Authors
Impaired glucose tolerance precedes type 2 diabetes and is characterized by hyperinsulinemia, which develops to balance peripheral insulin resistance. To gain insight into the deleterious effects of hyperinsulinemia on skeletal muscle, we studied the consequences of prolonged insulin treatment of L6 myoblasts on insulin-dependent signaling pathways. A 24-h long insulin treatment desensitized the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) and p42/p44 MAPK pathways toward a second stimulation with insulin or insulin-like growth factor-1 and led to decreased insulin-induced glucose uptake. Desensitization was correlated to a reduction in insulin receptor substrate (IRS)-1 and IRS-2 protein levels, which was reversed by the PI3K inhibitor LY294002. Co-treatment of cells with insulin and LY294002, while reducing total IRS-1 phosphorylation, increased its phosphotyrosine content, enhancing IRS-1/PI3K association. PDK1, mTOR, and MAPK inhibitors did not block insulin-induced reduction of IRS-1, suggesting that the PI3K serine-kinase activity causes IRS-1 serine phosphorylation and its commitment to proteasomal degradation. Contrarily, insulin-induced IRS-2 down-regulation occurred via a PI3K/mTOR pathway. Suppression of IRS-1/2 down-regulation by LY294002 rescued the responsiveness of PKB and MAPK toward acute insulin stimulation. Conversely, adenoviral-driven expression of constitutively active PI3K induced an insulin-independent reduction in IRS-1/2 protein levels. IRS-2 appears to be the chief molecule responsible for MAPK and PKB activation by insulin, as knockdown of IRS-2 (but not IRS-1) by RNA interference severely impaired activation of both kinases. In summary, (i) PI3K mediates insulin-induced reduction of IRS-1 by phosphorylating it while a PI3K/mTOR pathway controls insulin-induced reduction of IRS-2, (ii) in L6 cells, IRS-2 is the major adapter molecule linking the insulin receptor to activation of PKB and MAPK, (iii) the mechanism of IRS-1/2 down-regulation is different in L6 cells compared with 3T3-L1 adipocytes. In conclusion, the reduction in IRS proteins via different PI3K-mediated mechanisms contributes to the development of an insulin-resistant state in L6 myoblasts. Impaired glucose tolerance precedes type 2 diabetes and is characterized by hyperinsulinemia, which develops to balance peripheral insulin resistance. To gain insight into the deleterious effects of hyperinsulinemia on skeletal muscle, we studied the consequences of prolonged insulin treatment of L6 myoblasts on insulin-dependent signaling pathways. A 24-h long insulin treatment desensitized the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) and p42/p44 MAPK pathways toward a second stimulation with insulin or insulin-like growth factor-1 and led to decreased insulin-induced glucose uptake. Desensitization was correlated to a reduction in insulin receptor substrate (IRS)-1 and IRS-2 protein levels, which was reversed by the PI3K inhibitor LY294002. Co-treatment of cells with insulin and LY294002, while reducing total IRS-1 phosphorylation, increased its phosphotyrosine content, enhancing IRS-1/PI3K association. PDK1, mTOR, and MAPK inhibitors did not block insulin-induced reduction of IRS-1, suggesting that the PI3K serine-kinase activity causes IRS-1 serine phosphorylation and its commitment to proteasomal degradation. Contrarily, insulin-induced IRS-2 down-regulation occurred via a PI3K/mTOR pathway. Suppression of IRS-1/2 down-regulation by LY294002 rescued the responsiveness of PKB and MAPK toward acute insulin stimulation. Conversely, adenoviral-driven expression of constitutively active PI3K induced an insulin-independent reduction in IRS-1/2 protein levels. IRS-2 appears to be the chief molecule responsible for MAPK and