Tyrosine Phosphorylation Of Insulin Receptor Substrate-1 Research Articles

Arsenic-induced IGF-1 signaling impairment and neurite shortening: The protective roles of IGF-1 through the PI3K/Akt axis.

We recently reported that arsenic caused insulin resistance in differentiated human neuroblastoma SH-SY5Y cells. Herein, we further investigated the effects of sodium arsenite on IGF-1 signaling, which shares downstream signaling with insulin. A time-course experiment revealed that sodium arsenite began to decrease IGF-1-stimulated Akt phosphorylation on Day 3 after treatment, indicating that prolonged sodium arsenite treatment disrupted the neuronal IGF-1 response. Additionally, sodium arsenite decreased IGF-1-stimulated tyrosine phosphorylation of the IGF-1 receptor β (IGF-1Rβ) and its downstream target, insulin receptor substrate 1 (IRS1). These results suggested that sodium arsenite impaired the intrinsic tyrosine kinase activity of IGF-1Rβ, ultimately resulting in a reduction in tyrosine-phosphorylated IRS1. Sodium arsenite also reduced IGF-1 stimulated tyrosine phosphorylation of insulin receptor β (IRβ), indicating the potential inhibition of IGF-1R/IR crosstalk by sodium arsenite. Interestingly, sodium arsenite also induced neurite shortening at the same concentrations that caused IGF-1 signaling impairment. A 24-h IGF-1 treatment partially rescued neurite shortening caused by sodium arsenite. Moreover, the reduction in Akt phosphorylation by sodium arsenite was attenuated by IGF-1. Inhibition of PI3K/Akt by LY294002 diminished the protective effects of IGF-1 against sodium arsenite-induced neurite retraction. Together, our findings suggested that sodium arsenite-impaired IGF-1 signaling, leading to neurite shortening through IGF-1/PI3K/Akt.

Reduced Tyrosine and Serine-632 Phosphorylation of Insulin Receptor Substrate-1 in the Gastrocnemius Muscle of Obese Zucker Rat.

Obesity has become a serious health problem in the world, with increased morbidity, mortality, and financial burden on patients and health-care providers. The skeletal muscle is the most extensive tissue, severely affected by a sedentary lifestyle, which leads to obesity and type 2 diabetes. Obesity disrupts insulin signaling in the skeletal muscle, resulting in decreased glucose disposal, a condition known as insulin resistance. Although there is a large body of evidence on obesity-induced insulin resistance in various skeletal muscles, the molecular mechanism of insulin resistance due to a disruption in insulin receptor signaling, specifically in the gastrocnemius skeletal muscle of obese Zucker rats (OZRs), is not fully understood. This study subjected OZRs to a glucose tolerance test (GTT) to analyze insulin sensitivity. In addition, immunoprecipitation and immunoblotting techniques were used to determine the expression and tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and insulin receptor-β (IRβ), and the activation of serine-632-IRS-1 phosphorylation in the gastrocnemius muscle of Zucker rats. The results show that the GTT in the OZRs was impaired. There was a significant decrease in IRS-1 levels, but no change was observed in IRβ in the gastrocnemius muscle of OZRs, compared to Zucker leans. Obese rats had a higher ratio of tyrosine phosphorylation of IRS-1 and IRβ than lean rats. In obese rats, however, insulin was unable to induce tyrosine phosphorylation. Moreover, insulin increased the phosphorylation of serine 632-IRS-1 in the gastrocnemius muscle of lean rats. However, obese rats had a low basal level of serine-632-IRS-1 and insulin only mildly increased serine phosphorylation in obese rats, compared to those without insulin. Thus, we addressed the altered steps of the insulin receptor signal transduction in the gastrocnemius muscle of OZRs. These findings may contribute to a better understanding of human obesity and type 2 diabetes.

Extracellular vesicle biomarkers for cognitive impairment in Parkinson's disease.

