Classical Insulin Target Tissues Research Articles

β-Cell Insulin Resistance Plays a Causal Role in Fat-Induced β-Cell Dysfunction In Vitro and In Vivo.

In the classical insulin target tissues of liver, muscle, and adipose tissue, chronically elevated levels of free fatty acids (FFA) impair insulin signaling. Insulin signaling molecules are also present in β-cells where they play a role in β-cell function. Therefore, inhibition of the insulin/insulin-like growth factor 1 pathway may be involved in fat-induced β-cell dysfunction. To address the role of β-cell insulin resistance in FFA-induced β-cell dysfunction we co-infused bisperoxovanadate (BPV) with oleate or olive oil for 48 hours in rats. BPV, a tyrosine phosphatase inhibitor, acts as an insulin mimetic and is devoid of any antioxidant effect that could prevent β-cell dysfunction, unlike most insulin sensitizers. Following fat infusion, rats either underwent hyperglycemic clamps for assessment of β-cell function in vivo or islets were isolated for ex vivo assessment of glucose-stimulated insulin secretion (GSIS). We also incubated islets with oleate or palmitate and BPV for in vitro assessment of GSIS and Akt (protein kinase B) phosphorylation. Next, mice with β-cell specific deletion of PTEN (phosphatase and tensin homolog; negative regulator of insulin signaling) and littermate controls were infused with oleate for 48 hours, followed by hyperglycemic clamps or ex vivo evaluation of GSIS. In rat experiments, BPV protected against fat-induced impairment of β-cell function in vivo, ex vivo, and in vitro. In mice, β-cell specific deletion of PTEN protected against oleate-induced β-cell dysfunction in vivo and ex vivo. These data support the hypothesis that β-cell insulin resistance plays a causal role in FFA-induced β-cell dysfunction.

Acute and chronic metabolic effects of carvedilol in high-fructose, high-fat diet-fed mice: implication of β-arrestin2 pathway.

We aimed to investigate the acute and chronic effects of carvedilol on insulin resistance in high-fructose, high-fat diet (HFrHFD) - fed mice and the implication of the β-arrestin2 pathway. The acute effect of carvedilol (10mg/kg, i.p.) on glucose tolerance and hepatic lipid signaling in normal and insulin resistant mice was investigated. Then, the chronic effect of carvedilol on insulin resistance and dyslipidemia in HFrHFD-fed mice was examined. Changes in β-arrestin2 and its downstream signals in liver, skeletal muscle, and adipose tissue were measured. This involved measuring phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG) levels and protein kinase B (AKT) activity. Carvedilol acutely reduced fasting blood glucose levels in both normal and insulin resistant mice without significantly affecting the glucose tolerance. These acute effects were associated with increased hepatic PIP2 but decreased hepatic DAG levels. Chronic administration of carvedilol significantly ameliorated insulin resistance and dyslipidemia in HFrHFD-fed mice. These chronic effects were associated with increased β-arrestin2, PIP2, and AKT activity levels but decreased DAG levels in the classical insulin target tissues. In conclusion, carvedilol acutely maintains glucose homeostasis and chronically ameliorates insulin resistance and dyslipidemia in HFrHFD-fed mice. The insulin sensitizing effects of carvedilol are highly correlated with the upregulation of β-arrestin2 pathway.

Pioglitazone Enhances β-Arrestin2 Signaling and Ameliorates Insulin Resistance in Classical Insulin Target Tissues

Introduction: Pioglitazone is a thiazolidinedione oral antidiabetic agent. This study aimed to investigate the effects of pioglitazone as insulin sensitizer on β-arrestin2 signaling in classical insulin target tissues. Methods: Experiments involved three groups of mice; the first one involved mice fed standard chow diet for 16 weeks; the second one involved mice fed high-fructose, high-fat diet (HFrHFD) for 16 weeks; and the third one involved mice fed HFrHFD for 16 weeks and received pioglitazone (30 mg/kg/day, orally) in the last four weeks of feeding HFrHFD. Results: The results showed significant improvement in the insulin sensitivity of pioglitazone-treated mice as manifested by significant reduction in the insulin resistance index. This improvement in insulin sensitivity was associated with significant increases in the β-arrestin2 levels in the adipose tissue, liver, and skeletal muscle. Moreover, pioglitazone significantly increased β-arrestin2 signaling in all the examined tissues as estimated from significant increases in phosphatidylinositol 4,5 bisphosphate and phosphorylation of Akt at serine 473 and significant decrease in diacylglycerol level. Conclusion: To the best of our knowledge, our work reports a new mechanism of action for pioglitazone through which it can enhance the insulin sensitivity. Pioglitazone increases β-arrestin2 signaling in the adipose tissue, liver, and skeletal muscle of HFrHFD-fed mice.

