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.     

The effect of creatine supplementation on mass and performance of rat skeletal muscle

This study investigated the effect of dietary creatine supplementation on hypertrophy and performance of rat skeletal muscle. Male Sprague-Dawley rats underwent either tibialis anterior ablation or partial ablation of the plantaris/gastrocnemius to induce compensatory hypertrophy of the extensor digitorum longus (EDL) or soleus respectively, or sham surgery. Creatine (300 mg/kg) was administered to one half of each group for 5 weeks, after which force production was measured. With the leg fixed at the knee and ankle, the distal tendon of the EDL or soleus was attached to a force transducer and the muscle was electrically stimulated via the sciatic nerve. Synergist ablation resulted in a significant increase in EDL mass and in soleus mass relative to control muscles. However, no effect of creatine supplementation on muscle mass or performance was found between control and either group of creatine-treated rats. Despite an apparent increase in muscle creatine content, creatine supplementation did not augment muscle hypertrophy or force production in rat EDL or soleus muscle, providing evidence that the potential benefits of creatine supplementation are not due to a direct effect on muscle but rather to an enhanced ability to train.     

Histochemical demonstration of ATP: creatine phosphotransferase in rat skeletal muscle

Summary An attempt was made to locate the ATP: creatine phosphotransferase (creatine kinase, CKase) in rat skeletal muscle by a lead precipitation method. The muscle is not stained at all with creatine phosphate (CP), and only weakly with adenosine diphosphate (ADP) as substrate, while it hydrolyzes adenosine triphosphate (ATP) actively. Taking advantage of this fact, it is possible to demonstrate the CKase activity using both ADP and CP as substrate. The CKase activity thus obtained was located in various profiles of sarcoplasmic reticulum as well as in A bands, the staining being comparable to that obtained with ATP as substrate.A weak activity was found only in cisternal dilatations of sarcoplasmic reticulum when sections were incubated with ADP as substrate.     

Quantitative histochemistry of creatine kinase in rat myocardium and skeletal muscle

Summary Creatine kinase (ec 2.7.3.2) activity was demonstrated in rat myocardium using a polyvinyl alcohol-containing incubation medium and auxiliary enzymes. The activity was quantified by microdensitometry using both endpoint measurements and kinetic measurements. Control reactions were performed in the absence of creatine phosphate and ADP.The linear regression lines of the absorbances of reduced Nitro BT at the isobestic wavelength (585 nm) on incubation time were highly significant for both endpoint and kinetic measurements. The activity obtained from endpoint measurements was about 40% lower. This was caused by loss of the formazan reaction product from the tissue sections when the incubation medium was removed at the end of the reaction. The relationship between creatine kinase activity (test minus control reaction) and section thickness was not linear for either myocardium or skeletal muscle; control reactions, however, showed linear relationships with section thickness for both tissues. Limited penetration of auxiliary enzymes into the sections may be responsible for this disporportionality. Therefore, care should be taken in the interpretation of quantitative data obtained with different tissues.In conclusion, multi-step enzyme reactions can be used for quantitative histochemical purposes provided it is taken into account that the reactivity is not proportional to section thickness.     

Compartmentation of high-energy phosphates in resting and working rat skeletal muscle

The subcellular distribution of high-energy phosphates in various types of skeletal muscle of the rat was analysed by subfractionation of tissues in non-aqueous solvents. Different glycolytic and oxidative capacities were calculated from the ratio of phosphoglycerate kinase and citrate synthase activities, ranging from 25 in m. soleus to 130 in m. tensor fasciae latae. In the resting state, the subcellular contents of ATP, creatine phosphate and creatine were similar in m. soleus, m. vastus intermedius, m. gastrocnemius and m. tensor fasciae latae but, significantly, a higher extramitochondrial ADP-content was found in m. soleus. A similar observation was made in isometrically and isotonically working m. gastrocnemius. The extramitochondrial, bound ADP accounted fully for actin-binding sites in resting fast-twitch muscles, but an excess of bound ADP was found in m. soleus and working m. gastrocnemius. The amount of non-actin-bound ADP reached maximal values of approx. 1.2 nmol/mg total protein. It could not be enhanced further by prolonged isotonic stimulation or by increased isometric force development. It is suggested that non-actin-bound ADP is accounted for by actomyosin-ADP complexes generated during the contraction cycle. Binding of extramitochondrial ADP to actomyosin complexes in working muscles thus acts as a buffer for cytosolic ADP in addition to the creatine system, maintaining a high cytosolic phosphorylation potential also at increasing rates of ATP hydrolysis during muscle contraction.     