PKB activation by insulin, as knockdown of IRS-2 (but not IRS-1) by RNA interference severely impaired activation of both kinases. In summary, (i) PI3K mediates insulin-induced reduction of IRS-1 by phosphorylating it while a PI3K/mTOR pathway controls insulin-induced reduction of IRS-2, (ii) in L6 cells, IRS-2 is the major adapter molecule linking the insulin receptor to activation of PKB and MAPK, (iii) the mechanism of IRS-1/2 down-regulation is different in L6 cells compared with 3T3-L1 adipocytes. In conclusion, the reduction in IRS proteins via different PI3K-mediated mechanisms contributes to the development of an insulin-resistant state in L6 myoblasts. insulin receptor insulin receptor substrate phosphoinositide 3-kinase N α-tosyl-l-phenylalanyl chloromethyl ketone mitogen-activated protein kinase protein kinase B insulin-like growth factor 1 platelet-derived growth factor Dulbecco's modified Eagle's medium fetal calf serum double stranded RNA interference short interfering double stranded RNA multiplicity of infection Type 2 diabetes is caused by a progressive decrease in insulin action and gradual development of chronic hyperglycemia. Initial peripheral insulin resistance, i.e. the failure of adipose and muscle tissues to properly dispose of circulating glucose, and the failure of liver to control glucose production, is compensated by increased insulin secretion from pancreatic β-cells, leading to hyperinsulinemia. In the long-term, β-cell failure to compensate for peripheral insulin resistance leads to type 2 diabetes (1Saltiel A.R. Cell. 2001; 104: 517-529Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). Hyperinsulinemia, in association with increased circulating levels of fatty acids (2Kraegen E.W. Cooney G.J. Ye J. Thompson A.L. Exp. Clin. Endocrinol. Diabetes. 2001; 109: 516-526Crossref PubMed Scopus (102) Google Scholar), which exacerbate peripheral insulin resistance, is thought to be a major factor contributing to progression to type 2 diabetes. Activation of the insulin receptor (IR)1 by hormone binding activates a number of cellular responses such as translocation of the glucose transporter GLUT4 to the plasma membrane in muscle and adipocytes (3Pessin J.E. Thurmond D.C. Elmendorf J.S. Coker K.J. Okada S. J. Biol. Chem. 1999; 274: 2593-2596Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar), hepatic glycogen synthesis (4Bollen M. Keppens S. Stalmans W. Biochem. J. 1998; 336: 19-31Crossref PubMed Scopus (324) Google Scholar), lipogenesis (5Kersten S. EMBO Rep. 2001; 2: 282-286Crossref PubMed Scopus (490) Google Scholar), and modulation of gene expression (6O'Brien R.M. Granner D.K. Physiol. Rev. 1996; 76: 1109-1161Crossref PubMed Scopus (439) Google Scholar). Insulin signaling is initiated by the recruitment of intracellular molecules to the activated receptor and their ensuing tyrosine phosphorylation. These molecules include IRS-1/2/3/4, Cbl, and the adapter proteins Shc (7White M.F. Diabetologia. 1997; 40 Suppl. 2: S2-S17Crossref PubMed Scopus (465) Google Scholar). Phosphorylated IRSs then engage the phosphoinositide 3-kinase (PI3K) p85/p110 heterodimer by binding the SH2 domains of the p85 adapter to specific pYMXM motifs (8Backer J.M. Myers Jr., M.G. Shoelson S.E. Chin D.J. Sun X.J. Miralpeix M. Hu P. Margolis B. Skolnik E.Y. Schlessinger J. EMBO J. 1992; 11: 3469-3479Crossref PubMed Scopus (822) Google Scholar). Once captured by IRS, PI3K becomes activated and produces the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate, which stimulates the serine/threonine kinase cascade PDK-1-PKB/Akt-p70 S6 kinase (9Chan T.O. Rittenhouse S.E. Tsichlis P.N. Annu. Rev. Biochem. 