Regulation of mitochondrial uncoupling respiration during exercise in rat heart: role of reactive oxygen species (ROS) and uncoupling protein 2

The physiological significance of cardiac mitochondrial uncoupling protein 2 (UCP2)-mediated uncoupling respiration in exercise is unknown. In the current study, mitochondrial respiratory function, UCP2 mRNA level, UCP2-mediated respiration (UCR), and reactive oxygen species (ROS) generation, as well as manganese superoxide dismutase (MnSOD) activity were determined in rat heart with or without endurance training after an acute bout of exercise of different duration. In the untrained rats, state 4 respiration and UCR-independent respiration rates were progressively increased with exercise time and were 64 and 70% higher, respectively, than resting rate at 150 min, whereas UCR was elevated by 86% with no significant change in state 3 respiration. UCP2 mRNA level showed a 5- and 4-fold increase, respectively, after 45 and 90 min of exercise, but returned to resting level at 120 and 150 min. Mitochondrial ROS production and membrane potential (Deltapsi) increased progressively until 120 min, followed by a decrease to the resting level at 150 min. MnSOD mRNA abundance showed a 2-fold increase at 120 min but MnSOD activity did not change with exercise. Training significantly increased mitochondrial ATP synthetase activity, ADP to oxygen consumption (P/O) ratio, respiratory control ratio, and MnSOD activity, whereas exercise-induced state 4 respiration, UCR, ROS production, and Deltapsi were attenuated in the trained rats. We conclude that (1) UCP2 mRNA expression and activity in rat heart can be upregulated during prolonged exercise, which may reduce cross-membrane Deltapsi and thus ROS production; and (2) endurance training can blunt exercise-induced UCP2 and UCR, and improve mitochondrial efficiency of oxidative phosphorylation due to increased removal of ROS.     

Response of mitochondrial fusion and fission protein gene expression to exercise in rat skeletal muscle

The purpose of this study was to investigate the changes in the gene expression of Mitofusion (Mfn) 1 and 2 and Fission 1 (Fis1) and mitochondrial energy metabolism in response to altered energy demand during prolonged exercise in rat skeletal muscle. Male Sprague–Dawley rats were subjected to an acute bout of treadmill running at various durations and killed immediately or during recovery. Mfn1/2 and Fis1 mRNA and protein contents, reactive oxygen species (ROS) generation, state 3 and state 4 respiration rates, trans-innermembrane potential and ATP synthase activity were measured in isolated muscle mitochondria. We found that (1) Mfn1/2 mRNA contents were progressively decreased during 150 min of exercise, along with decreased Mfn 1 protein levels. Fis1 mRNA and protein contents showed significant increases after 120–150 min of exercise. These changes persisted through the recovery period up to 24 h. (2) Mitochondrial ROS generation and state 4 respiration showed progressive increases up to 120 min, but dropped at 150 min of exercise. (3) State 3 respiration rate and respiratory control index were unchanged initially but decreased at 150 and 120 min of exercise, respectively, whereas ATP synthase activity was elevated at 45 min and returned to resting level thereafter. Our data suggested that the gene expression of mitochondrial fusion and fission proteins in skeletal muscle can respond rapidly to increased metabolic demand during prolonged exercise, which could significantly affect the efficiency of oxidative phosphorylation.     

Expression of uncoupling protein-3 and mitochondrial activity in the transition from hypothyroid to hyperthyroid state in rat skeletal muscle

We sought a correlation between rat skeletal muscle triiodothyronine (T3)-mediated regulation of uncoupling protein-3 (UCP3) expression and mitochondrial activity. UCP3 mRNA expression increased strongly during the hypothyroid-hyperthyroid transition. The rank order of mitochondrial State 3 and State 4 respiration rates was hypothyroid < euthyroid < hyperthyroid. The State 4 increase may have been due to the increased UCP3 expression, as the proton leak kinetic was stimulated in the hypothyroid-hyperthyroid transition and a good correlation exists between the State 4 and UCP3 mRNA level. As a significant proportion of an organism's resting oxygen consumption is dedicated to opposing the proton leak, skeletal muscle mitochondrial UCP3 may mediate part of T3's effect on energy metabolism.     