Besides motor symptoms, many individuals with Parkinson's disease develop cognitive impairment perhaps due to coexisting α-synuclein and Alzheimer's disease pathologies and impaired brain insulin signalling. Discovering biomarkers for cognitive impairment in Parkinson's disease could help clarify the underlying pathogenic processes and improve Parkinson's disease diagnosis and prognosis. This study used plasma samples from 273 participants: 103 Parkinson's disease individuals with normal cognition, 121 Parkinson's disease individuals with cognitive impairment (81 with mild cognitive impairment, 40 with dementia) and 49 age- and sex-matched controls. Plasma extracellular vesicles enriched for neuronal origin were immunocaptured by targeting the L1 cell adhesion molecule, then biomarkers were quantified using immunoassays. α-Synuclein was lower in Parkinson's disease compared to control individuals (P = 0.004) and in cognitively impaired Parkinson's disease individuals compared to Parkinson's disease with normal cognition (P < 0.001) and control (P < 0.001) individuals. Amyloid-β42 did not differ between groups. Phosphorylated tau (T181) was higher in Parkinson's disease than control individuals (P = 0.003) and in cognitively impaired compared to cognitively normal Parkinson's disease individuals (P < 0.001) and controls (P < 0.001). Total tau was not different between groups. Tyrosine-phosphorylated insulin receptor substrate-1 was lower in Parkinson's disease compared to control individuals (P = 0.03) and in cognitively impaired compared to cognitively normal Parkinson's disease individuals (P = 0.02) and controls (P = 0.01), and also decreased with increasing motor symptom severity (P = 0.005); serine312-phosphorylated insulin receptor substrate-1 was not different between groups. Mechanistic target of rapamycin was not different between groups, whereas phosphorylated mechanistic target of rapamycin trended lower in cognitively impaired compared to cognitively normal Parkinson's disease individuals (P = 0.05). The ratio of α-synuclein to phosphorylated tau181 was lower in Parkinson's disease compared to controls (P = 0.001), in cognitively impaired compared to cognitively normal Parkinson's disease individuals (P < 0.001) and decreased with increasing motor symptom severity (P < 0.001). The ratio of insulin receptor substrate-1 phosphorylated serine312 to insulin receptor substrate-1 phosphorylated tyrosine was higher in Parkinson's disease compared to control individuals (P = 0.01), in cognitively impaired compared to cognitively normal Parkinson's disease individuals (P = 0.02) and increased with increasing motor symptom severity (P = 0.003). α-Synuclein, phosphorylated tau181 and insulin receptor substrate-1 phosphorylated tyrosine contributed in diagnostic classification between groups. These findings suggest that both α-synuclein and tau pathologies and impaired insulin signalling underlie Parkinson's disease with cognitive impairment. Plasma neuronal extracellular vesicles biomarkers may inform cognitive prognosis in Parkinson's disease.

IL-4 and IL-13 Promote Proliferation of Mammary Epithelial Cells through STAT6 and IRS-1.

T helper (Th)2 cytokines such as interleukin (IL)-4 and IL-13 control immune function by acting on leukocytes. They also regulate multiple responses in non-hematopoietic cells. During pregnancy, IL-4 and IL-13 facilitate alveologenesis of mammary glands. This particular morphogenesis generates alveoli from existing ducts and requires substantial cell proliferation. Using 3D cultures of primary mouse mammary epithelial cells, we demonstrate that IL-4 and IL-13 promote cell proliferation, leading to enlargement of mammary acini with partially filled lumens. The mitogenic effects of IL-4 and IL-13 are mediated by STAT6 as inhibition of STAT6 suppresses cell proliferation and improves lumen formation. In addition, IL-4 and IL-13 stimulate tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). Prolonged treatment with these cytokines leads to increased IRS-1 abundance, which, in turn, amplifies IL-4- and IL-13-stimulated IRS-1 tyrosine phosphorylation. Through signaling crosstalk between IL-4/IL-13 and insulin, a hormone routinely included in mammary cultures, IRS-1 tyrosine phosphorylation is further enhanced. Lowering IRS-1 expression reduces cell proliferation, suggesting that IRS-1 is involved in IL-4- and IL-13-stimulated cell proliferation. Thus, a Th2-dominant cytokine milieu during pregnancy confers mammary gland development by promoting cell proliferation.

Mechanism of Electroacupuncture Regulating IRS-1 Phosphorylation in Skeletal Muscle to Improve Insulin Sensitivity.

Objective To explore the possible mechanism of electroacupuncture to improve insulin sensitivity in type 2 diabetes rats. Methods Fourteen Zucker Diabetic Fatty (ZDF) rats were randomly divided into two groups: a model group and an electroacupuncture group, with 7 rats in each group. Seven Zucker Lean (ZL) rats served as a control group. All rats were fed with Purina #5008 for 4 weeks, and the electroacupuncture group received 4-week electroacupuncture intervention, while the control group and model group received no intervention. We measured fasting blood glucose (FBG) on the fourth weekend. After 4 weeks of intervention, the expression levels of insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, IRS-1 serine/threonine phosphorylation, and GLUT4 in quadriceps femoris muscles were detected by western Blot. Results Compared with the model group, the electroacupuncture group had a lower level of fasting blood glucose, serum insulin level, and insulin resistance index (P < 0.05). The electroacupuncture group had lower IRS-1 serine/threonine phosphorylation than the model group, with the difference showing statistical significance (P < 0.05). Furthermore, the mean score (MS) of the control group showed the lowest phosphorylation expression, followed by the electroacupuncture group, while the model group had the highest level of phosphorylated protein expression. The level of IRS-1 tyrosine phosphorylation at Tyr895 sites was compared, and the result showed that there was no significant difference between the electroacupuncture group and the control group (P > 0.05), and the electroacupuncture group had higher phosphorylation expression than the model group (P < 0.05). Compared with the control group and the model group, the expression level of GLUT4 protein in the electroacupuncture group was significantly increased (P < 0.05). Conclusion Electroacupuncture has the effect to improve the insulin sensitivity of type 2 diabetic ZDF rats by reducing fasting blood glucose, insulin level, and insulin resistance index, effectively up regulating the expression of GLUT4 protein in quadriceps femoris muscle. The mechanism is related to the regulation of skeletal muscle IRS-1 serine/threonine and tyrosine phosphorylation levels.

