Resting metabolic rate and skeletal muscle SERCA and Na+ /K+ ATPase activities are not affected by fish oil supplementation in healthy older adults.

Omega-3 polyunsaturated fatty acids (PUFAs) have unique properties purported to influence several aspects of metabolism, including energy expenditure and protein function. Supplementing with n-3 PUFAs may increase whole-body resting metabolic rate (RMR), by enhancing Na+ /K+ ATPase (NKA) activity and reducing the efficiency of sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA) activity by inducing a Ca2+ leak-pump cycle. The purpose of this study was to examine the effects of fish oil (FO) on RMR, substrate oxidation, and skeletal muscle SERCA and NKA pump function in healthy older individuals. Subjects (n = 16 females; n = 8 males; 65 ± 1 years) were randomly assigned into groups supplemented with either olive oil (OO) (5 g/day) or FO (5 g/day) containing 2 g/day eicosapentaenoic acid and 1 g/day docosahexaenoic acid for 12 weeks. Participants visited the laboratory for RMR and substrate oxidation measurements after an overnight fast at weeks 0 and 12. Skeletal muscle biopsies were taken during weeks 0 and 12 for analysis of NKA and SERCA function and protein content. There was a main effect of time with decrease in RMR (5%) and fat oxidation (18%) in both the supplementation groups. The kinetic parameters of SERCA and NKA maximal activity, as well as the expression of SR and NKA proteins, were not affected after OO and FO supplementation. In conclusion, these results suggest that FO supplementation is not effective in altering RMR, substrate oxidation, and skeletal muscle SERCA and NKA protein levels and activities, in healthy older men and women.

Assessment of Na+/K+ ATPase Activity in Small Rodent and Human Skeletal Muscle Samples.

INTRODUCTION: In skeletal muscle, the Na/K ATPase (NKA) plays essential roles in processes linked to muscle contraction, fatigue, and energy metabolism; however, very little information exists regarding the regulation of NKA activity. The scarcity of information regarding NKA function in skeletal muscle likely stems from methodological constraints, as NKA contributes minimally to total cellular ATP utilization, and therefore contamination from other ATPases prevents the assessment of NKA activity in muscle homogenates. Here we introduce a method that improves accuracy and feasibility for the determination of NKA activity in small rodent muscle samples (5-10 mg) and in human skeletal muscle. METHODS: Skeletal muscle homogenates from mice (n = 6) and humans (n = 3) were used to measure NKA and sarcoplasmic reticulum Ca ATPase (SERCA) activities with the addition of specific ATPase inhibitors to minimize "background noise." RESULTS: We observed that myosin ATPase activity was the major interfering factor for estimation of NKA activity in skeletal muscle homogenates, as the addition of 25 µM of blebbistatin, a specific myosin ATPase inhibitor, considerably minimized "background noise" (threefold) and enabled the determination of NKA maximal activity with values three times higher than previously reported. The specificity of the assay was demonstrated after the addition of 2 mM ouabain, which completely inhibited NKA. On the other hand, the addition of blebbistatin did not affect the ability to measure SERCA function. The coefficient of variation for NKA and SERCA assays were 6.2% and 4.4%, respectively. CONCLUSION: The present study has improved the methodology to determine NKA activity. We further show the feasibility of measuring NKA and SERCA activities from a common muscle homogenate. This methodology is expected to aid in our long-term understanding of how NKA affects skeletal muscle metabolic homeostasis and contractile function in diverse situations.

Incorporation of Omega-3 Fatty Acids Into Human Skeletal Muscle Sarcolemmal and Mitochondrial Membranes Following 12 Weeks of Fish Oil Supplementation.

Fish oil (FO) supplementation in humans results in the incorporation of omega-3 fatty acids (FAs) eicosapentaenoic acid (EPA; C20:5) and docosahexaenoic acid (DHA; C20:6) into skeletal muscle membranes. However, despite the importance of membrane composition in structure-function relationships, a paucity of information exists regarding how different muscle membranes/organelles respond to FO supplementation. Therefore, the purpose of the present study was to determine the effects 12 weeks of FO supplementation (3g EPA/2g DHA daily) on the phospholipid composition of sarcolemmal and mitochondrial fractions, as well as whole muscle responses, in healthy young males. FO supplementation increased the total phospholipid content in whole muscle (57%; p < 0.05) and the sarcolemma (38%; p = 0.05), but did not alter the content in mitochondria. The content of omega-3 FAs, EPA and DHA, were increased (+3-fold) in whole muscle, and mitochondrial membranes, and as a result the omega-6/omega-3 ratios were dramatically decreased (-3-fold), while conversely the unsaturation indexes were increased. Intriguingly, before supplementation the unsaturation index (UI) of sarcolemmal membranes was ∼3 times lower (p < 0.001) than either whole muscle or mitochondrial membranes. While supplementation also increased DHA within sarcolemmal membranes, EPA was not altered, and as a result the omega-6/omega-3 ratio and UI of these membranes were not altered. All together, these data revealed that mitochondrial and sarcolemmal membranes display unique phospholipid compositions and responses to FO supplementation.

