Evidence against regulation of AMP-activated protein kinase and LKB1/STRAD/MO25 activity by creatine phosphate

Muscle contraction results in phosphorylation and activation of the AMP-activated protein kinase (AMPK) by an AMPK kinase (AMPKK). LKB1/STRAD/MO25 (LKB1) is the major AMPKK in skeletal muscle; however, the activity of LKB1 is not increased by muscle contraction. This finding suggests that phosphorylation of AMPK by LKB1 is regulated by allosteric mechanisms. Creatine phosphate is depleted during skeletal muscle contraction to replenish ATP. Thus the concentration of creatine phosphate is an indicator of cellular energy status. A previous report found that creatine phosphate inhibits AMPK activity. The purpose of this study was to determine whether creatine phosphate would inhibit 1) phosphorylation of AMPK by LKB1 and 2) AMPK activity after phosphorylation by LKB1. We found that creatine phosphate did not inhibit phosphorylation of either recombinant or purified rat liver AMPK by LKB1. We also found that creatine phosphate did not inhibit 1) active recombinant alpha1beta1gamma1 or alpha2beta2gamma2 AMPK, 2) AMPK immunoprecipitated from rat liver extracts by either the alpha1 or alpha2 subunit, or 3) AMPK chromatographically purified from rat liver. Inhibition of skeletal muscle AMPK by creatine phosphate was greatly reduced or eliminated with increased AMPK purity. In conclusion, these results suggest that creatine phosphate is not a direct regulator of LKB1 or AMPK activity. Creatine phosphate may indirectly modulate AMPK activity by replenishing ATP at the onset of muscle contraction.     

The polysome as a terminal for the creatine phosphate energy shuttle

The role of the creatine phosphate shuttle in the energetics of muscle protein synthesis in isolated polysomes, from rat hindlimb muscle, was studied. Triton X-100-treated polysomes, following their centrifugation through a 1 M sucrose gradient, contained 38 mU/mg RNA of bound creatine kinase. In the presence of pH 5 enzyme (obtained from rat liver), 0.5 mM ATP, and 1 microM GTP, amino acid (leucine) incorporation by polysomes in the presence of 8 mM creatine phosphate was twice that in the presence of an exogenous ATP regenerating system of 10 mM phospho(enol)pyruvate and 10 U/ml pyruvate kinase. Since added creatine kinase had no effect on incorporation supported by creatine phosphate it is clear that endogenous creatine kinase allows sufficient regeneration of ATP. These data also suggest that nucleoside diphosphokinase must have been associated with the polysome for phosphate was transferred to GTP from [33P]creatine phosphate, and the specific activities of ATP and GTP increased at equal rates, reaching the specific activity of creatine phosphate at 8 min. We conclude that skeletal muscle polysomes have bound creatine kinase activity and they act as terminals for the creatine phosphate energy shuttle. Creatine phosphate regenerates GTP, probably through an intermediate reaction catalyzed by nucleoside diphosphokinase. This provided an added support for the hypothesis of compartmentation of enzymes and substrates and that the transport form of energy between the mitochondria and energy utilizing sites in muscle is creatine phosphate rather than ATP, which extends the general role of the creatine phosphate energy shuttle.     

Energy conservation attenuates the loss of skeletal muscle excitability during intense contractions

High-frequency stimulation of skeletal muscle has long been associated with ionic perturbations, resulting in the loss of membrane excitability, which may prevent action potential propagation and result in skeletal muscle fatigue. Associated with intense skeletal muscle contractions are large changes in muscle metabolites. However, the role of metabolites in the loss of muscle excitability is not clear. The metabolic state of isolated rat extensor digitorum longus muscles at 30 degrees C was manipulated by decreasing energy expenditure and thereby allowed investigation of the effects of energy conservation on skeletal muscle excitability. Muscle ATP utilization was reduced using a combination of the cross-bridge cycling blocker N-benzyl-p-toluene sulfonamide (BTS) and the SR Ca2+ release channel blocker Na-dantrolene, which reduce activity of the myosin ATPase and SR Ca2+-ATPase. Compared with control muscles, the resting metabolites ATP, phosphocreatine, creatine, and lactate, as well as the resting muscle excitability as measured by M-waves, were unaffected by treatment with BTS plus dantrolene. Following 20 or 30 s of continuous 60-Hz stimulation, BTS-plus-dantrolene-treated muscles showed a 25% lower ATP utilization compared with control muscles. Furthermore, the ability of muscles to maintain excitability during high-frequency stimulation was significantly improved in BTS-plus-dantrolene-treated muscles, indicating a strong link between metabolites, energetic state, and the excitability of the muscle.     

