Impacts of rat hindlimb Fndc5/irisin overexpression on muscle and adipose tissue metabolism.

Myokines, such as irisin, have been purported to exert physiological effects on skeletal muscle in an autocrine/paracrine fashion. In this study, we aimed to investigate the mechanistic role of in vivo fibronectin type III domain-containing 5 (Fndc5)/irisin upregulation in muscle. Overexpression (OE) of Fndc5 in rat hindlimb muscle was achieved by in vivo electrotransfer, i.e., bilateral injections of Fndc5 harboring vectors for OE rats (n = 8) and empty vector for control rats (n = 8). Seven days later, a bolus of D2O (7.2 mL/kg) was administered via oral gavage to quantify muscle protein synthesis. After an overnight fast, on day 9, 2-deoxy-d-glucose-6-phosphate (2-DG6P; 6 mg/kg) was provided during an intraperitoneal glucose tolerance test (2 g/kg) to assess glucose handling. Animals were euthanized, musculus tibialis cranialis muscles and subcutaneous fat (inguinal) were harvested, and metabolic and molecular effects were evaluated. Muscle Fndc5 mRNA increased with OE (~2-fold; P = 0.014), leading to increased circulating irisin (1.5 ± 0.9 to 3.5 ± 1.2 ng/mL; P = 0.049). OE had no effect on protein anabolism or mitochondrial biogenesis; however, muscle glycogen was increased, along with glycogen synthase 1 gene expression (P = 0.04 and 0.02, respectively). In addition to an increase in glycogen synthase activation in OE (P = 0.03), there was a tendency toward increased glucose transporter 4 protein (P = 0.09). However, glucose uptake (accumulation of 2-DG6P) was identical. Irisin elicited no endocrine effect on mitochondrial biogenesis or uncoupling proteins in white adipose tissue. Hindlimb overexpression led to physiological increases in Fndc5/irisin. However, our data indicate limited short-term impacts of irisin in relation to muscle anabolism, mitochondrial biogenesis, glucose uptake, or adipose remodeling.

Maximal-intensity exercise does not fully restore muscle pyruvate dehydrogenase complex activation after 3 days of high-fat dietary intake.

BACKGROUND & AIMS: Exercise activates muscle pyruvate dehydrogenase complex (PDC), but moderate intensity exercise fails to fully activate muscle PDC after high-fat diet [1]. We investigated whether maximal intensity exercise overcomes this inhibition. METHODS: Quadriceps femoris muscle biopsy samples were obtained from healthy males at rest, and after 46 and 92 electrically-evoked maximal intermittent isometric contractions, which were preceded by 3 days of either low- (18%) or high- (69%) isocaloric dietary fat intake (LFD and HFD, respectively). RESULTS: The ratio of PDCa (active form) to total PDCt (fully activated) at rest was 50% less after HFD (0.32 ± 0.01 vs 0.15 ± 0.01; P < 0.05). This ratio increased to 0.77 ± 0.06 after 46 contractions (P < 0.001) and to 0.98 ± 0.07 after 92 contractions (P < 0.001) in LFD. The corresponding values after HFD were less (0.54 ± 0.06; P < 0.01 and 0.70 ± 0.07; P < 0.01, respectively). Resting muscle acetyl-CoA and acetylcarnitine content was greater after HFD than LFD (both P < 0.05), but their rate of accumulation in the former was reduced during contraction. Muscle lactate content after 92 contractions was 30% greater after HFD (P < 0.05). Muscle force generation during contraction was no different between interventions, but HFD lengthened muscle relaxation time (P < 0.05). Daily urinary total carnitine excretion after HFD was 2.5-fold greater than after LFD (P < 0.01). CONCLUSIONS: A bout of maximal intense exercise did not overcome dietary fat-mediated inhibition of muscle pyruvate dehydrogenase complex activation, and was associated with greater muscle lactate accumulation, as a result of lower PDC flux, and increased muscle relaxation time.

The effect of age and unilateral leg immobilization for 2 weeks on substrate utilization during moderate-intensity exercise in human skeletal muscle.