Mild Dehydration Does Not Influence Performance Or Skeletal Muscle Metabolism During Simulated Ice Hockey Exercise In Men.

This study determined whether mild dehydration influenced skeletal muscle glycogen use, core temperature or performance during high-intensity, intermittent cycle-based exercise in ice hockey players vs. staying hydrated with water. Eight males (21.6 ± 0.4 yr, 183.5 ± 1.6 cm, 83.9 ± 3.7 kg, 50.2 ± 1.9 ml·kg-1·min-1) performed two trials separated by 7 days. The protocol consisted of 3 periods (P) containing 10 × 45-s cycling bouts at ~133% VO2max, followed by 135 s of passive rest. Subjects drank no fluid and dehydrated during the protocol (NF), or maintained body mass by drinking WATER. Muscle biopsies were taken at rest, immediately before and after P3. Subjects were mildly dehydrated (-1.8% BM) at the end of P3 in the NF trial. There were no differences between the NF and WATER trials for glycogen use (P1+P2; 350.1 ± 31.9 vs. 413.2 ± 33.2, P3; 103.5 ± 16.2 vs. 131.5 ± 18.9 mmol·kg dm-1), core temperature (P1; 37.8 ± 0.1 vs. 37.7 ± 0.1, P2; 38.2 ± 0.1 vs. 38.1 ± 0.1, P3; 38.3 ± 0.1 vs. 38.2 ± 0.1 °C) or performance (P1; 156.3 ± 7.8 vs. 154.4 ± 8.2, P2; 150.5 ± 7.8 vs. 152.4 ± 8.3, P3; 144.1 ± 8.7 vs. 148.4 ± 8.7 kJ). This study demonstrated that typical dehydration experienced by ice hockey players (~1.8% BM loss), did not affect glycogen use, core temperature, or voluntary performance vs. staying hydrated by ingesting water during a cycle-based simulation of ice hockey exercise in a laboratory environment.

Pulmonary gas exchange and acid-base balance during exercise.

As the first step in the oxygen-transport chain, the lung has a critical task: optimizing the exchange of respiratory gases to maintain delivery of oxygen and the elimination of carbon dioxide. In healthy subjects, gas exchange, as evaluated by the alveolar-to-arterial PO2 difference (A-aDO2), worsens with incremental exercise, and typically reaches an A-aDO2 of approximately 25 mmHg at peak exercise. While there is great individual variability, A-aDO2 is generally largest at peak exercise in subjects with the highest peak oxygen consumption. Inert gas data has shown that the increase in A-aDO2 is explained by decreased ventilation-perfusion matching, and the development of a diffusion limitation for oxygen. Gas exchange data does not indicate the presence of right-to-left intrapulmonary shunt developing with exercise, despite recent data suggesting that large-diameter arteriovenous shunt vessels may be recruited with exercise. At the same time, multisystem mechanisms regulate systemic acid-base balance in integrative processes that involve gas exchange between tissues and the environment and simultaneous net changes in the concentrations of strong and weak ions within, and transfer between, extracellular and intracellular fluids. The physicochemical approach to acid-base balance is used to understand the contributions from independent acid-base variables to measured acid-base disturbances within contracting skeletal muscle, erythrocytes and noncontracting tissues. In muscle, the magnitude of the disturbance is proportional to the concentrations of dissociated weak acids, the rate at which acid equivalents (strong acid) accumulate and the rate at which strong base cations are added to or removed from muscle.

Mitochondrial creatine kinase activity and phosphate shuttling are acutely regulated by exercise in human skeletal muscle.

