Insulin Receptor Substrate-1 Function Research Articles

THE ASSOCIATION BETWEEN THE GLY972ARG POLYMORPHISM IN IRS1 GENE AND THE RISK OF PREDIABETES AMONG VIETNAMESE WOMEN

Insulin receptor substrate 1 (IRS1) is a ligand of the insulin receptor tyrosine kinase and participates in the insulin receptor signal transduction pathway. Dysregulations in IRS1 expression and function increase incidence of insulin-resistant states such as prediabetes and type 2 diabetes. The study aimed to investigate the association of the Gly972Arg (rs1801278) polymorphism in IRS1 gene with prediabetes in Northern Vietnamese women. The case-control study consisted of 1617 women (250 prediabetic cases and 1367 normoglycemic controls). The IRS1 Gly972Arg polymorphism was genotyped in these subjects using polymerase chain reaction–restriction fragment length polymorphism. The frequency of the ‘‘A’’ allele of the Gly972Arg (G>A) polymorphism was similar between the normal glucose and prediabetic subjects (2.7% and 2.6%, respectively). There was no significant difference in the genotypic frequency between the control and prediabetic cases (P = 0.673). The IRS1 Gly972Arg polymorphism was not associated with the risk of prediabetes in Vietnamese women before and after adjusted for socio-economic, lifestyles and clinical factors (P > 0.05).

Type 1 Insulin-Like Growth Factor Receptor/Insulin Receptor Substrate 1 Signaling Confers Pathogenic Activity on Breast Tumor Cells Lacking REST.

Loss of repressor element 1 silencing transcription factor (REST) occurs in 20% of breast cancers and correlates with a poor patient prognosis. However, the molecular basis for enhanced malignancy in tumors lacking REST (RESTless) is only partially understood. We used multiplatform array data from the Cancer Genome Atlas to identify consistent changes in key signaling pathways. Of the proteins screened in the reverse-phase protein array, we found that insulin receptor substrate 1 (IRS1) is the most highly upregulated protein in RESTless breast tumors. Analysis of breast tumor cell lines showed that REST directly represses IRS1, and cells lacking REST have increased levels of IRS1 mRNA and protein. We find that the upregulation of IRS1 function is both necessary and sufficient for enhanced signaling and growth in breast cancer cells lacking REST. IRS1 overexpression is sufficient to phenocopy the enhanced activation of the signaling hubs AKT and mitogen-activated protein kinase (MAPK) of MCF7 cells lacking REST. Loss of REST renders MCF7 and MDA-MB-231 breast tumor cells dependent on IRS1 activity for colony formation in soft agar. Inhibition of the type 1 insulin-like growth factor receptor (IGF1R) reduces the enhanced signaling, growth, and migration in breast tumor cells that occur upon REST loss. We show that loss of REST induces a pathogenic program that works through the IGF1R/IRS1 pathway.

4-Hydroxyisoleucine ameliorates fatty acid-induced insulin resistance and inflammatory response in skeletal muscle cells

The 4-hydroxyisoleucine (4-HIL), an unusual amino acid isolated from the seeds of Trigonella foenum-graecum was investigated for its metabolic effects to ameliorate free fatty acid-induced insulin resistance in skeletal muscle cells. An incubation of L6 myotubes with palmitate inhibited insulin stimulated-glucose uptake and -translocation of glucose transporter 4 (GLUT4) to the cell surface. Addition of 4-HIL strongly prevented this inhibition. We then examined the insulin signaling pathway, where 4-HIL effectively inhibited the ability of palmitate to reduce insulin-stimulated phosphorylation of insulin receptor substrate-1 (IRS-1), protein kinase B (PKB/AKT), AKT substrate of 160 kD (AS160) and glycogen synthase kinase 3β (GSK-3β) in L6 myotubes. Moreover, 4-HIL presented strong inhibition on palmitate-induced production of reactive oxygen species (ROS) and associated inflammation, as the activation of NF-κB, JNK1/2, ERK1/2 and p38 MAPK was greatly reduced. 4-HIL also inhibited inflammation-stimulated IRS-1 serine phosphorylation and restored insulin-stimulated IRS-1 tyrosine phosphorylation in the presence of palmitate, leading to enhanced insulin sensitivity. These findings suggested that 4-HIL could inhibit palmitate-induced, ROS-associated inflammation and restored insulin sensitivity through regulating IRS-1 function.

