Creatine uptake and creatine transporter expression among rat skeletal muscle fiber types

Total creatine (Cr(total) = phosphocreatine + creatine) concentrations differ substantially among mammalian skeletal muscle. Because the primary means to add Cr(total) to muscle is uptake of creatine through the sodium-dependent creatine transporter (CrT), differences in creatine uptake and CrT expression could account for the variations in [Cr(total)] among muscle fiber types. To test this hypothesis, hindlimbs of adult rats were perfused with 0.05-1 mM [(14)C]creatine for up to 90 min. Creatine uptake rates at 1 mM creatine were greatest in the soleus (140 +/- 8.8 nmol x h(-1) x g(-1)), less in the red gastrocnemius (117 +/- 8.3), and least in the white gastrocnemius (97 +/- 10.7). These rates were unaltered by time, insulin concentration, or increased perfusate sodium concentration. Conversely, creatine uptake rates were correspondingly decreased among fiber types by lower creatine and sodium concentrations. The CrT protein content by Western blot analysis was similarly greatest in the soleus, less in the red gastrocnemius, and least in the white gastrocnemius, whereas CrT mRNA was not different. Creatine uptake rates differ among skeletal muscle fiber sections in a manner reasonably assigned to the 58-kDa band of the CrT. Furthermore, creatine uptake rates scale inversely with creatine content, with the lowest uptake rate in the fiber type with the highest Cr(total) and vice versa. This suggests that the creatine pool fractional turnover rate is not common across muscle phenotypes and, therefore, is differentially regulated.     

Creatine transporter activity and content in the rat heart supplemented by and depleted of creatine

The intracellular creatine concentration is an important bioenergetic parameter in cardiac muscle. Although creatine uptake is known to be via a NaCl-dependent creatine transporter (CrT), its localization and regulation are poorly understood. We investigated CrT kinetics in isolated perfused hearts and, by using cardiomyocytes, measured CrT content at the plasma membrane or in total lysates. Rats were fed control diet or diet supplemented with creatine or the creatine analog beta-guanidinopropionic acid (beta-GPA). Creatine transport in control hearts followed saturation kinetics with a K(m) of 70 +/- 13 mM and a V(max) of 3.7 +/- 0.07 nmol x min(-1) x g wet wt(-1). Creatine supplementation significantly decreased the V(max) of the CrT (2.7 +/- 0.17 nmol x min(-1) x g wet wt(-1)). This was matched by an approximately 35% decrease in the plasma membrane CrT; the total CrT pool was unchanged. Rats fed beta-GPA exhibited a >80% decrease in tissue creatine and increase in beta-GPA(total). The V(max) of the CrT was increased (6.0 +/- 0.25 nmol x min(-1) x g wet wt(-1)) and the K(m) decreased (39.8 +/- 3.0 mM). The plasma membrane CrT increased about fivefold, whereas the total CrT pool remained unchanged. We conclude that, in heart, creatine transport is determined by the content of a plasma membrane isoform of the CrT but not by the total cellular CrT pool.     

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.

     

Hypoxic pulmonary vasoconstriction is unaltered by creatine depletion induced by dietary beta-guanidino propionic acid

It has been suggested that a specific phosphagen pool might serve a sensor function, allowing direct detection of alveolar hypoxia by the pulmonary vascular smooth muscle. The possibility that phosphocreatine (PCr) levels could serve as such a sensor was assessed in isolated rat lungs. Pulmonary vascular reactivity to angiotensin II and alveolar hypoxia was assessed in lungs from control and PCr-depleted rats. PCr depletion was accomplished by feeding rats a diet containing 2% beta-guanidino propionic acid (beta-GPA), an competitive inhibitor of creatine uptake. Total creatine was depleted in beta-GPA lungs, compared to control lungs (p less than 0.05). Lung PCr levels were undetectable by the available 31P NMR spectroscopy system. PCr and creatine were depleted in hearts from beta-GPA rats relative to control hearts (p less than 0.001). Normoxic pulmonary artery pressure and the pressor responses to angiotensin II and hypoxia were not qualitatively or quantitatively altered by the diet indicating either that PCr is not a critical participant in hypoxic pulmonary vasoconstriction or that the degree of PCr depletion achieved was inadequate to expose its role in the hypoxic pressor response.     