KEY POINTS: This study aimed to provide molecular insight into the differential effects of age and physical inactivity on the regulation of substrate metabolism during moderate-intensity exercise. Using the arteriovenous balance technique, we studied the effect of immobilization of one leg for 2 weeks on leg substrate utilization in young and older men during two-legged dynamic knee-extensor moderate-intensity exercise, as well as changes in key proteins in muscle metabolism before and after exercise. Age and immobilization did not affect relative carbohydrate and fat utilization during exercise, but the older men had higher uptake of exogenous fatty acids, whereas the young men relied more on endogenous fatty acids during exercise. Using a combined whole-leg and molecular approach, we provide evidence that both age and physical inactivity result in intramuscular lipid accumulation, but this occurs only in part through the same mechanisms. ABSTRACT: Age and inactivity have been associated with intramuscular triglyceride (IMTG) accumulation. Here, we attempt to disentangle these factors by studying the effect of 2 weeks of unilateral leg immobilization on substrate utilization across the legs during moderate-intensity exercise in young (n = 17; 23 ± 1 years old) and older men (n = 15; 68 ± 1 years old), while the contralateral leg served as the control. After immobilization, the participants performed two-legged isolated knee-extensor exercise at 20 ± 1 W (∼50% maximal work capacity) for 45 min with catheters inserted in the brachial artery and both femoral veins. Biopsy samples obtained from vastus lateralis muscles of both legs before and after exercise were used for analysis of substrates, protein content and enzyme activities. During exercise, leg substrate utilization (respiratory quotient) did not differ between groups or legs. Leg fatty acid uptake was greater in older than in young men, and although young men demonstrated net leg glycerol release during exercise, older men showed net glycerol uptake. At baseline, IMTG, muscle pyruvate dehydrogenase complex activity and the protein content of adipose triglyceride lipase, acetyl-CoA carboxylase 2 and AMP-activated protein kinase (AMPK)γ3 were higher in young than in older men. Furthermore, adipose triglyceride lipase, plasma membrane-associated fatty acid binding protein and AMPKγ3 subunit protein contents were lower and IMTG was higher in the immobilized than the contralateral leg in young and older men. Thus, immobilization and age did not affect substrate choice (respiratory quotient) during moderate exercise, but the whole-leg and molecular differences in fatty acid mobilization could explain the age- and immobilization-induced IMTG accumulation.

Temporal changes in the involvement of pyruvate dehydrogenase complex in muscle lactate accumulation during lipopolysaccharide infusion in rats.

A characteristic manifestation of sepsis is muscle lactate accumulation. This study examined any putative (causative) association between pyruvate dehydrogenase complex (PDC) inhibition and lactate accumulation in the extensor digitorum longus (EDL) muscle of rats infused with lipopolysaccharide (LPS), and explored the involvement of increased transcription of muscle-specific pyruvate dehydrogenase kinase (PDK) isoenzymes. Conscious, male Sprague-Dawley rats were infused i.v. with saline (0.4 ml h(-1), control) or LPS (150 mug kg(-1) h(-1)) for 2 h, 6 h or 24 h (n = 6-8). Muscle lactate concentration was elevated after 2, 6 and 24 h LPS infusion. Muscle PDC activity was the same at 2 h and 6 h, but was 65% lower after 24 h of LPS infusion (P < 0.01), when there was a 47% decrease in acetylcarnitine concentration (P < 0.05), and a 24-fold increase in PDK4 mRNA expression (P < 0.001). These changes were preceded by marked increases in tumour necrosis factor-alpha and interleukin-6 mRNA expression at 2 h. The findings indicate that the early (2 and 6 h) elevation in muscle lactate concentration during LPS infusion was not attributable to limited muscle oxygen availability or ATP production (evidenced by unchanged ATP and phosphocreatine (PCr) concentrations) or to PDC inhibition, whereas after 24 h, muscle lactate accumulation appears to have resulted from PDC activation status limiting pyruvate flux, most probably due to cytokine-mediated up-regulation of PDK4 transcription.

Chronic treatment with the beta(2)-adrenoceptor agonist prodrug BRL-47672 impairs rat skeletal muscle function by inducing a comprehensive shift to a faster muscle phenotype.

