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...