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Phosphorylation of IRS1 at Serine 307 in Response to Insulin in Human Adipocytes Is Not Likely to be Catalyzed by p70 Ribosomal S6 Kinase
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, Obstetrics and gynecology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Gynecology and Obstetrics in Linköping.
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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. Vol. 8, no 4
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-93257DOI: 10.1371/journal.pone.0059725ISI: 000317717300032OAI: oai:DiVA.org:liu-93257DiVA, id: diva2:623774
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: 2018-04-27
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. p. 64
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)
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Available from: 2013-07-08 Created: 2013-07-08 Last updated: 2013-10-23Bibliographically approved
2. Insulin Signalling in Human Adipocytes and its Interplay with beta-Adrenergic Control of Lipolysis
Open this publication in new window or tab >>Insulin Signalling in Human Adipocytes and its Interplay with beta-Adrenergic Control of Lipolysis
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [sv]

The prevalence of obesity has over the last 40 years nearly tripled and obesity is one of the major risk factors of developing type 2 diabetes. Type 2 diabetes was formerly called adultonset diabetes but today, probably due to the rise in childhood obesity, it is also seen in children and adolescents. Type 2 diabetes is diagnosed when the body no longer can control the glucose levels in the blood. This is due to an insulin resistant state in the insulin responding tissues, liver, adipose and muscle and insufficient production of insulin in the pancreas. However, in spite of extensive research the mechanisms behind insulin resistance is still not known.

The adipose tissue is believed to play a major role in the development of whole body insulin resistance. Adipocytes are the most important sites for storage of the high energy containing triacylglycerols. Insulin stimulation causes the adipocyte to increase the uptake of glucose and to reduce lipolysis: the hydrolysis of triacylglycerol and release of glycerol and fatty acids. The insulin signalling network is complex with numerous proteins involved. These signaling proteins not only transmit the insulin signal but also create negative and positive feedbackloops and induce cross talk between different parts of the network and with the signalling of other hormones. One important positive feedback in insulin signalling is the mTORC1 mediated feedback to phosphorylation of IRS1 at serine 307. In paper I we found that in human adipocytes this feedback is not likely catalysed by the assumed kinase S6K1. However we find an immunoprecipitate of mTOR to contain a ser307 phosphorylating kinase.

Scaffolding proteins serve as docking sites for several proteins to promote protein-protein interactions that facilitate signal transduction. In paper II we demonstrate the existence of the scaffolding protein IQGAP1 in human adipocytes and that the expression of IQGAP1 is downregulated in type 2 diabetes. We reveal that IQGAP1 co-localises with caveolae, invaginations of the plasma membrane where the insulin receptor is situated, and that this interaction is increased upon insulin stimulation.

In paper III we focus on the control of lipolysis, and sought to understand the interplay between insulin and beta-adrenergic stimulation. We demonstrated that the re-esterification of fatty acids is downregulated in type 2 diabetes causing an increased release of fatty acids from the cells. We showed that beta-adrenergic stimulation with isoproterenol induced a negative feedback via PKA/Epac1 -> PI3K -> PKB -> PDE3B that reduced the cAMP levels and thereby also reduced lipolysis. We also showed that insulin, in addition to its well-known anti-lipolytic effect, at high concentrations had a positive effect on lipolysis. In conclusion we reveal an intricate control of the stimulation as well as the inhibition of lipolysis induced by both isoproterenol and insulin.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2018. p. 49
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1624
National Category
Endocrinology and Diabetes Cell and Molecular Biology Cell Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:liu:diva-147618 (URN)10.3384/diss.diva-147618 (DOI)9789176852859 (ISBN)
Public defence
2018-06-05, Berzeliussalen, Campus US, Linköping, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2018-04-27 Created: 2018-04-27 Last updated: 2018-04-27Bibliographically approved

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Rohini Rajan, MeenuFagerholm, SiriKjölhede, PrebenTurkina, MariaStrålfors, Peter

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