Ronald N. Cortright

Mitochondrial H2O2 emission and cellular redox state link excess fat intake to insulin resistance in both rodents and humans

High dietary fat intake leads to insulin resistance in skeletal muscle, and this represents a major risk factor for type 2 diabetes and cardiovascular disease. Mitochondrial dysfunction and oxidative stress have been implicated in the disease process, but the underlying mechanisms are still unknown. Here we show that in skeletal muscle of both rodents and humans, a diet high in fat increases the H(2)O(2)-emitting potential of mitochondria, shifts the cellular redox environment to a more oxidized state, and decreases the redox-buffering capacity in the absence of any change in mitochondrial respiratory function. Furthermore, we show that attenuating mitochondrial H(2)O(2) emission, either by treating rats with a mitochondrial-targeted antioxidant or by genetically engineering the overexpression of catalase in mitochondria of muscle in mice, completely preserves insulin sensitivity despite a high-fat diet. These findings place the etiology of insulin resistance in the context of mitochondrial bioenergetics by demonstrating that mitochondrial H(2)O(2) emission serves as both a gauge of energy balance and a regulator of cellular redox environment, linking intracellular metabolic balance to the control of insulin sensitivity.

Energy Metabolism in Uncoupling Protein 3 Gene Knockout Mice

Uncoupling protein 3 (UCP3) is a member of the mitochondrial anion carrier superfamily. Based upon its high homology with UCP1 and its restricted tissue distribution to skeletal muscle and brown adipose tissue, UCP3 has been suggested to play important roles in regulating energy expenditure, body weight, and thermoregulation. Other postulated roles for UCP3 include regulation of fatty acid metabolism, adaptive responses to acute exercise and starvation, and prevention of reactive oxygen species (ROS) formation. To address these questions, we have generated mice lacking UCP3 (UCP3 knockout (KO) mice). Here, we provide evidence that skeletal muscle mitochondria lacking UCP3 are more coupled (i.e. increased state 3/state 4 ratio), indicating that UCP3 has uncoupling activity. In addition, production of ROS is increased in mitochondria lacking UCP3. This study demonstrates that UCP3 has uncoupling activity and that its absence may lead to increased production of ROS. Despite these effects on mitochondrial function, UCP3 does not seem to be required for body weight regulation, exercise tolerance, fatty acid oxidation, or cold-induced thermogenesis. The absence of such phenotypes in UCP3 KO mice could not be attributed to up-regulation of other UCP mRNAs. However, alternative compensatory mechanisms cannot be excluded. The consequence of increased mitochondrial coupling in UCP3 KO mice on metabolism and the possible role of yet unidentified compensatory mechanisms, remains to be determined.

Alterations in skeletal muscle protein-tyrosine phosphatase activity and expression in insulin-resistant human obesity and diabetes.

Obese human subjects have increased protein-tyrosine phosphatase (PTPase) activity in adipose tissue that can dephosphorylate and inactivate the insulin receptor kinase. To extend these findings to skeletal muscle, we measured PTPase activity in the skeletal muscle particulate fraction and cytosol from a series of lean controls, insulin-resistant obese (body mass index > 30) nondiabetic subjects, and obese individuals with non-insulin-dependent diabetes. PTPase activities in subcellular fractions from the nondiabetic obese subjects were increased to 140-170% of the level in lean controls (P < 0.05). In contrast, PTPase activity in both fractions from the obese subjects with non-insulin-dependent diabetes was significantly decreased to 39% of the level in controls (P < 0.05). By immunoblot analysis, leukocyte antigen related (LAR) and protein-tyrosine phosphatase 1B had the greatest increase (threefold) in the particulate fraction from obese, nondiabetic subjects, and immunodepletion of this fraction using an affinity-purified antibody directed at the cytoplasmic domain of leukocyte antigen related normalized the PTPase activity when compared to the activity from control subjects. These findings provide further support for negative regulation of insulin action by specific PTPases in the pathogenesis of insulin resistance in human obesity, while other regulatory mechanisms may be operative in the diabetic state.