Quercetin and quercetin‐3‐O‐glucuronide are equally effective in ameliorating endothelial insulin resistance through inhibition of reactive oxygen species‐associated inflammation

Quercetin represents antioxidative/antiinflammatory flavonoids widely distributed in the human diet. Quercetin is efficiently metabolized during absorption to quercetin-3-O-glucuronide. This study aims to parallelly investigate whether quercetin and quercetin-3-O-glucuronide exert protection against palmitate (PA)-induced inflammation and insulin resistance in the endothelium. Human umbilical vein endothelial cells were pretreated with quercetin and quercetin-3-O-glucuronide for 30 min, and then incubated with 100 μM PA for 30 min or 12 h with or without insulin. PA stimulation led to reactive oxygen species (ROS) production with collapse of mitochondrial membrane potential (Δψm). Quercetin and quercetin-3-O-glucuronide inhibited ROS overproduction and effectively restored Δψm, demonstrating their chemorpotection of mitochondrial function through antioxidative actions. Also, quercetin and quercetin-3-O-glucuronide inhibited ROS-associated inflammation by inhibition of interleukin-6 and tumor necrosis factor-α production with suppression of IKKβ/NF-κB phosphorylation. Inflammation impaired insulin PI3K signaling and reduced insulin-mediated nitric oxide (NO) production. Quercetin and quercetin-3-O-glucuronide facilitated PI3K signaling by positive regulation of serine/tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and restoration of downstream Akt/eNOS activation, leading to an increased insulin-mediated NO level. The above-mentioned evidence indicates that quercetin and quercetin-3-O-glucuronide are equally effective in inhibiting ROS-associated inflammation and ameliorating insulin resistant endothelial dysfunction by beneficial regulation of IRS-1 function.

Opposite Effects of Quercetin, Luteolin, and Epigallocatechin Gallate on Insulin Sensitivity Under Normal and Inflammatory Conditions in Mice

Flavonoids are polyphenolic compounds ubiquitous in plants. Quercetin, luteolin, and epigallocatechin gallate (EGCG) are flavonoids with a number of biochemical and cellular actions relevant to glucose homeostasis, but their regulation of insulin action is still uncertain. This study aims to evaluate the regulation of insulin action by quercetin, luteolin, and EGCG under normal and inflammatory conditions in mice. Oral administration of quercetin, luteolin, and EGCG impaired glucose tolerance and blunted the effect of insulin to low blood glucose. Luteolin and EGCG, but not quercetin, inhibited glucose load-induced insulin receptor substrate-1(IRS-1) tyrosine and Akt phosphorylation in adipose tissue. Meanwhile, insulin-stimulated glucose uptake was also inhibited by these flavonoids. We induced insulin resistance in mice by treatment with activated macrophages-derived conditioned medium (Mac-CM) and observed that quercetin, luteolin, and EGCG reversed glucose intolerance with improving insulin sensitivity. Quercetin, luteolin, and EGCG inhibited inflammation-evoked IKKβ activation and IRS-1 serine phosphorylation in adipose tissue, and thereby effectively restored glucose load-stimulated IRS-1 tyrosine and Akt phosphorylation, leading to an increase in insulin-mediated glucose uptake in adipocytes. The aforementioned results showed opposite effects of quercetin, luteolin, and EGCG on insulin sensitivity in mice. The different modulation of IRS-1 function by phosphorylating modification under normal and inflammatory conditions should be a key controlling for their action in regulation of insulin sensitivity.

Hepatitis C Virus Activates the mTOR/S6K1 Signaling Pathway in Inhibiting IRS-1 Function for Insulin Resistance

Hepatitis C virus (HCV) infection significantly increases the prevalence of type 2 diabetes mellitus (T2DM). Insulin receptor substrate 1 (IRS-1) plays a key role in insulin signaling, thus enabling metabolic regulation in mammalian cells. We have previously shown that HCV infection modulates phosphorylation of Akt, a downstream target of IRS-1. In this study, we further examined the status of total IRS-1 and the downstream regulation of the Akt pathway in understanding mTOR/S6K1 signaling using HCV genotype 2a (clone JFH1)-infected hepatocytes. Inhibition of IRS-1 expression was observed in HCV-infected hepatocytes compared to that in a mock-infected control. The status of the tuberous sclerosis complex (TSC-1/TSC-2) was significantly decreased after HCV infection of human hepatocytes, showing a modulation of the downstream Akt pathway. Subsequent study indicated an increased level of Rheb and mTOR expression in HCV-infected hepatocytes. Interestingly, the phosphoS6K1 level was higher in HCV-infected hepatocytes, suggesting a novel mechanism for IRS-1 inhibition. Ectopic expression of TSC-1/TSC-2 significantly recovered the IRS-1 protein expression level in HCV-infected hepatocytes. Further analyses indicated that HCV core protein plays a significant role in modulating the mTOR/S6K1 signaling pathway. Proteasome inhibitor MG 132 recovered IRS-1 and TSC1/2 expression, suggesting that degradation occurred via the ubiquitin proteasome pathway. A functional consequence of IRS-1 inhibition was reflected in a decrease in GLUT4 protein expression and upregulation of the gluconeogenic enzyme PCK2 in HCV-infected hepatocytes. Together, these observations suggested that HCV infection activates the mTOR/S6K1 pathway in inhibiting IRS-1 function and perturbs glucose metabolism via downregulation of GLUT4 and upregulation of PCK2 for insulin resistance.