1999; 68: 965-1014Crossref PubMed Scopus (876) Google Scholar). PKB/Akt and p70 S6 kinase are downstream kinases central to insulin action (10Alessi D.R. Downes C.P. Biochim. Biophys. Acta. 1998; 1436: 151-164Crossref PubMed Scopus (191) Google Scholar), the former controlling GLUT4 translocation, glycogen synthesis, and protein synthesis (11Hajduch E. Alessi D.R. Hemmings B.A. Hundal H.S. Diabetes. 1998; 47: 1006-1013Crossref PubMed Scopus (296) Google Scholar), and the latter being a key regulator of the growth promoting action of insulin (12Blume-Jensen P. Hunter T. Nature. 2001; 411: 355-365Crossref PubMed Scopus (3161) Google Scholar). Other SH2 domain containing proteins, including Grb2, SHP-2, and Nck associate with IRS to mediate insulin responses. In particular, Grb2 links insulin receptor activation to the p42/p44 mitogen-activated protein kinase (MAPK) cascade (7White M.F. Diabetologia. 1997; 40 Suppl. 2: S2-S17Crossref PubMed Scopus (465) Google Scholar). The occurrence of insulin-induced peripheral insulin resistance has been attributed to impairment of the tyrosine kinase activity of the insulin receptor, based on biochemical studies performed on skeletal muscle biopsies from type 2 diabetic patients (13Arner P. Pollare T. Lithell H. Livingston J.N. Diabetologia. 1987; 30: 437-440Crossref PubMed Scopus (164) Google Scholar, 14Nolan J.J. Freidenberg G. Henry R. Reichart D. Olefsky J.M. J. Clin. Endocrinol. Metab. 1994; 78: 471-477Crossref PubMed Scopus (87) Google Scholar, 15Maegawa H. Shigeta Y. Egawa K. Kobayashi M. Diabetes. 1991; 40: 815-819Crossref PubMed Google Scholar). 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S. 1998; PubMed Scopus Google Scholar). by with a of that to the and A and and IRS-1 was in E. with and by the into cells to by on and in 1 serum of was RNA was from cells of RNA by on a RNA was to a membrane with a and with a from The the of and was performed in as the for or by RNA interference L6 in in medium was with of and cells by the with double stranded RNA a of of with of of and of The was for to a and to the was with to insulin cells in In RNA interference occurred not with or of short interfering double stranded RNA to the in and of the IRS-1 and IRS-2 to was in 1 by the for 2 by to To the in intracellular signaling caused by a prolonged insulin treatment in muscle cells, L6 myoblasts for to insulin or not by a second treatment with 1 hormone for To the activation of the PI3K and the MAPK PI3K activity was in IRS-1 and MAPK activation was by with to of both PI3K and MAPK to a to 1 insulin was to a and in a in cells to a 24-h insulin and of the insulin signaling pathways. the an PI3K activity be the 24-h insulin MAPK activity was and insulin activates PI3K a MAPK activation is and becomes insulin stimulation not To PI3K activity a downstream in cells, we the activation of PKB by with to the active of PKB PKB activation PI3K activity the p70 S6 kinase activation an activation to that of as by insulin-induced not A of the in insulin responsiveness of PKB and MAPK that the down-regulation of both signaling pathways occurred in a with the MAPK signaling being PKB 2 The the PI3K activity and the p70 S6 kinase activation levels that the PI3K activity the PI3K activity in a 24-h insulin a in glucose as with in L6 R. 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Full Text PDF PubMed Google Scholar). the insulin receptor as a with the To L6 myoblasts to a prolonged insulin or cells with of insulin or for by a treatment with 1 insulin or a down-regulation of PKB and MAPK prolonged insulin of the for the second or a 24-h with induced a of PKB and MAPK cells for a to insulin or the of in L6 cells and the responsiveness of to insulin the 24-h to insulin the to PKB and MAPK down-regulation by on both PKB and MAPK down-regulation was to or interference with and to or interference with A and that (i) down-regulation