The lipid peroxidation end-product and oxidant 4-hydroxynonenal induces insulin resistance in rat slow-twitch skeletal muscle.

CONTEXT: The lipid peroxidation end-product and oxidant 4-hydroxynonenal (4-HNE) impairs cell function. However, the impact of 4-HNE on the glucose transport system in mammalian slow-twitch skeletal muscle is not known. OBJECTIVE: We assessed the effects of 4-HNE on insulin signalling and glucose transport activity in slow-twitch muscle by incubating soleus strips from lean Zucker rats with 4-HNE (50 µM) in the absence or presence of insulin (5 mU/ml) for up to 6 hr. RESULTS: Insulin-stimulated glucose transport activity was significantly (p<0.05) decreased by 4-HNE at 2 hr. AS160 Thr(642) phosphorylation was decreased at 2 hr, whereas Akt Ser473 phosphorylation and IRS-1 protein expression were not substantially changed until 4 hr. IRS-2 protein expression was slightly decreased only at 6 hr. CONCLUSIONS: The lipid peroxidation end-product and oxidant 4-HNE induces insulin resistance of glucose transport activity in rat slow-twitch skeletal muscle, initially associated with impaired phosphorylation of AS160.

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.

Effects of H2O2 on insulin signaling the glucose transport system in mammalian skeletal muscle.

Hydrogen peroxide (H2O2) is an important regulator of cellular events leading to glucose transport activation in mammalian skeletal muscle. In the absence of insulin, H2O2 in the low micromolar range engages the canonical IRS-1/PI3K/Akt-dependent insulin signaling pathway, as well as other signaling elements (AMPK and p38 MAPK), to increase basal glucose transport activity. In contrast, in the presence of insulin, H2O2 antagonizes insulin signaling by recruitment of various deleterious serine/threonine kinases, producing a state of insulin resistance. Here, we describe the H2O2 enzymatic-generating system, utilizing glucose oxidase, that has been used to investigate the impact of H2O2 on cellular signaling mechanisms related to glucose transport activity in isolated rat skeletal muscle preparations, such as the soleus. By varying the glucose oxidase concentration in the medium, target ranges of steady-state H2O2 concentrations (30-90 µM) can be attained for up to 6h, with subsequent assessment of cellular signaling and glucose transport activity.

The role of the renin-angiotensin system in the development of insulin resistance in skeletal muscle.

The canonical renin-angiotensin system (RAS) involves the initial action of renin to cleave angiotensinogen to angiotensin I (ANG I), which is then converted to ANG II by the angiotensin converting enzyme (ACE). ANG II plays a critical role in numerous physiological functions, and RAS overactivity underlies many conditions of cardiovascular dysregulation. In addition, ANG II, by acting on both endothelial and myocellular AT1 receptors, can induce insulin resistance by increasing cellular oxidative stress, leading to impaired insulin signaling and insulin-stimulated glucose transport activity. This insulin resistance associated with RAS overactivity, when coupled with progressive ß-cell dysfunction, eventually leads to the development of type 2 diabetes. Interventions that target RAS overactivity, including ACE inhibitors, ANG II receptor blockers, and, most recently, renin inhibitors, are effective both in reducing hypertension and in improving whole-body and skeletal muscle insulin action, due at least in part to enhanced Akt-dependent insulin signaling and insulin-dependent glucose transport activity. ANG-(1-7), which is produced from ANG II by the action of ACE2 and acts via Mas receptors, can counterbalance the deleterious actions of the ACE/ANG II/AT1 receptor axis on the insulin-dependent glucose transport system in skeletal muscle. This beneficial effect of the ACE2/ANG-(1-7)/Mas receptor axis appears to depend on the activation of Akt. Collectively, these findings underscore the importance of RAS overactivity in the multifactorial etiology of insulin resistance in skeletal muscle, and provide support for interventions that target the RAS to ameliorate both cardiovascular dysfunctions and insulin resistance in skeletal muscle tissue.

Obesity-related alterations in cardiac lipid profile and nondipping blood pressure pattern during transition to diastolic dysfunction in male db/db mice.