An Early Response Transcription Factor, Egr-1, Enhances Insulin Resistance in Type 2 Diabetes with Chronic Hyperinsulinism

One of the most important characteristics of type 2 diabetes is insulin resistance, during which the patients normally experienced hyperinsulinism stress that would alter insulin signal transduction in insulin target tissues. We have previously found that early growth responsive gene-1 (Egr-1), a zinc finger transcription factor, is highly expressed in db/db mice and in the fat tissue of individuals with type 2 diabetes. In this report, we found that chronic exposure to hyperinsulinism caused persistent Erk/MAPK activity in adipocytes and enhanced insulin resistance in an Egr-1-dependent manner. An elevation in Egr-1 augmented Erk1/2 activation via geranylgeranyl diphosphate synthase (GGPPS). Egr-1-promoted GGPPS transcription increased Ras prenylation and caused Erk1/2 activation. The sustained activation of Erk1/2 resulted in the phosphorylation of insulin receptor substrate-1 at Serine 612. Phosphorylation at this site impaired insulin signaling in adipocytes and reduced glucose uptake. The loss of Egr-1 function, knockdown of GGPPS, or inhibition of Erk1/2 activity in insulin-resistant adipocytes restored insulin receptor substrate-1 tyrosine phosphorylation and increased insulin sensitivity. Our results suggest a new mechanism by which the Egr-1/GGPPS/Erk1/2 pathway is responsible for insulin resistance during hyperinsulinism. This pathway provides a new therapeutic target for increasing insulin sensitivity: inhibiting the function of Egr-1.

Inhibitory effect on protein kinase Cθ by Crocetin attenuates palmitate-induced insulin insensitivity in 3T3-L1 adipocytes

Epidemiologic and experimental studies have pointed to an etiologic role of elevated plasma free fatty acids in insulin resistance, which is frequently associated with a state of low-grade inflammation. In this study, we investigated the effects of Crocetin, a unique carotenoid, on insulin resistance induced by palmitate in 3T3-L1 adipocytes. Exposure of palmitate led to an increase in insulin receptor substrate-1 (IRS-1) serine 307 phosphorylation as well as activation of c-Jun NH 2-terminal kinase (JNK) and inhibitor κB kinase β (IKKβ), concomitantly with reductions of IRS-1 function and glucose metabolism. Interestingly, pretreatment with Crocetin almost reversed all of these abnormalities in a dose-dependent manner. IRS-1 serine 307 phosphorylation was significantly reduced by JNK or IKKβ inhibitor, especially by combination of these two inhibitors. Moreover, palmitate treatment induced activation of protein kinase Cθ (PKCθ) while blocking PKCθ significantly inhibited JNK and IKKβ activation induced by palmitate or phorbol 12-myristate 13-acetate (PKC activator, PMA), and attenuated the palmitate-induced defects in insulin action. Crocetin demonstrated an impressive suppression in the activation of PKCθ induced not only by palmitate but also by PMA in a dose-dependent manner. Taken together, Crocetin inhibited JNK and IKKβ activation via suppression of PKCθ phosphorylation, attenuating insulin insensitivity induced by palmitate in 3T3-L1 adipocytes.

IRS1 Regulation by Wnt/β-Catenin Signaling and Varied Contribution of IRS1 to the Neoplastic Phenotype

Dysregulation of β-catenin levels and localization and constitutive activation of β-catenin/TCF (T cell factor)-regulated gene expression occur in many cancers, including the majority of colorectal carcinomas and a subset of ovarian endometrioid adenocarcinomas. Based on the results of microarray-based gene expression profiling we found the insulin receptor substrate 1 (IRS1) gene as one of the most highly up-regulated genes upon ectopic expression of a mutant, constitutively active form of β-catenin in the rat kidney epithelial cell line RK3E. We demonstrate expression of IRS1 can be directly activated by β-catenin, likely in part via β-catenin/TCF binding to TCF consensus binding elements located in the first intron and downstream of the IRS1 transcriptional start site. Consistent with the proposal that β-catenin is an important regulator of IRS1 expression in vivo, we observed that IRS1 is highly expressed in many cancers with constitutive stabilization of β-catenin, such as colorectal carcinomas and ovarian endometrioid adenocarcinomas. Using a short hairpin RNA approach to abrogate IRS1 expression and function, we found that IRS1 function is required for efficient de novo neoplastic transformation by β-catenin in RK3E cells. Our findings add to the growing body of data implicating IRS1 as a critical signaling component in cancer development and progression.

