Insulin receptor substrates (IRSs) are tyrosine-phosphorylated following stimulation with insulin, insulin-like growth factors (IGFs), and interleukins. A key question is whether different IRSs play different roles to mediate insulin's metabolic and growth-promoting effects. In a novel system of insulin receptor-deficient hepatocytes, insulin fails to (1) stimulate glucose phosphorylation; (2) enhance glycogen synthesis; (3) suppress glucose production, and (4) promote mitogenesis. However, insulin's ability to induce IRS-1 and gab-1 phosphorylation and binding to phosphatidylinositol (PI) 3-kinase is unaffected, by virtue of the compensatory actions of IGF-1 receptors. In contrast, phosphorylation of IRS-2 and generation of IRS-2/PI 3-kinase complexes are markedly reduced. Thus, absence of insulin receptors selectively reduces IRS-2, but not IRS-1 phosphorylation, and the impairment of IRS-2 activation is associated with lack of insulin effects. To address whether phosphorylation of additional IRSs is also affected, phosphotyrosine-containing proteins were analyzed in PI 3-kinase immunoprecipitates from insulin-treated cells. However, these experiments indicate that IRS-1 and IRS-2 are the main PI 3-kinase-bound proteins in hepatocytes. These data identify IRS-2 as the main effector of both the metabolic and growth-promoting actions of insulin through PI 3-kinase in hepatocytes, and IRS-1 as the main substrate mediating the mitogenic actions of IGF-1 receptors (Rother, 1999).

Insulin receptor substrates-1 and -2 (IRS-1 and -2) are important substrates of the insulin receptor tyrosine kinase. IRS-2 can mediate translocation of the insulin responsive glucose transporter GLUT4 in a physiologically relevant target cell for insulin action. Co-immunoprecipitation experiments performed on cell lysates derived from freshly isolated rat adipose cells incubated in the presence or absence of insulin indicate that twice as much phosphatidylinositol 3-kinase is associated with endogenous IRS-1 as with IRS-2 after insulin stimulation. When rat adipose cells in primary culture are transfected with expression vectors for IRS-1 or IRS-2, 40-fold overexpression of human IRS-1 or murine IRS-2 is observed. In addition, anti-phosphotyrosine immunoblotting experiments confirm that the recombinant substrates are phosphorylated in response to insulin stimulation. To examine the role of IRS-2 in insulin-stimulated translocation of GLUT4, the effects of overexpression of IRS-1 and -2 on translocation of a co-transfected epitope-tagged GLUT4 (GLUT4-HA) were studied. Overexpression of IRS-1 or IRS-2 in adipose cells results in a significant increase in the basal level of cell surface GLUT4 (in the absence of insulin). Interestingly, at maximally effective concentrations of insulin (60 nM), the level of cell surface GLUT4 in cells overexpressing IRS-1 or -2 significantly exceeds the maximal recruitment observed in the control cells (160% and 135% of control, respectively; p < 0.003). These data directly demonstrate that IRS-2, like IRS-1, is capable of participating in insulin signal transduction pathways leading to the recruitment of GLUT4. Thus, IRS-2 may provide an alternative pathway for critical metabolic actions of insulin (Zhou, 1997).

The insulin receptor initiates insulin action by phosphorylating multiple intracellular substrates. Insulin receptor substrates (IRS)-1 and -2 can mediate insulin's action to promote translocation of GLUT4 glucose transporters to the cell surface in rat adipose cells. Although IRS-1, -2, and -4 are similar in overall structure, IRS-3 is approximately 50% shorter and differs with respect to sites of tyrosine phosphorylation. Nevertheless both IRS-3 and IRS-4 can also stimulate translocation of GLUT4. Rat adipose cells were cotransfected with expression vectors for hemagglutinin (HA) epitope-tagged GLUT4 (GLUT4-HA) and human IRS-1, murine IRS-3, or human IRS-4. Overexpression of IRS-1 leads to a 2-fold increase in cell surface GLUT4-HA in cells incubated in the absence of insulin; overexpression of either IRS-3 or IRS-4 elicits a larger increase in cell surface GLUT4-HA. Indeed, the effect of IRS-3 in the absence of insulin is approximately 40% greater than the effect of a maximally stimulating concentration of insulin in cells not overexpressing IRS proteins. Because phosphatidylinositol (PI) 3-kinase is essential for insulin-stimulated translocation of GLUT4, a mutant IRS-3 molecule (IRS-3-F4) was studied in which Phe was substituted for Tyr in all four YXXM motifs (the phosphorylation sites predicted to bind to and activate PI 3-kinase). Interestingly, overexpression of IRS-3-F4 does not promote translocation of GLUT4-HA, but actually inhibits the ability of insulin to stimulate translocation of GLUT4-HA to the cell surface. These data suggest that IRS-3 and IRS-4 are capable of mediating PI 3-kinase-dependent metabolic actions of insulin in adipose cells, and that IRS proteins play a physiological role in mediating translocation of GLUT4 (Zhou, 1999).

