Insulin Receptor Substrate Proteins Research Articles

1835-P: Deletion of Insulin Receptor Substrate 2 in AGRP Neurons Causes Beta-Cell Dysfunction

In humans, type 2 diabetes progresses when beta-cells fail to compensate peripheral insulin resistance. Insulin receptor substrate (IRS) proteins mediate a variety of the metabolic and growth-promoting actions of insulin and IGF-1, and are important integrators of heterologous regulation. Systemic ablation of the Irs2 gene in mice causes β-cell dysfunction that prevents compensatory insulin secretion while insulin resistance progresses to life threatening hyperglycemia. Upregulation of IRS2 in pancreatic β-cell using rat insulin-2 promoter (Irs2RIP) promotes sufficient hyperinsulinemia to prevent diabetes. Targeted Irs2 deletion using CreRIP also causes beta-cell failure and hyperglycemia, but the mechanism is unclear because CreRIP drives deletion in beta-cells and hypothalamus—especially in the arcuate nucleus (ARC). AGRP neurons in the ARC play an important role in regulating energy homeostasis, but it is unknown whether IRS2 in the AGRP neurons might also affect beta-cell function. Here we show that disruption of Irs2 (but not Irs1) in AGRP neurons (Irs2f/fCreAgrp-mice) reduced islet mass by 45%. To test insulin secretory function, we subjected mice to hyperglycemic clamps. The glucose infusion rate to maintain steady-state hyperglycemia decreased significantly. Moreover, insulin secretion failed to respond to L-arginine treatment in aged Irs2f/fCreAgrp-mice. Moreover, Irs2f/fCreAgrp-mice displayed progressive deterioration of glucose homeostasis and a lack of beta -cell compensation during high fat diet. Overall, our results indicate that dysfunction of Irs2 in AGRP neurons might contribute to the pathophysiology of type 2 diabetes. Disclosure R. Tao: None. K.D. Copps: None. M.F. White: Advisory Panel; Self; Housey Pharmaceutical Research Laboratories. O. Stoehr: None. Funding National Institutes of Health (5R01DK098655-06)

Effects of Agriophyllum squarrosum extracts on glucose metabolism in KKAy mice and the associated underlying mechanisms

Ethnopharmacological relevanceAgriophyllum squarrosum (L.) Moq. is a traditional Mongol medicine commonly used in the treatment of diabetes. Aim of the studyTo examine the effects of Agriophyllum squarrosum extract (ASE) on glucose metabolism in type 2 diabetic KKAy mice, and to investigate the mechanisms underlying these effects. Material and methodsKKAy mice were divided into a model control group (MCG), a low-dose Agriophyllum squarrosum extract group (LASEG), a medium-dose Agriophyllum squarrosum extract group (MASEG), a high-dose Agriophyllum squarrosum extract group (HASEG), and a metformin group (MEG). Syngeneic C57BL/6 mice were used as a normal control group (NCG). Drugs were administered to all mice by gavage for 8 weeks. Random blood glucose levels were measured in the mice at baseline and after 2, 4, and 8 weeks of treatment. Glucose tolerance was measured after 6 weeks of drug administration. After 8 weeks, glycated serum proteins (GSP) and advanced glycation end-products (AGEs) in the serum of all mice were measured. Sections of mouse liver tissues were used for periodic acid-Schiff staining (PAS) and the content of hepatic glycogen was determined. Immunohistochemistry was used to determine the effects of ASE on liver phospho-insulin receptor substrate 2 (P-IRS2) protein expression. Western blotting was used to quantify the protein expression levels of phosphatidylinositol 3-kinase (PI3K), AKT, phospho-AKT (S473) (P-AKT), glycogen synthase kinase 3β (GSK3β), and glucose transporters 4 (GLUT4), while PCR was used to quantify the mRNA expression levels of insulin receptor substrate 2 (IRS2), PI3K, AKT, GSK3β, and GLUT4. ResultsASE treatment decreased random blood glucose levels in type 2 diabetic KKAy mice; increased glucose tolerance; decreased serum GSP and AGEs content; increased glycogen synthesis in liver tissues; upregulated the protein expression levels of PI3K, AKT, GLUT4, and P-IRS2; downregulated the protein expression level of GSK3β in liver tissues; upregulated the mRNA expression levels of IRS2, PI3K, AKT, and GLUT4; and downregulated the mRNA expression level of GSK3β in liver tissues. ConclusionASE treatment may increase glucose metabolism in KKAy mice and improve glucose tolerance. The underlying mechanisms of the beneficial effects of ASE may be associated with the increase of glycogen synthesis, the inhibition of AGEs production, the upregulation of IRS2, PI3K, AKT, and GLUT4 protein and mRNA expression, and the downregulation of GSK3β protein and mRNA expression.