DOWN-REGULATION OF AMP-ACTIVATED PROTEIN KINASE IN FETAL MUSCLE OF OBESE, OVERNOURISHED PREGNANT SHEEP

Estimates suggest that 18-35% of pregnant women in the USA, are clinically obese. This pregnancy-associated increase in overweight and obese women is accompanied by a 5-fold increase in gestational diabetes mellitus. Babies of obese mothers are macrosomic even in the absence of gestational diabetes. For ethical reasons it is impossible to evaluate the pathophysiological effects on specific signaling pathways in human fetal tissues that result from the adverse fetal metabolic environment due to maternal obesity. The objective of this study was to assess any alteration of AMP-activated protein kinase (AMPK) and insulin signalling in skeletal muscle of fetuses of obese ewes on a high caloric intake. METHODS: Non pregnant ewes were randomly assigned to a control (Con, 100% of NRC recommendations, n=7) or obesogenic (OB, 150% of NRC, n=7) diet from 60 days before to 75 days after conception at which time ewes were euthanized with an overdose of sodium pentobarbital. The fetal longissimus dorsi (Ld) muscle was collected at necropsy. Phosphorylated and unphosphorylated forms of key signaling proteins were measured by western analysis with appropriate antibodies. Insulin was measured by RIA. RESULTS: Body weight and crown rump length of fetuses of OB ewes were ∼30% greater (P<0.05) than fetuses from Con ewes (374 ± 10 g and 24.6 ± 0.2 cm vs. 268 ± 12 g and 22.1 ± 0.2 cm, respectively, P<0.05). While the weights of most fetal tissues from OB ewes were greater (P<0.05) than fetuses of CON ewes, only pancreatic weight was heavier as a percentage of fetal weight in fetuses from ewes on the OB versus the Con diet (P<0.05). At necropsy, fetal plasma glucose was 25.35 ± 2.10 mg/dl and 43.63 ± 5.58 mg/dl and plasma insulin was 1.14 ± 0.35 IU/ml and 7.29 ± 1.31 IU/ml in fetuses of Con and OB mothers respectively (p<0.05). Phosphorylated AMPK at Thr 172 (the active form) was down-regulated by 14.9 ± 4.2 % (p<0.05) in the fetal muscle of OB ewes. Phosphorylation of acetyl-CoA carboxylase (ACC), a down-stream target of AMPK was also reduced by 18.9 ± 7.1% (p<0.05) in OB compared to Con fetal muscle. The expression of PPARgamma, a marker of adipogenesis, was higher by 18.2 ± 3.4% (p<0.05) in the fetal muscle from OB compared to Con pregnancies. No alteration in the content of total insulin receptor in fetal muscle was observed but the phosphorylation of insulin receptor at Tyr 1146 was higher (85.4 ± 17.7%, p<0.01) in OB fetal muscle. Despite increased activation of the insulin receptor, down-stream insulin signaling was down-regulated in fetal muscle of OB ewes. The phosphorylation of Akt at Ser 473, a key mediator of insulin signaling, was 41.2 ± 6.7% lower in OB fetal muscle. mTOR phosphorylation at Ser 2448, a site mediated by Akt, was reduced for 29.1 ± 8.1%, as was 4E-BP1 (28.9 ± 5.9%, p<0.05). Phosphorylation of tuberous sclerosis 2 (TSC2) at Thr 1462, a mediator between Akt and mTOR, was also reduced for 27.5 ± 7.3% (p<0.05). CONCLUSIONS: These data clearly show that OB fetal muscle developed insulin resistance. Because AMPK phosphorylates insulin receptor substrate-1 (IRS-1) at Ser 789 which enhances the function of IRS-1, we propose that the low AMPK activity in OB fetal muscle reduced the insulin sensitivity by down-regulation of IRS-1 function, which could be at least partially responsible for the observed insulin resistance in OB fetal muscle. USDA 2006-55618-16914 and NIH INBRE 1P20RR16474 (platform)

Antidepressants induce cellular insulin resistance by activation of IRS-1 kinases

