Creatine supplementation spares muscle glycogen during high intensity intermittent exercise in rats

Background: The effects of creatine (CR) supplementation on glycogen content are still debatable. Thus, due to the current lack of clarity, we investigated the effects of CR supplementation on muscle glycogen content after high intensity intermittent exercise in rats. Methods: First, the animals were submitted to a high intensity intermittent maximal swimming exercise protocol to ensure that CR-supplementation was able to delay fatigue ( experiment 1). Then, the CR-mediated glycogen sparing effect was examined using a high intensity intermittent sub-maximal exercise test ( fixed number of bouts; six bouts of 30-second duration interspersed by two-minute rest interval) ( experiment 2). For both experiments, male Wistar rats were given either CR supplementation or placebo (Pl) for 5 days. Results: As expected, CR-supplemented animals were able to exercise for a significant higher number of bouts than Pl. Experiment 2 revealed a higher gastrocnemius glycogen content for the CR vs. the Pl group (33.59%). Additionally, CR animals presented lower blood lactate concentrations throughout the intermittent exercise bouts compared to Pl. No difference was found between groups in soleus glycogen content. Conclusion: The major finding of this study is that CR supplementation was able to spare muscle glycogen during a high intensity intermittent exercise in rats.

Branched-chain amino acids supplementation enhances exercise capacity and lipid oxidation during endurance exercise after muscle glycogen depletion

Aim. It has been demonstrated that branched-chain amino acids (BCAA) transaminase activation occurs simultaneously with exercise-induced muscle glycogen reduction, suggesting that BCAA supplementation might play an energetic role in this condition. This study aimed to test whether BCAA supplementation enhances exercise capacity and lipid oxidation in glycogen-depleted subjects. Methods. Using a double-blind cross-over design, volunteers (N.=7) were randomly assigned to either the BCAA (300 mg . kg . day (-1)) or the placebo (maltodextrine) for 3 days. On the second day, subjects were submitted to an exercise-induced glycogen depletion protocol. They then performed an exhaustive exercise test on the third day, after which time to exhaustion, respiratory exchange ratio (RER), plasma glucose, free fatty acids (HA), blood ketones and lactate were determined. BCAA supplementation promoted a greater resistance to fatigue when compared to the placebo (+17.2%). Moreover, subjects supplemented with BCAA showed reduced RER and higher plasma glucose levels during the exhaustive exercise test. Results. No significant differences appeared in FFA, blood ketones and lactate concentrations. Conclusion. In conclusion, BCAA supplementation increases resistance to fatigue and enhances lipid oxidation during exercise in glycogen-depleted subjects.

Ultracentrifugal studies of the effect of molecular crowding by trimethylamine N-oxide on the self-association of muscle glycogen phosphorylase b

The suitability of sedimentation equilibrium for characterizing the self-association of muscle glycogen phosphorylase b has been reappraised. Whereas sedimentation equilibrium distributions for phosphorylase b in 40 mM Hepes buffer (pH 6.8) supplemented with 1 mM AMP signify a lack of chemical equilibrium attainment, those in buffer supplemented additionally with potassium sulfate conform with the requirements of a dimerizing system in chemical as we:ll as sedimentation equilibrium. Because the rate of attainment of chemical equilibrium under the former conditions is sufficiently slow to allow resolution of the dimeric and tetrameric enzyme species by sedimentation velocity, this procedure has been used to examine the effects of thermodynamic nonideality arising from molecular crowding try trimethylamine N-oxide on the self-association behaviour of phosphorylase b. In those terms the marginally enhanced extent of phosphorylase b self-association observed in the presence of high concentrations of the cosolute is taken to imply that the effects of thermodynamic nonideality on the dimer-tetramer equilibrium are being countered by those displacing the T reversible arrow R isomerization equilibrium for dimer towards the smaller, nonassociating T state. Because the R state is the enzymically active form, an inhibitory effect is the predicted consequence of molecular crowding by high concentrations of unrelated solutes. Thermodynamic nonideality thus provides an alternative explanation for the inhibitory effects of high concentrations of glycerol, sucrose and ethylene glycol on phosphorylase b activity, phenomena that have been attributed to extremely weak interaction of these cryoprotectants with the T state of the enzyme.

