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Exercise induces signaling networks to improve muscle function and confer health benefits. To identify divergent and common signaling networks during and after different exercise modalities, we performed a phosphoproteomic analysis of human skeletal muscle from a cross-over intervention of endurance, sprint, and resistance exercise. This identified 5,486 phosphosites regulated during or after at least one type of exercise modality and only 420 core phosphosites common to all exercise. One of these core phosphosites was S67 on the uncharacterized protein C18ORF25, which we validated as an AMPK substrate. Mice lacking C18ORF25 have reduced skeletal muscle fiber size, exercise capacity, and muscle contractile function, and this was associated with reduced phosphorylation of contractile and Ca 2+ handling proteins. Expression of C18ORF25 S66/67D phospho-mimetic reversed the decreased muscle force production. This work defines the divergent and canonical exercise phosphoproteome across different modalities and identifies C18ORF25 as a regulator of exercise signaling and muscle function., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Crown Copyright © 2022. Published by Elsevier Inc. All rights reserved.)

The coordination of nutrient sensing, delivery, uptake and utilization is essential for maintaining cellular, tissue and whole-body homeostasis. Such synchronization can be achieved only if metabolic information is communicated between the cells and tissues of the entire organism. During intense exercise, the metabolic demand of the body can increase approximately 100-fold. Thus, exercise is a physiological state in which intertissue communication is of paramount importance. In this Review, we discuss the physiological processes governing intertissue communication during exercise and the molecules mediating such cross-talk.

New Findings: What is the central question of this study? Do circulating factors mediate exercise-induced effects on adipose tissue GLUT4 expression? What is the main finding and its importance? Serum (10%) obtained from human volunteers immediately after a single exercise bout increased GLUT4 protein levels in human adipocytes in culture. This result suggests that circulating factors might mediate the effects of exercise on adipose tissue GLUT4 and prompts further effort to identify the specific factor(s) and tissue(s) of origin., Abstract: In this study, we tested the hypothesis that circulating factors generated during exercise increase adipose tissue GLUT4 expression. Serum was obtained from eight healthy subjects before and after 60 min of cycling exercise, and primary adipocytes were cultured from stromal vascular fractions that were isolated from subcutaneous abdominal adipose tissue samples from one healthy, male volunteer. A 48 h exposure of human primary adipocytes to 10% serum obtained after exercise increased GLUT4 protein expression, on average, by 12% compared with exposure to 10% serum obtained at rest, before exercise. GLUT4 mRNA levels were increased after 12 h of exposure to exercise serum but were unchanged after 6 and 24 h of exposure. Our results suggest that circulating factors might mediate the effects of exercise on adipose tissue GLUT4 expression and encourage further efforts to identify the potential factor(s), tissue(s) of origin and physiological relevance., (© 2019 The Authors. Experimental Physiology © 2019 The Physiological Society.)

Triglycerides are stored within lipid droplets in skeletal muscle and can be hydrolyzed to produce fatty acids for energy production through β-oxidation and oxidative phosphorylation. While there was some controversy regarding the quantitative importance of intramyocellular triglyceride (IMTG) as a metabolic substrate, recent advances in proton magnetic resonance spectroscopy and confocal microscopy support earlier tracer and biopsy studies demonstrating a substantial contribution of IMTG to energy production, particularly during moderate-intensity endurance exercise. This review provides an update on the understanding of IMTG utilization during exercise, with a focus on describing the key regulatory proteins that control IMTG breakdown and how these proteins respond to acute exercise and in the adaptation to exercise training. This article is part of a Special Issue entitled: Recent Advances in Lipid Droplet Biology edited by Rosalind Coleman and Matthijs Hesselink., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Adipocytes are major regulators of metabolism, and endurance exercise training improves adipocyte function; however, the molecular mechanisms that regulate chronic adaptive responses remain unresolved. microRNAs (miRNAs) influence adipocyte differentiation and metabolism. Accordingly, we aimed to determine whether adipocyte miRNA expression is responsive to exercise training and to identify exercise-responsive miRNAs that influence adipocyte metabolism. Next-generation sequencing was used to profile miRNA expression of adipocytes that were isolated from abdominal subcutaneous (ABD) and gluteofemoral (GF) adipose tissue of overweight men before and after 6 wk of endurance exercise training. Differentially expressed miRNAs were overexpressed or silenced in 3T3-L1 adipocytes, and lipid metabolism was examined. Next-generation sequencing identified 526 miRNAs in adipocytes, and there were no statistical differences in miRNA expression when comparing pre- and post-training samples for ABD and GF adipocytes. miR-10b expression was increased in ABD compared with GF adipocytes, whereas miR-204, miR-3613, and miR-4532 were more highly expressed in GF compared with ABD adipocytes. Blocking miR-10b in adipocytes suppressed β-adrenergic lipolysis but generally had a minor effect on lipid metabolism. Thus, unlike their critical role in adipogenesis, stable changes in miRNA expression do not play a prominent role in the regulation of adipocyte function in response to endurance exercise training.-Tsiloulis, T., Pike, J., Powell, D., Rossello, F. J., Canny, B. J., Meex, R. C. R., Watt, M. J. Impact of endurance exercise training on adipocyte microRNA expression in overweight men., (© FASEB.)