Reproductive effects of irisin: Initial in vitro studies

The recently discovered myo- and adipokine irisin affects insulin sensitivity in classical insulin target tissues (adipose tissue, skeletal muscle and liver), but the reproductive effects of this hormone, if any, remain largely unexplored. We hypothesized that irisin may have effects on the hypothalamic-pituitary-gonadal axis. To test this hypothesis, we used murine pituitary mPit12 and human ovarian granulosa cells. GnRH treatment resulted in significant (up to 2.5-fold, p<0.0005) and dose-dependent stimulation of LH production by the mPit12 cells. Treating these cells with irisin alone showed a significant stimulatory effect on LH synthesis only at irisin concentration of 100ng/ml. When used together with GnRH, irisin abolished the stimulatory effect of GnRH on LH production. Human ovarian granulosa cells were treated with insulin, irisin or a combination of both and the estradiol (E2) production was measured. Both insulin or irisin stimulated granulosa cell E2 production (1.4-fold, p<0.05 and 2.5-fold, p=0.0002, respectively), but when insulin and irisin were used in combination, this stimulatory effect on E2 production was abolished. We conclude that irisin may have reproductive axis effects in the pituitary and in the ovary. Further studies are needed to confirm these initial observations and to explore the mechanisms of irisin effects in the reproductive system.

A Central Role for H2O2 in Insulin Signal Transduction in Cardiac Myocytes

Molecular mechanisms of insulin action have been extensively characterized in the classic insulin target tissues: fat, liver, and skeletal muscle. But in cardiac myocytes (CM), understanding of insulin‐modulated signaling pathways has lagged far behind. Early studies of insulin action implicated a physiological role for H2O2, but the relationships between insulin and H2O2 in CM signal transduction are almost entirely unexplored. In these studies, we identified a role for H2O2 in insulin signaling pathways in adult mouse CM. Insulin promoted the rapid (maximal at 5 min) robust (10‐ to 18‐fold) and reversible phosphorylation of insulin receptorβTyr1146/1150; IRS‐1 Tyr895; and Akt Ser473/308; these responses all had an EC50 of 1 nM insulin (n=3‐7). Insulin‐dependent phosphorylation of IRS‐1 Ser636/639, mTOR Ser2448, p70S6 Thr389/421, and S6 Ser235/Ser236 showed a slower time course (maximal at 30 min), again with an EC50 of 1 nM insulin and with robust phosphorylation responses (6‐ to 40‐fold increases from baseline; n=3‐7). All of these phosphorylation responses were blocked by the NADPH oxidase inhibitors apocynin and VAS2870, implicating NADPH oxidase‐derived reactive oxygen species in modulating insulin‐dependent phosphorylation responses. Importantly, the cell‐permeant derivatized enzyme PEG‐catalase (which degrades H2O2) attenuated all these insulin‐dependent phosphorylation responses. Our findings implicate NADPH oxidase‐generated H2O2 as a critical determinant of insulin‐dependent signaling responses in CM, and may have important implications for the abnormalities in cardiac function seen in disease states associated with oxidative stress. Research support: NIH and AHA.

The role of mouse Akt2 in insulin-dependent suppression of adipocyte lipolysis in vivo.