Discovering approaches to maintain or improve muscle function (fatigue resistance) in patients with cachexia, postoperative weakness, and sarcopenia is of clinical importance. beta(2)-Agonist treatment increases muscle mass, yet it alters fiber proportions such that the net consequences on muscle function remain unclear. In the present study, we focus on the contractile and metabolic consequences of chronic treatment with the beta(2)-agonist prodrug BRL-47672 (BRL). Gastrocnemius-plantaris-soleus (GPS) muscles were harvested at rest and studied for fatigue characteristics during 4 and 20 s of isometric stimulation (30 Hz; 10 V; 200 ms) using the perfused hind limb model. BRL treatment increased GPS mass by 21% (P < 0.05), whereas greater fatigue occurred during 20 s of contraction (45% less work; P < 0.05). Phenotypically, BRL resulted in 17% more type IIb myosin heavy chain protein expression (P < 0.001) and greater adenine nucleotide catabolism during 20 s of contraction (P < 0.05). Chronic BRL treatment impaired maximal lipid oxidation capacity by 30% (P < 0.05) and reduced glutamate dehydrogenase activity by 15% (P < 0.05). We conclude that beta(2)-agonist induced muscle hypertrophy may be clinically limited as impaired energy metabolism and function occur, presumably as a consequence of the shift in muscle phenotype.

Metabolic inertia in contracting skeletal muscle: a novel approach for pharmacological intervention in peripheral vascular disease.

Peripheral vascular disease (PVD) is generally accepted to result in the failure of skeletal muscle blood flow to increase adequately at the onset of muscular work. There are currently no routine pharmacological interventions towards the treatment of PVD, however, recent Phase III trials in the USA have demonstrated the clinical potential of the phosphodiesterase III inhibitor Cilostazol for pain-free and maximal walking distances in patients with intermittent claudication. PVD is characterized by a marked reliance on oxygen-independent routes of ATP regeneration (phosphocreatine hydrolysis and glycolysis) in skeletal muscle during contraction and the rapid onset of muscular pain and fatigue. The accumulation of metabolic by-products of oxygen-independent ATP production (hydrogen and lactate ions and inorganic phosphate) has long been associated with an inhibition in contractile function in both healthy volunteers and PVD patients. Therefore, any strategy that could reduce the reliance upon ATP re-synthesis from oxygen-independent routes, and increase the contribution of oxygen-dependent (mitochondrial) ATP re-synthesis, particularly at the onset of exercise, might be expected to improve functional capacity and be of considerable therapeutic value. Historically, the increased contribution of oxygen-independent ATP re-synthesis to total ATP generation at the onset of exercise has been attributed to a lag in muscle blood flow limiting oxygen delivery during this period. However, recent evidence suggests that limited inertia is present at the level of oxygen delivery, whilst considerable inertia exists at the level of mitochondrial enzyme activation and substrate supply. In support of this latter hypothesis, we have reported on a number of occasions that activation of the pyruvate dehydrogenase complex, using pharmacological interventions, can markedly reduce the dependence on ATP re-synthesis from oxygen-independent routes at the onset of muscle contraction. This review will focus on these findings and will highlight the pyruvate dehydrogenase complex as a novel therapeutic target towards the treatment of peripheral vascular disease, or any other disease state where premature muscular fatigue is prevalent due to metabolite accumulation.

An acetyl group deficit limits mitochondrial ATP production at the onset of exercise.

The oxygen deficit at the onset of submaximal exercise represents a period when the energy demand of contraction cannot be met solely by mitochondrial ATP generation, and as a consequence there is an acceleration of ATP re-synthesis from oxygen-independent routes (phosphocreatine hydrolysis and glycolysis). Historically, the origin of the oxygen deficit has been attributed to a lag in muscle blood flow and oxygen availability at the onset of exercise which limits mitochondrial respiration. However, more recent evidence suggests that considerable inertia exists at the level of mitochondrial enzyme activation and substrate supply. In support of this latter hypothesis, we have reported on a number of occasions that pharmacological activation of the pyruvate dehydrogenase complex (and consequent stockpiling of acetyl groups), using dichloroacetate or exercise interventions, can markedly reduce the degree of ATP re-synthesis from oxygen-independent routes during the rest-to-work transition period. This review will focus on these findings, and will offer the hypothesis that acetyl group delivery to the tricarboxylic acid cycle limits mitochondrial flux at the onset of exercise--the so-called acetyl group deficit.

