Negative Regulator Of Insulin Signaling Research Articles

Enhanced insulin signaling in density-enhanced phosphatase-1 (DEP-1) knockout mice.

ObjectiveInsulin resistance can be triggered by enhanced dephosphorylation of the insulin receptor or downstream components in the insulin signaling cascade through protein tyrosine phosphatases (PTPs). Downregulating density-enhanced phosphatase-1 (DEP-1) resulted in an improved metabolic status in previous analyses. This phenotype was primarily caused by hepatic DEP-1 reduction.MethodsHere we further elucidated the role of DEP-1 in glucose homeostasis by employing a conventional knockout model to explore the specific contribution of DEP-1 in metabolic tissues. Ptprj−/− (DEP-1 deficient) and wild-type C57BL/6 mice were fed a low-fat or high-fat diet. Metabolic phenotyping was combined with analyses of phosphorylation patterns of insulin signaling components. Additionally, experiments with skeletal muscle cells and muscle tissue were performed to assess the role of DEP-1 for glucose uptake.ResultsHigh-fat diet fed-Ptprj−/− mice displayed enhanced insulin sensitivity and improved glucose tolerance. Furthermore, leptin levels and blood pressure were reduced in Ptprj−/− mice. DEP-1 deficiency resulted in increased phosphorylation of components of the insulin signaling cascade in liver, skeletal muscle and adipose tissue after insulin challenge. The beneficial effect on glucose homeostasis in vivo was corroborated by increased glucose uptake in skeletal muscle cells in which DEP-1 was downregulated, and in skeletal muscle of Ptprj−/− mice.ConclusionTogether, these data establish DEP-1 as novel negative regulator of insulin signaling.

Deletion of protein tyrosine phosphatase 1B rescues against myocardial anomalies in high fat diet-induced obesity: Role of AMPK-dependent autophagy

Obesity-induced cardiomyopathy may be mediated by alterations in multiple signaling cascades involved in glucose and lipid metabolism. Protein tyrosine phosphatase-1B (PTP1B) is an important negative regulator of insulin signaling. This study was designed to evaluate the role of PTP1B in high fat diet-induced cardiac contractile anomalies. Wild-type and PTP1B knockout mice were fed normal (10%) or high (45%) fat diet for 5months prior to evaluation of cardiac function. Myocardial function was assessed using echocardiography and an Ion-Optix MyoCam system. Western blot analysis was employed to evaluate levels of AMPK, mTOR, raptor, Beclin-1, p62 and LC3-II. RT-PCR technique was employed to assess genes involved in hypertrophy and lipid metabolism. Our data revealed increased LV thickness and LV chamber size as well as decreased fractional shortening following high fat diet intake, the effect was nullified by PTP1B knockout. High fat diet intake compromised cardiomyocyte contractile function as evidenced by decreased peak shortening, maximal velocity of shortening/relengthening, intracellular Ca2+ release as well as prolonged duration of relengthening and intracellular Ca2+ decay, the effects of which were alleviated by PTP1B knockout. High fat diet resulted in enlarged cardiomyocyte area and increased lipid accumulation, which were attenuated by PTP1B knockout. High fat diet intake dampened myocardial autophagy as evidenced by decreased LC3-II conversion and Beclin-1, increased p62 levels as well as decreased phosphorylation of AMPK and raptor, the effects of which were significantly alleviated by PTP1B knockout. Pharmacological inhibition of AMPK using compound C disengaged PTP1B knockout-conferred protection against fatty acid-induced cardiomyocyte contractile anomalies. Taken together, our results suggest that PTP1B knockout offers cardioprotection against high fat diet intake through activation of AMPK. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.

184 Temporospatial Effects of Endothelium Specific Inhibition of Shc Homology 2 -containing Inositol 5´ Phosphatase 2 on Nitric Oxide Bioavailability and Whole Body Insulin Sensitivity

IntroductionAgeing is an important risk factor for diabetes and cardiovascular disease. Integrity of the endothelium plays a critical role in cardiovascular pathophysiology. Insulin signalling in endothelial cells modulates the generation...

G protein-coupled receptor kinase 2: a link between myocardial contractile function and cardiac metabolism.