Adipose tissue is a major regulator of metabolism in health and disease. The prominent roles of adipose tissue are to sequester fatty acids in times of energy excess and to release fatty acids via the process of lipolysis during times of high-energy demand, such as exercise. The fatty acids released during lipolysis are utilized by skeletal muscle to produce adenosine triphosphate to prevent fatigue during prolonged exercise. Lipolysis is controlled by a complex interplay between neuro-humoral regulators, intracellular signaling networks, phosphorylation events involving protein kinase A, translocation of proteins within the cell, and protein-protein interactions. Herein, we describe in detail the cellular and molecular regulation of lipolysis and how these processes are altered by acute exercise. We also explore the processes that underpin adipocyte adaptation to endurance exercise training, with particular focus on epigenetic modifications, control by microRNAs and mitochondrial adaptations. Finally, we examine recent literature describing how exercise might influence the conversion of traditional white adipose tissue to high energy-consuming "brown-like" adipocytes and the implications that this has on whole-body energy balance., (© 2015 Elsevier Inc. All rights reserved.)

Lipolysis involves the sequential breakdown of fatty acids from triacylglycerol and is increased during energy stress such as exercise. Adipose triglyceride lipase (ATGL) is a key regulator of skeletal muscle lipolysis and perilipin (PLIN) 5 is postulated to be an important regulator of ATGL action of muscle lipolysis. Hence, we hypothesized that non-genomic regulation such as cellular localization and the interaction of these key proteins modulate muscle lipolysis during exercise. PLIN5, ATGL and CGI-58 were highly (>60%) colocated with Oil Red O (ORO) stained lipid droplets. PLIN5 was significantly colocated with ATGL, mitochondria and CGI-58, indicating a close association between the key lipolytic effectors in resting skeletal muscle. The colocation of the lipolytic proteins, their independent association with ORO and the PLIN5/ORO colocation were not altered after 60 min of moderate intensity exercise. Further experiments in cultured human myocytes showed that PLIN5 colocation with ORO or mitochondria is unaffected by pharmacological activation of lipolytic pathways. Together, these data suggest that the major lipolytic proteins are highly expressed at the lipid droplet and colocate in resting skeletal muscle, that their localization and interactions appear to remain unchanged during prolonged exercise, and, accordingly, that other post-translational mechanisms are likely regulators of skeletal muscle lipolysis.