Obesity and a nondipping circadian blood pressure (BP) pattern are associated with diastolic dysfunction. Ectopic lipid accumulation is increasingly recognized as an important metabolic abnormality contributing to diastolic dysfunction. However, little is known about the contribution of different lipids and the composition of lipid analytes to diastolic dysfunction. We have performed functional and structural studies and analyzed cardiac lipid profile at two time points during progression to diastolic dysfunction in a genetic model of obesity. Serial cardiac magnetic resonance imaging and telemetric measures of BP between 12 and 15 wk of age in obese male db/db mice indicated a nondipping circadian BP pattern and normal diastolic function at 12 wk that progressed to a deteriorating nondipping pattern and onset of diastolic dysfunction at 15 wk of age. Lipidomic analysis demonstrated elevated fatty acids and ceramides in db/db at 12 wk, but their levels were decreased at 15 wk, and this was accompanied by persistent mitochondrial ultrastructural abnormalities in concert with evidence of increased fatty acid oxidation and enhanced production of reactive oxygen species. Triacylglyceride and diacylglyceride levels were elevated at both 12 and 15 wk, but their composition changed to consist of more saturated and less unsaturated fatty acyl at 15 wk. An increase in the lipid droplets was apparent at both time points, and this was associated with increases in phosphatidycholine. In conclusion, a distinct pattern of myocardial lipid remodeling, accompanied by oxidative stress, is associated with the onset of diastolic dysfunction in obese, insulin-resistant db/db mice.

ANG-(1-7) reduces ANG II-induced insulin resistance by enhancing Akt phosphorylation via a Mas receptor-dependent mechanism in rat skeletal muscle.

The nonapeptide angiotensin II (ANG II) induces vasoconstriction via the ANG II type I receptor, while its splice product ANG-(1-7) elicits an antihypertensive effect via the Mas receptor. Although a critical role of ANG II in the etiology of skeletal muscle insulin resistance is well documented, the role of the ANG-(1-7)/Mas receptor axis in this context is poorly understood. Therefore, we determined whether ANG-(1-7) is effective in ameliorating the negative effects of ANG II on insulin-stimulated insulin signaling and glucose transport activity in isolated soleus muscle from normotensive lean Zucker rats. ANG II alone (500 nM for 2 h) decreased insulin-stimulated glucose transport activity by 45% (P < 0.05). In the presence of 500-1000 nM ANG-(1-7), insulin-stimulated glucose transport activity in muscle exposed to ANG II improved by ~30% (P < 0.05). Moreover, ANG-(1-7) treatment increased Akt Ser(473) phosphorylation (47%, P < 0.05) without an effect on glycogen synthase kinase-3ß Ser(9) phosphorylation. The dependence of ANG-(1-7) action on the Mas receptor was assessed using A779 peptide, a selective Mas receptor antagonist. The positive effects of ANG-(1-7) on insulin-stimulated glucose transport activity and Akt Ser(473) phosphorylation in soleus muscle were completely prevented in presence of 1000 nM A779. In conclusion, the present study demonstrates that ANG-(1-7), via a Mas receptor-dependent mechanism, can ameliorate the inhibitory effect of ANG II on glucose transport activity in mammalian skeletal muscle, associated with enhanced Akt phosphorylation. These results provide further evidence supporting the targeting of the renin-angiotensin system for interventions designed to reduce insulin resistance in skeletal muscle tissue.

Contribution of the serine kinase c-Jun N-terminal kinase (JNK) to oxidant-induced insulin resistance in isolated rat skeletal muscle.

The specific and direct contribution of the stress-activated serine kinase c-Jun N-terminal kinase (JNK) in the development of oxidative stress-induced insulin resistance of the glucose transport system in mammalian skeletal muscle is not fully understood. We assessed the specific role of JNK in the development of insulin resistance caused by in vitro exposure of rat soleus muscle to low levels (30-40 µM) of the oxidant hydrogen peroxide (H(2)O(2)) for up to 6 h. Oxidant exposure caused significant (p < 0.05) decreases in insulin-stimulated glucose transport activity (up to 42%) and Akt Ser(473) phosphorylation (up to 67%), and increased (up to 74%) phosphorylation (Thr(183)/Tyr(185)) of JNK1 and JNK2/3 isoforms. Importantly, insulin-stimulated glucose transport activity in the presence of H(2)O(2) was moderately improved with the selective JNK inhibitor SP600125. These results indicate that activation of the serine kinase JNK contributes, at least in part, to oxidative stress-induced insulin resistance in isolated mammalian skeletal muscle.