Heart failure (HF) causes a tremendous burden on the worldwide healthcare system, affecting >23 million people. There are many cardiovascular disorders that contribute to the development of HF and multiple risk factors that accelerate its occurrence, but regardless of its underlying cause, HF is characterized by a marked decrease in myocardial contractility and loss of pump function. One biomarker molecule consistently shown to be upregulated in human HF and several animal models is G protein-coupled receptor kinase-2 (GRK2), a kinase originally discovered to be involved in G protein-coupled receptor desensitization, especially β-adrenergic receptors. Higher levels of GRK2 can impair β-adrenergic receptor-mediated inotropic reserve and its inhibition, or molecular reduction has shown to improve pump function in several animal models including a preclinical pig model of HF. Recently, nonclassical roles for GRK2 in cardiovascular disease have been described, including negative regulation of insulin signaling, a role in myocyte cell survival and apoptotic signaling, and it has been shown to be localized in/on mitochondria. These new roles of GRK2 suggest that GRK2 may be a nodal link in the myocyte, influencing both cardiac contractile function and cell metabolism and survival and contributing to HF independent of its canonical role in G protein-coupled receptor desensitization. In this review, classical and nonclassical roles for GRK2 will be discussed, focusing on recently discovered roles for GRK2 in cardiomyocyte metabolism and the effects that these roles may have on myocardial contractile function and HF development.

A Central Role for GRB10 in Regulation of Islet Function in Man

Variants in the growth factor receptor-bound protein 10 (GRB10) gene were in a GWAS meta-analysis associated with reduced glucose-stimulated insulin secretion and increased risk of type 2 diabetes (T2D) if inherited from the father, but inexplicably reduced fasting glucose when inherited from the mother. GRB10 is a negative regulator of insulin signaling and imprinted in a parent-of-origin fashion in different tissues. GRB10 knock-down in human pancreatic islets showed reduced insulin and glucagon secretion, which together with changes in insulin sensitivity may explain the paradoxical reduction of glucose despite a decrease in insulin secretion. Together, these findings suggest that tissue-specific methylation and possibly imprinting of GRB10 can influence glucose metabolism and contribute to T2D pathogenesis. The data also emphasize the need in genetic studies to consider whether risk alleles are inherited from the mother or the father.

SHIP2: A “NEW” Insulin Pathway Target for Aging Research

Strong evidence suggests that systemic inflammation and central adiposity contribute to and perpetuate metabolic syndrome. All of these alterations predispose individuals to type 2 diabetes mellitus (T2DM), cardiovascular disease, as well as Alzheimer's disease (AD), all characterized by chronic inflammatory status. On the other hand, extensive abnormalities in insulin and insulin-like growth factor I (IGF-I) and IGF-II signaling mechanisms in brains with AD have been demonstrated, suggesting that AD could be a third form of diabetes. The Src homology domain-containing inositol 5-phosphatase 2 (SHIP2) has an important role in the insulin pathway because its over-expression causes impairment of insulin/IGF-1 signaling. Because some single-nucleotide polymorphisms (SNP) of the gene encoding SHIP2 were significantly associated in T2DM patients with metabolic syndrome and some related conditions, we decided to conduct a case-control study on this gene, analyzing AD and T2DM subjects as cases and young, old, and centenarians as controls. Our results suggest a putative correlation between the the rs144989913 SNP and aging, both successful and unsuccessful, rather than age-related diseases. Because this SNP is an insertion/deletion of 28 bp, it might cause an alteration in SHIP2 expression. It is noteworthy that SHIP2 has been demonstrated to be a potent negative regulator of insulin signaling and insulin sensitivity. Many studies demonstrated the association of the insulin/IGF1 pathway with aging and longevity, so it is tempting to speculate that the found association with SHIP2 and aging might depend on its effect on the insulin/IGF-1 pathway.

Insulin signaling regulates neurite growth during metamorphic neuronal remodeling

