RESUMEN
This study utilized N-benzyl-p-toluene sulfonamide (BTS), a potent inhibitor of cross-bridge cycling, to measure 1) the relative metabolic costs of cross-bridge cycling and activation energy during contraction, and 2) oxygen uptake kinetics in the presence and absence of myosin ATPase activity, in isolated Xenopus laevis muscle fibers. Isometric tension development and either cytosolic Ca2+ concentration ([Ca2+]c) or intracellular Po2 (PiO2) were measured during contractions at 20 degrees C in control conditions (Con) and after exposure to 12.5 microM BTS. BTS attenuated tension development to 5+/-0.4% of Con but did not affect either resting or peak [Ca2+]c during repeated isometric contractions. To determine the relative metabolic cost of cross-bridge cycling, we measured the fall in PiO2) (DeltaPiO2; a proxy for Vo2) during contractions in Con and BTS groups. BTS attenuated DeltaP(iO2) by 55+/-6%, reflecting the relative ATP cost of cross-bridge cycling. Thus, extrapolating DeltaPiO2 to a value that would occur at 0% tension suggests that actomyosin ATP requirement is approximately 58% of overall ATP consumption during isometric contractions in mixed fiber types. BTS also slowed the fall in PiO2) (time to 63% of overall DeltaPiO2) from 75+/-9 s (Con) to 101+/-9 s (BTS) (P<0.05), suggesting an important role of the products of ATP hydrolysis in determining the Vo2 onset kinetics. These results demonstrate in isolated skeletal muscle fibers that 1) activation energy accounts for a substantial proportion (approximately 42%) of total ATP cost during isometric contractions, and 2) despite unchanged [Ca2+]c transients, a reduced rate of ATP consumption results in slower Vo2 onset kinetics.
Asunto(s)
Metabolismo Energético/fisiología , Contracción Isométrica/fisiología , Fibras Musculares Esqueléticas/metabolismo , Fosforilación Oxidativa , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Metabolismo Energético/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Femenino , Técnicas In Vitro , Contracción Isométrica/efectos de los fármacos , Cinética , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/fisiología , Miosinas/antagonistas & inhibidores , Oxígeno/metabolismo , Presión Parcial , Sulfonamidas/farmacología , Tolueno/análogos & derivados , Tolueno/farmacología , Xenopus laevisRESUMEN
Ageing is associated with reduced transport and utilization of O(2), diminishing exercise tolerance. Reductions may occur in cardiac output (delivery), and skeletal muscle oxidative capacity (utilization). To determine the reversibility of the declines in the muscular determinants of these limitations, skeletal muscle morphological, angiogenic and biochemical responses to acute exercise and endurance training were investigated in female Fischer 344 rats (n = 42; seven groups of six rats) aged 6 (Y) and 24 (O) months compared with resting untrained controls (Y(C), O(C)). Treadmill training lasted 8 weeks (10 deg incline, 1 h per day, 5 days per week). Two groups ran at maximum tolerated speeds (Y(TR), O(TR)), while an additional Y group (Y(TM)) trained at O(TR) speed. There was no effect of age on vascular endothelial growth factor gene expression in gastrocnemius muscles after acute exercise. Similarly, age did not impair the effects of training, with increases (P < 0.05; +/-s.e.m.) occurring in all of the following: 1 h exercise running speed (Y(TR) 92 +/- 4% versus O(TR) 140 +/- 25%); citrate synthase (Y(TR) 37 +/- 8% versus O(TR) 97 +/- 33%) and beta-hydroxyacyl-CoA-dehydrogenase (Y(TR) 31 +/- 7%, versus O(TR) 72 +/- 24%) activities; and capillary-to-fibre ratio (Y(TR) 5.2 +/- 0.2% versus O(TR) 8.1 +/- 0.2%). However, Y(TM) muscle was unchanged in each measure compared with Y(C). In conclusion, these muscular responses to training were (1) not reduced by ageing, but (2) dependent on relative and not absolute work rate, since, at the same speed, O(TR) rats showed greater changes than Y(TM). Therefore, increases in exercise tolerance and muscle adaptations are not impaired in female rats up to 24 months of age, and require a smaller absolute exercise stimulus (than young) to be manifest.