To understand the role of the insulin receptor pathway in beta-cell function, stable beta-cells (betaIRS1-A) were created that overexpress by 2-fold the insulin receptor substrate-1 (IRS-1) and they were compared to vector-expressing controls. IRS-1 overexpression dramatically increases basal cytosolic Ca2+ levels from 81 to 278 nM, but it does not affect Ca2+ response to glucose. Overexpression of the insulin receptor also causes an increase in cytosolic Ca2+. Increased cytosolic Ca2+ is due to inhibition of Ca2+ uptake by the endoplasmic reticulum, because endoplasmic reticulum Ca2+ uptake and content are reduced in betaIRS1-A cells. Fractional insulin secretion is significantly increased 2-fold, and there was a decrease in betaIRS1-A insulin content and insulin biosynthesis. Steady-state insulin mRNA levels and glucose-stimulated ATP are unchanged. High IRS-1 levels also reduce beta-cell proliferation. These data demonstrate a direct link between the insulin receptor signaling pathway and the Ca2+-dependent pathways regulating insulin secretion of beta-cells. It is postulated that during regulated insulin secretion, released insulin binds the beta-cell insulin receptor and activates IRS-1, thus further increasing cytosolic Ca2+ by reducing Ca2+ uptake. The existence of a novel pathway of autocrine regulation of intracellular Ca2+ homeostasis and insulin secretion in the beta-cell of the endocrine pancreas is suggested (Xu, 1999).

The contribution of the insulin receptor substrate proteins (IRS-1 and IRS-2) to insulin/insulin like growth factor I (IGF-I)-signaling pathways was investigated in fetal rat brown adipocytes, a model that expresses both insulin and IGF-I receptors. Insulin/IGF-I rapidly stimulates IRS-1 and IRS-2 tyrosine phosphorylation, their association with p85alpha, and IRS-1- and IRS-2-associated phosphatidylinositol (PI) 3-kinase activation to the same extent, the effect of insulin being stronger than the effect of IGF-I at the same physiological dose (10 nM). Furthermore, insulin/IGF-I stimulates IRS-1-associated Grb-2 phosphorylation. However, IRS-2-associated Grb-2 phosphorylation is barely detected. Pull-down experiments with glutathione-S-transferase-fusion proteins containing SH2-domains of p85alpha reveal a strong association between IRS-1 and IRS-2 with p85alpha in response to insulin/IGF-I, the insulin effect being stronger than IGF-I. However, the Grb-2-SH2 domain shows functional differences. While a strong association between IRS-1/Grb-2 is found, IRS-2/Grb-2 association is virtually absent in response to insulin/IGF-I, as also demonstrated in competition studies with a phosphopeptide containing the phosphotyrosine 895 residue within the putative Grb-2-binding domain. Finally, insulin/IGF-I stimulates tyrosine phosphorylation of the three SHC proteins (46, 52, and 66 kDa). Moreover, insulin/IGF-I markedly increases the amount of Grb-2-associated SHC proteins by the same extent. These results suggest that both IRS-1 and IRS-2 are required for phosphatidylinositol 3-kinase activation, which leads to adipogenic and thermogenic differentiation of fetal brown adipose tissue; meanwhile, IRS-1 and SHC, but not IRS-2, associate with Grb-2, leading to the ras-mitogen-activated protein kinase-signaling pathway required for fetal brown adipocyte proliferation (Valverde, 1998).