SummaryAlthough the growth capacity of mature neurons is often limited, some neurons can shift through largely unknown mechanisms from stable maintenance growth to dynamic, organizational growth (e.g. to repair injury, or during development transitions). During insect metamorphosis, many terminally differentiated larval neurons undergo extensive remodeling, involving elimination of larval neurites and outgrowth and elaboration of adult-specific projections. Here, we show in the fruit fly, Drosophila melanogaster (Meigen), that a metamorphosis-specific increase in insulin signaling promotes neuronal growth and axon branching after prolonged stability during the larval stages. FOXO, a negative effector in the insulin signaling pathway, blocked metamorphic growth of peptidergic neurons that secrete the neuropeptides CCAP and bursicon. RNA interference and CCAP/bursicon cell-targeted expression of dominant-negative constructs for other components of the insulin signaling pathway (InR, Pi3K92E, Akt1, S6K) also partially suppressed the growth of the CCAP/bursicon neuron somata and neurite arbor. In contrast, expression of wild-type or constitutively active forms of InR, Pi3K92E, Akt1, Rheb, and TOR, as well as RNA interference for negative regulators of insulin signaling (PTEN, FOXO), stimulated overgrowth. Interestingly, InR displayed little effect on larval CCAP/bursicon neuron growth, in contrast to its strong effects during metamorphosis. Manipulations of insulin signaling in many other peptidergic neurons revealed generalized growth stimulation during metamorphosis, but not during larval development. These findings reveal a fundamental shift in growth control mechanisms when mature, differentiated neurons enter a new phase of organizational growth. Moreover, they highlight strong evolutionarily conservation of insulin signaling in neuronal growth regulation.

PKCδ is activated in the liver of obese Zucker rats and mediates diet-induced whole body insulin resistance and hepatocyte cellular insulin resistance

Insulin resistance can arise when pathological levels of free fatty acids (FFAs) and proinflammatory cytokines disrupt insulin signaling. Protein kinase C delta (PKCδ) is a FFA- and a proinflammatory cytokine-regulated protein kinase that is associated with inhibition of insulin signaling and action. To gain insight into the role of PKCδ in insulin resistance, PKCδ activation was studied in a genetic model of obesity-linked insulin resistance. PKCδ was found to be activated in the liver of obese insulin-resistant Zucker rats and in isolated cultured hepatocytes. PKCδ was further studied in PKCδ-null mice and their wild-type littermates fed a high-fat or control diet for 10 weeks. PKCδ-null mice on a high-fat diet had improved insulin sensitivity and hepatic insulin signaling compared to wild-type littermates. Additionally, the deleterious effect of a high-fat diet on glucose tolerance in wild-type mice was completely blocked in PKCδ-null mice. To directly test the role of PKCδ in cellular insulin resistance, primary hepatocytes from the high-fat diet mice were isolated and stimulated with insulin. Primary hepatocytes from PKCδ-null mice had improved insulin-stimulated Akt and FOXO phosphorylation compared to hepatocytes from wild-type littermates. Consistent with this result, tumor necrosis factor alpha-mediated inhibition of insulin signaling was blocked in PKCδ knockdown primary hepatocytes. These results indicate that PKCδ plays a role in insulin resistance and is consistent with the hypothesis that PKCδ is a negative regulator of insulin signaling and thus may be a therapeutic target for the treatment of type 2 diabetes.

Protein tyrosine phosphatase 1B and insulin resistance: role of endoplasmic reticulum stress/reactive oxygen species/nuclear factor kappa B axis.

Obesity-induced endoplasmic reticulum (ER) stress has been proposed as an important pathway in the development of insulin resistance. Protein-tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signaling and is tethered to the ER-membrane. The aim of the study was to determine the mechanisms involved in the crosstalk between ER-stress and PTP1B. PTP1B whole body knockout and C57BL/6J mice were subjected to a high-fat or normal chow-diet for 20 weeks. High-fat diet feeding induced body weight gain, increased adiposity, systemic glucose intolerance, and hepatic steatosis were attenuated by PTP1B deletion. High-fat diet- fed PTP1B knockout mice also exhibited improved glucose uptake measured using [3H]-2-deoxy-glucose incorporation assay and Akt phosphorylation in the skeletal muscle tissue, compared to their wild-type control mice which received similar diet. High-fat diet-induced upregulation of glucose-regulated protein-78, phosphorylation of eukaryotic initiation factor 2α and c-Jun NH2-terminal kinase-2 were significantly attenuated in the PTP1B knockout mice. Mice lacking PTP1B showed decreased expression of the autophagy related protein p62 and the unfolded protein response adaptor protein NCK1 (non-catalytic region of tyrosine kinase). Treatment of C2C12 myotubes with the ER-stressor tunicamycin resulted in the accumulation of reactive oxygen species (ROS), leading to the activation of protein expression of PTP1B. Furthermore, tunicamycin-induced ROS production activated nuclear translocation of NFκB p65 and was required for ER stress-mediated expression of PTP1B. Our data suggest that PTP1B is induced by ER stress via the activation of the ROS-NFκB axis which is causes unfolded protein response and mediates insulin resistance in the skeletal muscle under obese condition.

