Impaired aerobic glycolysis in muscle phosphofructokinase deficiency results in biphasic post-exercise phosphocreatine recovery in 31P magnetic resonance spectroscopy

Using 31P magnetic resonance spectroscopy, energy metabolism in calf muscles of two patients with biochemically and genetically proven muscular phosphofructokinase deficiency, and an asymptomatic heterozygote was monitored during isometric foot plantarflexion performed under aerobic and anaerobic conditions and in the aerobic recovery phases. In the heterozygote only a moderate alteration from normal was found in terms of an elevated ATP demand during exercise. In the homozygote, hexose phosphates, indicated as phosphomonoesters, increased dramatically during contraction. Phosphomonoester accumulation resulted in consumption of free inorganic phosphate (P(i)). During ischemic exercise the absence of glycolytic ATP formation resulted in a linear time course of phosphocreatine breakdown and a moderate alkalinization. During the recovery, phosphocreatine resynthesis showed a biphasic time course, indicating that mitochondrial function itself was not directly affected. At first glance, the early depletion of P(i) below initial resting levels and the rate of phosphate splitting from sugar phosphates seemed to become the limiting factor for the rate of the oxidative phosphorylation and creatine kinase reaction. However, the actual concentrations of P(i) and ADP estimated at the onset of delay were too high to exclusively explain the dramatic delay in PCr resynthesis. For this reason, a reduced turnover of the citric acid cycle was assumed, which was caused by the complete absence of glycolysis in PFK deficiency patients. Furthermore, results from PFK deficiency patients were compared with previous findings from myophosphorylase deficiency patients in the literature.     

Mathematical model of compartmentalized energy transfer: its use for analysis and interpretation of 31P-NMR studies of isolated heart of creatine kinase deficient mice

A mathematical model of the compartmentalized energy transfer in cardiac cells is described and used for interpretation of novel experimental data obtained by using phosphorus NMR for determination of the energy fluxes in the isolated hearts of transgenic mice with knocked out creatine kinase isoenzymes. These experiments were designed to study the meaning and importance of compartmentation of creatine kinase isoenzymes in the cells in vivo. The model was constructed to describe quantitatively the processes of energy production, transfer, utilization, and feedback between these processes. It describes the production of ATP in mitochondrial matrix space by ATP synthase, use of this ATP for phosphocreatine production in the mitochondrial creatine kinase reaction coupled to the adenine nucleotide translocation, diffusional exchange of metabolites in the cytoplasmic space, and use of phosphocreatine for resynthesis of ATP in the myoplasmic creatine kinase reaction. It accounts also for the recently discovered phenomenon of restricted diffusion of adenine nucleotides through mitochondrial outer membrane porin pores (VDAC). Practically all parameters of the model were determined experimentally. The analysis of energy fluxes between different cellular compartments shows that in all cellular compartments of working heart cells the creatine kinase reaction is far from equilibrium in the systolic phase of the contraction cycle and approaches equilibrium only in cytoplasm and only in the end-diastolic phase of the contraction cycle. Experimental determination of the relationship between energy fluxes by a 31P-NMR saturation transfer method and workload in isolated and perfused heart of transgenic mice deficient in MM isoenzyme of the creatine kinase, MM-/-showed that in the hearts from wild mice, containing all creatine kinase isoenzymes, the energy fluxes determined increased 3-4 times with elevation of the workload. By contrast, in the hearts in which only the mitochondrial creatine kinase was active, the energy fluxes became practically independent of the workload in spite of the preservation of 26% of normal creatine kinase activity. These results cannot be explained on the basis of the conventional near-equilibrium theory of creatine kinase in the cells, which excludes any difference between creatine kinase isoenzymes. However, these apparently paradoxical experimental results are quantitatively described by a mathematical model of the compartmentalized energy transfer based on the steady state kinetics of coupled creatine kinase reactions, compartmentation of creatine kinase isoenzymes in the cells, and the kinetics of ATP production and utilization reactions. The use of this model shows that: (1) in the wild type heart cells a major part of energy is transported out of mitochondria via phosphocreatine, which is used for complete regeneration of ATP locally in the myofibrils--this is the quantitative estimate for PCr pathway; (2) however, in the absence of MM-creatine kinase in the myofibrils in transgenic mice the contraction results in a very rapid rise of ADP in cytoplasmic space, that reverses the mitochondrial creatine kinase reaction in the direction of ATP production. In this way, because of increasing concentrations of cytoplasmic ADP, mitochondrial creatine kinase is switched off functionally due to the absence of its counterpart in PCr pathway, MM-creatine kinase. This may explain why the creatine kinase flux becomes practically independent from the workload in the hearts of transgenic mouse without MM-CK. Thus, the analysis of the results of studies of hearts of creatine kinase-deficient transgenic mice, based on the use of a mathematical model of compartmentalized energy transfer, show that in the PCr pathway of intracellular energy transport two isoenzymes of creatine kinase always function in a coordinated manner out of equilibrium, in the steady state, and disturbances in functioning of one of them inevitably result     