Phosphocreatine degradation in type I and type II muscle fibres during submaximal exercise in man: effect of carbohydrate ingestion.

1. The aim of this study was to examine the effect of carbohydrate (CHO) ingestion on changes in ATP and phosphocreatine (PCr) concentrations in different muscle fibre types during prolonged running and relate those changes to the degree of glycogen depletion. 2. Five male subjects performed two runs at 70 % maximum oxygen uptake (.V(O2,max)), 1 week apart. Each subject ingested 8 ml (kg body mass (BM))(-1) of either a placebo (Con trial) or a 5.5 % CHO solution (CHO trial) immediately before each run and 2 ml (kg BM)(-1) every 20 min thereafter. In the Con trial, the subjects ran to exhaustion (97.0 +/- 6.7 min). In the CHO trial, the run was terminated at the time coinciding with exhaustion in the Con trial. Muscle samples were obtained from the vastus lateralis before and after each trial. 3. Carbohydrate ingestion did not affect ATP concentrations. However, it attenuated the decline in PCr concentration by 46 % in type I fibres (CHO: 20 +/- 8 mmol (kg dry matter (DM))(-1); Con: 34 +/- 6 mmol (kg DM)(-1); P < 0.05) and by 36 % in type II fibres (CHO: 30 +/- 5 mmol (kg DM)(-1); Con: 48 +/- 6 mmol (kg DM)(-1); P < 0.05). 4. A 56 % reduction in glycogen utilisation in type I fibres was observed in CHO compared with Con (117 +/- 39 vs. 240 +/- 32 mmol glucosyl units (kg DM)(-1), respectively; P < 0.01), but no difference was observed in type II fibres. 5. It is proposed that CHO ingestion during exhaustive running attenuates the decline in oxidative ATP resynthesis in type I fibres, as indicated by sparing of both PCr and glycogen breakdown. The CHO-induced sparing of PCr, but not glycogen, in type II fibres may reflect differential recruitment and/or role of PCr between fibre types.

The effects of increasing exercise intensity on muscle fuel utilisation in humans.

1. Contemporary stable isotope methodology was applied in combination with muscle biopsy sampling to accurately quantify substrate utilisation and study the regulation of muscle fuel selection during exercise. 2. Eight cyclists were studied at rest and during three consecutive 30 min stages of exercise at intensities of 40, 55 and 75 % maximal workload (W(max)). A continuous infusion of [U-(13)C]palmitate and [6,6-(2)H(2)]glucose was administered to determine plasma free fatty acid (FFA) oxidation and estimate plasma glucose oxidation, respectively. Biopsy samples were collected before and after each exercise stage. 3. Muscle glycogen and plasma glucose oxidation rates increased with every increment in exercise intensity. Whole-body fat oxidation increased to 32 +/- 2 kJ min(-1) at 55 % W(max), but declined at 75 % W(max) (19 +/- 2 kJ min(-1)). This decline involved a decrease in the oxidation rate of both plasma FFA and triacylglycerol fat sources (sum of intramuscular plus lipoprotein-derived triacylglycerol), and was accompanied by increases in muscle pyruvate dehydrogenase complex activation and acetylation of the carnitine pool, resulting in a decline in muscle free carnitine concentration. 4. We conclude that the most likely mechanism for the reduction in fat oxidation during high-intensity exercise is a downregulation of carnitine palmitoyltransferase I, either by this marked decline in free carnitine availability or by a decrease in intracellular pH.

Glutamine supplementation promotes anaplerosis but not oxidative energy delivery in human skeletal muscle.