is caused by to and (ii) prolonged to insulin acute signaling and we that a down-regulation a insulin and toward their and are not to with or with MAPK activation by was a 24-h insulin prolonged the and signaling to treatment by PKB and MAPK are of activated we insulin signaling down-regulation is by or it be caused by prolonged to an kinase we cells for to and with insulin or we of PKB and MAPK and down-regulation of insulin signaling that down-regulation of insulin signaling on a of the receptor signaling pathway. studies on fatty and an and insulin-resistant decreased IRS-1 expression levels in skeletal muscle E. Miralpeix M. M.F. J. Clin. 1992; PubMed Scopus Google Scholar, M. M. T. J. K. H. Y. Y. M. Y. T. Diabetes. 1998; 47: PubMed Scopus Google Scholar). IRS-1 was to be in adipose from diabetic patients P. S. 1997; PubMed Scopus Google and in 3T3-L1 adipocytes with insulin J.M. E. Y. Diabetologia. PubMed Scopus Google Scholar). we the protein levels of IRS-1 and IRS-2 in 24-h L6 myoblasts. IRS-1 and IRS-2 decreased a 24-h to insulin in a of controls with 1 insulin A and The decrease in IRS-1 was we did not decreased expression of the IRS-1 insulin was the insulin-induced effects J. K. J. Jr., J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google the protein levels of p42/p44 MAPK and p42/p44 expression was by prolonged insulin treatment the PKB expression increased to in a and of IRS-1 is by the pathway X.J. J.J. Diabetes. 1999; PubMed Scopus Google Scholar, R. J.J. J. Sun X.J. Cell. Biol. PubMed Scopus Google serine/threonine phosphorylation T. T. J. Egawa K. Olefsky J.M. Kobayashi M. Endocrinol. PubMed Scopus Google Scholar). we to which to IRS-1 serine/threonine phosphorylation. Co-treatment of cells with insulin and LY294002 in the of IRS-1 and in a of its a decreased serine/threonine phosphorylation. LY294002 insulin-induced IRS-2 The action of LY294002 the 24-h treatment was by with active PKB LY294002 insulin-induced IRS-1 down-regulation that PI3K IRS-1 serine/threonine phosphorylation, or via activation of downstream of To the of kinases in IRS-1 24-h L6 cells with the which both PKB and p70 S6 kinase by signaling B.A. E. J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In to LY294002, both PKB and p70 S6 kinase activation was in insulin-induced IRS-1 down-regulation To the of p42/p44 MAPK and mTOR, which have been to IRS-1 H. T. J.E. S. 2001; PubMed Scopus Google Scholar, K. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), we L6 myoblasts with or 1 insulin for in the of LY294002, or In the of insulin, LY294002, not or induced a toward a that IRS-1 phosphorylation is by LY294002, not or IRS-1 the treatment of cells with LY294002 or of insulin led to an of IRS-2, that a activity of the pathway to a IRS-2 pathway. is in with the of J. D. J. R. Cell. Biol. 2001; PubMed Scopus Google by the phosphatidylinositol in cells, a of IRS-2 and protein To gain insight into the in insulin-induced IRS-1 24-h L6 cells with in the of the inhibitors and IRS-1 phosphorylation was in IRS-1 LY294002 and not induced a decrease in IRS-1 phosphorylation LY294002 (but not the insulin-induced IRS-1 decrease A and it is PI3K that the on IRS-1 phosphorylation in 24-h L6 cells, LY294002 (but not of protein kinase inhibitors of a to in increased IRS-1 tyrosine phosphorylation and increased PI3K a 24-h insulin treatment phosphatidylinositol downstream signaling in the active as by increased IRS-1/PI3K IRS-1 tyrosine phosphorylation, and IRS-1 serine/threonine phosphorylation To the of PI3K in controlling IRS-1/2 L6 myoblasts multiplicity of infection with a constitutively active PI3K as a a of (but not activated the PI3K downstream PKB and p70 S6 kinase G. Cell. Biol. 1996; PubMed Scopus Google Scholar), and induced a