Ecotoxicology and landscape planning

Relationships between pH and the concentrations of phosphocreatine (PCr), inorganic phosphate (Pi), and lactate during ischemic exercise depend on passive buffering, proton consumption as a consequence of net PCr breakdown, the control of glycogenolysis, (particularly in relation to the concentration of Pi, a substrate of glycogen phosphorylase that is produced by net PCr breakdown), and the creatine kinase equilibrium. The author analyzes the implications of these relationships for the interpretation of 31P-magnetic resonance spectroscopic data and for the control of glycogenolysis. For realistic adenosine diphosphate (ADP) concentrations, given the constraints of the creatine kinase equilibrium, the pH must be near-linear with lactate, with an apparent buffer capacity (i.e., the ratio of lactate accumulation to pH change) that is nearly twice the true buffer capacity (i.e., the ratio of net proton loading to pH change). The implications for glycogenolytic control depend on adenosine triphosphate (ATP) turnover, but an upper limit of activation of glycogen phosphorylase (i.e., the amount of the a form) that would permit no increase in ADP concentration can be calculated. Phosphorylase activation during ischemic exercise seems approximately proportional to the power output, consistent with calcium stimulation of phosphorylase b kinase. In simulations, ADP concentration is highly sensitive to this proportionality, as (unlike in purely oxidative exercise) ADP concentration is not known to participate in any closed feedback loops in ischemic exercise.     

Creatine supplementation in health and disease. Effects of chronic creatine ingestion in vivo: down-regulation of the expression of creatine transporter isoforms in skeletal muscle

Interest in creatine (Cr) as a nutritional supplement and ergogenic aid for athletes has surged over recent years. After cellular uptake, Cr is phosphorylated to phosphocreatine (PCr) by the creatine kinase (CK) reaction using ATP. At subcellular sites with high energy requirements, e.g. at the myofibrillar apparatus during muscle contraction, CK catalyzes the transphosphorylation of PCr to ADP to regenerate ATP, thus preventing a depletion of ATP levels. PCr is thus available as an immediate energy source, serving not only as an energy buffer but also as an energy transport vehicle. Ingestion of creatine increases intramuscular Cr, as well as PCr concentrations, and leads to exercise enhancement, especially in sprint performance. Additional benefits of Cr supplementation have also been noticed for high-intensity long-endurance tasks, e.g. shortening of recovery periods after physical exercise. The present article summarizes recent findings on the influence of Cr supplementation on energy metabolism, and introduces the Cr transporter protein (CreaT), responsible for uptake of Cr into cells, as one of the key-players for the multi-faceted regulation of cellular Cr homeostasis. Furthermore, it is suggested that patients with disturbances in Cr metabolism or with different neuro-muscular diseases may benefit from Cr supplementation as an adjuvant therapy to relieve or delay the onset of symptoms. Although it is still unclear how Cr biosynthesis and transport are regulated in health and disease, so far there are no reports of harmful side effects of Cr loading in humans. However, in this study, we report that chronic Cr supplementation in rats down-regulates in vivo the expression of the CreaT. In addition, we describe the presence of CreaT isoforms most likely generated by alternative splicing.     

