Muscle Citrate Synthase Activity Research Articles

Lack of β2‐AR improves exercise capacity and skeletal muscle oxidative phenotype in mice

β(2)-adrenergic receptor (β(2)-AR) agonists have been used as ergogenics by athletes involved in training for strength and power in order to increase the muscle mass. Even though anabolic effects of β(2)-AR activation are highly recognized, less is known about the impact of β(2)-AR in endurance capacity. We presently used mice lacking β(2)-AR [β(2)-knockout (β(2) KO)] to investigate the role of β(2)-AR on exercise capacity and skeletal muscle metabolism and phenotype. β(2) KO mice and their wild-type controls (WT) were studied. Exercise tolerance, skeletal muscle fiber typing, capillary-to-fiber ratio, citrate synthase activity and glycogen content were evaluated. When compared with WT, β(2) KO mice displayed increased exercise capacity (61%) associated with higher percentage of oxidative fibers (21% and 129% of increase in soleus and plantaris muscles, respectively) and capillarity (31% and 20% of increase in soleus and plantaris muscles, respectively). In addition, β(2) KO mice presented increased skeletal muscle citrate synthase activity (10%) and succinate dehydrogenase staining. Likewise, glycogen content (53%) and periodic acid-Schiff staining (glycogen staining) were also increased in β(2) KO skeletal muscle. Altogether, these data provide evidence that disruption of β(2)-AR improves oxidative metabolism in skeletal muscle of β(2) KO mice and this is associated with increased exercise capacity.

Role of Skeletal Muscle Mitochondrial Density on Exercise‐Stimulated Lipid Oxidation

Reduced skeletal muscle mitochondrial density is proposed to lead to impaired muscle lipid oxidation and increased lipid accumulation in sedentary individuals. We assessed exercise-stimulated lipid oxidation by imposing a prolonged moderate-intensity exercise in men with variable skeletal muscle mitochondrial density as measured by citrate synthase (CS) activity. After a 2-day isoenergetic high-fat diet, lipid oxidation was measured before and during exercise (650 kcal at 50% VO(2)max) in 20 healthy men with either high (HI-CS = 24 ± 1; mean ± s.e.) or low (LO-CS = 17 ± 1 nmol/min/mg protein) muscle CS activity. Vastus lateralis muscle biopsies were obtained before and immediately after exercise. Respiratory exchange data and blood samples were collected at rest and throughout the exercise. HI-CS subjects had higher VO(2)max (50 ± 1 vs. 44 ± 2 ml/kg fat free mass/min; P = 0.01), lower fasting respiratory quotient (RQ) (0.81 ± 0.01 vs. 0.85 ± 0.01; P = 0.04) and higher ex vivo muscle palmitate oxidation (866 ± 168 vs. 482 ± 78 nmol/h/mg muscle; P = 0.05) compared to LO-CS individuals. However, whole-body exercise-stimulated lipid oxidation (20 ± 2 g vs. 19 ± 1 g; P = 0.65) and plasma glucose, lactate, insulin, and catecholamine responses were similar between the two groups. In conclusion, in response to the same energy demand during a moderate prolonged exercise bout, reliance on lipid oxidation was similar in individuals with high and low skeletal muscle mitochondrial density. This data suggests that decreased muscle mitochondrial density may not necessarily impair reliance on lipid oxidation over the course of the day since it was normal under a high-lipid oxidative demand condition. Twenty-four-hour lipid oxidation and its relationship with mitochondrial density need to be assessed.

Overfeeding Reduces Insulin Sensitivity and Increases Oxidative Stress, without Altering Markers of Mitochondrial Content and Function in Humans