Energetic driving forces are maintained in resting rat skeletal muscle after dietary creatine supplementation

The total creatine(TCr) pool of skeletal muscle is composed of creatine (Cr) andphosphocreatine (PCr). In resting skeletal muscle, the ratio ofPCr to TCr (PCr/TCr; PCr energy charge) is ~0.6-0.8, dependingon the fiber type. PCr/TCr is linked to the cellular free energy of ATPhydrolysis by the Cr kinase equilibrium. Dietary Cr supplementationincreases TCr in skeletal muscle. However, many previous studies havereported data indicating that PCr/TCr falls after supplementation,which would suggest that Cr supplementation alters the restingenergetic state of myocytes. This study investigated the effect of Crsupplementation on the energy phosphates of resting skeletal muscle.Male rats were fed either rodent chow (control) or chow supplementedwith 2% (wt/wt) Cr. After 2 wk on the diet, the gastrocnemius andsoleus muscles were freeze clamped and removed from anesthetizedanimals. Cr supplementation increased TCr, PCr, and Cr levels in thegastrocnemius by 20, 22, and 17%, respectively (P < 0.05). A numerical 6% higher mean soleus TCr in Cr-supplemented ratswas not statistically significant. All other energy phosphate concentrations, free energy of ATP hydrolysis, and PCr/TCr were notdifferent between the two groups in either muscle. We conclude that Crsupplementation simply increased TCr in fast-twitch rat skeletal musclebut did not otherwise alter resting cellular energetic state.

     

Kinetic characterization of human heart and skeletal muscle CK isoenzymes

The purpose of this study was to investigate the kinetic properties of human creatine kinase (CK) isoenzymes partially purified from heart and skeletal muscle. Utilizing the backward CK-catalyzed reaction of creatine phosphate + ADP in equilibrium creatine + ATP, Km values for heart and skeletal muscle CK MM (3.7 mmol/l) were significantly (p less than 0.05) greater than CK MB (2.1 mmol/l) which were significantly (p less than 0.05) greater than mitochondrial CK (1.8 mmol/l) at variable creatine phosphate and fixed ADP concentrations. However, Km values for similar isoenzymes from the two different tissues, i.e., CK MB from heart vs. skeletal muscle, were not different. These results show that kinetic analysis of CK isoenzymes cannot differentiate the tissue source of elevated blood CK isoenzymes after the acute stress of long distance running or after acute myocardial infarction.     

Phosphorus NMR studies on perfused heart

Phosphorus nuclear magnetic resonance measurements at 129MHz have been made on small beating rat hearts, perfused by the Langendorff technique. Good spectra, giving the levels of ATP, creatine phosphate and inorganic phosphate can be collected in as little as 10–20min and the heart can be maintained in a steady state in the spectrometer for at least 5 hours. In a good preparation, the ratios of the β-phosphate of ATP to creatine phosphate and inorganic phosphate are 1:1.8:1.8 which compare well with the data obtained by analyzing a freeze-clamped extract by NMR. The recovery of metabolites, after the induction of global ischaemia, has been followed.     

Relating structure to mechanism in creatine kinase

Found in all vertebrates, creatine kinase catalyzes the reversible reaction of creatine and ATP forming phosphocreatine and ADP. Phosphocreatine may be viewed as a reservoir of "high-energy phosphate" which is able to supply ATP, the primary energy source in bioenergetics, on demand. Consequently, creatine kinase plays a significant role in energy homeostasis of cells with intermittently high energy requirements. The enzyme is of clinical importance and its levels are routinely used as an indicator of myocardial and skeletal muscle disorders and for the diagnosis of acute myocardial infarction. First identified in 1928, the enzyme has undergone intensive investigation for over 75 years. There are four major isozymes, two cytosolic and two mitochondrial, which form dimers and octamers, respectively. Depending on the pH, the enzyme operates by a random or an ordered bimolecular mechanism, with the equilibrium lying towards phosphocreatine production. Evidence suggests that conversion of creatine to phosphocreatine occurs via the in-line transfer of a phosphoryl group from ATP. A recent X-ray structure of creatine kinase bound to a transition state analog complex confirmed many of the predictions based on kinetic, spectroscopic, and mutagenesis studies. This review summarizes and correlates the more significant mechanistic and structural studies on creatine kinase.     

