Robyn M. Murphy

Creatine and the creatine transporter: A review

The cellular role of creatine (Cr) and Cr phosphate (CrP) has been studied extensively in neural, cardiac and skeletal muscle. Several studies have demonstrated that alterations in the cellular total Cr (Cr + CrP) concentration in these tissues can produce marked functional and/or structural change. The primary aim of this review was to critically evaluate the literature that has examined the regulation of cellular total Cr content. In particular, the review focuses on the regulation of the activity and gene expression of the Cr transporter (CreaT), which is primarily responsible for cellular Cr uptake. Two CreaT genes (CreaT1 and CreaT2) have been identified and their chromosomal location and DNA sequencing have been completed. From these data, putative structures of the CreaT proteins have been formulated. Transcription products of the CreaT2 gene are expressed exclusively in the testes, whereas CreaT1 transcripts are found in a variety of tissues. Recent research has measured the expression of the CreaT1 protein in several tissues including neural, cardiac and skeletal muscle. There is very little information available about the factors regulating CreaT gene expression. There is some evidence that suggests the intracellular Cr concentration may be involved in the regulatory process but there is much more to learn before this process is understood. The activity of the CreaT protein is controlled by many factors. These include substrate concentration, transmembrane Na+ gradients, cellular location, and various hormones. It is also likely that transporter activity is influenced by its phosphorylation state and by its interaction with other plasma membrane proteins. The extent of CreaT protein glycosylation may vary within cells, the functional significance of which remains unclear.

Creatine supplementation increases glycogen storage but not GLUT-4 expression in human skeletal muscle

It has been speculated that creatine supplementation affects muscle glucose metabolism in humans by increasing muscle glycogen storage and up-regulating GLUT-4 protein expression. In the present study, we assessed the effects of creatine loading and prolonged supplementation on muscle glycogen storage and GLUT-4 mRNA and protein content in humans. A total of 20 subjects participated in a 6-week supplementation period during which creatine or a placebo was ingested. Muscle biopsies were taken before and after 5 days of creatine loading (20 g.day(-1)) and after 6 weeks of continued supplementation (2 g.day(-1)). Fasting plasma insulin concentrations, muscle creatine, glycogen and GLUT-4 protein content as well as GLUT-4, glycogen synthase-1 (GS-1) and glycogenin-1 (Gln-1) mRNA expression were determined. Creatine loading significantly increased total creatine, free creatine and creatine phosphate content with a concomitant 18 +/- 5% increase in muscle glycogen content (P<0.05). The subsequent use of a 2 g.day(-1) maintenance dose for 37 days did not maintain total creatine, creatine phosphate and glycogen content at the elevated levels. The initial increase in muscle glycogen accumulation could not be explained by an increase in fasting plasma insulin concentration, muscle GLUT-4 mRNA and/or protein content. In addition, neither muscle GS-1 nor Gln-1 mRNA expression was affected. We conclude that creatine ingestion itself stimulates muscle glycogen storage, but does not affect muscle GLUT-4 expression.

