AB3046 Sigma-AldrichAnti-Uncoupling Protein 3 Antibody

We explored whether altered expression of factors tuning mitochondrial metabolism contributes to muscular adaptations with endurance training in the condition of lowered ambient oxygen concentration (hypoxia) and whether these adaptations relate to oxygen transfer as reflected by subsarcolemmal mitochondria and oxygen metabolism in muscle.Male volunteers completed 30 bicycle exercise sessions in normoxia or normobaric hypoxia (4,000 m above sea level) at 65% of the respective peak aerobic power output. Myoglobin content, basal oxygen consumption, and re-oxygenation rates upon reperfusion after 8 min of arterial occlusion were measured in vastus muscles by magnetic resonance spectroscopy. Biopsies from vastus lateralis muscle, collected pre and post a single exercise bout, and training, were assessed for levels of transcripts and proteins being associated with mitochondrial metabolism.Hypoxia specifically lowered the training-induced expression of markers of respiratory complex II and IV (i.e. SDHA and isoform 1 of COX-4; COX4I1) and preserved fibre cross-sectional area. Concomitantly, trends (p less than 0.10) were found for a hypoxia-specific reduction in the basal oxygen consumption rate, and improvements in oxygen repletion, and aerobic performance in hypoxia. Repeated exercise in hypoxia promoted the biogenesis of subsarcolemmal mitochondria and this was co-related to expression of isoform 2 of COX-4 with higher oxygen affinity after single exercise, de-oxygenation time and myoglobin content (r ≥ 0.75). Conversely, expression in COX4I1 with training correlated negatively with changes of subsarcolemmal mitochondria (r less than -0.82).Hypoxia-modulated adjustments of aerobic performance with repeated muscle work are reflected by expressional adaptations within the respiratory chain and modified muscle oxygen metabolism.

We hypothesised that load-sensitive expression of costameric proteins, which hold the sarcomere in place and position the mitochondria, contributes to the early adaptations of antigravity muscle to unloading and would depend on muscle fibre composition and chymotrypsin activity of the proteasome. Biopsies were obtained from vastus lateralis (VL) and soleus (SOL) muscles of eight men before and after 3 days of unilateral lower limb suspension (ULLS) and subjected to fibre typing and measures for costameric (FAK and FRNK), mitochondrial (NDUFA9, SDHA, UQCRC1, UCP3, and ATP5A1), and MHCI protein and RNA content. Mean cross-sectional area (MCSA) of types I and II muscle fibres in VL and type I fibres in SOL demonstrated a trend for a reduction after ULLS (0.05 ≤ P less than 0.10). FAK phosphorylation at tyrosine 397 showed a 20% reduction in VL muscle (P = 0.029). SOL muscle demonstrated a specific reduction in UCP3 content (-23%; P = 0.012). Muscle-specific effects of ULLS were identified for linear relationships between measured proteins, chymotrypsin activity and fibre MCSA. The molecular modifications in costamere turnover and energy homoeostasis identify that aspects of atrophy and fibre transformation are detectable at the protein level in weight-bearing muscles within 3 days of unloading.

The basal metabolic rate (BMR) is referred to as the minimal rate of metabolism required to support basic body functions. It is well known that individual BMR varies greatly, even when correcting for body weight, fat content, and thyroid hormone levels, but the mechanistic determinants of this phenomenon remain unknown. Here, we show in humans that mass-related BMR correlates strongly to the mitochondrial oxygen affinity (p50(mito); R(2)=0.66, P=0.0004) measured in isolated skeletal muscle mitochondria. A similar relationship was found for oxygen affinity and efficiency during constant-load submaximal exercise (R(2)=0.46, P=0.007). In contrast, BMR did not correlate to overall mitochondrial density or to proton leak. Mechanistically, part of the p50(mito) seems to be controlled by the excess of cytochrome c oxidase (COX) protein and activity relative to other mitochondrial proteins. This is illustrated by the 5-fold increase in p50(mito) after partial cyanide inhibition of COX at doses that do not affect maximal mitochondrial electron flux through the ETS. These data suggest that the interindividual variation in BMR in humans is primarily explained by differences in mitochondrial oxygen affinity. The implications of these findings are discussed in terms of a trade-off between aerobic efficiency and power.

We recently showed that a week-long, high-fat diet reduced whole body exercise efficiency in sedentary men by greater than 10% (Edwards LM, Murray AJ, Holloway CJ, Carter EE, Kemp GJ, Codreanu I, Brooker H, Tyler DJ, Robbins PA, Clarke K. FASEB J 25: 1088-1096, 2011). To test if a similar dietary regime would blunt whole body efficiency in endurance-trained men and, as a consequence, hinder aerobic exercise performance, 16 endurance-trained men were given a short-term, high-fat (70% kcal from fat) and a moderate carbohydrate (50% kcal from carbohydrate) diet, in random order. Efficiency was assessed during a standardized exercise task on a cycle ergometer, with aerobic performance assessed during a 1-h time trial and mitochondrial function later measured using (31)P-magnetic resonance spectroscopy. The subjects then underwent a 2-wk wash-out period, before the study was repeated with the diets crossed over. Muscle biopsies, for mitochondrial protein analysis, were taken at the start of the study and on the 5th day of each diet. Plasma fatty acids were 60% higher on the high-fat diet compared with moderate carbohydrate diet (P less than 0.05). However, there was no change in whole body efficiency and no change in mitochondrial function. Endurance exercise performance was significantly reduced (P less than 0.01), most probably due to glycogen depletion. Neither diet led to changes in citrate synthase, ATP synthase, or mitochondrial uncoupling protein 3. We conclude that prior exercise training blunts the deleterious effect of short-term, high-fat feeding on whole body efficiency.