Asunto(s)
Adaptación Fisiológica/fisiología , Envejecimiento/fisiología , Músculo Esquelético/fisiología , Neovascularización Fisiológica/fisiología , Condicionamiento Físico Animal/fisiología , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , Animales , Citrato (si)-Sintasa/metabolismo , Femenino , Mitocondrias/enzimología , Fibras Musculares Esqueléticas/enzimología , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/citología , ARN Mensajero , Ratas , Ratas Endogámicas F344 , Carrera/fisiología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
We tested the hypotheses that: (1) Ca(2+) handling and force production would be irreversibly altered in skeletal muscle during steady-state contractions when subjected to severe, prolonged hypoxia and subsequent reoxygenation; and (2) application of the cardio-protective drug trimetazidine would attenuate these alterations. Single, living skeletal muscle fibres from Xenopus laevis were injected with the Ca(2+) indicator fura 2, and incubated for 1 h prior to stimulation in 100 micro M TMZ-Ringer solution (TMZ; n = 6) or standard Ringer solution (CON; n = 6). Force and relative free cytosolic Ca(2+) concentration ([Ca(2+)](c)) were measured during continuous tetanic contractions produced every 5 s as fibres were sequentially perfused in the following manner: 3 min high extracellular P(O(2)) (159 mmHg), 15 min hypoxic perfusion (3-5 mmHg) then 3 min high P(O(2)). Hypoxia caused a decrease in force and peak [Ca(2+)](c) in both the TMZ and CON fibres, with no significant (P < 0.05) difference between groups. However, basal [Ca(2+)](c) was significantly lower during hypoxia in the TMZ group vs. the CON group. While reoxygenation generated only modest recovery of relative force and peak [Ca(2+)](c) in both groups, basal [Ca(2+)](c) remained significantly less in the TMZ group. These results demonstrated that in contracting, single skeletal muscle fibres, TMZ prevented increases in basal [Ca(2+)](c) generated during a severe hypoxic insult and subsequent reoxygenation, yet failed to protect the cell from the deleterious effects of prolonged hypoxia followed by reoxygenation.
Asunto(s)
Calcio/metabolismo , Hipoxia/metabolismo , Contracción Muscular/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Trimetazidina/farmacología , Vasodilatadores/farmacología , Animales , Citosol/metabolismo , Femenino , Contracción Muscular/fisiología , Fibras Musculares Esqueléticas/efectos de los fármacos , Xenopus laevisRESUMEN
There is currently some controversy regarding the manner in which skeletal muscle intracellular PO(2) changes with work intensity. Therefore, this study investigated the relationship between intracellular PO(2) and stimulation frequency in intact, isolated, single skeletal muscle fibers. Single, living muscle fibers (n = 7) were microdissected from the lumbrical muscles of Xenopus and injected with the oxygen-sensitive probe palladium-meso-tetra(4-carboxyphenyl)porphine (0.5 mM). Fibers were mounted with platinum clips to a force transducer in a chamber, which was continuously perfused with Ringer solution (pH = 7.0) at a PO(2) of approximately 30 Torr. Fibers were then stimulated sequentially for 3 min, followed by a 3-min rest, at each of five contraction frequencies (0.15, 0.2, 0.25, 0.33, and 0.5 Hz), in a random order, using tetanic contractions. Resting intracellular PO(2) averaged 31.2 +/- 0.9 Torr. During steady-state stimulation, intracellular PO(2) declined to 21.2 +/- 2.3, 17.1 +/- 2.4, 15.3 +/- 1.9, 9.8 +/- 2.0, and 5.8 +/- 1.4 Torr for 0.15, 0.2, 0.25, 0.33, and 0.5-Hz stimulation, respectively. Significant fatigue, as defined by a decrease in force to <50% of the initial force, occurred only at the highest (0.5 Hz) stimulation frequency in five of the cells and at 0.33 Hz in the other two. Regression analysis demonstrated that there was a significant (P < 0.0001, r = 0.82) negative correlation between intracellular PO(2) and contraction frequency in these isolated, single cells. The linear decrease in intracellular PO(2) with stimulation frequency, and thus energy demand, suggests that a fall in intracellular PO(2) correlates with increased oxygen uptake in these single contracting cells.
