Acetyl group accumulation and pyruvate dehydrogenase activity in human muscle during incremental exercise.

The changes in the muscle contents of CoASH and carnitine and their acetylated forms, lactate and the active form of pyruvate dehydrogenase complex were studied during incremental dynamic exercise. Eight subjects exercised for 3-4 minutes on a bicycle ergometer at work loads corresponding to 30, 60 and 90% of their VO2max. Muscle samples were obtained by percutaneous needle biopsy technique at rest, at the end of each work period and after 10 minutes of recovery. During the incremental exercise test there was a continuous increase in muscle lactate, from a basal value of 4.5 mmol kg-1 dry weight to 83 mmol kg-1 at the end of the final period. The active form of pyruvate dehydrogenase complex increased from 0.37 mmol acetyl-CoA formed per minute per kilogram wet weight at rest to 0.80 at 30% VO2max, 1.28 and 1.25 at 60 and 90% VO2max, respectively. Both acetyl-CoA and acetylcarnitine increased at the two highest work loads. The increase of acetyl-CoA was from 12.5 mumol kg-1 dry weight at rest to 27.3 after the highest work load and for acetylcarnitine from 6.0 mmol kg-1 dry weight to 15.2. The CoASH and free carnitine contents fell correspondingly. There was a close relationship between acetyl-CoA and acetylcarnitine accumulation in muscle during exercise, with a binding of approximately 500 mol acetyl groups to carnitine for each mole of acetyl-CoA accumulated. The results imply that the carnitine store in muscle functions as a buffer for excess formation of acetyl groups from pyruvate catalyzed by the pyruvate dehydrogenase complex.     

Heterogeneity of response to exercise of rat muscle pyruvate dehydrogenase complex

Muscle glucose uptake is greatly stimulated by moderate exercise, but full oxidation of the glucose to CO₂ depends on the activity of the pyruvate dehydrogenase (PDH) complex. Our aim was to determine how PDH complex in different muscle groups responds to varying periods of moderate exercise. Rats were run on a motor-driven treadmill for 5–30 min and muscle PDH complex activity was determined in heart, diaphragm and red quadriceps muscles after isolation of mitochondria in the presence of inhibitors of PDH complex interconversion. In heart and diaphragm muscle, exercise caused an increase in PDH complex activity after 5 min, but this was followed by a significant decrease in activity as exercise progressed. In red quadriceps muscle, PDH complex activity was reduced after 5 min of exercise and was decreased further as exercise continued. We conclude that increased duration of exercise can lead to reduced PDH complex activity in rat muscles. We propose that this is a consequence of elevated fatty acid oxidation, the products of which stimulate PDH kinase. This implies that increased glycolysis to lactate and increased fatty acid oxidation can simultaneously provide energy for contracting muscle.     

Lactate accumulation in muscle and blood during submaximal exercise

Muscle and blood lactate concentration was studied in 10 healthy males during cycling exercise. For each subject the exercise intensity corresponding to a blood lactate concentration of 4 mmol I^-1 (OBLA_w) was assessed by a step-wise increased exercise intensity protocol. In a second series of experiments the same protocol was performed but exercise was terminated at OBLA_w and a muscle biopsy for subsequent analysis of lactate concentration was obtained from m. vastus lateralis. Biopsies were also taken at rest for histo-chemical determination of fiber type composition and capillary supply. The exercise intensity, which corresponded to OBLA_w averaged 159(117–216) W, equal to 65 (range 55–84)% of VO_2max, and was found to be correlated to capillaries per fiber of the exercising muscle (r=0.83, p>0.01). Muscle lactate concentration averaged 6.9 (range 2.1–12.6) mmol kg^-1 W.W. The change in blood lactate concentration (prior to and I min post exercise) was correlated to muscle lactate concentration (r=0.71, p<0.05). It is concluded that great individual variations in the musclelblood lactate gradient do exist during submaximal steady-state exercise, performed at a certain blood lactate level.     

