Dumitru Constantin-Teodosiu

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 (Wmax). A continuous infusion of [U‐13C]palmitate and [6,6‐2H2]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 % Wmax, but declined at 75 % Wmax (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.

New insights concerning the role of carnitine in the regulation of fuel metabolism in skeletal muscle

In skeletal muscle, carnitine plays an essential role in the translocation of long‐chain fatty‐acids into the mitochondrial matrix for subsequent β‐oxidation, and in the regulation of the mitochondrial acetyl‐CoA/CoASH ratio. Interest in these vital metabolic roles of carnitine in skeletal muscle appears to have waned over the past 25 years. However, recent research has shed new light on the importance of carnitine as a regulator of muscle fuel selection. It has been established that muscle free carnitine availability may be limiting to fat oxidation during high intensity submaximal exercise. Furthermore, increasing muscle total carnitine content in resting healthy humans (via insulin‐mediated stimulation of muscle carnitine transport) reduces muscle glycolysis, increases glycogen storage and is accompanied by an apparent increase in fat oxidation. By increasing muscle pyruvate dehydrogenase complex (PDC) activity and acetylcarnitine content at rest, it has also been established that PDC flux and acetyl group availability limits aerobic ATP re‐synthesis at the onset of exercise (the acetyl group deficit). Thus, carnitine plays a vital role in the regulation of muscle fuel metabolism. The demonstration that its availability can be readily manipulated in humans, and impacts on physiological function, will result in renewed business and scientific interest in this compound.

Radioisotopic assays of CoASH and carnitine and their acetylated forms in human skeletal muscle

Radioisotopic assays for the determination of acetyl-CoA, CoASH, and acetylcarnitine have been modified for application to the amount of human muscle tissue that can be obtained by needle biopsy. In the last step common to all three methods, acetyl-CoA is condensed with [14C]oxaloacetate by citrate synthase to give [14C]-citrate. For determination of CoASH, CoASH is reacted with acetylphosphate in a reaction catalyzed by phosphotransacetylase to yield acetyl-CoA. In the assay for acetylcarnitine, acetylcarnitine is reacted with CoASH in a reaction catalyzed by carnitine acetyltransferase to form acetyl-CoA. Inclusion of new simple steps in the acetylcarnitine assay and conditions affecting the reliability of all three methods are also described. Acetylcarnitine and free carnitine levels in human rectus abdominis muscle were 3.0 +/- 1.5 (SD) and 13.5 +/- 4.0 mumol/g dry wt, respectively. Values for acetyl-CoA and CoASH were about 500-fold lower, 6.7 +/- 1.8 and 21 +/- 8.9 nmol/g dry wt, respectively. A strong correlation between acetylcarnitine (y) and short-chain acylcarnitine (x), determined as the difference between total and free carnitine, was found in biopsies from the vastus lateralis muscle obtained during intense muscular effort, y = 1.0x + 0.5; r = 0.976.

A sensitive radioisotopic assay of pyruvate dehydrogenase complex in human muscle tissue

A radioactive assay for the determination of pyruvate dehydrogenase complex activity in muscle tissue has been developed. The assay measures the rate of acetyl-CoA formation from pyruvate in a reaction mixture containing NAD+ and CoASH. The acetyl-CoA is determined as [14C]citrate after condensation with [14C]-oxaloacetate by citrate synthase. The method is specific and sensitive to the picomole range of acetyl-CoA formed. In eleven normal subjects, the active form of pyruvate dehydrogenase (PDCa) in resting human skeletal muscle samples obtained using the needle biopsy technique was 0.44 +/- 0.16 (SD) mumol acetyl-CoA.min-1.g-1 wet wt. Total pyruvate dehydrogenase complex (PDCt) activity was determined after activation by pretreating the muscle homogenate with Ca2+, Mg2+, dichloroacetate, glucose, and hexokinase. The mean value for PDCt was 1.69 +/- 0.32 mumol acetyl-CoA.min-1.g-1 wet wt, n = 11. The precision of the method was determined by analyzing 4-5 samples of the same muscle piece. The coefficient of variation for PDCa was 8% and for PDCt 5%.

Oxygen uptake on-kinetics in dog gastrocnemius in situ following activation of pyruvate dehydrogenase by dichloroacetate.