Structural Basis for the Interaction of the Adaptor Protein Grb14 with Activated Ras

Grb14, a member of the Grb7-10-14 family of cytoplasmic adaptor proteins, is a tissue-specific negative regulator of insulin signaling. Grb7-10-14 contain several signaling modules, including a Ras-associating (RA) domain, a pleckstrin-homology (PH) domain, a family-specific BPS (between PH and SH2) region, and a C-terminal Src-homology-2 (SH2) domain. We showed previously that the RA and PH domains, along with the BPS region and SH2 domain, are necessary for downregulation of insulin signaling. Here, we report the crystal structure at 2.4-Å resolution of the Grb14 RA and PH domains in complex with GTP-loaded H-Ras (G12V). The structure reveals that the Grb14 RA and PH domains form an integrated structural unit capable of binding simultaneously to small GTPases and phosphoinositide lipids. The overall mode of binding of the Grb14 RA domain to activated H-Ras is similar to that of the RA domains of RalGDS and Raf1 but with important distinctions. The integrated RA-PH structural unit in Grb7-10-14 is also found in a second adaptor family that includes Rap1-interacting adaptor molecule (RIAM) and lamellipodin, proteins involved in actin-cytoskeleton rearrangement. The structure of Grb14 RA-PH in complex with H-Ras represents the first detailed molecular characterization of tandem RA-PH domains bound to a small GTPase and provides insights into the molecular basis for specificity.

Gene deletion of protein tyrosine phosphatase 1B limits cardiovascular dysfunction and mortality during endotoxinic shock

Sepsis-associated cardiovascular dysfunction is one of the main causes of mortality in critically ill patients. PTP1B is negative regulator of insulin signaling and endothelial Nitric Oxide (NO) production, two mechanisms likely involved in the pathogenesis of sepsis. We recently showed that gene deletion or pharmacological inhibition of PTP1B improves endothelial dysfunction and reduces the severity of heart failure. The aim of the present study was to assess the effect of PTP1B gene deletion on lipopolysaccharide (LPS)-induced cardiovascular dysfunction and mortality in mice. PTP1B-/- or wild-type (WT) mice received LPS (15mg/kg) or vehicle followed by subcutaneous saline resuscitation (30ml/kg). LPS significantly enhanced cardiac and arterial PTP1B mRNA expression. Compared with WT, PTP1B-/- mice displayed markedly reduced mortality (Survival 4 days after LPS: WT: 0%; PTP1B-/- 70%, p<0.01), associated with a higher locomotor activity. Evaluation of vascular function 8h after LPS in isolated mesenteric resistance arteries (arteriograph system) showed that PTP1B-/- LPS mice had improved contractile response to phenylephrine, and restored flow mediated, NO dependent-dilatation (% dilatation to 200μl/min: WT 6,3±3; PTP1B-/- 16,5±2, p<0.05) associated with significant increases in phosphorylation of Akt and eNOS, and reduced mRNA expression of VCAM-1, ICAM-1, COX-2, IL-1β, iNOS and Gp91phox (PCR). Evaluation of left ventricular (LV) function by echocardiography, and pressure-volume curves 8h after LPS revealed reduced cardiac dysfunction in PTP1B-/- mice as shown by an increased LV fractional shortening (WT 17,3±1; PTP1B-/- 24±1, p<0.05) and LV end systolic pressure-volume relation (WT 16±2; PTP1B-/- 20±2, p<0.05) together with reduced LV end diastolic pressure-volume relation (WT 4,7±1,7; PTP1B-/- 2.4±0.9 mmHg/RVU, p<0.05). Improved cardiac contractility was also observed ex-vivo in hearts isolated and perfused 8h after LPS (LV Developed Pressure: WT 25±5; PTP1B-/- 50±5 mmHg, p<0.05). Improved cardiac function was associated with significant decreases in myocardial markers of inflammation and oxidative stress (VCAM-1, ICAM-1, CD45, IL-1β, TNF-α and Gp91phox), reduced P38 and ERK1/2 phosphorylation and limited decrease of phospholamban phosphorylation (WT -42%; PTP1B-/- -22.8, p<0.05). Thus, we demonstrate for the first time that PTP1B gene deletion protects against lipopolysaccharide-induced cardiovascular dysfunction and mortality. These results suggest that PTP1B is an attractive target for the treatment of sepsis.

Inducible liver-specific knockdown of protein tyrosine phosphatase 1B improves glucose and lipid homeostasis in adult mice.

Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin signalling. Hepatic PTP1B deficiency, using the Alb-Cre promoter to drive Ptp1b deletion from birth in mice, improves glucose homeostasis, insulin sensitivity and lipid metabolism. The aim of this study was to investigate the therapeutic potential of decreasing liver PTP1B levels in obese and insulin-resistant adult mice. Inducible Ptp1b liver-specific knockout mice were generated using SA-Cre-ER(T2) mice crossed with Ptp1b floxed (Ptp1b(fl/fl)) mice. Mice were fed a high-fat diet (HFD) for 12 weeks to induce obesity and insulin resistance. Tamoxifen was administered in the HFD to induce liver-specific deletion of Ptp1b (SA-Ptp1b(-/-) mice). Body weight, glucose homeostasis, lipid homeostasis, serum adipokines, insulin signalling and endoplasmic reticulum (ER) stress were examined. Despite no significant change in body weight relative to HFD-fed Ptp1b(fl/fl) control mice, HFD-fed SA-Ptp1b(-/-) mice exhibited a reversal of glucose intolerance as determined by improved glucose and pyruvate tolerance tests, decreased fed and fasting blood glucose and insulin levels, lower HOMA of insulin resistance, circulating leptin, serum and liver triacylglycerols, serum NEFA and decreased HFD-induced ER stress. This was associated with decreased glycogen synthase, eukaryotic translation initiation factor-2α kinase 3, eukaryotic initiation factor 2α and c-Jun NH2-terminal kinase 2 phosphorylation, and decreased expression of Pepck. Inducible liver-specific PTP1B knockdown reverses glucose intolerance and improves lipid homeostasis in HFD-fed obese and insulin-resistant adult mice. This suggests that knockdown of liver PTP1B in individuals who are already obese/insulin resistant may have relatively rapid, beneficial therapeutic effects.

The Role of PTEN in Chronic Growth Hormone-Induced Hepatic Insulin Resistance

Chronic growth hormone (GH) therapy has been shown to cause insulin resistance, but the mechanism remains unknown. PTEN, a tumor suppressor gene, is a major negative regulator of insulin signaling. In this study, we explored the effect of chronic GH on insulin signaling in the context of PTEN function. Balb/c healthy mice were given recombinant human or bovine GH intraperitoneally for 3 weeks. We found that phosphorylation of Akt was significantly decreased in chronic GH group and the expression of PTEN was significantly increased. We further examined this effect in the streptozotocin-induced Type I diabetic mouse model, in which endogenous insulin secretion was disrupted. Insulin/PI3K/Akt signaling was impaired. However, different from the observation in healthy mice, the expression of PTEN did not increase. Similarly, PTEN expression did not significantly increase in chronic GH-treated mice with hypoinsulinemia induced by prolonged fasting. We conducted in-vitro experiments in HepG2 cells to validate our in-vivo findings. Long-term exposure to GH caused similar resistance of insulin/PI3K/Akt signaling in HepG2 cells; and over-expression of PTEN enhanced the impairment of insulin signaling. On the other hand, disabling the PTEN gene by transfecting the mutant PTEN construct C124S or siPTEN, disrupted the chronic GH induced insulin resistance. Our data demonstrate that PTEN plays an important role in chronic-GH-induced insulin resistance. These findings may have implication in other pathological insulin resistance.