Validation of the in vivo assessment of pyruvate dehydrogenase activity using hyperpolarised 13C MRS

Many diseases of the heart are characterised by changes in substrate utilisation, which is regulated in part by the activity of the enzyme pyruvate dehydrogenase (PDH). Consequently, there is much interest in the in vivo evaluation of PDH activity in a range of physiological and pathological states to obtain information on the metabolic mechanisms of cardiac diseases. Hyperpolarised [1‐¹³C]pyruvate, detected using MRS, is a novel technique for the noninvasive evaluation of PDH flux. PDH flux has been assumed to directly reflect in vivo PDH activity, although to date this assumption remains unproven. Control animals and animals undergoing interventions known to modulate PDH activity, namely high fat feeding and dichloroacetate infusion, were used to investigate the relationship between in vivo hyperpolarised MRS measurements of PDH flux and ex vivo measurements of PDH enzyme activity (PDH_a). Further, the plasma concentrations of pyruvate and other important metabolites were evaluated following pyruvate infusion to assess the metabolic consequences of pyruvate infusion during hyperpolarised MRS experiments. Hyperpolarised MRS measurements of PDH flux correlated significantly with ex vivo measurements of PDH_a, confirming that PDH activity influences directly the in vivo flux of hyperpolarised pyruvate through cardiac PDH. The maximum plasma concentration of pyruvate reached during hyperpolarised MRS experiments was approximately 250 µM , equivalent to physiological pyruvate concentrations reached during exercise or with dietary interventions. The concentrations of other metabolites, including lactate, glucose and β‐hydroxybutyrate, did not vary during the 60 s following pyruvate infusion. Hence, during the 60‐s data acquisition period, metabolism was minimally affected by pyruvate infusion. Copyright © 2010 John Wiley & Sons, Ltd.     

Influence of sodium bicarbonate ingestion on plasma ammonia accumulation during incremental exercise in man

Summary This investigation evaluated the influence of metabolic alkalosis on plasma ammonia (NH₃) accumulation during incremental exercise. On two occasions separated by at least 6 days, six healthy men cycled at 70, 80, and 90%g of maximum oxygen consumption ( ) for 5 min; each exercise period was followed by 5 min of seated recovery. Exercise was then performed at 100% until exhaustion. Beginning 3 h prior to exercise, subjects ingested 3.6 mmol · kg body mass^– NaHCO₃ (test, T) or 3.0 mmol · kg body mass^–1 CaCO₃ (placebo, P) (both equivalent to 0.3 g · kg^–1) over a 2-h period. Trials were performed after an overnight fast and the order of treatments was randomized. Arterialized venous blood samples for the determination of acid-base status, blood lactate and plasma NH₃ concentrations were obtained at rest before treatment, 15 s prior to each exercise bout (Pre 70%, Pre 80%, Pre 90%, and Pre 100%), and at 0, 5 (5Post), and 10 (10'Post) min after exhaustion. Additional samples for blood lactate and plasma NH₃ determination were obtained immediately after each exercise bout (Post 70%, Post 80%, Post 90%) and at 15 min after exercise (15Post). Time to exhaustion at 100% of was not significantly different between treatments [mean (SE): 173 (42) s and 184 (44) s for T and P respectively]. A significant treatment effect was observed for plasma pH with values being significantly higher on T than on P Pre 70% [7.461 (0.007) vs 7.398 (0.008)], Pre 90% [7.410 (0.010) vs 7.340 (0.016)], and 10'Post [7.317 (0.032) vs 7.242 (0.036)]. The change in plasma pH was significantly greater following the 90%g bout (Pre 100% Pre 90%) for T [–0.09 (0.02)] than for P [–0.06 (0.01)]. Blood base excess and plasma bicarbonate concentrations were significantly higher for T than P before each exercise bout but not at the point of exhaustion. During recovery, base excess was higher for T than P at 5Post and 10Post while the bicarbonate concentration was higher for T than P at 10Post. A significant treatment effect was observed for the blood lactate concentration with T on the average being higher than P [7.0 (1.0) and 6.3 (1.1) mmol · l^–1 for T and P averaged across the 12 sampling times]. Plasma NH₃ accumulation was not different between treatments at any point in time. In addition, no differences were observed between treatments in blood alanine accumulation. The results suggest that under the conditions of the present investigation metabolic alkalosis does not influence plasma NH₃ accumulation or endurance capacity during intense incremental exercise.     