Analysis of compartmentation of ATP in skeletal and cardiac muscle using 31P nuclear magnetic resonance saturation transfer.

We have developed a model for the analysis of the forward creatine kinase reaction in muscle as measured by the nuclear magnetic resonance (NMR) technique of magnetization transfer. The model, accounting for the double-exponential behavior observed in some NMR magnetization transfer data, allows for the existence of two ATP pools, one that is NMR-visible (NMR-VIS) and another that is NMR-invisible (NMR-INVIS). We have applied the model to experimental data for the forward creatine kinase reaction in skeletal and cardiac muscles to study the dependence of the creatine kinase rate constants and fluxes on workload and to account for the differences between heart and skeletal muscle. The results suggest that an NMR-distinct ATP pool exists in both heart and skeletal muscles, and that phosphate exchange with this pool catalyzed by creatine kinase increases with increased workload. The results also agree with previously published estimates of the rates of mitochondrial translocase and net ATP synthesis obtained by traditional biochemical methods.     

Ghrelin: Impact on Muscle Energy Metabolism in Sepsis

We investigated the effects of exogenous ghrelin on energy levels and tissue histology in skeletal muscle in experimentally lipopolysaccharide (LPS) induced septic rats. Male Wistar albino rats 200–250 g were separated into four groups; Control, LPS (5 mg/kg), Ghrelin (10 nmol/kg i.v.), and ghrelin+LPS. Gastrocnemius muscle tissue was taken and stained using modified Gomori trichrome (MGT), succinic dehydrogenase (SDH), and cytochrome oxidase (COX) and hematoxylin and eosin. In stained sections, histological score value was calculated according to the intensity and the distribution for MGT, SDH and COX stainings. Creatine, creatine phosphate, adenosine triphosphate (ATP), adenosine monophosphate (AMP) levels, and the ratios of AMP/ATP and CreaP/ATP were investigated using high performance liquid chromatography (HPLC) in muscle tissue. Significances between experimental groups were calculated with an analysis of variance (ANOVA) followed by Tukey’s tests. Myopathic changes were seen in the 50% of rats in the LPS group as rounding of muscle fibers and fiber size variation. In the ghrelin+LPS group, ghrelin treatment was reduced damage in skeletal muscle structure. There was no change in creatine or AMP levels between the groups. Ghrelin treatment significantly increased ATP values (P?<?0.01) and improved tissue histology in septic rats. Ratios of both AMP/ATP and CreaP/ATP were found increased in the septic group, but there were decreaments in both the ghrelin and ghrelin-treated septic groups. Ghrelin could play an important role in energy balance and muscle morphology in skeletal muscle during sepsis.     

Histochemical demonstration of ATP: creatine phosphotransferase in rat skeletal muscle

Summary An attempt was made to locate the ATP: creatine phosphotransferase (creatine kinase, CKase) in rat skeletal muscle by a lead precipitation method. The muscle is not stained at all with creatine phosphate (CP), and only weakly with adenosine diphosphate (ADP) as substrate, while it hydrolyzes adenosine triphosphate (ATP) actively. Taking advantage of this fact, it is possible to demonstrate the CKase activity using both ADP and CP as substrate. The CKase activity thus obtained was located in various profiles of sarcoplasmic reticulum as well as in A bands, the staining being comparable to that obtained with ATP as substrate.A weak activity was found only in cisternal dilatations of sarcoplasmic reticulum when sections were incubated with ADP as substrate.     