241 ENDOTHELIAL SHIP2 CONFERS AGE-DEPENDENT CONTRASTING AFFECTS ON WHOLE BODY GLUCOSE HOMEOSTASIS AND VASCULAR FUNCTION

<h3>Introduction</h3> Aging is an important risk factor for diabetes and cardiovascular disease and integrity of the endothelium plays a critical role in cardiovascular pathophysiology. Although the vascular implications of endothelial insulin resistance are well understood, the effect of <i>enhanced</i> endothelial insulin signaling on whole body glucose regulation and vascular function remains poorly characterized. We therefore generated mice with endothelial-specific downregulation of the lipid phosphatase SHIP2 (a negative regulator of insulin signaling) to investigate whether enhanced insulin signaling in the endothelium modulates vascular function and whole body glucose regulation. <h3>Methods</h3> Exons 18–19 of the ship 2 gene were deleted using Cre-Lox technology under control of the Tie2 promoter generating a catalytically inactivate protein. Male heterozygotes for the inactive protein (EC-SHIP2^+/−) were compared to sex-matched littermate controls. <h3>Results</h3> EC-SHIP^+/− mice were morphologically indistinguishable from controls, exhibiting normal development. At 8 weeks of age EC-SHIP2^+/− mice displayed increased glucose tolerance after glucose challenge (P=0.03) and improved insulin sensitivity (P=0.02) after insulin challenge compared to controls. Surprisingly however, by 40 weeks of age this phenotype was reversed; EC-SHIP2^+/− mice revealed significant insulin resistance 60 min after insulin challenge (P=&lt;0.05). This phenotype was confirmed by euglycemic hyperinsulinemic clamping showing whole body insulin resistance in EC-SHIP^+/− mice (decreased glucose infusion rate of 26% P&lt;0.05). In young mice <i>ex vivo</i> aortic vasomotor studies in both controls and EC-SHIP^+/− revealed similar contractile responses to phenylephrine and displayed decreased contraction after insulin incubation (Emax 0.88±0.05g vs 0.62±0.05g P=0.002 and 0.83±0.05g vs 0.69±0.05g P=0.025 respectively). Both groups displayed increased contraction after NO synthase inhibitor LNMMA incubation (Emax 0.88±0.05g vs 1.31±0.11g P=0.01 and 0.83±0.05g vs 1.28±0.02g P=&lt;0.0001 respectively). However, at 40 weeks old in EC-SHIP2^+/− mice the vasodilatory aortic ring response to insulin was abolished (Emax controls 0.59±0.04g vs 0.47±0.03g P=0.04, EC-SHIP2^+/− 0.64±0.04g vs 0.63±0.06g P=0.9) and EC-SHIP2^+/− displayed no increase in contraction to LNMMA incubation (Emax controls 0.59±0.04g vs 0.82±0.08g P=0.02, EC-SHIP2^+/− 0.64±0.04g vs 0.69±0.07g P=0.5) indicating insulin resistance and lower basal NO production, suggesting the change in glucose homeostasis may be mediated by changes in NO bioavailability. <h3>Conclusion</h3> Endothelial functional downregulation of SHIP2 augments whole body glucose disposal in young mice but attenuates whole body glucose disposal in older mice. Although further studies are required to elucidate the molecular mechanisms our data suggest a previously unrecognised age dependent role for the vascular endothelium in whole body glucose regulation.

Abstract 4312: Grb10, a negative regulator of insulin signaling, is a tumor suppressor in prostate cancer.

Abstract Prostate cancer (PCa) is manageable in its initial stages and when localized, but as it progresses it metastasizes and also becomes resistant to conventional chemotherapies. Identifying mechanisms that are related to the rapid progression of the disease could result in development of appropriate intervention strategies. We analyzed protein and mRNA expression of Grb10 (also known as insulin receptor-binding protein) in various PCa cell lines and found a significant decrease when compared to normal prostate epithelial cells. We also analyzed the expression pattern of Grb10 in a human PCa tissue array and found significant loss of Grb10 mRNA in more than 90% of the samples. Based on Gleason Score, the loss of Grb10 mRNA correlated with the stage and grade of the disease. These initial observations led us to hypothesize that Grb10 may act as a tumor suppressor whose expression is lost during the progression of human PCa. Using immuno-histochemical analysis of human PCa tissue samples we found loss of Grb10 protein with increasing tumor grade. We next analyzed various components of insulin signaling, based on the evidence that Grb10 expression is negatively correlated with insulin signaling. We observed significant increase at both mRNA and protein levels of IR, IGF-1R, IRS-1 and IRS-2 in PCa cells and tissues that directly correlated with loss of Grb10. Recently, Grb10 expression was shown to be negatively correlated with PTEN levels in different cancer types including PCa. We determined the expression pattern of Grb10 mRNA in prostate tissue samples from 15-weeks old PTEN wild type (WT) and PTEN knockout (KO) mice and observed that Grb10 levels were significantly down-regulated in PTEN-KO mice suggesting that loss of PTEN results in loss of Grb10. Further analysis suggested increased insulin signaling in PTEN-KO mice compared to PTEN-WT mice confirming negative regulation of Grb10 with insulin signaling. To further understand the role of Grb10 in PCa, we overexpressed Grb10 in human PCa DU145 (PTEN-mutated) cells. Overexpression of Grb10 resulted in decrease in colony formation, migration and invasion that was associated with down-regulation of insulin signaling. In summary, our findings suggest that Grb10 is a negative regulator of insulin signaling whose expression is lost in PCa in humans and correlates well with disease progression and aggressiveness. We conclude that Grb10 is a tumor suppressor in human PCa whose loss promotes rapid progression of the disease. Identification of this novel role of Grb10 could open up new strategies for the management of human PCa. Citation Format: Mohammad Imran Khan, Vaqar M. Adhami, Rahul Kumar Lall, Deeba Nadeem Syed, Imtiaz Ahmad Siddiqui, Bilal Bin Hafeez, Ajit Kumar Verma, Hasan Mukhtar. Grb10, a negative regulator of insulin signaling, is a tumor suppressor in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4312. doi:10.1158/1538-7445.AM2013-4312

