Implementation of periodic acid-thiosemicarbazide-silver proteinate staining for ultrastructural assessment of muscle glycogen utilization during exercise

Summary Distribution of glycogen particles in semithin and ultrathin sections of biopsy samples from human muscles subjected to either short- or long-term running were investigated using PAS and Periodic Acid-ThioSemiCarbazide-Silver Proteinate (PA-TSC-SP) staining methods. Glycogen particles were predominantly found immediately under the sarcolemma or aligned along the myofibrillar Iband. After long-term exhaustive exercise type-1 fibers with a few or no glycogen particles in the core of the fibers were frequently observed. The subsarcolemmal glycogen stores of these depleted type-1 fibers were about three times as large as after exhaustive short-time exercise. Another indication of utilization of subsarcolemmal glycogen stores during anaerobic exercise was that many particles displayed a pale, rudimentary shape. This observation suggests fragmental metabolization of glycogen. Thus, depending on type of exercise and type of fiber differential and sequential glycogen utilization patterns can be observed.     

Menstrual cycle phase and sex influence muscle glycogen utilization and glucose turnover during moderate-intensity endurance exercise

Numerous studies from our and other laboratories have shown that women have a lower respiratory exchange ratio (RER) during exercise than equally trained men, indicating a greater reliance on fat oxidation. Differences in estrogen concentration between men and women likely play a role in this sex difference. Differing estrogen and progesterone concentrations during the follicular (FP) and luteal (LP) phases of the female menstrual cycle suggest that fuel use may also vary between phases. The purpose of the current study was to determine the effect of menstrual cycle phase and sex upon glucose turnover and muscle glycogen utilization during endurance exercise. Healthy, recreationally active young women (n = 13) and men (n = 11) underwent a primed constant infusion of [6,6-2H]glucose with muscle biopsies taken before and after a 90-min cycling bout at 65% peak O2 consumption. LP women had lower glucose rate of appearance (Ra, P = 0.03), rate of disappearance (Rd, P = 0.03), and metabolic clearance rate (MCR, P = 0.04) at 90 min of exercise and lower proglycogen (P = 0.04), macroglycogen (P = 0.04), and total glycogen (P = 0.02) utilization during exercise compared with FP women. Men had a higher RER (P = 0.02), glucose Ra (P = 0.03), Rd (P = 0.03), and MCR (P = 0.01) during exercise compared with FP women, and men had a higher RER at 75 and 90 min of exercise (P = 0.04), glucose Ra (P = 0.01), Rd (P = 0.01), and MCR (P = 0.001) and a greater PG utilization (P = 0.05) compared with LP women. We conclude that sex, and to a lesser extent menstrual cycle, influence glucose turnover and glycogen utilization during moderate-intensity endurance exercise.     

Regulation of glycogen phosphorylase and PDH during exercise in human skeletal muscle during hypoxia

The present study examined the acute effects of hypoxia on the regulation of skeletal muscle metabolism at rest and during 15 min of submaximal exercise. Subjects exercised on two occasions for 15 min at 55% of their normoxic maximal oxygen uptake while breathing 11% O(2) (hypoxia) or room air (normoxia). Muscle biopsies were taken at rest and after 1 and 15 min of exercise. At rest, no effects on muscle metabolism were observed in response to hypoxia. In the 1st min of exercise, glycogenolysis was significantly greater in hypoxia compared with normoxia. This small difference in glycogenolysis was associated with a tendency toward a greater concentration of substrate, free P(i), in hypoxia compared with normoxia. Pyruvate dehydrogenase activity (PDH(a)) was lower in hypoxia at 1 min compared with normoxia, resulting in a reduced rate of pyruvate oxidation and a greater lactate accumulation. During the last 14 min of exercise, glycogenolysis was greater in hypoxia despite a lower mole fraction of phosphorylase a. The greater glycogenolytic rate was maintained posttransformationally through significantly higher free [AMP] and [P(i)]. At the end of exercise, PDH(a) was greater in hypoxia compared with normoxia, contributing to a greater rate of pyruvate oxidation. Because of the higher glycogenolytic rate in hypoxia, the rate of pyruvate production continued to exceed the rate of pyruvate oxidation, resulting in significant lactate accumulation in hypoxia compared with no further lactate accumulation in normoxia. Hence, the elevated lactate production associated with hypoxia at the same absolute workload could in part be explained by the effects of hypoxia on the activities of the rate-limiting enzymes, phosphorylase and PDH, which regulate the rates of pyruvate production and pyruvate oxidation, respectively.     