Certain selective serotonin reuptake inhibitors (SSRIs) induce the clinical and biochemical manifestations of a metabolic syndrome by as yet unknown mechanism. Here we demonstrate that incubation (1 h) of rat hepatoma Fao cells with the SSRIs paroxetine and sertraline, but not with the atypical antipsychotic drug olanzapine, inhibited the insulin-stimulated Tyr phosphorylation of the insulin receptor substrate-1 (IRS-1) with half-maximal effects at ∼ 10 μM. This inhibition correlated with a rapid phosphorylation and activation of a number of Ser/Thr IRS-1 kinases including JNK, S6K1, ERK and p38 MAPK, but not PKB (Akt). JNK appears as a key player activated by SSRIs because specific JNK inhibitors partially eliminated the effects of these drugs. The SSRIs induced the phosphorylation of IRS-1 on S307 and S408, which inhibits IRS-1 function and insulin signaling. These results implicate selected SSRIs as inhibitors of insulin signaling and as potential inducers of cellular insulin resistance.

IRS Cancer Connection

Two groups (Dearth et al . and Ma et al .) examined the role of insulin receptor substrate (IRS) in mouse mammary gland tumorigenesis and metastasis. Dearth et al . used transgenic mice forced to overexpress IRS-1 or IRS-2, or an immortalized but nontumorigenic cell line (MCF-10A) also forced to overexpress one of these two proteins. In the MCF-10A cells, overexpression of either protein caused an increase in phosphorylation (and activation) of Akt and an increase in proliferation in response to insulin growth factor-1 (IGF-1). Only the IRS-1-overexpressing cells showed enhanced phosphorylation of extracellular signal-related kinases 1 and 2. Both IRS proteins disrupted acinar formation in three-dimensional Matrigel cultures, and both IRS proteins coimmunoprecipitated with β-catenin. In the transgenic mice, there was no effect on lactational capacity, but both groups of mice formed tumors in the mammary glands. The IRS-2-overexpressing mice had more rapid tumor formation than did the IRS-1-overexpressing mice; however, IRS-2 was overexpressed to a greater extent than was IRS-1. Both groups exhibited lung metastasis. The histology of the tumors was quite diverse, and many contained cells that were highly differentiated, which is similar to phenotypes associated with activation of β-catenin. In the tumors from the IRS-1- or IRS-2-overexpressing mice, β-catenin coimmunoprecipitated with the IRS proteins, and there was increased abundance of cyclin D, which is encoded by a β-catenin target gene. In the overexpressing MCF-10A cells, IGF-1 stimulated the association between IRS-1 or IRS-2 and β-catenin. Ma et al . examined the different functions of IRS-1 and IRS-2 in tumorigenesis and metastasis by generating tumors in IRS-1-deficient mice using polyoma virus middle-T (PyV-MT:: Irs1 −/− ) and comparing these tumors to those formed in wild-type mice (PyV-MT) or in IRS-2-deficient mice (PyV-MT:: Irs2 −/− ). It was previously known that mammary tumors formed in PyV-MT:: Irs2 −/− mice exhibited decreased metastasis. The tumors from the PyV-MT:: Irs1 −/− mice showed increased lung metastasis compared with PyV-MT mice. This effect was intrinsic to the PyV-MT:: Irs1 −/− tumors, because the tumors grew faster and showed increased lung metastasis even when injected into wild-type mice. The PyV-MT:: Irs1 −/− tumors showed elevated microvascular density compared with the PyV-MT tumors and the PyV-MT:: Irs1 −/− tumors, and serum from these mice had increased concentrations of vascular endothelial growth factor A (VEGF-A). The tumors from the PyV-MT:: Irs1 −/− mice also showed increased signaling through the mTOR pathway, which regulates protein production and may explain the increase in VEGF-A production. The tumors from the PyV-MT:: Irs1 −/− mice also showed decreased abundance of apoptotic cells compared with tumors from the PyV-MT mice. IRS-2 abundance and tyrosine phosphorylation were increased in the tumors from the PyV-MT:: Irs1 −/− mice, and there was increased interaction between IRS-2 and the p85 regulatory subunit of phosphinositide 3-kinase, suggesting that in the absence of IRS-1, signaling through IRS-2 is enhanced. Further, these results suggest that IRS-1 may have an inhibitory effect of tumor metastasis, yet promote tumor formation and growth. Metastatic tumors from PyV-MT:: Irs2 −/− or PyV-MT mice had increased serine phosphorylation of IRS-1, which prevents tyrosine phosphorylation and thereby inhibits IRS-1 function. Small interfering RNA (SiRNA) used to decrease IRS-1 in cells from PyV-MT tumors increased IRS-2 abundance in those cells, suggesting that IRS-1 may inhibit IRS-2 signaling, and siRNA of IRS-2 in cells from PyV-MT:: Irs1 −/− decreased the phosphorylation of S6 kinase, a marker for activation of the mTOR pathway. Thus, it appears that IRS-1 and IRS-2 antagonize each other's activity and that inactivation of IRS-1 may contribute to the increased metastatic potential of mammary tumors. R. K. Dearth, X. Cui, H.-J. Kim, I. Kuiatse, N. A. Lawrence, X. Zhang, J. Divisova, O. L. Britton, S. Mohsin, D. C. Allred, D. L. Hadsell, A. V. Lee, Mammary tumorigenesis and metastasis caused by overexpression of insulin receptor substrate 1 (IRS-1) or IRS-2. Mol. Cell. Biol. 26 , 9302-9314 (2006). [Abstract] [Full Text] Z. Ma, S. L. Gibson, M. A. Byrne, J. Zhang, M. F. White, L. M. Shaw, Suppression of insulin receptor substrate 1 (IRS-1) promotes mammary tumor metastasis. Mol. Cell. Biol. 26 , 9338-9351 (2006). [Abstract] [Full Text]