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.     

Inflammatory cells in rat skeletal muscle are elevated after electrically stimulated contractions.

We determined the effect of muscle contractions resulting from high-frequency electrical stimulation (HFES) on inflammatory cells in rat tibialis anterior (TA), plantaris (Pln), and soleus (Sol) muscles at 6, 24, and 72 h post-HFES. A minimum of four and a maximum of seven rats were analyzed at each time point. HFES, applied to the sciatic nerve, caused the Sol and Pln to contract concentrically and the TA to contract eccentrically. Neutrophils were higher (P < 0.05) at 6 and 24 h after HFES in the Sol, Pln, and TA muscles relative to control muscles. ED1(+) macrophages in the Pln were elevated at 6 and 24 h after HFES and were also elevated in the Sol and TA after HFES relative to controls. ED2(+) macrophages in the Sol and TA were elevated at 24 and 72 h after HFES, respectively, and were also elevated in the Pln after HFES relative to controls. In contrast to the TA muscles, the Pln and Sol muscles showed no gross histological abnormalities. Collectively, these data indicate that both eccentric and concentric contractions can increase inflammatory cells in muscle, regardless of whether overt histological signs of injury are apparent.     

Adaptive responses to creatine loading and exercise in fast-twitch rat skeletal muscle

We investigated the effects of chronic creatine loading and voluntary running (Run) on muscle fiber types, proteins that regulate intracellular Ca2+, and the metabolic profile in rat plantaris muscle to ascertain the bases for our previous observations that creatine loading results in a higher proportion of myosin heavy chain (MHC) IIb, without corresponding changes in contractile properties. Forty Sprague-Dawley rats were assigned to one of four groups: creatine-fed sedentary, creatine-fed run-trained, control-fed sedentary, and control-fed run-trained animals. Proportion and cross-sectional area increased 10% and 15% in type IIb fibers and the proportion of type IIa fibers decreased 11% in the creatine-fed run-trained compared with the control-fed run-trained group (P < 0.03). No differences were observed in fast Ca2+-ATPase isoform SERCA1 content (P > 0.49). Creatine feeding alone induced a 41% increase (P < 0.03) in slow Ca2+-ATPase (SERCA2) content, which was further elevated by 33% with running (P < 0.02). Run training alone reduced parvalbumin content by 50% (P < 0.05). By comparison, parvalbumin content was dramatically decreased by 75% (P < 0.01) by creatine feeding alone but was not further reduced by run training. These adaptive changes indicate that elevating the capacity for high-energy phosphate shuttling, through creatine loading, alleviates the need for intracellular Ca2+ buffering by parvalbumin and increases the efficiency of Ca2+ uptake by SERCAs. Citrate synthase and 3-hydroxyacyl-CoA dehydrogenase activities were elevated by run training (P < 0.003) but not by run training + creatine feeding. This indicates that creatine loading during run training supports a faster muscle phenotype that is adequately supported by the existing glycolytic potential, without changes in the capacity for terminal substrate oxidation.     

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.     

Regulation of creatine kinase during steady-state isometric twitch contraction in rat skeletal muscle

In vivo 31P-NMR saturation transfer measurements of the creatine kinase exchange flux in the direction creatine phosphate----ATP were made in the gastrocnemius muscle of rats at rest and during steady-state isometric twitch contraction at frequencies from 0.25 to 2 Hz. There was no correlation between creatine kinase exchange flux and either free [ADP] or oxygen consumption, both of which increase with stimulation frequency. The flux was found to be nearly constant over all conditions at about 16 mM X s-1, 10-times greater than the highest estimated ATP turnover in this study. The kinetic properties of skeletal muscle creatine kinase in vivo are similar to, but not completely predictable from, the equilibrium exchange fluxes measured on the isolated enzyme. These results are not consistent with strong functional coupling between ATP synthesis and mitochondrial creatine kinase.     

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.     