Reduced efficiency, but increased fat oxidation, in mitochondria from human skeletal muscle after 24-h ultraendurance exercise.

The hypothesis that ultraendurance exercise influences muscle mitochondrial function has been investigated. Athletes in ultraendurance performance performed running, kayaking, and cycling at 60% of their peak O(2) consumption for 24 h. Muscle biopsies were taken preexercise (Pre-Ex), postexercise (Post-Ex), and after 28 h of recovery (Rec). Respiration was analyzed in isolated mitochondria during state 3 (coupled to ATP synthesis) and state 4 (noncoupled respiration), with fatty acids alone [palmitoyl carnitine (PC)] or together with pyruvate (Pyr). Electron transport chain activity was measured with NADH in permeabilized mitochondria. State 3 respiration with PC increased Post-Ex by 39 and 41% (P < 0.05) when related to mitochondrial protein and to electron transport chain activity, respectively. State 3 respiration with Pyr was not changed (P > 0.05). State 4 respiration with PC increased Post-Ex but was lower than Pre-Ex at Rec (P < 0.05 vs. Pre-Ex). Mitochondrial efficiency [amount of added ADP divided by oxygen consumed during state 3 (P/O ratio)] decreased Post-Ex by 9 and 6% (P < 0.05) with PC and PC + Pyr, respectively. P/O ratio remained reduced at Rec. Muscle uncoupling protein 3, measured with Western blotting, was not changed Post-Ex but tended to decrease at Rec (P = 0.07 vs. Pre-Ex). In conclusion, extreme endurance exercise decreases mitochondrial efficiency. This will increase oxygen demand and may partly explain the observed elevation in whole body oxygen consumption during standardized exercise (+13%). The increased mitochondrial capacity for PC oxidation indicates plasticity in substrate oxidation at the mitochondrial level, which may be of advantage during prolonged exercise.     

The effect of UCP3 overexpression on mitochondrial ROS production in skeletal muscle of young versus aged mice

Uncoupling protein 3 (UCP3) is suggested to protect mitochondria against aging and lipid-induced damage, possibly via modulation of reactive oxygen species (ROS) production. Here we show that mice overexpressing UCP3 (UCP3Tg) have a blunted age-induced increase in ROS production, assessed by electron spin resonance spectroscopy, but only after addition of 4-hydroxynonenal (4-HNE). Mitochondrial function, assessed by respirometry, on glycolytic substrate was lower in UCP3Tg mice compared to wild types, whereas this tended to be higher on fatty acids. State 4o respiration was higher in UCP3Tg animals. To conclude, UCP3 overexpression leads to increased state 4o respiration and, in presence of 4-HNE, blunts the age-induced increase in ROS production.     

UCP2 and UCP3 rise in starved rat skeletal muscle but mitochondrial proton conductance is unchanged

The relationship between UCP2 and UCP3 expression and mitochondrial proton conductance of rat skeletal muscle was examined. Rats were starved for 24 h and the levels of UCP2 and UCP3 mRNA and UCP3 protein were determined by Northern and Western blots. Proton conductance was measured by titrating mitochondrial respiration rate and membrane potential with malonate. Starvation increased UCP2 and UCP3 mRNA levels more than 5-fold and 4-fold, respectively, and UCP3 protein levels by 2-fold. However, proton conductance remained unchanged. These results suggest either that Northern and Western blots do not reflect the levels of active protein or that these UCPs do not catalyse the basal proton conductance in skeletal muscle mitochondria.     

Muscle Na+-K+-ATPase activity and isoform adaptations to intense interval exercise and training in well-trained athletes.