Myosin motor Myo1c and its receptor NEMO/IKK-γ promote TNF-α–induced serine307 phosphorylation of IRS-1

Tumor necrosis factor-α (TNF-α) signaling through the IκB kinase (IKK) complex attenuates insulin action via the phosphorylation of insulin receptor substrate 1 (IRS-1) at Ser307. However, the precise molecular mechanism by which the IKK complex phosphorylates IRS-1 is unknown. In this study, we report nuclear factor κB essential modulator (NEMO)/IKK-γ subunit accumulation in membrane ruffles followed by an interaction with IRS-1. This intracellular trafficking of NEMO requires insulin, an intact actin cytoskeletal network, and the motor protein Myo1c. Increased Myo1c expression enhanced the NEMO–IRS-1 interaction, which is essential for TNF-α– induced phosphorylation of Ser307–IRS-1. In contrast, dominant inhibitory Myo1c cargo domain expression diminished this interaction and inhibited IRS-1 phosphorylation. NEMO expression also enhanced TNF-α–induced Ser307–IRS-1 phosphorylation and inhibited glucose uptake. In contrast, a deletion mutant of NEMO lacking the IKK-β–binding domain or silencing NEMO blocked the TNF-α signal. Thus, motor protein Myo1c and its receptor protein NEMO act cooperatively to form the IKK–IRS-1 complex and function in TNF-α–induced insulin resistance.

RNAi‐mediated silencing of insulin receptor substrate 1 (IRS‐1) enhances tamoxifen‐induced cell death in MCF‐7 breast cancer cells

Insulin receptor substrate 1 (IRS-1) is a major downstream signaling protein for insulin and insulin-like growth factor I (IGF-I) receptors, conveying signals to PI-3K/Akt and ERK1/2 pathways. In breast cancer, IRS-1 overexpression has been associated with tumor development, hormone-independence and antiestrogen-resistance. In part, these effects are related to potentiation of IRS-1/PI-3K/Akt signaling. In estrogen sensitive breast cancer cell lines, tamoxifen treatment reduces IRS-1 expression and function; consequently, inhibiting IRS-1/PI-3K signaling. We tested whether anti-IRS1 siRNA could inhibit growth and survival of estrogen-sensitive MCF-7 breast cancer cells, when used alone or in combination with TAM. Our results indicated: (a) out of four tested anti-IRS1 siRNAs, two siRNAs reduced IRS-1 protein by approximately three-fold in both growing and IGF-I-stimulated cells without affecting a closely related protein, IRS-2; (b) these effects paralleled IRS1 mRNA downregulation by approximately three-fold, measured by quantitative real time-polymerase chain reaction; (c) action of anti-IRS1 siRNAs induced the apoptotic response, observed by altered mitochondrial membrane potential coupled with downregulation of NF-kappaB target Bcl-xL and reduced cell viability; (d) anti-IRS1 siRNA treatment enhanced the cytotoxic effects of TAM by approximately 20%. In summary, anti-IRS1 RNAi strategy could become a potent tool to induce breast cancer cell death, especially if combined with standard TAM therapy.