Inhibition of enzyme function by human autoantibodies to an autoantigen pyruvate dehydrogenase E2: different epitope for spontaneous human and induced rabbit autoantibodies.

Antibodies to the mitochondrial autoantigen M2, characteristic of the autoimmune liver disease primary biliary cirrhosis (PBC), react with the E2 subunit of the pyruvate dehydrogenase enzyme (PDH-E2). We examined the effect of disease sera on the enzyme activation catalysed by the PDH complex. Inhibition of enzyme activity was observed in 19 of 24 sera of patients with PBC with a level of greater than 90% inhibition in 14 at a serum dilution of 1/50. The onset of inhibition by serum was rapid, within the time of mixing, and the inhibitory activity was shown to reside in the immunoglobulin fraction of the serum. The immunoglobulin fraction of control sera from patients with other liver diseases (n = 26) and healthy persons (n = 8) failed to produce inhibitory activity. In addition sera from four rabbits, intensively immunized with a recombinant human M2 autoantigen, gave anti-M2 reactions by fluorescence, ELISA and immunoblotting, but did not inhibit the activity of PDH. The failure of experimentally induced M2 antibodies in rabbits to inhibit is interesting in view of the reactivity of the natural M2 autoantibodies of PBC with the highly conserved site on the enzyme which carries the essential lipoic acid cofactor.     

Formation of acetylcarnitine in muscle of horse during high intensity exercise

Summary To study the changes in carnitine in muscle with sprint exercise, two Thoroughbred horses performed two treadmill exercise tests. Biopsies of the middle gluteal were taken before, after exercise and after 12 min recovery. Resting mean muscle total carnitine content was 29.5 mmol · kg⁻¹ dry muscle (d. m.). Approximately 88% was free carnitine, 7% acetylcarnitine and acylcarnitine was estimated at 5%. Exercise did not affect total carnitine, but resulted in a marked fall in free carnitine and almost equivalent rise in acetylcarnitine. The results are consistent with a role for carnitine in the regulation of the acetyl-CoA/CoA ratio during sprint exercise in the Thoroughbred horse by buffering excess production of acetyl units.     

Effect of infusing branched-chain amino acid during incremental exercise with reduced muscle glycogen content

Summary The aim of this study was to investigate whether, when muscle glycogen is reduced, a pre-exercise infusion of branched-chain amino acids (BCAA) modifies exercise performance or the metabolic and respiratory responses to incremental exercise. Six moderately trained volunteers took part in the following protocol on two occasions. On day 1, at 9 a.m. in the postabsorptive state, they performed a graded incremental exercise (increases of 35 W every 4 min) to exhaustion (Ex-1). A meal of 1,000 kcal (4,200 kJ; 60% protein, 40% fat) was consumed at 12 p.m. No food was then allowed until the end of the experiment (20–21 h later). A 90-min period of exercise at alternating high and moderate intensities, designed to deplete muscle glycogen, was performed between 6 p.m. and 7.30 p.m. The morning after (day 2), the subjects randomly received either a mixed solution of BCAA (260 mg × kg^–1 × h^–1 for 70 min), or saline. They then repeated the graded incremental exercise to exhaustion (Ex-2). Metabolic and respiratory measurements suggested a muscle glycogen-depleted state had been achieved. No significant differences were observed in total work performed, maximal oxygen uptake or plasma ammonia, alanine, and blood pyruvate concentrations in the two treatments. After BCAA infusion, higher blood lactate concentrations were observed at maximal power output in comparison with those during saline [BCAA 4.97 (SEM 0.41) mmol × l^–1, Saline 3.88 (SEM 0.47) mmol × l^–1,P < 0.05]. In summary, in conditions of reduced muscle glycogen content, after a short period of fasting, BCAA infusion had no significant effect on the total work that could be performed during a graded incremental exercise.     