The Na+ -K+ -ATPase enzyme is vital in skeletal muscle function. We investigated the effects of acute high-intensity interval exercise, before and following high-intensity training (HIT), on muscle Na+ -K+ -ATPase maximal activity, content, and isoform mRNA expression and protein abundance. Twelve endurance-trained athletes were tested at baseline, pretrain, and after 3 wk of HIT (posttrain), which comprised seven sessions of 8 x 5-min interval cycling at 80% peak power output. Vastus lateralis muscle was biopsied at rest (baseline) and both at rest and immediately postexercise during the first (pretrain) and seventh (posttrain) training sessions. Muscle was analyzed for Na+ -K+ -ATPase maximal activity (3-O-MFPase), content ([3H]ouabain binding), isoform mRNA expression (RT-PCR), and protein abundance (Western blotting). All baseline-to-pretrain measures were stable. Pretrain, acute exercise decreased 3-O-MFPase activity [12.7% (SD 5.1), P < 0.05], increased alpha1, alpha2, and alpha3 mRNA expression (1.4-, 2.8-, and 3.4-fold, respectively, P < 0.05) with unchanged beta-isoform mRNA or protein abundance of any isoform. In resting muscle, HIT increased (P < 0.05) 3-O-MFPase activity by 5.5% (SD 2.9), and alpha3 and beta3 mRNA expression by 3.0- and 0.5-fold, respectively, with unchanged Na+ -K+ -ATPase content or isoform protein abundance. Posttrain, the acute exercise induced decline in 3-O-MFPase activity and increase in alpha1 and alpha3 mRNA each persisted (P < 0.05); the postexercise 3-O-MFPase activity was also higher after HIT (P < 0.05). Thus HIT augmented Na+ -K+ -ATPase maximal activity despite unchanged total content and isoform protein abundance. Elevated Na+ -K+ -ATPase activity postexercise may contribute to reduced fatigue after training. The Na+ -K+ -ATPase mRNA response to interval exercise of increased alpha- but not beta-mRNA was largely preserved posttrain, suggesting a functional role of alpha mRNA upregulation.     

Antioxidant supplementation enhances the exercise-induced increase in mitochondrial uncoupling protein 3 and endothelial nitric oxide synthase mRNA content in human skeletal muscle

The effects of acute exercise on the mRNA content of selected genes were examined during control conditions and after oral intake of antioxidants. In addition, to provide evidence for formation of reactive oxygen species (ROS) in human skeletal muscle during exercise, cytochrome c reduction was measured in microdialysate from the muscle. For the study on the effects of antioxidants on mRNA content, seven healthy, habitually active, male subjects participated in a double-blinded experimental design in which they, on one occasion, received a placebo and, on another, a mixture of antioxidants containing 1500 mg vitamin C, 120 mg coenzyme Q, and 345 mg alpha-tocopherol every day for 7 days before the experiment. On the experimental day the subjects cycled for 90 min and muscle biopsies were taken preexercise and at 1, 3, and 5 h after exercise. Exercise induced an increase in the eNOS, UCP3, PGC-1alpha, VEGF, Hsp72, and HO-1 mRNA content (p < 0.001), whereas there was no change in the Hsc70 mRNA level. Prior antioxidant treatment further enhanced (p < 0.05) the eNOS and UCP3 mRNA content after exercise. Moreover, the overall level of Hsc70 mRNA tended (p = 0.07) to be higher after antioxidant treatment. In another group of healthy male subjects, cytochrome c reduction was determined in microdialysate from the thigh muscle at rest and during knee extensor exercise to determine ROS formation. There was a significant increase in cytochrome c reduction with exercise both at 14 ( approximately 25%) and at 30 W ( approximately 50%). The data show that ROS are formed within skeletal muscle during exercise and that oral intake of antioxidants can enhance the exercise-induced adaptive mRNA responses of eNOS and UCP3.     

Insulin and contraction directly stimulate UCP2 and UCP3 mRNA expression in rat skeletal muscle in vitro