Theoretical modelling of some spatial and temporal aspects of the mitochondrion/creatine kinase/myofibril system in muscle

After discussing approaches to the modelling of mitochondrial regulation in muscle, we describe a model that takes account, in a simplified way, of some aspects of the metabolic and physical structure of the energy production/usage system. In this model, high-energy phosphates (ATP and phosphocreatine) and low energy metabolites (ADP and creatine) diffuse between the mitochondrion and the myofibrillar ATPase, and can be exchanged at any point by creatine kinase. Creatine kinase is not assumed to be at equilibrium, so explicit account can be taken of substantial changes in its activity of the sort that can now be achieved by transgenic technology in vivo. The ATPase rate is the input function. Oxidative ATP synthesis is controlled by juxtamitochondrial ADP concentration. To allow for possible functional 'coupling' between the components of creatine kinase associated with the mitochondrial adenine nucleotide translocase and the myofibrillar ATPase, we define parameters phi and psi that set the fraction of the total flux carried by ATP rather than phosphocreatine out of the mitochondrial unit and into the ATPase unit, respectively. This simplification is justified by a detailed analysis of the interplay between the mitochondrial outer membrane porin proteins, mitochondrial creatine kinase and the adenine nucleotide translocase. As both processes of possible 'coupling' are incorporated into the model as quantitative parameters, their effect on the energetics of the whole cell model can be explicitly assessed. The main findings are as follows: (1) At high creatine kinase activity, the hyperbolic relationship of oxidative ATP synthesis rate to spatially averaged ADP concentration at steady state implies also a near-linear relationship to creatine concentration, and a sigmoid relation to free energy of ATP hydrolysis. At high creatine kinase activity, the degree of functional coupling at either the mitochondrial or ATPase end has little effect on these relationships. However, lowering the creatine kinase activity raises the mean steady state ADP and creatine concentrations, and this is exaggerated when phi or psi is near unity (i.e. little coupling). (2) At high creatine kinase activity, the fraction of flow at steady state carried in the middle of the model by ATP is small, unaffected by the degree of functional coupling, but increases with ADP concentration and rate of ATP turnover. Lowering the creatine kinase activity raises this fraction, and this is exaggerated when psi or psi is near unity. (3) Both creatine and ADP concentrations show small gradients decreasing towards the mitochondrion (in the direction of their net flux), while ATP and phosphocreatine concentration show small gradients decreasing towards the myosin ATPase. Unless phi = psi = 0 (i.e. complete coupling), there is a gradient of net creatine kinase flux that results from the need to transform some of the 'adenine nucleotide flux' at the ends of the model into 'creatine flux' in the middle; the overall net flux is small, but only zero if phi = psi. A reduction in cytosolic creatine kinase activity decreases ADP concentration at the mitochondrial end and increases it at the ATPase end. (4) During work-jump transitions, spatial average responses exhibit exponential kinetics similar to those of models of mitochondrial control that assume equilibrium conditions for creatine kinase. (5) In response to a step increase in ATPase activity, concentration changes start at the ATPase end and propagate towards the mitochondrion, damped in time and space. This simplified model embodies many important features of muscle in vivo, and accommodates a range of current theories as special cases. We end by discussing its relationship to other approaches to mitochondrial regulation in muscle, and some possible extensions of the model.     

Exercise metabolism in Duchenne muscular dystrophy: a biochemical and [31P]-nuclear magnetic resonance study of mdx mice

Intracellular pH, ratios of phosphocreatine (PCr) to ATP and PCr to inorganic phosphate (Pi) as well as isometric tension were measured during 1 Hz sciatic nerve stimulation and during recovery in the calf muscles of mdx (a model of Duchenne muscular dystrophy) and control mice. Tension did not decline significantly in either strain. The ratio of PCr/(PCr + Pi) was significantly reduced in mdx as against control muscle during exercise and recovery, but the ratio of PCr/ATP and the half-time for PCr recovery were similar in both strains. A reduction in the maximal activities of succinate dehydrogenase and succinate-cytochrome c reductase suggests that mitochondrial metabolism may be impaired. The similarity in PCr recovery times suggests that the muscle has adapted, making any impairment of oxidative metabolism negligible in the intact system. The rate of pH recovery is prolonged in mdx muscle and provides strong evidence for a decline in the capacity of dystrophic muscle to extrude proton equivalents. These data are compared with a previous study which used 10 Hz stimulation and also observed a slow pH recovery. The slow pH recovery could be explained by an elevation in intracellular sodium.     