The aims of the present study were twofold: first to investigate whether TCA cycle intermediate (TCAI) pool expansion at the onset of moderate-intensity exercise in human skeletal muscle could be enhanced independently of pyruvate availability by ingestion of glutamine or ornithine alpha-ketoglutarate, and second, if it was, whether this modification of TCAI pool expansion had any effect on oxidative energy status during subsequent exercise. Seven males cycled for 10 min at approximately 70% maximal O2) uptake 1 h after consuming either an artificially sweetened placebo (5 ml/kg body wt solution, CON), 0.125 g/kg body wt L-(+)-ornithine alpha-ketoglutarate dissolved in 5 ml/kg body wt solution (OKG), or 0.125 g/kg body wt L-glutamine dissolved in 5 ml/kg body wt solution (GLN). Vastus lateralis muscle was biopsied 1 h postsupplement and after 10 min of exercise. The sum of four measured TCAI (SigmaTCAI; citrate, malate, fumarate, and succinate, approximately 85% of total TCAI pool) was not different between conditions 1 h postsupplement. However, after 10 min of exercise, SigmaTCAI (mmol/kg dry muscle) was greater in the GLN condition (4.90 +/- 0.61) than in the CON condition (3.74 +/- 0.38, P < 0.05) and the OKG condition (3.85 +/- 0.28). After 10 min of exercise, muscle phosphocreatine (PCr) content was significantly reduced (P < 0.05) in all conditions, but there was no significant difference between conditions. We conclude that the ingestion of glutamine increased TCAI pool size after 10 min of exercise most probably because of the entry of glutamine carbon at the level of alpha-ketoglutarate. However, this increased expansion in the TCAI pool did not appear to increase oxidative energy production, because there was no sparing of PCr during exercise.

Carbohydrate ingestion prior to exercise augments the exercise-induced activation of the pyruvate dehydrogenase complex in human skeletal muscle.

This study examined the effect of pre-exercise carbohydrate (CHO) ingestion on pyruvate dehydrogenase complex (PDC) activation, acetyl group availability and substrate level phosphorylation (glycogenolysis and phosphocreatine (PCr) hydrolysis) in human skeletal muscle during the transition from rest to steady-state exercise. Seven male subjects performed two 10 min treadmill runs at 70 % maximum oxygen uptake (VO2,max), 1 week apart. Each subject ingested 8 ml (kg body mass (BM))-1 of either a placebo solution (CON trial) or a 5.5 % CHO solution (CHO trial) 10 min before each run. Muscle biopsy samples were obtained from the vastus lateralis at rest and immediately after each trial. Muscle PDC activity was higher at the end of exercise in the CHO trial compared with the CON trial (1.78+/-0.18 and 1.27+/-0.16 mmol min(-1) (kg wet matter (WM))(-1), respectively; P 0.05) and this was accompanied by lower acetylcarnitine (7.1+/-1.2 and 9.1+/-1.1 mmol kg(-1) (dry matter (DM))(-1) in CHO and CON, respectively; P<0.05) and citrate concentrations (0.73+/-0.05 and 0.91+/-0.10 mmol (kg DM)(-1) in CHO and CON, respectively; P<0.05). No difference was observed between trials in the rates of muscle glycogen and PCr breakdown and lactate accumulation. This is the first study to demonstrate that CHO ingestion prior to exercise augments the exercise-induced activation of muscle PDC and reduces acetylcarnitine accumulation during the transition from rest to steady-state exercise. However, those changes did not affect the contribution of substrate level phosphorylation to ATP resynthesis.

The importance of pyruvate availability to PDC activation and anaplerosis in human skeletal muscle.

No studies have singularly investigated the relationship between pyruvate availability, pyruvate dehydrogenase complex (PDC) activation, and anaplerosis in skeletal muscle. This is surprising given the functional importance attributed to these processes in normal and disease states. We investigated the effects of changing pyruvate availability with dichloroacetate (DCA), epinephrine, and pyruvate infusions on PDC activation and accumulation of acetyl groups and tricarboxylic acid (TCA) cycle intermediates (TCAI) in human muscle. DCA increased resting PDC activity sixfold (P < 0.05) but decreased the muscle TCAI pool (mmol/kg dry muscle) from 1.174 +/- 0.042 to 0.747 +/- 0.055 (P < 0.05). This was probably a result of pyruvate being diverted to acetyl-CoA and acetylcarnitine after near-maximal activation of PDC by DCA. Conversely, neither epinephrine nor pyruvate activated PDC. However, both increased the TCAI pool (1.128 +/- 0.076 to 1.614 +/- 0.188, P < 0.05 and 1.098 +/- 0.059 to 1.385 +/- 0.114, P < 0.05, respectively) by providing a readily available pool of pyruvate for anaplerosis. These data support the hypothesis that TCAI pool expansion is principally a reflection of increased muscle pyruvate availability and, together with our previous work (J. A. Timmons, S. M. Poucher, D. Constantin-Teodosiu, V. Worrall, I. A. Macdonald, and P. L. Greenhaff. J. Clin. Invest. 97: 879-883, 1996), indicate that TCA cycle expansion may be of little functional significance to TCA cycle flux. It would appear therefore that the primary effect of DCA on oxidative ATP provision is to provide a readily available pool of acetyl groups to the TCA cycle at the onset of exercise rather than increasing TCA cycle flux by expanding the TCAI pool.