Yunpi Heluo decoction attenuates insulin resistance by regulating liver miR-29a-3p in Zucker diabetic fatty rats

Background and objectiveYunpiheluo (YPHL) decoction is a Chinese herbal formula with unique advantages for the treatment of type 2 diabetes mellitus (T2DM). The aim of the present study was to investigate changes in miRNA expression and downstream gene expression in Zucker diabetic fatty (ZDF) rats treated with YPHL to determine whether YPHL could be used as an adjuvant treatment of T2DM. MethodsSerum and liver total cholesterol (TC) and triglycerides (TG) levels, insulin resistance index (IR) and differentially expressed miRNAs were detected in a T2DM ZDF rat model. miRNA target prediction was based on bioinformatic algorithms and dual luciferase reporter assay. Protein expression of genes in the insulin receptor signaling pathway was detected by Western blot. The IR cell model was established and the effects of lyophilized YPHL powder on the protein expressions were observed by transfecting specific miRNA mimics and inhibitors. ResultsThe miR-29a-3p expression level was significantly increased in the liver of ZDF rats. Insulin receptor substrate 1 (IRS1) was the target gene of miR-29a-3p. IRS1 mRNA and protein expressions of IRS1, IRS1 (phospho S307), protein kinase B (Akt), Akt (phosphor ser473) and pyruvate dehydrogenase lipoamide kinase isozyme 1 (PDK1) were decreased significantly. miR-29a-3p over-expression decrease IRS1 and the others protein expressions in the HepG2 IR cell model while anti-miR-29a-3p showed the opposite result. The miR-29a-3p level was decreased, and the expressions of IRS1 mRNA and the above proteins were all increased after YPHL treatment. ConclusionmiR-29a-3p played a functional role in insulin receptor signaling in the liver of ZDF rats. YPHL decoction attenuated IR in T2DM probably by down-regulating or maintaining the miR-29a-3p level, increasing the expression of IRS1 mRNA and its phosphorylated proteins, and regulating the expression of insulin receptor signaling-related proteins. YPHL may prove to be an alternative treatment for T2DM.

Hesperidin ameliorates insulin resistance by regulating the IRS1-GLUT2 pathway via TLR4 in HepG2 cells.

The aim of this study was to evaluate the effect and mechanism of hesperidin (HES) on insulin resistance (IR) in the human hepatocellular carcinoma cell line (HepG2 cells). HepG2 cells were induced with lipopolysaccharide (LPS) as a model of IR and treated with HES at three dosages. Next, the levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), the glucose content, and glucose uptake were evaluated by enzyme-linked immunosorbent assay, glucose oxidase-peroxidase method (GOD-POD), or (2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-2-deoxyglucose) (2-NBDG). Moreover, the protein expression of toll-like receptors 4 (TLR4), insulin receptor substrate 1 (IRS1), nuclear factor kappa B (NF-κB), and glucose transporter 2 (GLUT2) in HepG2 cells treated with HES were assessed via western blotting analysis. In addition, GLUT2 protein expression exposed to HES was detected following treatment with TLR4 inhibitor (HTA125). Our results demonstrated that HES decreased the levels of TNF-α and IL-6, attenuated the glucose content in culture medium and increased glucose uptake in insulin-resistant HepG2 cells in vitro. Moreover, HES upregulated the expression of IRS1 and GLUT2 protein and downregulated the protein expression of TLR4 and NF-κB in insulin-resistant HepG2 cells. The expression of GLUT2 protein had no significant changes when treated with HES after blockade of TLR4. HES attenuated IR in LPS-inducedinsulin-resistant HepG2 cells. Therefore, regulating the IRS1-GLUT2 pathway via TLR4 represents a potential mechanism of HES on IR in HepG2 cells.