Early metabolic adaptations of rabbit fast-twitch muscle to chronic low-frequency stimulation

To investigate early adaptive responses to chronic low-frequency stimulation (CLFS), rabbit tibialis anterior (TA) muscles were continuously stimulated at 10 Hz for 8 days, allowed to rest for 1 h, and then subjected to a 15-min fatigue test at 10-Hz stimulation. The contralateral TA muscles which had not been exposed to CLFS, served as controls during the fatigue test. Compared to the controls, the initial tension output of the 8-day prestimulated muscles was reduced by 25%. However, these muscles maintained higher tensions during the fatigue test than the controls. Citrate synthase activity, an indicator of aerobic-oxidative capacity, was only slightly elevated (40%) in the 8-day stimulated muscles. Unlike the controls, the prestimulated muscles failed to produce potentiation during the fatigue test. Control muscles responded to the fatigue test with pronounced reductions in contents of adenosine 5'-triphosphate (ATP), phosphocreatine (PCr), and glycogen, as well as with large increases in contents of inosine monophosphate (IMP), inorganic phosphate (Pi), creatine (Cr), and lactate. Under the same conditions contents of ATP, PCr, Cr, glycogen, lactate, Pi, and IMP were unaltered in the 8-day prestimulated muscles. These findings demonstrated that CLFS for 8 days elicited pronounced alterations in energy metabolism and contractile properties. These adaptive changes occurred prior to fibre type transitions and substantial increases in aerobic-oxidative potential.     

Mitochondrial intermembrane inclusion bodies: the common denominator between human mitochondrial myopathies and creatine depletion, due to impairment of cellular energetics

Mitochondrial inclusion bodies are often described in skeletal muscle of patients suffering diseases termed mitochondrial myopathies. A major component of these structures was discovered as being mitochondrial creatine kinase. Similar creatine kinase enriched inclusion bodies in the mitochondria of creatine depleted adult rat cardiomyocytes have been demonstrated. Structurally similar inclusion bodies are observed in mitochondria of ischemic and creatine depleted rat skeletal muscle. This paper describes the various methods for inducing mitochondrial inclusion bodies in rodent skeletal muscle, and compares their effects on muscle metabolism to the metabolic defects of mitochondrial myopathy muscle. We fed rats with a creatine analogue guanidino propionic acid and checked their solei for mitochondrial inclusion bodies, with the electron microscope. The activity of creatine kinase was analysed by measuring creatine stimulated oxidative phosphorylation in soleus skinned fibres using an oxygen electrode. The guanidino propionic acid-rat soleus mitochondria displayed no creatine stimulation, whereas control soleus did, even though the GPA solei had a five fold increase in creatine kinase protein per mitochondrial protein. The significance of these results in light of their relevance to human mitochondrial myopathies and the importance of altered cell energetics and metabolism in the formation of these crystalline structures are discussed.     

Muscle creatine kinase-deficient mice. II. Cardiac and skeletal muscles exhibit tissue-specific adaptation of the mitochondrial function

Functional properties of in situ mitochondria and of mitochondrial creatine kinase were studied in saponin-skinned fibers taken from normal and M-creatine kinase-deficient mice. In control animals, apparent Km values of mitochondrial respiration for ADP in cardiac (ventricular) and slow-twitch (soleus) muscles (137 +/- 16 microM and 209 +/- 10 microM, respectively) were manyfold higher than that in fast-twitch (gastrocnemius) muscle (7.5 +/- 0.5 microM). Creatine substantially decreased the Km values only in cardiac and slow-twitch muscles (73 +/- 11 microM and 131 +/- 21 microM, respectively). As compared to control, in situ mitochondria in transgenic ventricular and slow-twitch muscles showed two times lower Km values for ADP, and the presence of creatine only slightly decreased the Km values. In mutant fast-twitch muscle, a decrease rather than increase in mitochondrial sensitivity to ADP occurred, but creatine still had no effect. Furthermore, in these muscles, relatively low oxidative capacity was considerably elevated. It is suggested that in the mutant mice, impairment of energy transport function in ventricular and slow-twitch muscles is compensated by a facilitation of adenine nucleotide transportation between mitochondria and cellular ATPases; in fast-twitch muscle, mainly energy buffering function is depressed, and that is overcome by an increase in energy-producing potential.     