The release of fatty acids from adipocytes, i.e. lipolysis, is maintained under tight control, primarily by the opposing actions of catecholamines and insulin. A widely accepted model is that insulin antagonises catecholamine-dependent lipolysis through phosphorylation and activation of cAMP phosphodiesterase 3B (PDE3B) by the serine-threonine protein kinase Akt (protein kinase B). Recently, this hypothesis has been challenged, as in cultured adipocytes insulin appears, under some conditions, to suppress lipolysis independently of Akt. To address the requirement for Akt2, the predominant isoform expressed in classic insulin target tissues, in the suppression of fatty acid release in vivo, we assessed lipolysis in mice lacking Akt2. In the fed state and following an oral glucose challenge, Akt2 null mice were glucose intolerant and hyperinsulinaemic, but nonetheless exhibited normal serum NEFA and glycerol levels, suggestive of normal suppression of lipolysis. Furthermore, insulin partially inhibited lipolysis in Akt2 null mice during an insulin tolerance test (ITT) and hyperinsulinaemic-euglycaemic clamp, respectively. In support of these in vivo observations, insulin antagonised catecholamine-induced lipolysis in primary brown fat adipocytes from Akt2-deficient mice. These data suggest that suppression of lipolysis by insulin in hyperinsulinaemic states can take place in the absence of Akt2.

Endocrine role of bone: recent and emerging perspectives beyond osteocalcin.

Recent developments in endocrinology, made possible by the combination of mouse genetics, integrative physiology and clinical observations have resulted in rapid and unanticipated advances in the field of skeletal biology. Indeed, the skeleton, classically viewed as a structural scaffold necessary for mobility, and regulator of calcium-phosphorus homoeostasis and maintenance of the haematopoietic niche has now been identified as an important regulator of male fertility and whole-body glucose metabolism, in addition to the classical insulin target tissues. These seminal findings confirm bone to be a true endocrine organ. This review is intended to detail the key events commencing from the elucidation of osteocalcin (OC) in bone metabolism to identification of new and emerging candidates that may regulate energy metabolism independently of OC.

Nucleobindin-2 is a positive regulator for insulin-stimulated glucose transporter 4 translocation in fenofibrate treated E11 podocytes.

The physiology of insulin signaling under normal and disease conditions is well studied in classical insulin target tissues, but not in podocytes. To examine insulin stimulation of podocyte GLUT4 translocation, we established a protocol involving treatment with the PPARα agonist fenofibrate to induce E11 podocyte differentiation within 48 hours rather than 7-10 days, which is required for differentiation under the reported protocol. This allowed us to transiently introduce GLUT4 reporter cDNA and RNAi and thereby to examine the regulatory pathway involved. Here we demonstrate that treatment with 200 μM fenofibrate for 36 hours following transfection had a dramatic effect on podocyte morphology, induced several podocyte specific protein expression markers (G protein-coupled receptor 137B, chloride intracellular channel 5, and nephrin) and resulted in insulin-stimulated GLUT4 translocation. In addition, Nucleobindin-2 was found to constitutively associate with Septin 7 (the repressor of GLUT4 translocation), and knockdown of Nucleobindin-2 was found to completely abrogate insulin-stimulated GLUT4 translocation. Together, these data suggest that Nucleobindin-2 may repress Septin7-induced inhibition of insulin-stimulated GLUT4 translocation in podocytes.

Insulin Resistance and the Polycystic Ovary Syndrome Revisited: An Update on Mechanisms and Implications

Polycystic ovary syndrome (PCOS) is now recognized as an important metabolic as well as reproductive disorder conferring substantially increased risk for type 2 diabetes. Affected women have marked insulin resistance, independent of obesity. This article summarizes the state of the science since we last reviewed the field in the Endocrine Reviews in 1997. There is general agreement that obese women with PCOS are insulin resistant, but some groups of lean affected women may have normal insulin sensitivity. There is a post-binding defect in receptor signaling likely due to increased receptor and insulin receptor substrate-1 serine phosphorylation that selectively affects metabolic but not mitogenic pathways in classic insulin target tissues and in the ovary. Constitutive activation of serine kinases in the MAPK-ERK pathway may contribute to resistance to insulin's metabolic actions in skeletal muscle. Insulin functions as a co-gonadotropin through its cognate receptor to modulate ovarian steroidogenesis. Genetic disruption of insulin signaling in the brain has indicated that this pathway is important for ovulation and body weight regulation. These insights have been directly translated into a novel therapy for PCOS with insulin-sensitizing drugs. Furthermore, androgens contribute to insulin resistance in PCOS. PCOS may also have developmental origins due to androgen exposure at critical periods or to intrauterine growth restriction. PCOS is a complex genetic disease, and first-degree relatives have reproductive and metabolic phenotypes. Several PCOS genetic susceptibility loci have been mapped and replicated. Some of the same susceptibility genes contribute to disease risk in Chinese and European PCOS populations, suggesting that PCOS is an ancient trait.

