Attenuation of Insulin-stimulated Insulin Receptor Substrate-1 Serine 307 Phosphorylation in Insulin Resistance of Type 2 Diabetes

Insulin resistance is a primary characteristic of type 2 diabetes and likely causally related to the pathogenesis of the disease. It is a result of defects in signal transduction from the cell surface receptor of insulin to target effects. We found that insulin-stimulated phosphorylation of serine 307 (corresponding to serine 302 in the murine sequence) in the immediate downstream mediator protein of the insulin receptor, insulin receptor substrate-1 (IRS1), is required for efficient insulin signaling and that this phosphorylation is attenuated in adipocytes from patients with type 2 diabetes. Inhibition of serine 307 phosphorylation by rapamycin mimicked type 2 diabetes and reduced the sensitivity of IRS1 tyrosine phosphorylation in response to insulin, while stimulation of the phosphorylation by okadaic acid, in cells from patients with type 2 diabetes, rescued cells from insulin resistance. EC50 for insulin-stimulated phosphorylation of serine 307 was about 0.2 nm with a t1/2 of about 2 min. The amount of IRS1 was similar in cells from non-diabetic and diabetic subjects. These findings identify a molecular mechanism for insulin resistance in non-selected patients with type 2 diabetes. Insulin resistance is a primary characteristic of type 2 diabetes and likely causally related to the pathogenesis of the disease. It is a result of defects in signal transduction from the cell surface receptor of insulin to target effects. We found that insulin-stimulated phosphorylation of serine 307 (corresponding to serine 302 in the murine sequence) in the immediate downstream mediator protein of the insulin receptor, insulin receptor substrate-1 (IRS1), is required for efficient insulin signaling and that this phosphorylation is attenuated in adipocytes from patients with type 2 diabetes. Inhibition of serine 307 phosphorylation by rapamycin mimicked type 2 diabetes and reduced the sensitivity of IRS1 tyrosine phosphorylation in response to insulin, while stimulation of the phosphorylation by okadaic acid, in cells from patients with type 2 diabetes, rescued cells from insulin resistance. EC50 for insulin-stimulated phosphorylation of serine 307 was about 0.2 nm with a t1/2 of about 2 min. The amount of IRS1 was similar in cells from non-diabetic and diabetic subjects. These findings identify a molecular mechanism for insulin resistance in non-selected patients with type 2 diabetes. The incidence of type 2 diabetes is rapidly increasing in all parts of the world that allows a sedentary and affluent lifestyle, as the disease is closely associated with obesity. It has been estimated that over 200 million people will be afflicted with the disease by the end of this decade. The pathogenesis of diabetes is not understood in any great detail, but it is generally believed that insulin resistance in skeletal muscle and especially in adipose tissue is an early, if not primary, event. The peripheral insulin resistance is compensated for by increased concentrations of circulating insulin, masking development to diabetes. Eventually the pancreatic β-cells fail to compensate for the insulin resistance, and type 2 diabetes can be diagnosed. The insulin resistance of muscle and fat tissues is the result of impaired signal transduction from the cell surface receptor of insulin to metabolic effects, such as to increase glucose transport and inhibit lipolysis in adipocytes (1Saltiel A.R. Kahn C.R. Nature. 2001; 414: 799-806Crossref PubMed Scopus (3935) Google Scholar, 2Birnbaum M.J. J. Clin. Inv. 2001; 108: 655-659Crossref PubMed Scopus (83) Google Scholar). 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This site has also been reported to be phosphorylated in response to tumor necrosis factor-α (24Aguirre V. Uchida T. Yenush L. Davis R. White M.F. J. Biol. Chem. 2000; 275: 9047-9054Abstract Full Text Full Text PDF PubMed Scopus (1173) Google Scholar) and in animal models of type 2 diabetes (19Werner E.D. Lee J. Hansen L. Yuan M. Shoelson S.E. J. Biol. Chem. 2004; 279: 35298-35305Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 25Um S.H. Frigerio F. Watanabe M. Picard F. Joaquin M. Sticker M. Fumagalli S. Allegrini P.R. Kozma S.C. Auwerx J. Thomas G. Nature. 