Mechanism for Improved Insulin Sensitivity after Gastric Bypass Surgery

CONTEXT Surgical treatments of obesity have been shown to induce rapid and prolonged improvements in insulin sensitivity. OBJECTIVE The aim of the study was to investigate the effects of gastric bypass surgery and the mechanisms that explain the improvement in insulin sensitivity. DESIGN We performed a cross-sectional, nonrandomized, controlled study. SETTING This study was conducted jointly between the Departments of Exercise Science and Physiology at East Carolina University in Greenville, North Carolina. SUBJECTS Subjects were recruited into four groups: 1) lean [body mass index (BMI) < 25 kg/m(2); n = 93]; 2) weight-matched (BMI = 25 to 35 kg/m(2); n = 310); 3) morbidly obese (BMI > 35 kg/m(2); n = 43); and 4) postsurgery patients (BMI approximately 30 kg/m(2); n = 40). Postsurgery patients were weight stable 1 yr after surgery. MAIN OUTCOME MEASURES Whole-body insulin sensitivity, muscle glucose transport, and muscle insulin signaling were assessed. RESULTS Postsurgery subjects had insulin sensitivity index values that were similar to the lean and higher than morbidly obese and weight-matched control subjects. Glucose transport was higher in the postsurgery vs. morbidly obese and weight-matched groups. IRS1-pSer(312) in the postsurgery group was lower than morbidly obese and weight-matched groups. Inhibitor kappaBalpha was higher in the postsurgery vs. the morbidly obese and weight-matched controls, indicating reduced inhibitor of kappaB kinase beta activity. CONCLUSIONS Insulin sensitivity and glucose transport are greater in the postsurgery patients than predicted from the weight-matched group, suggesting that improved insulin sensitivity after bypass is due to something other than, or in addition to, weight loss. Improved insulin sensitivity is related to reduced inhibitor of kappaB kinase beta activity and enhanced insulin signaling in muscle.

Regulation of skeletal muscle UCP-2 and UCP-3 gene expression by exercise and denervation

The factors that regulate gene expression of uncoupling proteins 2 and 3 (UCP-2 and UCP-3) in skeletal muscle are poorly understood, but both genes are clearly responsive to the metabolic state of the organism. Therefore, we tested the hypothesis that denervation and acute and/or chronic exercise (factors that profoundly affect metabolism) would alter UCP-2 and UCP-3 gene expression. For the denervation studies, the sciatic nerve of rat and mouse hindlimb was sectioned in one leg while the contralateral limb served as control. Northern blot analysis revealed that denervation was associated with a 331% increase (P < 0.001) in UCP-3 mRNA and a 200% increase (P < 0. 01) in UCP-2 mRNA levels in rat mixed gastrocnemius (MG) muscle. In contrast, denervation caused a 53% decrease (P < 0.001) in UCP-3 and a 63% increase (P < 0.01) in UCP-2 mRNA levels in mouse MG. After acute exercise (2-h treadmill running), rat UCP-3 mRNA levels were elevated (vs. sedentary control) 252% (P < 0.0001) in white gastrocnemius and 63% (P < 0.05) in red gastrocnemius muscles, whereas UCP-2 levels were unaffected. To a lesser extent, elevations in UCP-3 mRNA (22%; P < 0.01) and UCP-2 mRNA (55%; P < 0.01) levels were observed after acute exercise in the mouse MG. There were no changes in either UCP-2 or UCP-3 mRNA levels after chronic exercise (9 wk of wheel running). These results indicate that acute exercise and denervation regulate gene expression of skeletal muscle UCPs.The factors that regulate gene expression of uncoupling proteins 2 and 3 (UCP-2 and UCP-3) in skeletal muscle are poorly understood, but both genes are clearly responsive to the metabolic state of the organism. Therefore, we tested the hypothesis that denervation and acute and/or chronic exercise (factors that profoundly affect metabolism) would alter UCP-2 and UCP-3 gene expression. For the denervation studies, the sciatic nerve of rat and mouse hindlimb was sectioned in one leg while the contralateral limb served as control. Northern blot analysis revealed that denervation was associated with a 331% increase ( P < 0.001) in UCP-3 mRNA and a 200% increase ( P < 0.01) in UCP-2 mRNA levels in rat mixed gastrocnemius (MG) muscle. In contrast, denervation caused a 53% decrease ( P< 0.001) in UCP-3 and a 63% increase ( P < 0.01) in UCP-2 mRNA levels in mouse MG. After acute exercise (2-h treadmill running), rat UCP-3 mRNA levels were elevated (vs. sedentary control) 252% ( P < 0.0001) in white gastrocnemius and 63% ( P < 0.05) in red gastrocnemius muscles, whereas UCP-2 levels were unaffected. To a lesser extent, elevations in UCP-3 mRNA (22%; P < 0.01) and UCP-2 mRNA (55%; P < 0.01) levels were observed after acute exercise in the mouse MG. There were no changes in either UCP-2 or UCP-3 mRNA levels after chronic exercise (9 wk of wheel running). These results indicate that acute exercise and denervation regulate gene expression of skeletal muscle UCPs.