Hexokinase 2, Glycogen Synthase and Phosphorylase Play a Key Role in Muscle Glycogen Supercompensation

Background: Glycogen-depleting exercise can lead to supercompensation of muscle glycogen stores, but the biochemical mechanisms of this phenomenon are still not completely understood. Methods: Using chronic low-frequency stimulation (CLFS) as an exercise model, the tibialis anterior muscle of rabbits was stimulated for either 1 or 24 hours, inducing a reduction in glycogen of 90% and 50% respectively. Glycogen recovery was subsequently monitored during 24 hours of rest. Results: In muscles stimulated for 1 hour, glycogen recovered basal levels during the rest period. However, in those stimulated for 24 hours, glycogen was supercompensated and its levels remained 50% higher than basal levels after 6 hours of rest, although the newly synthesized glycogen had fewer branches. This increase in glycogen correlated with an increase in hexokinase-2 expression and activity, a reduction in the glycogen phosphorylase activity ratio and an increase in the glycogen synthase activity ratio, due to dephosphorylation of site 3a, even in the presence of elevated glycogen stores. During supercompensation there was also an increase in 59-AMP-activated protein kinase phosphorylation, correlating with a stable reduction in ATP and total purine nucleotide levels. Conclusions: Glycogen supercompensation requires a coordinated chain of events at two levels in the context of decreased cell energy balance: First, an increase in the glucose phosphorylation capacity of the muscle and secondly, control of the enzymes directly involved in the synthesis and degradation of the glycogen molecule. However, supercompensated glycogen has fewer branches.

Muscle glycogen metabolism changes in rats fed early postnatal a fructose-rich diet after maternal protein malnutrition: effects of acute physical exercise at the maximal lactate steady-state intensity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of diets with varying starch content on muscle glycogen concentrations during training and replenishment after highintensity exercise

Pós-graduação em Zootecnia - FCAV

Muscle glycogen resynthesis during recovery from cycle exercise: no effect of additional protein ingestion

[EN] In the present study, we have investigated the effect of carbohydrate and protein hydrolysate ingestion on muscle glycogen resynthesis during 4 h of recovery from intense cycle exercise. Five volunteers were studied during recovery while they ingested, immediately after exercise, a 600-ml bolus and then every 15 min a 150-ml bolus containing 1) 1.67 g. kg body wt(-1). l(-1) of sucrose and 0.5 g. kg body wt(-1). l(-1) of a whey protein hydrolysate (CHO/protein), 2) 1.67 g. kg body wt(-1). l(-1) of sucrose (CHO), and 3) water. CHO/protein and CHO ingestion caused an increased arterial glucose concentration compared with water ingestion during 4 h of recovery. With CHO ingestion, glucose concentration was 1-1.5 mmol/l higher during the first hour of recovery compared with CHO/protein ingestion. Leg glucose uptake was initially 0.7 mmol/min with water ingestion and decreased gradually with no measurable glucose uptake observed at 3 h of recovery. Leg glucose uptake was rather constant at 0.9 mmol/min with CHO/protein and CHO ingestion, and insulin levels were stable at 70, 45, and 5 mU/l for CHO/protein, CHO, and water ingestion, respectively. Glycogen resynthesis rates were 52 +/- 7, 48 +/- 5, and 18 +/- 6 for the first 1.5 h of recovery and decreased to 30 +/- 6, 36 +/- 3, and 8 +/- 6 mmol. kg dry muscle(-1). h(-1) between 1.5 and 4 h for CHO/protein, CHO, and water ingestion, respectively. No differences could be observed between CHO/protein and CHO ingestion ingestion. It is concluded that coingestion of carbohydrate and protein, compared with ingestion of carbohydrate alone, did not increase leg glucose uptake or glycogen resynthesis rate further when carbohydrate was ingested in sufficient amounts every 15 min to induce an optimal rate of glycogen resynthesis.

Enzymatic phosphorylation of muscle glycogen synthase: a mechanism for maintenance of metabolic homeostasis.

We recently analyzed experimental studies of mammalian muscle glycogen synthesis using metabolic control analysis and concluded that glycogen synthase (GSase) does not control the glycogenic flux but rather adapts to the flux which is controlled bv the activity of the proximal glucose transport and hexokinase steps. This model did not provide a role for the well established relationship between GSase fractional activity, determined by covalent phosphorylation, and the rate of glycogen synthesis. Here we propose that the phosphorylation of GSase, which alters the sensitivity to allosteric activation by glucose 6-phosphate (G6P), is a mechanism for controlling the concentration of G6P instead of controlling the flux. When the muscle cell is exposed to conditions which favor glycogen synthesis such as high plasma insulin and glucose concentrations the fractional activity of GSase is increased in coordination with increases in the activity of glucose transport and hexokinase. This increase in GSase fractional activity helps to maintain G6P homeostasis by reducing the G6P concentration required to activate GSase allosterically to match the flux determined by the proximal reactions. This role for covalent phosphorylation also provides a novel solution to the Kacser and Acarenza paradigm which requires coordinated activity changes of the enzymes proximal and distal to a shared intermediate, to avoid unwanted flux changes.

