Nava Bashan

Positive and Negative Regulation of Insulin Signaling by Reactive Oxygen and Nitrogen Species

Regulated production of reactive oxygen species (ROS)/reactive nitrogen species (RNS) adequately balanced by antioxidant systems is a prerequisite for the participation of these active substances in physiological processes, including insulin action. Yet, increasing evidence implicates ROS and RNS as negative regulators of insulin signaling, rendering them putative mediators in the development of insulin resistance, a common endocrine abnormality that accompanies obesity and is a risk factor of type 2 diabetes. This review deals with this dual, seemingly contradictory, function of ROS and RNS in regulating insulin action: the major processes for ROS and RNS generation and detoxification are presented, and a critical review of the evidence that they participate in the positive and negative regulation of insulin action is provided. The cellular and molecular mechanisms by which ROS and RNS are thought to participate in normal insulin action and in the induction of insulin resistance are then described. Finally, we explore the potential usefulness and the challenges in modulating the oxidant-antioxidant balance as a potentially promising, but currently disappointing, means of improving insulin action in insulin resistance-associated conditions, leading causes of human morbidity and mortality of our era.

Oxidative Stress Disrupts Insulin-induced Cellular Redistribution of Insulin Receptor Substrate-1 and Phosphatidylinositol 3-Kinase in 3T3-L1 Adipocytes A PUTATIVE CELLULAR MECHANISM FOR IMPAIRED PROTEIN KINASE B ACTIVATION AND GLUT4 TRANSLOCATION

In a recent study we have demonstrated that 3T3-L1 adipocytes exposed to low micromolar H2O2 concentrations display impaired insulin stimulated GLUT4 translocation from internal membrane pools to the plasma membrane (Rudich, A., Tirosh, A., Potashnik, R., Hemi, R., Kannety, H., and Bashan, N. (1998) Diabetes 47, 1562–1569). In this study we further characterize the cellular mechanisms responsible for this observation. Two-hour exposure to ∼25 μm H2O2 (generated by adding glucose oxidase to the medium) resulted in disruption of the normal insulin stimulated insulin receptor substrate (IRS)-1 and phosphatidylinositol (PI) 3-kinase cellular redistribution between the cytosol and an internal membrane pool (low density microsomal fraction (LDM)). This was associated with reduced insulin-stimulated IRS-1 and p85-associated PI 3-kinase activities in the LDM (84 and 96% inhibition, respectively). The effect of this finding on the downstream insulin signal was demonstrated by a 90% reduction in insulin stimulated protein kinase B (PKB) serine 473 phosphorylation and impaired activation of PKBα and PKBγ. Both control and oxidized cells exposed to heat shock displayed a wortmannin insensitive PKB serine phosphorylation and activity. These data suggest that activation of PKB and GLUT4 translocation are insulin signaling events dependent upon a normal insulin induced cellular compartmentalization of PI 3-kinase and IRS-1, which is oxidative stress-sensitive. These findings represent a novel cellular mechanism for the induction of insulin resistance in response to changes in the extracellular environment.

Altered autophagy in human adipose tissues in obesity.

CONTEXT Autophagy is a housekeeping mechanism, involved in metabolic regulation and stress response, shown recently to regulate lipid droplets biogenesis/breakdown and adipose tissue phenotype. OBJECTIVE We hypothesized that in human obesity autophagy may be altered in adipose tissue in a fat depot and distribution-dependent manner. SETTING AND PATIENTS Paired omental (Om) and subcutaneous (Sc) adipose tissue samples were used from obese and nonobese (n = 65, cohort 1); lean, Sc-obese and intraabdominally obese (n = 196, cohort 2); severely obese persons without diabetes or obesity-associated morbidity, matched for being insulin sensitive or resistant (n = 60, cohort 3). RESULTS Protein and mRNA levels of the autophagy genes Atg5, LC3A, and LC3B were increased in Om compared with Sc, more pronounced among obese persons, particularly with intraabdominal fat accumulation. Both adipocytes and stromal-vascular cells contribute to the expression of autophagy genes. An increased number of autophagosomes and elevated autophagic flux assessed in fat explants incubated with lysosomal inhibitors were observed in obesity, particularly in Om. The degree of visceral adiposity and adipocyte hypertrophy accounted for approximately 50% of the variance in omental Atg5 mRNA levels by multivariate regression analysis, whereas age, sex, measures of insulin sensitivity, inflammation, and adipose tissue stress were excluded from the model. Moreover, in cohort 3, the autophagy marker genes were increased in those who were insulin resistant compared with insulin sensitive, particularly in Om. CONCLUSIONS Autophagy is up-regulated in adipose tissue of obese persons, especially in Om, correlating with the degree of obesity, visceral fat distribution, and adipocyte hypertrophy. This may co-occur with insulin resistance but precede the occurrence of obesity-associated morbidity.