To study the regulation of the mitochondrial uncoupling protein 2 and 3 (UCP2 and UCP3), we studied the effect of insulin and muscle contraction on UCP mRNA expression in rat skeletal muscle in vitro. Insulin dose-dependently increased skeletal muscle UCP2 and UCP3 mRNA expression in m. extensor digitorum longus (EDL) with maximal stimulation obtained at around 0.6-6 nM. The concentration of insulin giving half-maximal stimulation was 60 pM for the UCP2 and 48 pM for the UCP3 mRNA expression. The effect of insulin was maximal after 2 h and the effect was sustained during the whole study period (6 h). The insulin-induced increase in UCP mRNA was independent of the glucose uptake (as UCP mRNA was stimulated even in incubations without glucose). In addition, electrically induced contractions (in vitro) increased UCP2 and UCP3 mRNA expression 60-120 min after a single bout of contraction (for 10 min). Both the increment of UCP2 and UCP3 mRNA were sustained throughout the study period (4 h) (153 +/- 62 and 216 +/- 71% above basal, P < 0.05 respectively). Finally, 5-aminoimidazole-4-carboxamid-ribosid (AICAR), an activator of the AMP-activated protein kinase (AMPK), that is activated during exercise, was able to mimic the increase in UCP2 and UCP3 mRNA expression. In conclusion, UCP2 and UCP3 mRNA expression in skeletal muscle are stimulated rapidly by insulin and contraction in vitro, thus the stimulation is direct and not caused by changes in other hormones or metabolites. Even a brief bout of contraction induces an increase in UCP2 and UCP3 expression, an effect that could be mimicked by activation of the AMP-activated protein kinase by AICAR.     

Long-term caloric restriction increases UCP3 content but decreases proton leak and reactive oxygen species production in rat skeletal muscle mitochondria

Calorie restriction (CR) without malnutrition increases life span and delays the onset of a variety of diseases in a wide range of animal species. However, the mechanisms responsible for the retardation of aging with CR are poorly understood. We proposed that CR may act, in part, by inducing a hypometabolic state characterized by decreased reactive oxygen species (ROS) production and mitochondrial proton leak. Here, we examine the effects of long-term CR on whole animal energetics as well as muscle mitochondrial energetics, ROS production, and ROS damage. CR was initiated in male FBNF1 rats at 6 mo of age and continued for 12 or 18 mo. Mean whole body VO2 was 34.6 (P < 0.01) and 35.6% (P < 0.001) lower in CR rats than in controls after 12 and 18 mo of CR, respectively. Body mass-adjusted VO2 was 11.1 and 29.5% lower (both P < 0.05) in CR rats than in controls after 12 and 18 mo of CR. Muscle mitochondrial leak-dependent (State 4) respiration was decreased after 12 mo compared with controls; however, after 18 mo of CR, there were slight but not statistically significant differences. Proton leak kinetics were affected by 12 mo of CR such that leak-dependent respiration was lower in CR mitochondria only at protonmotive force values exceeding 170 mV. Mitochondrial H2O2 production and oxidative damage were decreased by CR at both time points and increased with age. Muscle UCP3 protein content increased with long-term CR, consistent with a role in protection from ROS but inconsistent with the observed decrease or no change in proton leak.     

Genipin-induced inhibition of uncoupling protein-2 sensitizes drug-resistant cancer cells to cytotoxic agents

Uncoupling protein-2 (UCP2) is known to suppress mitochondrial reactive oxygen species (ROS) production and is employed by drug-resistant cancer cells to mitigate oxidative stress. Using the drug-sensitive HL-60 cells and the drug-resistant MX2 subline as model systems, we show that genipin, a UCP2 inhibitor, sensitizes drug-resistant cells to cytotoxic agents. Increased MX2 cell death was observed upon co-treatment with genipin and different doses of menadione, doxorubicin, and epirubicin. DCFH-DA fluorimetry revealed that the increase in MX2 cell death was accompanied by enhanced cellular ROS levels. The drug-induced increase in ROS was linked to genipin-mediated inhibition of mitochondrial proton leak. State 4 and resting cellular respiratory rates were higher in the MX2 cells in comparison to the HL-60 cells, and the increased respiration was readily suppressed by genipin in the MX2 cells. UCP2 accounted for a remarkable 37% of the resting cellular oxygen consumption indicating that the MX2 cells are functionally reliant on this protein. Higher amounts of UCP2 protein were detected in the MX2 versus the HL-60 mitochondria. The observed effects of genipin were absent in the HL-60 cells pointing to the selectivity of this natural product for drug-resistant cells. The specificity of genipin for UCP2 was confirmed using CHO cells stably expressing UCP2 in which genipin induced an ～22% decrease in state 4 respiration. These effects were absent in empty vector CHO cells expressing no UCP2. Thus, the chemical inhibition of UCP2 with genipin sensitizes multidrug-resistant cancer cells to cytotoxic agents.     