Energy transfer between mitochondrial and cytosolic compartments is predominantly achieved by creatine-dependent phosphate shuttling (PCr/Cr) involving mitochondrial creatine kinase (miCK). However, ADP/ATP diffusion through adenine nucleotide translocase (ANT) and voltage-dependent anion carriers (VDACs) is also involved in this process. To determine if exercise alters the regulation of this system, ADP-stimulated mitochondrial respiratory kinetics were assessed in permeabilized muscle fibre bundles (PmFBs) taken from biopsies before and after 2 h of cycling exercise (60% ) in nine lean males. Concentrations of creatine (Cr) and phosphocreatine (PCr) as well as the contractile state of PmFBs were manipulated in situ. In the absence of contractile signals (relaxed PmFBs) and miCK activity (no Cr), post-exercise respiratory sensitivity to ADP was reduced in situ (up to 126% higher apparent K(m) to ADP) suggesting inhibition of ADP/ATP diffusion between matrix and cytosolic compartments (possibly ANT and VDACs). However this effect was masked in the presence of saturating Cr (no effect of exercise on ADP sensitivity). Given that the role of ANT is thought to be independent of Cr, these findings suggest ADP/ATP, but not PCr/Cr, cycling through the outer mitochondrial membrane (VDACs) may be attenuated in resting muscle after exercise. In contrast, in contracted PmFBs, post-exercise respiratory sensitivity to ADP increased with miCK activation (saturating Cr; 33% lower apparent K(m) to ADP), suggesting prior exercise increases miCK sensitivity in situ. These observations demonstrate that exercise increases miCK-dependent respiratory sensitivity to ADP, promoting mitochondrial-cytosolic energy exchange via PCr/Cr cycling, possibly through VDACs. This effect may mask an underlying inhibition of Cr-independent ADP/ATP diffusion. This enhanced regulation of miCK-dependent phosphate shuttling may improve energy homeostasis through more efficient coupling of oxidative phosphorylation to perturbations in cellular energy charge during subsequent bouts of contraction.

Effects of gas exchange on acid-base balance.

This paper describes the interactions between ventilation and acid-base balance under a variety of conditions including rest, exercise, altitude, pregnancy, and various muscle, respiratory, cardiac, and renal pathologies. We introduce the physicochemical approach to assessing acid-base status and demonstrate how this approach can be used to quantify the origins of acid-base disorders using examples from the literature. The relationships between chemoreceptor and metaboreceptor control of ventilation and acid-base balance summarized here for adults, youth, and in various pathological conditions. There is a dynamic interplay between disturbances in acid-base balance, that is, exercise, that affect ventilation as well as imposed or pathological disturbances of ventilation that affect acid-base balance. Interactions between ventilation and acid-base balance are highlighted for moderate- to high-intensity exercise, altitude, induced acidosis and alkalosis, pregnancy, obesity, and some pathological conditions. In many situations, complete acid-base data are lacking, indicating a need for further research aimed at elucidating mechanistic bases for relationships between alterations in acid-base state and the ventilatory responses.

Exercise training increases sarcolemmal and mitochondrial fatty acid transport proteins in human skeletal muscle.

Fatty acid oxidation is highly regulated in skeletal muscle and involves several sites of regulation, including the transport of fatty acids across both the plasma and mitochondrial membranes. Transport across these membranes is recognized to be primarily protein mediated, limited by the abundance of fatty acid transport proteins on the respective membranes. In recent years, evidence has shown that fatty acid transport proteins move in response to acute and chronic perturbations; however, in human skeletal muscle the localization of fatty acid transport proteins in response to training has not been examined. Therefore, we determined whether high-intensity interval training (HIIT) increased total skeletal muscle, sarcolemmal, and mitochondrial membrane fatty acid transport protein contents. Ten untrained females (22 +/- 1 yr, 65 +/- 2 kg; .VO(2peak): 2.8 +/- 0.1 l/min) completed 6 wk of HIIT, and biopsies from the vastus lateralis muscle were taken before training, and following 2 and 6 wk of HIIT. Training significantly increased maximal oxygen uptake at 2 and 6 wk (3.1 +/- 0.1, 3.3 +/- 0.1 l/min). Training for 6 wk increased FAT/CD36 at the whole muscle (10%) and mitochondrial levels (51%) without alterations in sarcolemmal content. Whole muscle plasma membrane fatty acid binding protein (FABPpm) also increased (48%) after 6 wk of training, but in contrast to FAT/CD36, sarcolemmal FABPpm increased (23%), whereas mitochondrial FABPpm was unaltered. The changes on sarcolemmal and mitochondrial membranes occurred rapidly, since differences (< or =2 wk) were not observed between 2 and 6 wk. This is the first study to demonstrate that exercise training increases fatty acid transport protein content in whole muscle (FAT/CD36 and FABPpm) and sarcolemmal (FABPpm) and mitochondrial (FAT/CD36) membranes in human skeletal muscle of females. These results suggest that increases in skeletal muscle fatty acid oxidation following training are related in part to changes in fatty acid transport protein content and localization.

Prior heavy exercise elevates pyruvate dehydrogenase activity and muscle oxygenation and speeds O2 uptake kinetics during moderate exercise in older adults.

