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.

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. Uncoupling protein 3 (UCP3)1 (1-3) is a member of the mitochondrial anion carrier superfamily with high homology (57%) to UCP1, a well characterized uncoupling protein (4, 5). UCP3 together with UCP1, UCP2 (6, 7), and possibly BMCP1 (brain mitochondrial carrier protein) (8) and UCP4 (9), form a family of uncoupling proteins located in the inner mitochondrial membrane. The evidence supporting the uncoupling activity of these proteins comes from studies where UCPs have been heterologously expressed in yeast or reconstituted into proteoliposomes. The expression of UCP2 and -3 decreases the mitochondrial membrane potential, as assessed by uptake of fluorescent membrane potential-sensitive dyes in whole yeast. They also increase state 4 respiration in isolated mitochondria, which serves as an indicator of inner membrane proton leak (3, 6, 10). More recently, reconstitution of UCPs into liposomes has shown that UCP2 and UCP3, like UCP1, mediate proton transport across bilipid layers (11). It is well established that UCP1 is exclusively expressed in brown fat, where it plays a key role in facultative thermogenesis in rodents. Although there is controversy about the molecular mechanisms involved (12-16), it is clear that activated UCP1 catalyzes a proton leak across the mitochondrial inner membrane leading to thermogenesis. The activity of UCP1 is highly regulated, facilitated by fatty acids and inhibited by purine ribose di-and trinucleotides (ATP, ADP, GTP, GDP) (17). UCP1 is also highly regulated at the transcriptional level (18) by cat...

The purpose of this study was to discern cellular mechanisms that contribute to the suppression of lipid oxidation in the skeletal muscle of obese individuals. Muscle was obtained from obese [body mass index (BMI), 38.3 +/- 3.1 kg/m(2)] and lean (BMI, 23.8 +/- 0.9 kg/m(2)) women, and fatty acid oxidation was studied by measuring (14)CO(2) production from (14)C-labeled fatty acids. Palmitate oxidation, which is at least partially dependent on carnitine palmitoyltransferase-1 (CPT-1) activity, was depressed (P < 0.05) by approximately 50% with obesity (6.8 +/- 2.2 vs. 13.7 +/- 1.4 nmole CO(2).g(-1).h(-1)). The CPT-1-independent event of palmitoyl carnitine oxidation was also depressed (P < 0.01) by approximately 45%. There were significant negative relationships (P < 0.05) for adiposity with palmitate (r = -0.76) and palmitoyl carnitine (r = -0.82) oxidation. Muscle CPT-1 and citrate synthase activity, an index of mitochondrial content, were also significantly (P < 0.05) reduced ( approximately 35%) with obesity. CPT-1 (r = -0.48) and citrate synthase (r = -0.65) activities were significantly (P < 0.05) related to adiposity. These data suggest that lesions at CPT-1 and post-CPT-1 events, such as mitochondrial content, contribute to the reduced reliance on fat oxidation evident in human skeletal muscle with obesity.

