Acetyl Group Availability Research Articles

Muscular Metabolism in Aged Rats Under Exhaustive Exercise: Effect of a Modified Alkaline Supplementation

A modified alkalizing supplementation (MAS) was tested on skeletal muscle metabolism in aged rats undergoing exhaustive exercise. Aged Wistar rats were allocated into two groups: saline (A) and saline added with 16 mg of MAS (B) before treadmill exercise. Blood and gastrocnemius and soleus muscle were analyzed after exercise for succinate dehydrogenase (SDH), acetylcarnitine (ALCAR), and glycogen. Lactic acid (LA), creatin-phosphokinase (CPK), and gas analysis were tested in the blood. Exercise caused a significant increase of LA and CPK and muscle glycogen fall. Arterial desaturation at exhaustion was prevented in the B group (p < 0.05). Exercise-induced increase of SDH and ALCAR was further enhanced in B rats (p < 0.05). This study suggests that MAS can improve fast and endurance muscle metabolism in aged rats by increasing cellular acetyl group availability and tricarboxylic acid turnover.

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 beta-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.

Sodium acetate ingestion perturbs substrate utilisation at rest and during the early stages of prolonged exercise in man

Sodium acetate administration has been shown to increase muscle acetyl-CoA and acetylcarnitine content at rest and during the early stages of exercise. We therefore investigated the effect of sodium acetate ingestion at rest on subsequent substrate utilisation during prolonged exercise. With local ethics approval six healthy males ingested either sodium acetate (NaAc) or sodium bicarbonate (NaHCO3) at a dose of 4 mmol.kg−1 body mass in a randomised order over 90 min prior to 120 min of exercise at 50% V̇O2max. NaAc ingestion decreased resting fat oxidation (0.09 ± 0.02 vs. 0.07 ± 0.02 g.min−1 pre- and post-ingestion respectively, P<0.05) with no effect upon carbohydrate utilisation. In contrast, NaHCO3 ingestion did not affect substrate utilisation at rest. Fat and CHO oxidation increased in both trials when exercise was undertaken, but fat oxidation was lower (0.16 ± 0.10 vs. 0.30 ± 0.10 g.min−1, P<0.05) and carbohydrate oxidation higher (1.75 ± 0.36 vs. 1.50 ± 0.23 g.min−1, P<0.05) in the NaAc compared with the NaHCO3 trial during the first 15 min of exercise. Plasma acetate concentration had returned to baseline after 30 min of exercise in the NaAc trial with no difference in either fat or carbohydrate utilisation between the trials over the last 90 min of exercise. These results demonstrate that increasing plasma acetate concentration suppresses fat oxidation at rest and at the onset of exercise possibly due to changes in acetyl group availability.

Prior heavy exercise eliminates slow component and reduces efficiency during submaximal exercise in humans

We investigated the hypothesis that the pulmonary oxygen uptake (VO2) slow component is related to a progressive increase in muscle lactate concentration and that prior heavy exercise (PHE) with pronounced acidosis alters VO2 kinetics and reduces work efficiency. Subjects (n= 9) cycled at 75% of the peak VO2 (VO2peak) for 10 min before (CON) and after (AC) PHE. VO2 was measured continuously (breath-by-breath) and muscle biopsies were obtained prior to and after 3 and 10 min of exercise. Muscle lactate concentration was stable between 3 and 10 min of exercise but was 2- to 3-fold higher during AC (P < 0.05 versus CON). Acetylcarnitine (ACn) concentration was 6-fold higher prior to AC and remained higher during exercise. Phosphocreatine (PCr) concentration was similar prior to exercise but the decrease was 2-fold greater during AC than during CON. The time constant for the initial VO2 kinetics (phase II) was similar but the asymptote was 14% higher during AC. The slow increase in VO2 between 3 and 10 min of exercise during CON (+7.9 +/- 0.2%) was not correlated with muscle or blood lactate levels. PHE eliminated the slow increase in VO2 and reduced gross exercise efficiency during AC. It is concluded that the VO2 slow component cannot be explained by a progressive acidosis because both muscle and blood lactate levels remained stable during CON. We suggest that both the VO2 slow component during CON and the reduced gross efficiency during AC are related to impaired contractility of the working fibres and the necessity to recruit additional motor units. Despite a pronounced stockpiling of ACn during AC, initial VO2 kinetics were not affected by PHE and PCr concentration decreased to a lower plateau. The discrepancy with previous studies, where initial oxidative ATP generation appears to be limited by acetyl group availability, might relate to remaining fatiguing effects of PHE.