The adaptation of pulmonary oxygen uptake (VO(2)(p)) kinetics during the transition to moderate-intensity exercise is slowed in older compared with younger adults; however, this response is faster following a prior bout of heavy-intensity exercise. We have examined VO(2)(p) kinetics, pyruvate dehydrogenase (PDH) activation, muscle metabolite contents, and muscle deoxygenation in older adults [n = 6; 70 +/- 5 (67-74) yr] during moderate-intensity exercise (Mod(1)) and during moderate-intensity exercise preceded by heavy-intensity warm-up exercise (Mod(2)). The phase 2 VO(2)(p) time constant (tauVO(2)(p)) was reduced (P < 0.05) in Mod(2) (29 +/- 5 s) compared with Mod(1) (39 +/- 14 s). PDH activity was elevated (P < 0.05) at baseline prior to Mod(2) (2.1 +/- 0.6 vs. 1.2 +/- 0.3 mmol acetyl-CoA x min(-1) x kg wet wt(-1)), and the delay in attaining end-exercise activity was abolished. Phosphocreatine breakdown during exercise was reduced (P < 0.05) at both 30 s and 6 min in Mod(2) compared with Mod(1). Near-infrared spectroscopy-derived indices of muscle oxygenation were elevated both prior to and throughout Mod(2), while muscle deoxygenation kinetics were not different between exercise bouts consistent with elevated perfusion and O(2) availability. These results suggest that in older adults, faster VO(2)(p) kinetics following prior heavy-intensity exercise are likely a result of prior activation of mitochondrial enzyme activity in combination with elevated muscle perfusion and O(2) availability.

Carbohydrate refeeding after a high-fat diet rapidly reverses the adaptive increase in human skeletal muscle PDH kinase activity.

Pyruvate dehydrogenase (PDH) regulates oxidative carbohydrate disposal in skeletal muscle and is downregulated by reversible phosphorylation catalyzed by PDH kinase (PDK). Previous work has demonstrated increased PDK activity and PDK4 expression in human skeletal muscle following a high-fat low-carbohydrate (HF) diet, which leads to decreased PDH in the active form (PDHa activity) and carbohydrate oxidation. The purpose of this study was to examine the time course of changes in PDK and PDHa activities with refeeding of carbohydrates after an HF diet in human skeletal muscle. Healthy male volunteers (n = 8) consumed a standardized 3-day Pre-diet with the same energy content as their habitual diet, followed by a eucaloric 6-day HF diet (Pre-diet: 50:30:20%; HF diet: 5:75:20%; carbohydrate/fat/protein). Muscle biopsies were taken before and after the HF diet and at 45 min and 3 h after carbohydrate refeeding with a single high-glycemic index carbohydrate meal (88:5:7% carbohydrate/fat/protein) representing approximately one third of the individual subject's habitual energy intake. PDK activity increased from 0.08 +/- 0.01 Pre- to 0.25 +/- 0.02 min (P < 0.001) Post-HF diet, and decreased with carbohydrate refeeding to 0.17 +/- 0.05 (P = 0.014) and 0.11 +/- 0.01 min (P = 0.006) at 45 min and 3 h, respectively. PDHa decreased from 0.89 +/- 0.20 to 0.32 +/- 0.05 (P = 0.007) mmol x min(-1) x kg wet wt(-1) following the HF diet, and was increased transiently with refeeding at 45 min, but returned to lower values by 3 h (P = 0.025 compared with Pre). The potential mechanism(s) for this attenuation of PDHa activity remains unclear. These data demonstrate that in human skeletal muscle, the adaptive increase in PDK activity following an HF diet is rapidly reversed to Pre-diet activity levels within 45 min to 3 h, and this is accompanied by a short-term increase in PDHa activity.

High-intensity aerobic interval training increases fat and carbohydrate metabolic capacities in human skeletal muscle.