. Skeletal muscle lipid metabolism with obesity. Am J Physiol Endocrinol Metab 284: E741-E747, 2003. First published December 27, 2002 10.1152/ajpendo.00514.2002The objectives of this study were to 1) examine skeletal muscle fatty acid oxidation in individuals with varying degrees of adiposity and 2) determine the relationship between skeletal muscle fatty acid oxidation and the accumulation of long-chain fatty acyl-CoAs. Muscle was obtained from normal-weight [n ϭ 8; body mass index (BMI) 23.8 Ϯ 0.58 kg/m 2 ], overweight/obese (n ϭ 8; BMI 30.2 Ϯ 0.81 kg/m 2 ), and extremely obese (n ϭ 8; BMI 53.8 Ϯ 3.5 kg/m 2 ) females undergoing abdominal surgery. Skeletal muscle fatty acid oxidation was assessed in intact muscle strips. Long-chain fatty acyl-CoA concentrations were measured in a separate portion of the same muscle tissue in which fatty acid oxidation was determined. Palmitate oxidation was 58 and 83% lower in skeletal muscle from extremely obese (44.9 Ϯ 5.2 nmol ⅐ g Ϫ1 ⅐ h Ϫ1 ) patients compared with normal-weight (71.0 Ϯ 5.0 nmol ⅐ g Ϫ1 ⅐ h Ϫ1 ) and overweight/obese (82.2 Ϯ 8.7 nmol ⅐ g Ϫ1 ⅐ h Ϫ1 ) patients, respectively. Palmitate oxidation was negatively (R ϭ Ϫ0.44, P ϭ 0.003) associated with BMI. Long-chain fatty acyl-CoA content was higher in both the overweight/obese and extremely obese patients compared with normal-weight patients, despite significantly lower fatty acid oxidation only in the extremely obese. No associations were observed between long-chain fatty acyl-CoA content and palmitate oxidation. These data suggest that there is a defect in skeletal muscle fatty acid oxidation with extreme obesity but not overweight/obesity and that the accumulation of intramyocellular long-chain fatty acyl-CoAs is not solely a result of reduced fatty acid oxidation.long-chain fatty acyl-coenzyme A; intramyocellular triacylglycerol; fatty acids THE PREVALENCE OF OVERWEIGHT/OBESITY and insulin resistance is continually increasing and is associated with increased risk for the development of non-insulin-dependent diabetes mellitus (NIDDM), hypertension, and cardiovascular disease (5,11,24). The cellular mechanisms responsible for insulin resistance with overweight and obesity are not yet clear. Data have shown that intramyocellular triacylglycerols (IMTG) are increased with obesity and NIDDM (14,19,21). In addition, the accumulation of IMTG is associated with skeletal muscle insulin resistance (3,13,15,19,23,28,29,31,36,39). It is believed, however, that the accumulation of IMTG is not the direct cause of the development of insulin resistance but that IMTG is an inert marker for the presence of other lipid intermediates (diacylglycerol, fatty acyl-CoAs, or ceramide, etc.), which have been directly linked to defects in insulin signaling (8,17,25,32,37).To date, the mechanism(s) responsible for the accretion of IMTG and intermediates of lipid metabolism in intact skeletal muscle are not evident. Two possibilities include an increase in lipid synthesis and/or a reduction in fatty acid oxidation, both of which may res...

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 Ͼ 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 Ͻ 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 Ͻ 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. (

We recently reported that collaborative testing (i.e., group test taking) increased student performance on quizzes. It is unknown, however, whether collaborative testing improves student retention of course content. Therefore, this study was designed to test the hypotheses that collaborative-group testing improves student retention of course content. To test this hypothesis, our undergraduate exercise physiology class of 38 students was randomly divided into two groups: group A (n = 19) and group B (n = 19). During exam 1, students from both groups answered questions in the traditional format as individuals. Immediately after completing the exam as individuals, students from group A answered a randomly selected subset of questions from exam 1 in groups of two (1 group had 3 students) to test the effectiveness of collaborative-group testing on test performance and level of student retention. On the next exam (exam 2, 4 wk later), students from both groups answered questions in the traditional format as individuals and responded to the same subset of questions from exam 1. The subset of questions was analyzed to determine the level of retention of the original test material. In addition, immediately after completing the exam as individuals, students from group B answered a randomly selected subset of questions from exam 2 in groups of two (1 group had 3 students). Finally, on the next exam (exam 3, 4 wk later), students from both groups answered questions in the traditional format as individuals and responded to the same subset of questions from exam 2. This protocol followed a randomized crossover design to control for time and order effects. Student retention of course content was reduced when students completed the original examinations individually. In sharp contrast, student retention was improved (P < 0.05) when students completed the original examinations in groups. Results suggest that collaborative testing is an effective strategy to enhance learning and increase student retention of course content.