The aim of the present study was to determine whether the activation of the pyruvate dehydrogenase complex (PDC) by dichloroacetate (DCA) is associated with faster O2 uptake (V̇O2) on‐kinetics. V̇O2 on‐kinetics was determined in isolated canine gastrocnemius muscles in situ (n= 6) during the transition from rest to 4 min of electrically stimulated isometric tetanic contractions, corresponding to ∼60–70 % of peak V̇O2. Two conditions were compared: (1) control (saline infusion, C); and (2) DCA infusion (300 mg (kg body mass)−1, 45 min before contraction). Muscle blood flow (Q̇) was measured continuously in the popliteal vein; arterial and popliteal vein O2 contents were measured at rest and at 5–7 s intervals during the transition. Muscle V̇O2 was calculated as Q̇ multiplied by the arteriovenous O2 content difference. Muscle biopsies were taken before and at the end of contraction for determination of muscle metabolite concentrations. DCA activated PDC at rest, as shown by the 9‐fold higher acetylcarnitine concentration in DCA (vs. C; P < 0.0001). Phosphocreatine degradation and muscle lactate accumulation were not significantly different between C and DCA. DCA was associated with significantly less muscle fatigue. Resting and steady‐state V̇O2 values during contraction were not significantly different between C and DCA. The time to reach 63 % of the V̇O2 difference between the resting baseline and the steady‐state V̇O2 values during contraction was 22.3 ± 0.5 s in C and 24.5 ± 1.4 s in DCA (n.s.). In this experimental model, activation of PDC by DCA resulted in a stockpiling of acetyl groups at rest and less muscle fatigue, but it did not affect ‘anaerobic’ energy provision and V̇O2 on‐kinetics.

A potential role for Akt/FOXO signalling in both protein loss and the impairment of muscle carbohydrate oxidation during sepsis in rodent skeletal muscle

Sepsis causes muscle atrophy and insulin resistance, but the underlying mechanisms are unclear. Therefore, the present study examined the effects of lipopolysaccharide (LPS)‐induced endotoxaemia on the expression of Akt, Forkhead Box O (FOXO) and its downstream targets, to identify any associations between changes in FOXO‐dependent processes influencing muscle atrophy and insulin resistance during sepsis. Chronically instrumented male Sprague–Dawley rats received a continuous intravenous infusion of LPS (15 μg kg−1 h−1) or saline for 24 h at 0.4 ml h−1. Animals were terminally anaesthetized and the extensor digitorum longus muscles from both hindlimbs were removed and snap‐frozen. Measurements were made of mRNA and protein expression of selected signalling molecules associated with pathways regulating protein synthesis and degradation and carbohydrate metabolism. LPS infusion induced increases in muscle tumour necrosis factor‐α (8.9‐fold, P < 0.001) and interleukin‐6 (8.4‐fold, P < 0.01), paralleled by reduced insulin receptor substrate‐1 mRNA expression (−0.7‐fold, P < 0.01), and decreased Akt1 protein and cytosolic FOXO1 and FOXO3 phosphorylation. These changes were accompanied by significant increases in muscle atrophy F‐box mRNA (5.5‐fold, P < 0.001) and protein (2‐fold, P < 0.05) expression, and pyruvate dehydrogenase kinase 4 mRNA (15‐fold, P < 0.001) and protein (1.6‐fold, P < 0.05) expression. There was a 29% reduction in the muscle protein: DNA ratio, a 56% reduction in pyruvate dehydrogenase complex (PDC) activity (P < 0.05), and increased glycogen degradation and lactate accumulation. The findings of this study suggest a potential role for Akt/FOXO in the simultaneous impairment of carbohydrate oxidation, at the level of PDC, and up‐regulation of muscle protein degradation, in LPS‐induced endotoxaemia.