The tricarboxylic acid cycle in human skeletal muscle: is there a role for nutritional intervention?

The tricarboxylic acid (TCA) cycle is essential for oxidative energy production. The expansion (anaplerosis) of the intermediates of the TCA cycle is achieved via a number of pathways, and is known to be influenced by metabolic status and nutritional and pharmacological interventions. Contraction is associated with anaplerosis in skeletal muscle, and some authors have suggested that the rate of anaplerosis can limit oxidative energy delivery. The results of more recent studies, however, are consistent with the idea that expansion of the muscle TCA intermediate pool is principally a reflection of muscle pyruvate availability, and is of little functional importance to TCA cycle flux, thereby indicating that any intervention aimed at increasing TCA intermediates expansion will be of little practical value.

Regulation of skeletal muscle carbohydrate oxidation during steady-state contraction.

Pyruvate dehydrogenase complex (PDC) activation status has been described as being central in the regulation of tissue substrate oxidation as outlined by the glucose fatty-acid cycle. In the present study we examined the effects of reduced lipolysis, with use of nicotinate, and increased PDC activation, with use of dichloroacetate (DCA), on substrate utilization during 20 min of submaximal steady-state contraction (approximately 80% of maximal O2 uptake) in canine gracilis skeletal muscle. At rest, PDC activation was unchanged by nicotinate but was approximately 2.5-fold higher in the DCA group than in the control group (P < 0.05). During contraction, PDC activation status increased to 3.5 mmol acetyl-CoA.min-1.kg-1 at 37 degrees C in the control group, remained at 4.5 mmol acetyl-CoA.min-1.kg-1 at 37 degrees C in the DCA group, but only increased to 2.2 mmol acetyl-CoA.min-1.kg-1 at 37 degrees C in the nicotinate group (P < 0.05). However, the estimated amount of carbohydrate oxidized during the 20-min contraction was similar across groups and did not follow the degree of PDC activation (81.2 +/- 22.9, 95.9 +/- 11.7, and 89.3 +/- 18.9 mmol glucosyl units/kg dry muscle for control, nicotinate, and DCA, respectively). Thus it would appear that, during steady-state contraction, PDC activation status does not determine the rate of carbohydrate oxidation in skeletal muscle.

Dual effects of dichloroacetate on cardiac ischaemic preconditioning in the rat isolated perfused heart.

1. Ischaemic cardiac preconditioning represents an important cardioprotective mechanism which limits myocardial ischaemic damage. The aim of this investigation was to assess the impact of dichloroacetate (DCA), a pyruvate dehydrogenase complex activator, on preconditioning. 2. Rat isolated hearts were perfused by use of the Langendorff technique, and were subjected to either preconditioning (3 x 4 or 3 x 6 min ischaemia) or continuous perfusion, followed by 30 min global ischaemia and 60 min reperfusion. DCA (3 mM) was either given throughout the protocol (pretreatment), during reperfusion only (post-treatment), or not at all. Throughout reperfusion mechanical performance was assessed as the rate-pressure product (RPP: left ventricular developed pressure x heart rate). 3. In non-preconditioned control hearts, mechanical performance was substantially (P < 0.001) depressed on reperfusion (the RPP after 60 min of reperfusion (RPP(t=60)) was 4,246+/-974 mmHg beats min(-1) compared to baseline value of 21,297+/-1,728 mmHg beats min(-1)). Preconditioning with either 3 x 4 min or 3 x 6 min cycles caused significant protection, as shown by enhanced recovery (RPP(t=60) = 7,818+/-1,138, P < 0.05, and 11,123+/-587 mmHg beats min(-1), P < 0.001, respectively). 4. Addition of DCA (3 mM) to hearts under baseline conditions significantly (P < 0.001) enhanced systolic function with an increased left ventricular developed pressure of 108+/-5 mmHg compared to 88.3+/-3.0 mmHg in the controls. 5. Pretreatment with 3 mM DCA had no effect on recovery of mechanical performance in the non-preconditioned hearts (RPP(t=60) = 3,640+/-1,235 mmHg beats min(-1)) while the beneficial effects of preconditioning were reduced in the preconditioned hearts (3 x 4 min: RPP(t=60) = 2,919+/-1,060 mmHg beats min(-1); 3 x 6 min: RPP(t=60) = 8,032+/-1,367 mmHg beats min(-1)). Therefore, DCA had increased the threshold for preconditioning. 6. By contrast, post-treatment of hearts with 3 mM DCA substantially improved recovery on reperfusion in all groups (RPP(t=60) = 5,827+/-1,328 (non-preconditioned), 14,022+/-3,743 (3 x 4 min; P < 0.01) and 23,219+/-1,374 (3 x 6 min; P < 0.001) mmHg beats min(-1)). 7. The results of the present investigation clearly show that pretreatment with DCA enhances baseline cardiac mechanical performance but increases the threshold for cardiac preconditioning. However, post-treatment with DCA substantially augments the beneficial effects of preconditioning.