Chronic renin inhibition with aliskiren improves glucose tolerance, insulin sensitivity, and skeletal muscle glucose transport activity in obese Zucker rats.

We have demonstrated previously that overactivity of the renin-angiotensin system (RAS) is associated with whole body and skeletal muscle insulin resistance in obese Zucker (fa/fa) rats. Moreover, this obesity-associated insulin resistance is reduced by treatment with angiotensin-converting enzyme inhibitors or angiotensin receptor (type 1) blockers. However, it is currently unknown whether specific inhibition of renin itself, the rate-limiting step in RAS functionality, improves insulin action in obesity-associated insulin resistance. Therefore, the present study assessed the effect of chronic, selective renin inhibition using aliskiren on glucose tolerance, whole body insulin sensitivity, and insulin action on the glucose transport system in skeletal muscle of obese Zucker rats. Obese Zucker rats were treated for 21 days with either vehicle or aliskiren (50 mg/kg body wt ip). Renin inhibition was associated with a significant lowering (10%, P < 0.05) of resting systolic blood pressure and induced reductions in fasting plasma glucose (11%) and free fatty acids (46%) and homeostatic model assessment for insulin resistance (13%). Glucose tolerance (glucose area under the curve) and whole body insulin sensitivity (inverse of the glucose-insulin index) during an oral glucose tolerance test were improved by 15% and 16%, respectively, following chronic renin inhibition. Moreover, insulin-stimulated glucose transport activity in isolated soleus muscle of renin inhibitor-treated animals was increased by 36% and was associated with a 2.2-fold greater Akt Ser(473) phosphorylation. These data provide evidence that chronic selective inhibition of renin activity leads to improvements in glucose tolerance and whole body insulin sensitivity in the insulin-resistant obese Zucker rat. Importantly, chronic renin inhibition is associated with upregulation of insulin action on skeletal muscle glucose transport, and it may involve improved Akt signaling. These data support the strategy of targeting the RAS to improve both blood pressure regulation and insulin action in conditions of insulin resistance.

Prenatal Programming and Epigenetics in the Genesis of the Cardiorenal Syndrome.

The presence of a group of interacting maladaptive factors, including hypertension, insulin resistance, metabolic dyslipidemia, obesity, and microalbuminuria and/or reduced renal function, collectively constitutes the cardiorenal metabolic syndrome (CRS). Nutritional and other environmental cues during fetal development can permanently affect the composition, homeostatic systems, and functions of multiple organs and systems; this process has been referred to as 'programming'. Since the original formulation of the notion that low birth weight is a proxy for 'prenatal programming' of adult hypertension and cardiovascular disease, evidence has also emerged for programming of kidney disease, insulin resistance, obesity, metabolic dyslipidemia, and other chronic diseases. The programming concept was initially predicated on the notion that in utero growth restriction due to famine was responsible for increased hypertension, and cardiovascular and renal diseases. On the other hand, we are now more commonly exposed to increasing rates of maternal obesity. The current review will discuss the overarching role of maternal overnutrition, as well as fetal undernutrition, in epigenetic programming in relation to the pathogenesis of the CRS in children and adults.

The Physiology undergraduate major in the University of Arizona College of Medicine: past, present, and future.

The American Physiological Society (APS) and APS Council encourage the teaching of physiology at the undergraduate, graduate, and medical school levels to support the continued prominence of this area of science. One area identified by the APS Council that is of particular importance for the development of future physiologists (the "physiology pipeline") is the teaching of physiology and physiology-related topics at the undergraduate level. In this article, we describe the historical development and implementation of an undergraduate program offered through the Department of Physiology, a basic science department in the College of Medicine at the University of Arizona, culminating in a Bachelor of Science in Health Sciences degree with a major in Physiology. Moreover, we discuss the current Physiology curriculum offered at our institution and explain how this program prepares our students for successful entry into a variety of postbaccalaureate professional programs, including medical school and numerous other programs in health professions, and in graduate study in the Masters and Doctoral programs in biomedical sciences. Finally, we cover the considerable challenges that we have faced, and continue to face, in developing and sustaining a successful physiology undergraduate major in a college of medicine. We hope that the information provided on the Physiology major offered by the Department of Physiology in the College of Medicine at the University of Arizona will be helpful for individuals at other institutions who may be contemplating the development and implementation of an undergraduate program in Physiology.