BackgroundMitochondrial dysfunction and increased oxidative stress are associated with obesity and type 2 diabetes. High fat feeding induces insulin resistance and increases skeletal muscle oxidative stress in rodents, but there is controversy as to whether skeletal muscle mitochondrial biogenesis and function is altered.Methodology and Principal FindingsForty (37±2 y) non-obese (25.6±0.6 kg/m2) sedentary men (n = 20) and women (n = 20) were overfed (+1040±100 kcal/day, 46±1% of energy from fat) for 28 days. Hyperinsulinemic-euglycemic clamps were performed at baseline and day 28 of overfeeding and skeletal muscle biopsies taken at baseline, day 3 and day 28 of overfeeding in a sub cohort of 26 individuals (13 men and 13 women) that consented to having all 3 biopsies performed. Weight increased on average in the whole cohort by 0.6±0.1 and 2.7±0.3 kg at days 3 and 28, respectively (P<0.0001, without a significant difference in the response between men and women (P = 0.4). Glucose infusion rate during the hyperinsulinemic-euglycemic clamp decreased from 54.8±2.8 at baseline to 50.3±2.5 µmol/min/kg FFM at day 28 of overfeeding (P = 0.03) without a significant difference between men and women (P = 0.4). Skeletal muscle protein carbonyls and urinary F2-isoprostanes increased with overfeeding (P<0.05). Protein levels of muscle peroxisome proliferator-activated receptor gamma coactivator-1α (PGC1α) and subunits from complex I, II and V of the electron transport chain were increased at day 3 (all P<0.05) and returned to basal levels at day 28. No changes were detected in muscle citrate synthase activity or ex vivo CO2 production at either time point.ConclusionsPeripheral insulin resistance was induced by overfeeding, without reducing any of the markers of mitochondrial content that were examined. Oxidative stress was however increased, and may have contributed to the reduction in insulin sensitivity observed.Trial Registration ClinicalTrials.gov NCT00562393

NADPH oxidase mutation protects against skeletal muscle insulin resistance in high fat fed mice

In the present study, we hypothesized that NADPH oxidase‐dependent increases in reactive oxygen species (ROS) associated with diet‐induced obesity impair skeletal muscle insulin action and mitochondrial function. Pathogen free B6(Cg)‐Ncf1m1J/J mice ( Jackson Laboratory), which harbor an inactivating mutation for the p47phox subunit of NADPH oxidase, and C57/BL6J (C57) control mice were maintained on either a high fat diet (HF; 45%) or low fat normal chow diet (LF, 10%) for 10 weeks. Final body weights were significantly greater for mice fed HF compared to LF diets in both C57 (29%) and B6(Cg)‐Ncf1m1J/J (30%) mice. Insulin‐stimulated glucose uptake in soleus muscle (ex vivo) was significantly lower in HF vs. LF fed C57 mice (−41%; p&lt;0.05), but not in HF B6(Cg)‐Ncf1m1J/J mice (−6%; p&gt;0.05), suggesting that NADPH oxidase activation is integral to HF diet‐induced muscle insulin resistance. Hindlimb muscle cytochrome oxidase (COX) activity increased with HF feeding in the B6(Cg)‐Ncf1m1J/J mice (38%; p&lt;0.05), but not in HF fed C57 mice. There was no significant change in muscle citrate synthase activity with HF feeding in either the B6(Cg)‐Ncf1m1J/J or C57 mice. In summary, the adverse effects of HF feeding on skeletal muscle insulin sensitivity and mitochondrial COX activity were abrogated in mice with a p47phox subunit inactivating mutation.Supported by an Endocrine Society Research Award.

Lack of myostatin alters intermyofibrillar mitochondria activity, unbalances redox status, and impairs tolerance to chronic repetitive contractions in muscle

Loss of myostatin (mstn) function leads to a decrease in mitochondrial content, a reduced expression of cytochrome c oxidase, and a lower citrate synthase activity in skeletal muscle. These data suggest functional or ultrastructural mitochondrial abnormalities that can impact on muscle endurance characteristics in such phenotype. To address this issue, we investigated subsarcolemmal and intermyofibrillar (IMF) mitochondrial activities, skeletal muscle redox homeostasis, and muscle fiber endurance quality in mstn-deficient mice [mstn knockout (KO)]. We report that lack of mstn induced a decrease in the coupling of IMF mitochondria respiration, with significantly higher basal oxygen consumption. No lysis of mitochondrial cristae or excessive swelling were observed in mstn KO mice compared with wild-type (WT) mice. Concerning redox status, mstn KO gastrocnemius exhibited a significant decrease in lipid peroxidation levels (-56%; P < 0.01 vs. WT) together with a significant upregulation of the antioxidant glutathione system. In contrast, superoxide dismutase and catalase activities were altered in mstn KO, gastrocnemius and soleus with a reduction of up to 80% compared with WT animals. The force production observed after contractile endurance test was significantly lower in extensor digitorum longus and soleus muscles of mstn KO mice compared with the controls (17 ± 3 and 36 ± 5% vs. 28 ± 4 and 56 ± 5%, respectively, P < 0.05). Together, these findings indicate that, besides an increased skeletal muscle mass, genetic mstn inhibition has differential effects on redox homeostasis and mitochondrial function that would have functional consequences on muscle response to endurance exercise.