Asunto(s)
Contracción Muscular/fisiología , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/fisiología , Oxígeno/metabolismo , Análisis de Varianza , Animales , Calibración , Estimulación Eléctrica/métodos , Femenino , Técnicas In Vitro , Líquido Intracelular/fisiología , Oxígeno/análisis , Presión Parcial , Análisis de Regresión , Factores de Tiempo , Xenopus laevisRESUMEN
Reperfusion following prolonged ischemia induces cellular damage in whole skeletal muscle models. Ischemic preconditioning attenuates the deleterious effects. We tested whether individual skeletal muscle fibers would be similarly affected by severe hypoxia and reoxygenation (H/R) in the absence of extracellular factors and whether cellular damage could be alleviated by hypoxic preconditioning. Force and free cytosolic Ca2+ ([Ca2+]c) were monitored in Xenopus single muscle fibers (n = 24) contracting tetanically at 0.2 Hz during 5 min of severe hypoxia and 5 min of reoxygenation. Twelve cells were preconditioned by a shorter bout of H/R 1 h before the experimental trial. In preconditioned cells, force relative to initial maximal values (P/P(o)) and relative peak [Ca2+]c fell (P < 0.05) during 5 min of hypoxia and recovered during reoxygenation. In contrast, P/P(o) and relative peak [Ca2+]c fell more during hypoxia (P < 0.05) and recovered less during reoxygenation (P < 0.05) in control cells. The ratio of force to [Ca2+]c was significantly higher in the preconditioned cells during severe hypoxia, suggesting that changes in [Ca2+]c were not solely responsible for the loss in force. We conclude that 1) isolated skeletal muscle fibers contracting in the absence of extracellular factors are susceptible to H/R injury associated with changes in Ca2+ handling; and 2) hypoxic preconditioning improves contractility, Ca2+ handling, and cell recovery during subsequent hypoxic insult.
Asunto(s)
Precondicionamiento Isquémico , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Oxígeno/metabolismo , Animales , Calcio/metabolismo , Hipoxia de la Célula/fisiología , Femenino , Contracción Muscular/fisiología , Músculo Esquelético/irrigación sanguínea , Daño por Reperfusión/metabolismo , Daño por Reperfusión/fisiopatología , Xenopus laevisRESUMEN
This study examined the relationship between force and cytosolic free calcium concentration ([Ca2+]c) in different fiber types from Xenopus before, during, and after cells underwent postcontractile depression (PCD). During a standardized fatigue run, force in the two fast fatiguing (FF) fiber types (types 1 and 2, n = 10) fell more quickly (5.8 vs. 8.1 min) and to a greater degree [0.36 vs. 0.51 of initial (P(o))] than in the slow fatiguing (SF) fiber type (type 3, n = 11). After the initial fatigue run, both FF and SF experienced a drop in force to <15% P(o) (PCD) at a similar time (20.6 vs. 21.4 min). A second stimulation period, undertaken during PCD, produced significant recovery of force in both groups, but significantly more so in SF than FF (64 +/- 7 vs. 29 +/- 2% P(o)). This force recovery during PCD was accompanied by a significant increase in peak [Ca2+]c, particularly in SF. However, despite the significant recovery of force during stimulation while in PCD, the amount of force produced for a given peak [Ca2+]c was significantly lower in both groups during PCD than at any other point in the experiment. A final stimulation period, initiated when all fibers had recovered from PCD, demonstrated a recovery of both force and peak [Ca2+]c in both groups, but this recovery was significantly greater in SF vs. FF. These data demonstrate that with continuous electrical stimulation, it is possible to produce a significant recovery of force production during the normally quiescent period of PCD, but that it occurs with a decreased muscle force production for a given peak [Ca2+]c. This suggests that factors other than structural alterations of the sarcoplasmic reticulum are likely the cause of PCD in these fibers.