Although the literature contains many studies on the function of UCP3, its role is still being debated. It has been hypothesized that UCP3 may mediate lipid hydroperoxide (LOOH) translocation across the mitochondrial inner membrane (MIM), thus protecting the mitochondrial matrix from this very aggressive molecule. However, no experiments on mitochondria have provided evidence in support of this hypothesis. Here, using mitochondria isolated from UCP3-null mice and their wild-type littermates, we demonstrate the following. (i) In the absence of free fatty acids, proton conductance did not differ between wild-type and UCP3-null mitochondria. Addition of arachidonic acid (AA) to such mitochondria induced an increase in proton conductance, with wild-type mitochondria showing greater enhancement. In wild-type mitochondria, the uncoupling effect of AA was significantly reduced both when the release of O2* in the matrix was inhibited and when the formation of LOOH was inhibited. In UCP3-null mitochondria, however, the uncoupling effect of AA was independent of the above mechanisms. (ii) In the presence of AA, wild-type mitochondria released significantly more LOOH compared with UCP3-null mitochondria. This difference was abolished both when UCP3 was inhibited by GDP and under a condition in which there was reduced LOOH formation on the matrix side of the MIM. These data demonstrate that UCP3 is involved both in mediating the translocation of LOOH across the MIM and in LOOH-dependent mitochondrial uncoupling.

An uncoupling protein (cUCP) was identified in heart and skeletal muscle mitochondria of canary birds. cUCP was immunodetected using polyclonal antibodies raised against murine UCP2. Its molecular mass was similar to those of mammalian UCPs (32 kDa). The activity of cUCP was stimulated by palmitic acid (PA) and inhibited by GTP mainly in state 3 respiration. Additions of PA augmented state 4 respiration and lowered the ADP/O ratio. Thus, the activity of cUCP diverted energy from oxidative phosphorylation in state 3 respiration. cUCP in heart and skeletal muscles of canary birds might have implications in thermogenesis as well as protection against free radical production.

Uncoupling protein-3 (UCP3) is expressed in liver only under conditions of high fatty acid catabolism. However, the specific role of UCP3 in liver mitochondria and overall hepatic function is still poorly known.

Uncoupling protein 3 (UCP3) is a mitochondrial inner membrane protein, which is hypothesized to shuttle nonmetabolized fatty acids, particularly when excessive fatty acids are present. PURPOSE: Obese Zucker rats (OZR) have systematically elevated levels of fatty acids, with decreased fatty acid metabolism. We hypothesized that basal UCP3 protein expression levels would be elevated in the skeletal muscles of the OZR compared with the lean Zucker rats (LZR). In addition, because aerobic exercise training has been shown to elevate the ability of skeletal muscle to metabolize lipids, we also hypothesized that aerobic exercise training would decrease skeletal muscle UCP3 protein expression and that this would be more pronounced in the skeletal muscles of the OZR. METHODS: OZR and LZR were aerobically trained on a motorized treadmill for 55 min x d(-1), 5 d x wk(-1), for 9 wk. UCP3 and oxidative enzymes were measured in plantaris, gastrocnemius, and soleus muscles. RESULTS: Basal UCP3 protein expression was elevated approximately eightfold in the plantaris muscles and threefold in the gastrocnemius muscles of the OZR compared with the LZR (P 0.05). However, there was no difference in UCP3 protein expression in the soleus muscles of the OZR compared with the LZR (P = 0.34). Furthermore, aerobic exercise training did not significantly alter UCP3 protein expression in the soleus, plantaris, or gastrocnemius muscles of the LZR; however, UCP3 protein expression levels decreased in trained OZR soleus and gastrocnemius muscles compared with controls. CONCLUSIONS: The decrease in UCP3 with aerobic exercise training was most notable in the soleus of the OZR. These data demonstrate that the exercise-induced adaptations of UCP3 protein levels are muscle specific in obese animals compared with lean animals.

Isolated perfused hearts from type 2 diabetic (db/db) mice show impaired ventricular function, as well as altered cardiac metabolism. Assessment of the relationship between myocardial oxygen consumption (MVO(2)) and ventricular pressure-volume area (PVA) has also demonstrated reduced cardiac efficiency in db/db hearts. We hypothesized that lowering the plasma fatty acid supply and subsequent normalization of altered cardiac metabolism by chronic treatment with a peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist will improve cardiac efficiency in db/db hearts. Rosiglitazone (23 mg/kg body weight/day) was administered as a food admixture to db/db mice for five weeks. Ventricular function and PVA were assessed using a miniaturized (1.4 Fr) pressure-volume catheter; MVO(2) was measured using a fibre-optic oxygen sensor. Chronic rosiglitazone treatment of db/db mice normalized plasma glucose and lipid concentrations, restored rates of cardiac glucose and fatty acid oxidation, and improved cardiac efficiency. The improved cardiac efficiency was due to a significant decrease in unloaded MVO(2), while contractile efficiency was unchanged. Rosiglitazone treatment also improved functional recovery after low-flow ischemia. In conclusion, the present study demonstrates that in vivo PPARgamma-treatment restores cardiac efficiency and improves ventricular function in perfused hearts from type 2 diabetic mice.