Increased mitochondrial proton leak in skeletal muscle mitochondria of UCP1-deficient mice

Mice having targeted inactivation of uncoupling protein 1 (UCP1) are cold sensitive but not obese (Enerb?ck S, Jacobsson A, Simpson EM, Guerra C, Yamashita H, Harper M-E, and Kozak LP. Nature 387: 90-94, 1997). Recently, we have shown that proton leak in brown adipose tissue (BAT) mitochondria from UCP1-deficient mice is insensitive to guanosine diphosphate (GDP), a well known inhibitor of UCP1 activity (Monemdjou S, Kozak LP, and Harper M-E. Am J Physiol Endocrinol Metab 276: E1073-E1082, 1999). Moreover, despite a fivefold increase of UCP2 mRNA in BAT of UCP1-deficient mice, we found no differences in the overall kinetics of this GDP-insensitive proton leak between UCP1-deficient mice and controls. Based on these findings, which show no adaptive increase in UCP1-independent leak in BAT, we hypothesized that adaptive thermogenesis may be occurring in other tissues of the UCP1-deficient mouse (e.g., skeletal muscle), thus allowing them to maintain their normal resting metabolic rate, feed efficiency, and adiposity. Here, we report on the overall kinetics of the mitochondrial proton leak, respiratory chain, and ATP turnover in skeletal muscle mitochondria from UCP1-deficient and heterozygous control mice. Over a range of mitochondrial protonmotive force (Deltap) values, leak-dependent oxygen consumption is higher in UCP1-deficient mice compared with controls. State 4 (maximal leak-dependent) respiration rates are also significantly higher in the mitochondria of mice deficient in UCP1, whereas state 4 Deltap is significantly lower. No significant differences in state 3 respiration rates or Deltap values were detected between the two groups. Thus the altered kinetics of the mitochondrial proton leak in skeletal muscle of UCP1-deficient mice indicate a thermogenic mechanism favoring the lean phenotype of the UCP1-deficient mouse.     

UCP1 muscle gene transfer and mitochondrial proton leak mediated thermogenesis

Mitochondrial uncoupling protein 1 (UCP1) mediates the thermogenic transport of protons through the inner mitochondrial membrane. This proton leak uncouples respiration from ATP synthesis. The current study assessed the possible contribution of UCP1 muscle gene transfer to impair mitochondrial respiration in a tissue lacking UCP1 gene expression. Rats received an intramuscular injection of plasmid pXC1 containing UCP1 cDNA in the right tibialis muscles, while left tibialis muscles were injected with empty plasmid as control. Ten days after DNA injection, mitochondria from tibialis anterior muscles were isolated and analyzed. UCP1 gene transfer resulted in protein expression as analyzed by inmunoblotting. Mitochondria isolated from UCP1-injected muscles showed a significant increase in state 2 and state 4 oxygen consumption rates and a decreased respiration control ratio in comparison to mitochondria from control muscles. Furthermore, UCP1-containing mitochondria had a lower membrane potential in those states (2 and 4) when compared with control mitochondria. Our results revealed that UCP1 muscle gene transfer is associated with an induced mitochondrial proton leak, which could contribute to increase energy expenditure.     

The basal proton conductance of skeletal muscle mitochondria from transgenic mice overexpressing or lacking uncoupling protein-3.

The ability of native uncoupling protein-3 (UCP3) to uncouple mitochondrial oxidative phosphorylation is controversial. We measured the expression level of UCP3 and the proton conductance of skeletal muscle mitochondria isolated from transgenic mice overexpressing human UCP3 (UCP3-tg) and from UCP3 knockout (UCP3-KO) mice. The concentration of UCP3 in UCP3-tg mitochondria was approximately 3 microg/mg protein, approximately 20-fold higher than the wild type value. UCP3-tg mitochondria had increased nonphosphorylating respiration rates, decreased respiratory control, and approximately 4-fold increased proton conductance compared with the wild type. However, this increased uncoupling in UCP3-tg mitochondria was not caused by native function of UCP3 because it was not proportional to the increase in UCP3 concentration and was neither activated by superoxide nor inhibited by GDP. UCP3 was undetectable in mitochondria from UCP3-KO mice. Nevertheless, UCP3-KO mitochondria had unchanged respiration rates, respiratory control ratios, and proton conductance compared with the wild type under a variety of assay conditions. We conclude that uncoupling in UCP3-tg mice is an artifact of transgenic expression, and that UCP3 does not catalyze the basal proton conductance of skeletal muscle mitochondria in the absence of activators such as superoxide.     