DPP-4 inhibition resembles exercise in preventing type 2 diabetes development by inhibiting hepatic protein kinase Cε expression in a mouse model of hyperinsulinemia.

ObjectiveInterventions for hyperinsulinemia (HINS), an early indicator of type 2 diabetes mellitus (T2DM), can significantly reduce the T2DM risk. This study aims to determine how dipeptidyl peptidase-4 (DPP-4) inhibition prevents HINS progression to T2DM through ameliorating hepatic steatosis.MethodsKKay mice were used as a HINS model and they underwent exercise or received a DPP-4 inhibitor, MK0626. Hepatic steatosis was examined and liver diacylglycerol levels were determined. Human hepatic cells (LO2) were treated with MK0626 or transfected with DPP-4 siRNA. Protein kinase C ε isoform (PKCε) and DPP-4 expression and insulin receptor substrate 1 (IRS-1) phosphorylation were assessed using immunohistochemistry and western blot.ResultsKKay mice developed HINS spontaneously at 7 weeks of age. Similar to exercise, MK0626 ameliorated hepatic steatosis and reduced the liver triglyceride and diacylglycerol content. Both exercise and MK0626 suppressed diacylglycerol-induced PKCε expression and restored insulin signaling, which was shown by tyrosine phosphorylation of IRS-1, in the livers of KKay mice. Additionally, silencing DPP-4 or MK0626 treatment decreased PKCε expression in LO2 cells.ConclusionsOur data demonstrate that DPP-4 inhibition resembles exercise and effectively delays T2DM onset by suppressing hepatic PKCε expression in the HINS mouse model.

SUN-651 Murine Cecal Ligation and Puncture (CLP) Perturbs Phosphorylation of Insulin Receptor Substrate 2 (IRS-2)

Hyperglycemia is a characteristic finding in sepsis, and its presence worsens outcome (1). Patients with sepsis need larger doses of insulin to reduce glucose levels. This abnormality has been termed “insulin resistance” but the molecular mechanism by which sepsis attenuates the insulin signaling pathway is unknown. Previous work has shown impairment of phosphorylation in several intracellular signaling pathways following CLP, a well-validated murine model of sepsis (2). Phosphorylation of tyrosine in IRS-2 is essential for functional insulin signaling in hepatocytes (3). Therefore the aim of this study was to investigate the effects of CLP on IRS-2 phosphorylation. Hypothesis: CLP attenuates phosphorylation of IRS-2. Methods: All studies were approved by the Feinstein IACUC and conformed to ARRIVE guidelines. CLP was performed on C57Bl6 mice. Before CLP, animals were identified for sacrifice at specific post-procedure time points. To stimulate phosphorylation of IRS-2, insulin was injected in control and CLP mice at 24 and 48 hours post CLP. Following sacrifice, protein was isolated from liver tissue. Protein abundance was determined using immunoblotting. The detection of the phosphorylated form of these proteins was determined by enzyme-linked immunosorbent assay (ELISA) with a phospho-insulin receptor antibody. Statistical significance was determined using ANOVA for repeated measures with a Sidak post-hoc correction. Results: Relative to the control, tyrosine phosphorylation of IRS-2 was significantly (p<0.05) reduced at 24 and 48 hours following CLP. Conclusions: Tyrosine phosphorylation of hepatic IRS2 is attenuated at early time points following CLP. These results are consistent with other studies examining the effects of CLP on intra-cellular signal transduction pathways (1). Further, these results provide evidence that changes in the insulin signaling transduction underlie CLP-induced “insulin resistance”.

Insulin receptor substrate 1 overexpression promotes survival of glioblastoma cells through AKT1 activation.

