The regulation of total creatine content in a myoblast cell line

Abstract

Total cellular creatine content is an important bioenergetic parameter in skeletal muscle. To understand its regulation we investigated creatine transport and accumulation in the G8 cultured skeletal myoblast line. Like other cell types, these contain a creatine transporter, whose activity, measured using a radiolabelling technique, was saturable (Km = 110 +/- 25 microM) and largely dependent on extracellular [Na+]. To study sustained influences on steady state creatine concentration we measured total cellular creatine content using a fluorimetric method in 48 h incubations. We found that the total cellular creatine content was relatively independent of extracellular creatine concentration, consistent with high affinity sodium-dependent uptake balanced by slow passive efflux. Accordingly, in creatine-free incubations net creatine efflux was slow (5 +/- 1% of basal creatine content per day over 6 days), while creatine content in 48 h incubations was reduced by 28 +/- 13% of control by the Na+, K(+)-ATPase inhibitor ouabain. Creatine accumulation after 48 h was stimulated by treatment with the mixed alpha- and beta-adrenergic agonist noradrenaline, the beta-adrenergic agonist isoproterenol, the beta 2-agonist clenbuterol and the cAMP analogue N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate, but was unaffected by the alpha 1 adrenergic agonist methoxamine. The noradrenaline enhancement of creatine accumulation at 48 h was inhibited by the mixed alpha- and beta-antagonist labetalol and by the beta-antagonist propranolol, but was unaffected by the alpha 2 antagonist phentolamine; greater inhibition was caused by the beta 2 antagonist butoxamine than the beta 1 antagonist atenolol. Creatine accumulation at 48 h was increased to 230 +/- 6% of control by insulin and by 140 +/- 13% by IGF-I (both at 3 nM). Creatine accumulation at 48 h was also increased to 280 +/- 40% of control by 3,3',5-triiodothyronine (at 70 microM) and to 220 +/- 35% of control by amylin (60 nM). As 3,3', 5-triiodothyronine, amylin and isoproterenol all stimulate the Na+, K(+)-ATPase, we suggest that they stimulate Na(+)-creatine cotransport indirectly by increasing the transmembrane [Na+] concentration gradient and membrane potential.

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

“…The second, slower component (k 2 ) has a half-time of 4.2 min and may be the washout of the interstitial space or some nonmuscle tissue or cells. This slower component is not likely an efflux from the intracellular muscle creatine pool because that rate of loss, which actually appears as creatinine, has been measured to have a half-time of many days (7,11,33). Perfusate creatine concentrations had no significant effect on the washout rate (k 1 or k 2 ; data not shown); therefore, the data were pooled, and the single curve is presented in Fig.…”

“…Creatine uptake rates measured in cell lines overexpressing the cloned CrT (14,20,32,37), cultured myoblasts (13,27,33), giant sarcolemmal vesicles (47), and incubated muscle (51) reveal an apparent MichaelisMenten constant (K m) for transport of 30-188 M. Because this is near or below the typical plasma creatine concentration for mammals, it is probable that the transporter may be nearly saturated in vivo. This implies that creatine uptake would be proportional to the CrT content of the myocyte.…”

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

“…For example, PCr or Cr total content may be decreased in muscle because of neuromuscular disease (36), heart failure (29), or by Cr analog feeding (13,38). Conversely, PCr and/or Cr total content may be increased in humans and animals by dietary Cr supplementation (19,23,26) or in myocyte culture by increasing extracellular Cr concentration (24,30). Despite these observations and the integral role that Cr and PCr play in energy management, it is still unclear how the Cr total of skeletal muscle is controlled.…”

Human Physiology

“…in vitro work indicates that supraphysiological concentrations of insulin are required to augment muscle Cl' accumulation (Odoom et al 1996). The purpose of the present study, therefore, was to determine the effect of insulin availability on Cl' accumulation in human skeletal muscle.…”