Characterization of Toscana virus-defective interfering particles generated in vivo

Adenylate kinase (AK), which catalyzes the equilibrium reaction among AMP, ADP, and ATP, is considered to participate in the homeostasis of energy metabolism in cells. Among three vertebrate isozymes, AK isozyme 1 (AK1) is present prominently in the cytosol of skeletal muscle and brain. When mouse embryonal carcinoma P19 cells were differentiated by retinoic acid into neural cells, the amount of AK1 protein and enzyme activity increased about fivefold concomitantly with neurofilament (NF). Double-immunofluorescence staining showed that both AK1 and NF were located in neuronal processes as well as the perinuclear regions in neuron-like cells, but not in glia-like cells. The amount of brain-type creatine kinase increased only twofold during P19 differentiation. The AK isozyme 2, which was not detected in adult mouse brain, was found in P19 cells and did not increase during the differentiation. Mitochondrial AK isozyme 3, which uses GTP instead of ATP as a phosphate donor, was increased significantly. Immunohistochemical analysis with the primary cultured cells from rat cerebral cortex showed similar cellular localization of AK1 to those observed with differentiated P19 cells. These results suggest an important role of this enzyme in neuronal functions and in neuronal differentiation.     

Metabolic response to halothane in piglets susceptible to malignant hyperthermia: an in vivo 31P-NMR study

Using in vivo 31P-nuclear magnetic resonance spectroscopy, we studied the skeletal muscle metabolism of 17 anesthetized malignant hyperthermia-susceptible piglets and 25 control piglets during and after a halothane stress test. At rest, the phosphocreatine- (PCr) to-ATP ratio was 12% higher in the anesthetized piglets than in the control piglets, which may reflect a higher proportion of fast glycolytic fibers in the former. About 15 min of halothane administration sufficed to provoke onset of a reaction, which was characterized by a reciprocal drop in PCr and an increase in Pi with commencing intracellular acidosis. Halothane was withdrawn after a 20% drop in PCr. Within the next few minutes, intracellular pH dropped sharply and phosphomonoesters (PME) accumulated excessively. ATP was observed to decrease in 8 of the 17 animals. Halothane inhalation provoked a switch of metabolism toward glycolysis. Accumulation of PME suggests a mismatch between glycogenolysis and glycolysis. Despite severe acidification, glycolysis was not completely halted. Recovery of PCr and Pi started approximately 5 min after halothane withdrawal, with a longer time constant for recovery of the former. PME and intracellular pH aberrations lingered and started to recover later. Lost ATP was never restored within the observed recovery period of approximately 20 min.     

Quantitative studies of enzyme-substrate compartmentation, functional coupling and metabolic channelling in muscle cells

Some historical aspects of development of the concepts of functional coupling, metabolic channelling, compartmentation and energy transfer networks are reviewed. Different quantitative approaches, including kinetic and mathematical modeling of energy metabolism, intracellular energy transfer and metabolic regulation of energy production and fluxes in the cells in vivo are analyzed. As an example of the system with metabolic channelling, thermodynamic aspects of the functioning the mitochondrial creatine kinase functionally coupled to the oxidative phosphorylation are considered. The internal thermodynamics of the mitochondrial creatine kinase reaction is similar to that for other isoenzymes of creatine kinase, and the oxidative phosphorylation process specifically influences steps of association and dissociation of MgATP with the enzyme due to channelling of ATP from adenine nucleotide translocase. A new paradigm of muscle bioenergetics-the paradigm of energy transfer and feedback signaling networks based on analysis of compartmentation phenomena and structural and functional interactions in the cell is described. Analysis of the results of mathematical modeling of the compartmentalized energy transfer leads to conclusion that both calcium and ADP, which concentration changes synchronously in contraction cycle, may simultaneously activate oxidative phosphorylation in the muscle cells in vivo. The importance of the phosphocreatine circuit among other pathways of intracellular energy transfer network is discussed on the basis of the recent data published in the literature, with some experimental demonstration. The results of studies of perfused rat hearts with completely inhibited creatine kinase show significantly decreased work capacity and respectively, energy fluxes, in these hearts in spite of significant activation of adenylate kinase system (Dzeja et al. this volume). These results, combined with those of mathematical analysis of the energy metabolism of hearts of transgenic mice with switched off creatine kinase isoenzymes confirm the importance of phosphocreatine pathway for energy transfer for cell function and energetics in mature heart and many other types of cells, as one of major parts of intracellular energy transfer network and metabolic regulation.     

Modification of ischemia and reperfusion damage of skeletal muscles with allopurinol: in vivo 31P MR spectroscopy of the posterior limb of the rat

The effect of Allopurinol on energy metabolism (re-utilisation of hypoxanthine) was studied in a in vivo skeletal muscle ischemia rat model by 31-P-MR spectroscopy. Allopurinol-treatment showed no benefit to the kinetics of PCr/(Pi + PCr) and ATP/(Pi + PCr). The role of re-utilisation of hypoxanthine has to be further investigated.     