Regulation of UCP1 and UCP3 in arctic ground squirrels and relation with mitochondrial proton leak.

Uncoupling protein (UCP) 1 (UCP1) catalyzes a proton leak in brown adipose tissue (BAT) mitochondria that results in nonshivering thermogenesis (NST), but the extent to which UCP homologs mediate NST in other tissues is controversial. To clarify the role of UCP3 in mediating NST in a hibernating species, we measured Ucp3 expression in skeletal muscle of arctic ground squirrels in one of three activity states (not hibernating, not hibernating and fasted for 48 h, or hibernating) and housed at 5 degrees C or -10 degrees C. We then compared Ucp3 mRNA levels in skeletal muscle with Ucp1 mRNA and UCP1 protein levels in BAT in the same animals. Ucp1 mRNA and UCP1 protein levels were increased on cold exposure and decreased with fasting, with the highest UCP1 levels in thermogenic hibernators. In contrast, Ucp3 mRNA levels were not affected by temperature but were increased 10-fold during fasting and >3-fold during hibernation. UCP3 protein levels were increased nearly fivefold in skeletal muscle mitochondria isolated from fasted squirrels compared with nonhibernators, but proton leak kinetics in the presence of BSA were unchanged. Proton leak in BAT mitochondria also did not differ between fed and fasted animals but did show classical inhibition by the purine nucleotide GDP. Levels of nonesterified fatty acids were highest during hibernation, and tissue temperatures during hibernation were related to Ucp1, but not Ucp3, expression. Taken together, these results do not support a role for UCP3 as a physiologically relevant mediator of NST in muscle.     

A single bout of exercise with high mechanical loading induces the expression of Cyr61/CCN1 and CTGF/CCN2 in human skeletal muscle.

High mechanical loading was hypothesized to induce the expression of angiogenic and/or lymphangiogenic extracellular matrix (ECM) proteins in skeletal muscle. Eight men performed a strenuous exercise protocol, which consisted of 100 unilateral maximal drop jumps followed by submaximal jumping until exhaustion. Muscle biopsies were taken 30 min and 48 h postexercise from the vastus lateralis muscle and analyzed for the following parameters: mRNA and protein expression of ECM-associated CCN proteins [cysteine-rich angiogenic protein 61 (Cyr61)/CCN1, connective tissue growth factor (CTGF)/CCN2], and mRNA expression of vascular endothelial growth factors (VEGFs) and hypoxia-inducible factor-1alpha. The mRNA expression of Cyr61 and CTGF increased 30 min after the exercise (14- and 2.5-fold, respectively; P < 0.001). Cyr61 remained elevated 48 h postexercise (threefold; P < 0.05). The mRNA levels of VEGF-A, VEGF-B, VEGF-C, VEGF-D, or hypoxia-inducible factor-1alpha did not change significantly at either 30 min or 48 h postexercise; however, the variation between subjects increased markedly in VEGF-A and VEGF-B mRNA. Cyr61 protein levels were higher at both 30 min and 48 h after the exercise compared with the control (P < 0.05). Cyr61 and CTGF proteins were localized to muscle fibers and the surrounding ECM by immunohistochemistry. Fast fibers stained more intensively than slow fibers. In conclusion, mechanical loading induces rapid expression of CCN proteins in human skeletal muscle. This may be one of the early mechanisms involved in skeletal muscle remodeling after exercise, since Cyr61 and CTGF regulate the expression of genes involved in angiogenesis and ECM remodeling.     