Molecular Mechanisms of Hypothalamic Insulin Resistance.

Insulin exists in the central nervous system, where it executes two important functions in the hypothalamus: the suppression of food intake and the improvement of glucose metabolism. Recent studies have shown that both are exerted robustly in rodents and humans. If intact, these functions exert beneficial effects on obesity and diabetes, respectively. Disruption of both occurs due to a condition known as hypothalamic insulin resistance, which is caused by obesity and the overconsumption of saturated fat. An enormous volume of literature addresses the molecular mechanisms of hypothalamic insulin resistance. IKKβ and JNK are major players in the inflammation pathway, which is activated by saturated fatty acids that induce hypothalamic insulin resistance. Two major tyrosine phosphatases, PTP-1B and TCPTP, are upregulated in chronic overeating. They dephosphorylate the insulin receptor and insulin receptor substrate proteins, resulting in hypothalamic insulin resistance. Prolonged hyperinsulinemia with excessive nutrition activates the mTOR/S6 kinase pathway, thereby enhancing IRS-1 serine phosphorylation to induce hypothalamic insulin resistance. Other mechanisms associated with this condition include hypothalamic gliosis and disturbed insulin transport into the central nervous system. Unveiling the precise molecular mechanisms involved in hypothalamic insulin resistance is important for developing new ways of treating obesity and type 2 diabetes.

Insulin and Insulin Receptors in Adipose Tissue Development.

Insulin is a major endocrine hormone also involved in the regulation of energy and lipid metabolism via the activation of an intracellular signaling cascade involving the insulin receptor (INSR), insulin receptor substrate (IRS) proteins, phosphoinositol 3-kinase (PI3K) and protein kinase B (AKT). Specifically, insulin regulates several aspects of the development and function of adipose tissue and stimulates the differentiation program of adipose cells. Insulin can activate its responses in adipose tissue through two INSR splicing variants: INSR-A, which is predominantly expressed in mesenchymal and less-differentiated cells and mainly linked to cell proliferation, and INSR-B, which is more expressed in terminally differentiated cells and coupled to metabolic effects. Recent findings have revealed that different distributions of INSR and an altered INSR-A:INSR-B ratio may contribute to metabolic abnormalities during the onset of insulin resistance and the progression to type 2 diabetes. In this review, we discuss the role of insulin and the INSR in the development and endocrine activity of adipose tissue and the pharmacological implications for the management of obesity and type 2 diabetes.

Treatment of type 2 diabetes mellitus with the stem and leaf of blacktartary buckwheat and its effects on the pancreas and spleen