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.     

PGC-1α and PGC-1β increase CrT expression and creatine uptake in myotubes via ERRα

Intramuscular creatine plays a crucial role in maintaining skeletal muscle energy homeostasis, and its entry into the cell is dependent upon the sodium chloride dependent Creatine Transporter (CrT; Slc6a8). CrT activity is regulated by a number of factors including extra- and intracellular creatine concentrations, hormones, changes in sodium concentration, and kinase activity, however very little is known about the regulation of CrT gene expression. The present study aimed to investigate how Creatine Transporter (CrT) gene expression is regulated in skeletal muscle. Within the first intron of the CrT gene, we identified a conserved sequence that includes the motif recognized by the Estrogen-related receptor α (ERRα), also known as an Estrogen-related receptor response element (ERRE). Additional ERREs confirming to the known consensus sequence were also identified in the region upstream of the promoter. When partnered with peroxisome proliferator-activated receptor-gamma co-activator-1alpha (PGC-1α) or beta (PGC-1β), ERRα induces the expression of many genes important for cellular bioenergetics. We therefore hypothesized that PGC-1 and ERRα could also regulate CrT gene expression and creatine uptake in skeletal muscle. Here we show that adenoviral overexpression of PGC-1α or PGC-1β in L6 myotubes increased CrT mRNA (2.1 and 1.7-fold, P < 0.0125) and creatine uptake (1.8 and 1.6-fold, P < 0.0125), and this effect was inhibited with co-expression of shRNA for ERRα. Overexpression of a constitutively active ERRα (VP16-ERRα) increased CrT mRNA approximately 8-fold (P < 0.05), resulting in a 2.2-fold (P < 0.05) increase in creatine uptake. Lastly, chromatin immunoprecipitation assays revealed that PGC-1α and ERRα directly interact with the CrT gene and increase CrT gene expression.     

Phosphocreatine content of freeze-clamped muscle: influence of creatine kinase inhibition.

The study of cellular energetics is critically dependent on accurate measurement of high-energy phosphates. Muscle values of phosphocreatine (PCr) vary greatly between in vivo measurements (i.e., by nuclear magnetic resonance) and chemical measurements determined from muscles isolated and quick-frozen. The source of this difference has not been experimentally identified. A likely cause is activation of ATPases and phosphotransfer from PCr to ADP. Therefore, rat hindlimb skeletal muscle was perfused either with or without 2 mM iodoacetamide, a creatine kinase inhibitor, and muscle was freeze-clamped either at rest or after contraction. Creatine kinase inhibition resulted in approximately 6 micromol/g higher PCr and lower creatine in the freeze-clamped soleus, red gastrocnemius, and white gastrocnemius. This PCr content difference was reduced when the initial PCr content was decreased with prior contractions. Therefore, the amount of PCr artifact appears to scale with initial PCr content within a fiber-type section. This artifact directly affects the measurement and, thus, the calculations of muscle energetic parameters from studies using isolated and frozen muscle.     

Reduced growth of Ehrlich ascites tumor cells in creatine depleted mice fed beta-guanidinopropionic acid

The effect of implantation of Ehrlich ascites tumor (EAT) cells on creatine distribution was investigated. It was also studied how depletion of creatine by feeding creatine-analogue beta-guanidinopropionic acid (beta-GPA) affects the growth of EAT cells in mice. Enhanced mobilization of creatine from host tissues to EAT cells against a greater concentration gradient was observed. The creatine (but not creatinine) level in blood plasma was lowered to 22% of the normal value by beta-GPA feeding alone and assimilation of 14C-creatine into EAT cells was inhibited. The growth of EAT cells was significantly reduced and the duration of survival of mice after implantation of EAT cells was extended when the creatine concentration was decreased. A decrease in daily food consumption and the degree of muscle atrophy after implantation of EAT cells was less in beta-GPA than control groups. In the creatine-depleted mice, the rate of increase in total EAT cell number and the volume of abdominal ascites were approximately half of the control values, and more dead EAT cells were observed. These results suggest that supplementation of beta-GPA inhibits creatine transfer to EAT cells and reduces the growth of cancer cells.     