Metformin overdose, but not lactic acidosis per se, inhibits oxygen consumption in pigs

IntroductionHepatic mitochondrial dysfunction may play a critical role in the pathogenesis of metformin-induced lactic acidosis. However, patients with severe metformin intoxication may have a 30 to 60% decrease in their global oxygen consumption, as for generalized inhibition of mitochondrial respiration. We developed a pig model of severe metformin intoxication to validate this clinical finding and assess mitochondrial function in liver and other tissues.MethodsTwenty healthy pigs were sedated and mechanically ventilated. Ten were infused with a large dose of metformin (4 to 8 g) and five were not (sham controls). Five others were infused with lactic acid to clarify whether lactic acidosis per se diminishes global oxygen use. Arterial pH, lactatemia, global oxygen consumption (VO2) (metabolic module) and delivery (DO2) (cardiac output by thermodilution) were monitored for nine hours. Oxygen extraction was computed as VO2/DO2. Activities of the main components of the mitochondrial respiratory chain (complex I, II and III, and IV) were measured with spectrophotometry (and expressed relative to citrate synthase activity) in heart, kidney, liver, skeletal muscle and platelets taken at the end of the study.ResultsPigs infused with metformin (6 ± 2 g; final serum drug level 77 ± 45 mg/L) progressively developed lactic acidosis (final arterial pH 6.93 ± 0.24 and lactate 18 ± 7 mmol/L, P < 0.001 for both). Their VO2 declined over time (from 115 ± 34 to 71 ± 30 ml/min, P < 0.001) despite grossly preserved DO2 (from 269 ± 68 to 239 ± 51 ml/min, P = 0.58). Oxygen extraction accordingly fell from 43 ± 10 to 30 ± 10% (P = 0.008). None of these changes occurred in either sham controls or pigs infused with lactic acid (final arterial pH 6.86 ± 0.16 and lactate 22 ± 3 mmol/L). Metformin intoxication was associated with inhibition of complex I in the liver (P < 0.001), heart (P < 0.001), kidney (P = 0.003), skeletal muscle (P = 0.012) and platelets (P = 0.053). The activity of complex II and III diminished in the liver (P < 0.001), heart (P < 0.001) and kidney (P < 0.005) while that of complex IV declined in the heart (P < 0.001).ConclusionsMetformin intoxication induces lactic acidosis, inhibits global oxygen consumption and causes mitochondrial dysfunction in liver and other tissues. Lactic acidosis per se does not decrease whole-body respiration.

Chronic cold acclimation increases thermogenic capacity, non-shivering thermogenesis and muscle citrate synthase activity in both wild-type and brown adipose tissue deficient mice

The purpose of this study was to determine whether chronic cold exposure would increase the aerobic capacity of skeletal muscle in UCP-dta mice, a transgenic line lacking brown adipose tissue (BAT). Wild type and UCP-dta mice were acclimated to either warm (23°C), or cold (4°C) conditions. Cold increased muscle oxidative capacity nearly equivalently in wild-type and UCP-dta mice, but did not affect the respiratory function of isolated mitochondria. Summit metabolism (V˙O2summit) and norepinephrine-induced thermogenesis (V˙O2NST) were significantly lower in UCP-dta mice relative to wild-type mice regardless of temperature treatment, but both were significantly higher in cold relative to warm acclimated mice. BAT mass was significantly higher in the cold relative to warm acclimated wild-type mice, but not in cold acclimated UCP-dta mice. BAT citrate synthase activity was lower in transgenic animals regardless of acclimation temperature and BAT citrate synthase activity per depot was significantly higher only in the cold acclimated wild-type mice. Muscle citrate synthase activity was increased in both genotypes. As defects in muscle oxidative function have been observed with obesity and type 2 diabetes, these results suggest that chronic cold exposure is a useful intervention to drive skeletal muscle oxidative capacity in mouse models of obesity.