Regular exercise reverses sensory hypersensitivity in a rat neuropathic pain model: role of endogenous opioids.

BACKGROUND: Exercise is often prescribed as a therapy for chronic pain. Short-term exercise briefly increases the production of endogenous analgesics, leading to transient antinociception. In limited studies, exercise produced sustained increases in endogenous opioids, sustained analgesia, or diminished measures of chronic pain. This study tests the hypothesis that regular aerobic exercise leads to sustained reversal of neuropathic pain by activating endogenous opioid-mediated pain modulatory systems. METHODS: After baseline measurements, the L5 and L6 spinal nerves of male Sprague-Dawley rats were tightly ligated. Animals were randomized to sedentary or 5-week treadmill exercise-trained groups. Thermal and tactile sensitivities were assessed 23 h after exercise, using paw withdrawal thresholds to von Frey filaments and withdrawal latencies to noxious heat. Opioid receptor antagonists were administered by subcutaneous, intrathecal, or intracerebroventricular injection. Opioid peptides were quantified using immunohistochemistry with densitometry. RESULTS: Exercise training ameliorated thermal and tactile hypersensitivity in spinal nerve-ligated animals within 3 weeks. Sensory hypersensitivity returned 5 days after discontinuation of exercise training. The effects of exercise were reversed by using systemically or intracerebroventricularly administered opioid receptor antagonists and prevented by continuous infusion of naltrexone. Exercise increased ß-endorphin and met-enkephalin content in the rostral ventromedial medulla and the mid-brain periaqueductal gray area. CONCLUSIONS: Regular moderate aerobic exercise reversed signs of neuropathic pain and increased endogenous opioid content in brainstem regions important in pain modulation. Exercise effects were reversed by opioid receptor antagonists. These results suggest that exercise-induced reversal of neuropathic pain results from an up-regulation of endogenous opioids.

Critical role of the transient activation of p38 MAPK in the etiology of skeletal muscle insulin resistance induced by low-level in vitro oxidant stress.

Increased cellular exposure to oxidants may contribute to the development of insulin resistance and type 2 diabetes. Skeletal muscle is the primary site of insulin-dependent glucose disposal in the body; however, the effects of oxidative stress on insulin signaling and glucose transport activity in mammalian skeletal muscle are not well understood. We therefore studied the effects of a low-level in vitro oxidant stress (30-40 µM H2O2) on basal and insulin-stimulated (5 mU/ml) glucose transport activity and insulin signaling at 2, 4, and 6 h in isolated rat soleus muscle. H2O2 increased basal glucose transport activity at 2 and 4 h, but not at 6 h. This low-level oxidant stress significantly impaired insulin-stimulated glucose transport activity at all time points, and was associated with inhibition of insulin-stimulated phosphorylation of Akt Ser473 and GSK-3ß Ser9. In the presence of insulin, H2O2 decreased total protein expression of IRS-1 at 6 h and IRS-2 at 4 and 6 h. Phosphorylation of p38 MAPK Thr180/Tyr182 was transiently increased by H2O2 in the presence and absence of insulin at 2 and 4 h, but not at 6 h. Selective inhibition of p38 MAPK with A304000 partially rescued the H2O2-induced reduction in insulin-stimulated glucose transport activity. These results indicate that direct in vitro exposure of isolated mammalian skeletal muscle to a low-level oxidant stress impairs distal insulin signaling and insulin-stimulated glucose transport activity, at least in part, due to a p38 MAPK-dependent mechanism.

Oxidative stress and the etiology of insulin resistance and type 2 diabetes.