The effect of creatine supplementation on glucose uptake in rat skeletal muscle

Glucose transport in muscle is a function of the muscle metabolic state, as evidenced by the increase in glucose transport which occurs with conditions of altered aerobic metabolism such as hypoxia or contractile activity. The energy state of the muscle can be determined by the muscle phosphocreatine concentration. Dietary supplementation of creatine has been shown to increase both phosphocreatine (PCr) and creatine (TCr) levels in muscle, although not in the same proportion, so that the PCr/TCr ratio falls suggesting an altered energy state in the cell. The purpose of this study was to determine the effect of increased creatine content on glucose uptake in muscle. PCr and TCr were determined in plantaris muscles from rats following five weeks of dietary supplementation of creatine monohydrate (300 mg/kg/day). (3)H-2-deoxyglucose uptake was measured in epitrochlearis muscles incubated in the presence or absence of a maximally stimulating dose of insulin. Despite a significant increase in creatine content in muscle, neither basal nor insulin-stimulated glucose uptake was altered in creatine supplemented rats. Since PCr levels were not increased with creatine supplementation, these results suggest that the actual concentration of PCr is a more important determinant of glucose uptake than the PCr/TCr ratio.     

Effect of a short-term dietary creatine supplementation on high-energy phosphates in the rat myocardium.

The aim of this study was to find out whether creatine (Cr) feeding affects total creatine (TCr), phosphocreatine (PCr), adenine nucleotide contents and beta-hydroxy-acyl-CoA-dehydrogenase (HAD) activity in myocardium as compared to red skeletal muscle. Ten adult Wistar rats received Cr (2.5% of diet weight) for 7 days. In Cr fed rats, PCr was increased (by approx. 20%) in cardiac and in soleus muscles with ATP elevated in myocardium and TCr and free Cr in soleus. In both muscles, Cr feeding enhanced HAD activity. It is concluded, that dietary Cr does increase cardiac muscle high energy phosphate reserves and its oxidative potential.     

Acute creatine monohydrate supplementation: a descriptive physiological profile of responders vs. nonresponders

The purpose of this study was to describe the physiological profile of responders (>20 mmol.kg(-1) dry weight [dw] increase in total intramuscular creatine monohydrate [Cr] + phosphorylated creatine [PCr]) versus nonresponders (<10 mmol.kg(-1) dw increase) to a 5-day Cr load (0.3 g.kg(-1).d(-1)) in 11 healthy men (mean age = 22.7 years). Pre-post 5-day cellular measures included total resting Cr content (Cr + PCr), fiber type composition, and fiber type cross-sectional area (CSA) determined from muscle biopsies of the vastus lateralis. Body mass, daily dietary intake, 24-hour urine outputs, urinary Cr and creatinine (CrN), and strength performance measures (1 repetition maximum [1RM] bench and leg press) were also assessed before and after the 5-day loading period. Results indicated that there were 3 levels of response to the 5-day supplementation: responders (R), quasi responders (QR), and nonresponders (NR) with mean changes in resting Cr + PCr of 29.5 mmol.kg(-1) dw (n = 3), 14.9 mmol.kg(-1) dw (n = 5), and 5.1 mmol.kg(-1) dw (n = 3), respectively. The results support a person-by-treatment interaction to acute Cr supplementation with R possessing a biological profile of lowest initial levels of Cr + PCr, greatest percentage of type II fibers, and greatest preload muscle fiber CSA and fat-free mass. Responders also showed improvement in 1RM leg press scores following the 5-day loading period. NR had higher preload levels of Cr + PCr, less type II muscle fibers, small preload muscle CSA, and lower fat-free mass and displayed no improvements in 1RM strength scores. The results suggest that to be considered a responder to acute oral supplementation, a favorable preexisting biological profile may determine the final extent to which an individual responds to supplementation. Physiologic profiles of nonresponders appear to be different and may limit their ability to uptake Cr. This may help partially explain the reported equivocal performance findings in the Cr supplementation literature.     

Adaptation of rat skeletal muscle to creatine depletion: AMP deaminase and AMP deamination.