Simvastatin impairs ADP-stimulated respiration and increases mitochondrial oxidative stress in primary human skeletal myotubes

Statins, the widely prescribed cholesterol-lowering drugs for the treatment of cardiovascular disease, cause adverse skeletal muscle side effects ranging from fatigue to fatal rhabdomyolysis. The purpose of this study was to determine the effects of simvastatin on mitochondrial respiration, oxidative stress, and cell death in differentiated primary human skeletal muscle cells (i.e., myotubes). Simvastatin induced a dose-dependent decrease in viability of proliferating and differentiating primary human muscle precursor cells, and a similar dose-dependent effect was noted in differentiated myoblasts and myotubes. Additionally, there were decreases in myotube number and size following 48 h of simvastatin treatment (5 μM). In permeabilized myotubes, maximal ADP-stimulated oxygen consumption, supported by palmitoylcarnitine+malate (PCM, complex I and II substrates) and glutamate+malate (GM, complex I substrates), was 32-37% lower (P<0.05) in simvastatin-treated (5 μM) vs control myotubes, providing evidence of impaired respiration at complex I. Mitochondrial superoxide and hydrogen peroxide generation were significantly greater in the simvastatin-treated human skeletal myotube cultures compared to control. In addition, simvastatin markedly increased protein levels of Bax (proapoptotic, +53%) and Bcl-2 (antiapoptotic, +100%, P<0.05), mitochondrial PTP opening (+44%, P<0.05), and TUNEL-positive nuclei in human skeletal myotubes, demonstrating up-regulation of mitochondrial-mediated myonuclear apoptotic mechanisms. These data demonstrate that simvastatin induces myotube atrophy and cell loss associated with impaired ADP-stimulated maximal mitochondrial respiratory capacity, mitochondrial oxidative stress, and apoptosis in primary human skeletal myotubes, suggesting that mitochondrial dysfunction may underlie human statin-induced myopathy.

Skeletal Muscle Fat Oxidation Is Increased in African-American and White Women after 10 days of Endurance Exercise Training

Objective: Obesity is associated with lower rates of skeletal muscle fatty acid oxidation (FAO), which is linked to insulin resistance. FAO is reduced further in obese African‐American (AAW) vs. white women (CW) and may also be lower in lean AAW vs. CW. In lean CW, endurance exercise training (EET) elevates the oxidative capacity of skeletal muscle. Therefore, we determined whether EET would elevate skeletal muscle FAO similarly in AAW and CW with a lower lipid oxidative capacity.