To investigate the role of insulin receptor substrate 1 (IRS-1) and IRS-2, the two ubiquitously expressed IRS proteins, in adipocyte differentiation, embryonic fibroblast cells were established with four different genotypes, i.e., wild-type, IRS-1 deficient (IRS-1^-/-), IRS-2 deficient (IRS-2^-/-), and IRS-1 IRS-2 double deficient (IRS-1^-/- IRS-2^-/-), from mouse embryos of the corresponding genotypes. The abilities of IRS-1^-/- cells and IRS-2^-/- cells to differentiate into adipocytes are approximately 60% and 15%, respectively, lower than that of wild-type cells, at day 8 after induction and, surprisingly, IRS-1^-/- IRS-2^-/- cells have no ability to differentiate into adipocytes. The expression of CCAAT/enhancer binding protein alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma (PPARgamma) is severely decreased in IRS-1^-/- IRS-2^-/- cells at both the mRNA and the protein level, and the mRNAs of lipoprotein lipase and adipocyte fatty acid binding protein are severely decreased in IRS-1^-/- IRS-2^-/- cells. Phosphatidylinositol 3-kinase (PI 3-kinase) activity that increases during adipocyte differentiation is almost completely abolished in IRS-1^-/- IRS-2^-/- cells. Treatment of wild-type cells with a PI 3-kinase inhibitor, LY294002, markedly decreases the expression of C/EBPalpha and PPARgamma, a result that is associated with a complete block of adipocyte differentiation. Moreover, histologic analysis of IRS-1^-/- IRS-2^-/- double-knockout mice 8 h after birth reveals severe reduction in white adipose tissue mass. These results suggest that IRS-1 and IRS-2 play a crucial role in the upregulation of the C/EBPalpha and PPARgamma expression and adipocyte differentiation (Miki, 2001).

To characterize the contribution of glycogen synthase kinase 3beta (GSK3beta) inactivation to insulin-stimulated glucose metabolism, wild-type (WT-GSK), catalytically inactive (KM-GSK), and uninhibitable (S9A-GSK) forms of GSK3beta were expressed in insulin-responsive 3T3-L1 adipocytes using adenovirus technology. WT-GSK, but not KM-GSK, reduces basal and insulin-stimulated glycogen synthase activity without affecting the stimulation of the enzyme by insulin. S9A-GSK similarly decreases cellular glycogen synthase activity, but also partially blocks insulin stimulation of the enzyme. S9A-GSK expression also markedly inhibits insulin stimulation of IRS-1-associated phosphatidylinositol 3-kinase activity, but only weakly inhibits insulin-stimulated Akt/PKB phosphorylation and glucose uptake, with no effect on GLUT4 translocation. To further evaluate the role of GSK3beta in insulin signaling, the GSK3beta inhibitor lithium was used to mimic the consequences of insulin-stimulated GSK3beta inactivation. Although lithium stimulates the incorporation of glucose into glycogen and glycogen synthase enzyme activity, the inhibitor is without effect on GLUT4 translocation and pp70 S6 kinase. Lithium stimulation of glycogen synthesis is insensitive to wortmannin, which is consistent with its acting directly on GSK3beta downstream of phosphatidylinositol 3-kinase. These data support the hypothesis that GSK3beta contributes to insulin regulation of glycogen synthesis, but is not responsible for the increase in glucose transport (Summers, 1999).

Inflammation associates with peripheral insulin resistance, which dysregulates nutrient homeostasis and leads to diabetes. Inflammation induces the expression of suppressors of cytokine signaling (SOCS) proteins. SOCS1 and SOCS3 target IRS1 and IRS2, two critical signaling molecules for insulin action, for ubiquitin-mediated degradation. SOCS1 or SOCS3 bind both recombinant and endogenous IRS1 and IRS2 and promote their ubiquitination and subsequent degradation in multiple cell types. Mutations in the conserved SOCS box of SOCS1 abrogate its interaction with the elongin BC ubiquitin-ligase complex without affecting its binding to IRS1 or IRS2. The SOCS1 mutants also fail to promote the ubiquitination and degradation of either IRS1 or IRS2. Adenoviral-mediated expression of SOCS1 in mouse liver dramatically reduce hepatic IRS1 and IRS2 protein levels and cause glucose intolerance; by contrast, expression of the SOCS1 mutants has no effect. Thus, SOCS-mediated degradation of IRS proteins, presumably via the elongin BC ubiquitin-ligase, might be a general mechanism of inflammation-induced insulin resistance, providing a target for therapy (Rui, 2002).