Asunto(s)
Contracción Muscular/fisiología , Fibras Musculares de Contracción Rápida/fisiología , Fibras Musculares de Contracción Lenta/fisiología , Músculo Esquelético/fisiología , Animales , Calcio/fisiología , Citosol/fisiología , Estimulación Eléctrica , Femenino , Miembro Posterior , Técnicas In Vitro , Fatiga Muscular/fisiología , Estrés Mecánico , Tendones/fisiología , Factores de Tiempo , Xenopus laevisRESUMEN
PURPOSE: This paper examines the production of lactate in human skeletal muscle over a range of power outputs (35-250% VO2max) from an enzymatic flux point of view. The conversion of pyruvate and NADH to lactate and NAD in the cytoplasm of muscle cells is catalyzed by the near-equilibrium enzyme lactate dehydrogenase (LDH). As flux through LDH is increased by its substrates, pyruvate and NADH, the factors governing the production of these substrates will largely dictate how much lactate is produced at any exercise power output. In an attempt to understand lactate production, flux rates through the enzymes that regulate glycogenolysis/glycolysis, the transfer of cytoplasmic reducing equivalents into the mitochondria, and the various fates of pyruvate have been measured or estimated. RESULTS: At low power outputs, the rates of pyruvate and NADH production in the cytoplasm are low, and pyruvate dehydrogenase (PDH) and the shuttle system enzymes (SS) metabolize the majority of these substrates, resulting in little or no lactate production. At higher power outputs (65, 90, and 250% VO2max), the mismatch between the ATP demand and aerobic ATP provision at the onset of exercise increases as a function of intensity, resulting in increasing accumulations of the glycogenolytic/glycolytic activators (free ADP, AMP, and Pi). The resulting glycolytic flux, and NADH and pyruvate production, is progressively greater than can be handled by the SS and PDH, and lactate is produced at increasing rates. Lactate production during the onset of exercise and 10 min of sustained aerobic exercise may be a function of adjustments in the delivery of O2 to the muscles, adjustments in the activation of the aerobic ATP producing metabolic pathways and/or substantial glycogenolytic/glycolytic flux through a mass action effect.
Asunto(s)
Ejercicio Físico/fisiología , Ácido Láctico/biosíntesis , Músculo Esquelético/enzimología , Adenosina Trifosfato/metabolismo , Glucógeno/metabolismo , Glucólisis/fisiología , Humanos , Oxígeno/metabolismoRESUMEN
The present study examined the sensitivity of carnitine palmitoyltransferase I (CPT I) activity to its inhibitor malonyl-CoA (M-CoA), and simulated metabolic conditions of rest and exercise, in aerobically trained and untrained humans. Maximal CPT I activity was measured in mitochondria isolated from resting human skeletal muscle. Mean CPT I activity was 492.8 +/- 72.8 and 260.8 +/- 33.6 micromol. min(-1). kg wet muscle(-1) in trained and untrained subjects, respectively (pH 7.0, 37 degrees C). The sensitivity to M-CoA was greater in trained muscle; the IC(50) for M-CoA was 0.17 +/- 0.04 and 0.49 +/- 0.17 microM in trained and untrained muscle, respectively. The presence of acetyl-CoA, free coenzyme A (CoASH), and acetylcarnitine, in concentrations simulating rest and exercise conditions did not release the M-CoA-induced inhibition of CPT I activity. However, CPT I activity was reduced at pH 6.8 vs. pH 7.0 in both trained and untrained muscle in the presence of physiological concentrations of M-CoA. The results of this study indicate that aerobic training is associated with an increase in the sensitivity of CPT I to M-CoA. Accumulations of acetyl-CoA, CoASH, and acetylcarnitine do not counteract the M-CoA-induced inhibition of CPT I activity. However, small decreases in pH produce large reductions in the activity of CPT I and may contribute to the decrease in fat metabolism that occurs during moderate and intense aerobic exercise intensities.
Asunto(s)
Carnitina O-Palmitoiltransferasa/metabolismo , Malonil Coenzima A/farmacología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/enzimología , Educación y Entrenamiento Físico , Acetilcoenzima A/farmacología , Acetilcarnitina/farmacología , Adulto , Coenzima A/farmacología , Femenino , Humanos , Concentración de Iones de Hidrógeno , Masculino , Mitocondrias Musculares/efectos de los fármacos , Mitocondrias Musculares/enzimología , Valores de ReferenciaRESUMEN
This study investigated whether increased provision of oxidative substrate would reduce the reliance on nonoxidative ATP production and/or increase power output during maximal sprint exercise. The provision of oxidative substrate was increased at the onset of exercise by the infusion of acetate (AC; increased resting acetylcarnitine) or dichloroacetate [DCA; increased acetylcarnitine and greater activation of pyruvate dehydrogeanse (PDH-a)]. Subjects performed 10 s of maximal cycling on an isokinetic ergometer on three occasions after either DCA, AC, or saline (Con) infusion. Resting PDH-a with DCA was increased significantly over AC and Con trials (3.58 +/- 0.4 vs. 0.52 +/- 0.1 and 0.74 +/- 0.1 mmol. kg wet muscle(-1). min(-1)). DCA and AC significantly increased resting acetyl-CoA (35.2 +/- 4.4 and 22.7 +/- 2.9 vs. 10.2 +/- 1.3 micromol/kg dry muscle) and acetylcarnitine (12.9 +/- 1.4 and 11.0 +/- 1.0 vs. 3.3 +/- 0.6 mmol/kg dry muscle) over Con. Resting contents of phosphocreatine, lactate, ATP, and glycolytic intermediates were not different among trials. Average power output and total work done were not different among the three 10-s sprint trials. Postexercise, PDH-a in AC and Con trials had increased significantly but was still significantly lower than in DCA trial. Acetyl-CoA did not increase in any trial, whereas acetylcarnitine increased significantly only in DCA. Exercise caused identical decreases in ATP and phosphocreatine and identical increases in lactate, pyruvate, and glycolytic intermediates in all trials. These data suggest that there is an inability to utilize extra oxidative substrate (from either stored acetylcarnitine or increased PDH-a) during exercise at this intensity, possibly because of O(2) and/or metabolic limitations.