A Second Chance for a PPARγ Targeted Therapy?

### Antidiabetic Actions of a Non-Agonist PPARγ Ligand Blocking Cdk5-Mediated Phosphorylation Choi et al Nature . 2011;477:477–481. A new class of non-agonist ligands target the transcription factor PPARγ and promote expression of insulin-sensitizing adipokines. They have potent antidiabetic actions, yet they lack several of the adverse effects commonly associated with thiazolidinediones. The ligands may represent a new class of anti-diabetes medications that preserve the most beneficial effects of PPARγ activation without imparting major side effects, which have limited the clinical usefulness of thiazolidinediones . Once thought to be a magic bullet for the treatment of type 2 diabetes, thiazolidinedione (TZD) drugs such as rosiglitazone and pioglitazone are now recognized to have adverse effects, which have limited their clinical usefulness. TZDs were originally thought to improve insulin sensitivity by robustly activating the transcriptional activity of the ligand-activated nuclear receptor PPARγ. A study by the Spiegelman laboratory published in the July 22, 2010 issue of Nature revealed that rosiglitazone also inhibits the obesity-induced phosphorylation of PPARγ by the cyclin-dependent kinase 5 (Cdk5).1 The inhibition of CDK5-mediated phosphorylation of PPARγ could also be mediated by PPARγ ligands, which lack full agonist activity suggesting a novel action of TZDs on PPARγ. Now, in the September 22, 2011 issue of Nature , the same research team reports that a new class of PPARγ ligands, which do not act as classical agonists as they lack robust transcriptional activation, effectively block Cdk5-mediated phosphorylation of PPARγ.2 Importantly, these compounds have potent antidiabetic actions, but they do not cause fluid retention nor inhibit bone formation, major adverse effects of TZDs. The study provides an important proof-of-principle that new non-TZD drugs targeting PPARγ could be designed that preserve its most beneficial actions while eliminating its most detrimental side effects. PPARγ is the target of the TZD class of antidiabetic agents, which improves glycemic control by increasing sensitivity to insulin. An ideal type 2 diabetes drug …

Insulin stimulation of PKCδ triggers its rapid degradation via the ubiquitin-proteasome pathway

Insulin rapidly upregulates protein levels of PKCδ in classical insulin target tissues skeletal muscle and liver. Insulin induces both a rapid increase in de novo synthesis of PKCδ protein. In this study we examined the possibility that insulin may also inhibit degradation of PKCδ. Experiments were performed on L6 skeletal muscle myoblasts or myotubes in culture. Phorbol ester (PMA)- and insulin-induced degradation of PKCδ were abrogated by proteasome inhibition. Both PMA and insulin induced ubiquitination of PKCδ, but not of that PKCα or PKCε and increased proteasome activity within 5 min. We examined the role of tyrosine phosphorylation of PKCδ in targeting PKCδ for degradation by the ubiquitin-proteasome pathway. Transfection of cells with PKCδY 311F, which is not phosphorylated, resulted in abolition of insulin-induced ubiquitination of PKCδ and increase in proteasome activity. We conclude that insulin induces degradation of PKCδ via the ubiquitin-proteasome system, and that this effect requires phosphorylation on specific tyrosine residues for targeting PKCδ for degradation by the ubiquitin-proteasome pathway. These studies provide additional evidence for unique effects of insulin on regulation of PKCδ protein levels.