Carbohydrate supercompensation and muscle glycogen utilization during exhaustive running in highly trained athletes

Three female and three male highly trained endurance runners with mean maximal oxygen uptake (VO2max) values of 60.5 and 71.5 ml.kg-1.min-1, respectively, ran to exhaustion at 75%-80% of VO2max on two occasions after an overnight fast. One experiment was performed after a normal diet and training regimen (Norm), the other after a diet and training programme intended to increase muscle glycogen levels (Carb). Muscle glycogen concentration in the gastrocnemius muscle increased by 25% (P less than 0.05) from 581 mmol.kg-1 dry weight, SEM 50 to 722 mmol.kg-1 dry weight, SEM 34 after Carb. Running time to exhaustion, however, was not significantly different in Carb and Norm, 77 min, SEM 13 vs 70 min, SEM 8, respectively. The average glycogen concentration following exhaustive running was 553 mmol.kg-1 dry weight, SEM 70 in Carb and 434 mmol.kg-1 dry weight, SEM 57 in Norm, indicating that in both tests muscle glycogen stores were decreased by about 25%. Periodic acid-Schiff staining for semi-quantitative glycogen determination in individual fibres confirmed that none of the fibres appeared to be glycogen-empty after exhaustive running. The steady-state respiratory exchange ratio was higher in Carb than in Norm (0.92, SEM 0.01 vs 0.89, SEM 0.01; P less than 0.05). Since muscle glycogen utilization was identical in the two tests, the indication of higher utilization of total carbohydrate appears to be related to a higher utilization of liver glycogen. We have concluded that glycogen depletion of the gastrocnemius muscle is unlikely to be the cause of fatigue during exhaustive running at 75%-80% of VO2max in highly trained endurance runners. Furthermore, diet- and training-induced carbohydrate super-compensation does not appear to improve endurance capacity in such individuals.     

Ingestion of a high-glycemic index meal increases muscle glycogen storage at rest but augments its utilization during subsequent exercise.

The aim of this study was to compare the effect of preexercise breakfast containing high- and low-glycemic index (GI) carbohydrate (CHO) (2.5g CHO/kg body mass) on muscle glycogen metabolism. On two occasions, 14 days apart, seven trained men ran at 71% maximal oxygen uptake for 30 min on a treadmill. Three hours before exercise, in a randomized order, subjects consumed either isoenergetic high- (HGI) or low-GI (LGI) CHO breakfasts that provided (per 70 kg body mass) 3.43 MJ energy, 175 g CHO, 21 g protein, and 4 g fat. The incremental areas under the 3-h plasma glucose and serum insulin response curves after the HGI meal were 3.9- (P < 0.05) and 1.4-fold greater (P < 0.001), respectively, than those after the LGI meal. During the 3-h postprandial period, muscle glycogen concentration increased by 15% (P < 0.05) after the HGI meal but remained unchanged after the LGI meal. Muscle glycogen utilization during exercise was greater in the HGI (129.1 +/- 16.1 mmol/kg dry mass) compared with the LGI (87.9 +/- 15.1 mmol/kg dry mass; P < 0.01) trial. Although the LGI meal contributed less CHO to muscle glycogen synthesis in the 3-h postprandial period compared with the HGI meal, a sparing of muscle glycogen utilization during subsequent exercise was observed in the LGI trial, most likely as a result of better maintained fat oxidation.     