Context: The accumulation of intramyocellular lipids (IMCLs) and mitochondrial dysfunction in skeletal muscle have been associated with insulin resistance in obesity. Endurance training (ET) increases mitochondrial content/activity and IMCL content in young, active men and women. We have previously shown that ET alters the size, number, and physical juxtaposition of IMCLs and mitochondria., Objective: The purpose of this study was to determine the effects of obesity and ET on mitochondrial function, IMCL content, and IMCL-mitochondria juxtaposition in sedentary lean and obese women. DESIGN, SETTING, SUBJECTS, INTERVENTION, AND MAIN OUTCOME MEASURES: Obese (n = 11) and lean (n = 12), sedentary women were recruited using local advertisements and underwent 12 weeks of ET in our training facility at McMaster University. Blood and muscle biopsy samples (vastus lateralis) were collected before and after ET to measure IMCL and mitochondrial ultrastructure, mitochondrial oxidative capacity, lipid oxidation capacity, and lipid metabolism by-products., Results: Obese women were insulin resistant (homeostasis model assessment of insulin resistance) compared with lean women. ET did not change body weight but increased mitochondrial oxidative and β-oxidation capacity in both groups. ET mediated reorganization of the muscle architecture, whereby IMCL content in the subsarcolemmal region was reduced with a concomitant increase in intermyofibrillar IMCLs. ET increased the percentage of IMCLs in direct contact with mitochondria and did not alter diacylglycerol and ceramide content in either group., Conclusions: ET mediated positive changes in mitochondrial function and lipid oxidation and induced intracellular IMCL reorganization, which is reflective of greater IMCL turnover capacity in both lean and obese women.

Pigment epithelium-derived factor (PEDF) is upregulated in obese rodents and is involved in the development of insulin resistance (IR). We aim to explore the relationships between PEDF, adiposity, insulin sensitivity, and cardiovascular risk factors in obese women with polycystic ovary syndrome (PCOS) and weight-matched controls and to examine the impact of endurance exercise training on PEDF. This prospective cohort intervention study was based at a tertiary medical center. Twenty obese PCOS women and 14 non-PCOS weight-matched women were studied at baseline. PEDF, cardiometabolic markers, detailed body composition, and euglycemic-hyperinsulinemic clamps were performed and measures were repeated in 10 PCOS and 8 non-PCOS women following 12 weeks of intensified aerobic exercise. Mean glucose infusion rate (GIR) was 31.7% lower (P = 0.02) in PCOS compared to controls (175.6 ± 96.3 and 257.2 ± 64.3 mg.m(-2).min(-1)) at baseline, yet both PEDF and BMI were similar between groups. PEDF negatively correlated to GIR (r = -0.41, P = 0.03) and high-density lipoprotein (HDL) (r = -0.46, P = 0.01), and positively to cardiovascular risk factors, systolic (r = 0.41, P = 0.02) and diastolic blood pressure (r = 0.47, P = 0.01) and triglycerides (r = 0.49, P = 0.004). The correlation with GIR was not significant after adjusting for fat mass (P = 0.07). Exercise training maintained BMI and increased GIR in both groups; however, plasma PEDF was unchanged. In summary, PEDF is not elevated in PCOS, is not associated with IR when adjusted for fat mass, and is not reduced by endurance exercise training despite improved insulin sensitivity. PEDF was associated with cardiovascular risk factors, suggesting PEDF may be a marker of cardiovascular risk status.

Intramyocellular triacylglycerol provides fatty acid substrate for ATP generation in contracting muscle. The protein adipose triglyceride lipase (ATGL) is a key regulator of triacylglycerol lipolysis and whole body energy metabolism at rest and during exercise, and ATGL activity is reported to be enhanced by 5'-AMP-activated protein kinase (AMPK)-mediated phosphorylation at Ser(406) in mice. This is a curious observation, because AMPK activation reduces lipolysis in several cell types. We investigated whether the phosphorylation of ATGL Ser(404) (corresponding to murine Ser(406)) was increased during exercise in human skeletal muscle and with pharmacological AMPK activation in myotubes in vitro. In human experiments, skeletal muscle and venous blood samples were obtained from recreationally active male subjects before and at 5 and 60 min during exercise. ATGL Ser(404) phosphorylation was not increased from rest during exercise, but ATGL Ser(404) phosphorylation correlated with myosin heavy chain 1 expression, suggesting a possible fiber type dependency. ATGL Ser(404) phosphorylation was not related to increases in AMPK activity, and immunoprecipitation experiments indicated no interaction between AMPK and ATGL. Rather, ATGL Ser(404) phosphorylation was associated with protein kinase A (PKA) signaling. ATGL Ser(406) phosphorylation in C(2)C(12) myotubes was unaffected by 5-aminoimidazole-4-carboxaminde-1-β-d-ribofuranoside, an AMPK activator, and the PKA activator forskolin. Our results demonstrate that ATGL Ser(404) phosphorylation is not increased in mixed skeletal muscle during moderate-intensity exercise and that AMPK does not appear to be an activating kinase for ATGL Ser(404/406) in skeletal muscle.