Acetyl group availability influences phosphocreatine degradation even during intense muscle contraction

We previously established that activation of the pyruvate dehydrogenase complex (PDC) using dichloroacetate (DCA) reduced the reliance on substrate-level phosphorylation (SLP) at the onset of exercise, with normal and reduced blood flow. PDC activation also reduced fatigue development during contraction with reduced blood flow. Since these observations, several studies have re-evaluated our observations. One study demonstrated a performance benefit without a reduction in SLP, raising a question mark over PDC's role in the regulation of ATP regeneration and our interpretation of fatigue mechanisms. Using a model of muscle contraction similar to the conflicting study (i.e. tetanic rather than twitch stimulation), we re-examined this question. Using canine skeletal muscle, one group was infused with saline while the other was pretreated with 300 mg (kg body mass)(-1) DCA. Muscle biopsies were taken at rest, peak tension (1 min) and after 6 min of tetanic electrical stimulation (75 ms on-925 ms off per second) and blood flow was limited to 25% of normal values observed during contraction. DCA reduced phosphocreatine (PCr) degradation by 40% during the first minute of contraction, but did not prevent the almost complete depletion of PCr stores at 6 min, while muscle fatigue did not differ between the two groups. During intermittent tetanic stimulation PCr degradation was 75% greater than with our previous 3 Hz twitch contraction protocol, despite a similar rate of oxygen consumption at 6 min. Thus, in the present study enhanced acetyl group availability altered the time course of PCr utilization but did not prevent the decline towards depletion. Consistent with our earlier conclusions, DCA pretreatment reduces muscle fatigue only when SLP is attenuated. The present study and our met-analysis indicates that enhanced acetyl group availability results in a readily measurable reduction in SLP when the initial rate of PCr utilization is approximately 1 mmol (kg dry mass)(-1) s(-1) or less (depending on intrinsic mitochondrial capacity). When measured early during an uninterrupted period of muscle contraction, acetyl group availability is likely to influence SLP under any condition where mitochondria are responsible for a significant proportion of ATP regeneration.

The acetyl group deficit at the onset of contraction in ischaemic canine skeletal muscle.

Considerable debate surrounds the identity of the precise cellular site(s) of inertia that limit the contribution of mitochondrial ATP resynthesis towards a step increase in workload at the onset of muscular contraction. By detailing the relationship between canine gracilis muscle energy metabolism and contractile function during constant-flow ischaemia, in the absence (control) and presence of pyruvate dehydrogenase complex activation by dichloroacetate, the present study examined whether there is a period at the onset of contraction when acetyl-coenzyme A (acetyl-CoA) availability limits mitochondrial ATP resynthesis, i.e. whether a limitation in mitochondrial acetyl group provision exists. Secondly, assuming it does exist, we also aimed to identify the mechanism by which dichloroacetate overcomes this "acetyl group deficit". No increase in pyruvate dehydrogenase complex activation or acetyl group availability occurred during the first 20 s of contraction in the control condition, with strong trends for both acetyl-CoA and acetylcarnitine to actually decline (indicating the existence of an acetyl group deficit). Dichloroacetate increased resting pyruvate dehydrogenase complex activation, acetyl-CoA and acetylcarnitine by approximately 20-fold (P < 0.01), approximately 3-fold (P < 0.01) and approximately 4-fold (P < 0.01), respectively, and overcame the acetyl group deficit at the onset of contraction. As a consequence, the reliance upon non-oxidative ATP resynthesis was reduced by approximately 40 % (P < 0.01) and tension development was increased by approximately 20 % (P < 0.05) following 5 min of contraction. The present study has demonstrated, for the first time, the existence of an acetyl group deficit at the onset of contraction and has confirmed the metabolic and functional benefits to be gained from overcoming this inertia.