JNK1 phosphorylation of HNF4α represses miR‐122, which causes PTP1B induction

MicroRNAs have emerged as key regulators of energy metabolism. The miR‐122 is the most abundant microRNA in the liver, and may affect cholesterol metabolism. Nevertheless, the role of miR‐122 in insulin sensitivity and blood glucose control had not been studied. This study investigated whether dysregulation of miR‐122 contributes to hepatic insulin resistance using animal and cell models. The levels of miR‐122 were down‐regulated in the liver of high‐fat diet (HFD)‐fed mice. miR‐122 was also repressed in hepatocytes treated with either TNFα or palmitate. We identified protein tyrosine phosphatase 1B (PTP1B), a key negative regulator of insulin signaling, as a novel target of miR‐122; decrease of miR‐122 caused PTP1B induction, as indicated by the results of miR‐122 mimic and its inhibitor experiments. JNK1 facilitated serine/threonine phosphorylation of HNF4α (i.e., HNF4α inhibition), which was responsible for miR‐122 down‐regulation in hepatocytes. Consistently, functional inhibition of JNK by licorice flavonoid treatment caused not only the restoration of miR‐122 levels, but also the repression of PTP1B. Moreover, JNK1 inhibition enabled cells to restore tyrosine‐phosphorylation of IR or IRS1/2, lowering blood glucose contents in HFD‐fed or ob/ob mice. In conclusion, miR‐122 dysregulation may increase insulin resistance through PTP1B induction, which depends on HNF4α phosphorylation by JNK1.

Modulation of cellular insulin signaling and PTP1B effects by lipid metabolites in skeletal muscle cells

Normal glucose regulation is achieved by having adequate insulin secretion and effective glucose uptake/disposal. Excess lipids in peripheral tissues — skeletal muscle, liver and adipose tissue — may attenuate insulin signaling through the protein kinase B (AKt) pathway and up-regulate protein tyrosine phosphatase 1B (PTP1B), a negative regulator of insulin signaling. We studied accumulation of lipid metabolites [triglycerides (TAGs), diglycerides (DAGs)] and ceramides in relation to insulin signaling and expression and phosphorylation of PTP1B by preincubating rat skeletal muscle cells (L6 myotubes) with three saturated and three unsaturated free fatty acids (FFAs) (200 μM). Cells were also evaluated in the presence of wortmannin, an inhibitor of phosphatidylinositol 3-kinases and thus AKt (0–100 nM). Unsaturated FFAs increased DAGs, TAGs and PTP1B expression significantly, but cells remained insulin sensitive as assessed by robust AKt and PTP1B phosphorylation at serine (Ser) 50, Ser 398 and tyrosine 152. Saturated palmitic and stearic acids increased ceramides, up-regulated PTP1B, and had AKt and PTP1B phosphorylation at Ser 50 impaired. We show a significant correlation between phosphorylation levels of AKt and of PTP1B at Ser 50 (R2=0.84, P<.05). The same was observed with increasing wortmannin dose (R2=0.73, P<.05). Only FFAs that increased ceramides caused impairment of AKt and PTP1B phosphorylation at Ser 50. PTP1B overexpression in the presence of excess lipids may not directly cause insulin resistance unless it is accompanied by decreased PTP1B phosphorylation. A clear relationship between PTP1B phosphorylation levels at Ser 50 and its negative effect on insulin signaling is shown.