Glioblastoma multiforme (GBM) is the most common and malignant type of central nervous system tumours in adults. Strict regulation of glucose homeostasis has a significant role in GBM pathogenesis. Insulin receptor substrate 1 (IRS1) protein is the most important adaptor molecule involved in the regulation of glucose metabolism. It interacts with many cancer-related receptors and its overexpression is strongly associated with cell proliferation and survival. Our study was aimed to understand the role of IRS1 proteins in GBM cell viability. U-87 MG cells were transfected with pcDNA3.1-flagtagged-human IRS1 expression vector. Insulin induced phosphorylation levels of IRS1, AKT1 and ERK1/2 and Grb2 expression were examined to determine the effects of ectopic IRS1 overexpression on insulin signalling and the viability levels of U-87 MG cells were determined by MTT analysis. Overexpression of IRS1 in U-87 MG cells led to an increase in cell viability. Its overexpression also increased Grb2 expression and phosphorylation of AKT1 through elevation of IRS1 tyrosine phosphorylation in IRS1-transfected U-87 MG cells compared to control and mock transfected groups. Our study showed that increased IRS1 expression and activation may promote the cell viability via AKT1 activation. IRS1 signalling may be considered as a therapeutic target for further studies.

Utility of Neuronal-Derived Exosomes to Examine Molecular Mechanisms That Affect Motor Function in Patients With Parkinson Disease

Exenatide, a glucagon-like peptide 1 agonist used in type 2 diabetes, was recently found to have beneficial effects on motor function in a randomized, placebo-controlled trial in Parkinson disease (PD). Accumulating evidence suggests that impaired brain insulin and protein kinase B (Akt) signaling play a role in PD pathogenesis; however, exploring the extent to which drugs engage with putative mechnisms in vivo remains a challenge. To assess whether participants in the Exenatide-PD trial have augmented activity in brain insulin and Akt signaling pathways. Serum samples were collected from 60 participants in the single-center Exenatide-PD trial (June 18, 2014, to June 16, 2016), which compared patients with moderate PD randomized to 2 mg of exenatide once weekly or placebo for 48 weeks followed by a 12-week washout period. Serum extracellular vesicles, including exosomes, were extracted, precipitated, and enriched for neuronal source by anti-L1 cell adhesion molecule antibody absorption, and proteins of interest were evaluated using electrochemiluminescence assays. Statistical analysis was performed from May 1, 2017, to August 31, 2017. The main outcome was augmented brain insulin signaling that manifested as a change in tyrosine phosphorylated insulin receptor substrate 1 within neuronal extracellular vesicles at the end of 48 weeks of exenatide treatment. Additional outcome measures were changes in other insulin receptor substrate proteins and effects on protein expression in the Akt and mitogen-activated protein kinase pathways. Sixty patients (mean [SD] age, 59.9 [8.4] years; 43 [72%] male) participated in the study: 31 in the exenatide group and 29 in the placebo group (data from 1 patient in the exenatide group were excluded). Patients treated with exenatide had augmented tyrosine phosphorylation of insulin receptor substrate 1 at 48 weeks (0.27 absorbance units [AU]; 95% CI, 0.09-0.44 AU; P = .003) and 60 weeks (0.23 AU; 95% CI, 0.05-0.41 AU; P = .01) compared with patients receiving placebo. Exenatide-treated patients had elevated expression of downstream substrates, including total Akt (0.35 U/mL; 95% CI, 0.16-0.53 U/mL; P < .001) and phosphorylated mechanistic target of rapamycin (mTOR) (0.22 AU; 95% CI, 0.04-0.40 AU; P = .02). Improvements in Movement Disorders Society Unified Parkinson's Disease Rating Scale part 3 off-medication scores were associated with levels of total mTOR (F4,50 = 5.343, P = .001) and phosphorylated mTOR (F4,50 = 4.384, P = .04). The results of this study are consistent with target engagement of brain insulin, Akt, and mTOR signaling pathways by exenatide and provide a mechanistic context for the clinical findings of the Exenatide-PD trial. This study suggests the potential of using exosome-based biomarkers as objective measures of target engagement in clinical trials using drugs that target neuronal pathways.

Effect of AMPK/mTOR/S6K1 pathways and the insulin-sensitizing effect for adiponectin in endometrial cancer cells