Respiratory uncoupling by UCP1 and UCP2 and superoxide generation in endothelial cell mitochondria

Mitochondria represent a major source of reactive oxygen species (ROS), particularly during resting or state 4 respiration wherein ATP is not generated. One proposed role for respiratory mitochondrial uncoupling proteins (UCPs) is to decrease mitochondrial membrane potential and thereby protect cells from damage due to ROS. This work was designed to examine superoxide production during state 4 (no ATP production) and state 3 (active ATP synthesis) respiration and to determine whether uncoupling reduced the specific production of this radical species, whether this occurred in endothelial mitochondria per se, and whether this could be modulated by UCPs. Superoxide formation by isolated bovine aortic endothelial cell (BAE) mitochondria, determined using electron paramagnetic resonance spectroscopy, was approximately fourfold greater during state 4 compared with state 3 respiration. UCP1 and UCP2 overexpression both increased the proton conductance of endothelial cell mitochondria, as rigorously determined by the kinetic relationship of respiration to inner membrane potential. However, despite uncoupling, neither UCP1 nor UCP2 altered superoxide formation. Antimycin, known to increase mitochondrial superoxide, was studied as a positive control and markedly enhanced the superoxide spin adduct in our mitochondrial preparations, whereas the signal was markedly impaired by the powerful chemical uncoupler p-(trifluoromethoxyl)-phenyl-hydrazone. In summary, we show that UCPs do have uncoupling properties when expressed in BAE mitochondria but that uncoupling by UCP1 or UCP2 does not prevent acute substrate-driven endothelial cell superoxide as effluxed from mitochondria respiring in vitro.     

Insulin-resistant muscle is exercise resistant: evidence for reduced response of nuclear-encoded mitochondrial genes to exercise

Mitochondrial dysfunction, associated with insulin resistance, is characterized by low expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) and nuclear-encoded mitochondrial genes. This deficit could be due to decreased physical activity or a decreased response of gene expression to exercise. The objective of this study was to investigate whether a bout of exercise induces the same increase in nuclear-encoded mitochondrial gene expression in insulin-sensitive and insulin-resistant subjects matched for exercise capacity. Seven lean and nine obese subjects took part. Insulin sensitivity was assessed by an 80 mU.m(-2).min(-1) euglycemic clamp. Subjects were matched for aerobic capacity and underwent a single bout of exercise at 70 and 90% of maximum heart rate with muscle biopsies at 30 and 300 min postexercise. Quantitative RT-PCR and immunoblot analyses were used to determine the effect of exercise on gene expression and protein abundance and phosphorylation. In the postexercise period, lean subjects immediately increased PGC-1alpha mRNA level (reaching an eightfold increase by 300 min postexercise) and protein abundance and AMP-dependent protein kinase phosphorylation. Activation of PGC-1alpha was followed by increase of nuclear respiratory factor-1 and cytochrome c oxidase (subunit VIc). However, in insulin-resistant subjects, there was a delayed and reduced response in PGC-1alpha mRNA and protein, and phosphorylation of AMP-dependent protein kinase was transient. None of the genes downstream of PGC-1alpha was increased after exercise in insulin resistance. Insulin-resistant subjects have a reduced response of nuclear-encoded mitochondrial genes to exercise, and this could contribute to the origin and maintenance of mitochondrial dysfunction.     

Uncoupling proteins and sleep deprivation

In both humans and animals sleep deprivation (SD) produces an increase in food intake and in energy expenditure (EE). The increase in EE is a core element of the SD syndrome and, in rats, is negatively correlated with survival rate. However, the mechanisms involved are not understood. A large component of resting EE is accounted for by the mitochondrial proton leak, which is mediated by uncoupling proteins (UCPs). We measured UCP2, UCP3, and UCP5 mRNA levels in rats during the spontaneous sleep/waking cycle and after short (8 hours) and long (7 days) SD. During spontaneous sleep and waking there was no change in the level of mitochondrial uncoupling as measured by UCPs expression, either in the brain or in peripheral tissues. During SD, by contrast, UCP3 expression in skeletal muscle was elevated, but the increase was similar, compared to sleep, after both short-term and long-term SD. UCP2 expression, on the other hand, was strongly increased in the liver and skeletal muscle of long-term sleep deprived animals and much less so, or not at all, in yoked controls or in rats that lost only 8 hours of sleep. Since the skeletal muscle is the largest tissue in the body, an elevated muscular expression of UCP2 is likely to affect the overall resting EE and may thus contribute to its increase after SD.