Asunto(s)
Acetatos/farmacología , Ácido Dicloroacético/farmacología , Ejercicio Físico/fisiología , Músculo Esquelético/metabolismo , Carrera/fisiología , Acetatos/administración & dosificación , Acetilcoenzima A/metabolismo , Acetilación , Acetilcarnitina/metabolismo , Adenosina Trifosfato/metabolismo , Adulto , Ciclo del Ácido Cítrico/efectos de los fármacos , Ácido Dicloroacético/administración & dosificación , Metabolismo Energético/efectos de los fármacos , Ergometría , Femenino , Humanos , Masculino , Músculo Esquelético/efectos de los fármacos , Oxidación-Reducción , Consumo de Oxígeno/efectos de los fármacos , Complejo Piruvato Deshidrogenasa/metabolismo , Descanso/fisiologíaRESUMEN
This study investigated whether dichloroacetate (DCA) decreases the reliance on substrate level phosphorylation during the transition from rest to moderate-intensity exercise in humans. Nine subjects cycled at approximately 65% of maximal oxygen uptake (VO(2 max)) after a saline or DCA (100 mg/kg body wt) infusion, with muscle biopsies taken at rest and at 30 s and 2 and 10 min of exercise. DCA infusion increased pyruvate dehydrogenase (PDH) activation at rest (4.0 +/- 0.3 vs. 0.9 +/- 0.1 mmol. kg wet wt(-1). min(-1)) and at 30 s (3.6 +/- 0.2 vs. 2.5 +/- 0.4 mmol. kg(-1). min(-1)) of exercise. As a result, acetyl-CoA (45.9 +/- 5.9 vs. 11.3 +/- 1.5 micromol/kg dry wt) and acetylcarnitine (13.1 +/- 1.0 vs. 1.6 +/- 0.3 mmol/kg dry wt) were markedly increased by DCA infusion at rest. These differences were maintained at 30 s and 2 min for both acetyl-CoA and acetylcarnitine. Resting muscle lactate and phosphocreatine (PCr) were not different between trials, but DCA infusion resulted in lower lactate accumulation throughout exercise, especially at 2 min (21.6 +/- 3.1 vs. 44.6 +/- 8.0 mmol/kg dry wt). PCr utilization in the initial 30 s (16.9 +/- 0.4 vs. 31.7 +/- 2.6 mmol/kg dry wt) and 2 min (27.8 +/- 4.7 vs. 45.1 +/- 2.6 mmol/kg dry wt) of exercise was decreased with DCA. This resulted in a lower accumulation of free inorganic phosphate at 30 s (25.4 +/- 2.0 vs. 36.4 +/- 2.8 mmol/kg dry wt) and 2 min (34.6 +/- 4.7 vs. 50.5 +/- 2.2 mmol/kg dry wt) with DCA and decreased glycogenolysis over 10 min. The data from this study support the hypothesis that increased provision of substrate by DCA infusion increases oxidative metabolism during the rest-to-work transition, resulting in decreased PCr utilization and an improved cellular energy state at the onset of exercise. The transitory improvement in energy state decreased glycogenolysis and lactate accumulation during moderate-intensity exercise.