The acetyl group deficit at the onset of contraction in ischaemic canine skeletal muscle

Considerable debate surrounds the identity of the precise cellular site(s) of inertia that limit the contribution of mitochondrial ATP resynthesis towards a step increase in workload at the onset of muscular contraction. By detailing the relationship between canine gracilis muscle energy metabolism and contractile function during constant-flow ischaemia, in the absence (control) and presence of pyruvate dehydrogenase complex activation by dichloroacetate, the present study examined whether there is a period at the onset of contraction when acetyl-coenzyme A (acetyl-CoA) availability limits mitochondrial ATP resynthesis, i.e. whether a limitation in mitochondrial acetyl group provision exists. Secondly, assuming it does exist, we also aimed to identify the mechanism by which dichloroacetate overcomes this ‘acetyl group deficit’. No increase in pyruvate dehydrogenase complex activation or acetyl group availability occurred during the first 20 s of contraction in the control condition, with strong trends for both acetyl-CoA and acetylcarnitine to actually decline (indicating the existence of an acetyl group deficit). Dichloroacetate increased resting pyruvate dehydrogenase complex activation, acetyl-CoA and acetylcarnitine by ≈20-fold (P < 0.01), ≈3-fold (P < 0.01) and ≈4-fold (P < 0.01), respectively, and overcame the acetyl group deficit at the onset of contraction. As a consequence, the reliance upon non-oxidative ATP resynthesis was reduced by ≈40 % (P < 0.01) and tension development was increased by ≈20 % (P < 0.05) following 5 min of contraction. The present study has demonstrated, for the first time, the existence of an acetyl group deficit at the onset of contraction and has confirmed the metabolic and functional benefits to be gained from overcoming this inertia.

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.

Skeletal muscle VO2 on-kinetics: set by O2 delivery or by O2 utilization? New insights into an old issue.

Recent work conducted by our group has expanded knowledge on some basic issues related to pulmonary and skeletal muscle O2 uptake (VO2) on-kinetics. We demonstrated that, in exercising humans during transitions from unloaded pedaling to loaded pedaling below the ventilatory threshold, alveolar VO2 on-kinetics can be taken as a rather close approximation of skeletal muscle VO2 on-kinetics. Experiments conducted on the isolated in situ dog gastrocnemius preparation have shown that, during transitions from rest to contractions corresponding to approximately 70% of the muscle peak VO2, convective O2 delivery to muscle, intramuscular blood flow (Q) versus VO2 maldistribution, and peripheral O2 diffusion are not limiting factors for skeletal muscle VO2 on-kinetics. The latter, therefore, appears to be mainly determined by an intrinsic inertia of skeletal muscle oxidative metabolism, possibly related to acetyl group availability within mitochondria, to regulatory effects on intracellular respiration related to phosphocreatine splitting, and/or to other still not precisely identified control mechanism(s). Evidence from the literature suggests that the limiting factors for skeletal muscle VO2 on-kinetics may vary according to the intensity of muscular contractions or of exercise.

Muscle acetyl group availability is a major determinant of oxygen deficit in humans during submaximal exercise.

