The yeast sir2 gene and its orthologues in Drosophila and C. elegans have well-established roles in lifespan determination and response to caloric restriction. We have studied mice carrying two null alleles for SirT1, the mammalian orthologue of sir2, and found that these animals inefficiently utilize ingested food. These mice are hypermetabolic, contain inefficient liver mitochondria, and have elevated rates of lipid oxidation. When challenged with a 40% reduction in caloric intake, normal mice maintained their metabolic rate and increased their physical activity while the metabolic rate of SirT1-null mice dropped and their activity did not increase. Moreover, CR did not extend lifespan of SirT1-null mice. Thus, SirT1 is an important regulator of energy metabolism and, like its orthologues from simpler eukaryotes, the SirT1 protein appears to be required for a normal response to caloric restriction.

Oxidative stress in skeletal muscle is a hallmark of various pathophysiologic states that also feature increased reliance on long-chain fatty acid (LCFA) substrate, such as insulin resistance and exercise. However, little is known about the mechanistic basis of the LCFA-induced reactive oxygen species (ROS) burden in intact mitochondria, and elucidation of this mechanistic basis was the goal of this study. Specific aims were to determine the extent to which LCFA catabolism is associated with ROS production and to gain mechanistic insights into the associated ROS production. Because intermediates and byproducts of LCFA catabolism may interfere with antioxidant mechanisms, we predicted that ROS formation during LCFA catabolism reflects a complex process involving multiple sites of ROS production as well as modified mitochondrial function. Thus, we utilized several complementary approaches to probe the underlying mechanism(s). Using skeletal muscle mitochondria, our findings indicate that even a low supply of LCFA is associated with ROS formation in excess of that generated by NADH-linked substrates. Moreover, ROS production was evident across the physiologic range of membrane potential and was relatively insensitive to membrane potential changes. Determinations of topology and membrane potential as well as use of inhibitors revealed complex III and the electron transfer flavoprotein (ETF) and ETF-oxidoreductase, as likely sites of ROS production. Finally, ROS production was sensitive to matrix levels of LCFA catabolic intermediates, indicating that mitochondrial export of LCFA catabolic intermediates can play a role in determining ROS levels. Reactive oxygen species (ROS)2 are generated in mitochondria as a normal byproduct of aerobic metabolism; 0.2-2% of O 2 consumption is estimated to be lost as superoxide under normal conditions (1, 2). Within the mitochondrial matrix, a suite of enzymes manages the ROS load by converting ROS to less toxic species or by mitigating their formation. Nevertheless, mitochondria are significant sources of cellular ROS, and oxidative stress is a hallmark of various physiological and pathological states, including exercise, insulin resistance, and atherosclerosis (3-10).Inhibitor studies in mitochondria utilizing NADH-or FADH 2 -linked substrates suggest that complexes I and III of the electron transport chain (ETC) are predominant sites of superoxide production (11-13). Whether this is the case under physiologic conditions is not well appreciated. ROS generation by the ETC is critically dependent upon ETC redox state, such that ROS production is low until the inner membrane is significantly polarized and then rises steeply with small increments in membrane potential (14). However, a membrane potential-independent component of ROS production has been observed in brain mitochondria (15)(16)(17). Indeed, electrons may escape from sites other than the respiratory complexes, such as from the electron transfer flavoprotein (ETF) and ETF-ubiquinone oxidoreductase (ETF-QO) (18) or the ␣-ket...