To investigate the therapeutic effects of the black buckwheat leaf (BBL) in type 2 diabetes mellitus mice and its effects on pancreas and spleen. Forty male C57 / B16 mice (SPF) were randomly divided into normal control (NC) group (n=10) and the experimental group (n=30), the experimental group were fed with high sugar and high fat, combined with intraperitoneal injection of streptozotocin in small dose to establish the model of type 2 diabetes mellitus (T2DM). Those thirty model mice were randomly divided into 3 groups (n=10), diabetes mellitus group (DM), low dose of BBL (DM+L) treated group, high dose of BBL (DM+H) treated group. The mice in the NC group and the DM group were given normal saline per day, and the DM+L group and DM+H group were treated with black tartary buckwheat at the doses of 0.21g/kg·d-1 and 0.42g/kg·d-1 respectively. After 14 days. All mice were executed by cervical dislocation, then blood samples were collected, pancreas and spleen were removed for subsequent experiments. The serum levels of fasting blood glucose (FBG), triglyceride (TG), total cholesterol (TCH) and insulin were detected. TNF-α protein in spleen tissue was detected by ELISA kit. The morphology of pancreas tissue was observed by HE staining, and the spleen coefficient was calculated. The expression levels of insulin receptor substrate-1(IRS-1) mRNA and IRS-1 protein in pancreatic tissue were detected. Compared with the control group, the serum levels of FBG, TC and TCH in the model group were increased significantly, while the serum level of insulin was decreased significantly (P＜0.05), the expression of TNF-α protein in spleen tissues was obviously raised, the expressions of IRS-1 mRNA and IRS-1 protein in pancreatic tissue in model group were decreased significantly (P＜0.05). Compared with the model group, the serum levels of FBG, TC and TCH were decreased significantly in the BBL treated groups. The serum insulin level, spleen coefficient, TNF-α protein expression level in spleen tissue, IRS-1 mRNA expression and IRS-1 protein expression levels in pancreatic tissue in BBL treated group were increased significantly (P＜ 0.05). High-dose black tartary buckwheat leaves (0.42g/kg·d-1) exerted a more significant effect. Stem and leaf of black bitter buckwheat has significant therapeutic effects on reducing blood sugar and blood fat in type 2 diabetic mice, and has certain protective effects on pancreas and spleen of diabetic mice.

Three Novel Mutations I65S, R66S, and G86R Divulge Significant Conformational Variations in the PTB Domain of the IRS1 Gene.

Insulin receptor substrate 1 (IRS1) is one of the major substrates for the IR, and their interaction mediates several downstream insulin signaling pathways. In this study, we have identified three novel mutations in the IRS1 gene of type 2 diabetic (T2D) patients, which reflected in the amino acid changes as I65S, R66S, and G86R in the phosphotyrosine binding domain of the IRS1 protein. The impact of these mutations on the structure and function of the IRS1 protein was evaluated through molecular modeling studies, and distinct conformational fluctuations were recorded. The variable binding affinities and positional displacement of these mutant models were observed in the ligand-binding cleft of IR. The mutant IRS1 models triggered conformational changes in the L1 domain of IR upon their binding. Such structural variations in IRS1 and IR structures due to mutations resulted in variable molecular interactions that could lead to altered insulin transduction, followed by insulin resistance and T2D.

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.

Involvement of insulin receptor substrates in cognitive impairment and Alzheimer's disease.

Type 2 diabetes—associated with impaired insulin/insulin-like growth factor-1 (IGF1) signaling (IIS)—is a risk factor for cognitive impairment and dementia including Alzheimer’s disease (AD). The insulin receptor substrate (IRS) proteins are major components of IIS, which transmit upstream signals via the insulin receptor and/or IGF1 receptor to multiple intracellular signaling pathways, including AKT/protein kinase B and extracellular-signal-regulated kinase cascades. Of the four IRS proteins in mammals, IRS1 and IRS2 play key roles in regulating growth and survival, metabolism, and aging. Meanwhile, the roles of IRS1 and IRS2 in the central nervous system with respect to cognitive abilities remain to be clarified. In contrast to IRS2 in peripheral tissues, inactivation of neural IRS2 exerts beneficial effects, resulting in the reduction of amyloid β accumulation and premature mortality in AD mouse models. On the other hand, the increased phosphorylation of IRS1 at several serine sites is observed in the brains from patients with AD and animal models of AD or cognitive impairment induced by type 2 diabetes. However, these serine sites are also activated in a mouse model of type 2 diabetes, in which the diabetes drug metformin improves memory impairment. Because IRS1 and IRS2 signaling pathways are regulated through complex mechanisms including positive and negative feedback loops, whether the elevated phosphorylation of IRS1 at specific serine sites found in AD brains is a primary response to cognitive dysfunction remains unknown. Here, we examine the associations between IRS1/IRS2-mediated signaling in the central nervous system and cognitive decline.