Activation of the c-Jun N-terminal kinase (JNK) by proinflammatory cytokines inhibits insulin signaling, at least in part, by stimulating phosphorylation of rat/mouse insulin receptor substrate 1 (Irs1) at Ser(307) [Ser(312) in human IRS1]. JNK mediated feedback inhibition of the insulin signal has been demonstrated in mouse embryo fibroblasts, 3T3-L1 adipocytes, and 32D(IR) cells. Insulin stimulation of JNK activity requires phosphatidylinositol 3-kinase and Grb2 signaling. Moreover, activation of JNK by insulin is inhibited by a cell-permeable peptide that disrupts the interaction of JNK with cellular proteins. However, the direct binding of JNK to Irs1 is not required for its activation by insulin, whereas direct binding is required for Ser(307) phosphorylation of Irs1. Insulin-stimulated Ser(307) phosphorylation was reduced 80% in cells lacking JNK1 and JNK2 or in cells expressing a mutant Irs1 protein lacking the JNK binding site. Reduced Ser(307) phosphorylation is directly related to increased insulin-stimulated tyrosine phosphorylation, Akt phosphorylation, and glucose uptake. These results support the hypothesis that JNK is a negative feedback regulator of insulin action by phosphorylating Ser(307) in Irs1 (Lee, 2003).

The principal substrate for the insulin and insulin-like growth factor-1 (IGF-1) receptors is the cytoplasmic protein insulin-receptor substrate-1 (IRS-1/pp185). After tyrosine phosphorylation at several sites, IRS-1 binds to and activates phosphatidylinositol-3'-OH kinase (PI(3)K) and several other proteins containing SH2 (Src-homology 2) domains. To elucidate the role of IRS-1 in insulin/IGF-1 action, IRS-1-deficient mice were created by targeted gene mutation. These mice have no IRS-1 and show no evidence of IRS-1 phosphorylation or IRS-1-associated PI(3)K activity. They also have a 50% reduction in intrauterine growth, impaired glucose tolerance, and a decrease in insulin/IGF-1-stimulated glucose uptake in vivo and in vitro. The residual insulin/IGF-1 action correlates with the appearance of a new tyrosine-phosphorylated protein (IRS-2) which binds to PI(3)K, but is slightly larger than and immunologically distinct from IRS-1. These results provide evidence for IRS-1-dependent and IRS-1-independent pathways of insulin/IGF-1 signaling and for the existence of an alternative substrate of these receptor kinases (Araki, 1994).

Mice with a targeted disruption of the insulin receptor substrate 1 (IRS-1) gene exhibit growth retardation and have resistance to the glucose-lowering effect of insulin. Insulin initiates its biological effects by activating at least two major signaling pathways, one involving phosphatidylinositol 3-kinase (PI3-kinase) and the other involving a ras/mitogen-activated protein kinase (MAP kinase) cascade. In this study the roles of IRS-1 and IRS-2 in the biological action in the physiological target organs of insulin was investigated by comparing the effects of insulin in wild-type and IRS-1-deficient mice. In muscles from IRS-1-deficient mice, the responses to insulin-induced PI3-kinase activation, glucose transport, p70 S6 kinase and MAP kinase activation, mRNA translation, and protein synthesis are significantly impaired compared with those in wild-type mice. Insulin-induced protein synthesis is wortmannin insensitive in IRS-1 deficient mice. However, in another target organ, the liver, the responses to insulin-induced PI3-kinase and MAP kinase activation are not significantly reduced. The amount of tyrosine-phosphorylated IRS-2 (in IRS-1-deficient mice) is roughly equal to that of IRS-1 (in wild-type mice) in the liver, whereas it only 20% to 30% of that of IRS-1 in the muscles. In conclusion, (1) IRS-1 plays central roles in two major biological actions of insulin in muscles, glucose transport and protein synthesis; (2) the insulin resistance of IRS-1-deficient mice is mainly due to resistance in the muscles, and (3) the degree of compensation for IRS-1 deficiency appears to be correlated with the amount of tyrosine-phosphorylated IRS-2 (in IRS-1-deficient mice) relative to that of IRS-1 (in wild-type mice) (Yamauchi, 1996).

Insulin resistance is often associated with atherosclerotic diseases in subjects with obesity and impaired glucose tolerance. This study examined the effects of insulin resistance on coronary risk factors in IRS-1 deficient mice, a nonobese animal model of insulin resistance. Blood pressure and plasma triglyceride levels are significantly higher in IRS-1 deficient mice than in normal mice. Impaired endothelium-dependent vascular relaxation is also observed in IRS-1 deficient mice. Furthermore, lipoprotein lipase activity is lower than in normal mice, suggesting impaired lipolysis to be involved in the increase in plasma triglyceride levels under insulin-resistant conditions. Thus, insulin resistance plays an important role in the clustering of coronary risk factors which may accelerate the progression of atherosclerosis in subjects with insulin resistance (Abe, 1998).