AMP deaminase catalyzes deamination of the AMP formed in contracting muscles to inosine 5'-monophosphate (IMP). Slow-twitch muscle has only approximately 30% as high a level of AMP deaminase activity as fast-twitch muscle in the rat, and rates of IMP formation during intense contractile activity are much lower in slow-twitch muscle. We found that feeding the creatine analogue beta-guanidinopropionic acid (beta-GPA) to rats, which results in creatine depletion, causes a large decrease in muscle AMP deaminase. This adaptation was used to evaluate the role of AMP deaminase activity level in accounting for differences in IMP production in slow-twitch and fast-twitch muscles. beta-GPA feeding for 3 wk lowered AMP deaminase activity in fast-twitch epitrochlearis muscle to a level similar to that found in the normal slow-twitch soleus muscle but had no effect on the magnitude of the increase in IMP in response to intense contractile activity. Despite a similar decrease in ATP in the normal soleus and the epitrochlearis from beta-GPA-fed rats, the increase in IMP was only approximately 30% as great in the soleus in response to intense contractile activity. These results demonstrate that the accumulation of less IMP in slow- compared with fast-twitch skeletal muscle during contractile activity is not due to the lower level of AMP deaminase in slow-twitch muscle.     

Changes in creatine transporter function during cardiac maturation in the rat

Background  

It is well established that the immature myocardium preferentially utilises non-oxidative energy-generating pathways. It exhibits low energy-transfer capacity via the creatine kinase (CK) shuttle, reflected in phosphocreatine (PCr), total creatine and CK levels that are much lower than those of adult myocardium. The mechanisms leading to gradually increasing energy transfer capacity during maturation are poorly understood. Creatine is not synthesised in the heart, but taken up exclusively by the action of the creatine transporter protein (CrT). To determine whether this transporter is ontogenically regulated, the present study serially examined CrT gene expression pattern, together with creatine uptake kinetics and resulting myocardial creatine levels, in rats over the first 80 days of age.     

Presence of normal creatine in the muscle of a patient with a mutation in the creatine transporter: A case study

To date, more than seven families have been reported who carry a mutation in the X-linked creatine-transporter (CrT) gene. The resulting lack of creatine in the brain is associated with mental retardation, severe expressive language disorder, mild epilepsy, and a complete absence of Cr in the brain (measured using MRS). Conversely, these patients had no observable cardiac or musculo-skeletal deficits. In this case study, a 22-year-old patient underwent surgical repair for scoliosis. Proton MRS of this patient's brain demonstrated the near-absence of creatine and phosphocreatine within the cerebral white and deep gray matter structures. Cerebral atrophy was noted with serial MRI examinations. Subsequent genetic and metabolic analysis showed some biochemical anomalies consistent with a CrT deficiency. The mutation in this patient was identified as a deletion at phenylalanine 107 (delF107). Control muscle biopsies were obtained from archived samples, which had been taken with informed consent during routine muscle biopsies for diagnostic purposes. We determined that the total Cr concentration in the skeletal muscle biopsy was 39.3 +/- 2.94 mmol/kg wet wt., which is not significantly different from non-CrT controls, n = 3 (43.3 +/- 3.57 mmol/kg wet wt.). We conclude that the brain appears to lack the ability to transport creatine when there is a mutation in the CrT gene. However, the muscle utilizes another mechanism for maintaining normal creatine levels. Identifying this alternative creatine-transport mechanism may be useful in treating the neurologic and cognitive impairments of patients with creatine-transporter deficiency.     