Role of Mitochondrial Dysfunction in Insulin Resistance

Insulin resistance is characteristic of obesity, type 2 diabetes, and components of the cardiometabolic syndrome, including hypertension and dyslipidemia, that collectively contribute to a substantial risk for cardiovascular disease. Metabolic actions of insulin in classic insulin target tissues (eg, skeletal muscle, fat, and liver), as well as actions in nonclassic targets (eg, cardiovascular tissue), help to explain why insulin resistance and metabolic dysregulation are central in the pathogenesis of the cardiometabolic syndrome and cardiovascular disease. Glucose and lipid metabolism are largely dependent on mitochondria to generate energy in cells. Thereby, when nutrient oxidation is inefficient, the ratio of ATP production/oxygen consumption is low, leading to an increased production of superoxide anions. Reactive oxygen species formation may have maladaptive consequences that increase the rate of mutagenesis and stimulate proinflammatory processes. In addition to reactive oxygen species formation, genetic factors, aging, and reduced mitochondrial biogenesis all contribute to mitochondrial dysfunction. These factors also contribute to insulin resistance in classic and nonclassic insulin target tissues. Insulin resistance emanating from mitochondrial dysfunction may contribute to metabolic and cardiovascular abnormalities and subsequent increases in cardiovascular disease. Furthermore, interventions that improve mitochondrial function also improve insulin resistance. Collectively, these observations suggest that mitochondrial dysfunction may be a central cause of insulin resistance and associated complications. In this review, we discuss mechanisms of mitochondrial dysfunction related to the pathophysiology of insulin resistance in classic insulin-responsive tissue, as well as cardiovascular tissue.

Cardiovascular Actions of Insulin

Insulin has important vascular actions to stimulate production of nitric oxide from endothelium. This leads to capillary recruitment, vasodilation, increased blood flow, and subsequent augmentation of glucose disposal in classical insulin target tissues (e.g., skeletal muscle). Phosphatidylinositol 3-kinase-dependent insulin-signaling pathways regulating endothelial production of nitric oxide share striking parallels with metabolic insulin-signaling pathways. Distinct MAPK-dependent insulin-signaling pathways (largely unrelated to metabolic actions of insulin) regulate secretion of the vasoconstrictor endothelin-1 from endothelium. These and other cardiovascular actions of insulin contribute to coupling metabolic and hemodynamic homeostasis under healthy conditions. Cardiovascular diseases are the leading cause of morbidity and mortality in insulin-resistant individuals. Insulin resistance is typically defined as decreased sensitivity and/or responsiveness to metabolic actions of insulin. This cardinal feature of diabetes, obesity, and dyslipidemia is also a prominent component of hypertension, coronary heart disease, and atherosclerosis that are all characterized by endothelial dysfunction. Conversely, endothelial dysfunction is often present in metabolic diseases. Insulin resistance is characterized by pathway-specific impairment in phosphatidylinositol 3-kinase-dependent signaling that in vascular endothelium contributes to a reciprocal relationship between insulin resistance and endothelial dysfunction. The clinical relevance of this coupling is highlighted by the findings that specific therapeutic interventions targeting insulin resistance often also ameliorate endothelial dysfunction (and vice versa). In this review, we discuss molecular mechanisms underlying cardiovascular actions of insulin, the reciprocal relationships between insulin resistance and endothelial dysfunction, and implications for developing beneficial therapeutic strategies that simultaneously target metabolic and cardiovascular diseases.

Hyperglycemia and insulin resistance: possible mechanisms.

Sustained hyperglycemia impairs insulin-stimulated glucose utilization and glycogen synthesis in human and rat skeletal muscles, a phenomenon referred to clinically as glucose toxicity. In rat extensor digitorum longus (EDL) muscle preparations preincubated for 2-4 h in a hyperglycemic medium (25 mM vs. 0 mM glucose), we have shown that the ability of insulin to stimulate glucose incorporation into glycogen is impaired. Interestingly, this was associated with a decreased activation of Akt/PKB, but not its upstream regulator, PI3-kinase. A similar pattern of signaling abnormalities has been observed in adipocytes, L6 muscle cells, C2C12 cells, and (as reported here) EDL incubated with C(2)-ceramide. On the other hand, no increase was observed in ceramide mass in EDL incubated with 25 mM glucose. Hyperglycemia-induced insulin resistance also has been described in adipocytes, where it has been linked to activation of novel and conventional protein kinase C isoforms that phosphorylate the insulin receptor and IRS. In addition, we have recently shown that hyperglycemia causes insulin resistance in cultured human umbilical vein endothelial cells (HUVEC). Here, it was associated with an increased propensity to apoptosis and, as in muscle, with an impaired ability of insulin to activate Akt. Interestingly, these effects of hyperglycemia and an increase in diacylglycerol synthesis, which is also caused, were prevented by adding AICAR, an activator of AMP-activated protein kinase (AMPK), to the incubation medium. These results suggest that hyperglycemia causes insulin resistance in cells other than those in classic insulin target tissues. Whether AMPK activation can reverse or prevent insulin resistance in all of these cells remains to be determined.