一次性力竭运动对大鼠PHB1表达及线粒体功能的影响

目的：观察一次性力竭运动后大鼠脑、心、骨骼肌组织和线粒体中PHB1含量的变化及对大鼠线粒体功能的影响,探寻PHB1与线粒体功能和能量代谢的关系。方法：健康雄性SD大鼠40只,随机分为2组（n=20）：对照组和一次性力竭运动组,大鼠进行一次性急性跑台运动建立力竭运动模型。收集各组大鼠的心、脑和骨骼肌组织样品并提取线粒体,检测其呼吸功能和ROS的变化。用Western blot方法检测组织和线粒体中PHB1蛋白表达水平;用分光光度计检测各器官中ATP含量以及线粒体中复合体V活性（ATP合酶活性）。结果：①一次性力竭运动后脑、心肌、骨骼肌中ATP含量显著性降低;②一次性力竭运动后脑、心肌、骨骼肌线粒体中复合体V活性、RCR、ROS显著性降低,ST4均显著性升高,ST3无显著性差异。③一次性力竭运动后心、脑、骨骼肌线粒体中PHB1的表达显著性减少。④通过相关性分析得出：一次性力竭运动后心、脑、骨骼肌中ATP含量与心、脑、骨骼肌中复合体V活性呈正相关;心、脑、骨骼肌中ATP含量和心、脑骨骼肌中PHB1的表达呈正相关。结论：一次性力竭运动后,降低线粒体氧化磷酸化功能,使大鼠脑、骨骼肌线粒体内ROS生成增加,PHB1的表达、ATP含量和复合体V活性均下降。一次性力竭运动使得大鼠线粒体内PHB1表达降低,线粒体功能减弱,机体能量代谢降低。     

A 31P-nuclear magnetic resonance study of skeletal muscle metabolism in rats depleted of creatine with the analogue β-guanidinopropionic acid

Rats were fed a diet containing 1% β-guanidinopropionic acid (GPA) for 6–10 weeks to deplete their skeletal muscle of creatine. ³¹P-NMR was used to monitor metabolic changes in the gastrocnemius muscle at rest, during stimulated steady-state isometric contraction at 4 Hz and during recovery from stimulation. In resting muscles, the [creatine phosphate] was reduced to 10% (2.8 μmol·g^?1) and the [ATP] to 50% (3.3 μmol·g^?1) of those found in rats fed a control diet. The concentration of the phosphorylated form of the analogue (PGPA) was 23 μmol·g^?1. There was no significant difference in muscle performance or in the relative changes in the [ATP] during stimulation. Intracellular pH decreased rapidly on stimulation and recovered during the stimulation period to near resting values in both groups. In control rats, the initial decrease in pH was greater and the time to recovery was longer than in GPA-fed rats. The rate at which PGPA supplied energy to the contracting muscle (0.027 mM·s^?1) was insignificant relative to the minimum estimated rate of ATP turnover (1 mM·s^?1). The rate of PGPA resynthesis during recovery (0.018 mM·s^?1) is enzyme-limited and provides an independent estimate of creatine kinase flux during this period (18.9 mM·s^?1). The creatine kinase flux (creatine phosphate → ATP) in the resting muscle of GPA-fed rats was 12-fold less than in control animals, 1.3 vs. 15.7 mM·s^?1. These results demonstrate that neither the [creatine phosphate] nor the activity of creatine kinase is critical for aerobic metabolism. Skeletal muscle appears to adapt to a diminished creatine pool by enhancing its aerobic capacity.     

Differential extractability of creatine phosphate and ATP from cardiac muscle with ethanol and perchloric acid solution.

To compare the extractability of creatine phosphate with that of ATP by alcohol extraction, both compounds were extracted from normal perfused rat heart tissues by using various stepwise concentrations of ethanol and 0.4 M HClO4. Powdered samples (6-15 mg wet wt) from the freeze-clamped tissues were homogenized in 2 ml of the ethanol solutions. After centrifugation, the supernatant was removed; each centrifuged sediment was rehomogenized with 2 ml of 0.4 M HClO4 and centrifuged. The supernatant was neutralized with 0.4 m KHCO3. The same powdered samples were directly homogenized with 2 ml of 0.4 M HClO4 and treated in the same manner. Only a small amount of ATP in the tissues was extracted by an 85% or higher concentration of ethanol. Further, about 13% of the tissue ATP was not extractable by the subsequent perchloric acid extraction. In contrast to ATP, creatine phosphate in the tissues was partially extracted by 95% ethanol and nearly all of the tissue creatine phosphate was extracted by 70% ethanol. The total creatine phosphate obtained by 70% ethanol and by subsequent perchloric acid extraction was significantly higher than that obtained by direct perchloric acid extraction. From these results, it was concluded that the extractability of creatine phosphate in the tissue by alcohol extraction is clearly different from that of ATP. Additionally, the stepwise extraction is recommended as a useful method for the extraction of energy metabolites in perfused rat heart tissue.     

Phosphorus nuclear magnetic resonance studies of phosphorus metabolites in frog muscle.