2004; 431: 200-205Crossref PubMed Scopus (1368) Google Scholar). Recently, phosphorylation of IRS1 on serine 302 in murine 32D cells (corresponding to serine 307 in human IRS1) was reported to be stimulated by insulin and abolishing of this phosphorylation by introduction of an alanine residue in place of serine 302 impaired insulin-stimulated tyrosine-specific phosphorylation of IRS1 (26Giraud J. Leshan R. Lee Y-H. White M.F. J. Biol. Chem. 2004; 279: 3447-3454Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). However, negative effects by serine 302 phosphorylation on insulin stimulation of IRS1 tyrosine phosphorylation (27Harrington L.S. Findlay G.M. Gray A. Tolkacheva T. Wigfield S. Rebbholz H. Barnett J. Leslie N.R. Cheng S. Shepherd P.R. Gout I. Downes C.P. Lamb R.F. J. Cell Biol. 2004; 166: 213-223Crossref PubMed Scopus (927) Google Scholar) and disruption of insulin receptor-IRS1 interaction using a yeast two-hybrid method (19Werner E.D. Lee J. Hansen L. Yuan M. Shoelson S.E. J. Biol. Chem. 2004; 279: 35298-35305Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar) have also been reported. Herein we have examined the effect of IRS1 serine 307 phosphorylation on IRS1 tyrosine phosphorylation in response to insulin and the ability of insulin to induce phosphorylation of serine 307 in primary adipocytes from patients with type 2 diabetes. Subjects—Subject characteristics are described in the respective figure legends. Informed consent from patients and approval by the Ethics Committee were obtained. Materials—Mouse anti-phosphotyrosine (PY20) monoclonal antibodies were from Transduction Laboratories (Lexington, KY). Rabbit anti-IRS1 and goat anti-actin-(1–19) polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-phospho-serine 307-IRS1 antibodies were from Cell Signaling Technology (Beverly, MA). Insulin, rapamycin, okadaic acid, and other chemicals were from Sigma or as indicated in the text. Isolation and Incubation of Adipocytes—Subcutaneous adipose tissue was obtained as described in the figure legends to Figs. 1 and 4. Adipocytes were isolated by collagenase (type 1, Worthington) digestion as described (28Strålfors P. Honnor R.C. Eur. J. Biochem. 1989; 182: 379-385Crossref PubMed Scopus (116) Google Scholar). Tissue pieces from biopsies performed with local anesthesia using xylocain were extensively washed in large volumes of 0.15 m NaCl before digestion with collagenase. At a final concentration of 100 μl packed cell volume per ml, cells were incubated in Krebs-Ringer solution (0.12 m NaCl, 4.7 mm KCl, 2.5 mm CaCl2, 1.2 mm MgSO4, 1.2 mm KH2PO4) containing 20 mm Hepes, pH 7.40, 1% (w/v) fatty acid-free bovine serum albumin, 100 nm phenylisopropyladenosine, 0.5 unit·ml–1 adenosine deaminase with 2 mm glucose, at 37 °C on a shaking water bath. For analysis after 20–24-h incubation cells were incubated at 37 °C, 10% CO2 in the same solution mixed with an equal volume of Dulbecco's modified Eagle's medium containing 7% (w/v) albumin, 200 nm phenylisopropyladenosine, 20 mm Hepes, 50 UI/ml penicillin, 50 μg/ml streptomycin, pH 7.40 (3Danielsson A. Öst A. Lystedt E. Kjolhede P. Gustavsson J. Nystrom F.H. Strålfors P. FEBS J. 2005; 272: 141-151Crossref PubMed Scopus (64) Google Scholar). Before analysis cells were washed and transferred to the Krebs-Ringer solution.FIGURE 4Effects of okadaic acid on insulin-resistant adipocytes from patients with type 2 diabetes. Abdominal subcutaneous adipocytes were obtained from Caucasian male type 2 diabetic subjects (61 ± 2 years; BMI: 36 ± 4 kg/m2, HbA1c: 5.9 ± 0.2%) after local anesthesia with xylocain and needle biopsy through the skin. Cells were incubated with (filled circles) or without (open circles) 1 μm okadaic acid for 15 min when insulin at the indicated concentration was added for 10 min, and