NMR studies of muscle glycogen synthesis in insulin-resistant offspring of parents with non-insulin-dependent diabetes mellitus immediately after glycogen-depleting exercise.

To examine the impact of insulin resistance on the insulin-dependent and insulin-independent portions of muscle glycogen synthesis during recovery from exercise, we studied eight young, lean, normoglycemic insulin-resistant (IR) offspring of individuals with non-insulin-dependent diabetes mellitus and eight age-weight matched control (CON) subjects after plantar flexion exercise that lowered muscle glycogen to approximately 25% of resting concentration. After approximately 20 min of exercise, intramuscular glucose 6-phosphate and glycogen were simultaneously monitored with 31P and 13C NMR spectroscopies. The postexercise rate of glycogen resynthesis was nonlinear. Glycogen synthesis rates during the initial insulin independent portion (0-1 hr of recovery) were similar in the two groups (IR, 15.5 +/- 1.3 mM/hr and CON, 15.8 +/- 1.7 mM/hr); however, over the next 4 hr, insulin-dependent glycogen synthesis was significantly reduced in the IR group [IR, 0.1 +/- 0.5 mM/hr and CON, 2.9 +/- 0.2 mM/hr; (P < or = 0.001)]. After exercise there was an initial rise in glucose 6-phosphate concentrations that returned to baseline after the first hour of recovery in both groups. In summary, we found that following muscle glycogen-depleting exercise, IR offspring of parents with non-insulin-dependent diabetes mellitus had (i) normal rates of muscle glycogen synthesis during the insulin-independent phase of recovery from exercise and (ii) severely diminished rates of muscle glycogen synthesis during the subsequent recovery period (2-5 hr), which has previously been shown to be insulin-dependent in normal CON subjects. These data provide evidence that exercise and insulin stimulate muscle glycogen synthesis in humans by different mechanisms and that in the IR subjects the early response to stimulation by exercise is normal.

In vivo regulation of muscle glycogen synthase and the control of glycogen synthesis.

The activity of glycogen synthase (GSase; EC 2.4.1.11) is regulated by covalent phosphorylation. Because of this regulation, GSase has generally been considered to control the rate of glycogen synthesis. This hypothesis is examined in light of recent in vivo NMR experiments on rat and human muscle and is found to be quantitatively inconsistent with the data under conditions of glycogen synthesis. Our first experiments showed that muscle glycogen synthesis was slower in non-insulin-dependent diabetics compared to normals and that their defect was in the glucose transporter/hexokinase (GT/HK) part of the pathway. From these and other in vivo NMR results a quantitative model is proposed in which the GT/HK steps control the rate of glycogen synthesis in normal humans and rat muscle. The flux through GSase is regulated to match the proximal steps by "feed forward" to glucose 6-phosphate, which is a positive allosteric effector of all forms of GSase. Recent in vivo NMR experiments specifically designed to test the model are analyzed by metabolic control theory and it is shown quantitatively that the GT/HK step controls the rate of glycogen synthesis. Preliminary evidence favors the transporter step. Several conclusions are significant: (i) glucose transport/hexokinase controls the glycogen synthesis flux; (ii) the role of covalent phosphorylation of GSase is to adapt the activity of the enzyme to the flux and to control the metabolite levels not the flux; (iii) the quantitative data needed for inferring and testing the present model of flux control depended upon advances of in vivo NMR methods that accurately measured the concentration of glucose 6-phosphate and the rate of glycogen synthesis.

