Muscle creatine uptake and creatine transporter expression in response to creatine supplementation and depletion.

The total creatine pool size [Cr(total); creatine (Cr) + phosphocreatine (PCr)] is crucial for optimal energy utilization in skeletal muscle, especially at the onset of exercise and during intense contractions. The Cr(total) likely is controlled by long-term modulation of Cr uptake via the sodium-dependent Cr transporter (CrT). To test this hypothesis, adult male Sprague-Dawley rats were fed 1% Cr, their muscle Cr(total) was reduced by approximately 85% [1% beta-guanidinoproprionic acid (beta-GPA)], or their muscle Cr(total) was repleted (1% Cr after beta-GPA depletion). Cr uptake was assessed by skeletal muscle (14)C-Cr accumulation to Cr and PCr by using hindlimb perfusion, and CrT protein content was assessed by Western blot. Cr uptake rate decreased with dietary Cr supplementation in the white gastrocnemius (WG; 45%) only. Depletion of muscle Cr(total) to approximately 15% of normal increased Cr uptake in the soleus (21%) and red gastrocnemius (22%), corresponding to 70-150% increases in muscle CrT content. In contrast, the inherently lower Cr uptake rate in the WG was unchanged with depletion of muscle Cr(total) even though CrT band density was increased by 230%. Thus there was no direct relationship between apparent muscle CrT abundance and Cr uptake rates. However, Cr uptake rates scaled inversely with decreases in muscle Cr(total) in the high-oxidative muscle types but not in the WG. This implies that factors controlling Cr uptake are different among fiber types. These observations may help explain the influence of initial muscle Cr(total), time dependency, and variations in muscle Cr(total) accumulation during Cr supplementation.     

ATP nucleotidylation of creatine kinase.

Creatine kinase (CK) will autoincorporate radiolabel from [gamma32P]ATP and has thus been reported to be autophosphorylated. Also, in contrast to normal brain enzyme, CK in Alzheimer-diseased brain homogenate shows greatly decreased activity, abolished photolabeling with [32P]8N3ATP, and no detectable autoincorporation of radiolabel by [gamma32P]ATP. Surprisingly, our studies with both human brain and purified CK showed that [alpha32P]ATP, [gamma32P]ATP, [alpha32P]ADP, [2,8H3]ATP, [gamma32P]2',3'-O-(2,4, 6-trinitrophenyl)-ATP, and [gamma32P]benzophenone-gammaATP all autoincorporate radiolabel into CK with good efficiency. This demonstrates that the gamma-phosphate and the 2' and 3' hydroxyls are not involved in the covalent linkage and that all three phosphates, the ribose and base of the ATP molecule are retained upon autoincorporation (nucleotidylation). Treatment with NaIO3 to break the 2'-3' linkage effected total loss of radiolabel indicating that nucleotidylation resulted in opening of the ribose ring at the C1' position. Nucleotidylation with increasing [alpha32P]ATP at 37 degrees C gives an approximate k0.5 of 125 microM and saturates at 340 microM nucleotide. Modification of 8-10% of the copy numbers occurs at saturation, and CK activity is inhibited to approximately the same degree. Low micromolar levels of native substrates such as ADP, ATP, and phosphocreatine substantially reduce [alpha32P]ATP nucleotidylation. In contrast, AMP, GTP, GMP, NADH, and creatine did not effectively reduce nucleotidylation. When [alpha32P]ATP-nucleotidylated or [alpha32P]8N3ATP-photolabeled CK is treated with trypsin a single, identical radiolabeled peptide (V279-R291) is generated that comigrates on reverse phase HPLC and Tris-tricine electrophoresis. Nucleotidylation into this peptide was prevented 86% by the presence of ATP. We conclude that CK is nucleotidylated within the active site by modification at the C1'position and that autophosphorylation of this enzyme does not occur.     

Direct antioxidant properties of creatine.

Creatine is the most popular supplement proposed to be an ergogenic aid. There is some evidence in the literature that creatine supplementation increases lean body mass, muscular strength, and sprint power. However, the efficacy of creatine has not been consistent, and the potential mechanisms are unresolved. While limited evidence that suggests that creatine could possess an antioxidant effect this has not been tested directly. Because oxidants such as free radicals can affect muscle fatigue and protein turnover, it is important to know whether creatine can neutralize free radicals and other reactive oxygen species. We tested the hypothesis that creatine would remove superoxide anions (O(*-)(2)), peroxynitrite (OONO-), hydrogen peroxide, and lipid peroxides (t-butyl hydroperoxide). We also determined whether creatine displayed a significant antioxidant scavenging capacity (ASC) using 2,2'-azino-bis(3-ethylbenzothiazolamine-6-sulfonic acid) (ABTS+) quenching as a marker. Creatine did not significantly reduce levels of hydrogen peroxide or lipid peroxidation. In contrast, creatine displayed a significant ability to remove ABTS+, O(*-)(2), and OONO- when compared with controls. Creatine quenching of ABTS+ was less than physiological levels of reduced glutathione (0.375 mM). To our knowledge, this is the first evidence that creatine has the potential to act as a direct antioxidant against aqueous radical and reactive species ions.     