Lipoic acid protects against oxidative stress induced impairment in insulin stimulation of protein kinase B and glucose transport in 3T3-L1 adipocytes

Aims/hypothesis. Oxidative stress has been shown to impair insulin-stimulated glucose transporter 4 translocation in 3T3-L1 adipocytes. This study explores the potential of the antioxidant lipoic acid to protect the cells against the induction of insulin resistance when given before exposure to oxidative stress. Methods. 3T3-LI were exposed for 16 h to lipoic acid after which cells were exposed for 2 h to continuous production of H2O2 by adding glucose oxidase to the culture medium. Results. These conditions resulted in a 50–70 % reduction in insulin-stimulated glucose transport activity associated with a decrease in reduced glutathione content from 37.4 ± 3.1 to 26.4 ± 4.9 nmol/mg protein, (p < 0.005). Lipoic acid pretreatment increased insulin-stimulated glucose transport following oxidative stress, reaching 84.8 ± 4.4 % of the control, associated with an increase in reduced glutathione content. Oxidation impaired the 4.89 ± 0.36-fold insulin-stimulated increase in glucose transporter 4 content in plasma membrane lawns of control cells. Lipoic acid pretreatment was, however, associated with preserved insulin-induced glucose transporter 4 translocation in cells exposed to oxidation, yielding 80 % of its content in controls. Although tyrosine phosphorylation patterns were not affected by lipoic acid pretreatment, insulin-stimulated protein kinase B/Akt serine 473 phosphorylation and activity were considerably impaired by oxidation but protected by lipoic acid pretreatment. A protective effect was not observed with either troglitazone, its isolated vitamin E moiety, or with vitamin C. Conclusion/interpretation. This study shows the ability of lipoic acid to provide partial protection against the impaired insulin-stimulated glucose transporter 4 translocation and protein kinase B/Akt activation induced by oxidative stress, potentially by its capacity to maintain intracellular redox state. [Diabetologia (1999) 42: 949–957]

Lipoic acid reduces glycemia and increases muscle GLUT4 content in streptozotocin-diabetic rats

Alpha lipoic acid (lipoate [LA]), a cofactor of alpha-ketodehydrogenase, exhibits unique antioxidant properties. Recent studies suggest a direct effect of LA on glucose metabolism in both human and experimental diabetes. This study examines the possibility that LA positively affects glucose homeostasis in streptozotocin (STZ)-induced diabetic rats by altering skeletal muscle glucose utilization. Blood glucose concentration in STZ-diabetic rats following 10 days of intraperitoneal (i.p.) injection of LA 30 mg/kg was reduced compared with that in vehicle-treated diabetic rats (495 +/- 131 v 641 +/- 125 mg/dL in fed state, P = .003, and 189 +/- 48 v 341 +/- 36 mg/dL after 12-hour fast, P = .001). No effect of LA on plasma insulin was observed. Gastrocnemius muscle crude membrane GLUT4 protein was elevated both in control and in diabetic rats treated with LA by 1.5- and 2.8-fold, respectively, without significant changes in GLUT4 mRNA levels. Gastrocnemius lactic acid was increased in diabetic rats (19.9 +/- 5.5 v 10.4 +/- 2.8 mumol/g muscle, P < .05 v nondiabetic rats), and was normal in LA-treated diabetic rats (9.1 +/- 5.0 mumol/g muscle). Insulin-stimulated 2-deoxyglucose (2 DG) uptake into isolated soleus muscle was reduced in diabetic rats compared with the control group (474 +/- 15 v 568 +/- 52 pmol/mg muscle 30 min, respectively, P = .05). LA treatment prevented this reduction, resulting in insulin-stimulated glucose uptake comparable to that of nondiabetic animals. These results suggest that daily LA treatment may reduce blood glucose concentrations in STZ-diabetic rats by enhancing muscle GLUT4 protein content and by increasing muscle glucose utilization.