Creatine transporters: A reappraisal

Creatine (Cr) plays a key role in cellular energy metabolism and is found at high concentrations in metabolically active cells such as skeletal muscle and neurons. These, and a variety of other cells, take up Cr from the extra cellular fluid by a high affinity Na+/Cl−-dependent creatine transporter (CrT). Mutations in the crt gene, found in several patients, lead to severe retardation of speech and mental development, accompanied by the absence of Cr in the brain.In order to characterize CrT protein(s) on a biochemical level, antibodies were raised against synthetic peptides derived from the N- and C-terminal cDNA sequences of the putative CrT-1 protein. In total homogenates of various tissues, both antibodies, directed against these different epitopes, recognize the same two major polypetides on Western blots with apparent Mr of 70 and 55 kDa. The C-terminal CrT antibody (α-CrTCOOH) immunologically reacts with proteins located at the inner membrane of mitochondria as determined by immuno-electron microscopy, as well as by subfractionation of mitochondria. Cr-uptake experiments with isolated mitochondria showed these organelles were able to transport Cr via a sulfhydryl-reagent-sensitive transporter that could be blocked by anti-CrT antibodies when the outer mitochondrial membrane was permeabilized. We concluded that mitochondria are able to specifically take-up Cr from the cytosol, via a low-affinity CrT, and that the above polypeptides would likely represent mitochondrial CrT(s). However, by mass spectrometry techniques, the immunologically reactive proteins, detected by our anti-CrT antibodies, were identified as E2 components of the α-keto acid dehydrogenase multi enzyme complexes, namely pyruvate dehydrogenase (PDH), branched chain keto acid dehydrogenase (BC-KADH) and α-ketoglutarate dehydrogenase (α-KGDH). The E2 components of PDH are membrane associated, whilst it would be expected that a mitochondrial CrT would be a transmembrane protein. Results of phase partitioning by Triton X-114, as well as washing of mitochondrial membranes at basic pH, support that these immunologically cross-reactive proteins are, as expected for E2 components, membrane associated rather than transmembrane. On the other hand, the fact that mitochondrial Cr uptake into intact mitoplast could be blocked by our α-CrTCOOH antibodies, indicate that our antisera contain antibodies reactive to proteins involved in mitochondrial transport of Cr. The presence of specific antibodies against CrT is also supported by results from plasma membrane vesicles isolated from human and rat skeletal muscle, where both 55 and 70 kDa polypeptides disappeared and a single polypeptide with an apparent electrophoretic mobility of ~ 60 kDa was enriched This latter is most likely representing the genuine plasma membrane CrT.Due to the fact that all anti-CrT antibodies that were independently prepared by several laboratories seem to cross-react with non-CrT polypeptides, specifically with E2 components of mitochondrial dehydrogenases, further research is required to characterise on a biochemical/biophysical level the CrT polypeptides, e.g. to determine whether the ~ 60 kDa polypeptide is indeed a bona-fide CrT and to identify the mitochondrial transporter that is able to facilitate Cr-uptake into these organelles. Therefore, the anti-CrT antibodies available so far should only be used with these precautions in mind. This holds especially true for quantitation of CrT polypeptides by Western blots, e.g. when trying to answer whether CrTs are up- or down-regulated by certain experimental interventions or under pathological conditions.In conclusion, we still hold to the scheme that besides the high-affinity and high-efficiency plasmalemma CrT there exists an additional low affinity high Km Cr uptake mechanism in mitochondria. However, the exact biochemical nature of this mitochondrial creatine transport, still remains elusive. Finally, similar to the creatine kinase (CK) isoenzymes, which are specifically located at different cellular compartments, also the substrates of CK are compartmentalized in cytosolic and mitochondrial pools. This is in line with 14C-Cr-isotope tracer studies and a number of [31P]-NMR magnetization transfer studies, as well as with recent [1H]-NMR spectroscopy data.

Intense exercise up-regulates Na+,K+-ATPase isoform mRNA, but not protein expression in human skeletal muscle.

Characterization of expression of, and consequently also the acute exercise effects on, Na+,K+‐ATPase isoforms in human skeletal muscle remains incomplete and was therefore investigated. Fifteen healthy subjects (eight males, seven females) performed fatiguing, knee extensor exercise at ∼40% of their maximal work output per contraction. A vastus lateralis muscle biopsy was taken at rest, fatigue and 3 and 24 h postexercise, and analysed for Na+,K+‐ATPase α1, α2, α3, β1, β2 and β3 mRNA and crude homogenate protein expression, using Real‐Time RT‐PCR and immunoblotting, respectively. Each individual expressed gene transcripts and protein bands for each Na+,K+‐ATPase isoform. Each isoform was also expressed in a primary human skeletal muscle cell culture. Intense exercise (352 ± 69 s; mean ±s.e.m.) immediately increased α3 and β2 mRNA by 2.4‐ and 1.7‐fold, respectively (P < 0.05), whilst α1 and α2 mRNA were increased by 2.5‐ and 3.5‐fold at 24 h and 3 h postexercise, respectively (P < 0.05). No significant change occurred for β1 and β3 mRNA, reflecting variable time‐dependent responses. When the average postexercise value was contrasted to rest, mRNA increased for α1, α2, α3, β1, β2 and β3 isoforms, by 1.4‐, 2.2‐, 1.4‐, 1.1‐, 1.0‐ and 1.0‐fold, respectively (P < 0.05). However, exercise did not alter the protein abundance of the α1–α3 and β1–β3 isoforms. Thus, human skeletal muscle expresses each of the Na+,K+‐ATPase α1, α2, α3, β1, β2 and β3 isoforms, evidenced at both transcription and protein levels. Whilst brief exercise increased Na+,K+‐ATPase isoform mRNA expression, there was no effect on isoform protein expression, suggesting that the exercise challenge was insufficient for muscle Na+,K+‐ATPase up‐regulation.

Factors influencing creatine loading into human skeletal muscle.

SNOW, R. J., and R. M. MURPHY. Factors influencing creatine loading into human skeletal muscle. Exerc. Sport Sci. Rev., Vol. 31, No. 3, pp. 154–158, 2003. This review describes several factors involved in regulating skeletal muscle creatine uptake and total creatine content. Skeletal muscle total creatine content increases with oral creatine supplementation, although the response is variable. Factors that may account for this variation are carbohydrate intake, physical activity, training status, and possibly fiber type.