The insulin-mimetic effect of Morin: A promising molecule in diabetes treatment

BackgroundType-2 diabetes is a worldwidely diffuse disease characterized by insulin resistance that arises from alterations of receptor and/or post-receptor events of insulin signalling. Studies performed with PTP1B-deficent mice demonstrated that PTP1B is the main negative regulator of insulin signalling. Inhibition or down regulation of this enzyme causes enhanced insulin sensitivity. Hence this enzyme represents the most attractive target for development of innovative anti-diabetic drugs. MethodsSelection of new PTP1B inhibitors among an in house library of polyphenolic compounds was carried out screening their activity. The inhibition mechanism of Morin was determined by kinetic analyses. The cellular action of Morin was assayed on HepG2 cells. Analyses of the insulin signalling pathways was carried out by Western blot methods, glycogen synthesis was estimated by measuring the incorporation of [3H]-glucose, gluconeogenesis rate was assayed by measuring the glucose release in the cell medium. Cell growth was estimated by cell count. Docking analysis was conducted with SwissDock program. ResultsWe demonstrated that Morin: i) is a non-competitive inhibitor of PTP1B displaying a Ki in the μM range; ii) increases the phosphorylation of the insulin receptor and Akt; iii) inhibits gluconeogenesis and enhances glycogen synthesis. Morin does not enhance cell growth. ConclusionsWe have identified Morin as a new small molecular non-competitive inhibitor of PTP1B, which behaves as an activator and sensitizer of the insulin receptor stimulating the metabolic pathways only. General significanceOur study suggests that Morin is a useful lead for development of new low Mr compounds potentially active as antidiabetic drugs.

The Novel Angiotensin II Receptor Blocker Azilsartan Medoxomil Ameliorates Insulin Resistance Induced by Chronic Angiotensin II Treatment in Rat Skeletal Muscle

Angiotensin receptor (type 1) blockers (ARBs) can reduce both hypertension and insulin resistance induced by local and systemic activation of the renin-angiotensin-aldosterone system. The effectiveness of azilsartan medoxomil (AZIL-M), a novel imidazole-based ARB, to facilitate metabolic improvements in conditions of angiotensin II (Ang II)-associated insulin resistance is currently unknown. The aim of this study was to determine the impact of chronic AZIL-M treatment on glucose transport activity and key insulin signaling elements in red skeletal muscle of Ang II-treated rats. Male Sprague-Dawley rats were treated for 8 weeks with or without Ang II (200 ng/kg/min) combined with either vehicle or AZIL-M (1 mg/kg/day). Ang II induced significant (p < 0.05) increases in blood pressure, which were completely prevented by AZIL-M. Furthermore, Ang II reduced insulin-mediated glucose transport activity in incubated soleus muscle, and AZIL-M co-treatment increased this parameter. Moreover, AZIL-M treatment of Ang II-infused animals increased the absolute phosphorylation of insulin signaling molecules, including Akt [both Ser⁴⁷³ (81%) and Thr³⁰⁸ (23%)] and AS160 Thr⁶⁴² (42%), in red gastrocnemius muscle frozen in situ. Absolute AMPKα (Thr¹⁷²) phosphorylation increased (98%) by AZIL-M treatment, and relative Thr³⁸⁹ phosphorylation of p70 S6K1, a negative regulator of insulin signaling, decreased (51%) with AZIL-M treatment. These results indicate that ARB AZIL-M improves the in vitro insulin action on glucose transport in red soleus muscle and the functionality of the Akt/AS160 axis in red gastrocnemius muscle in situ in Ang II-induced insulin-resistant rats, with the latter modification possibly associated with enhanced AMPKα and suppressed p70 S6K1 activation.

A secreted decoy of InR antagonizes insulin/IGF signaling to restrict body growth in Drosophila

Members of the insulin peptide family have conserved roles in the regulation of growth and metabolism in a wide variety of metazoans. Drosophila insulin-like peptides (Dilps) promote tissue growth through the single insulin-like receptor (InR). Despite the important role of Dilps in nutrient-dependent growth control, the molecular mechanism that regulates the activity of circulating Dilps is not well understood. Here, we report the function of a novel secreted decoy of InR (SDR) as a negative regulator of insulin signaling. SDR is predominantly expressed in glia and is secreted into the hemolymph. Larvae lacking SDR grow at a faster rate, thereby increasing adult body size. Conversely, overexpression of SDR reduces body growth non-cell-autonomously. SDR is structurally similar to the extracellular domain of InR and interacts with several Dilps in vitro independent of Imp-L2, the ortholog of the mammalian insulin-like growth factor-binding protein 7 (IGFBP7). We further demonstrate that SDR is constantly secreted into the hemolymph independent of nutritional status and is essential for adjusting insulin signaling under adverse food conditions. We propose that Drosophila uses a secreted decoy to fine-tune systemic growth against fluctuations of circulating insulin levels.