IRS1 degradation and increased serine phosphorylation cannot predict the degree of metabolic insulin resistance induced by oxidative stress

Aim/hypothesisOxidative stress was shown to selectively induce impaired metabolic response to insulin, raising the possible involvement of alterations in Insulin-Receptor-Substrate (IRS) proteins. This study was conducted to assess whether oxidative stress induced IRS protein degradation and enhanced serine phosphorylation, and to assess their functional importance.Methods3T3-L1 adipocytes and rat hepatoma cells (FAO) were exposed to micro-molar H2O2 by adding glucose oxidase to the culture medium, and IRS1 content, its serine phosphorylation and downstream metabolic insulin effects were measured.ResultsCells exposed to oxidative stress exhibited decreased IRS1 (but not IRS2) content, and increased serine phosphorylation of both proteins. Total protein ubiquitination was increased in oxidized cells, but not in cells exposed to prolonged insulin treatment. Yet, lactacystin and MG132, two unrelated proteasome inhibitors, prevented IRS1 degradation induced by prolonged insulin but not by oxidative stress. The PI 3-kinase inhibitor LY294002 and the mTOR inhibitor rapamycin, but not the MEK1 inhibitor PD98059, could prevent IRS1 changes in oxidized cells. Rapamycin, which protected against IRS1 degradation and serine phosphorylation was not associated with improved response to acute insulin stimulation. Moreover, the antioxidant alpha lipoic acid, while protecting against oxidative stress-induced insulin resistance in 3T3-L1 adipocytes, could not prevent IRS1 degradation and serine phosphorylation.Conclusion/interpretationOxidative stress induces serine phosphorylation of IRS1 and increases its degradation by a proteasome-independent pathway; yet, these changes do not correlate with the induction of impaired metabolic response to insulin.

The effect of the duration of the second stage of labour on the acid‐base state of the fetus

A prospective study was made in 153 patients with a second stage of labour lasting 1 to 120 min. Maternal venous, umbilical artery and vein blood were analysed for acid‐base variables and lactate levels. Blood was obtained immediately at delivery. Fetal heart rate monitoring data and blood test results were related to the duration of the second stage of labour and clinical outcome. In primiparae and multiparae there was a steady rise in umbilical artery lactate levels, with duration of second stage of labour, P>0·025 and P= 0·023, respectively. The median umbilical artery pH decreased significantly from 7·31 in patients with a second stage of labour that lasted ≤15 min to a pH of 7·25 in patients with a second stage of labour that lasted more than 30 min. Changes in Po2 and Pco2 were not statistically significant.

Interleukin-1β regulates fat-liver crosstalk in obesity by auto-paracrine modulation of adipose tissue inflammation and expandability.

The inflammasome has been recently implicated in obesity-associated dys-metabolism. However, of its products, the specific role of IL-1β was clinically demonstrated to mediate only the pancreatic beta-cell demise, and in mice mainly the intra-hepatic manifestations of obesity. Yet, it remains largely unknown if IL-1β, a cytokine believed to mainly function locally, could regulate dysfunctional inter-organ crosstalk in obesity. Here we show that High-fat-fed (HFF) mice exhibited a preferential increase of IL-1β in portal compared to systemic blood. Moreover, portally-drained mesenteric fat transplantation from IL-1βKO donors resulted in lower pyruvate-glucose flux compared to mice receiving wild-type (WT) transplant. These results raised a putative endocrine function for visceral fat-derived IL-1β in regulating hepatic gluconeogenic flux. IL-1βKO mice on HFF exhibited only a minor or no increase in adipose expression of pro-inflammatory genes (including macrophage M1 markers), Mac2-positive crown-like structures and CD11b-F4/80-double-positive macrophages, all of which were markedly increased in WT-HFF mice. Further consistent with autocrine/paracrine functions of IL-1β within adipose tissue, adipose tissue macrophage lipid content was increased in WT-HFF mice, but significantly less in IL-1βKO mice. Ex-vivo, adipose explants co-cultured with primary hepatocytes from WT or IL-1-receptor (IL-1RI)-KO mice suggested only a minor direct effect of adipose-derived IL-1β on hepatocyte insulin resistance. Importantly, although IL-1βKOs gained weight similarly to WT-HFF, they had larger fat depots with similar degree of adipocyte hypertrophy. Furthermore, adipogenesis genes and markers (pparg, cepba, fabp4, glut4) that were decreased by HFF in WT, were paradoxically elevated in IL-1βKO-HFF mice. These local alterations in adipose tissue inflammation and expansion correlated with a lower liver size, less hepatic steatosis, and preserved insulin sensitivity. Collectively, we demonstrate that by promoting adipose inflammation and limiting fat tissue expandability, IL-1β supports ectopic fat accumulation in hepatocytes and adipose-tissue macrophages, contributing to impaired fat-liver crosstalk in nutritional obesity.