Rats selectively bred for high endurance running capacity (HCR) have higher insulin sensitivity and improved metabolic health compared with those bred for low endurance capacity (LCR). We investigated several skeletal muscle characteristics, in vitro and in vivo, that could contribute to the metabolic phenotypes observed in sedentary LCR and HCR rats. After 16 generations of selective breeding, HCR had approximately 400% higher running capacity (P < 0.001), improved insulin sensitivity (P < 0.001), and lower fasting plasma glucose and triglycerides (P < 0.05) compared with LCR. Skeletal muscle ceramide and diacylglycerol content, basal AMP-activated protein kinase (AMPK) activity, and basal lipolysis were similar between LCR and HCR. However, the stimulation of lipolysis in response to 10 mum isoproterenol was 70% higher in HCR (P = 0.004). Impaired isoproterenol sensitivity in LCR was associated with lower basal triacylglycerol lipase activity, Ser660 phosphorylation of HSL, and beta2-adrenergic receptor protein content in skeletal muscle. Expression of the orphan nuclear receptor Nur77, which is induced by beta-adrenergic signaling and is associated with insulin sensitivity, was lower in LCR (P < 0.05). Muscle protein content of Nur77 target genes, including uncoupling protein 3, fatty acid translocase/CD36, and the AMPK gamma3 subunit were also lower in LCR (P < 0.05). Our investigation associates whole-body insulin resistance with impaired beta-adrenergic response and reduced expression of genes that are critical regulators of glucose and lipid metabolism in skeletal muscle. We identify impaired beta-adrenergic signal transduction as a potential mechanism for impaired metabolic health after artificial selection for low intrinsic exercise capacity.

We determined the effect of coingestion of caffeine (Caff) with carbohydrate (CHO) on rates of muscle glycogen resynthesis during recovery from exhaustive exercise in seven trained subjects who completed two experimental trials in a randomized, double-blind crossover design. The evening before an experiment subjects performed intermittent exhaustive cycling and then consumed a low-CHO meal. The next morning subjects rode until volitional fatigue. On completion of this ride subjects consumed either CHO [4 g/kg body mass (BM)] or the same amount of CHO + Caff (8 mg/kg BM) during 4 h of passive recovery. Muscle biopsies and blood samples were taken at regular intervals throughout recovery. Muscle glycogen levels were similar at exhaustion [ approximately 75 mmol/kg dry wt (dw)] and increased by a similar amount ( approximately 80%) after 1 h of recovery (133 +/- 37.8 vs. 149 +/- 48 mmol/kg dw for CHO and Caff, respectively). After 4 h of recovery Caff resulted in higher glycogen accumulation (313 +/- 69 vs. 234 +/- 50 mmol/kg dw, P < 0.001). Accordingly, the overall rate of resynthesis for the 4-h recovery period was 66% higher in Caff compared with CHO (57.7 +/- 18.5 vs. 38.0 +/- 7.7 mmol x kg dw(-1) x h(-1), P < 0.05). After 1 h of recovery plasma Caff levels had increased to 31 +/- 11 microM (P < 0.001) and at the end of the recovery reached 77 +/- 11 microM (P < 0.001) with Caff. Phosphorylation of CaMK(Thr286) was similar after exercise and after 1 h of recovery, but after 4 h CaMK(Thr286) phosphorylation was higher in Caff than CHO (P < 0.05). Phosphorylation of AMP-activated protein kinase (AMPK)(Thr172) and Akt(Ser473) was similar for both treatments at all time points. We provide the first evidence that in trained subjects coingestion of large amounts of Caff (8 mg/kg BM) with CHO has an additive effect on rates of postexercise muscle glycogen accumulation compared with consumption of CHO alone.