Objective: To explore effect of AMP-activated protein kinase (AMPK) /mammalian target of rapamycin (mTOR) /ribosomal protein S6 kinase-1 (S6K1) signaling pathways and the insulin-sensitizing effect by adiponectin in endometrial cancer HEC-1B cells. Methods: The experiments were divided into 4 groups, adiponectin (Ad) group (HEC-1B cells treated with 20 μg/ml adiponectin for 30 minutes) , inhibitor group (HEC-1B cells treated with 10 μmol/L compound C for 30 minutes) , inhibitor+ Ad group (HEC-1B cells treated with 10 μmol/L compound C for 30 minutes following incubation with 20 μg/ml adiponectin for 30 minutes) , control group (only added the culture medium without serum DMEM) . (1) Real-time quantitative PCR and western blot analysis were used to detect the level of mRNA and protein of adiponectin receptor (AdipoR) 1 and AdipoR2. (2) Western blot analysis were used to detect phosphorylation of AMPK, mTOR, S6K1 or insulin receptor substrate 1 (IRS1) protein expression with stimulation in different concentrations of adiponectin (2.5, 5, 10, and 20 μg/ml) , or following incubation with insulin 50 nmol/L for 5 minutes; or treated with 20 μg/ml adiponectin for different times (15, 30, 45, and 60 minutes) , or following incubation with insulin 50 nmol/L for 5 minutes. (3) Cell counting kit-8 (CCK-8) assay was performed to investigate the cell proliferation, and transwell chamber assay was used to detect the cell migration in different groups. Results: (1) The relative expression level of AdipoR1 mRNA and protein were higher than AdipoR2 in HEC-1B cell (8.50±0.09 to 1.00±0.00, and 0.91±0.03 to 0.69±0.03; P<0.05) . (2) The phosphorylation level of p-AMPK was significantly induced, and the phosphorylation level of p-mTOR and p-S6K1 proteins, and 20 μg/ml adiponectin at 30 minutes, AMPK protein had the highest level of activation. (3) Adiponectin induces increased tyrosine phosphorylation of IRS1 in a time-and concentration-dependent manner. (4) The proliferation inhibition ratio in Ad group (0.68±0.34) % was much more than that in inhibitor+Ad group (0.24±0.04) % (t=17.88, P<0.05) . The number of cell migration in Ad group (77±8) was much more than that in inhibitor+Ad group (132±13; t=-7.34, P<0.05) . Conclusions: Adiponectin maybe inhibit proliferation and migration of endometrial cancer cells through AMPK/mTOR/S6K1 signal pathway. Adiponectin insensitizes insulin signaling may by regulating by the AMPK/S6K1/IRS1 pathway.

Effect of ursolic acid on obesity-induced insulin resistance in rat liver

Purpose: To determine the expression of protein tyrosine phosphatase-1B (PTP-1B) and insulin receptor substrate-2 (IRS-2) in the liver tissue of obesity-induced insulin-resistant rats.Methods: Insulin resistance (IR) was induced in Wistar rats by placing them on a high fat diet for 6weeks, and ursolic acid (UA) was administered. Metformin served as positive control drug. The rats were divided into 5 groups based on the treatments given: normal group, positive control group, metformin group, high-dose UA group, and low-dose UA group. The general conditions of the rats were assessed 4 and 8 weeks after the various treatments. Liver glycogen levels were measured, and liver histological examination carried out after tissue processing and staining with hematoxylin and eosin (H &amp; E). Real-time polymerase chain reaction (RT-PCR) was employed for the determination of hepatic expressions of PTP-1B and IRS-2 mRNAs, while expressions of PTP-1B protein and IRS-2 protein, and phosphorylation of IRS-2 tyrosine were assayed by Western blotting.Results: Liver glycogen levels were significantly increased in the UA-treated groups (p &lt; 0.05). Moreover, UA provoked reductions in the expression of PTP-1B protein (p &lt; 0.05), but up-regulated the expression of IRS-2 protein (p &lt; 0.05), and enhanced IRS-2 tyrosine phosphorylation (p &lt; 0.05).Conclusion: These results suggest that UA mitigates IR through blockage of PTP-1B expression and up-regulation of the expression of IRS-2 mRNA. Therefore, PTP-1B is a potential target for the treatment of type 2 diabetes.Keywords: Ursolic acid, Insulin resistance, Liver, Protein tyrosine phosphatase-1B, Insulin receptor substrate-

Effects of Modified Sanzi Yangqin Decoction on Tyrosine Phosphorylation of IRS-1 in Skeletal Muscle of Type 2 Diabetic Rats.

This study aimed to investigate the effect of Modified Sanzi Yangqin Decoction on tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) in skeletal muscle of type 2 diabetic rats. The rat model of type 2 diabetes was induced by high-fat diet and multiple low-dose streptozotocin injections. Diabetic model rats were randomly divided into 5 groups: the model control group, the metformin group, and Modified Sanzi Yangqin Decoction groups of low, medium, and high doses. OGTT was conducted every two weeks during treatment period. At the end of the treatment, the fasting blood glucose (FBG) level and the fasting C-peptide level were measured to calculate insulin resistance index. The levels of IRS-1, p-IRS-1(Tyr895), and protein tyrosine phosphates 1B (PTP1B) in skeletal muscle were also measured. Modified Sanzi Yangqin Decoction significantly reduced the FBG level, increased the fasting C-peptide level, and lowered the insulin resistance index in type 2 diabetic rats. It also significantly increased the protein level of p-IRS-1(Tyr895) and reduced the PTP1B protein level in skeletal muscle of type 2 diabetic rats. Modified Sanzi Yangqin Decoction increases tyrosine phosphorylation of IRS-1 in skeletal muscle of type 2 diabetic rats, which results from the increase of p-IRS-1(Tyr895) protein and is related to the suppression of PTP1B protein.