Substrate availability limits human skeletal muscle oxidative ATP regeneration at the onset of ischemic exercise.

We have demonstrated previously that dichloroacetate can attenuate skeletal muscle fatigue by up to 35% in a canine model of peripheral ischemia (Timmons, J.A., S.M. Poucher, D. Constantin-Teodosiu, V. Worrall, I.A. Macdonald, and P.L. Greenhaff. 1996. J. Clin. Invest. 97:879-883). This was thought to be a consequence of dichloroacetate increasing acetyl group availability early during contraction. In this study we characterized the metabolic effects of dichloroacetate in a human model of peripheral muscle ischemia. On two separate occasions (control-saline or dichloroacetate infusion), nine subjects performed 8 min of single-leg knee extension exercise at an intensity aimed at achieving volitional exhaustion in approximately 8 min. During exercise each subject's lower limbs were exposed to 50 mmHg of positive pressure, which reduces blood flow by approximately 20%. Dichloroacetate increased resting muscle pyruvate dehydrogenase complex activation status by threefold and elevated acetylcarnitine concentration by fivefold. After 3 min of exercise, phosphocreatine degradation and lactate accumulation were both reduced by approximately 50% after dichloroacetate pretreatment, when compared with control conditions. However, after 8 min of exercise no differences existed between treatments. Therefore, it would appear that dichloroacetate can delay the accumulation of metabolites which lead to the development of skeletal muscle fatigue during ischemia but does not alter the metabolic profile when a maximal effort is approached.

Metabolic responses from rest to steady state determine contractile function in ischemic skeletal muscle.

Skeletal muscle contraction during ischemia, such as that experienced by peripheral vascular disease patients, is characterized by rapid fatigue. Using a canine gracilis model, we tested the hypothesis that a critical factor determining force production during ischemia is the metabolic response during the transition from rest to steady state. Dichloroacetate (DCA) administration before gracilis muscle contraction increased pyruvate dehydrogenase complex activation and resulted in acetylation of 80% of the free carnitine pool to acetylcarnitine. After 1 min of contraction, phosphocreatine (PCr) degradation in the DCA group was approximately 50% lower than in the control group (P < 0.05) during conditions of identical force production. After 6 min of contraction, steady-state force production was approximately 30% higher in the DCA group (P < 0.05), and muscle ATP, PCr, and glycogen degradation and lactate accumulation were lower (P < 0.05 in all cases). It appears, therefore, that an important determinant of contractile function during ischemia is the mechanisms by which ATP regeneration occurs during the period of rest to steady-state transition.

Anaerobic energy production in human skeletal muscle in intense contraction: a comparison of 31P magnetic resonance spectroscopy and biochemical techniques.