Asunto(s)
Ácido Dicloroacético/farmacología , Ejercicio Físico/fisiología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Adulto , Sangre/metabolismo , Femenino , Humanos , Masculino , Músculo Esquelético/enzimología , Consumo de Oxígeno/efectos de los fármacos , Complejo Piruvato Deshidrogenasa/metabolismo , Respiración/efectos de los fármacosRESUMEN
To characterize human skeletal muscle enzymatic adaptation to a low-carbohydrate, high-fat, and high-protein diet (LCD), subjects consumed a eucaloric diet consisting of 5% of the total energy intake from carbohydrate, 63% from fat, and 33% from protein for 6 days compared with their normal diet (52% carbohydrate, 33% fat, and 14% protein). Biopsies were taken from the vastus lateralis before and after 3 and 6 days on a LCD. Intact mitochondria were extracted from fresh muscle and analyzed for pyruvate dehydrogenase (PDH) kinase, total PDH, and carnitine palmitoyltransferase I activities and mitochondrial ATP production rate (using carbohydrate and fat substrates). beta-Hydroxyacyl CoA dehydrogenase, active PDH (PDHa), and citrate synthase activities were also measured on whole muscle homogenates. PDH kinase (PDHK) was calculated as the absolute value of the apparent first-order rate constant of the inactivation of PDH in the presence of 0.3 mM Mg2+-ATP. PDHK increased dramatically from 0.10 +/- 0.02 min-1 to 0.35 +/- 0.09 min-1 at 3 days and 0.49 +/- 0. 06 min-1 after 6 days. Resting PDHa activity decreased from 0.63 +/- 0.17 to 0.17 +/- 0.04 mmol. min-1. kg-1 after 6 days on the diet, whereas total PDH activity did not change. Activities for all other enzymes were unaltered by the LCD. In summary, severe deficiency of dietary carbohydrate combined with a twofold increase in dietary fat and protein caused a rapid three- to fivefold increase in PDHK activity in human skeletal muscle. The increased PDHK activity downregulated the amount of PDH in its active form at rest and decreased carbohydrate metabolism. However, an increase in the activities of enzymes involved in fatty acid oxidation did not occur.
Asunto(s)
Carbohidratos de la Dieta/administración & dosificación , Músculo Esquelético/enzimología , Proteínas Quinasas/metabolismo , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , Adenosina Trifosfato/biosíntesis , Adulto , Carnitina O-Palmitoiltransferasa/metabolismo , Citrato (si)-Sintasa/metabolismo , Carbohidratos de la Dieta/farmacología , Femenino , Humanos , Masculino , Mitocondrias Musculares/metabolismo , Proteínas Serina-Treonina Quinasas , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Complejo Piruvato Deshidrogenasa/metabolismo , Factores de TiempoRESUMEN
This study investigated the transformational and posttransformational control of skeletal muscle glycogen phosphorylase and pyruvate dehydrogenase (PDH) at three exercise power outputs [35, 65, and 90% of maximal oxygen uptake (VO2 max)]. Seven untrained subjects cycled at one power output for 10 min on three separate occasions, with muscle biopsies at rest and 1 and 10 min of exercise. Glycogen phosphorylase in the more active (a) form was not significantly different at any time across power outputs (21. 4-29.6%), with the exception of 90%, where it fell significantly to 15.3% at 10 min. PDH transformation increased significantly from rest (average 0.53 mmol . kg wet muscle-1 . min-1) to 1 min of exercise as a function of power output (1.60 +/- 0.26, 2.77 +/- 0.29, and 3.33 +/- 0.31 mmol . kg wet muscle-1 . min-1 at 35, 65, and 90%, respectively) with a further significant increase at 10 min (4.45 +/- 0.35) at 90% VO2 max. Muscle lactate, acetyl-CoA, acetylcarnitine, and free ADP, AMP, and Pi were unchanged from rest at 35% VO2 max but rose significantly at 65 and 90%, with accumulations at 90% being significantly higher than 65%. The results of this study indicate that glycogen phosphorylase transformation is independent of increasing power outputs, despite increasing glycogenolytic flux, suggesting that flux through glycogen phosphorylase is matched to the demand for energy by posttransformational factors, such as free Pi and AMP. Conversely, PDH transformation is directly related to the increasing power output and the calculated flux through the enzyme. The rise in PDH transformation is likely due to increased Ca2+ concentration and/or increased pyruvate. These results demonstrate that metabolic signals related to contraction and the energy state of the cell are sensitive to the exercise intensity and coordinate the increase in carbohydrate use with increasing power output.