decrease in IRS-1 and IRS-2 protein that IRS-1/2 down-regulation be induced in a and is by To a of insulin signaling prolonged to insulin and decreased levels of IRS L6 myoblasts for to insulin in the of LY294002, down-regulation of the inhibitor was and the cells with 1 insulin, as in A 24-h with insulin PKB and MAPK 1 and However, PKB be activated by a to 1 a 24-h with or that LY294002 was a activation of MAPK be induced treatment with insulin and LY294002, and a activation be insulin was The occurrence of (i) of IRS-1/2 down-regulation by PI3K and (ii) activation of PKB and MAPK by acute insulin stimulation prolonged that the of insulin signaling on the decreased protein levels of IRS-1/2 To the that the decrease in IRS-1 in the down-regulation of insulin we of the a protein be by of J. W. K. T. Nature. 2001; 411: PubMed Scopus Google Scholar). stranded to knockdown IRS-1 or IRS-2 in L6 cells their expression by Insulin stimulation of cells for IRS-1 led to an activation of PKB and MAPK with that of cells not to the of IRS-2 both PKB and MAPK to insulin, that the major adapter molecule linking the activated insulin receptor to downstream PKB and MAPK activation is IRS-2 in L6 cells by knockdown of IRS-2, we a decrease in IRS-1 levels it not be that the impaired activation of PKB and MAPK was of the decrease in To levels of IRS-1 the of IRS-2, L6 cells in which to IRS-2 been performed a of an these IRS-1 expression was with the multiplicity of However, insulin stimulation of PKB or MAPK was that a IRS-1 for these the of IRS-2 in L6 Insulin resistance from the of tissues to properly to the state is with hyperinsulinemia, and increased insulin secretion becomes to glucose chronic and type 2 diabetes To the induced by hyperinsulinemia in muscle cells, we L6 cells for with different hormone by a second acute hormone These a a increased insulin secretion is an insulin In PI3K recruitment to IRS-1 and activation of and MAPK pathways by acute insulin stimulation in a by the 24-h insulin with a down-regulation of both pathways hormone The responses of the pathways to the 24-h insulin MAPK activity being and the activation a of the hormone The 24-h insulin-induced down-regulation of in a decreased acute insulin-induced uptake. reduction is to be of a insulin-induced GLUT4 translocation the plasma membrane R. W. D. Diabetes. PubMed Scopus Google Scholar). the as to down-regulation of insulin receptor number kinase activity is responsible for the insulin-resistant state we the and of tyrosine phosphorylation of the insulin receptor of insulin with to the receptor a reduction of in both receptor and receptor tyrosine phosphorylation a 24-h to that receptor and activation a we intracellular downstream be in the down-regulation of insulin-induced intracellular responses. L6 cells a number of receptor molecules molecules 1992; PubMed Scopus Google Scholar). of the of a of the effects of insulin and on PKB and MAPK by prolonged with insulin or by a second to insulin or PKB and MAPK down-regulation acute or insulin of the in the 24-h that insulin and the MAPK a prolonged insulin treatment These that the and MAPK down-regulation on a signaling downstream the in insulin resistance have been including of IRS-1, decreased IRS-2 levels, decreased GLUT4 protein expression levels X.J. J.J. Diabetes. 1999; PubMed Scopus Google Scholar, J. M.F. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, E. J. 2001; PubMed Scopus Google M. Y. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar), as as of and MAPK activation via down-regulation S. T. S. Olefsky J.M. Cell. Biol. PubMed Scopus Google Scholar). we the insulin-induced insulin signaling down-regulation on in the levels of IRS molecules the downstream kinases PKB and an insulin-induced down-regulation of IRS protein levels that the down-regulation of and IRS-1 down-regulation be by PI3K with LY294002, not by PDK1, mTOR, or MAPK is the that insulin-induced IRS-1 was not by an inhibitor of and activates downstream including p70 S6 and protein kinase P. PubMed Scopus Google Scholar), the of these kinases in IRS-1 phosphorylation in L6 cells be In 3T3-L1 IRS-1 is by a pathway T. T. J. Egawa K. Olefsky J.M. Kobayashi M. Endocrinol. PubMed Scopus Google Scholar). the in as as in cells R. J.J. J. Sun X.J. Cell. Biol. PubMed Scopus Google Scholar), insulin-induced IRS-1 was not by that kinases of mTOR, PI3K or kinases of are in L6 and cells to IRS-1 degradation. to IRS-1, IRS-2 levels decreased prolonged insulin treatment in a In to IRS-1 which was on both LY294002 and insulin-induced IRS-2 of cells to LY294002 or induced an in the IRS-2 expression IRS-1 a PI3K activity to mediate IRS-2 via the that PI3K is the chief molecule controlling insulin-induced of both IRS-1 and insulin-dependent of IRS-2 levels in by of the IRS-2 gene J. J. Y. S. 2001; PubMed Scopus Google Scholar), we that of L6 cells, in the of different inhibitors or in a into IRS-1, a tyrosine phosphorylation, in that the PI3K serine kinase activity IRS-1 on serine/threonine its degradation. In of is that IRS-1 from L6 is in an in kinase in a and S. the caused a reduction in was not to IRS-1 from being that phosphorylation not not in IRS-1 degradation. of the of PI3K in IRS-1/2 down-regulation by an constitutively active PI3K of the its to the of the protein to the plasma membrane it P. S. J. EMBO J. 1997; PubMed Scopus Google Scholar). In the of insulin, expression of type (but not was to in a multiplicity of and induced a decrease in both IRS-1 and IRS-2 as in 3T3-L1 adipocytes K. H. Y. R. Olefsky J.M. PubMed Scopus Google Scholar). To insulin-induced down-regulation of and MAPK pathways and of IRS proteins, we the of insulin-induced IRS by a 24-h treatment with insulin in the of LY294002, IRS-1 and IRS-2 expression levels insulin stimulation medium to LY294002 and PI3K PKB stimulation and MAPK responsiveness the insulin to MAPK activation insulin is of the that insulin a of the which be reversed by insulin Okada S. J.E. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google and the of MAPK down-regulation as compared with PKB in 2 the IRS-1 and down-regulation we to the of by RNA interference knockdown of IRS-1 knockdown on the activation of PKB and MAPK, we that knockdown of IRS-2 induced an total of insulin activation of PKB and MAPK, IRS-2 as the chief molecule insulin downstream in L6 In summary, that prolonged insulin treatment of L6 muscle cells the effects of hyperinsulinemia, leading to down-regulation of both and MAPK signaling pathways and glucose via a decrease in IRS-1/2 that PI3K is the key molecule controlling the decrease in IRS-1/2 as down-regulation of IRS-1/2 is by PI3K induced by expression of a constitutively active we for the of a IRS-1/2 decrease and down-regulation of the and MAPK with a by IRS-2 as by in L6 cells, insulin-induced of IRS molecules is by for IRS-1 a with PI3K as a IRS-1 serine/threonine appears to be IRS-2 is by a mechanisms control IRS as IRS-1 in adipocytes appears to be T. T. J. Egawa K. Olefsky J.M. Kobayashi M. Endocrinol. PubMed Scopus Google in L6 and cells R. J.J. J. Sun X.J. Cell. Biol. PubMed Scopus Google Scholar). of IRS proteins is by serine/threonine phosphorylation Diabetes. 2001; PubMed Scopus Google and a to insulin resistance Y. Biol. 2001; 11: Full Text PDF PubMed Scopus Google Scholar), it be of to the of serine/threonine kinase the not the PI3K toward IRS serine/threonine phosphorylation. J. for the B. Hemmings for to and S. for the S. for the
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