Cerebral energetic effects of creatine supplementation in humans

There has been considerable interest in the use of creatine (Cr) supplementation to treat neurological disorders. However, in contrast to muscle physiology, there are relatively few studies of creatine supplementation in the brain. In this report, we use high-field MR (31)P and (1)H spectroscopic imaging of human brain with a 7-day protocol of oral Cr supplementation to examine its effects on cerebral energetics (phosphocreatine, PCr; ATP) and mitochondrial metabolism (N-acetyl aspartate, NAA; and Cr). We find an increased ratio of PCr/ATP (day 0, 0.80 +/- 0.10; day 7, 0.85 +/- 09), with this change largely due to decreased ATP, from 2.7 +/- 0.3 mM to 2.5 +/- 0.3 mM. The ratio of NAA/Cr also decreased (day 0, 1.32 +/- 0.17; day 7 1.18 +/- 0.13), primarily from increased Cr (9.6 +/- 1.9 to 10.1 +/- 2.0 mM). The Cr-induced changes significantly correlated with the basal state, with the fractional increase in PCr/ATP negatively correlating with the basal PCr/ATP value (R = -0.74, P < 0.001). As NAA is a measure of mitochondrial function, there was also a significant negative correlation between basal NAA concentrations with the fractional change in PCr and ATP. Thus healthy human brain energetics is malleable and shifts with 7 days of Cr supplementation, with the regions of initially low PCr showing the largest increments in PCr. Overall, Cr supplementation appears to improve high-energy phosphate turnover in healthy brain and can result in either a decrease or an increase in high-energy phosphate concentrations.     

Changes of tissue creatine concentrations upon oral supplementation of creatine-monohydrate in various animal species

Creatine is a nutritional supplement with major application as ergogenic and neuroprotective substrate. Varying supplementation protocols differing in dosage and duration have been applied but systematic studies of total creatine (creatine and phosphocreatine) content in the various organs of interest are lacking. We investigated changes of total creatine concentrations in brain, muscle, heart, kidney, liver, lung and venous/portal plasma of guinea pigs, mice and rats in response to 2-8 weeks oral creatine-monohydrate supplementation (1.3-2 g/kg/d; 1.4-2.8% of dietary intake). Analysis of creatine and phosphocreatine content was performed by high performance liquid chromatography. Total creatine was determined as the sum of creatine and phosphocreatine. Presupplementation total creatine concentrations were high in brain, skeletal and heart muscle (10-22 micromol/g wet weight), and low in liver, kidney and lung (5-8 micromol/g wet weight). During creatine supplementation, the relative increase of total creatine was low (15-55% of presupplementation values) in organs with high presupplementation concentrations, and high (260-500% of presupplementation values) in organs with low presupplementation concentrations. The increase of total creatine concentrations was most pronounced after 4 weeks of supplementation. In muscle, brain, kidney and lungs, an additional increase (p<0.01) was observed between 2-4 and 2-8 weeks of supplementation. Absolute concentrations of phosphocreatine increased, but there was no increase of the relative (percentual) proportion of phosphocreatine (14-45%) during supplementation. Statistical comparison of total creatine concentrations across the species revealed no systematically differences in organ distribution and in time points of supplementation. Results suggest that in organs with low presupplementation creatine levels (liver, kidney), a major determinant of creatine uptake is an extra-intracellular concentration gradient. In organs with high presupplementation total creatine levels like brain, skeletal and heart muscle, the maximum capacity of creatine accumulation is low compared to other organs. A supplementation period of 2 to 4 weeks is necessary for significant augmentation of the creatine pool in these organs.     

An (1)H-MRS evaluation of the phosphocreatine/creatine pool (tCr) in human muscle

The human gastrocnemius was examined with and without creatine supplementation under the conditions of rest, ischemic fatigue (IF), and recovery to perturb the pool sizes and equilibrium between phosphocreatine (PCr) and creatine (Cr). (1)H- and (31)P-magnetic resonance spectroscopy (MRS) were used to examine the total creatine (tCr) pool in each of the metabolic states. (31)P-MRS monitored the depletion of the PCr peak during IF to <5% of that at rest. (1)H-MRS focused on the tCr methyl peak at 3.02 ppm (dipolar coupled triplet), at which point it was expected that the triplet peak intensity would be similar both in IF and rest. Initial (1)H-MRS data showed the peak intensity during IF decreased, suggesting a change in tCr pool size. Subsequent studies of transverse relaxation time (T(2)) revealed that this decline was primarily due to a more rapid T(2) decay of the tCr peak in IF (T(2) approximately 40 ms) compared with at rest (T(2) approximately 162 ms). Because Cr is the major contributor to tCr in IF, it is possible that there is a pool of Cr displaying reduced mobility in vivo. Moreover, the residual dipolar coupled triplet observed at rest collapsed into a broad singlet during IF, suggestive of significant changes in the ordered environment experienced at rest for PCr compared with when it is converted to Cr during IF. In addition, these data suggest that in (1)H-MRS studies whose goals include quantitative estimates of tCr pool sizes, standardized metabolic conditions or careful T(2) evaluations will be required.     