Hormone-sensitive lipase (HSL) catalyzes the hydrolysis of intramuscular triacylglycerol (IMTG); however, its regulation in skeletal muscle is poorly understood. To examine the effects of reduced free fatty acid (FFA) availability on HSL activity in skeletal muscle during aerobic exercise, 11 trained men exercised at 55% maximal O2 uptake for 40 min after the ingestion of nicotinic acid (NA) or nothing (control). Muscle biopsies were taken at rest and 5, 20, and 40 min of exercise. Plasma FFA were suppressed (P < 0.05) in NA during exercise ( approximately 0.40 +/- 0.04 vs. approximately 0.07 +/- 0.01 mM). The respiratory exchange ratio (RER) was increased throughout exercise (0.020 + 0.008) after NA ingestion. However, the provision of energy from fat oxidation only decreased from 33% of the total in the control trial to 26% in the NA trial, suggesting increased IMTG oxidation in the NA trial. Mean HSL activity was 2.25 + 0.15 mmol x kg dry mass(-1) x min(-1) at rest and increased (P < 0.05) to 2.94 +/- 0.20 mmol x kg dry mass(-1) x min(-1) at 5 min in control. Contrary to the hypothesis, mean HSL was not activated to a greater extent in the NA trial during exercise (2.20 + 0.28 at rest to 2.88 + 0.21 mmol x kg dry mass(-1) x min(-1) at 5 min). No further HSL increases were observed at 20 or 40 min in both trials. There was variability in the response to NA ingestion, as some subjects experienced a large increase in RER and decrease in fat oxidation, whereas other subjects experienced no shift in RER and maintained fat oxidation despite the reduced FFA availability in the NA trial. However, even in these subjects, HSL activity was not further increased during the NA trial. In conclusion, reduced plasma FFA availability accompanied by increased epinephrine concentration did not further activate HSL beyond exercise alone.

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.

IL-6 induces lipolysis when administered to humans. Consequently, it has been hypothesized that IL-6 is released from skeletal muscle during exercise to act in a "hormonelike" manner and increase lipolysis from adipose tissue to supply the muscle with substrate. In the present study, we hypothesized that suppressing lipolysis, and subsequent free fatty acid (FFA) availability, would result in a compensatory elevation in IL-6 at rest and during exercise. First, we had five healthy men ingest nicotinic acid (NA) at 30-min intervals for 120 min at rest [10 mg/kg body mass (initial dose), 5 mg/kg body mass (subsequent doses)]. Plasma was collected and analyzed for FFA and IL-6. After 120 min, plasma FFA concentration was attenuated (0 min: 0.26 +/- 0.05 mmol/l; 120 min: 0.09 +/- 0.02 mmol/l; P < 0.01), whereas plasma IL-6 was concomitantly increased approximately eightfold (0 min: 0.75 +/- 0.18 pg/ml; 120 min: 6.05 +/- 0.89 pg/ml; P < 0.001). To assess the effect of lipolytic suppression on the exercise-induced IL-6 response, seven active, but not specifically trained, men performed two experimental exercise trials with (NA) or without [control (Con)] NA ingestion 60 min before (10 mg/kg body mass) and throughout (5 mg/kg body mass every 30 min) exercise. Blood samples were obtained before ingestion, 60 min after ingestion, and throughout 180 min of cycling exercise at 62 +/- 5% of maximal oxygen consumption. IL-6 gene expression, in muscle and adipose tissue sampled at 0, 90, and 180 min, was determined by using semiquantitative real-time PCR. IL-6 mRNA increased in Con (rest vs. 180 min; P < 0.01) approximately 13-fold in muscle and approximately 42-fold in fat with exercise. NA increased (rest vs. 180 min; P < 0.01) IL-6 mRNA 34-fold in muscle, but the treatment effect was not statistically significant (Con vs. NA, P = 0.1), and 235-fold in fat (Con vs. NA, P < 0.01). Consistent with the study at rest, NA completely suppressed plasma FFA (180 min: Con, 1.42 +/- 0.07 mmol/l; NA, 0.10 +/- 0.01 mmol/l; P < 0.001) and increased plasma IL-6 (180 min: Con, 9.81 +/- 0.98 pg/ml; NA, 19.23 +/- 2.50 pg/ml; P < 0.05) during exercise. In conclusion, these data demonstrate that circulating IL-6 is markedly elevated at rest and during prolonged moderate-intensity exercise when lipolysis is suppressed.