Different types of exercise induce differential effects on neuronal adaptations and memory performance

Different exercise paradigms show differential effects on various forms of memory. We hypothesize that the differential effects of exercises on memory performance are caused by different neuroplasticity changes in relevant brain regions in response to different exercise trainings. We examined the effects of treadmill running (TR) and wheel running (WR) on the Pavlovian fear conditioning task that assesses learning and memory performance associated with the amygdala (cued conditioning) and both the amygdala and hippocampus (contextual conditioning). The skeletal muscle citrate synthase activity, an indicator of aerobic capacity, was elevated in rats received 4w of TR, but not WR. While both TR and WR elevated the contextual conditional response, only TR facilitated the cued conditional response. Using a single-neuron labeling technique, we found that while both TR and MR enlarged the dendritic field and increased the spine density in hippocampal CA3 neurons, only TR showed these effects in basolateral amygdalar neurons. Moreover, both types of exercise upregulated synaptic proteins (i.e. TrkB and SNAP-25) in the hippocampus; however only TR showed similar effects in the amygdala. Injection of K252a, a TrkB kinase inhibitor, in the dorsal hippocampus or basolateral amygdala abolished the exercise-facilitated contextual or cued fear learning and memory performance, respectively, regardless of the types of exercise. In summary, our results supported that different types of exercise affect the performance of learning and memory via BDNF-TrkB signaling and neuroplasticity in specific brain regions. The brain region-specific neuronal adaptations are possibly induced by various levels of intensity/stress elicited by different types of exercise.

Ontogenetic changes in citrate synthase and lactate dehydrogenase activity in the jumping muscle of the American locust (Schistocerca americana)

Intraspecific studies have repeatedly shown that muscle-specific oxidative enzyme activities scale negatively with body mass while muscle-specific glycolytic enzyme activities scale positively. However, most of these studies have not included juveniles. In this study, we examined how citrate synthase (CS, EC 2.3.3.1) and lactate dehydrogenase (LDH; EC 1.1.1.27) activity in the jumping muscle of Schistocerca americana grasshoppers varied with ontogeny across a 40-fold increase in body size. In contrast to the pattern observed when adult conspecifics are compared, we show that jumping muscle CS activity increased more than 2-fold from 2nd instars to adults, while jumping muscle LDH activity increased more than 5-fold. The increased LDH activity in older grasshoppers supports previous data that older grasshoppers have a reduced jumping endurance. The increased CS activity with age may help older grasshoppers efficiently produce aerobic ATP to bend cuticular springs for energy storage before a jump or alternatively recover from anaerobic metabolism after jumping. Metabolic changes in S. americana jumping muscle are similar to other developing taxa and highlight the importance of including juveniles within intraspecific studies. When compared to adults, juvenile locomotion may have increased selection pressure because of both greater energetic demands during growth and higher predation rates.

The relationship between human skeletal muscle pyruvate dehydrogenase phosphatase activity and muscle aerobic capacity

Pyruvate dehydrogenase (PDH) is a mitochondrial enzyme responsible for regulating the conversion of pyruvate to acetyl-CoA for use in the tricarboxylic acid cycle. PDH is regulated through phosphorylation and inactivation by PDH kinase (PDK) and dephosphorylation and activation by PDH phosphatase (PDP). The effect of endurance training on PDK in humans has been investigated; however, to date no study has examined the effect of endurance training on PDP in humans. Therefore, the purpose of this study was to examine differences in PDP activity and PDP1 protein content in human skeletal muscle across a range of muscle aerobic capacities. This association is important as higher PDP activity and protein content will allow for increased activation of PDH, and carbohydrate oxidation. The main findings of this study were that 1) PDP activity (r(2) = 0.399, P = 0.001) and PDP1 protein expression (r(2) = 0.153, P = 0.039) were positively correlated with citrate synthase (CS) activity as a marker for muscle aerobic capacity; 2) E1α (r(2) = 0.310, P = 0.002) and PDK2 protein (r(2) = 0.229, P =0.012) are positively correlated with muscle CS activity; and 3) although it is the most abundant isoform, PDP1 protein content only explained ∼ 18% of the variance in PDP activity (r(2) = 0.184, P = 0.033). In addition, PDP1 in combination with E1α explained ∼ 38% of the variance in PDP activity (r(2) = 0.383, P = 0.005), suggesting that there may be alternative regulatory mechanisms of this enzyme other than protein content. These data suggest that with higher muscle aerobic capacity (CS activity) there is a greater capacity for carbohydrate oxidation (E1α), in concert with higher potential for PDH activation (PDP activity).

