without affecting insulin receptor phosphorylation or endocytosis. Immunocapture of endosomal vesicles with antibodies against the insulin receptor co-captured caveolin-1 and immunocapture with antibodies against tyrosine(14)-phosphorylated caveolin-1 co-captured the insulin receptor, demonstrating that the insulin receptor was endocytosed together with tyrosine(14)-phosphorylated caveolin-1. By immunogold electron microscopy the insulin receptor and caveolin-1 were colocalized in endosome vesicles that resembled caveosomes. Clathrin was not endocytosed with the insulin receptor and the inhibitor of clathrin-coated pit-mediated endocytosis, chlorpromazine, did not inhibit internalization of the insulin receptor, while transferrin receptor internalization was inhibited. Conclusion: It is concluded that in response to insulin stimulation the autophosphorylated insulin receptor in primary adipocytes is rapidly endocytosed in a caveolae-mediated process, involving tyrosine phosphorylation of caveolin-1.

Place, publisher, year, edition, pages
2009. Vol. 4, no 6, e5985- p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-21319DOI: 10.1371/journal.pone.0005985OAI: oai:DiVA.org:liu-21319DiVA: diva2:241059
Note
Original Publication: Siri Fagerholm, Unn Örtegren Kugelberg, M. Karlsson, I. Ruishalme and Peter Strålfors, Rapid insulin-dependent endocytosis of the insulin receptor by caveolae in primary adipocytes, 2009, PLoS ONE, (4), 6, e5985. http://dx.doi.org/10.1371/journal.pone.0005985 Available from: 2009-09-30 Created: 2009-09-30 Last updated: 2013-07-08
In thesis
1. Insulin signaling in primary adipocytes in insulin sensitive and insulin resistant states
Open this publication in new window or tab >>Insulin signaling in primary adipocytes in insulin sensitive and insulin resistant states
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Increasing numbers of people world-wide develops the disease type 2 diabetes. Development of type 2 diabetes is characterized by a shift from an insulin sensitive state to an insulin resistant state in peripheral insulin responding organs, which originates from the development of insulin resistance in the adipose tissue. Insulin resistance in combination with reduced pancreatic insulin secretion lead to overt type 2 diabetes.

In this thesis, the insulin signaling network in primary adipocytes was analyzed. Key proteins and mechanisms were studied to gain deeper knowledge of signaling both in the insulin sensitive state and in the insulin resistant state produced by rapid weight gain as well as in type 2 diabetes.

The surface of the adipocyte is dotted with invaginations in the cell membrane called caveolae that act as important metabolic and signaling platforms in adipocytes, and also harbor the insulin receptor. In paper I we show that insulin stimulation of primary adipocytes results in a rapid phosphorylation of the insulin receptor and caveolin-1, and that internalization of the proteins is mediated by endocytosis of caveolae.

Weight gain due to overfeeding and obesity has been associated with the development of insulin resistance in insulin sensitive tissues such as the adipose tissue. In paper II we show that short-term overfeeding for one month of lean subjects results in an insulin resistant state. At the end of the study, the subjects had developed a mild systemic insulin resistance. Moreover, in isolated subcutaneous adipocytes we found several alterations of the insulin signaling pathway that mimicked alterations found in isolated subcutaneous adipocytes from subjects with type 2 diabetes.

In paper III we present a first dynamic mathematical model of the insulin signaling network in human adipocytes that are based on experimental data acquired in a consistent fashion. The model takes account of insulin signaling in both the healthy, insulin sensitive state and in the insulin resistant state of type 2 diabetes. We show that attenuated mTORC1-mediated positive feedback to control of phosphorylation of IRS1 at Ser307 is an essential component of the insulin resistant state of type 2 diabetes. A future application of the model is the identification and evaluation of drug targets for the treatment of insulin resistance and type 2 diabetes.

In paper IV we examine the protein kinase that catalyzes the insulin stimulated mTORC1- mediated feedback to IRS1. We find that the phosphorylation of IRS1 at Ser307 is not likely to be catalyzed by the kinases S6K1, mTOR or PKB. However, a catalyzing protein kinase for the in vitro phosphorylation of IRS1 at Ser307 was found to be associated with the complex mTORC1.

In conclusion, this thesis provide new insights and characterize mechanisms of the intrinsically complex insulin signaling network of primary adipocytes, both in insulin sensitive and insulin resistant states.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2013. 64 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1369
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-95562 (URN)978-91-7519-577-3 (ISBN)
Public defence
2013-08-30, Berzeliussalen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2013-07-08 Created: 2013-07-08 Last updated: 2013-10-23Bibliographically approved

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Fagerholm, SiriÖrtegren Kugelberg, UnnStrålfors, Peter

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