Taurine supplementation decreases oxidative stress in skeletal muscle after eccentric exercise

Infrequent exercise, typically involving eccentric actions, has been shown to cause oxidative stress and to damage muscle tissue. High taurine levels are present in skeletal muscle and may play a role in cellular defences against free radical‐mediated damage. This study investigates the effects of taurine supplementation on oxidative stress biomarkers after eccentric exercise (EE). Twenty‐four male rats were divided into the following groups (n = 6): control; EE; EE plus taurine (EE + Taurine); EE plus saline (EE + Saline). Taurine was administered as a 1‐ml 300 mg kg^?1 per body weight (BW) day^?1 solution in water by gavage, for 15 consecutive days. Starting on the 14th day of supplementation, the animals were submitted to one 90‐min downhill run session and constant velocity of 1·0 km h^?1. Forty‐eight hours after the exercise session, the animals were killed and the quadriceps muscles were surgically removed. Production of superoxide anion, creatine kinase (CK) levels, lipoperoxidation, carbonylation, total thiol content and antioxidant enzyme were analysed. Taurine supplementation was found to decrease superoxide radical production, CK, lipoperoxidation and carbonylation levels and increased total thiol content in skeletal muscle, but it did not affect antioxidant enzyme activity after EE. The present study suggests that taurine affects skeletal muscle contraction by decreasing oxidative stress, in association with decreased superoxide radical production. Copyright © 2010 John Wiley & Sons, Ltd.     

Triacylglycerol metabolism in rat skeletal muscle after exercise

The temporal relationships between triacylglycerol (TG) content and TG lipase activity in slow-twitch (STR) and fast-twitch red (FTR) muscles were determined in rats during recovery from a 2-h swim. Immediately after the exercise, plasma free fatty acid (FFA) was elevated and glycogen concentrations were decreased. TG content was decreased 40% in STR muscle and reduced 45% in FTR muscle. The TG concentration of STR muscle increased in a linear fashion throughout recovery so that control levels were reached within the first 24 h after exercise. TG lipase activity of STR muscle was elevated 36% above control immediately after the swim and continued to increase to 84% above control 24 h after the work. In STR muscle there was a net synthesis of TG, while lipase activity was elevated above that measured in muscle of control rats. TG content of FTR muscle remained 45% below control throughout the first 24 h of recovery, and TG lipase activity increased from 26% (P greater than 0.05) greater than control immediately after exercise to threefold above control 24 h after work. All parameters returned to control levels by 48 h of recovery. These data indicated that a net TG synthesis occurs in STR muscle when lipolytic activity is elevated. In FTR muscle, however, a gradual increase in TG lipase activity that occurs during the first 24 h of recovery accompanies a TG concentration well below the control level throughout this same time frame.(ABSTRACT TRUNCATED AT 250 WORDS)     

The pressure-flow relation in resting rat skeletal muscle perfused with pure erythrocyte suspensions

In spite of numerous investigations of erythrocyte rheology, there is limited information about the influence of erythrocyte suspensions on whole organ pressure-flow relationships. In this study, we present whole organ pressure-flow curves for resting vasodilated gracilis muscle of the rat, in which the microanatomy and vessel properties have been determined previously. For pure erythrocyte suspensions from donor rats, the organ resistance increases only mildly with perfusion time (less than a 5% shift over a one-hour perfusion time), while in contrast, erythrocyte suspensions containing leukocytes show an increases of resistance near 100% over a period of 25 min. Variation in pressure-flow curves in the muscle at the same arterial hematocrit between different rats is less than 15%. The pressure-flow relation for pure erythrocyte suspensions depends on hematocrit. Shear thinning is exhibited at high hematocrits, while Newtonian behavior is approached at arterial hematocrits below 15%. The whole organ apparent viscosity for pure erythrocyte suspensions (normalized by cell-free plasma resistance) is a non-linear function of hematocrit; at physiological pressures, it reaches values comparable to those of apparent viscosities measured in rotational viscometers or in in vitro tube flow (diameters greater than 0.8 mm). The apparent viscosities estimated from the whole organ experiments tend to be higher than those measured in straight tubes under in vitro conditions. The pressure-flow curves for pure erythrocyte suspensions are shifted towards lower pressures than the curves for mixed suspensions of erythrocytes at the same hematocrit and with leukocytes at physiological cell counts. These acute experiments show that pure erythrocyte suspensions yield highly reproducible resistances in the skeletal muscle microcirculation with dilated arterioles. Relative apparent viscosities measured in vivo are higher than those measured in straight glass tubes of comparable dimesions.     