Effects of Different High Fat Diets on FABP Content in Rat Skeletal Muscles

Long-term feeding of high fat diet increases both oxidative enzymes in skeletal muscle and endurance capacity in rats, whereas it is well-known to cause obesity. PURPOSE: The purpose of this study was to examine the effects of high fat diet containing long-chain diacylglycerol (DAG), medium-chain triacylglycerol (MTG) or long-chain triacylglycerol (TAG) on oxidative enzyme activities and fatty acid binding protein (FABP) contents in rat skeletal muscles. METHODS: Male Wistar rats, 5 weeks old, were assigned to 4 groups; low fat diet (LF; n=6, 12% calories as fat), TAG high fat diet (n=6, 60% calories as fat), DAG high fat diet (n=6, 60% calories as fat) and MTG high fat diet (n=7, 60% calories as fat) groups. Animals were fed their diets and water ad libitum for 5 weeks. Citrate synthase (CS) activity was measured by a spectrophotometric assay, and FABP content was measured with an ELISA kit. RESULTS: Epidydimal fat pad was much smaller in MTG group than in HTG and DAG groups (P<0.05). TAG and MTG groups were increased CS activity (TAG, 40±2.5 mol·min-1·g-1; MTG, 44±2.8 mol·min-1·g-1) in soleus muscle (Sol) compared with LF (35±4.1 mol·min-1·g-1) group (p < 0.01). FABP contents of Sol and red gastrocnemius muscle (Gas) were significantly higher in TAG (Sol, 62 ±7.3; red Gas, 59±21.8 ng/ml) and DAG (Sol, 58±9.1; red Gas, 51±5.8 ng/ml) groups than in MTG (Sol, 28±2.8; red Gas, 28±6.2 ng/ml) group (p < 0.05). On the other hand, FABP contents of white Gas were, albeit small, significantly higher in TAG (1.8±0.3 ng/ml), DAG (2.4±0.3 ng/ml) and MTG (1.5±0.2 ng/ml) groups than in LF (0.3±0.1 ng/ml) group (p < 0.05). CONCLUSION: Thus, the 5-week-feeding of DAG high fat diet increased FABP content in Sol and red/white Gas, and also the MTG high fat diet increased both FABP content in white Gas and oxidative enzymes without fat accumulation, suggesting that "MTG plus DAG" high fat diet may enhance fat metabolism and exercise performance without obesity.

Recovered insulin response by 2 weeks of leptin administration in high-fat fed rats is associated with restored AS160 activation and decreased reactive lipid accumulation

Leptin is an adipokine that increases fatty acid (FA) oxidation, decreases intramuscular lipid stores, and improves insulin response in skeletal muscle. In an attempt to elucidate the underlying mechanisms by which these metabolic changes occur, we administered leptin (Lep) or saline (Sal) by miniosmotic pumps to rats during the final 2 wk of a 6-wk low-fat (LF) or high-fat (HF) diet. Insulin-stimulated glucose transport was impaired by the HF diet (HF-Sal) but was restored with leptin administration (HF-Lep). This improvement was associated with restored phosphorylation of Akt and AS160 and decreased in reactive lipid species (ceramide, diacylglycerol), known inhibitors of the insulin-signaling cascade. Total muscle citrate synthase (CS) activity was increased by both leptin and HF diet, but was not additive. Leptin increased subsarcolemmal (SS) and intramyofibrillar (IMF) mitochondria CS activity. Total muscle, sarcolemmal, and mitochondrial (SS and IMF) FA transporter (FAT/CD36) protein content was significantly increased with the HF diet, but not altered by leptin. Therefore, the decrease in reactive lipid stores and subsequent improvement in insulin response, secondary to leptin administration in rats fed a HF diet was not due to a decrease in FA transport protein content or altered cellular distribution.

