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The glycogen content of muscle determines not only our capacity for exercise but also the signaling events that occur in response to exercise. The result of the shift in signaling is that frequent training in a low-glycogen state results in improved fat oxidation during steady-state submaximal exercise. This review will discuss how the amount or localization of glycogen particles can directly or indirectly result in this differential response to training. The key direct effect discussed is carbohydrate binding, whereas the indirect effects include the metabolic shift toward fat oxidation, the increase in catecholamines, and osmotic stress. Although our understanding of the role of glycogen in response to training has expanded exponentially over the past 5 years, there are still many questions remaining as to how stored carbohydrate affects the muscular adaptation to exercise.

Women exhibit an enhanced capability for lipid metabolism during endurance exercise compared with men. The underlying regulatory mechanisms behind this sex-related difference are not well understood but may comprise signaling through a myocyte enhancer factor 2 (MEF2) regulatory pathway. The primary purpose of this study, therefore, was to investigate the protein signaling of MEF2 regulatory pathway components at rest and during 90 min of bicycling exercise at 60% Vo(2peak) in healthy, moderately trained men (n = 8) and women (n = 9) to elucidate the potential role of these proteins in substrate utilization during exercise. A secondary purpose was to screen for mRNA expression of MEF2 isoforms and myogenic regulatory factor (MRF) family members of transcription factors at rest and during exercise. Muscle biopsies were obtained before and immediately after exercise. Nuclear AMP-activated protein kinase-alpha (alphaAMPK) Thr(172) (P < 0.001), histone deacetylase 5 (HDAC5) Ser(498) (P < 0.001), and MEF2 Thr (P < 0.01) phosphorylation increased with exercise. No significant sex differences were observed at rest or during exercise. At rest, no significant sex differences were observed in mRNA expression of the measured transcription factors. mRNA for transcription factors MyoD, myogenin, MRF4, MEF2A, MEF2C, MEF2D, and peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC1alpha) were significantly upregulated by exercise. Of these, MEF2A mRNA increased 25% specifically in women (P < 0.05), whereas MEF2D mRNA tended to increase in men (P = 0.11). Although minor sex differences in mRNA expression were observed, the main finding of the present study was the implication of a joint signaling action of AMPK, HDAC5, and PGC1alpha on MEF2 in the immediate regulatory response to endurance exercise. This signaling response was independent of sex.

As substrate for evaluation of metabolic diseases, we developed novel rat models that contrast for endurance exercise capacity. Through two-way artificial selection, we created rodent phenotypes of intrinsically low-capacity runners (LCR) and high-capacity runners (HCR) that also differed markedly for cardiovascular and metabolic disease risk factors. Here, we determined skeletal muscle proteins with putative roles in lipid and carbohydrate metabolism to better understand the mechanisms underlying differences in whole body substrate handling between phenotypes. Animals (generation 16) differed for endurance running capacity by 295%. LCR animals had higher resting plasma glucose (6.58 +/- 0.45 vs. 6.09 +/- 0.45 mmol/l), insulin (0.48 +/- 0.03 vs. 0.32 +/- 0.02 ng/ml), nonesterified fatty acid (0.57 +/- 0.14 v 0.35 +/- 0.05 mM), and triglyceride (TG; 0.47 +/- 0.11 vs. 0.25 +/- 0.08 mmol/l) concentrations (all P < 0.05). Muscle TG (72.3 +/- 14.7 vs. 38.9 +/- 6.2 mmol/kg dry muscle wt; P < 0.05) and diacylglycerol (96 +/- 28 vs. 42 +/- 8 pmol/mg dry muscle wt; P < 0.05) contents were elevated in LCR vs. HCR rats. Accompanying the greater lipid accretion in LCR was increased fatty acid translocase/CD36 content (1,014 +/- 80 vs. 781 +/- 70 arbitrary units; P < 0.05) and reduced TG lipase activity (0.158 +/- 0.0125 vs. 0.274 +/- 0.018 mmol.min(-1).kg dry muscle wt(-1); P < 0.05). Muscle glycogen, GLUT4 protein, and basal phosphorylation states of AMP-activated protein kinase-alpha1, AMP-activated protein kinase-alpha2, and acetyl-CoA carboxylase were similar in LCR and HCR. In conclusion, rats with low intrinsic aerobic capacity demonstrate abnormalities in lipid-handling capacity. These disruptions may, in part, be responsible for the increased risk of metabolic disorders observed in this phenotype.