Hepatitis C virus infection, genotypes and mechanism of insulin resistance

Background: Hepatitis C virus (HCV) infection is a major global public health problem that causes profound metabolic abnormalities, primarily in insulin-sensitive target tissues, notably the phenomenon of steatosis or fatty liver. The route of transmission and genetic mutation of HCV, together with the lack of reliable nation-specific epidemiological data on the distribution of genotypes and sub-genotypes of this RNA virus, provide significant challenges to correct diagnosis and effective treatment. Issue: HCV-induced insulin resistance in HCV-infected individuals is independent of the occurrence of metabolic syndrome and diabetes mellitus, primarily type 2 diabetes mellitus, that causes insulin resistance. Some but not all HCV genotypes exert a steatotic effect. However, the molecular mechanism(s) by which HCV infection causes insulin resistance in insulin target tissues or hepatic steatosis is not elucidated clearly. Findings: Mechanisms proposed by experimental studies include interference with insulin signaling pathways, upregulation of genes controlling gluconeogenesis, phosphorylation of insulin receptor substrate proteins, and induction and overexpression of inflammatory cytokines that interact closely with host lipid metabolism. Conclusion: We review HCV genotypes and subtypes, the mechanisms by which HCV infection induces insulin resistance, the virus genotypes and subtypes that are implicated in this and those that are steatotic. We conclude by discussing proposed mechanisms of steatosis and considering HCV laboratory investigation methods from traditional to current techniques.

Therapeutic effect of catalpol on type 2 diabetic mice induced by STZ and high-fat diet and its possible mechanism

To explore the therapeutic effect and possible mechanism of catalpol on type 2 diabetic mice. Twenty-four C57/BL6 male mice were randomly divided into four groups—normal control group (CON, n = 6), diabetic model group (DM, n = 6), the lower dose catalpol treatment (80 mg/kg body weight) group (DM+L, n = 6), and the higher dose catalpol treatment (160 mg/kg body weight) group (DM+H, n = 6). Intraperitoneal glucose tolerance test was performed after 30 days of treatment. Fasting blood glucose (FBG), triglyceride (TG), and total cholesterol (TC) in serum were detected by full automatic biochemical instrument. Enzyme-linked immunosorbent assay was used to detect insulin levels in serum. Insulin resistance level was calculated by trapezoid rule. The morphological changes of pancreatic tissue were observed through HE staining. The protein levels of insulin receptor substrate 1 (IRS1) and glucose transporter type 4 (GLUT4) in the liver and muscle were measured by Western blot (WB). Compared with the DM group mice, the glucose tolerance and insulin resistance in the DM+H group and the DM+L group were improved, the levels of TG, TC, and FBG decreased (p < 0.01). The blood insulin levels were elevated in the DM+H group and the DM+L group (p < 0.01) and the insulin resistance level decreased (p < 0.01, p < 0.05). IRS1 and GLUT4 protein levels in the liver and muscle increased in the DM+H group and the DM+L group (p < 0.01, p < 0.05). Catalpol could be potential medicine to treat type 2 diabetes. Its therapeutic mechanism might be improving insulin-stimulated glucose uptake in the liver and muscle.