Exercise training and selenium or a combined treatment ameliorates aberrant expression of glucose and lactate metabolic proteins in skeletal muscle in a rodent model of diabetes

Exercise training (ET) and selenium (SEL) were evaluated either individually or in combination (COMBI) for their effects on expression of glucose (AMPK, PGC-1α, GLUT-4) and lactate metabolic proteins (LDH, MCT-1, MCT-4, COX-IV) in heart and skeletal muscles in a rodent model (Goto-Kakisaki, GK) of diabetes. Forty GK rats either remained sedentary (SED), performed ET, received SEL, (5 µmol·kg body wt-1·day-1) or underwent both ET and SEL treatment for 6 wk. ET alone, SEL alone, or COMBI resulted in a significant lowering of lactate, glucose, and insulin levels as well as a reduction in HOMA-IR and AUC for glucose relative to SED. Additionally, ET alone, SEL alone, or COMBI increased glycogen content and citrate synthase (CS) activities in liver and muscles. However, their effects on glycogen content and CS activity were tissue-specific. In particular, ET alone, SEL alone, or COMBI induced upregulation of glucose (AMPK, PGC-1α, GLUT-4) and lactate (LDH, MCT-1, MCT-4, COX-IV) metabolic proteins relative to SED. However, their effects on glucose and lactate metabolic proteins also appeared to be tissue-specific. It seemed that glucose and lactate metabolic protein expression was not further enhanced with COMBI compared to that of ET alone or SEL alone. These data suggest that ET alone or SEL alone or COMBI represent a practical strategy for ameliorating aberrant expression of glucose and lactate metabolic proteins in diabetic GK rats.

A decreasedn-6/n-3 ratio in the fat-1 mouse is associated with improved glucose tolerance

A reduction in skeletal muscle fatty acid oxidation (FAO), manifested as a reduction in mitochondrial content and (or) FAO within mitochondria, may contribute to the development of insulin resistance. n-3 polyunsaturated fatty acids (PUFA) have been observed to increase the capacity for FAO and improve insulin sensitivity. We used the fat-1 mouse model, a transgenic animal capable of synthesizing n-3 PUFA from n-6 PUFA, to examine this relationship. Fat-1 mice exhibited a approximately 20-fold decrease in the n-6/n-3 ratio in skeletal muscle, and plasma glucose and the area under the glucose curve were significantly (p < 0.05) lower in fat-1 mice during a glucose challenge test. The improvement in whole-body glucose tolerance in the fat-1 mouse was associated with a approximately 21% (p < 0.05) decrease in whole-muscle citrate synthase (CS) activity (in red muscle only), without alterations in CS activity of isolated mitochondria (either red or white muscle; p > 0.05). These data suggest that the fat-1 mouse has decreased skeletal muscle mitochondrial content. However, the intrinsic ability of mitochondria to oxidize fatty acids was not altered in the fat-1 mouse, as rates of palmitate oxidation in isolated mitochondria from both red and white muscle were unchanged. Overall, this study demonstrates that a decrease in the n-6/n-3 ratio can enhance glucose tolerance in healthy animals, independent of changes in mitochondrial content.

Knockdown of Natural Voluntary Running's Caloric Expenditure causes Obesity in High-fat Fed C57BL/6 Mice

Western lifestyle, characterized by physical inactivity and a high-fat (HF) diet, are some of the environmental factors responsible for increased risk of overweight/obesity. However, some claim that obesity is solely due to increased caloric intake. It is, therefore, vital to determine whether removal of natural physical activity decreases caloric expenditure, and is associated with increased percentage of body fat in high-fat-fed mice. PURPOSE: The purpose of this study was to determine whether daily, natural, voluntary running increases energy expenditure while mice consume a HF diet as compared to cohorts without voluntary running. METHODS: C57BL/6 (n=25, 9-10 wks) began being fed either a chow (Formulab 5008, 6.5% energy from fat) or HF chow (TestDiet, 45% energy from fat) for ten weeks. HF-fed mice were either given access to voluntary running wheels (HFRun, n=7) or limited to cage activity (HFSed, n=12). Chow-fed animals (LFSed, n=6) served as low-fat sedentary controls. Oxygen consumption (VO2) was measured in Columbus Instruments metabolic chambers with and without voluntary running wheels. RESULTS: HFSed weighed 24% more (P=0.01) and had a correspondingly higher percentage fat mass (P=0.0002) that that of HFRun or LFSed groups. Despite lower body weights for HFRun, HFRun consumed 21% more calories than HFSed (P=0.001) while consuming comparable calories as LFSed. The increase in expended calories (as measured by VO2 and corrected by Respiratory Exchange Ratio [RER]) in the HFRun, compared to the HFSed, resulted in the maintenance of body weight in HFRun. The lack of increased percentage body fat might be attributed to 1) greater 22-hour whole-body O2 consumption (P=0.02); 2) greater sleep-cycle fatty acid oxidation exhibited by 9% lower RER (P= 0.015), and 3) 28% greater mitochondrial concentration as indicated by greater gastrocnemius muscle citrate synthase activity (P=0.048). CONCLUSIONS: Loss of caloric expenditure from knockdown of natural voluntary running in female, C57BL/6 mice as they age from 10 to 20 weeks of age, rather than increased caloric intake from a HF diet, is associated with an increased percentage of body fat. The results are similar to clinical studies, which indicate that high levels of physical activity are characteristic of persons who maintain a reduced body weight over prolonged periods of time. The data from our mouse model is relevant to humans since both mice and humans have similar adaptive mechanisms when increased physical activity enhances caloric expenditure.