There is evidence that increasing carbohydrate (CHO) availability during exercise by raising preexercise muscle glycogen levels attenuates the activation of AMPKalpha2 during exercise in humans. Similarly, increasing glucose levels decreases AMPKalpha2 activity in rat skeletal muscle in vitro. We examined the effect of CHO ingestion on skeletal muscle AMPK signaling during exercise in nine active male subjects who completed two 120-min bouts of cycling exercise at 65 +/- 1% V(O2 peak). In a randomized, counterbalanced order, subjects ingested either an 8% CHO solution or a placebo solution during exercise. Compared with the placebo trial, CHO ingestion significantly (P < 0.05) increased plasma glucose levels and tracer-determined glucose disappearance. Exercise-induced increases in muscle-calculated free AMP (17.7- vs. 11.8-fold), muscle lactate (3.3- vs. 1.8-fold), and plasma epinephrine were reduced by CHO ingestion. However, the exercise-induced increases in skeletal muscle AMPKalpha2 activity, AMPKalpha2 Thr(172) phosphorylation and acetyl-CoA Ser(222) phosphorylation, were essentially identical in the two trials. These findings indicate that AMPK activation in skeletal muscle during exercise in humans is not sensitive to changes in plasma glucose levels in the normal range. Furthermore, the rise in plasma epinephrine levels in response to exercise was greatly suppressed by CHO ingestion without altering AMPK signaling, raising the possibility that epinephrine does not directly control AMPK activity during muscle contraction under these conditions in vivo.

We compared in human skeletal muscle the effect of absolute vs. relative exercise intensity on AMP-activated protein kinase (AMPK) signaling and substrate metabolism under normoxic and hypoxic conditions. Eight untrained males cycled for 30 min under hypoxic conditions (11.5% O(2), 111 +/- 12 W, 72 +/- 3% hypoxia Vo(2 peak); 72% Hypoxia) or under normoxic conditions (20.9% O(2)) matched to the same absolute (111 +/- 12 W, 51 +/- 1% normoxia Vo(2 peak); 51% Normoxia) or relative (to Vo(2 peak)) intensity (171 +/- 18 W, 73 +/- 1% normoxia Vo(2 peak); 73% Normoxia). Increases (P < 0.05) in AMPK activity, AMPKalpha Thr(172) phosphorylation, ACCbeta Ser(221) phosphorylation, free AMP content, and glucose clearance were more influenced by the absolute than by the relative exercise intensity, being greatest in 73% Normoxia with no difference between 51% Normoxia and 72% Hypoxia. In contrast to this, increases in muscle glycogen use, muscle lactate content, and plasma catecholamine concentration were more influenced by the relative than by the absolute exercise intensity, being similar in 72% Hypoxia and 73% Normoxia, with both trials higher than in 51% Normoxia. In conclusion, increases in muscle AMPK signaling, free AMP content, and glucose disposal during exercise are largely determined by the absolute exercise intensity, whereas increases in plasma catecholamine levels, muscle glycogen use, and muscle lactate levels are more closely associated with the relative exercise intensity.