Non-insulin-dependent diabetes mellitus (NIDDM) is considered a polygenic disorder in which insulin resistance and insulin secretory defect are the major etiologic factors. Homozygous mice with insulin receptor substrate-1 (IRS-1) gene knockout show normal glucose tolerance associated with insulin resistance and compensatory hyperinsulinemia. Heterozygous mice with beta cell glucokinase (GK) gene knockout show impaired glucose tolerance due to decreased insulin secretion to glucose. To elucidate the interplay between insulin resistance and insulin secretory defect for the development of NIDDM, double knockout mice were generated with disruption of IRS-1 and beta cell GK genes by crossing the mice with each of the single gene knockout. The double knockout mice develope overt diabetes. Blood glucose levels 120 min after intraperitoneal glucose load (1.5 mg/g body wt) are 108 +/- 24 (wild type), 95 +/- 26 (IRS-1 knockout), 159 +/- 68 (GK knockout), and 210 +/- 38 (double knockout) mg/dl (mean +/- SD) (double versus wild type, IRS-1, or GK; P < 0.01). The double knockout mice show fasting hyperinsulinemia and selective hyperplasia of the beta cells as the IRS-1 knockout mice (fasting insulin levels: 0.38 +/- 0.30 [double knockout], 0.35 +/- 0.27 [IRS-1 knockout] versus 0.25 +/- 0.12 [wild type] ng/ml) (proportion of areas of insulin-positive cells to the pancreas: 1.18 +/- 0.68%; P < 0.01 [double knockout], 1.20 +/- 0.93%; P < 0.05 [IRS-1 knockout] versus 0.54 +/- 0.26% [wild type]), but impaired insulin secretion to glucose (the ratio of increment of insulin to that of glucose during the first 30 min after load: 31 [double knockout] versus 163 [wild type] or 183 [IRS-1 knockout] ng insulin/mg glucose x 10³). In conclusion, the genetic abnormalities, each of which is nondiabetogenic by itself, cause overt diabetes if they coexist. This report provides the first genetic reconstitution of NIDDM as a polygenic disorder in mice (Terauchi, 1997).

Insulin receptor substrate-1 (IRS-1) is rapidly phosphorylated on multiple tyrosine residues in response to insulin and binds several Src homology 2 domain-containing proteins, thereby initiating downstream signaling. To assess the tyrosine phosphorylation sites that mediate relevant downstream signaling and biological effects, site-directed mutants of IRS-1 were created and they were overexpressed in the Xenopus laevis oocyte. In oocytes overexpressing IRS-1 or IRS-1-895F (Tyr-895 replaced with phenylalanine), insulin activatea phosphatidylinositol (PI) 3-kinase, p70 S6 kinase, and mitogen-activated protein kinase and induces oocyte maturation. In contrast, in oocytes overexpressing IRS-1-4F (Tyr-460, Tyr-608, Tyr-939, and Tyr-987 of IRS-1 replaced with phenylalanine), insulin does not activate PI 3-kinase, p70 S6 kinase, and mitogen-activated protein kinase and fails to induce oocyte maturation. These observations indicate that in X. laevis oocytes overexpressing IRS-1, the association of PI 3-kinase rather than Grb2 (growth factor-bound protein 2) with IRS-1 plays a major role in insulin-induced oocyte maturation. Activation of PI 3-kinase may lie upstream of mitogen-activated protein kinase activation and p70 S6 kinase activation in response to insulin (Yamamoto-Honda, 1996).

Based on the phenotypes of knockout mice and cell lines, as well as pathway-specific analysis, the insulin receptor substrates IRS-1, IRS-2, IRS-3, and IRS-4 have been shown to play unique roles in insulin signal transduction. To investigate possible functional complementarity within the IRS family, mice with double knockout of the genes for IRS-1/IRS-3 and IRS-1/IRS-4 were generated. Mice with a combined deficiency of IRS-1 and IRS-4 showed no differences from Irs1^-/- mice with respect to growth and glucose homeostasis. In contrast, mice with a combined deficiency of IRS-1 and IRS-3 developed early-onset severe lipoatrophy associated with marked hyperglycemia, hyperinsulinemia, and insulin resistance. However, in contrast to other models of lipoatrophic diabetes, there was no accumulation of fat in liver or muscle. Furthermore, plasma leptin levels were markedly decreased, and adenovirus-mediated expression of leptin in liver reversed the hyperglycemia and hyperinsulinemia. The results indicate that IRS-1 and IRS-3 play important complementary roles in adipogenesis and establish the Irs1^-/-/Irs3^-/- double knockout mouse as a novel model of lipoatrophic diabetes (Laustsen, 2002).