Dohm GL, Cortright RN, Lust RM. Artificial selection for high-capacity endurance running is protective against high-fat diet-induced insulin resistance. Am J Physiol Endocrinol Metab 293: E31-E41, 2007. First published March 6, 2007; doi:10.1152/ajpendo.00500.2006.-Elevated oxidative capacity, such as occurs via endurance exercise training, is believed to protect against the development of obesity and diabetes. Rats bred both for low (LCR)-and high (HCR)-capacity endurance running provide a genetic model with inherent differences in aerobic capacity that allows for the testing of this supposition without the confounding effects of a training stimulus. The purpose of this investigation was to determine the effects of a high-fat diet (HFD) on weight gain patterns, insulin sensitivity, and fatty acid oxidative capacity in LCR and HCR male rats in the untrained state. Results indicate chow-fed LCR rats were heavier, hypertriglyceridemic, less insulin sensitive, and had lower skeletal muscle oxidative capacity compared with HCR rats. Upon exposure to an HFD, LCR rats gained more weight and fat mass, and their insulin resistant condition was exacerbated, despite consuming similar amounts of metabolizable energy as chow-fed controls. These metabolic variables remained unaltered in HCR rats. The HFD increased skeletal muscle oxidative capacity similarly in both strains, whereas hepatic oxidative capacity was diminished only in LCR rats. These results suggest that LCR rats are predisposed to obesity and that expansion of skeletal muscle oxidative capacity does not prevent excess weight gain or the exacerbation of insulin resistance on an HFD. Elevated basal skeletal muscle oxidative capacity and the ability to preserve liver oxidative capacity may protect HCR rats from HFD-induced obesity and insulin resistance. fatty acid; lipid metabolism; liver; heart; skeletal muscle THE INCIDENCE OF METABOLIC DISEASES such as obesity and type II diabetes is increasing dramatically and is strongly linked to the rise in cardiovascular disease. In 2002, ϳ64% of the population in the United States was classified as overweight or obese (22), and health care costs attributable to these conditions exceeded $78 billion dollars (13). Although type II diabetes afflicts a substantially lower percentage (ϳ6.3%) of the population (9), this disease accounts for $132 billion in annual health care costs (24). With the increase in the incidence of such metabolic diseases reaching epidemic proportions and the threat of health care costs spiraling out of control, much research has been focused toward elucidating the mechanisms involved in the etiology of these conditions in hopes of ultimately discovering better treatments. Several therapies are currently used to alleviate symptoms of these diseases, but other than dietary modifications, endurance exercise is the only universally prescribed treatment.Enhanced aerobic capacity has long been associated with diminished morbidity and improvements in functional living, yet all the physiological mechanisms ...

Skeletal muscle contains two populations of mitochondria that appear to be differentially affected by disease and exercise training. It remains unclear how these mitochondrial subpopulations contribute to fiber type-related and/or training-induced changes in fatty acid oxidation and regulation of carnitine palmitoyltransferase-1beta (CPT1beta), the enzyme that controls mitochondrial fatty acid uptake in skeletal muscle. To this end, we found that fatty acid oxidation rates were 8.9-fold higher in subsarcolemmal mitochondria (SS) and 5.3-fold higher in intermyofibrillar mitochondria (IMF) that were isolated from red gastrocnemius (RG) compared with white gastrocnemius (WG) muscle, respectively. Malonyl-CoA (10 muM), a potent inhibitor of CPT1beta, completely abolished fatty acid oxidation in SS and IMF mitochondria from WG, whereas oxidation rates in the corresponding fractions from RG were inhibited only 89% and 60%, respectively. Endurance training also elicited mitochondrial adaptations that resulted in enhanced fatty acid oxidation capacity. Ten weeks of treadmill running differentially increased palmitate oxidation rates 100% and 46% in SS and IMF mitochondria, respectively. In SS mitochondria, elevated fatty acid oxidation rates were accompanied by a 48% increase in citrate synthase activity but no change in CPT1 activity. Nonlinear regression analyses of mitochondrial fatty acid oxidation rates in the presence of 0-100 muM malonyl-CoA indicated that IC(50) values were neither dependent on mitochondrial subpopulation nor affected by exercise training. However, in IMF mitochondria, training reduced the Hill coefficient (P < 0.05), suggesting altered CPT1beta kinetics. These results demonstrate that endurance exercise provokes subpopulation-specific changes in mitochondrial function that are characterized by enhanced fatty acid oxidation and modified CPT1beta-malonyl-CoA dynamics.