High-intensity aerobic interval training (HIIT) is a compromise between time-consuming moderate-intensity training and sprint-interval training requiring all-out efforts. However, there are few data regarding the ability of HIIT to increase the capacities of fat and carbohydrate oxidation in skeletal muscle. Using untrained recreationally active individuals, we investigated skeletal muscle and whole-body metabolic adaptations that occurred following 6 weeks of HIIT (~1 h of 10 x 4 min intervals at ~90% of peak oxygen consumption (VO2 peak), separated by 2 min rest, 3 d.week-1). A VO2 peak test, a test to exhaustion (TE) at 90% of pre-training VO2 peak, and a 1 h cycle at 60% of pre-training VO2 peak were performed pre- and post-HIIT. Muscle biopsies were sampled during the TE at rest, after 5 min, and at exhaustion. Training power output increased by 21%, and VO2 peak increased by 9% following HIIT. Muscle adaptations at rest included the following: (i) increased cytochrome c oxidase IV content (18%) and maximal activities of the mitochondrial enzymes citrate synthase (26%), beta-hydroxyacyl-CoA dehydrogenase (29%), aspartate-amino transferase (26%), and pyruvate dehydrogenase (PDH; 21%); (ii) increased FAT/CD36, FABPpm, GLUT 4, and MCT 1 and 4 transport proteins (14%-30%); and (iii) increased glycogen content (59%). Major adaptations during exercise included the following: (i) reduced glycogenolysis, lactate accumulation, and substrate phosphorylation (0-5 min of TE); (ii) unchanged PDH activation (carbohydrate oxidation; 0-5 min of TE); (iii) ~2-fold greater time during the TE; and (iv) increased fat oxidation at 60% of pre-training VO2 peak. This study demonstrated that 18 h of repeated high-intensity exercise sessions over 6 weeks (3 d.week-1) is a powerful method to increase whole-body and skeletal muscle capacities to oxidize fat and carbohydrate in previously untrained individuals.

Seven days of oral taurine supplementation does not increase muscle taurine content or alter substrate metabolism during prolonged exercise in humans.

This study examined 1) the plasma taurine response to acute oral taurine supplementation (T), and 2) the effects of 7 days of T on muscle amino acid content and substrate metabolism during 2 h of cycling at approximately 60% peak oxygen consumption (VO2peak). In the first part of the study, after an overnight fast, 7 volunteers (28+/-3 yr, 184+/-2 cm, 88.0+/-6.6 kg) ingested 1.66 g oral taurine doses with breakfast (8 AM) and lunch (12 noon), and blood samples were taken throughout the day. In the second part of the study, eight men (22+/-1 yr, 181+/-1 cm, 80.9+/-3.8 kg, 4.21+/-0.16 l/min VO2peak) cycled for 2 h after 7 days of placebo (P) ingestion (6 g glucose/day) and again following 7 days of T (5 g/day). In the first part of the study, plasma taurine was 64+/-4 microM before T and rose rapidly to 778+/-139 microM by 10 AM and remained elevated at noon (359+/-56 microM). Plasma taurine reached 973+/-181 microM at 1 PM and was 161+/-31 microM at 4 PM. In the second part of the study, seven days of T had no effect on muscle taurine content (mmol/kg dry muscle) at rest (P, 44+/-15 vs. T, 42+/-15) or after exercise (P, 43+/-12 vs. T, 43+/-11). There was no difference in muscle glycogen or other muscle metabolites between conditions, but there were notable interaction effects for muscle valine, isoleucine, leucine, cystine, glutamate, alanine, and arginine amino acid content following exercise after T. These data indicate that 1) acute T produces a 13-fold increase in plasma taurine concentration; 2) despite the ability to significantly elevate plasma taurine for extended periods throughout the day, 7 days of T does not alter skeletal muscle taurine content or carbohydrate and fat oxidation during exercise; and 3) T appears to have some impact on muscle amino acid response to exercise.

Adrenergic regulation of HSL serine phosphorylation and activity in human skeletal muscle during the onset of exercise.

Skeletal muscle hormone-sensitive lipase (HSL) activity is increased by contractions and increases in blood epinephrine (EPI) concentrations and cyclic AMP activation of the adrenergic pathway during prolonged exercise. To determine the importance of hormonal stimulation of HSL activity during the onset of moderate- and high-intensity exercise, nine men [age 24.3 +/- 1.2 yr, 80.8 +/- 5.0 kg, peak oxygen consumption (VO2 peak) 43.9 +/- 3.6 ml x kg(-1) x min(-1)] cycled for 1 min at approximately 65% VO2 peak, rested for 60 min, and cycled at approximately 90% VO2 peak for 1 min. Skeletal muscle biopsies were taken pre- and postexercise, and arterial blood was sampled throughout exercise. Arterial EPI increased (P < 0.05) postexercise at 65% (0.45 +/- 0.10 to 0.78 +/- 0.27 nM) and 90% VO2 peak (0.57 +/- 0.34 to 1.09 +/- 0.50 nM). HSL activity increased (P < 0.05) following 1 min of exercise at 65% VO2 peak [1.05 +/- 0.39 to 1.78 +/- 0.54 mmol x min(-1) x kg dry muscle (dm)(-1)] and 90% VO2 peak (1.07 +/- 0.24 to 1.91 +/- 0.62 mmol x min(-1) x kg dm(-1)). Cyclic AMP content also increased (P < 0.05) at both exercise intensities (65%: 1.52 +/- 0.67 to 2.75 +/- 1.12, 90%: 1.85 +/- 0.65 to 2.64 +/- 0.93 micromol/kg dm). HSL Ser660 phosphorylation (approximately 55% increase) and ERK1/2 phosphorylation ( approximately 33% increase) were augmented following exercise at both intensities, whereas HSL Ser563 and Ser565 phosphorylation were not different from rest. The results indicate that increases in arterial EPI concentration during the onset of moderate- and high-intensity exercise increase cyclic AMP content, which results in the phosphorylation of HSL Ser660. This adrenergic stimulation contributes to the increase in HSL activity that occurs in human skeletal muscle in the first minute of exercise at 65% and 90% VO2 peak.