Effect of osmolytes on the interaction of flavin adenine dinucleotide with muscle glycogen phosphorylase b

The effect of three osmolytes, trimethylamine N-oxide (TMAO), betaine and proline, on the interaction of muscle glycogen phosphorylase b with allosteric inhibitor FAD has been examined. In the absence of osmolyte, the interaction is described by a single intrinsic dissociation constant (17.8 muM) for two equivalent and independent binding sites on the dimeric enzyme. However, the addition of osmolytes gives rise to sigmoidal dependencies of fractional enzyme-site saturation upon free inhibitor concentration. The source of this cooperativity has been shown by difference sedimentation velocity to be an osmolyte-mediated isomerization of phosphorylase b to a smaller dimeric state with decreased affinity for FAD. These results thus have substantiated a previous inference that the tendency for osmolyte-enhanced self-association of dimeric glycogen phosphorylase b in the presence of AMP was being countered by the corresponding effect of molecular crowding on an isomerization of dimer to a smaller, nonassociating state. (C) 2004 Elsevier Ltd. Inc. All rights reserved.

Muscle glycogen inharmoniously regulates glycogen synthase activity, glucose uptake, and proximal insulin signaling

Muscle glycogen inharmoniously regulates glycogen synthase activity, glucose uptake, and proximal insulin signaling. Am J Physiol Endocrinol Metab 290: E154-E162, 2006. First published August 23, 2005; doi:10.1152/ajpendo. 00330.2005.-Insulin-stimulated glucose uptake and incorporation of glucose into skeletal muscle glycogen contribute to physiological regulation of blood glucose concentration. In the present study, glucose handling and insulin signaling in isolated rat muscles with low glycogen (LG, 24-h fasting) and high glycogen (HG, refed for 24 h) content were compared with muscles with normal glycogen (NG, rats kept on their normal diet). In LG, basal and insulin-stimulated glycogen synthesis and glycogen synthase activation were higher and glycogen synthase phosphorylation (Ser645, Ser649, Ser653, Ser657) lower than in NG. GLUT4 expression, insulin-stimulated glucose uptake, and PKB phosphorylation were higher in LG than in NG, whereas insulin receptor tyrosyl phosphorylation, insulin receptor substrate-1-associated phosphatidylinositol 3-kinase activity, and GSK-3 phosphorylation were unchanged. Muscles with HG showed lower insulin-stimulated glycogen synthesis and glycogen synthase activation than NG despite similar dephosphorylation. Insulin signaling, glucose uptake, and GLUT4 expression were similar in HG and NG. This discordant regulation of glucose uptake and glycogen synthesis in HG resulted in higher insulin-stimulated glucose 6-phosphate concentration, higher glycolytic flux, and intracellular accumulation of nonphosphorylated 2-deoxyglucose. In conclusion, elevated glycogen synthase activation, glucose uptake, and GLUT4 expression enhance glycogen resynthesis in muscles with low glycogen. High glycogen concentration per se does not impair proximal insulin signaling or glucose uptake. Insulin resistance is observed at the level of glycogen synthase, and the reduced glycogen synthesis leads to increased levels of glucose 6-phosphate, glycolytic flux, and accumulation of nonphosphorylated 2-deoxyglucose.

Correlations of glycogen synthase and phosphorylase activities with glycogen concentration in human muscle biopsies. Evidence for a double-feedback mechanism regulating glycogen synthesis and breakdown.

The purpose of this study was to verify in man the relationships of muscle glycogen synthase and phosphorylase activities with glycogen concentration that were reported in animal studies. The upper level of glycogen concentration in muscle is known to be tightly controlled, and glycogen concentration was reported to have an inhibitory effect on synthase activity and a stimulatory effect on phosphorylase activity. Glycogen synthase and phosphorylase activity and glycogen concentration were measured in muscle biopsies in a group of nine normal subjects after stimulating an increase of their muscle glycogen concentration through either an intravenous glucose-insulin infusion to stimulate glycogen synthesis, or an Intralipid (Vitrum, Stockholm, Sweden) infusion in the basal state to inhibit glycogen mobilization by favoring lipid oxidation at the expense of glucose oxidation. Phosphorylase activity increased from 71.3 +/- 21.0 to 152.8 +/- 20.0 nmol/min/mg protein (P < .005) after the glucose-insulin infusion. Phosphorylase activity was positively correlated with glycogen concentration (P = .005 and P = .0001) after the glucose-insulin and Intralipid infusions, respectively. Insulin-stimulated glycogen synthase activity was significantly negatively correlated with glycogen concentration at the end of the Intralipid infusion (P < .005). In conclusion, by demonstrating a negative correlation of glycogen concentration with glycogen synthase and a positive correlation with phosphorylase, this study might confirm in man the double-feedback mechanism by which changes in glycogen concentration regulate glycogen synthase and phosphorylase activities. It suggests that this mechanism might play an important role in the regulation of glucose storage.