The condition of oxidative stress arises when oxidant production exceeds antioxidant activity in cells and plasma. The overabundance of oxidants is mechanistically connected to the multifactorial etiology of insulin resistance, primarily in skeletal muscle tissue, and the subsequent development of type 2 diabetes. Two important mechanisms for this oxidant excess are (1) the mitochondrial overproduction of hydrogen peroxide and superoxide ion under conditions of energy surplus and (2) the enhanced activation of cellular NADPH oxidase via angiotensin II receptors. Several recent studies are reviewed that support the concept that direct exposure of mammalian skeletal muscle to an oxidant stress (including hydrogen peroxide) results in stimulation of the serine kinase p38 mitogen-activated protein kinase (p38 MAPK), and that the engagement of this stress-activated p38 MAPK signaling is mechanistically associated with diminished insulin-dependent stimulation of insulin signaling elements and glucose transport activity. The beneficial interactions between the antioxidant α-lipoic acid and the advanced glycation end-product inhibitor pyridoxamine that ameliorate oxidant stress-associated defects in whole-body and skeletal-muscle insulin action in the obese Zucker rat, a model of prediabetes, are also addressed. Overall, this review highlights the importance of oxidative stress in the development of insulin resistance in mammalian skeletal muscle tissue, at least in part via a p38-MAPK-dependent mechanism, and indicates that interventions that reduce this oxidative stress and oxidative damage can improve insulin action in insulin-resistant animal models. Strategies to prevent and ameliorate oxidative stress remain important in the overall treatment of insulin resistance and type 2 diabetes.

Overnutrition and the Cardiorenal Syndrome: Use of a Rodent Model to Examine Mechanisms.

Obesity has reached epidemic proportions with far-reaching health care and economic implications. Overnutrition, characterized by excess intake of carbohydrates and fats, has been associated with end-organ damage in several tissues, including the heart and the kidney. Furthermore, overnutrition is one of the most important modifiable and preventable causes of morbidity and mortality associated with cardiovascular and kidney diseases. Insulin resistance and compensatory hyperinsulinemia as well as associated mechanisms, including enhanced renin-angiotensin-aldosterone system activity, inflammation, and oxidative stress, have been implicated in obesity-related cardiorenal injury. In this review, the effect of overnutrition on heart and kidney disease is assessed in a rodent model of overnutrition and obesity, the Zucker obese rat.

Effects of Chronic Antagonism of Endocannabinoid-1 Receptors on Glucose Tolerance and Insulin Action in Skeletal Muscles of Lean and Obese Zucker Rats.

BACKGROUND/AIMS: Antagonism of the endocannabinoid receptor-1 (CB1R) directly improves whole-body metabolic parameters of insulin resistance. The present investigation determined the effects of chronic CB1R antagonism on whole-body and skeletal-muscle insulin action in insulin-sensitive lean and insulin-resistant obese Zucker rats. METHODS: Animals were either fed ad libitum or in pairs, or treated with SR141716 (10 mg/kg i.p. for 14 days). RESULTS: Food intake was significantly reduced (p < 0.05) after initial SR141716 treatment and remained decreased in both lean and obese animals until day 13. Fasting plasma glucose decreased (24%) and insulin increased (43%) in lean SR141716-treated (24%) rats compared to lean ad libitum-fed controls, but not in the corresponding obese groups. Fasting plasma free fatty acids were reduced by CB1R antagonism in lean (21%) and obese (42%) animals. Whole-body insulin sensitivity was increased (36%) in obese SR141716-treated rats compared to obese ad libitum-fed controls, which was associated with reduced insulin secretion during an oral glucose tolerance test. Insulin-stimulated glucose transport activity in the soleus was greatest in the respective SR141716-treated lean and obese groups compared to the corresponding ad libitum- and pair-fed controls. Chronic SR141716 treatment did not induce alterations in signaling factors associated with the regulation of glucose transport [protein kinase B (Akt), glycogen synthase kinase-3ß, 5'-AMP-dependent protein kinase, or p38 mitogen-activated protein kinase] in the soleus. CONCLUSIONS: These results indicate that, while the chronic treatment with CB1R antagonism markedly diminished food intake in lean and obese Zucker rats, there are also significant metabolic improvements in whole-body and skeletal-muscle insulin action mediated by CB1R antagonism through mechanisms independent of reduced caloric intake.

Dysregulation of glycogen synthase kinase-3 in skeletal muscle and the etiology of insulin resistance and type 2 diabetes.