Endurance training in obese humans improves glucose tolerance and mitochondrial fatty acid oxidation and alters muscle lipid content.

Muscle fatty acid (FA) metabolism is impaired in obesity and insulin resistance, reflected by reduced rates of FA oxidation and accumulation of lipids. It has been suggested that interventions that increase FA oxidation may enhance insulin action by reducing these lipid pools. Here, we examined the effect of endurance training on rates of mitochondrial FA oxidation, the activity of carnitine palmitoyltransferase I (CPT I), and the lipid content in muscle of obese individuals and related these to measures of glucose tolerance. Nine obese subjects completed 8 wk of moderate-intensity endurance training, and muscle biopsies were obtained before and after training. Training significantly improved glucose tolerance, with a reduction in the area under the curve for glucose (P < 0.05) and insulin (P = 0.01) during an oral glucose tolerance test. CPT I activity increased 250% (P = 0.001) with training and became less sensitive to inhibition by malonyl-CoA. This was associated with an increase in mitochondrial FA oxidation (+120%, P < 0.001). Training had no effect on muscle triacylglycerol content; however, there was a trend for training to reduce both the total diacylglcyerol (DAG) content (-15%, P = 0.06) and the saturated DAG-FA species (-27%, P = 0.06). Training reduced both total ceramide content (-42%, P = 0.01) and the saturated ceramide species (-32%, P < 0.05). These findings suggest that the improved capacity for mitochondrial FA uptake and oxidation leads not only to a reduction in muscle lipid content but also a to change in the saturation status of lipids, which may, at least in part, provide a mechanism for the enhanced insulin action observed with endurance training in obese individuals.

Effects of 7 wk of endurance training on human skeletal muscle metabolism during submaximal exercise.

This is the first study to examine the effects of endurance training on the activation state of glycogen phosphorylase (Phos) and pyruvate dehydrogenase (PDH) in human skeletal muscle during exercise. We hypothesized that 7 wk of endurance training (Tr) would result in a posttransformationally regulated decrease in flux through Phos and an attenuated activation of PDH during exercise due to alterations in key allosteric modulators of these important enzymes. Eight healthy men (22 +/- 1 yr) cycled to exhaustion at the same absolute workload (206 +/- 5 W; approximately 80% of initial maximal oxygen uptake) before and after Tr. Muscle biopsies (vastus lateralis) were obtained at rest and after 5 and 15 min of exercise. Fifteen minutes of exercise post-Tr resulted in an attenuated activation of PDH (pre-Tr: 3.75 +/- 0.48 vs. post-Tr: 2.65 +/- 0.38 mmol.min(-1).kg wet wt(-1)), possibly due in part to lower pyruvate content (pre-Tr: 0.94 +/- 0.14 vs. post-Tr: 0.46 +/- 0.03 mmol/kg dry wt). The decreased pyruvate availability during exercise post-Tr may be due to a decreased muscle glycogenolytic rate (pre-Tr: 13.22 +/- 1.01 vs. post-Tr: 7.36 +/- 1.26 mmol.min(-1).kg dry wt(-1)). Decreased glycogenolysis was likely mediated, in part, by posttransformational regulation of Phos, as evidenced by smaller net increases in calculated muscle free ADP (pre-Tr: 111 +/- 16 vs. post-Tr: 84 +/- 10 micromol/kg dry wt) and P(i) (pre-Tr: 57.1 +/- 7.9 vs. post-Tr: 28.6 +/- 5.6 mmol/kg dry wt). We have demonstrated for the first time that several signals act to coordinately regulate Phos and PDH, and thus carbohydrate metabolism, in human skeletal muscle after 7 wk of endurance training.

Effect of endurance training on muscle TCA cycle metabolism during exercise in humans.