Asunto(s)
Músculo Esquelético/enzimología , Fosforilasas/metabolismo , Esfuerzo Físico/fisiología , Complejo Piruvato Deshidrogenasa/metabolismo , Acetilcoenzima A/metabolismo , Acetilcarnitina/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Adulto , Ejercicio Físico/fisiología , Prueba de Esfuerzo , Femenino , Glucosa/metabolismo , Glucosa-6-Fosfato/metabolismo , Homeostasis , Humanos , Lactatos/metabolismo , Masculino , Músculo Esquelético/metabolismo , Consumo de Oxígeno , Fosfatos/metabolismo , Fosforilasa a/metabolismo , Factores de TiempoRESUMEN
This study examined the effects of caffeine (Caf) ingestion on muscle glycogen use and the regulation of muscle glycogen phosphorylase (Phos) activity during intense aerobic exercise. In two separate trials, 12 untrained males ingested either placebo (Pl) or Caf (9 mg/kg body wt) 1 h before cycling at 80% maximum O2 consumption (VO2 max) for 15 min. Muscle biopsies were obtained from the vastus lateralis at 0, 3, and 15 min of exercise. In this study, glycogen "sparing" was defined as a 10% or greater reduction in muscle glycogen use during exercise after Caf ingestion compared with Pl. Muscle glycogen use decreased by 28% (Pl 255 +/- 38 vs. Caf 184 +/- 24 mmol/kg dry muscle) after Caf in six subjects [glycogen sparers (Sp)] but was unaffected by Caf in six other subjects [nonsparers (NSp), Pl 210 +/- 35 vs. Caf 214 +/- 37 mmol/kg dry muscle]. In both groups, Caf significantly increased resting free fatty acid concentration, significantly increased epinephrine concentration by twofold during exercise, and increased the Phos a mole fraction at 3 min of exercise compared with Pl, although not significantly. Caf improved the energy status of the muscle during exercise in the Sp group: muscle phosphocreatine (PCr) degradation was significantly reduced (Pl 47.9 +/- 3.6 vs. Caf 40.4 +/- 6.7 mmol/kg dry muscle at 3 min) and the accumulations of free ADP and free AMP (Pl 6.8 +/- 1.3 vs. Caf 3.1 +/- 1.4 micromol/kg dry muscle at 3 min; Pl 8.7 +/- 0.8 vs. Caf 4.7 +/- 1.1 micromol/kg dry muscle at 15 min) were significantly reduced. Caf had no effect on these measurements in the NSp group. It is concluded that the Caf-induced decrease in flux through Phos (glycogen-sparing effect) is mediated via an improved energy status of the muscle in the early stages of intense aerobic exercise. This may be related to an increased availability of fat and/or ability of mitochondria to oxidize fat during exercise preceded by Caf ingestion. It is presently unknown why the glycogen-sparing effect of Caf does not occur in all untrained individuals during intense aerobic exercise.
Asunto(s)
Cafeína/farmacología , Ejercicio Físico/fisiología , Glucógeno/metabolismo , Músculo Esquelético/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Adulto , Glucemia/metabolismo , Ácidos Grasos no Esterificados/sangre , Humanos , Lactatos/sangre , Lactatos/metabolismo , Masculino , Músculo Esquelético/efectos de los fármacos , Consumo de Oxígeno , Factores de TiempoRESUMEN
This study was designed to compare the activity of skeletal muscle carnitine palmitoyltransferase I (CPT I) in trained and inactive men (n = 14) and women (n = 12). CPT I activity was measured in intact mitochondria, isolated from needle biopsy vastus lateralis muscle samples ( approximately 60 mg). The variability of CPT I activity determined on two biopsy samples from the same leg on the same day was 4.4, whereas it was 7.0% on two biopsy samples from the same leg on different days. The method was sensitive to the CPT I inhibitor malonyl-CoA (88% inhibition) and therefore specific for CPT I activity. The mean CPT I activity for all 26 subjects was 141.1 +/- 10.6 micromol . min-1 . kg wet muscle (wm)-1 and was not different when all men vs. all women (140.5 +/- 15.7 and 142.2 +/- 14.5 micromol . min-1 . kg wm-1, respectively) were compared. However, CPT I activity was significantly higher in trained vs. inactive subjects for both men (176.2 +/- 21.1 vs. 104.1 +/- 13.6 micromol . min-1 . kg wm-1) and women (167.6 +/- 14.1 vs. 91.2 +/- 9.5 micromol . min-1 . kg wm-1). CPT I activity was also significantly correlated with citrate synthase activity (all subjects, r = 0.76) and maximal oxygen consumption expressed in milliliters per kilogram per minute (all subjects, r = 0.69). The results of this study suggest that CPT I activity can be accurately and reliably measured in intact mitochondria isolated from human muscle biopsy samples. CPT I activity was not affected by gender, and higher activities in aerobically trained subjects appeared to be the result of increased mitochondrial content in both men and women.