Novel mitochondrial creatine transport activity. Implications for intracellular creatine compartments and bioenergetics

Immunoblotting of isolated mitochondria from rat heart, liver, kidney, and brain with antibodies made against N- and C-terminal peptide sequences of the creatine transporter, together with in situ immunofluorescence staining and immunogold electron microscopy of adult rat myocardium, revealed two highly related polypeptides with molecular masses of approximately 70 and approximately 55 kDa in mitochondria. These polypeptides were localized by immunoblotting of inner and outer mitochondrial membrane fractions, as well as by immunogold labeling in the mitochondrial inner membrane. In addition, a novel creatine uptake via a mitochondrial creatine transport activity was demonstrated by [(14)C]creatine uptake studies with isolated mitochondria from rat liver, heart, and kidney showing a saturable low affinity creatine transporter, which was largely inhibited in a concentration-dependent manner by the sulfhydryl-modifying reagent NEM, as well as by the addition of the above anti-creatine transporter antibodies to partially permeabilized mitochondria. Mitochondrial creatine transport was to a significant part dependent on the energetic state of mitochondria and was inhibited by arginine, and to some extent also by lysine, but not by other creatine analogues and related compounds. The existence of an active creatine uptake mechanism in mitochondria indicates that not only creatine kinase isoenzymes, but also creatine transporters and thus a certain proportion of the creatine kinase substrates, might be subcellularly compartmentalized. Our data suggest that mitochondria, shown here to possess creatine transport activity, may harbor such a creatine/phosphocreatine pool.     

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.     

Monoclonal-antibody studies of creatine kinase. The proteinase K-cleavage site.

Proteinase K cleaves a small peptide from native muscle-specific creatine kinase. We present evidence, from the binding of two monoclonal antibodies to formic acid-cleavage fragments and proteinase K-digest fragments of chick muscle creatine kinase, that the proteinase K-cleavage site is in the C-terminal region of the molecule. This specificity of proteinase K, which is not normally a highly specific enzyme, and the continued association of the two peptide fragments after cleavage suggest an unusual conformational feature in the cleavage-site region. By applying predictive methods for hydrophobicity and secondary structure to an amino acid sequence in this region, we suggest possible structural features at the cleavage site that are evidently conserved across avian and mammalian species. The most likely site is next to, or within, a beta-turn on the surface of the molecule.     

Studies on the stability of creatine kinase isozymes.

Research on the stabilizing properties of creatine kinase isozymes CK-BB, CK-MB, and CK-MM showed that minor alteration of their sequence and structure influenced their stability significantly. An analysis of the stability of the isozymes in storage after freeze drying indicates that creatine kinase isozymes are all in monomer form because of the loss of subunit interactions. Freeze-drying leads to the oxidization of CK-BB and rearrangement of CK-MB. There are also differences in the unfolding of the isozymes in urea. CK-BB and CK-MB are unfolded in lower urea concentrations than CK-MM. Differences in the thermal unfolding were also examined by differential scanning calorimetry. This paper discusses the potential biological significance of these results.     

Effect of temperature on the creatine kinase equilibrium.

The effect of temperature on the apparent equilibrium constant of creatine kinase (ATP:creatine N-phosphotransferase (EC 2.7.3.2)) was determined. At equilibrium the apparent K' for the biochemical reaction was defined as [formula: see text] The symbol sigma denotes the sum of all the ionic and metal complex species of the reactant components in M. The K' at pH 7.0, 1.0 mM free Mg2+, and ionic strength of 0.25 M at experimental conditions was 177 +/- 7.0, 217 +/- 11, 255 +/- 10, and 307 +/- 13 (n = 8) at 38, 25, 15, and 5 degrees C, respectively. The standard apparent enthalpy or heat of the reaction at the specified conditions (delta H' degree) was calculated from a van't Hoff plot of log10K' versus 1/T, and found to be -11.93 kJ mol-1 (-2852 cal mol-1) in the direction of ATP formation. The corresponding standard apparent entropy of the reaction (delta S' degree) was +4.70 J K-1 mol-1. The linear function (r2 = 0.99) between log10 K' and 1/K demonstrates that both delta H' degree and delta S' degree are independent of temperature for the creatine kinase reaction, and that delta Cp' degree, the standard apparent heat capacity of products minus reactants in their standard states, is negligible between 5 and 38 degrees C. We further show from our data that the sign and magnitude of the standard apparent Gibbs energy (delta G' degree) of the creatine kinase reaction was comprised mostly of the enthalpy of the reaction, with 11% coming from the entropy T delta S' degree term. The thermodynamic quantities for the following two reference reactions of creatine kinase were also determined. [formula: see text] The delta H degree for Reaction 2 was -16.73 kJ mol-1 (-3998 cal mol-1) and for Reaction 3 was -23.23 kJ mol-1 (-5552 cal mol-1) over the temperature range 5-38 degrees C. The corresponding delta S degree values for the reactions were +110.43 and +83.49 J K-1 mol-1, respectively. Using the delta H' degree of -11.93 kJ mol-1, and one K' value at one temperature, a second K' at a second temperature can be calculated, thus permitting bioenergetic investigations of organs and tissues using the creatine kinase equilibria over the entire physiological temperature range.     