We tested the theory that links the capacity to perform prolonged exercise with the size of the muscle tricarboxylic acid (TCA) cycle intermediate (TCAI) pool. We hypothesized that endurance training would attenuate the exercise-induced increase in TCAI concentration ([TCAI]); however, the lower [TCAI] would not compromise cycle endurance capacity. Eight men (22 +/- 1 yr) cycled at approximately 80% of initial peak oxygen uptake before and after 7 wk of training (1 h/day, 5 days/wk). Biopsies (vastus lateralis) were obtained during both trials at rest, after 5 min, and at the point of exhaustion during the pretraining trial (42 +/- 6 min). A biopsy was also obtained at the end of exercise during the posttraining trial (91 +/- 6 min). In addition to improved performance, training increased (P < 0.05) peak oxygen uptake and citrate synthase maximal activity. The sum of four measured TCAI was similar between trials at rest but lower after 5 min of exercise posttraining [2.7 +/- 0.2 vs. 4.3 +/- 0.2 mmol/kg dry wt (P < 0.05)]. There was a clear dissociation between [TCAI] and endurance capacity because the [TCAI] at the point of exhaustion during the pretraining trial was not different between trials (posttraining: 2.9 +/- 0.2 vs. pretraining: 3.5 +/- 0.2 mmol/kg dry wt), and yet cycle endurance time more than doubled in the posttraining trial. Training also attenuated the exercise-induced decrease in glutamate concentration (posttraining: 4.5 +/- 0.7 vs. pretraining: 7.7 +/- 0.6 mmol/kg dry wt) and increase in alanine concentration (posttraining: 3.3 +/- 0.2 vs. pretraining: 5.6 +/- 0.3 mmol/kg dry wt; P < 0.05), which is consistent with reduced carbon flux through alanine aminotransferase. We conclude that, after aerobic training, cycle endurance capacity is not limited by a decrease in muscle [TCAI].

Effects of aerobic training on pyruvate dehydrogenase and pyruvate dehydrogenase kinase in human skeletal muscle.

This study examined the effects of short- and long-term aerobic training on the stable up-regulation of pyruvate dehydrogenase (PDH) and PDH kinase (PDK) in human skeletal muscle. We hypothesized that 8 weeks, but not 1 week, of aerobic training would increase total PDH (PDHt) and PDK activities compared to pretraining, and this would be detectable at the level of gene transcription (mRNA) and/or gene translation (protein). Resting muscle biopsies were taken before and after 1 and 8 weeks of aerobic cycle exercise training. PDHt and PDK activities, and their respective protein and mRNA expression, did not differ after 1 week of aerobic training. PDHt activity increased 31% after 8 weeks and this may be partially due to a 1.3-fold increase in PDH-E(1)alpha protein expression. PDK activity approximately doubled after 8 weeks of aerobic training and this was attributed to a 1.3-fold increase in PDK2 isoform protein expression. Similar to 1 week, no changes were observed at the mRNA level after 8 weeks of training. These findings suggest that aerobically trained human skeletal muscle has an increased maximal capacity to utilize carbohydrates, evident by increased PDHt, but increased metabolic control sensitivity to pyruvate through increased contribution of PDK2 to total PDK activity.

Palmitate movement across red and white muscle membranes of rainbow trout.

We examined the movement of [3H]palmitate across giant sarcolemmal vesicles prepared from red and white muscle of rainbow trout (Oncorhynchus mykiss). Red and white muscle fatty acid carriers have similar affinities for palmitate (apparent Km = 26 +/- 6 and 33 +/- 8 nM, respectively); however, red muscle has a higher maximal uptake compared with white muscle (Vmax = 476 +/- 41 vs. 229 +/- 23 pmol.mg protein-1.s-1, respectively). Phloretin (250 microM) inhibited palmitate influx in red and white muscle vesicles by approximately 40%, HgCl2 (2.5 mM) inhibited palmitate uptake by 20-30%, and the anion-exchange inhibitor DIDS (250 microM) inhibited palmitate influx in red and white muscle vesicles by approximately 15 and 30%, respectively. Western blot analysis of red and white muscle vesicles did not detect a mammalian-type fatty acid transporter (FAT); however, preincubation of vesicles with sulfo-N-succinimidyloleate, a specific inhibitor of FAT in rats, reduced palmitate uptake in red and white muscle vesicles by approximately 15 and 25%, respectively. A mammalian-type plasma membrane fatty acid-binding protein was identified in trout muscle using Western blotting, but the protein differed in size between red and white muscle. At low concentrations of free palmitate (2.5 nM), addition of high concentrations (111 microM total) of oleate (18:0) caused approximately 50% reduction in palmitate uptake by red and white muscle vesicles, but high concentrations (100 microM) of octanoate (8:0) caused no inhibition of uptake. Five days of aerobic swimming at approximately 2 body lengths/s and 9 days of chronic cortisol elevation in vivo, both of which stimulate lipid metabolism, had no effect on the rate of palmitate movement in red or white muscle vesicles.