Myocyte vascular endothelial growth factor is required for exercise-induced skeletal muscle angiogenesis

We have previously shown, using a Cre-LoxP strategy, that vascular endothelial growth factor (VEGF) is required for the development and maintenance of skeletal muscle capillarity in sedentary adult mice. To determine whether VEGF expression is required for skeletal muscle capillary adaptation to exercise training, gastrocnemius muscle capillarity was measured in myocyte-specific VEGF gene-deleted (mVEGF(-/-)) and wild-type (WT) littermate mice following 6 wk of treadmill running (1 h/day, 5 days/wk) at the same running speed. The effect of training on metabolic enzyme activity levels and whole body running performance was also evaluated in mVEGF(-/-) and WT mice. Posttraining capillary density was significantly increased by 59% (P < 0.05) in the deep muscle region of the gastrocnemius in WT mice but did not change in mVEGF(-/-) mice. Maximal running speed and time to exhaustion during submaximal running increased by 20 and 13% (P < 0.05), respectively, in WT mice after training but were unchanged in mVEGF(-/-) mice. Training led to increases in skeletal muscle citrate synthase (CS) and phosphofructokinase (PFK) activities in both WT and mVEGF(-/-) mice (P < 0.05), whereas β-hydroxyacyl-CoA dehydrogenase (β-HAD) activity was increased only in WT mice. These data demonstrate that skeletal muscle capillary adaptation to physical training does not occur in the absence of myocyte-expressed VEGF. However, skeletal muscle metabolic adaptation to exercise training takes place independent of myocyte VEGF expression.

Effects of exercise training on vasodilatory protein expression and activity in rats

Increased endothelium-dependent vasodilatation is associated with endurance exercise training. The purpose of this study was to test the hypothesis that increased endothelial nitric oxide synthase (eNOS) protein function, but not increased vascular smooth muscle sensitivity to NO, underlies augmented endothelium-dependent dilatation with training. To test these hypotheses, rats ran on a treadmill at 30m/min (10% grade) for 60min/day, 5days/week, over 8-12weeks (Trn). Training efficacy was demonstrated by greater (P<0.05) hindlimb muscle citrate synthase activity and left ventricular mass-body mass ratio in Trn compared with sedentary control rats (Sed). Expression of eNOS protein in the aorta was increased with training (Sed, 1.00±0.18 normalized units; Trn, 1.55±0.23; P<0.05). Aortic NOS activity was, however, unchanged by training (Sed, 1,505±288fmol/h/mg protein; Trn, 1,650±247; n.s.). Expression of heat shock protein 90 and protein kinase B/Akt was not different between groups, nor was their association with eNOS. In follow-up series of rats, phosphorylated eNOS content (Serine 1177) was similar for Sed and Trn in both the aorta and gastrocnemius feed artery. Aortic NOS activity with eNOS phosphorylation status preserved was also similar between groups. Finally, cGMP concentration with a NO donor did not differ between groups (Sed, 73.0±20.2pmol/mg protein; Trn, 62.5±12.9; n.s.). These findings indicate that training-induced increases in eNOS protein expression are not coupled to augmented function, illustrating the complexity of eNOS regulation. Further, they show that vascular sensitivity to NO is not altered by exercise training.