Shortening of muscle relaxation time after creatine loading.

The effect of creatine (Cr) supplementation on muscle isometric torque generation and relaxation was investigated in healthy male volunteers. Maximal torque (Tmax), contraction time (CT) from 0.25 to 0.75 of Tmax, and relaxation time (RT) from 0.75 to 0.25 of Tmax were measured during 12 maximal isometric 3-s elbow flexions interspersed by 10-s rest intervals. Between the pretest and the posttest, subjects ingested Cr monohydrate (4 x 5 g/day; n = 8) or placebo (n = 8) for 5 days. Pretest Tmax, CT, and RT were similar in Cr and placebo groups. Also in the posttest, Tmax and CT were similar between groups. However, posttest RT was decreased consistently by approximately 20% (P < 0.05) in the Cr group from the first to the last of the 12 contractions. In addition, the mean decrease in RT after Cr loading was positively correlated with pretest RT (r = 0.82). It is concluded that Cr loading facilitates the rate of muscle relaxation during brief isometric muscle contractions without affecting torque production.     

Structural changes of creatine kinase upon substrate binding.

Small-angle x-ray scattering was used to investigate structural changes upon binding of individual substrates or a transition state analog complex (TSAC; Mg-ADP, creatine, and KNO3) to creatine kinase (CK) isoenzymes (dimeric muscle-type (M)-CK and octameric mitochondrial (Mi)-CK) and monomeric arginine kinase (AK). Considerable changes in the shape and the size of the molecules occurred upon binding of Mg-nucleotide or TSAC. The radius of gyration of Mi-CK was reduced from 55.6 A (free enzyme) to 48.9 A (enzyme plus Mg-ATP) and to 48.2 A (enzyme plus TSAC). M-CK showed similar changes from 28.0 A (free enzyme) to 25.6 A (enzyme plus Mg-ATP) and to 25.5 A (enzyme plus TSAC). Creatine alone did not lead to significant changes in the radii of gyration, nor did free ATP or ADP. AK also showed a change of the radius of gyration from 21.5 A (free enzyme) to 19.7 A (enzyme plus Mg-ATP), whereas with arginine alone only a minor change could be observed. The primary change in structure as seen with monomeric AK seems to be a Mg-nucleotide-induced domain movement relative to each other, whereas the effect of substrate may be of local order only. In CK, however, additional movements have to be involved.     

Two-state irreversible thermal denaturation of muscle creatine kinase.

Thermal denaturation of creatine kinase from rabbit skeletal muscle has been studied by differential scanning calorimetry. The excess heat capacity vs. temperature profiles were independent of protein concentration, but strongly temperature scanning rate-dependent. It has been shown that thermal denaturation of creatine kinase satisfies the previously proposed validity criteria for the two-state irreversible model [Kurganov et al., Biophys. Chem.70 (1997) 125]. The energy activation value has been calculated to be 461.0 +/- 0.7 kJ/mol.     

Effect of creatine loading on neuromuscular fatigue threshold.

The purpose of this investigation was to determine the effect of creatine (Cr) loading on the onset of neuromuscular fatigue by monitoring electromyographic fatigue curves from the vastus lateralis muscle using the physical working capacity at the fatigue threshold (PWC(FT)) test. Using a double-blind random design, 15 women athletes [mean age 19.0 +/- 2.0 (SD) yr] from the university crew team received a placebo (n = 8; 20 g glucose) or Cr (n = 7; 5 g Cr monohydrate + 20 g glucose) four times per day for 5 consecutive days. Analysis of covariance was used to analyze the data (covaried for presupplementation PWC(FT) values). The adjusted mean postsupplementation PWC(FT) value for the Cr group (mean = 186 W) was significantly (P < 0.05) higher than that of the placebo group (mean = 155 W). These findings suggest that Cr loading may delay the onset of neuromuscular fatigue.