TAZ enhances mammary cell proliferation in 3D culture through transcriptional regulation of IRS1

WW domain-containing transcriptional regulator 1 (TAZ) is a transcriptional co-activator and effector of the Hippo signaling pathway. In certain breast cancer subtypes, Hippo signaling is dysregulated leading to activation of TAZ and altered expression of TAZ transcriptional targets. Over the past decade, we and others have found that TAZ transcriptionally regulates genes that affect multiple aspects of breast cancer cell behaviour. However, while cancer cell-intrinsic oncogenic functions of TAZ have emerged, less is known about whether TAZ might also contribute to tumourigenesis by sensitizing tumour cells to factors present in the tumour microenvironment or in systemic circulation. Here, we show that TAZ directly regulates the expression of insulin receptor substrate 1 (IRS1) in breast cancer cells. TAZ or IRS1 overexpression induces a similar proliferative transformation phenotype in MCF10A mammary epithelial cells. TAZ enhances IRS1 mRNA, protein levels and downstream signaling in MCF10A. Mechanistically, TAZ interacts with the IRS1 promoter through the TEAD family of transcription factors and enhances its activity. Critically, TAZ-induced IRS1 upregulation contributes to the proliferation of TAZ-overexpressing MCF10A in 3-dimensional (3D) Matrigel culture. Therefore, we offer compelling evidence that TAZ regulates signaling through the insulin pathway in breast cancer cells. These findings highlight an additional mechanism by which TAZ may promote breast cancer tumourigenesis and progression by modulating cancer cell responses to exogenously produced factors.

Insulin-like growth factor 1 receptor stabilizes the ETV6–NTRK3 chimeric oncoprotein by blocking its KPC1/Rnf123-mediated proteasomal degradation

Many oncogenes, including chimeric oncoproteins, require insulin-like growth factor 1 receptor (IGF1R) for promoting cell transformation. The ETS variant 6 (ETV6)-neurotrophic receptor tyrosine kinase 3 (NTRK3) (EN) chimeric tyrosine kinase is expressed in mesenchymal, epithelial, and hematopoietic cancers and requires the IGF1R axis for transformation. However, current models of IGF1R-mediated EN activation are lacking mechanistic detail. We demonstrate here that IGF-mediated IGF1R stimulation enhances EN tyrosine phosphorylation and that blocking IGF1R activity or decreasing protein levels of the adaptor protein insulin receptor substrate 1/2 (IRS1/2) results in rapid EN degradation. This was observed both in vitro and in vivo in fibroblast and breast epithelial cell line models and in MO91, an EN-expressing human leukemia cell line. Stable isotope labeling with amino acids in cell culture (SILAC)-based MS analysis identified the E3 ligase RING-finger protein 123 (Rnf123, more commonly known as KPC1) as an EN interactor upon IGF1R/insulin receptor (INSR) inhibitor treatment. KPC1/Rnf123 ubiquitylated EN in vitro, and its overexpression decreased EN protein levels. In contrast, KPC1/Rnf123 knockdown rendered EN resistant to IGF1R inhibitor-mediated degradation. These results support a critical function for IGF1R in protecting EN from KPC1/Rnf123-mediated proteasomal degradation. Attempts to therapeutically target oncogenic chimeric tyrosine kinases have traditionally focused on blocking kinase activity to restrict downstream activation of essential signaling pathways. In this study, we demonstrate that IGF1R inhibition results in rapid ubiquitylation and degradation of the EN oncoprotein through a proteasome-dependent mechanism that is reversible, highlighting a potential strategy for targeting chimeric tyrosine kinases in cancer.

Potential activities and mechanisms of extracellular polysaccharopeptides from fermented Trametes versicolor on regulating glucose homeostasis in insulin-resistant HepG2 cells.