Dissociation of AMPK activity and ACCbeta phosphorylation in human muscle during prolonged exercise

During prolonged, low intensity exercise, the type of substrate utilized varies with time. If 5' AMP-activated protein kinase (AMPK) regulates muscle metabolism during exercise, signaling through AMPK would be expected to change in concordance with changes in substrate utilization. Six healthy, young males cycled (approximately 45% VO(2peak)) until exhaustion (approximately 3.5h). During exercise, leg glucose uptake and rate of glycogenolysis gradually decreased whereas free fatty acid uptake gradually increased. In the thigh muscle, the alpha AMPK subunits became progressively more phosphorylated on Thr(172) during exercise eliciting a parallel increase in alpha2 but not alpha1 AMPK activity. In contrast, after 1h of exercise, Ser(221) phosphorylation of acetyl-CoA carboxylase-beta (ACCbeta) peaked at 1h of exercise and returned to resting levels at exhaustion. Protein expression of alpha2 AMPK, alpha1 AMPK or ACCbeta did not change with time. These data suggest that AMPK signaling is not a key regulatory system of muscle substrate combustion during prolonged exercise and that marked activation of AMPK via phosphorylation is not sufficient to maintain an elevated ACCbeta Ser(221) phosphorylation during prolonged exercise.     

Effects of pre-exercise carbohydrate feedings on muscle glycogen use during exercise in well-trained runners

The purpose of this study was to examine the effects of pre-exercise glucose and fructose feedings on muscle glycogen utilization during exercise in six well-trained runners (VO2max = 68.2 +/- 3.4 ml X kg-1 X min-1). On three separate occasions, the runners performed a 30 min treadmill run at 70% VO2max. Thirty minutes prior to exercise each runner ingested 75 g of glucose (trial G), 75 g of fructose (trial F) or 150 ml of a sweetened placebo (trial C). During exercise, no differences were observed between any of the trials for oxygen uptake, heart rate or perceived exertion. Serum glucose levels were elevated as a result of the glucose feeding (P less than 0.05) reaching peak levels at 30 min post-feeding (7.90 +/- 0.24 mmol X l-1). With the onset of exercise, glucose levels dropped to a low of 5.89 +/- 0.85 mmol X l-1 at 15 min of exercise in trial G. Serum glucose levels in trials F and C averaged 6.21 +/- 0.31 mmol X l-1 and 5.95 +/- 0.23 mmol X l-1 respectively, and were not significantly different (P less than 0.05). There were also no differences in serum glucose levels between any of the trials at 15 and 30 min of exercise.     

Alpha-adrenergic receptor-mediated restraint of skeletal muscle blood flow during prolonged exercise.

Sympathetic nervous system restraint of skeletal muscle blood flow during dynamic exercise has been well documented. However, whether sympathetic restraint of muscle blood flow persists and is constant throughout prolonged exercise has not been established. We hypothesized that both alpha1- and alpha2-adrenergic receptors would restrain skeletal muscle blood flow throughout prolonged constant-load exercise and that the restraint would increase as a function of exercise duration. Mongrel dogs were instrumented chronically with transit-time flow probes on the external iliac arteries and an indwelling catheter in a branch of the femoral artery. Flow-adjusted doses of selective alpha1- (prazosin) and alpha2-adrenergic receptor (rauwolscine) antagonists were infused after 5, 30, and 50 min of treadmill exercise at 3 and 6 miles/h. During mild-intensity exercise (3 miles/h), prazosin infusion resulted in a greater (P < 0.05) increase in vascular conductance (VC) after 5 [42% (SD 6)], compared with 30 [28% (SD 6)] and 50 [28% (SD 8)] min of running. In contrast, prazosin resulted in a similar increase in VC after 5 [29% (SD 10)], 30 [24% (SD 9)], and 50 [22% (SD 9)] min of moderate-intensity (6 miles/h) exercise. Rauwolscine infusion resulted in a greater (P < 0.05) increase in VC after 5 [39% (SD 14)] compared with 30 [26% (SD 9)] and 50 [22% (SD 4)] min of exercise at 3 miles/h. Rauwolscine infusion produced a similar increase in VC after 5 [19% (SD 3)], 30 [15% (SD 6)], and 50 [16% (SD 2)] min of exercise at 6 miles/h. These results suggest that the ability of alpha1- and alpha2-adrenergic receptors to produce vasoconstriction and restrain blood flow to active muscles may be influenced by both the intensity and duration of exercise.     