PKCδ and mTOR interact to regulate stress and IGF-I induced IRS-1 Ser312 phosphorylation in breast cancer cells

IRS-1 (Insulin Receptor Substrate-1) is an adaptor protein important for insulin and IGF-I receptor (Insulin-like Growth Factor-IR) transduction to downstream targets. One mechanism recently identified to downregulate IGF-I or insulin receptor signaling in diabetic models is IRS-1 Ser(312) phosphorylation. To date, the importance of this residue in cancer is unknown. This paper identifies mechanisms leading to Ser(312) regulation in MCF-7 breast cancer cells. Whereas IGF-I phosphorylation of IRS(312) is PI (phosphatidylinositol) 3-kinase dependent, anisomycin stress treatment requires JNK activation to induce phosphorylation of IRS(312). We show that both IGF-I and anisomycin stress treatment converge downstream onto mTOR (Mammalian Target of Rapamycin) and PKCdelta (Protein Kinase C-delta) to induce IRS-1 Ser(312) phosphorylation. mTOR associates with IRS-1 and is primarily required for Ser(312) phosphorylation in response to stress or IGF-I treatment. PKCdelta binds to mTOR and its activity is also important for stress or IGF-I mediated Ser(312) phosphorylation. Thus, mTOR and PKCdelta convey diverse signals to regulate IRS-1 function.

Serine phosphorylation proximal to its phosphotyrosine binding domain inhibits insulin receptor substrate 1 function and promotes insulin resistance.

Ser/Thr phosphorylation of insulin receptor substrate (IRS) proteins negatively modulates insulin signaling. Therefore, the identification of serine sites whose phosphorylation inhibit IRS protein functions is of physiological importance. Here we mutated seven Ser sites located proximal to the phosphotyrosine binding domain of insulin receptor substrate 1 (IRS-1) (S265, S302, S325, S336, S358, S407, and S408) into Ala. When overexpressed in rat hepatoma Fao or CHO cells, the mutated IRS-1 protein in which the seven Ser sites were mutated to Ala (IRS-1(7A)), unlike wild-type IRS-1 (IRS-1(WT)), maintained its Tyr-phosphorylated active conformation after prolonged insulin treatment or when the cells were challenged with inducers of insulin resistance prior to acute insulin treatment. This was due to the ability of IRS-1(7A) to remain complexed with the insulin receptor (IR), unlike IRS-1(WT), which underwent Ser phosphorylation, resulting in its dissociation from IR. Studies of truncated forms of IRS-1 revealed that the region between amino acids 365 to 430 is a main insulin-stimulated Ser phosphorylation domain. Indeed, IRS-1 mutated only at S408, which undergoes phosphorylation in vivo, partially maintained the properties of IRS-1(7A) and conferred protection against selected inducers of insulin resistance. These findings suggest that S408 and additional Ser sites among the seven mutated Ser sites are targets for IRS-1 kinases that play a key negative regulatory role in IRS-1 function and insulin action. These sites presumably serve as points of convergence, where physiological feedback control mechanisms, which are triggered by insulin-stimulated IRS kinases, overlap with IRS kinases triggered by inducers of insulin resistance to terminate insulin signaling.

Protein Kinase C-ζ-induced Phosphorylation of Ser318 in Insulin Receptor Substrate-1 (IRS-1) Attenuates the Interaction with the Insulin Receptor and the Tyrosine Phosphorylation of IRS-1

Insulin receptor substrate-1 (IRS-1) was recently identified as a novel upstream substrate for the insulin-activated protein kinase C (PKC)-zeta. This interaction down-regulates insulin signal transduction under hyper-insulinemic conditions. To clarify the molecular mechanism of this feedback loop, we sought to identify the PKC-zeta phosphorylation sites of IRS-1 and to investigate their biological significance. Upon incubation of recombinant IRS-1 fragments with PKC-zeta, we identified Ser(318) of rat IRS-1 (Ser(323) in human IRS-1) as the major in vitro phosphorylation site (confirmed by mutation of Ser(318) to alanine). To monitor phosphorylation of Ser(318) in cellular extracts, we prepared a polyclonal phosphosite-specific antibody. The biological significance was studied in baby hamster kidney cells stably expressing the insulin receptor (BHK(IR)). Using the phospho-Ser(318)-specific antibody we observed that insulin stimulates phosphorylation of Ser(318) in IRS-1, which is mediated, at least partially, by PKC-zeta. Moreover, we found that the previously described insulin-stimulated, PKC-zeta-mediated inhibition of the interaction of IRS-1 with the insulin receptor and the reduced tyrosine phosphorylation of IRS-1 was abrogated by mutation of IRS-1 Ser(318) to alanine. These results, generated in BHK(IR) cells, suggest that phosphorylation of Ser(318) by PKC-zeta might contribute to the inhibitory effect of prolonged hyperinsulinemia on IRS-1 function.