The subcellular localization of insulin signaling proteins is altered by various stimuli such as insulin, insulin-like growth factor I, and oxidative stress and is thought to be an important mechanism that can influence intracellular signal transduction and cellular function. This study examined the possibility that exercise may also alter the subcellular localization of insulin signaling proteins in human skeletal muscle. Nine untrained males performed 60 min of cycling exercise (approximately 67% peak pulmonary O2 uptake). Muscle biopsies were sampled at rest, immediately after exercise, and 3 h postexercise. Muscle was fractionated by centrifugation into the following crude fractions: cytosolic, nuclear, and a high-speed pellet containing membrane and cytoskeletal components. Fractions were analyzed for protein content of insulin receptor, insulin receptor substrate (IRS)-1 and -2, p85 subunit of phosphatidylinositol 3-kinase, Akt, and glycogen synthase kinase-3 (GSK-3). There was no significant change in the protein content of the insulin signaling proteins in any of the crude fractions after exercise or 3 h postexercise. Exercise had no significant effect on the phosphorylation of IRS-1 Tyr612 in any of the fractions. In contrast, exercise increased (P < 0.05) the phosphorylation of Akt Ser473 and GSK-3alpha/beta Ser9/21 in the cytosolic fraction only. In conclusion, exercise can increase phosphorylation of downstream insulin signaling proteins specifically in the cytosolic fraction but does not result in changes in the subcellular localization of insulin signaling proteins in human skeletal muscle. Change in the subcellular protein localization is therefore an unlikely mechanism to influence signal transduction pathways and cellular function in skeletal muscle after exercise.

Five days of a high-fat diet while training, followed by 1 day of carbohydrate (CHO) restoration, increases rates of whole body fat oxidation and decreases CHO oxidation during aerobic cycling. The mechanisms responsible for these shifts in fuel oxidation are unknown but involve up- and downregulation of key regulatory enzymes in the pathways of skeletal muscle fat and CHO metabolism, respectively. This study measured muscle PDH and HSL activities before and after 20 min of cycling at 70% VO2peak and 1 min of sprinting at 150% peak power output (PPO). Estimations of muscle glycogenolysis were made during the initial minute of exercise at 70% VO2peak and during the 1-min sprint. Seven male cyclists undertook this exercise protocol on two occasions. For 5 days, subjects consumed in random order either a high-CHO (HCHO) diet (10.3 g x kg(-1) x day(-1) CHO, or approximately 70% of total energy intake) or an isoenergetic high-fat (FAT-adapt) diet (4.6 g x kg(-1) x day(-1) FAT, or 67% of total energy) while undertaking supervised aerobic endurance training. On day 6 for both treatments, subjects ingested an HCHO diet and rested before their experimental trials on day 7. This CHO restoration resulted in similar resting glycogen contents (FAT-adapt 873 +/- 121 vs. HCHO 868 +/- 120 micromol glucosyl units/g dry wt). However, the respiratory exchange ratio was lower during cycling at 70% VO2peak in the FAT-adapt trial, which resulted in an approximately 45% increase and an approximately 30% decrease in fat and CHO oxidation, respectively. PDH activity was lower at rest and throughout exercise at 70% VO2peak (1.69 +/- 0.25 vs. 2.39 +/- 0.19 mmol x kg wet wt(-1) x min(-1)) and the 1-min sprint in the FAT-adapt vs. the HCHO trial. Estimates of glycogenolysis during the 1st min of exercise at 70% VO2peak and the 1-min sprint were also lower after FAT-adapt (9.1 +/- 1.1 vs. 13.4 +/- 2.1 and 37.3 +/- 5.1 vs. 50.5 +/- 2.7 glucosyl units x kg dry wt(-1) x min(-1)). HSL activity was approximately 20% higher (P = 0.12) during exercise at 70% VO2peak after FAT-adapt. Results indicate that previously reported decreases in whole body CHO oxidation and increases in fat oxidation after the FAT-adapt protocol are a function of metabolic changes within skeletal muscle. The metabolic signals responsible for the shift in muscle substrate use during cycling at 70% VO2peak remain unclear, but lower accumulation of free ADP and AMP after the FAT-adapt trial may be responsible for the decreased glycogenolysis and PDH activation during sprinting.

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.