Human muscle function following prolonged eccentric exercise

4 subjects performed repeated eccentric contractions with leg extensors during prolonged downhill walking (-25% gradient) at 6.44 km.h-1 until collapse due to muscle weakness (range of exercise duration 29 to 40 min). During the exercise oxygen uptake rose progressively from approximately 45% of the previously determined VO2max at 10 min to approximately 65% at the end of the exercise. Following the exercise there was an immediate, significant, and sustained reduction in maximal voluntary isometric contraction, and short term (anaerobic) power output measured concentrically on an isokinetic ergometer. These reductions in muscle function persisted for 96 hours post exercise, and were reflected by significant reductions in the tension generated at low frequency (20 Hz) relative to higher frequency (50 Hz) percutaneous stimulation of the quadriceps. All four subjects showed an increase in plasma levels of creatine kinase post eccentric exercise. Performing concentric contractions by walking uphill for one hour at a significantly greater metabolic cost failed to induce comparable reductions in muscle function. These results provide evidence for the consequences of prolonged eccentric work upon dynamic function which complements earlier reports of structural, enzymatic, and static function changes.     

Effect of epinephrine on glucose disposal during exercise in humans: role of muscle glycogen

This study examined the effect of epinephrine on glucose disposal during moderate exercise when glycogenolytic flux was limited by low preexercise skeletal muscle glycogen availability. Six male subjects cycled for 40 min at 59 +/- 1% peak pulmonary O2 uptake on two occasions, either without (CON) or with (EPI) epinephrine infusion starting after 20 min of exercise. On the day before each experimental trial, subjects completed fatiguing exercise and then maintained a low carbohydrate diet to lower muscle glycogen. Muscle samples were obtained after 20 and 40 min of exercise, and glucose kinetics were measured using [6,6-2H]glucose. Exercise increased plasma epinephrine above resting concentrations in both trials, and plasma epinephrine was higher (P < 0.05) during the final 20 min in EPI compared with CON. Muscle glycogen levels were low after 20 min of exercise (CON, 117 +/- 25; EPI, 122 +/- 20 mmol/kg dry matter), and net muscle glycogen breakdown and muscle glucose 6-phosphate levels during the subsequent 20 min of exercise were unaffected by epinephrine infusion. Plasma glucose increased with epinephrine infusion (i.e., 20-40 min), and this was due to a decrease in glucose disposal (R(d)) (40 min: CON, 33.8 +/- 3; EPI, 20.9 +/- 4.9 micromol. kg(-1). min(-1), P < 0.05), because the exercise-induced rise in glucose rate of appearance was similar in the trials. These results show that glucose R(d) during exercise is reduced by elevated plasma epinephrine, even when muscle glycogen availability and utilization are low. This suggests that the effect of epinephrine does not appear to be mediated by increased glucose 6-phosphate, secondary to enhanced muscle glycogenolysis, but may be linked to a direct effect of epinephrine on sarcolemmal glucose transport.     

Effect of infusing branched-chain amino acid during incremental exercise with reduced muscle glycogen content