The delay in skeletal muscle mitochondrial ATP production at the onset of exercise is thought to be a function of a limited oxygen supply. The delay, termed the oxygen deficit, can be quantified by assessing the above baseline oxygen consumption during the first few minutes of recovery from exercise. During submaximal exercise, the oxygen deficit is reflected by the extent of muscle phosphocreatine (PCr) breakdown. In the present study, nine male subjects performed 8 min of submaximal, single leg knee extension exercise after saline (Control) and dichloroacetate (DCA) infusion on two separate occasions. Administration of DCA increased resting skeletal muscle pyruvate dehydrogenase complex activation status threefold (Control = 0.4 +/- 0.1 vs. DCA = 1.3 +/- 0.1 mmol acetyl-CoA.min-1.kg wet muscle-1 at 37 degrees C, P < 0.01) and elevated acetylcarnitine concentration fivefold (Control = 2.2 +/- 0.5 vs. DCA = 10.9 +/- 1.2 mmol/kg dry mass, P < 0.01). During exercise, PCr degradation was reduced by approximately 50% after DCA (Control = 33.2 +/- 7.1 vs. DCA = 18.4 +/- 7.1 mmol/kg dry mass, P < 0.05). It would appear, therefore, that in humans acetyl group availability is a major determinant of the rate of increase in mitochondrial respiration at the onset of exercise and hence the oxygen deficit.

Pyruvate dehydrogenase complex activation status and acetyl group availability as a site of interchange between anaerobic and oxidative metabolism during intense exercise.

During high intensity muscular contraction ATP is supplied at near maximal rates by PCr degradation and glycolysis. However, as exercise duration increases, the contribution of anaerobic ATP turnover to energy delivery declines due to the depletion of PCr stores and a reduction in the rate of glycogenolysis, which together may be responsible for the parallel reduction in muscle force production and power output. The importance of oxidative phosphorylation to total ATP production during intense muscle contraction has been underestimated to date. Recent studies have, however, demonstrated that the reduction in work production during repeated bouts of maximal exercise is less than the reduction observed in anaerobic energy provision. This observation has been suggested to reflect an increased contribution from oxidative phosphorylation to total energy production; but the mechanism responsible for this increased contribution is poorly understood. Recent evidence has pointed to the activation status of the pyruvate dehydrogenase complex and/or acetyl group availability as being focal in dictating temporal changes in ADP flux at the onset of intense exercise and, hence, the relative contribution made by anaerobic and oxidative ATP regenerating pathways under these conditions. As might be expected, therefore, maximising the contribution from oxidative ATP regeneration at the onset of exercise (by pharmacologically activating the pyruvate dehydrogenase complex prior to exercise) has been shown to have substantial functional benefits during high intensity contraction. This body of work has also illustrated that, contrary to popular theory, a large proportion of muscle lactate accumulation at the onset of exercise is associated with a lag in the activation of oxidative ATP production rather than with a lag in oxygen delivery.

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.

Increased acetyl group availability enhances contractile function of canine skeletal muscle during ischemia.

Skeletal muscle contractile function is impaired during acute ischemia such as that experienced by peripheral vascular disease patients. We therefore, examined the effects of dichloroacetate, which can alter resting metabolism, on canine gracilis muscle contractile function during constant flow ischemia. Pretreatment with dichloroacetate increased resting pyruvate dehydrogenase complex activity and resting acetylcarnitine concentration by approximately 4- and approximately 10-fold, respectively. After 20-min contraction the control group had demonstrated an approximately 40% reduction in isomeric tension whereas the dichloroacetate group had fatigued by approximately 25% (P < 0.05). Dichloroacetate resulted in less lactate accumulation (10.3 +/- 3.0 vs 58.9 +/- 10.5 mmol.kg-1 dry muscle [dm], P < 0.05) and phosphocreatine hydrolysis (15.6 +/- 6.3 vs 33.8 +/- 9.0 mmol.kg-1 dm, P < 0.05) during contraction. Acetylcarnitine concentration fell during contraction by 5.4 +/- 1.8 mmol.kg-1 dm in the dichloroacetate group but increased by 10.0 +/- 1.9 mmol.kg-1 dm in the control group. In conclusion, dichloroacetate enhanced contractile function during ischemia, independently of blood flow, such that it appears oxidative ATP regeneration is limited by pyruvate dehydrogenase complex activity and acetyl group availability.