Hyperosmotic Stress Inhibits Insulin Receptor Substrate-1 Function by Distinct Mechanisms in 3T3-L1 Adipocytes

In 3T3-L1 adipocytes, hyperosmotic stress was found to inhibit insulin signaling, leading to an insulin-resistant state. We show here that, despite normal activation of insulin receptor, hyperosmotic stress inhibits both tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and IRS-1-associated phosphoinositide 3 (PI 3)-kinase activity in response to physiological insulin concentrations. Insulin-induced membrane ruffling, which is dependent on PI 3-kinase activation, was also markedly reduced. These inhibitory effects were associated with an increase in IRS-1 Ser307 phosphorylation. Furthermore, the mammalian target of rapamycin (mTOR) inhibitor rapamycin prevented the osmotic shock-induced phosphorylation of IRS-1 on Ser307. The inhibition of mTOR completely reversed the inhibitory effect of hyperosmotic stress on insulin-induced IRS-1 tyrosine phosphorylation and PI 3-kinase activation. In addition, prolonged osmotic stress enhanced the degradation of IRS proteins through a rapamycin-insensitive pathway and a proteasome-independent process. These data support evidence of new mechanisms involved in osmotic stress-induced cellular insulin resistance. Short-term osmotic stress induces the phosphorylation of IRS-1 on Ser307 by an mTOR-dependent pathway. This, in turn, leads to a decrease in early proximal signaling events induced by physiological insulin concentrations. On the other hand, prolonged osmotic stress alters IRS-1 function by inducing its degradation, which could contribute to the down-regulation of insulin action.

IRS-1 regulation in health and disease.

The global incidence of diabetes is increasing at epidemic rates. Estimates suggest there are currently 150 million people with diabetes and this number is expected to double in the next 20 years. Type 2 diabetes accounts for 95% of all cases and is characterized in part by impaired sensitivity to insulin or 'insulin resistance'. Defects in the insulin signalling pathways underpin this resistance. In the current article we discuss the regulation of Insulin Receptor Substrate-1 (IRS-1), a protein that plays a pivotal role in insulin signalling and whose function is impaired in subjects with insulin resistance. Coordination of IRS-1 function is multi-faceted, involving phosphorylation of IRS-1 at multiple serine/threonine residues. This controls many aspects of IRS-1, including its interaction with the insulin receptor and subsequent tyrosine phosphorylation, as well as its subcellular distribution and targeting for degradation by the proteasome. Such tight control ensures appropriate transduction and attenuation of the insulin signal, thereby regulating insulin action in healthy individuals. Emerging evidence indicates that 'diabetogenic factors' associated with insulin resistance, such as TNFalpha and elevated circulating fatty acids, impact on insulin signalling at the level of IRS-1 serine/threonine phosphorylation. The expression and/or activity of several kinases, such as IkappaB kinase beta (IKKbeta) and salt-induced kinase 2 (SIK2), and the phosphorylation of IRS-1 at key sites, such as Ser307 and Ser789, are increased in states of insulin resistance. Identifying the pathways by which such factors activate these and other kinases, and defining the precise roles of specific serine/ threonine phosphorylation events in IRS-1 regulation, represent important goals which may eventually provide a rationale for therapeutic intervention.

Deficient expression of insulin receptor substrate-1 (IRS-1) fails to block insulin-like growth factor-I (IGF-I) stimulation of brain growth and myelination

To determine whether insulin receptor substrate-1 (IRS-1) is essential in mediating insulin-like growth factor-I (IGF-I) stimulation of brain growth and myelination in vivo, we cross-bred IGF-I transgenic (Tg) mice with IRS-1 null mutant (IRS-1 −/−) mice and examined brain growth and expression of myelin-specific proteins in mice that overexpress IGF-I with or without IRS-1 expression. We found that while IGF-I overexpression stimulates a dramatic increase in brain weight (43%) by 7–8 weeks of age in the absence of IRS-1, it stimulates a greater increase (50%) with intact IRS-1 expression. To evaluate myelination we investigated IGF-I-stimulated expression of myelin basic protein (MBP) and proteolipid protein (PLP) in the cerebral cortex CTX and brainstem, and found similar increases in each region in IRS-1 −/− and wild type mice. In studies using mixed glial cultures derived from IRS-1 −/− mice, IGF-I also increased the abundance of MBP and PLP mRNA. To assess possible alternate mediators of IGF-I actions, we examined IRS-2 and IRS-4 and found that the abundance of each is increased in the CTX of IRS-1 −/− mice and IGF-I Tg mice. Our results suggest that IRS-1 is not essential in IGF-I promotion of oligodendrocyte development and myelination, and that IRS-2 and IRS-4 may compensate for the loss of IRS-1 expression and function in the cells of oligodendrocyte lineage. Nonetheless, the finding that IGF-I stimulates brain growth less well in the absence of IRS-1 suggests that IRS-1-mediated signaling may be more central to IGF-I action in cells other than glia and oligodendrocytes.