Polysaccharides derived from mushrooms have potential to control blood sugar, reduce insulin resistance and prevent diabetic complications. The intracellular polysaccharopeptides of Trametes versicolor (TV) have been used as immunologic and oncologic adjuvants. The aim of this study was to investigate the potential activities and mechanisms of extracellular polysaccharopeptides (ePSP) obtained from TV strain LH-1 on regulating glucose homeostasis. Human hepatoma HepG2 cells incubated with normal glucose (5.5 mM, NG model), high glucose (33 mM, HG model), or high glucose (33 mM) plus high insulin (10−7 M, HGI model) concentrations were administered with TV LH-1 ePSP (50, 100, and 1000 μg/ml) for 24 hr. Glucose uptake of HepG2 cells, determined by flow cytometry, was significantly decreased in the HG and HGI models with insulin stimulation, suggesting insulin resistance of these cells; however, ePSP reversed this decrease in a dose-dependent manner (one-way ANOVA, p<0.05). In the HG and HGI models, ePSP significantly increased glycogen content, insulin receptor substrate-2 protein and phosphorylated AMP-activated protein kinase (AMPK), as determined by western blot analysis. In addition, ePSP significantly increased glucokinase in the NG and HG models, increased membrane glucose transporter-1 and decreased glycogen synthase kinase-3β in the HGI model, and increased glucose-6-phosphatase in the NG and HGI models (one-way ANOVA, p<0.05). In summary, TV LH-1 ePSP may elevate cellular glucose uptake to regulate glucose homeostasis via the activation of AMPK and glycogen synthesis in an insulin-independent manner. These results suggest that TV LH-1 ePSP may be a nutraceutical with anti-hyperglycemic activity.

Identification of a Novel Invasion-Promoting Region in Insulin Receptor Substrate 2.

Although the insulin receptor substrate (IRS) proteins IRS1 and IRS2 share considerable homology and activate common signaling pathways, their contributions to breast cancer are distinct. IRS1 has been implicated in the proliferation and survival of breast tumor cells. In contrast, IRS2 facilitates glycolysis, invasion, and metastasis. To determine the mechanistic basis for IRS2-dependent functions, we investigated unique structural features of IRS2 that are required for invasion. Our studies revealed that the ability of IRS2 to promote invasion is dependent upon upstream insulin-like growth factor 1 receptor (IGF-1R)/insulin receptor (IR) activation and the recruitment and activation of phosphatidylinositol 3-kinase (PI3K), functions shared with IRS1. In addition, a 174-amino-acid region in the IRS2 C-terminal tail, which is not conserved in IRS1, is also required for IRS2-mediated invasion. Importantly, this "invasion (INV) region" is sufficient to confer invasion-promoting ability when swapped into IRS1. However, the INV region is not required for the IRS2-dependent regulation of glucose uptake. Bone morphogenetic protein 2-inducible kinase (BMP2K) binds to the INV region and contributes to IRS2-dependent invasion. Taken together, our data advance the mechanistic understanding of how IRS2 regulates invasion and reveal that IRS2 functions important for cancer can be independently targeted without interfering with the metabolic activities of this adaptor protein.

Egg white hydrolysate and peptide reverse insulin resistance associated with tumor necrosis factor-\u03b1 (TNF-\u03b1) stimulated mitogen-activated protein kinase (MAPK) pathway in skeletal muscle cells

PurposeExcessive formation of tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine, has been implicated in the development of insulin resistance in obesity and type-2 diabetes. In skeletal muscle, chronic exposure to TNF-α impairs insulin-stimulated glucose uptake and insulin signaling. The aim of this study is to investigate the effects of enzymatic egg white hydrolysate (EWH) and its responsible peptide, IRW, on TNF-α-induced insulin resistance and the underlying molecular mechanisms using rat skeletal muscle cells (L6 cells).MethodsInsulin resistance was induced by treating L6 cells with 5 ng/ml TNF-α for 24 h. Effects of EWH and IRW on glucose uptake were detected by glucose uptake assay, glucose transporter 4 (GLUT4) translocation by immunofluorescence, and western blot, while insulin-signaling pathway and mitogen-activated protein kinase (MAPK) pathway were investigated using western blot.ResultsAdding both EWH and IRW significantly improved glucose uptake in TNF-α-treated cells, increased activation of insulin receptor substrate (IRS-1) tyrosine residue and protein kinase B (Akt), whereas decreased activation of IRS-1 serine residue. In addition, TNF-α-induced activation of p38-mitogen-activated protein kinase (p38) and c-Jun N-terminal kinases (JNK) 1/2 were decreased by either EWH or IRW treatment.ConclusionEWH and IRW improve impaired insulin sensitivity by down-regulating the activation of p38 and JNK1/2 in TNF-α-treated skeletal muscle cells.