A pathogenic glutamate-to-aspartate substitution (D296E) in the pyruvate dehydrogenase E1 subunit gene PDHA1

In a patient with fatal neonatal lactic acidosis due to pyruvate dehydrogenase deficiency, the only potential mutation detected was c.888C>G in PDHA1, the gene for the E1alpha subunit of the complex. This would result in a substitution of glutamate for aspartate (D296E). Pathogenicity of this minor alteration in amino acid sequence was demonstrated by expression studies. By comparing the mutant sequence with the known structures of the E1 components of pyruvate dehydrogenase and the closely related branched chain alpha-ketoacid dehydrogenase, an explanation for the profound consequences of the mutation can be proposed.     

Arginine 302 mutations in the pyruvate dehydrogenase E1α subunit gene: Identification of further patients and in vitro demonstration of pathogenicity

Three further patients with mutations in the codon for arginine 302 of the E1α subunit of the pyruvate dehydrogenase complex have been identified. Mutations in this codon have now been found in nine patients with pyruvate dehydrogenase deficiency in seven unrelated families, in sharp contrast to the great majority of other PDH E1α mutations which have been described in single individuals only. Because of the relatively high frequency of this mutation and because very few PDH E1α mutations have been demonstrated to be causative, we have established a system for analysing the consequences of defined mutations using transfection of normal and mutant PDH E1α cDNA into transformed human fibroblasts which have no endogenous E1α mRNA or protein. Using this test system, we have demonstrated that the R302C mutation results in the production of PDH E1α protein which is devoid of enzymic activity. Hum Mutat 12:114–121, 1998. © 1998 Wiley-Liss, Inc.     

Lipoprotein lipase activity in the skeletal muscle during physical exercise in dogs

Changes in lipoportein lipase activity/LPLA/ in the quadriceps femoris muscle were followed in ten dogs during 3-hour treadmill exercise and 2-hour post-exercise recovery period. During the first hour of exercise muscle LPLA steeply increased. Subsequently the enzyme activity tended to plateau. After exercise LPLA decreased to the pre-exercise value within 1 hour. It is concluded that exercise increases the ability of skeletal muscles to remove triglycerides from the circulation.     

AMPK as a metabolic switch in rat muscle,liver and adipose tissue after exercise

An increasing body of evidence has revealed that activation of adenosine monophosphate (AMP)‐activated protein kinase (AMPK)‐activated protein kinase increases fatty acid oxidation by lowering the concentration of malonyl coenzyme A (CoA), an inhibitor of carnitine palmitoyl transferase 1. Studies carried out primarily in skeletal muscle suggest that AMPK modulates the concentration of malonyl CoA by concurrently phosphorylating and inhibiting acetyl CoA carboxylase (ACC), the rate limiting enzyme in malonyl CoA synthesis, and phosphorylating and activating malonyl CoA decarboxylase (MCD), an enzyme involved in its degradation. We have recently observed that AMPK and MCD activities are increased and ACC activity diminished in skeletal muscle, liver and, surprisingly, in adipose tissue 30 min following exercise (treadmill run) in normal rats. In liver and adipose tissue these changes were associated with a decrease in the activity of glycerol‐3‐phosphate acyltransferase (GPAT), which catalyses the first committed reaction in glycerolipid synthesis and, which like ACC, is phosphorylated and inhibited by AMPK. Similar changes in ACC, MCD and GPAT were observed following the administration of 5‐aminoimidazole 4‐carboxamide‐riboside (AICAR), further indicating that the exercise‐induced alterations in these enzymes were AMPK‐mediated. Conclusions: (1) AMPK plays a major role in regulating lipid metabolism in multiple tissues following exercise. (2) The net effect of its activation is to increase fatty acid oxidation and diminish glycerolipid synthesis. (3) The relevance of these findings to the regulation of muscle glycogen repletion in the post‐exercise state and to the demonstrated ability of AMPK activation to decrease adiposity and increase insulin sensitivity in rodents remains to be determined.     