31P-nuclear magnetic resonance was applied to living muscles of bullfrogs, and the time courses of metabolic changes of ATP, creatine phosphate, inorganic phosphate, and sugar phosphates were studied under anaerobic and aerobic conditions. A decrease in creatine phosphate was observed in the resting muscle under anaerobic conditions with a concomitant decrease in the intracellular pH, while the ATP level remained constant. With the use of 2,4-dinitro-1-fluorobenzene and iodoacetic acid, ATP disappeared quickly. When the resting muscle was perfused with oxygen-saturated glucose-Ringer's solution, the amount of creatine phosphate increased gradually. These findings indicate that anaerobic glycolysis is insufficient for even the resting energy consumption whereas oxidative phosphorylation is sufficient. The effects of tetanic stimulation on living muscles were also studied. When glycolysis and oxidative phosphorylation were suppressed, the intracellular energy store was depleted by the tetanic contraction. Anaerobic glycolysis produced rapid recovery of the energy store level, although it was insufficient to reach the initial level. Aerobic oxidative phosphorylation produced sufficient energy to reach the initial level, and this level was never exceeded. This finding suggests the existence of a regulatory mechanism for the energy store level.     

Use of inversion spin transfer to monitor creatine kinase kinetics in rat skeletal muscle in vivo

A simple multipulse sequence has been used to monitor creatine kinase kinetics in rat skeletal muscle in vivo. Using these procedures, the forward (ATP synthesis) and reverse fluxes (phosphocreatine synthesis) have been calculated to be 8.98 +/- 0.6 and 10.7 +/- 0.8 mumoles/g wet wt/s (n = 5) respectively. These results suggest that in resting skeletal muscle most of the gamma ATP observed in 31P NMR spectra is cytosolic and rapidly exchanging with phosphocreatine. The high flux rates reflect the high catalytic capacity of creatine kinase in skeletal muscle.     

Rhythmic change of myocardial phosphate metabolite content in cardiac cycle observed by depth-selected and EKG-gated in vivo 31P-NMR spectroscopy in a whole animal

The newly developed pulse width modulation method for the depth-selected in vivo NMR under high magnetic field (6.4 Tesla), sectional magnetic resonance (SMR), enabled us to selectively obtain and follow time sequence of P metabolism of rat heart in a whole body. An EKG-gated 31P-SMR spectroscopy at every 30 m sec, after the R wave, with calibrating the resonance intensity by an external standard, demonstrated a synchronous oscillation of both contents of creatine phosphate (CP) and beta-ATP: minimal at the early 2/3 of the systole as was identified by the aortic pressure measurement and maximal at the last 1/3 of the diastole, while inorganic phosphate content varied antiphasically to CP or ATP without obvious change of intracellular pH in cardiac cycle. This is the first report that described an in vivo detection of cyclic change of phosphate metabolites in the heart.     

Metabolic alterations associated with increased energy demand in fish white muscle

Summary Time course measurements of glycogen, lactate, creatine phosphate, the adenylates and ammonia contents were made during the transition from rest to various levels of activity in fish (Macrozoarces americanus) white muscle. The muscle was perturbed by direct electrical stimulation resulting in sustained tetanus, 60 contractions/min or 20 contractions/min. Increased ATP demand was invariably associated with decreases in creatine phosphate followed by increases in lactate levels. The contribution of creatine phosphate to anaerobic energy production was equivalent to that of anaerobic glycolysis. In addition, decreases in creatine phosphate content may play an important role in the facilitation of glycolytic flux presumably by relief of inhibition of phosphofructokinase. Under some conditions the work transition was associated with an initial transient increase in ATP content which could not be accounted for by decreases in ADP and AMP levels. Furthermore, ammonia content was noted to oscillate during the work period, a feature which is fundamentally different from that which occurs in mammalian muscle.     

Systems bioenergetics of creatine kinase networks: physiological roles of creatine and phosphocreatine in regulation of cardiac cell function