Uncoupling protein-3 (UCP3) is a mitochondrial inner membrane protein expressed most abundantly in skeletal muscle and to a lesser extent in heart and brown adipose tissue. Evidence supports a role for UCP3 in fatty acid oxidation (FAO); however, the underlying mechanism has not been explored. In 2001 we proposed a role for UCP3 in fatty acid export, leading to higher FAO rates (Himms-Hagen, J., and Uncoupling protein-3 (UCP3)3 is a member of the family of mitochondrial inner membrane anion carrier proteins which includes uncoupling protein-1 (UCP1), expressed exclusively in brown adipose tissue where it mediates adaptive thermogenesis via an inducible proton leak (1). UCP3 shares 57% amino acid homology with UCP1, has the same predicted tertiary structure, and like UCP1, possesses a nucleotide binding domain (2, 3). UCP3 protein is most abundant in skeletal muscle and is present to a lesser extent in brown adipose tissue and heart (4). In contrast to UCP1, the physiological role and underlying mechanism of action of UCP3 are as yet unresolved. A proposed function for UCP3 is in lipid metabolism (5, 6) and supportive evidence has accrued. In Gullah African-Americans an exon 6 splice junction polymorphism resulting in a truncated form of UCP3 was associated with lower fatty acid oxidation (FAO) as assessed by indirect calorimetry (7); similar results were found in Ucp3 Ϫ/Ϫ mice (8). Early overexpression studies in mice and human muscle cells reported increased FAO (9, 10); however, these results may be confounded by nonspecifically increased basal proton leak due to supraphysiological UCP3 overexpression (11,12). Physiological overexpression is associated with greater FAO in L6 myotubes (13) and elevated maximal activity of carnitine palmitoyl transferase-1, ␤-hydroxyacyl dehydrogenase, and citrate synthase and lower lipid storage in mouse skeletal muscle (14,15). Although these studies functionally link UCP3 and increased FAO, the underlying mechanism remains unexplored, including whether the association between UCP3 and FAO may be related to reduced oxidative stress (13, 16 -19).Two hypotheses of UCP3-mediated fatty acid handling propose that UCP3 transports fatty acid anions from the mitochondrial matrix (20,21). Schrauwen et al. (21) suggest the physiological outcome to be reduced matrix lipotoxicity. We, on the other hand, proposed a UCP3 export function that leads to increased FAO (20). Specifically, when fatty acyl supply is high, UCP3 would act in concert with mitochondrial thioesterase-1 (MTE-1), which cleaves long chain acyl-CoA into fatty acid anions and CoA (22, 23). Fatty acid anions, which cannot be reactivated in the matrix, would be exported by UCP3 to the cytosol to be reactivated then oxidized or esterified (Fig. 1). * This work was supported by the Canadian Institutes of Health Research(Institute of Nutrition, Metabolism, and Diabetes) (to M.-E. H.), the Canadian Diabetes Association (to E. L. S.), and the Natural Sciences and Engineering Research Council (to V. B.). The costs of publication ...

Background:The contribution of E2F4 to hypoxic/ischemic neuronal death is understood poorly. Results: Loss of E2F4 leads to an increase in B-Myb and contributes to hypoxic/ischemic neuronal death. Conclusion: E2F4 is important for survival following hypoxic/ischemic neuronal death. Significance: Targeting E2F4-repressive functions may be important in maintaining neuronal survival under hypoxic/ischemic conditions.

Exercise capacity and performance strongly associate with metabolic and biophysical characteristics of skeletal muscle, factors that also relate to overall disease risk. Despite its importance, the exact mechanistic features that connect aerobic metabolism with health status are unknown. To explore this, we applied artificial selection of rats for intrinsic (i.e., untrained) aerobic treadmill running to generate strains of low- and high-capacity runners (LCR and HCR, respectively), subsequently shown to diverge for disease risk. Concurrent breeding of LCR and HCR per generation allows the lines to serve as reciprocal controls for unknown environmental changes. Here we provide the first direct evidence in mitochondria isolated from skeletal muscle that intrinsic mitochondrial capacity is higher in HCR rats. Maximal phosphorylating respiration was ~40% greater in HCR mitochondria, independent of substrate and without altered proton leak or major changes in protein levels or muscle fiber type, consistent with altered control of phosphorylating respiration. Unexpectedly, H(2)O(2) emission was ~20% higher in HCR mitochondria, due to greater reduction of more harmful reactive oxygen species to H(2)O(2); indeed, oxidative modification of mitochondrial proteins was lower. When the higher mitochondrial yield was considered, phosphorylating respiration and H(2)O(2) emission were 70-80% greater in HCR muscle. Greater capacity of HCR muscle for work and H(2)O(2) signaling may result in enhanced and more immediate cellular repair, possibly explaining lowered disease risks.