Interleukin-1β May Mediate Insulin Resistance in Liver-Derived Cells in Response to Adipocyte Inflammation

Central obesity is frequently associated with adipose tissue inflammation and hepatic insulin resistance. To identify potential individual mediators in this process, we used in vitro systems and assessed if insulin resistance in liver cells could be induced by secreted products from adipocytes preexposed to an inflammatory stimulus. Conditioned medium from 3T3-L1 adipocytes pretreated without (CM) or with TNFalpha (CM-TNFalpha) was used to treat Fao hepatoma cells. ELISAs were used to assess the concentration of several inflammatory mediators in CM-TNFalpha. CM-TNFalpha-treated Fao cells exhibited about 45% diminution in insulin-stimulated phosphorylation of insulin receptor, insulin receptor substrate proteins, protein kinase B, and glycogen synthase kinase-3 as compared with CM-treated cells, without changes in the total abundance of these protein. Insulin increased glycogenesis by 2-fold in CM-treated Fao cells but not in cells exposed to CM-TNFalpha. Expression of IL-1beta mRNA was elevated 3-fold in TNFalpha-treated adipocytes, and CM-TNFalpha had 10-fold higher concentrations of IL-1beta but not TNFalpha or IL-1alpha. IL-1beta directly induced insulin resistance in Fao, HepG2, and in primary rat hepatocytes. Moreover, when TNFalpha-induced secretion/production of IL-1beta from adipocytes was inhibited by the IL-1 converting enzyme (ICE-1) inhibitor II (Ac-YVAD-CMK), insulin resistance was prevented. Furthermore, liver-derived cells treated with IL-1 receptor antagonist were protected against insulin resistance induced by CM-TNFalpha. Finally, IL-1beta secretion from human omental fat explants correlated with body mass index (R(2) = 0.639, P < 0.01), and the resulting CM induced insulin resistance in HepG2 cells, inhibitable by IL-1 receptor antagonist. Our results suggest that adipocyte-derived IL-1beta may constitute a mediator in the perturbed cross talk between adipocytes and liver cells in response to adipose tissue inflammation.

REACTIVE OXYGEN SPECIES ACTIVATE GLUCOSE TRANSPORT IN L6 MYOTUBES

Under oxidative stress, increased energy requirements are needed To induce repair mechanisms. As glucose is a major energy source in L6 myotubes, we evaluated glucose metabolism and transport, following exposure to glucose oxidase (H2O2 generating system), or xanthine oxidase (O2. and H2O2 generating system), added to the medium. Exposure for 24 h to 5 mM glucose and 50 mU/ml glucose oxidase, or to 50 microM xanthine and 20 mU/ml xanthine oxidase resulted in significant oxidant stress indicated by increased DNA binding activity of NF-kappa B. Under these conditions, approximately 2-fold increase in glucose consumption, lactate production and CO2 release were observed. 2-deoxyglucose uptake into myotubes increased time and dose dependently, reaching a 2.6 +/- 0.4-fold and 2.2 +/- 0.7-fold after 24 h exposure to glucose oxidase and xanthine oxidase, respectively. Peroxidase prevented this effect, indicating the role of H2O2 in mediating glucose uptake activation. The elevation in glucose uptake under oxidative stress was associated with increased expression of GLUT1 mRNA and protein. The observed 2-deoxyglucose uptake activation by oxidants was not limited to the L6 cell line and was observed in 3T3-L1 adipocytes as well.