Vitamin E supplementation modifies adaptive responses to training in rat skeletal muscle

Abstract

Aim of the present study was to test, by vitamin E treatment, the hypothesis that muscle adaptive responses to training are mediated by free radicals produced during the single exercise sessions. Therefore, we determined aerobic capacity of tissue homogenates and mitochondrial fractions, tissue content of mitochondrial proteins and expression of factors (PGC-1, NRF-1, and NRF-2) involved in mitochondrial biogenesis. Moreover, we determined the oxidative damage extent, antioxidant enzyme activities, and glutathione content in both tissue preparations, mitochondrial ROS production rate. Finally we tested mitochondrial ROS production rate and muscle susceptibility to oxidative stress. The metabolic adaptations to training, consisting in increased muscle oxidative capacity coupled with the proliferation of a mitochondrial population with decreased oxidative capacity, were generally prevented by antioxidant supplementation. Accordingly, the expression of the factors involved in mitochondrial biogenesis, which were increased by training, was restored to the control level by the antioxidant treatment. Even the training-induced increase in antioxidant enzyme activities, glutathione level and tissue capacity to oppose to an oxidative attach were prevented by vitamin E treatment. Our results support the idea that the stimulus for training-induced adaptive responses derives from the increased production, during the training sessions, of reactive oxygen species that stimulates the expression of PGC-1, which is involved in mitochondrial biogenesis and antioxidant enzymes expression. On the other hand, the observation that changes induced by training in some parameters are only attenuated by vitamin E treatment suggests that other signaling pathways, which are activated during exercise and impinge on PGC-1, can modify the response to the antioxidant integration.     

The deleterious effects of bed rest on human skeletal muscle fibers are exacerbated by hypercortisolemia and ameliorated by dietary supplementation

Prolonged inactivity associated with bed rest in a clinical setting or spaceflight is frequently associated with hypercortisolemia and inadequate caloric intake. Here, we determined the effect of 28 days of bed rest (BR); bed rest plus hypercortisolemia (BRHC); and bed rest plus essential amino acid (AA) and carbohydrate (CHO) supplement (BRAA) on the size and function of single slow- and fast-twitch muscle fibers. Supplementing meals, the BRAA group consumed 16.5 g essential amino acids and 30 g sucrose at 1100, 1600, and 2100 h, and the BRHC subjects received 5 daily doses of 10–15 mg of oral hydrocortisone sodium succinate throughout bed rest. Bed rest induced atrophy and loss of force (mN) and power (µN·FL·s^–1) in single fibers was exacerbated by hypercortisolemia where soleus peak force declined by 23% in the type I fiber from a prevalue of 0.78 ± 0.02 to 0.60 ± 0.02 mN post bed rest (compared to a 7% decline with bed rest alone) and 27% in the type II fiber (1.10 ± 0.08 vs. 0.81 ± 0.05 mN). In the BRHC group, peak power dropped by 19, 15, and 11% in the soleus type I, and vastus lateralis (VL) type I and II fibers, respectively. The AA/CHO supplement protected against the bed rest-induced loss of peak force in the type I soleus and peak power in the VL type II fibers. These results provide evidence that an AA/CHO supplement might serve as a successful countermeasure to help preserve muscle function during periods of relative inactivity. isotonic contractile properties; peak force and power; calcium sensitivity; essential amino acids     

Single chloride-selective channels active at resting membrane potentials in cultured rat skeletal muscle.

The patch-clamp technique was used to characterize channels that could contribute to the resting Cl-conductance in the surface membrane of cultured rat skeletal muscle. Two Cl- -selective channels, in addition to the Cl- -selective channel of large conductance described previously (Blatz and Magleby, 1983), were observed. One of these channels had fast kinetics and a conductance of 45 +/- 1.8 pS (SE) in symmetrical 100 mM KCl. The other had slow kinetics and a conductance of 61 +/- 2.4 pS. The channel with fast kinetics typically closed within 1 ms after opening and flickered between the open and shut states. The channel with slow kinetics typically closed within 10 ms after opening and displayed less flickering. Both channels were active in excised patches of membrane held at potentials similar to resting membrane potentials in intact cells, and both were open a greater percentage of time with depolarization. Under conditions of high ion concentrations, both channels exhibited nonideal selectivity for Cl- over K+ with the permeability ratio PK/PCl of 0.15-0.2. Additional experiments on the fast Cl- channel indicated that its activity decreased with lowered pHi and that SO2-4 and CH3SO-4 were ineffective charge carriers. These findings, plus the observation that the fast Cl- channel was also active in membrane patches on intact cells, suggest that the fast Cl- channel provides a molecular basis for at least some of the resting Cl- conductance. The extent to which the slow Cl- channel contributes is less clear as it was typically active only after excised patches of membrane had been exposed to high concentrations of KCl at the inner membrane surface.