Exercise-induced activation of the branched-chain 2-oxo acid dehydrogenase in human muscle

Summary The present study was conducted to investigate the metabolic regulation of the oxidation of branched-chain amino acids (BCAA) by exercise in human skeletal muscle. Five trained male volunteers were exercised on a cycle ergometer at 70%±10% (mean±SD) of their maximal oxygen consumption . Percutaneous quadriceps muscle biopsies were obtained under local anaesthesia at rest and after 30 and 120 min of exercise. In the muscle samples the active and total amount of the branched-chain 2-oxo acid dehydrogenase complex (BC-complex), the regulatory enzyme in the oxidative pathway of the BCAA, were measured. Glycogen content and activity of mitochondrial marker enzymes were also measured. Blood samples were obtained every 20 min for the measurement of metabolites. Heart rate and rated perceived exertion on the Borg scale were recorded every 10 min. At rest 4.0%±2.5% of the BC complex was active, after 30 min of exercise 9.9%±9.0% and after 120 min 17.5%±8.5% (mean±SD). Exercise did not change the total activity. The largest activation was seen in two of the subjects who developed higher blood lactates early on during exercise and decreased their muscle glycogen more (indications of anaerobic metabolism). These data demonstrate that in trained individuals significant increases in the activity of the BC-complex occur only after prolonged intense exercise. In spite of the 4-fold activation, the data support the classical view that amino acids and protein do not contribute substantially as an energy source during exercise, since increased more than 20-fold.     

Prolonged stage duration during incremental cycle exercise: effects on the lactate threshold and onset of blood lactate accumulation

The aim of this study was to investigate whether increasing the duration of workloads from 3 min to 8 min during incremental exercise would influence workload (W), oxygen consumption ( ) and heart rate (HR) at the lactate threshold (LT) and the onset of blood lactate accumulation(OBLA). Two groups of six male cyclists were assigned to a well-trained (WT) and recreational (REC) group on the basis of their performance in a maximal incremental ramp test. Each subject then performed two incremental lactate tests (EXT) consisting of six workloads of either 3 min (EXT_3-min) or 8 min (EXT_8-min) duration. At the completion of each workload whole capillary blood samples were obtained for the determination of blood lactate (BLa) concentration (mM). Power output (Watts, W), HR and were averaged in the final minute of each workload as well as in the third minute of the EXT_8-min. The workload, HR and at the LT and OBLA were subsequently determined from the data of EXT_3-min and EXT_8-min. The results demonstrate that workload and , but not HR, at the LT and OBLA were higher in the WT cyclists. At the same time, the workload at the LT obtained from the results of the EXT_3-min was significantly (P<0.05) higher then the value obtained in the EXT_8-min in the WT subjects but not the REC subjects. However, the workload, and HR at the OBLA, together with the and HR at the LT were not significantly different when calculated from data obtained from EXT_3-min or EXT_8-min. The data obtained in this study suggest that incremental exercise protocols using workloads of duration longer than 3 min have the effect of increasing the workload at the LT in well-trained cyclists. However, the OBLA determined in exercise tests using stage increments of either 3 min or 8 min is similar in cyclists of different training status. Electronic Publication     

Regulation of the Ascaris suum pyruvate dehydrogenase complex by phosphorylation and dephosphorylation