Physiological role of creatine (Cr) became first evident in the experiments of Belitzer and Tsybakova in 1939, who showed that oxygen consumption in a well-washed skeletal muscle homogenate increases strongly in the presence of creatine and with this results in phosphocreatine (PCr) production with PCr/O₂ ratio of about 5–6. This was the beginning of quantitative analysis in bioenergetics. It was also observed in many physiological experiments that the contractile force changes in parallel with the alteration in the PCr content. On the other hand, it was shown that when heart function is governed by Frank–Starling law, work performance and oxygen consumption rate increase in parallel without any changes in PCr and ATP tissue contents (metabolic homeostasis). Studies of cellular mechanisms of all these important phenomena helped in shaping new approach to bioenergetics, Molecular System Bioenergetics, a part of Systems Biology. This approach takes into consideration intracellular interactions that lead to novel mechanisms of regulation of energy fluxes. In particular, interactions between mitochondria and cytoskeleton resulting in selective restriction of permeability of outer mitochondrial membrane anion channel (VDAC) for adenine nucleotides and thus their recycling in mitochondria coupled to effective synthesis of PCr by mitochondrial creatine kinase, MtCK. Therefore, Cr concentration and the PCr/Cr ratio became important kinetic parameters in the regulation of respiration and energy fluxes in muscle cells. Decrease in the intracellular contents of Cr and PCr results in a hypodynamic state of muscle and muscle pathology. Many experimental studies have revealed that PCr may play two important roles in the regulation of muscle energetics: first by maintaining local ATP pools via compartmentalized creatine kinase reactions, and secondly by stabilizing cellular membranes due to electrostatic interactions with phospholipids. The second mechanism decreases the production of lysophosphoglycerides in hypoxic heart, protects the cardiac cells sarcolemma against ischemic damage, decreases the frequency of arrhythmias and increases the post-ischemic recovery of contractile function. PCr is used as a pharmacological product Neoton in cardiac surgery as one of the components of cardioplegic solutions for protection of the heart against intraoperational injury and injected intravenously in acute myocardial ischemic conditions for improving the hemodynamic response and clinical conditions of patients with heart failure.     

Oxidative Myocytes of Heart and Skeletal Muscle Express Abundant Sarcomeric Mitochondrial Creatine Kinase

Sarcomeric mitochondrial creatine kinase catalyzes the reversible transfer of a high energy phosphate between ATP and creatine. To study cellular distribution of the kinase, we performed immunocytochemical studies using a peptide antiserum specific for the kinase protein. Our results demonstrated that the sarcomeric mitochondrial creatine kinase gene is abundantly expressed in heart and skeletal muscle, with no protein detected in other tissues examined, including brain, lung, liver, spleen, kidney, bladder, testis, stomach, intestine, and colon. RNA blot study showed that there is no detectable expression of the kinase mRNA in the thymus gland. In heart and skeletal muscle, the kinase protein is expressed in atrial and ventricular cardiomyocytes and a subpopulation of skeletal myofibres. In skeletal muscle, fast myosin heavy chain co-localization studies demonstrated that the sarcomeric mitochondrial creatine kinase is highly expressed in type 1, slow-oxidative and type 2A, fast-oxidative-glycolytic myofibres. We conclude that the kinase gene is abundantly expressed in oxidative myocytes of heart and skeletal muscle and may contribute to oxidative capacity of these cells.     

A 31P-nuclear magnetic resonance study of skeletal muscle metabolism in rats depleted of creatine with the analogue beta-guanidinopropionic acid

Rats were fed a diet containing 1% beta-guanidinopropionic acid (GPA) for 6-10 weeks to deplete their skeletal muscle of creatine. 31P-NMR was used to monitor metabolic changes in the gastrocnemius muscle at rest, during stimulated steady-state isometric contraction at 4 Hz and during recovery from stimulation. In resting muscles, the [creatine phosphate] was reduced to 10% (2.8 mumol X g-1) and the [ATP] to 50% (3.3 mumol X g-1) of those found in rats fed a control diet. The concentration of the phosphorylated form of the analogue (PGPA) was 23 mumol X g-1. There was no significant difference in muscle performance or in the relative changes in the [ATP] during stimulation. Intracellular pH decreased rapidly on stimulation and recovered during the stimulation period to near resting values in both groups. In control rats, the initial decrease in pH was greater and the time to recovery was longer than in GPA-fed rats. The rate at which PGPA supplied energy to the contracting muscle (0.027 mM X s-1) was insignificant relative to the minimum estimated rate of ATP turnover (1 mM X s-1). The rate of PGPA resynthesis during recovery (0.018 mM X s-1) is enzyme-limited and provides an independent estimate of creatine kinase flux during this period (18.9 mM X s-1). The creatine kinase flux (creatine phosphate----ATP) in the resting muscle of GPA-fed rats was 12-fold less than in control animals, 1.3 vs. 15.7 mM X s-1. These results demonstrate that neither the [creatine phosphate] nor the activity of creatine kinase is critical for aerobic metabolism. Skeletal muscle appears to adapt to a diminished creatine pool by enhancing its aerobic capacity.