The creatine kinase system and pleiotropic effects of creatine

The pleiotropic effects of creatine (Cr) are based mostly on the functions of the enzyme creatine kinase (CK) and its high-energy product phosphocreatine (PCr). Multidisciplinary studies have established molecular, cellular, organ and somatic functions of the CK/PCr system, in particular for cells and tissues with high and intermittent energy fluctuations. These studies include tissue-specific expression and subcellular localization of CK isoforms, high-resolution molecular structures and structure–function relationships, transgenic CK abrogation and reverse genetic approaches. Three energy-related physiological principles emerge, namely that the CK/PCr systems functions as (a) an immediately available temporal energy buffer, (b) a spatial energy buffer or intracellular energy transport system (the CK/PCr energy shuttle or circuit) and (c) a metabolic regulator. The CK/PCr energy shuttle connects sites of ATP production (glycolysis and mitochondrial oxidative phosphorylation) with subcellular sites of ATP utilization (ATPases). Thus, diffusion limitations of ADP and ATP are overcome by PCr/Cr shuttling, as most clearly seen in polar cells such as spermatozoa, retina photoreceptor cells and sensory hair bundles of the inner ear. The CK/PCr system relies on the close exchange of substrates and products between CK isoforms and ATP-generating or -consuming processes. Mitochondrial CK in the mitochondrial outer compartment, for example, is tightly coupled to ATP export via adenine nucleotide transporter or carrier (ANT) and thus ATP-synthesis and respiratory chain activity, releasing PCr into the cytosol. This coupling also reduces formation of reactive oxygen species (ROS) and inhibits mitochondrial permeability transition, an early event in apoptosis. Cr itself may also act as a direct and/or indirect anti-oxidant, while PCr can interact with and protect cellular membranes. Collectively, these factors may well explain the beneficial effects of Cr supplementation. The stimulating effects of Cr for muscle and bone growth and maintenance, and especially in neuroprotection, are now recognized and the first clinical studies are underway. Novel socio-economically relevant applications of Cr supplementation are emerging, e.g. for senior people, intensive care units and dialysis patients, who are notoriously Cr-depleted. Also, Cr will likely be beneficial for the healthy development of premature infants, who after separation from the placenta depend on external Cr. Cr supplementation of pregnant and lactating women, as well as of babies and infants are likely to be of benefit for child development. Last but not least, Cr harbours a global ecological potential as an additive for animal feed, replacing meat- and fish meal for animal (poultry and swine) and fish aqua farming. This may help to alleviate human starvation and at the same time prevent over-fishing of oceans.     

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.     

Effect of creatine supplementation during high resistance training on mass,strength, and fatigue resistance in rat skeletal muscle

The aim of this study was to utilize a rodent model of resistance exercise to compare training with creatine supplementation with training alone. We tested the hypothesis that creatine supplementation during high resistance training would result in greater increases in muscle mass, contractile force, and superior resistance to fatigue compared with training alone. Two groups of rats underwent training of the tibialis anterior muscle (TA) for 4 weeks without creatine (NCr group) or with creatine (0.5 g.kg(-1).d(-1)) (CrT group). The relative loads in each animal were held constant during the training protocol. Training resulted in comparable significant increases in muscle contractile force in both the NCr and CrT groups. Creatine supplementation did not result in a significant increase in fatigue resistance and resulted in a significant decrease in postfatigue recovery compared with training alone. Training resulted in a significant increase in muscle dry weight in both groups, whereas muscle wet weight gains in the CrT group were double the gains in the NCr group. The data from this study suggest that for creatine to have a beneficial effect on muscle strength and mass beyond training alone, the workloads need to be adjusted. That is, any potential benefit of creatine to enable a greater lifting volume during resistance training needs to be incorporated into the training regime for creatine to be effective.