Regulation of CPT I activity in intermyofibrillar and subsarcolemmal mitochondria from human and rat skeletal muscle.

Carnitine palmitoyltransferase I (CPT I) is considered the rate-limiting enzyme in the transfer of long-chain fatty acids (LCFA) into the mitochondria and is reversibly inhibited by malonyl-CoA (M-CoA) in vitro. In rat skeletal muscle, M-CoA levels decrease during exercise, releasing the inhibition of CPT I and increasing LCFA oxidation. However, in human skeletal muscle, M-CoA levels do not change during moderate-intensity exercise despite large increases in fat oxidation, suggesting that M-CoA is not the sole regulator of increased CPT I activity during exercise. In the present study, we measured CPT I activity in intermyofibrillar (IMF) and subsarcolemmal (SS) mitochondria isolated from human vastus lateralis (VL), rat soleus (Sol), and red gastrocnemius (RG) muscles. We tested whether exercise-related levels ( approximately 65% maximal O2 uptake) of calcium and adenylate charge metabolites (free AMP, ADP, and Pi) could override the M-CoA-induced inhibition of CPT I activity and explain the increased CPT I flux during exercise. Protein content was approximately 25-40% higher in IMF than in SS mitochondria in all muscles. Maximal CPT I activity was similar in IMF and SS mitochondria in all muscles (VL: 282 +/- 46 vs. 280 +/- 51; Sol: 390 +/- 81 vs. 368 +/- 82; RG: 252 +/- 71 vs. 278 +/- 44 nmol.min-1.mg protein-1). Sensitivity to M-CoA did not differ between IMF and SS mitochondria in all muscles (25-31% inhibition in VL, 52-70% in Sol and RG). Calcium and adenylate charge metabolites did not override the M-CoA-induced inhibition of CPT I activity in mitochondria isolated from VL, Sol, and RG muscles. Decreasing pH from 7.1 to 6.8 reduced CPT I activity by approximately 34-40% in both VL mitochondrial fractions. In summary, this study reports no differences in CPT I activity or sensitivity to M-CoA between IMF and SS mitochondria isolated from human and rat skeletal muscles. Exercise-induced increases in calcium and adenylate charge metabolites do not appear responsible for upregulating CPT I activity in human or rat skeletal muscle during moderate aerobic exercise.

Hormone-sensitive lipase activity and fatty acyl-CoA content in human skeletal muscle during prolonged exercise.

Hormone-sensitive lipase (HSL) catalyzes the hydrolysis of intramuscular triacylglycerols (IMTGs), but HSL regulation is poorly understood in skeletal muscle. The present study measured human skeletal muscle HSL activity at rest and during 120 min of cycling at 60% of peak O2 uptake. Several putative HSL regulators were also measured, including muscle long-chain fatty acyl-CoA (LCFA CoA) and free AMP contents and plasma epinephrine and insulin concentrations. HSL activity increased from resting levels by 10 min of exercise (from 2.09 +/- 0.19 to 2.56 +/- 0.22 mmol. min-1x kg dry mass-1, P < 0.05), increased further by 60 min (to 3.12 +/- 0.27 mmol x min-1x kg dry mass-1, P < 0.05), and decreased to near-resting rates after 120 min of cycling. Skeletal muscle LCFA CoA increased (P < 0.05) above rest by 60 min (from 15.9 +/- 3.0 to 50.4 +/- 7.9 micromol/kg dry mass) and increased further by 120 min. Estimated free AMP increased (P < 0.05) from rest to 60 min and was approximately 20-fold greater than that at rest by 120 min. Epinephrine was increased above rest (P < 0.05) at 60 (1.47 +/- 0.15 nM) and 120 min (4.87 +/- 0.76 nM) of exercise. Insulin concentrations decreased rapidly and were lower than resting levels by 10 min and continued to decrease throughout exercise. In summary, HSL activity was increased from resting levels by 10 min, increased further by 60 min, and decreased to near-resting values by 120 min. The increased HSL activity at 60 min was associated with the stimulating effect of increased epinephrine and decreased insulin levels. After 120 min, the decreased HSL activity was associated with the proposed inhibitory effects of increased free AMP. The accumulation of LCFA CoA in the 2nd h of exercise may also have reduced the flux through HSL and accounted for the reduction in IMTG utilization previously observed late in prolonged exercise.