Metformin selectively attenuates mitochondrial H2O2 emission without affecting respiratory capacity in skeletal muscle of obese rats

Metformin is a widely prescribed drug for treatment of type 2 diabetes, although no cellular mechanism of action has been established. To determine whether in vivo metformin treatment alters mitochondrial function in skeletal muscle, respiratory O(2) flux and H(2)O(2) emission were measured in saponin-permeabilized myofibers from lean and obese (fa/fa) Zucker rats treated for 4 weeks with metformin. Succinate- and palmitoylcarnitine-supported respiration generated greater than twofold higher rates of H(2)O(2) emission in myofibers from untreated obese versus lean rats, indicative of an obesity-associated increased mitochondrial oxidant emitting potential. In conjunction with improved glycemic control, metformin treatment reduced H(2)O(2) emission in muscle from obese rats to rates near or below those observed in lean rats during both succinate- and palmitoylcarnitine-supported respiration. Surprisingly, metformin treatment did not affect basal or maximal rates of O(2) consumption in muscle from obese or lean rats. Ex vivo dose-response experiments revealed that metformin inhibits complex I-linked H(2)O(2) emission at a concentration approximately 2 orders of magnitude lower than that required to inhibit respiratory O(2) flux. These findings suggest that therapeutic concentrations of metformin normalize mitochondrial H(2)O(2) emission by blocking reverse electron flow without affecting forward electron flow or respiratory O(2) flux in skeletal muscle.

Skeletal muscle lipid metabolism: A frontier for new insights into fuel homeostasis

Abstract Although skeletal muscle is recognized as a primary site of lipid utilization, the study of muscle bioenergetics has focused mainly on carbohydrate, and consequently our understanding of the variables that regulate muscle lipid metabolism is comparably poor. This review focuses on the significance of muscle lipid metabolism in regulating whole-body energy homeostasis. Multiple pathways involved in controlling muscle lipid biochemistry are discussed, and comparisons with other tissues are described. Considerable evidence indicates that muscle lipid biochemistry is altered in disease states, and a number of metabolic disorders may be explained by dysregulation of muscle lipid metabolism. An understanding of the factors accounting for dysregulated muscle metabolism is a necessity in light of the increase in the incidence of disease syndromes such as obesity and diabetes, which collectively account for a high incidence of morbidity and mortality in the western society. Therefore, the purpose of this review is to describe the biochemical events involved in the regulation and dysregulation of skeletal muscle lipid metabolism and to encourage new investigation in muscle lipid research.

Progesterone increases skeletal muscle mitochondrial H2O2 emission in nonmenopausal women.

The luteal phase of the female menstrual cycle is associated with both 1) elevated serum progesterone (P4) and estradiol (E2), and 2) reduced insulin sensitivity. Recently, we demonstrated a link between skeletal muscle mitochondrial H(2)O(2) emission (mE(H2O2)) and insulin resistance. To determine whether serum levels of P4 and/or E(2) are related to mitochondrial function, mE(H2O2) and respiratory O(2) flux (Jo(2)) were measured in permeabilized myofibers from insulin-sensitive (IS, n = 24) and -resistant (IR, n = 8) nonmenopausal women (IR = HOMA-IR > 3.6). Succinate-supported mE(H2O2) was more than 50% greater in the IR vs. IS women (P < 0.05). Interestingly, serum P4 correlated positively with succinate-supported mE(H2O2) (r = 0. 53, P < 0.01). To determine whether P4 or E2 directly affect mitochondrial function, saponin-permeabilized vastus lateralis myofibers biopsied from five nonmenopausal women in the early follicular phase were incubated in P4 (60 nM), E2 (1.4 nM), or both. P4 alone inhibited state 3 Jo(2), supported by multisubstrate combination (P < 0.01). However, E2 alone or in combination with P4 had no effect on Jo(2). In contrast, during state 4 respiration, supported by succinate and glycerophosphate, mE(H2O2) was increased with P4 alone or in combination with E2 (P < 0.01). The results suggest that 1) P4 increases mE(H2O2) with or without E2; 2) P4 alone inhibits Jo(2) but not when E2 is present; and 3) P4 is related to the mE(H2O2) previously linked to skeletal muscle insulin resistance.