Nine endurance-trained men exercised on a cycle ergometer at approximately 68% peak O2 uptake to the point of volitional fatigue [232 +/- 14 (SE) min] while ingesting an 8% carbohydrate solution to determine how high glucose disposal could increase under physiological conditions. Plasma glucose kinetics were measured using a primed, continuous infusion of [6,6-2H]glucose and the appearance of ingested glucose, assessed from [3-3H]glucose that had been added to the carbohydrate drink. Plasma glucose was increased (P < 0.05) after 30 min of exercise but thereafter remained at the preexercise level. Glucose appearance rate (R(a)) increased throughout exercise, reaching its peak value of 118 +/- 7 micromol. kg(-1). min(-1) at fatigue, whereas gut R(a) increased continuously during exercise, peaking at 105 +/- 10 micromol. kg(-1). min(-1) at the point of fatigue. In contrast, liver glucose output never rose above resting levels at any time during exercise. Glucose disposal (R(d)) increased throughout exercise, reaching a peak value of 118 +/- 7 micromol. kg(-1). min(-1) at fatigue. If we assume 95% oxidation of glucose R(d), estimated exogenous glucose oxidation at fatigue was 1.36 +/- 0.08 g/min. The results of this study demonstrate that glucose uptake increases continuously during prolonged, strenuous exercise when carbohydrate is ingested and does not appear to limit exercise performance.

The effects of a single bout of exercise and exercise training on the expression of genes necessary for the transport and beta-oxidation of fatty acids (FA), together with the gene expression of transcription factors implicated in the regulation of FA homeostasis were investigated. Seven human subjects (3 male, 4 female, 28.9 +/- 3.1 yr of age, range 20-42 yr, body mass index 22.6 kg/m(2), range 17-26 kg/m(2)) underwent a 9-day exercise training program of 60 min cycling per day at 63% peak oxygen uptake (VO(2 peak); 104 +/- 14 W). On days 1 and 9 of the program, muscle biopsies were sampled from the vastus lateralis muscle at rest, at the completion of exercise, and again 3 h postexercise. Gene expression of key components of FA transport [FA translocase (FAT/CD36), plasma membrane-associated FA-binding protein], beta-oxidation [carntine palmitoyltransferase(CPT) I, beta-hydroxyacyl-CoA dehydrogenase] and transcriptional control [peroxisome proliferator-activated receptor (PPAR)alpha, PPAR gamma, PPAR gamma coactivator 1, sterol regulatory element-binding protein-1c] were unaltered by exercise when measured at the completion and at 3 h postexercise. Training increased total lipid oxidation by 24% (P < 0.05) for the 1-h cycling bout. This increased capacity for lipid oxidation was accompanied by an increased expression of FAT/CD36 and CPT I mRNA. Similarly, FAT/CD36 protein abundance was also upregulated by exercise training. We conclude that enhanced fat oxidation after exercise training is most closely associated with the genes involved in regulating FA uptake across the plasma membrane (FAT/CD36) and across the mitochondrial membrane (CPT I).

Several studies have demonstrated that oral glucose tolerance is impaired in the immediate postexercise period. A double-tracer technique was used to examine glucose kinetics during a 2-h oral glucose (75 g) tolerance test (OGTT) 30 min after exercise (Ex, 55 min at 71 +/- 2% of peak O(2) uptake) and 24 h after exercise (Rest) in endurance-trained men. The area under the plasma glucose curve was 71% greater in Ex than in Rest (P = 0.01). The higher glucose response occurred even though whole body rate of glucose disappearance was 24% higher after exercise (P = 0.04, main effect). Whole body rate of glucose appearance was 25% higher after exercise (P = 0.03, main effect). There were no differences in total (2 h) endogenous glucose appearance (R(a)E) or the magnitude of suppression of R(a)E, although R(a)E was higher from 15 to 30 min during the OGTT in Ex. However, the cumulative appearance of oral glucose was 30% higher in Ex (P = 0.03, main effect). There were no differences in glucose clearance rate or plasma insulin responses between the two conditions. These results suggest that adaptations in splanchnic tissues by prior exercise facilitate greater glucose output from the splanchnic region after glucose ingestion, resulting in a greater glycemic response and, consequently, a greater rate of whole body glucose uptake.