The pyruvate dehydrogenase complex isolated from ‘anaerobic’ mitochondria of Ascaris suum has a subunit composition similar to complexes isolated from most other eukaryotic organisms and is regulated by phosphorylation and dephosphorylation. Pyruvate dehydrogenase kinase activity is stimulated by NADH and a number of physiologically important acyl-CoA intermediates and is inhibited by CoA, propionate, tiglate and pyruvate. It is suggested that the elevated levels of pyruvate observed in the ascarid organelle may be important in maintaining the pyruvate dehydrogenase complex in an active state, even in the presence of a reduced pyridine nucleotide pool.     

Inhibition of carnitine palmitoyltransferase in normal human skeletal muscle and in muscle of patients with carnitine palmitoyltransferase deficiency by long- and short-chain acylcarnitine and acyl-coenzyme A

Summary The inhibition of total carnitine palmitoyltransferase (CPT) by short- and long-chain acylcarnitine and acyl-coenzyme A (acyl-CoA) was studied in muscle homogenates of normal controls and of five new patients with CPT deficiency using the isotope forward assay. Acetylcarnitine inhibited neither normal CPT activity nor the CPT of patients. d,l-Palmitoylcarnitine almost completely inhibited CPT in patients but only 55% of normal activity. In controls the CPT fraction sensitive to inhibition by palmitoylcarnitine appeared to be identical with the fraction sensitive to inhibition by malonyl-CoA and succinyl-CoA, which probably represents CPT II. The abnormal inhibition of CPT by palmitoylcarnitine was more likely due to product inhibition than to a detergent effect. Acetyl-CoA concentrations up to 0.4 mM and palmitoyl-CoA above optimal substrate concentrations up to 0.3 mM both inhibited normal CPT by about 25%, whereas the CPT of patients was significantly more inhibited by both substances than was normal CPT. The inhibition by acetyl-CoA was probably due to the structural relationship with malonyl-CoA and succinyl-CoA. The abnormal inhibition of CPT in patients by palmitoyl-CoA was due either to an abnormal substrate inhibition or to a detergent effect on CPT II similar to that of Triton X-100. The data indicate that in CPT deficiency total CPT activity is normal under optimal assay conditions. CPT II, however, is abnormally inhibited by fatty acid metabolites that accumulate during fasting.Abbreviations CoA coenzyme A - CPT carnitine palmitoyltransferase     

Comparison of muscle sympathetic nerve activity during exercise in dominant and nondominant forearm

Summary To determine whether or not muscle endurance training alters exercise-induced sympathetic nerve response, we recorded muscle sympathetic nerve activity (MSNA) microneurographically during forearm exercise and compared MSNA between dominant (D) and nondominant (ND) forearms of players of racket sports. Three kinds of forearm exercise were conducted on each side; static (SHG) and dynamic (DHG, at a rate of 1 Hz) handgrip exercise at a loading of 25°10 of maximal voluntary contraction until exhaustion, and 10-min submaximal dynamic handgrip (at a rate of 1 Hz) at an intensity of 0.9 W. Heart rate, ventilation and blood pressure were also monitored at rest and during SHG and DHG exercises. During the last minute of SHG exercise, MSNA burst rate had increased on average by 290 (SEM 46) % in D and 330 (SEM 46) % in ND, while during DHG it increased by 288 (SEM 38) % in D and 344 (SEM 36) % in ND, respectively. There were no significant differences in the MSNA responses between D and ND forearms in either exercise modes. Significant increases in heart rate, ventilation and blood pressure during the last minute of fatiguing SHG and DHG were observed, but there were no significant differences between the two forearms. During submaximal DHG, while MSNA increased significantly above control values in both D and ND, the MSNA response was less in D than that in ND forearm. The results would suggest that exercise-induced MSNA responsiveness is influenced little by muscle endurance training but the intensity of response may be due to the magnitude of metaboreceptor stimulation in the exercising muscle.