Histone deacetylase regulates insulin signaling via two pathways in pancreatic β cells.

Recent studies demonstrated that insulin signaling plays important roles in the regulation of pancreatic β cell mass, the reduction of which is known to be involved in the development of diabetes. However, the mechanism underlying the alteration of insulin signaling in pancreatic β cells remains unclear. The involvement of epigenetic control in the onset of diabetes has also been reported. Thus, we analyzed the epigenetic control of insulin receptor substrate 2 (IRS2) expression in the MIN6 mouse insulinoma cell line. We found concomitant IRS2 up-regulation and enhanced insulin signaling in MIN6 cells, which resulted in an increase in cell proliferation. The H3K9 acetylation status of the Irs2 promoter was positively associated with IRS2 expression. Treatment of MIN6 cells with histone deacetylase inhibitors led to increased IRS2 expression, but this occurred in concert with low insulin signaling. We observed increased IRS2 lysine acetylation as a consequence of histone deacetylase inhibition, a modification that was coupled with a decrease in IRS2 tyrosine phosphorylation. These results suggest that insulin signaling in pancreatic β cells is regulated by histone deacetylases through two novel pathways affecting IRS2: the epigenetic control of IRS2 expression by H3K9 promoter acetylation, and the regulation of IRS2 activity through protein modification. The identification of the histone deacetylase isoform(s) involved in these mechanisms would be a valuable approach for the treatment of type 2 diabetes.

Differential involvement of the microtubule cytoskeleton in insulin receptor substrate 1 (IRS-1) and IRS-2 signaling to AKT determines the response to microtubule disruption in breast carcinoma cells

The insulin receptor substrate (IRS) proteins serve as essential signaling intermediates for the activation of PI3K by both the insulin-like growth factor 1 receptor (IGF-1R) and its close family member, the insulin receptor (IR). Although IRS-1 and IRS-2 share significant homology, they regulate distinct cellular responses downstream of these receptors and play divergent roles in breast cancer. To investigate the mechanism by which signaling through IRS-1 and IRS-2 results in differential outcomes, we assessed the involvement of the microtubule cytoskeleton in IRS-dependent signaling. Treatment with drugs that either stabilize or disrupt microtubules reveal that an intact microtubule cytoskeleton contributes to IRS-2- but not IRS-1-mediated activation of AKT by IGF-1. Proximal IGF-1R signaling events, including IRS tyrosine phosphorylation and recruitment of PI3K, are not inhibited by microtubule disruption, indicating that IRS-2 requires the microtubule cytoskeleton at the level of downstream effector activation. IRS-2 colocalization with tubulin is enhanced upon Taxol-mediated microtubule stabilization, which, together with the signaling data, suggests that the microtubule cytoskeleton may facilitate access of IRS-2 to downstream effectors such as AKT. Of clinical relevance is that our data reveal that expression of IRS-2 sensitizes breast carcinoma cells to apoptosis in response to treatment with microtubule-disrupting drugs, identifying IRS-2 as a potential biomarker for the response of breast cancer patients to Vinca alkaloid drug treatment.

Regulation of skeletal muscle insulin-stimulated signaling through the MEK-REDD1-mTOR axis

Recent findings in adipocytes suggest that mitogen-activated protein kinase (MAPK)/extracellular-regulated signaling kinase (ERK) kinase 1/2 (MEK1/2) signaling regulates regulated in development and DNA damage 1 (REDD1) protein expression. Similarly, our previous work show that a lack of REDD1 protein expression, and associated hyperactive basal mechanistic target of rapamycin (mTOR) signaling, limits skeletal muscle's response to insulin. Therefore, we sought to determine: 1) if MEK1/2 inhibition is sufficient to reduce REDD1 protein expression and subsequently insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation via negative feedback of hyperactive mTOR in REDD1 wild-type (WT) mice and 2) if rapamycin-mediated mTOR inhibition is sufficient to improve IRS-1 tyrosine phosphorylation in REDD1 knockout (KO) mice. REDD1 WT mice were injected with 10 mg/kg BW of the MEK1/2 non-competitive inhibitor, PD184352, 3 h prior to acute insulin treatment. In separate studies, REDD1 KO mice were injected with 5 mg/kg BW of the mTOR inhibitor, rapamycin, 3 h prior to acute insulin treatment. Following the inhibitor treatment period, markers of insulin signaling activation (IRS-1 Y1222, MEK1/2 S217/221, ERK1/2 T202/Y204), REDD1, and mTOR signaling activation (S6K1 T389, rpS6 S240/244) were examined in skeletal muscle collected before and after a 10 min insulin treatment. PD184352 treatment reduced MEK/ERK phosphorylation and REDD1 protein expression, independent of insulin. This reduction in REDD1 protein expression was associated with elevated basal S6K1 and rpS6 phosphorylation and reduced insulin stimulated IRS-1 phosphorylation. Conversely, rapamycin inhibited S6K1 and rpS6 activation, and significantly improved insulin –stimulated activation of IRS-1 and MEK1/2 in KO mice. These data support that REDD1 is required for normal insulin-stimulated signaling, and that a subtle balance exists between MEK1/2, REDD1, and mTOR for the proper regulation of insulin signaling.