The aim of this study was to investigate whether, when muscle glycogen is reduced, a pre-exercise infusion of branched-chain amino acids (BCAA) modifies exercise performance or the metabolic and respiratory responses to incremental exercise. Six moderately trained volunteers took part in the following protocol on two occasions. On day 1, at 9 a.m. in the postabsorptive state, they performed a graded incremental exercise (increases of 35 W every 4 min) to exhaustion (Ex-1). A meal of 1,000 kcal (4,200 kJ; 60% protein, 40% fat) was consumed at 12 p.m. No food was then allowed until the end of the experiment (20–21 h later). A 90-min period of exercise at alternating high and moderate intensities, designed to deplete muscle glycogen, was performed between 6 p.m. and 7.30 p.m. The morning after (day 2), the subjects randomly received either a mixed solution of BCAA (260 mg × kg^–1 × h^–1 for 70 min), or saline. They then repeated the graded incremental exercise to exhaustion (Ex-2). Metabolic and respiratory measurements suggested a muscle glycogen-depleted state had been achieved. No significant differences were observed in total work performed, maximal oxygen uptake or plasma ammonia, alanine, and blood pyruvate concentrations in the two treatments. After BCAA infusion, higher blood lactate concentrations were observed at maximal power output in comparison with those during saline [BCAA 4.97 (SEM 0.41) mmol × l^–1, Saline 3.88 (SEM 0.47) mmol × l^–1,P < 0.05]. In summary, in conditions of reduced muscle glycogen content, after a short period of fasting, BCAA infusion had no significant effect on the total work that could be performed during a graded incremental exercise.     

Fiber type-specific muscle glycogen sparing due to carbohydrate intake before and during exercise.

The effect of carbohydrate intake before and during exercise on muscle glycogen content was investigated. According to a randomized crossover study design, eight young healthy volunteers (n = 8) participated in two experimental sessions with an interval of 3 wk. In each session subjects performed 2 h of constant-load bicycle exercise ( approximately 75% maximal oxygen uptake). On one occasion (CHO), they received carbohydrates before ( approximately 150 g) and during (1 g.kg body weight(-1).h(-1)) exercise. On the other occasion they exercised after an overnight fast (F). Fiber type-specific relative glycogen content was determined by periodic acid Schiff staining combined with immunofluorescence in needle biopsies from the vastus lateralis muscle before and immediately after exercise. Preexercise glycogen content was higher in type IIa fibers [9.1 +/- 1 x 10(-2) optical density (OD)/microm(2)] than in type I fibers (8.0 +/- 1 x 10(-2) OD/microm(2); P < 0.0001). Type IIa fiber glycogen content decreased during F from 9.6 +/- 1 x 10(-2) OD/microm(2) to 4.5 +/- 1 x 10(-2) OD/microm(2) (P = 0.001), but it did not significantly change during CHO (P = 0.29). Conversely, in type I fibers during CHO and F the exercise bout decreased glycogen content to the same degree. We conclude that the combination of carbohydrate intake both before and during moderate- to high-intensity endurance exercise results in glycogen sparing in type IIa muscle fibers.     

Rapid upregulation of pyruvate dehydrogenase kinase activity in human skeletal muscle during prolonged exercise.

Prolonged moderate-intensity exercise is characterized by a progressive reduction in carbohydrate oxidation and concomitant increase in fat oxidation. Pyruvate dehydrogenase (PDH) controls the entry of pyruvate into oxidative pathways and is a rate-limiting enzyme for carbohydrate metabolism. PDH is controlled by the activities of a kinase (PDK, inhibitory) and phosphatase (stimulatory). To test the hypothesis that increased PDK activity was associated with decreased PDH activity and carbohydrate oxidation during an acute exercise bout, seven recreationally active men completed 4 h of cycle exercise at 55% peak oxygen consumption. Muscle samples were obtained before and at 10 min and 4 h of exercise for the measurement of PDH activity and the extraction of intact mitochondria for the measurements of PDK activity and PDK-2 and PDK-4 protein expression. Carbohydrate oxidation was reduced (P < 0.05) with exercise duration. Muscle glycogen content was lower (P < or = 0.05) at 4 h compared with rest and there was no change in muscle pyruvate content from 10 to 240 min during exercise (10 min: 0.28 +/- 0.05; 240 min: 0.35 +/- 0.09 mmol/kg dry muscle). PDH activity increased (P < 0.05) above resting values at 10 min (2.86 +/- 0.26 mmol.min(-1).kg wet muscle(-1)), but was lower than 10 min after 4 h (2.23 +/- 0.24 mmol.min(-1).kg wet muscle(-1)) of exercise. PDK-2 and PDK-4 protein expression was not different from rest at 10 min and 4 h of exercise. PDK activity at rest averaged 0.081 +/- 0.016 min(-1), was similar at 10 min, and increased (P < 0.05) to 0.189 +/- 0.013 min(-1) at 4 h. Although reduced glycolytic flux may have played a role in decreasing carbohydrate oxidation, the results suggest that increased PDK activity contributed to the reduction in PDH activity and carbohydrate oxidation late in prolonged exercise. The increased PDK activity was independent of changes in intra-mitochondrial effectors, and PDK-2 and PDK-4 protein content, suggesting that it was caused by a change in the specific activity of the existing kinases.     