Mitochondria and diabetes. Genetic, biochemical, and clinical implications of the cellular energy circuit

Physiologically, a postprandial glucose rise induces metabolic signal sequences that use several steps in common in both the pancreas and peripheral tissues but result in different events due to specialized tissue functions. Glucose transport performed by tissue-specific glucose transporters is, in general, not rate limiting. The next step is phosphorylation of glucose by cell-specific hexokinases. In the beta-cell, glucokinase (or hexokinase IV) is activated upon binding to a pore protein in the outer mitochondrial membrane at contact sites between outer and inner membranes. The same mechanism applies for hexokinase II in skeletal muscle and adipose tissue. The activation of hexokinases depends on a contact site-specific structure of the pore, which is voltage-dependent and influenced by the electric potential of the inner mitochondrial membrane. Mitochondria lacking a membrane potential because of defects in the respiratory chain would thus not be able to increase the glucose-phosphorylating enzyme activity over basal state. Binding and activation of hexokinases to mitochondrial contact sites lead to an acceleration of the formation of both ADP and glucose-6-phosphate (G-6-P). ADP directly enters the mitochondrion and stimulates mitochondrial oxidative phosphorylation. G-6-P is an important intermediate of energy metabolism at the switch position between glycolysis, glycogen synthesis, and the pentose-phosphate shunt. Initiated by blood glucose elevation, mitochondrial oxidative phosphorylation is accelerated in a concerted action coupling glycolysis to mitochondrial metabolism at three different points: first, through NADH transfer to the respiratory chain complex I via the malate/aspartate shuttle; second, by providing FADH2 to complex II through the glycerol-phosphate/dihydroxy-acetone-phosphate cycle; and third, by the action of hexo(gluco)kinases providing ADP for complex V, the ATP synthetase. As cytosolic and mitochondrial isozymes of creatine kinase (CK) are observed in insulinoma cells, the phosphocreatine (CrP) shuttle, working in brain and muscle, may also be involved in signaling glucose-induced insulin secretion in beta-cells. An interplay between the plasma membrane-bound CK and the mitochondrial CK could provide a mechanism to increase ATP locally at the KATP channels, coordinated to the activity of mitochondrial CrP production. Closure of the KATP channels by ATP would lead to an increase of cytosolic and, even more, mitochondrial calcium and finally to insulin secretion. Thus in beta-cells, glucose, via bound glucokinase, stimulates mitochondrial CrP synthesis. The same signaling sequence is used in the opposite direction in muscle during exercise when high ATP turnover increases the creatine level that stimulates mitochondrial ATP synthesis and glucose phosphorylation via hexokinase. Furthermore, this cytosolic/mitochondrial cross-talk is also involved in activation of muscle glycogen synthesis by glucose. The activity of mitochondrially bound hexokinase provides G-6-P and stimulates UTP production through mitochondrial nucleoside diphosphate kinase. Pathophysiologically, there are at least two genetically different forms of diabetes linked to energy metabolism: the first example is one form of maturity-onset diabetes of the young (MODY2), an autosomal dominant disorder caused by point mutations of the glucokinase gene; the second example is several forms of mitochondrial diabetes caused by point and length mutations of the mitochondrial DNA (mtDNA) that encodes several subunits of the respiratory chain complexes. Because the mtDNA is vulnerable and accumulates point and length mutations during aging, it is likely to contribute to the manifestation of some forms of NIDDM.(ABSTRACT TRUNCATED)     

Caffeine counteracts the ergogenic action of muscle creatine loading

This study aimed to compare the effects of oral creatine (Cr) supplementation with creatine supplementation in combination with caffeine (Cr+C) on muscle phosphocreatine (PCr) level and performance in healthy male volunteers (n = 9). Before and after 6 days of placebo, Cr (0.5 g x kg-1 x day-1), or Cr (0.5 g x kg-1 x day-1) + C (5 mg x kg-1 x day-1) supplementation, 31P-nuclear magnetic resonance spectroscopy of the gastrocnemius muscle and a maximal intermittent exercise fatigue test of the knee extensors on an isokinetic dynamometer were performed. The exercise consisted of three consecutive maximal isometric contractions and three interval series of 90, 80, and 50 maximal voluntary contractions performed with a rest interval of 2 min between the series. Muscle ATP concentration remained constant over the three experimental conditions. Cr and Cr+C increased (P < 0.05) muscle PCr concentration by 4-6%. Dynamic torque production, however, was increased by 10-23% (P < 0.05) by Cr but was not changed by Cr+C. Torque improvement during Cr was most prominent immediately after the 2-min rest between the exercise bouts. The data show that Cr supplementation elevates muscle PCr concentration and markedly improves performance during intense intermittent exercise. This ergogenic effect, however, is completely eliminated by caffeine intake.     