cells were analyzed for IRS1 tyrosine phosphorylation (mean ± S.E., n = 4 subjects).View Large Image Figure ViewerDownload Hi-res image Download (PPT) SDS-PAGE and Immunoblotting—Cell incubations were terminated by separating cells from medium by centrifugation through dinonylphtalate. To minimize postincubation signaling modifications in the cells and protein modifications during immunoprecipitation, the cells were immediately dissolved in SDS and β-mercaptoethanol with protease and protein phosphatase inhibitors, frozen within 10 s, and thawed in boiling water (28Strålfors P. Honnor R.C. Eur. J. Biochem. 1989; 182: 379-385Crossref PubMed Scopus (116) Google Scholar). Equal amounts of cells as determined by lipocrit, which is total cell volume, was subjected to SDS-PAGE and immunoblotting. After SDS-PAGE and electrotransfer membranes were incubated with the indicated antibodies that were detected using with as and by pH SDS-PAGE and and IRS1, of the was determined to IRS1 (3Danielsson A. Öst A. Lystedt E. Kjolhede P. Gustavsson J. Nystrom F.H. Strålfors P. FEBS J. 2005; 272: 141-151Crossref PubMed Scopus (64) Google Scholar). of IRS1 primary subcutaneous adipocytes from a of non-diabetic subjects insulin stimulated the phosphorylation of IRS1 on serine 307 the of this phosphorylation in primary human However, in adipocytes from an of obese 1, and or male patients with type 2 diabetes this serine 307 phosphorylation of IRS1 was its in the disease. same diabetic patients the insulin-stimulated phosphorylation of IRS1 on tyrosine was at insulin concentrations A. Öst A. Lystedt E. Kjolhede P. Gustavsson J. Nystrom F.H. Strålfors P. FEBS J. 2005; 272: 141-151Crossref PubMed Scopus (64) Google of IRS1 in has been that the amount of IRS1 protein is reduced in adipocytes from patients with diabetes (12Carvalho E. Jansson P. Axelsen M. Eriksson J. Huang X. Groop L. Rondinone C. Sjöström L. Smith U. FASEB J. 1999; 13: 2173-2178Crossref PubMed Scopus (122) Google Scholar). We found the amount of IRS1 protein to be in the adipocytes from the of patients with type 2 diabetes and the non-diabetic However, it can be that were in the not have been and for of 307 was phosphorylated in response to insulin in a with EC50 = 0.2 nm in cells from non-diabetic subjects This is the insulin receptor = (3Danielsson A. Öst A. Lystedt E. Kjolhede P. Gustavsson J. Nystrom F.H. Strålfors P. FEBS J. 2005; 272: 141-151Crossref PubMed Scopus (64) Google Scholar) a downstream effect signal by protein for A. Öst A. Lystedt E. Kjolhede P. Gustavsson J. Nystrom F.H. Strålfors P. FEBS J. 2005; 272: 141-151Crossref PubMed Scopus (64) Google studies in cells from non-diabetic subjects a phosphorylation of serine 307 after about 2 min which was the tyrosine phosphorylation of IRS1, effect within 1 min of 307 in Cells from Cells protein phosphatase okadaic acid protein at 1 nm and protein at 1 μm concentrations in adipocytes D. Honnor R.C. Y. P. 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A. 2005; PubMed Scopus (147) Google Scholar), other effects of rapamycin downstream of IRS1 Science. 2005; PubMed Scopus Google Scholar), which it to the analysis of rapamycin effects on serine 307 phosphorylation in human adipocytes to effects on glucose transport This is a first of a of serine phosphorylation of IRS1 in primary human cells that also are target cells for in two of patients with type 2 diabetes findings identify a mechanism with the to the insulin resistance of the a phosphorylation of serine 307 the tyrosine phosphorylation of IRS1 at concentrations of 2 diabetes has a strong genetic and it that different genetic to the at phosphorylation of IRS1 on serine We used two different to serine 307 phosphorylation that effects on the sensitivity to tyrosine phosphorylation by of serine 307 by protein phosphatase in cells from patients with type 2 diabetes the cells adipocytes from non-diabetic subjects. Inhibition of serine 307 phosphorylation by rapamycin in adipocytes from non-diabetic subjects the cells cells from type 2 diabetic patients (3Danielsson A. Öst A. Lystedt E. Kjolhede P. Gustavsson J. Nystrom F.H. Strålfors P. FEBS J. 2005; 272: 141-151Crossref PubMed Scopus (64) Google Scholar). and okadaic acid the phosphorylation of phosphorylation sites in the tyrosine phosphorylation of the insulin receptor or IRS1, and have different of two different and in and diabetic the sensitivity of IRS1 to be in response to insulin was in a It is to findings as a between serine 307 phosphorylation and reduced insulin sensitivity in cells from patients with type 2 diabetes. To this it will be to IRS1 and it with serine 307 IRS1, to block in adipocytes and with serine 307 to in adipocytes from patients with type 2 diabetes. It be that sites are phosphorylated and with serine 307 to IRS1 to tyrosine of serine 302 to alanine in murine 32D that the phosphorylation of this serine is to inhibit IRS1 tyrosine phosphorylation in response to insulin (26Giraud J. Leshan R. Lee Y-H. White M.F. J. Biol. Chem. 2004; 279: 3447-3454Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The EC50 for insulin-stimulated phosphorylation of serine 307 downstream protein 307 is in a for phosphorylation by the downstream protein 2003; PubMed Scopus Google Scholar). The ability of rapamycin to inhibit the phosphorylation the of protein in the The has been to by phosphorylation of serine phosphorylation Science. 2005; PubMed Scopus Google Scholar). not to be by rapamycin, but of the inhibit the Science. 2005; PubMed Scopus Google Scholar). a for serine 307 phosphorylation the insulin receptor and downstream has been shown to IRS1 on serine 307 in (27Harrington L.S. Findlay G.M. Gray A. Tolkacheva T. Wigfield S. Rebbholz H. Barnett J. Leslie N.R. Cheng S. Shepherd P.R. Gout I. Downes C.P. Lamb R.F. J. Cell Biol. 2004; 166: 213-223Crossref PubMed Scopus (927) Google Scholar). the other phosphorylation of 307 in murine cells 312 in human a negative of insulin stimulation (23Greene M.W. Sakaue H. Wang L. Alessi D.R. Roth R.A. J. Biol. Chem. 2003; 278: 8199-8211Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 25Um S.H. Frigerio F. Watanabe M. Picard F. Joaquin M. Sticker M. Fumagalli S. Allegrini P.R. Kozma S.C. Auwerx J. Thomas G. Nature. 2004; 431: 200-205Crossref PubMed Scopus (1368) Google Scholar). This mechanism is different from the phosphorylation of serine 307 in human which IRS1 to activation by the insulin It be that negative (19Werner E.D. Lee J. Hansen L. Yuan M. Shoelson S.E. J. Biol. Chem. 2004; 279: 35298-35305Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, L.S. Findlay G.M. Gray A. Tolkacheva T. Wigfield S. Rebbholz H. Barnett J. Leslie N.R. Cheng S. Shepherd P.R. Gout I. Downes C.P. Lamb R.F. J. Cell Biol. 2004; 166: 213-223Crossref PubMed Scopus (927) Google Scholar) or (26Giraud J. Leshan R. Lee Y-H. White M.F. J. Biol. Chem. 2004; 279: 3447-3454Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) effects of murine serine 302 phosphorylation (corresponding to human serine on IRS1 tyrosine phosphorylation have been performed in cell the findings are from primary human The mechanism from insulin a protein the serine 307 phosphorylation be an of sensitivity through The mechanism be for insulin sensitivity in adipocytes of human The IRS1 serine 307 with tyrosine phosphorylation that serine 307 phosphorylation is not required for the tyrosine phosphorylation but for the of tyrosine phosphorylation at insulin determined after 10 min. This be by of of IRS1, as has been described for phosphorylation of the of and sequence) in IRS1 of cells K. H. Hemi R. LeRoith D. M. Kanety H. R. Zick Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). It will be to this mechanism is in other target tissues of insulin such as and in The protein and to be and are by insulin and the of the in type 2 diabetes. The of insulin to enhance the phosphorylation of serine 307 be a of insulin resistance and type 2 diabetes. The protein and the protein in serine 307 phosphorylation are for We Kjolhede and for biopsies of adipose we the of all diabetic and non-diabetic patients that to adipose