Serine 302 Phosphorylation of Mouse Insulin Receptor Substrate 1 (IRS1) Is Dispensable for Normal Insulin Signaling and Feedback Regulation by Hepatic S6 Kinase

Constitutive activation of the mammalian target of rapamycin complex 1 and S6 kinase (mTORC1→ S6K) attenuates insulin-stimulated Akt activity in certain tumors in part through "feedback" phosphorylation of the upstream insulin receptor substrate 1 (IRS1). However, the significance of this mechanism for regulating insulin sensitivity in normal tissue remains unclear. We investigated the function of Ser-302 in mouse IRS1, the major site of its phosphorylation by S6K in vitro, through genetic knock-in of a serine-to-alanine mutation (A302). Although insulin rapidly stimulated feedback phosphorylation of Ser-302 in mouse liver and muscle, homozygous A302 mice (A/A) and their knock-in controls (S/S) exhibited similar glucose homeostasis and muscle insulin signaling. Furthermore, both A302 and control primary hepatocytes from which Irs2 was deleted showed marked inhibition of insulin-stimulated IRS1 tyrosine phosphorylation and PI3K binding after emetine treatment to raise intracellular amino acids and activate mTORC1 → S6K signaling. To specifically activate mTORC1 in mouse tissue, we deleted hepatic Tsc1 using Cre adenovirus. Although it moderately decreased IRS1/PI3K association and Akt phosphorylation in liver, Tsc1 deletion failed to cause glucose intolerance or promote hyperinsulinemia in mixed background A/A or S/S mice. Moreover, Tsc1 deletion failed to stimulate phospho-Ser-302 or other putative S6K sites within IRS1, whereas ribosomal S6 protein was constitutively phosphorylated. Following acute Tsc1 deletion from hepatocytes, Akt phosphorylation, but not IRS1/PI3K association, was rapidly restored by treatment with the mTORC1 inhibitor rapamycin. Thus, within the hepatic compartment, mTORC1 → S6K signaling regulates Akt largely through IRS-independent means with little effect upon physiologic insulin sensitivity.

4-Hydroxyisoleucine attenuates the inflammation-mediated insulin resistance by the activation of AMPK and suppression of SOCS-3 coimmunoprecipitation with both the IR-β subunit as well as IRS-1.

It is known that 4-hydroxyisoleucine (4-HIL) from seeds of Trigonella foenum-graecum has beneficial effects on low-grade inflammation; therefore, the insulin signaling as well as the anti-inflammatory effects of 4-HIL in TNF-α-induced insulin resistance in C2C12 myotubes was studied with an aim to dissect out the mechanism(s) of the inflammation-mediated insulin resistance. TNF-α suppressed insulin-stimulated glucose transport rate and increased Ser-307 phosphorylation of insulin receptor substrate-1 (IRS-1). However, the treatment of 4-hydroxyisoleucine enhanced insulin-stimulated glucose transport rate via the activation of AMP-activated protein kinase (AMPK) in a dose-dependent manner. 4-HIL also increases the tyrosine phosphorylation of both IR-β and IRS-1. Moreover, coimmunoprecipitation (Co-IP) of insulin receptor-β (IR-β) subunit with IRS-1 was found to be increased by 4-hydroxyisoleucine. Concentration of SOCS-3 protein and coimmunoprecipitation of SOCS-3 protein with both the IR-β subunit as well as IRS-1 was found to be decreased by 4-HIL. We conclude that the 4-hydroxyisoleucine reverses the insulin resistance by the activation of AMPK and suppression of SOCS-3 coimmunoprecipitation with both the IR-β subunit as well as IRS-1.