Phosphocreatine and creatine kinase in piglet cerebral gray and white matter in situ

Rates of adenosine triphosphate (ATP) metabolism are higher in cerebral gray matter than in white matter. Like other excitable tissues, brain contains a phosphocreatine (PCr)/creatine kinase (CK)/ATP system including cytosolic (B-CK) and mitochondrial (Mi-CK) isozymes. High B-CK activity is present in white and gray matter while Mi-CK is mostly in gray matter. An in situ localizing 31P-NMR technique, one-dimensional chemical shift imaging (1D-CSI), has been used to study the PCr/CK/ATP system in these regions. In the metabolically mature 4-week-old piglet, the PCr/nucleoside triphosphate (NTP) ratio measured by the 1D-CSI technique is at least 50% higher in white than gray matter. Total creatine (Cr), ATP, and total NTP concentrations are the same in rapidly frozen rat white and gray matter, suggesting that PCr/Cr ratio is much higher in white matter. The PCr increases more in gray than white matter between 4 days and 4 weeks of age in piglet brain. The CK catalyzed reaction rate constant, measured by combining the saturation transfer experiment with the 1D-CSI, is also much higher in white than gray matter at both ages. The postnatal maturational increase in the CK rate constant is greater in gray matter. In summary, these differences in PCr concentration and CK reaction rates and isozymes characterize two physiologically different PCr/CK/ATP systems in gray and white matter.     

Contribution of phosphocreatine and aerobic metabolism to energy supply during repeated sprint exercise

This study examined the contribution of phosphocreatine (PCr) and aerobic metabolism during repeated bouts of sprint exercise. Eight male subjects performed two cycle ergometer sprints separated by 4 min of recovery during two separate main trials. Sprint 1 lasted 30 s during both main trials, whereas sprint 2 lasted either 10 or 30 s. Muscle biopsies were obtained at rest, immediately after the first 30-s sprint, after 3.8 min of recovery, and after the second 10- and 30-s sprints. At the end of sprint 1, PCr was 16.9 +/- 1.4% of the resting value, and muscle pH dropped to 6.69 +/- 0.02. After 3.8 min of recovery, muscle pH remained unchanged (6.80 +/- 0.03), but PCr was resynthesized to 78.7 +/- 3.3% of the resting value. PCr during sprint 2 was almost completely utilized in the first 10 s and remained unchanged thereafter. High correlations were found between the percentage of PCr resynthesis and the percentage recovery of power output and pedaling speed during the initial 10 s of sprint 2 (r = 0.84, P < 0.05 and r = 0.91, P < 0.01). The anaerobic ATP turnover, as calculated from changes in ATP, PCr, and lactate, was 235 +/- 9 mmol/kg dry muscle during the first sprint but was decreased to 139 +/- 7 mmol/kg dry muscle during the second 30-s sprint, mainly as a result of a approximately 45% decrease in glycolysis. Despite this approximately 41% reduction in anaerobic energy, the total work done during the second 30-s sprint was reduced by only approximately 18%. This mismatch between anaerobic energy release and power output during sprint 2 was partly compensated for by an increased contribution of aerobic metabolism, as calculated from the increase in oxygen uptake during sprint 2 (2.68 +/- 0.10 vs. 3.17 +/- 0.13 l/min; sprint 1 vs. sprint 2; P < 0.01). These data suggest that aerobic metabolism provides a significant part (approximately 49%) of the energy during the second sprint, whereas PCr availability is important for high power output during the initial 10 s.     