Mouse models of insulin resistance.

The hallmarks of type 2 diabetes are impaired insulin action in peripheral tissues and decreased pancreatic beta-cell function. Classically, the two defects have been viewed as separate entities, with insulin resistance arising primarily from impaired insulin-dependent glucose uptake in skeletal muscle, and beta-cell dysfunction arising from impaired coupling of glucose sensing to insulin secretion. Targeted mutagenesis and transgenesis involving components of the insulin action pathway have changed our understanding of these phenomena. It appears that the role of insulin signaling in the pathogenesis of type 2 diabetes has been overestimated in classic insulin target tissues, such as skeletal muscle, whereas it has been overlooked in liver, pancreatic beta-cells, and brain, which had been thought not to be primary insulin targets. We review recent progress and try to reconcile areas of apparent controversy surrounding insulin signaling in skeletal muscle and pancreatic beta-cells.

IRS proteins and beta-cell function.

Insulin receptor substrate (IRS) proteins mediate a variety of the metabolic and growth-promoting actions of insulin and IGF-1. After phosphorylation by activated receptors, these intracellular signaling molecules recruit various downstream effector pathways including phosphatidylinositol 3-kinase and Grb2. Ablation of the IRS-2 gene produces a diabetic phenotype; mice lacking IRS-2 display peripheral insulin resistance and beta-cell dysfunction characterized by a 50% reduction in beta-cell mass. In contrast, deletion of IRS-1 retards somatic growth and enhances beta-cell mass. IRS1-/- mice are 50% smaller than controls but have a twofold increase in pancreatic beta-cell mass. Thus, observations from these recently developed animal models implicate the IRS signaling systems in the response of classical insulin target tissues, and they suggest a critical role for these proteins in the regulation of beta-cell function. In humans, type 2 diabetes generally occurs when insulin-secretory reserves fail to compensate for peripheral insulin resistance. Study and identification of the signals downstream of IRS proteins in beta-cells may provide unique insights into the compensatory mechanisms by which these cells respond to insulin resistance. Therefore, the intent of this review is to summarize recent observations regarding the regulation of beta-cell function by members of the IRS protein family.

Insulin action and type 2 diabetes: lessons from knockout mice

Recent evidence from a number of knockout mouse models has not only provided novel insights into mechanisms by which insulin signal transduction pathways regulate glucose homeostasis in classical insulin target tissues, but has revealed previously unsuspected roles for these pathways in pancreatic beta cell function. A most striking example is that insulin receptor substrate 2-dependent signaling pathways, long implicated in peripheral insulin action, have now been demonstrated to mediate the beta cell compensatory mechanisms that occur in response to insulin resistance. Further evidence that insulin signaling pathways are important in beta cell function comes from the recent finding that beta cell-specific deletion of the insulin receptor results in defects in insulin secretion. Additional models aimed at abrogating insulin signaling in muscle and adipose tissue show that these defects are insufficient to result in a marked disturbance of glucose homeostasis. Taken together these new experimental models challenge the classical views of insulin signaling pathways in glucose disposal and identify novel mechanisms for the pathogenesis of type 2 diabetes.

Evidence for independent regulation of human erythrocyte and adipocyte insulin receptors.

Specific binding of [125I]-insulin was measured in erythrocytes and fat obtained at the time of mastectomy in twenty-three subjects. There was a significant negative correlation between the level of specific binding to fat and fasting insulin concentration (r=0.55, p less than 0.05, n=23) suggesting down-regulation of fat insulin level by insulin. Insulin binding to erythrocytes did not correlate with insulin nor was there any relationship between insulin binding to erythrocytes and fat from the same subject. Thus while the erythrocyte insulin receptor may be of interest in its own right, it may not be subject to the normal regulatory influences seen in classical insulin target tissues. Insulin binding to erythrocytes may therefore be invalid as a model to study down-regulation of insulin receptors.