Five subjects underwent twenty electrically evoked maximal isometric contractions of the anterior tibialis muscle of both legs (n = 10), with limb blood flow occluded. Measurements of muscle high-energy phosphates (ATP, ADP and phosphocreatine (PCr)), lactate and pH were made using both 31P magnetic resonance spectroscopy (MRS) and the biochemical analysis of biopsy samples obtained from directly below the MRS surface coil. The resting PCr concentration determined using MRS was similar to that measured in the biopsy material. Following contraction, MRS showed a greater decrease in ATP concentration compared with biochemical analysis (P < 0.05), but the decrease in PCr was similar. Good agreement was found when comparing resting muscle pH estimated by the two methods. Post-exercise muscle pH was, however, consistently lower with MRS and consequently the accumulation of muscle lactate estimated using MRS was markedly greater than the corresponding biochemical measurement (P < 0.05). As a result, MRS revealed an approximately 30% greater anaerobic ATP turnover during contraction, although this just failed to reach statistical significance (P > 0.05). The results of the present study indicate that there is little difference in the muscle concentration of PCr estimated by the two methods, but that there are differences in the estimates of ATP, pH and lactate changes during contraction. This latter discrepancy may lead to greater estimates of ATP turnover being made as a result of MRS analysis.

Energy expenditure using isotope-labelled water (2H218O), exercise performance, skeletal muscle enzyme activities and plasma biochemical parameters in humans during 95 days of endurance exercise with inadequate energy intake.

Two men, R.F. and M.S., pulled sledges each with starting masses of 222 kg, 2300 km across Antarctica. Exercise was performed for approximately 10 h each day for 95 days. Despite an average energy intake of 21.3 MJ.day-1 both subjects lost more than 25% of body weight. Energy expenditure was measured using energy balance data (EB) and isotope-labelled water (2H218O). Isotope doses were taken on day 0 and day 50 of the expedition. During the first 50 days both methods gave reasonable agreement, giving energy expenditures of 38.3 (EB) and 35.5 (2H218O) MJ.day-1 in R.F. and 28.6 (EB) and 29.1 (2H218O) MJ.day-1 in M.S. The isotope data for days 20-30 yielded exceptional values of 44.6 MJ.day-1 in R.F. and 48.7 MJ.day-1 in M.S. Estimates of energy expenditure between day 51 and day 96 were much lower and although the methods were in agreement for R.F.-24.1 (EB) and 23.1 (2H218O) MJ. day-1, there was poor agreement for MS-26.8 (EB) and 18.8 (2H218O) MJ.day-1. However, some practical difficulties occurred during this second period and there were also problems arising from marked increases in body water that made estimates of body mass and composition change difficult to interpret. The latter problems were probably due to malnutrition, which may have also been responsible for surprising increases in urinary excretion of 2H and 18O observed in both men at around day 81. These increases may reflect the release of label incorporated into molecules other than water which do not normally freely exchange with the body water pool under the circumstances of marked malnourishment. Following the expedition, both men showed declines in maximal O2 consumption (VO2max, 53.6 to 41.2 ml O2 kg-1.min-1 in R.F., 58.1-46.0 ml O2 kg-1.min-1 in M.S.); maximal voluntary isometric force production in different muscle groups (up to 19.9% in R.F. and 55.8% in M.S.) and both cytoplasmic and mitochondrial skeletal muscle enzyme activities (up to 56% in R.F. and 63% in M.S.). Plasma samples taken during the expedition showed low glucose levels, inappropriately high insulin levels, and declines in testosterone and luteinizing hormone. Thyroxine, cholesterol, albumin and triglyceride levels remained normal.

Creatine ingestion favorably affects performance and muscle metabolism during maximal exercise in humans.

Nine male subjects performed two bouts of 30-s maximal isokinetic cycling before and after ingestion of 20 g creatine (Cr) monohydrate/day for 5 days. Cr ingestion produced a 23.1 +/- 4.7 mmol/kg dry matter increase in the muscle total creatine (TCr) concentration. Total work production during bouts 1 and 2 increased by approximately 4%, and the cumulative increases in both peak and total work production over the two exercise bouts were positively correlated with the increase in muscle TCr. Cumulative loss of ATP was 30.7 +/- 12.2% less after Cr ingestion, despite the increase in work production. Resting phosphocreatine (PCr) increased in type I and II fibers. Changes in PCr before exercise bouts 1 and 2 in type II fibers were positively correlated with changes in PCr degradation during exercise in this fiber type and changes in total work production. The results suggest that improvements in performance were mediated via improved ATP resynthesis as a consequence of increased PCr availability in type II fibers.