The utilisation of creatine and its analogues by cytosolic and mitochondrial creatine kinase

We have investigated the utilisation of four analogues of creatine by cytosolic Creatine Kinase (CK), using 31P-NMR in the porcine carotid artery, and by mitochondrial CK (Mt-CK), using oxygen consumption studies in isolated heart mitochondria and skinned fibers. Porcine carotid arteries were superfused for 12 h with Krebs-Henseleit buffer at 22 degrees C, containing 11 mM glucose as substrate, and supplemented with either 20 mM beta-guanidinopropionic acid (beta-GPA), methyl-guanidinopropionic acid (m-GPA), guanidinoacetic acid (GA) or cyclocreatine (cCr). All four analogues entered the tissue and became phosphorylated by CK as seen by 31 P-NMR, Inhibition of oxidative metabolism by 1 mM cyanide after accumulation of the phosphorylated analogue resulted in the utilisation of PCr, beta-GPA-P, GA-P and GA-P over a similar time course (approximately 2 h), despite very different kinetic properties of these analogues in vitro. cCr-P was utilised at a significantly slower rate, but was rapidly dephosphorylated in the presence of both 1 mM iodoacetate and cyanide (to inhibit both glycolysis and oxidative metabolism respectively). The technique of creatine stimulated respiration was used to investigate the phosphorylation of the analogues by Mt-CK, Isolated mitochondria were subjected to increasing [ATP], whereas skinned fibres received a similar protocol with increasing [ADP]. There was a significant stimulation of respiration by creatine and cCr in isolated mitochondria (decreased K(m) and increased Vmax vs control), but none by GA, mGPA or beta-GPA (also in skinned fibres), indicating that these latter analogues were not utilised by Mt-CK. These results demonstrate differences in the phosphorylation and dephosphorylation of creatine and its analogues by cytosolic CK and Mt-CK in vivo and in vitro.     

Impaired cardiac energetics in mice lacking muscle-specific isoenzymes of creatine kinase

Our purpose was to determine whether hearts from mice bioengineered to lack either the M isoform of creatine kinase (MCK-/- mice) or both the M and mitochondrial isoforms (M/MtCK-/- mice) have deficits in cardiac contractile function and energetics, which have previously been reported in skeletal muscle from these mice. The phenotype of hearts with deleted creatine kinase (CK) genes is of clinical interest, since heart failure is associated with decreased total CK activity and changes in the relative amounts of the CK isoforms in the heart. We measured isovolumic contractile performance in isolated perfused hearts from wild-type, MCK-/-, and M/MtCK-/- mice simultaneously with cardiac energetics (31P-nuclear magnetic resonance spectroscopy) at baseline, during increased cardiac work, and during recovery. Hearts from wild-type, MCK-/-, and M/MtCK-/- mice had comparable baseline function and responded to 10 minutes of increased heart rate and perfusate Ca2+ with similar increases in rate-pressure product (48+/-5%, 42+/-6%, and 51+/-6%, respectively). Despite a similar contractile response, M/MtCK-/- hearts increased [ADP] by 95%, whereas wild-type and MCK-/- hearts maintained [ADP] at baseline levels. The free energy released from ATP hydrolysis decreased by 3.6 kJ/mol in M/MtCK-/- hearts during increased cardiac work but only slightly in wild-type (1.7 kJ/mol) and MCK-/- (1.5 kJ/mol) hearts. In contrast to what has been reported in skeletal muscle, M/MtCK-/- hearts were able to hydrolyze and resynthesize phosphocreatine. Taken together, our results demonstrate that when CK activity is lowered below a certain level, increases in cardiac work become more "energetically costly" in terms of high-energy phosphate use, accumulation of ADP, and decreases in free energy released from ATP hydrolysis, but not in terms of myocardial oxygen consumption.     

Non-insulin and non-exercise related increase of glucose utilization in rats and mice

The effects of high-energy phosphate contents in muscles on glucose tolerance and glucose uptake into tissues were studied in rats and mice. Enhanced glucose tolerance associated with depleted high-energy phosphates and elevated glycogen content in muscles and liver was observed in animals fed creatine analogue beta-guanidinopropionic acid (beta-GPA). Distribution of infused 2-[1-14C]deoxy-D-glucose in tissues especially in the soleus muscle, kidney, and brain was greater in mice fed beta-GPA than controls. The glucose uptake was decreased when the contents of ATP and glycogen were normalized following creatine supplementation. Plasma insulin in animals at rest was lower and its concentration after intraperitoneal glucose infusion tended to be less in animals fed beta-GPA than controls (p > 0.05), although the pattern of insulin response to glucose loading was similar to the control. The daily voluntary activity in beta-GPA fed mice was also less than controls. These results suggest that improved glucose tolerance is not related to elevated insulin concentration and/or decreased glycogen following exercise. Such improvement may be due to an increased mitochondrial energy metabolism caused by depletion of high-energy phosphates.