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Insulin signaling in primary adipocytes in insulin sensitive and insulin resistant states
Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
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: urn:nbn:se:liu:diva-95562ISBN: 978-91-7519-577-3 (print)OAI: oai:DiVA.org:liu-95562DiVA: diva2:636052
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
List of papers
1. Rapid insulin-dependent endocytosis of the insulin receptor by caveolae in primary adipocytes
Open this publication in new window or tab >>Rapid insulin-dependent endocytosis of the insulin receptor by caveolae in primary adipocytes
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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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-21319 (URN)10.1371/journal.pone.0005985 (DOI)
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
2. Short-Term Overeating Induces Insulin Resistance in Fat Cells in Lean Human Subjects
Open this publication in new window or tab >>Short-Term Overeating Induces Insulin Resistance in Fat Cells in Lean Human Subjects
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2009 (English)In: Molecular medicine (Cambridge, Mass. Print), ISSN 1076-1551, E-ISSN 1528-3658, Vol. 15, no 7-8, 228-234 p.Article in journal (Refereed) Published
Abstract [en]

Insulin resistance and type 2 diabetes (T2D) are closely linked to obesity. Numerous prospective studies have reported on weight gain, insulin resistance, and insulin signaling in experimental animals, but not in humans. We examined insulin signaling in adipocytes from lean volunteers, before and at the end of a 4-wk period of consuming a fast-food, high-calorie diet that led to weight gain. We also examined adipocytes from patients with T2D. During the high-calorie diet, subjects gained 10% body weight and 19% total body fat, but stayed lean (body mass index = 24.3 kg/m2) and developed moderate systemic insulin resistance. Similarly to the situation in T2D subjects, in subjects on the high-calorie diet, the amount of insulin receptors was reduced and phosphorylation of IRS1 at tyrosine and at serine-307 (human sequence, corresponding to murine serine-302) were impaired. The amount of insulin receptor substrate protein-1 (IRS1) and the phosphorylation of IRS1 at serine-312 (human sequence, corresponding to murine serine-307) were unaffected by the diet. Unlike the T2D subjects, in subjects on the high-calorie diet, likely owing to the ongoing weight-gain, phosphorylation of MAP-kinases ERK1/2 became hyperresponsive to insulin. To our knowledge this study is the first to investigate insulin signaling during overeating in humans, and it demonstrates that T2D effects on intracellular insulin signaling already occur after 4 wks of a high-calorie diet and that the effects in humans differ from those in laboratory animals.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-20893 (URN)10.2119/molmed.2009.00037 (DOI)000276043800004 ()
Available from: 2009-09-24 Created: 2009-09-24 Last updated: 2017-12-13
3. Insulin Signaling in Type 2 Diabetes: Experimental and Modeling Analyses Reveal Mechanisms of Insulin Resistance in Human Adipocytes
Open this publication in new window or tab >>Insulin Signaling in Type 2 Diabetes: Experimental and Modeling Analyses Reveal Mechanisms of Insulin Resistance in Human Adipocytes
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2013 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 14, 9867-9880 p.Article in journal (Refereed) Published
Abstract [en]

Type 2 diabetes originates in an expanding adipose tissue that for unknown reasons becomes insulin resistant. Insulin resistance reflects impairments in insulin signaling, but mechanisms involved are unclear because current research is fragmented. We report a systems-level mechanistic understanding of insulin resistance in humans. We developed a dynamic mathematical model of insulin signaling – normally and in diabetes – based on quantitative steady-state and dynamic time-course data on signaling intermediaries in human mature adipocytes. At the core of insulin resistance is attenuation of a positive feedback from mammalian target of rapamycin in complex with raptor (mTORC1) to the insulin receptor substrate-1 (IRS1), which explains reduced sensitivity and signal strength throughout the signaling network. We demonstrate the potential of the model for identification of drug targets, e.g. increasing the feedback restores insulin signaling. Our findings suggest that insulin resistance in an expanded adipose tissue results from cell growth restriction to prevent cell necrosis.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84999 (URN)10.1074/jbc.M112.432062 (DOI)000317114000027 ()
Available from: 2012-10-30 Created: 2012-10-30 Last updated: 2017-12-07Bibliographically approved
4. Phosphorylation of IRS1 at Serine 307 in Response to Insulin in Human Adipocytes Is Not Likely to be Catalyzed by p70 Ribosomal S6 Kinase
Open this publication in new window or tab >>Phosphorylation of IRS1 at Serine 307 in Response to Insulin in Human Adipocytes Is Not Likely to be Catalyzed by p70 Ribosomal S6 Kinase
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 4Article in journal (Refereed) Published
Abstract [en]

The insulin receptor substrate-1 (IRS1) is phosphorylated on serine 307 (human sequence, corresponding to murine serine 302) in response to insulin as part of a feedback loop that controls IRS1 phosphorylation on tyrosine residues by the insulin receptor. This in turn directly affects downstream signaling and is in human adipocytes implicated in the pathogenesis of insulin resistance and type 2 diabetes. The phosphorylation is inhibited by rapamycin, a specific inhibitor of mammalian target of rapamycin (mTOR) in complex with raptor (mTORC1). The mTORC1-downstream p70 ribosomal protein S6 kinase (S6K1), which is activated by insulin, can phosphorylate IRS1 at serine 307 in vitro and is considered the physiological protein kinase. Because the IRS1 serine 307-kinase catalyzes a critical step in the control of insulin signaling and constitutes a potential target for treatment of insulin resistance, it is important to know whether S6K1 is the physiological serine 307-kinase or not. We report that, by several criteria, S6K1 does not phosphorylate IRS1 at serine 307 in response to insulin in intact human primary adipocytes: (i) The time-courses for phosphorylation of S6K1 and its phosphorylation of S6 are not compatible with the phosphorylation of IRS1 at serine 307; (ii) A dominant-negative construct of S6K1 inhibits the phosphorylation of S6, without effect on the phosphorylation of IRS1 at serine 307; (iii) The specific inhibitor of S6K1 PF-4708671 inhibits the phosphorylation of S6, without effect on phosphorylation of IRS1 at serine 307. mTOR-immunoprecipitates from insulin-stimulated adipocytes contains an unidentified protein kinase specific for phosphorylation of IRS1 at serine 307, but it is not mTOR or S6K1.

Place, publisher, year, edition, pages
Public Library of Science, 2013
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-93257 (URN)10.1371/journal.pone.0059725 (DOI)000317717300032 ()
Note

Funding Agencies|Swedish Diabetes Fund||Novo Nordic Foundation||University of Linkoping||Swedish Research Council||

Available from: 2013-05-28 Created: 2013-05-28 Last updated: 2017-12-06

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