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-

The proximity-labeling technique BioID identifies sorting nexin 6 as a member of the insulin-like growth factor 1 (IGF1)–IGF1 receptor pathway

The insulin-like growth factor 1 receptor (IGF1R) is a receptor tyrosine kinase with critical roles in various biological processes. Recent results from clinical trials targeting IGF1R indicate that IGF1R signaling pathways are more complex than previously thought. Moreover, it has become increasingly clear that the function of many proteins can be understood only in the context of a network of interactions. To that end, we sought to profile IGF1R-protein interactions with the proximity-labeling technique BioID. We applied BioID by generating a HEK293A cell line that stably expressed the BirA* biotin ligase fused to the IGF1R. Following stimulation by IGF1, biotinylated proteins were analyzed by MS. This screen identified both known and previously unknown interactors of IGF1R. One of the novel interactors was sorting nexin 6 (SNX6), a protein that forms part of the retromer complex, which is involved in intracellular protein sorting. Using co-immunoprecipitation, we confirmed that IGF1R and SNX6 physically interact. SNX6 knockdown resulted in a dramatic diminution of IGF1-mediated ERK1/2 phosphorylation, but did not affect IGF1R internalization. Bioluminescence resonance energy transfer experiments indicated that the SNX6 knockdown perturbed the association between IGF1R and the key adaptor proteins insulin receptor substrate 1 (IRS1) and SHC adaptor protein 1 (SHC1). Intriguingly, even in the absence of stimuli, SNX6 overexpression significantly increased Akt phosphorylation. Our study confirms the utility of proximity-labeling methods, such as BioID, to screen for interactors of cell-surface receptors and has uncovered a role of one of these interactors, SNX6, in the IGF1R signaling cascade.

Elevated expression of IRS-1 associates with phosphorylated Akt expression and predicts poor prognosis of breast invasive ductal carcinoma

Overexpression of insulin receptor substrate 1 (IRS-1) has been reported to promote cell growth, atypical hyperplasia, and carcinogenesis, and phosphorylated Akt (p-Akt) is certified to be involved in many types of cancers such as breast invasive ductal carcinoma (BIDC). However, the relationship between IRS-1 and Akt, as well as the role of expression of IRS-1 in BIDC, has never been reported. The purpose of this research is to investigate the association between expression of IRS-1 and p-Akt proteins and clinicopathological features of BIDC by immunohistochemistry, as well as the survival status. The results showed that the percentage of either elevated expression of IRS-1 or positive p-Akt expression in BIDC was significantly higher than that in control breast tissue from noncancer patients (P < .001 and P = .001, respectively). Overexpression of IRS-1 was evidently associated with positive expression of p-Akt (r = 0.337, P < .001). Also, positive percentage of p-Akt expression was statistically different among different molecular subtypes of BIDC (highest in luminal B BIDC, P = .009). Furthermore, significantly worse overall survival was found in BIDC patients with high expression of IRS-1 and p-Akt than in patients with low expression (P = .006 and P = .004, respectively). The multivariate Cox proportional hazard regression analysis showed that high expression of IRS-1 and positive expression of p-Akt protein were independent poor prognostic factors for patients with BIDC (P = .022 and P = .046, respectively). In conclusion, we report for the first time that overexpression of IRS-1 protein is associated with expression of p-Akt, and overexpression of IRS-1 and positive expression of p-Akt might be independent biomarkers for poor prognosis in BIDC.