Exercise-induced changes in renal URAT1 activity and expression in rats

During exercise, the plasma urate levels and urinary excretion increase due to the enhanced purine degradation in skeletal muscle. Although urate transporter-1 (URAT1) is the main transporter responsible for the reabsorption of filtered urate, potential changes in its activity and expression during exercise have not been studied yet. Therefore, the effect of heavy muscle activity on renal URAT1 activity and expression was investigated in this study. Wistar rats were used in the study and the experimental design consisted of three groups: a control group, an exercise group where animals were exhausted once a day for 5 days, and a hyperuricemia group, which was induced by an uricase inhibitor, oxonic acid. URAT1 activity measurements were performed in isolated proximal tubule segments and expression of URAT1 mRNA and protein levels were determined by the reverse transcription polymerase chain reaction and western blot analyses, respectively. Increased citrate synthase activity in soleus muscle of exercised animals proved the efficiency of our exercise protocol. Proteinuria, glucosuria, and hypoglycemia were observed only in exercised animals; however, plasma and urinary urate levels were found to be elevated in both exercising and hyperuricemia groups. Moreover, in both of the groups URAT1 transporter activity was found to be increased despite the significant decrease in URAT1 protein levels. Considering the similar changes of urate metabolism observed in both exercising and hyperuricemic rats, our results suggest that exercise-induced changes in URAT1 expression and activity depend on the increased urate concentration in plasma.

Inhibition of goldfish mitochondrial metabolism by in vitro exposure to Cd, Cu and Ni

Although impairment of aerobic capacities has been reported in metal-contaminated wild fish, little is known about the direct toxicity of the metals themselves at the low concentrations found in the field compared to indirect consequences mediated by metal effects on ecological variables such as prey type and abundance, predation and competition. This study examined the in vitro effects of Cd, Cu and Ni on mitochondrial enzyme activity and maximal (State 3) mitochondrial oxygen consumption rate in goldfish ( Carassius auratus) tissues at concentrations representative of values reported in wild metal-contaminated fish. There was little effect of adding metals to liver or muscle homogenates on the activity of citrate synthase (CS), although a slight inhibition of liver CS was observed at the highest Cd concentration tested. In contrast, adding high concentrations of Ni to muscle homogenates increased muscle CS activity. Unlike CS, the metalloenzyme cytochrome C oxidase (CCO) was quite sensitive to metal additions; its activity was consistently enhanced by all three metals tested. When added to liver mitochondrial preparations, both Cd and Cu strongly inhibited State 3 respiration. In contrast, Ni did not affect mitochondrial respiration even at the highest concentration tested. Taken together, these results demonstrate that low concentrations of Cd, Cu and Ni have toxic effects on mitochondrial metabolism and enzyme activities and suggest that the inhibition of aerobic capacities frequently reported for wild metal-contaminated fish is at least partly due to metal effects on mitochondrial function, although the mechanisms probably do not involve direct enzyme inhibition.

PGC-1α is required for training-induced prevention of age-associated decline in mitochondrial enzymes in mouse skeletal muscle

The aim of the present study was to test the hypothesis that exercise training prevents an age-associated decline in skeletal muscle mitochondrial enzymes through a PGC-1α dependent mechanism. Whole body PGC-1α knock-out (KO) and littermate wildtype (WT) mice were submitted to long term running wheel exercise training or a sedentary lifestyle from 2 to 13 month of age. Furthermore, a group of approximately 4-month-old mice was used as young untrained controls. There was in both genotypes an age-associated ∼30% decrease in citrate synthase (CS) activity and superoxide dismutase (SOD)2 protein content in 13-month-old untrained mice compared with young untrained mice. However, training prevented the age-associated decrease in CS activity and SOD2 protein content only in WT mice, but long term exercise training did increase HKII protein content in both genotypes. In addition, while CS activity and protein expression of cytc and SOD2 were 50–150% lower in skeletal muscle of PGC-1α mice than WT mice, the expression of the pro-apoptotic protein Bax and the anti-apoptotic Bcl2 was ∼30% elevated in PGC-1α KO mice. In conclusion, the present findings indicate that PGC-1α is required for training-induced prevention of an age-associated decline in CS activity and SOD2 protein expression in skeletal muscle.