Regulation of human metabolism by hypoxia- inducible factor

The hypoxia-inducible factor (HIF) family of transcription factors directs a coordinated cellular response to hypoxia that includes the transcriptional regulation of a number of metabolic enzymes. Chuvash polycythemia (CP) is an autosomal recessive human disorder in which the regulatory degradation of HIF is impaired, resulting in elevated levels of HIF at normal oxygen tensions. Apart from the polycythemia, CP patients have marked abnormalities of cardiopulmonary function. No studies of integrated metabolic function have been reported. Here we describe the response of these patients to a series of metabolic stresses: exercise of a large muscle mass on a cycle ergometer, exercise of a small muscle mass (calf muscle) which allowed noninvasive in vivo assessments of muscle metabolism using 31P magnetic resonance spectroscopy, and a standard meal tolerance test. During exercise, CP patients had early and marked phosphocreatine depletion and acidosis in skeletal muscle, greater accumulation of lactate in blood, and reduced maximum exercise capacities. Muscle biopsy specimens from CP patients showed elevated levels of transcript for pyruvate dehydrogenase kinase, phosphofructokinase, and muscle pyruvate kinase. In cell culture, a range of experimental manipulations have been used to study the effects of HIF on cellular metabolism. However, these approaches provide no potential to investigate integrated responses at the level of the whole organism. Although CP is relatively subtle disorder, our study now reveals a striking regulatory role for HIF on metabolism during exercise in humans. These findings have significant implications for the development of therapeutic approaches targeting the HIF pathway.

Chronic oral ingestion of l‐carnitine and carbohydrate increases muscle carnitine content and alters muscle fuel metabolism during exercise in humans

Non‐technical summary After 30 years of endeavour, this is the first study to show that muscle carnitine content can be increased in humans by dietary means and, perhaps more importantly, that carnitine plays a dual role in skeletal muscle fuel metabolism that is exercise intensity dependent. Specifically, we have shown that increasing muscle total carnitine content reduces muscle carbohydrate use during low intensity exercise, consistent with an increase in muscle lipid utilisation. However, during high intensity exercise muscle carnitine loading results in a better matching of glycolytic, pyruvate dehydrogenase complex and mitochondrial flux, thereby reducing muscle anaerobic energy generation. Collectively, these metabolic effects resulted in a reduced perception of effort and increased work output during a validated exercise performance test. These findings have significant implications for athletic performance and pathophysiological conditions where fat oxidation is impaired or anaerobic ATP production is increased during exercise.

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 subjects 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.

Blunted Akt/FOXO signalling and activation of genes controlling atrophy and fuel use in statin myopathy

Statins are used clinically for cholesterol reduction, but statin therapy is associated with myopathic changes through a poorly defined mechanism. We used an in vivo model of statin myopathy to determine whether statins up‐regulate genes associated with proteasomal‐ and lysosomal‐mediated proteolysis and whether PDK gene expression is simultaneously up‐regulated leading to the impairment of muscle carbohydrate oxidation. Animals were dosed daily with 80 mg kg−1 day−1 simvastatin for 4 (n= 6) and 12 days (n= 5), 88 mg kg−1 day−1 simvastatin for 12 days (n= 4), or vehicle (0.5% w/v hydroxypropyl‐methylcellulose and 0.1% w/v polysorbate 80; Control, n= 6) for 12 days by oral gavage. We found, in biceps femoris muscle, decreased AktSer473, FOXO1Ser253 and FOXO3aSer253 phosphorylation in the cytosol (P < 0.05, P < 0.05, P < 0.001, respectively) and decreased phosphorylation of FOXO1 in the nucleus after 12 days simvastatin when compared to Control (P < 0.05). This was paralleled by a marked increase in the transcription of downstream targets of FOXO, i.e. MAFbx (P < 0.001), MuRF‐1 (P < 0.001), cathepsin‐L (P < 0.05), PDK2 (P < 0.05) and PDK4 (P < 0.05). These changes were accompanied by increased PPARα (P < 0.05), TNFα (P < 0.01), IL6 (P < 0.01), Mt1A (P < 0.01) mRNA and increased muscle glycogen (P < 0.05) compared to Control. RhoA activity decreased after 4 days simvastatin (P < 0.05); however, activity was no different from Control after 12 days. Simvastatin down‐regulated PI3k/Akt signalling, independently of RhoA, and up‐regulated FOXO transcription factors and downstream gene targets known to be implicated in proteasomal‐ and lysosomal‐mediated muscle proteolysis, carbohydrate oxidation, oxidative stress and inflammation in an in vivo model of statin‐induced myopathy. These changes occurred in the main before evidence of extensive myopathy or a decline in the muscle protein to DNA ratio.