Insulin Receptor Substrate 1, the Hub Linking Follicle-stimulating Hormone to Phosphatidylinositol 3-Kinase Activation

The ubiquitous phosphatidylinositol 3-kinase (PI3K) signaling pathway regulates many cellular functions. However, the mechanism by which G protein-coupled receptors (GPCRs) signal to activate PI3K is poorly understood. We have used ovarian granulosa cells as a model to investigate this pathway, based on evidence that the GPCR agonist follicle-stimulating hormone (FSH) promotes the protein kinase A (PKA)-dependent phosphorylation of insulin receptor substrate 1 (IRS1) on tyrosine residues that activate PI3K. We report that in the absence of FSH, granulosa cells secrete a subthreshold concentration of insulin-like growth factor-1 (IGF-1) that primes the IGF-1 receptor (IGF-1R) but fails to promote tyrosine phosphorylation of IRS1. FSH via PKA acts to sensitize IRS1 to the tyrosine kinase activity of the IGF-1R by activating protein phosphatase 1 (PP1) to promote dephosphorylation of inhibitory Ser/Thr residues on IRS1, including Ser(789). Knockdown of PP1β blocks the ability of FSH to activate PI3K in the presence of endogenous IGF-1. Activation of PI3K thus requires both PKA-mediated relief of IRS1 inhibition and IGF-1R-dependent tyrosine phosphorylation of IRS1. Treatment with FSH and increasing concentrations of exogenous IGF-1 triggers synergistic IRS1 tyrosine phosphorylation at PI3K-activating residues that persists downstream through protein kinase B (AKT) and FOXO1 (forkhead box protein O1) to drive synergistic expression of genes that underlies follicle maturation. Based on the ability of GPCR agonists to synergize with IGFs to enhance gene expression in other cell types, PP1 activation to relieve IRS1 inhibition may be a more general mechanism by which GPCRs act with the IGF-1R to activate PI3K/AKT.

Timosaponin B-II Ameliorates Palmitate-Induced Insulin Resistance and Inflammation via IRS-1/PI3K/Akt and IKK/NF-κB Pathways

This study aimed to investigate the effect of timosaponin B-II (TB-II) on palmitate (PA)-induced insulin resistance and inflammation in HepG2 cells, and probe the potential mechanisms. TB-II, a main ingredient of the traditional Chinese medicine Anemarrhena asphodeloides Bunge, notably ameliorated PA-induced insulin resistance and inflammation, and significantly improved cell viability, decreased PA-induced production of tumor necrosis factor-[Formula: see text] (TNF-[Formula: see text]) and interleukin-6 (IL-6) levels. Further, TB-II treatment notably decreased malondialdehyde (MDA) and lactate dehydrogenase (LDH) levels, and improved superoxide dismutase (SOD) and nitric oxide (NO). TB-II also reduced HepG2 cells apoptosis. Insulin receptor substrate-1 (IRS1)/phosphatidylinositol 3-kinase (PI3K)/Akt and inhibitor of nuclear factor [Formula: see text]-B kinase (IKK)/NF-[Formula: see text]B pathways-related proteins, and IKK[Formula: see text], p65 phosphorylation, serine phosphorylation of insulin receptor substrate-1 (IRS-1) at S307, tyrosine phosphorylation of IRS-1, and Akt activation were determined by Western blot. Compared to model group, TB-II significantly downregulated the expression of p-NF-[Formula: see text]Bp65, p-IKK[Formula: see text], p-IRS-1, p-PI3K and p-Akt. TB-II is a promising potential agent for the management of palmitate-induced insulin resistance and inflammation, which might be via IR/IRS-1/PI3K/Akt and IKK/NF-[Formula: see text]B pathways.

NOD2 activation induces oxidative stress contributing to mitochondrial dysfunction and insulin resistance in skeletal muscle cells

Nucleotide-binding oligomerization domain protein-2 (NOD2) activation in skeletal muscle cells has been associated with insulin resistance, but the underlying mechanisms are not yet clear. Here we demonstrate the implication of oxidative stress in the development of mitochondrial dysfunction and insulin resistance in response to NOD2 activation in skeletal muscle cells. Treatment with the selective NOD2 ligand muramyl dipeptide (MDP) increased mitochondrial reactive oxygen species (ROS) generation in L6 myotubes. MDP-induced ROS production was associated with increased levels of protein carbonyls and reduction in citrate synthase activity, cellular ATP level, and mitochondrial membrane potential, as well as altered expression of genes involved in mitochondrial function and metabolism. Antioxidant treatment attenuated MDP-induced ROS production and restored mitochondrial functions. In addition, the presence of antioxidant prevented NOD2-mediated activation of MAPK kinases and the inflammatory response. This was associated with reduced serine phosphorylation of insulin receptor substrate-1 (IRS-1) and improved insulin-stimulated tyrosine phosphorylation of IRS-1 and downstream activation of Akt phosphorylation. These data indicate that oxidative stress plays a role in NOD2 activation-induced inflammatory response and that MDP-induced oxidative stress correlates with impairment of mitochondrial functions and induction of insulin resistance in skeletal muscle cells.