Insulin resistance of glucose transport and metabolism in insulin-sensitive tissues is a primary defect leading to the development of type 2 diabetes. While the etiology of insulin resistance is multifactorial, one factor associated with reduced insulin action is enhanced activity of the serine/threonine kinase glycogen synthase kinase-3 (GSK-3) in skeletal muscle, liver, and adipose tissue. GSK-3 is involved in numerous cellular functions, including glycogen synthesis, protein synthesis, gene transcription, and cell differentiation. Evidence from muscle and fat cell lines and in skeletal muscle from a variety of obese rodent models and from type 2 diabetic humans supports a role of GSK-3 overactivity in the development of insulin resistance of glucose transport and glycogenesis. Studies utilizing highly selective GSK-3 inhibitors indicate that GSK-3 overactivity in obesity is associated with enhanced IRS-1 serine phosphorylation and defective IRS-1-dependent signaling, ultimately resulting in reduced GLUT-4 translocation and glucose transport activity in skeletal muscle. A role of GSK-3 overactivity in the exaggerated hepatic glucose production of type 2 diabetes has also been reported. Recent studies have demonstrated that oxidative stress, resulting from enhanced exposure to oxidants, causes impaired insulin signaling and insulin resistance of skeletal muscle glucose transport, in part due to reduced suppression of GSK-3 activity and increased IRS-1 Ser(307) phosphorylation. The evidence to date supports an important role of GSK-3 dysfunction in the multifactorial etiology of insulin resistance in skeletal muscle. GSK-3 remains an important target for interventions designed to improve insulin action in obesity-associated insulin resistance and type 2 diabetes.

Direct inhibition by angiotensin II of insulin-dependent glucose transport activity in mammalian skeletal muscle involves a ROS-dependent mechanism.

No previous study has investigated how the vaso-constrictive peptide Ang II impacts insulin action in isolated mammalian skeletal muscle. We investigated the molecular actions of Ang II on insulin signalling and glucose transport in skeletal muscle from lean Zucker rats. Soleus strips were incubated with insulin (5 mU/ml) and/or Ang II (500 nM) for 2 hours. Ang II caused significant (p < 0.05) inhibition of insulin-stimulated glucose transport (39%) and decreased phosphorylation of Akt Ser(473) (37%) and glycogen synthase kinase-3beta Ser(9) (42%) without affecting phosphorylation of IRS-1 Ser(307) or p38 MAPK. We used the superoxide dismutase mimetic, tempol (1 mM), to determine if reactive oxygen species (ROS) contribute to Ang II-mediated insulin resistance. Tempol partially reversed (42%) Ang II-induced inhibition of insulin-stimulated glucose transport. These results indicate that Ang II inhibits distal insulin signalling and insulin-stimulated glucose transport in isolated mammalian skeletal muscle, and that this effect is partially mediated by ROS.

Oxidant stress-induced loss of IRS-1 and IRS-2 proteins in rat skeletal muscle: role of p38 MAPK.

Oxidative stress is characterized as an imbalance between the cellular production of oxidants and the cellular antioxidant defenses and contributes to the development of numerous cardiovascular and metabolic disorders, including hypertension and insulin resistance. The effects of prolonged oxidant stress in vitro on the insulin-dependent glucose transport system in mammalian skeletal muscle are not well understood. This study examined the in vitro effects of low-level oxidant stress (60-90 microM, H(2)O(2)) for 4 h on insulin-stimulated (5 mU/ml) glucose transport activity (2-deoxyglucose uptake) and on protein expression of critical insulin signaling factors (insulin receptor (IR), IR substrates IRS-1 and IRS-2, phosphatidylinositol 3-kinase, Akt, and glycogen synthase kinase-3 (GSK-3)) in isolated soleus muscle of lean Zucker rats. This oxidant stress exposure caused significant (50%, p<0.05) decreases in insulin-stimulated glucose transport activity that were associated with selective loss of IRS-1 (59%) and IRS-2 (33%) proteins, increased (64%) relative IRS-1 Ser(307) phosphorylation, and decreased phosphorylation of Akt Ser(473) (50%) and GSK-3beta Ser(9) (43%). Moreover, enhanced (37%) phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was observed. Selective inhibition of p38 MAPK (10 microM A304000) prevented a significant portion (29%) of the oxidant stress-induced loss of IRS-1 (but not IRS-2) protein and allowed partial recovery of the impaired insulin-stimulated glucose transport activity. These results indicate that in vitro oxidative stress in mammalian skeletal muscle leads to substantial insulin resistance of distal insulin signaling and glucose transport activity, associated with a selective loss of IRS-1 protein, in part due to a p38 MAPK-dependent mechanism.