Asunto(s)
Carnitina O-Palmitoiltransferasa/metabolismo , Mitocondrias Musculares/enzimología , Músculo Esquelético/enzimología , Adulto , Citrato (si)-Sintasa/metabolismo , Ejercicio Físico/fisiología , Femenino , Humanos , Técnicas In Vitro , Masculino , Consumo de Oxígeno/fisiología , Aptitud Física/fisiología , Caracteres SexualesRESUMEN
To investigate the regulation of intramuscular fuel selection, we measured the malonyl-CoA (M-CoA) content in human skeletal muscle at three exercise power outputs [35, 65, and 90% maximal rate of O2 consumption (VO2 max)]. Four males and four females cycled for 10 min at one power output on three separate occasions with muscle biopsies sampled at rest and at 1 and 10 min. The respiratory exchange ratio was 0.84 +/- 0.03, 0.92 +/- 0.02, and >1.0 at 35, 65 and 90% VO2 max, respectively. Muscle lactate content increased and phosphocreatine content decreased as a function of power output. Pyruvate dehydrogenase a activity increased from 0.40-0.64 mmol . kg wet muscle-1 . min-1 at rest to 1.57 +/- 0.28, 2.80 +/- 0.41, and 3. 28 +/- 0.27 mmol . kg wet muscle-1 . min-1 after 1 min of cycling at the three power outputs, respectively. Mean resting M-CoA contents were similar at all power outputs (1.85-1.98 micromol/kg dry muscle). During exercise at 35% VO2 max, M-CoA decreased from rest at 1 min (1.85 +/- 0.29 to 1.20 +/- 0.12 micromol/kg dry muscle) but returned to rest level by 10 min (1.86 +/- 0.25 micromol/kg dry muscle). M-CoA content did not decrease during cycling at 65% VO2 max. At 90% VO2 max, M-CoA did not increase despite significant acetyl-CoA accumulation (the substrate for M-CoA synthesis). The data suggest that a decrease in M-CoA content is not required for the increase in free fatty acid uptake and oxidation that occurs during exercise at 35 and 65% VO2 max. Furthermore, M-CoA content does not increase during exercise at 90% VO2 max and does not contribute to the lower rate of fat oxidation at this power output.
Asunto(s)
Ejercicio Físico/fisiología , Malonil Coenzima A/metabolismo , Músculo Esquelético/metabolismo , Consumo de Oxígeno , Adenosina Trifosfato/metabolismo , Adulto , Ácidos Grasos no Esterificados/metabolismo , Femenino , Glucosa/metabolismo , Humanos , Ácido Láctico/metabolismo , Masculino , Oxidación-Reducción , Fosfocreatina/metabolismo , Intercambio Gaseoso Pulmonar , Complejo Piruvato Deshidrogenasa/metabolismoRESUMEN
A substrate cycle between citric acid cycle (CAC) intermediates isocitrate and 2-oxoglutarate, involving NAD+- and NADP+-linked isocitrate dehydrogenase (NAD-IDH and NADP-IDH, respectively) and mitochondrial transhydrogenase (H+-Thase), has recently been proposed. This cycle has been hypothesized to enhance mitochondrial respiratory control by increasing the sensitivity of NAD-IDH to its modulators and allowing for enhanced increases in flux through this step of the CAC during periods of increased ATP demand. The activities of the enzymes comprising the substrate cycle: NAD-IDH, forward and reverse NADP-IDH, and forward and reverse H+-Thase, along with the activity of a marker of mitochondrial content, citrate synthase (CS) were measured in mitochondria isolated from rabbit Type I and Type IIb muscles and in whole muscle homogenates, representing the various fiber types, from rats. In isolated rabbit muscle mitochondria, NAD-IDH had significantly higher (1.6 x ) activity in white muscle while forward NADP-IDH, forward and reverse H+-Thase, and CS all had significantly higher (1.2-1.6 x ) activities in red muscle. There was no difference in reverse NADP-IDH between fiber types. Similarly, in rat whole muscle enzyme activities normalized to CS, NAD-IDH had significantly higher activity in fast-twitch glycolytic (FG) fibers, while forward NADP-IDH and forward H+-Thase had significantly higher activities in slow-twitch oxidative (SO) fibers. These results suggest that differences in the activities of the substrate cycle enzymes between skeletal muscle fiber types could contribute to differences in respiratory control due to differential cycling rates and/or loci of control.