Hormone-sensitive lipase activity and fatty acyl-CoA content in human skeletal muscle during prolonged exercise.

Hormone-sensitive lipase (HSL) catalyzes the hydrolysis of intramuscular triacylglycerols (IMTGs), but HSL regulation is poorly understood in skeletal muscle. The present study measured human skeletal muscle HSL activity at rest and during 120 min of cycling at 60% of peak O2 uptake. Several putative HSL regulators were also measured, including muscle long-chain fatty acyl-CoA (LCFA CoA) and free AMP contents and plasma epinephrine and insulin concentrations. HSL activity increased from resting levels by 10 min of exercise (from 2.09 +/- 0.19 to 2.56 +/- 0.22 mmol. min-1x kg dry mass-1, P < 0.05), increased further by 60 min (to 3.12 +/- 0.27 mmol x min-1x kg dry mass-1, P < 0.05), and decreased to near-resting rates after 120 min of cycling. Skeletal muscle LCFA CoA increased (P < 0.05) above rest by 60 min (from 15.9 +/- 3.0 to 50.4 +/- 7.9 micromol/kg dry mass) and increased further by 120 min. Estimated free AMP increased (P < 0.05) from rest to 60 min and was approximately 20-fold greater than that at rest by 120 min. Epinephrine was increased above rest (P < 0.05) at 60 (1.47 +/- 0.15 nM) and 120 min (4.87 +/- 0.76 nM) of exercise. Insulin concentrations decreased rapidly and were lower than resting levels by 10 min and continued to decrease throughout exercise. In summary, HSL activity was increased from resting levels by 10 min, increased further by 60 min, and decreased to near-resting values by 120 min. The increased HSL activity at 60 min was associated with the stimulating effect of increased epinephrine and decreased insulin levels. After 120 min, the decreased HSL activity was associated with the proposed inhibitory effects of increased free AMP. The accumulation of LCFA CoA in the 2nd h of exercise may also have reduced the flux through HSL and accounted for the reduction in IMTG utilization previously observed late in prolonged exercise.     

Effects of a rapid change in muscle glycogen availability on metabolic and hormonal responses during exercise

To evaluate the metabolic and hormonal adaptations following a rapid change in muscle glycogen availability, 14 subjects had their muscle glycogen content increased in one leg (IG) and decreased in the other (DG). In group A (n = 7), subjects exercised on a bicycle ergometer at 70% maximal oxygen uptake for 20 min using the DG leg. Without resting these same subjects exercised another 20 min using the IG leg. Subjects in group B (n = 7) followed the same single-leg exercise protocol but in the reverse order. In order to get some information on the time sequence of these possible adaptations, blood samples were collected at rest and at the beginning and the end of each exercise period (min 5, 20, 25, and 40). Results indicated that 5 min after the switch from the DG leg to the IG leg, transient increases in plasma free fatty acids (1.20 to 1.39 meq X 1(-1)) and serum insulin (10.1 to 12 mu X 1(-1)) concentrations occurred. Between minute 25 and 40 of exercise, the DG to IG switch was accompanied by a decrease in free fatty acids and glycerol concentrations as well as an increase in lactate levels. An opposite response was observed in the IG to DG condition during the same time span. Plasma norepinephrine, epinephrine, glucagon, and serum cortisol concentrations were not significantly affected by the leg change. These results suggest a rapid preferential use of muscle glycogen when available and a time lag in the response of the extramuscular substrate mobilization factors.