Hormone-sensitive lipase (HSL) is believed to play a regulatory role in initiating the degradation of intramuscular triacylglycerol (IMTG) in skeletal muscle. A series of studies designed to characterise the response of HSL to three stimuli: exercise of varying intensities and durations; adrenaline infusions; altered fuel supply have recently been conducted in human skeletal muscle. In an attempt to understand the regulation of HSL activity the changes in the putative intramuscular and hormonal regulators of the enzyme have also been measured. In human skeletal muscle at rest there is a high constitutive level of HSL activity, which is not a function of biopsy freezing. The combination of low adrenaline and Ca(2+) levels and resting levels of insulin appear to dictate the level of HSL activity at rest. During the initial minute of low and moderate aerobic exercise HSL is activated by contractions in the apparent absence of increases in circulating adrenaline. During intense aerobic exercise, adrenaline may contribute to the early activation of HSL. The contraction-induced activation may be related to increased Ca(2+) and/or other unknown intramuscular activators. As low- and moderate-intensity exercise continues beyond a few minutes, activation by adrenaline through the cAMP cascade may also occur. With prolonged moderate-intensity exercise beyond 1-2 h and sustained high-intensity exercise, HSL activity decreases despite continuing increases in adrenaline, possibly as a result of increasing accumulations of free AMP, activation of AMP kinase and phosphorylation of inhibitory sites on HSL. The existing work in human skeletal muscle also suggests that there are numerous levels of control involved in the regulation of IMTG degradation, with control points downstream from HSL also being important. For example, it must be remembered that the actual flux (IMTG degradation) through HSL may be allosterically inhibited during prolonged exercise as a result of the accumulation of long-chain fatty acyl-CoA.

To examine the effect of attenuated epinephrine and elevated insulin on intramuscular hormone sensitivity lipase activity (HSLa) during exercise, seven men performed 120 min of semirecumbent cycling (60% peak pulmonary oxygen uptake) on two occasions while ingesting either 250 ml of a 6.4% carbohydrate (GLU) or sweet placebo (CON) beverage at the onset of, and at 15 min intervals throughout, exercise. Muscle biopsies obtained before and immediately after exercise were analyzed for HSLa. Blood samples were simultaneously obtained from a brachial artery and a femoral vein before and during exercise, and leg blood flow was measured by thermodilution in the femoral vein. Net leg glycerol and lactate release and net leg glucose and free fatty acid (FFA) uptake were calculated from these measures. Insulin and epinephrine were also measured in arterial blood before and throughout exercise. During GLU, insulin was elevated (120 min: CON, 11.4 +/- 2.4, GLU, 35.3 +/- 6.9 pM, P < 0.05) and epinephrine suppressed (120 min: CON, 6.1 +/- 2.5, GLU, 2.1 +/- 0.9 nM; P < 0.05) compared with CON. Carbohydrate feeding also resulted in suppressed (P < 0.05) HSLa relative to CON (120 min: CON, 1.71 +/- 0.18, GLU, 1.27 +/- 0.16 mmol.min-1.kg dry mass-1). There were no differences in leg lactate or glycerol release when trials were compared, but leg FFA uptake was lower (120 min: CON, 0.29 +/- 0.06, GLU, 0.82 +/- 0.09 mmol/min) and leg glucose uptake higher (120 min: CON, 3.16 +/- 0.59, GLU, 1.37 +/- 0.37 mmol/min) in GLU compared with CON. These results demonstrate that circulating insulin and epinephrine play a role in HSLa in contracting skeletal muscle.

This study examined the effect of combined alpha- and beta-adrenergic blockade on glucose kinetics during intense exercise. Six endurance-trained men exercised for 20 minutes at approximately 78% of their peak oxygen consumption (Vo(2)) following ingestion of a placebo (CON) or combined alpha- (prazosin hydrochloride) and beta- (timolol maleate) adrenoceptor antagonists (BLK). Plasma glucose increased during exercise in CON (0 minutes: 5.5 +/- 0.1; 20 minutes: 6.5 +/- 0.3 mmol. L(-1), P <.05). In BLK, the exercise-induced increase in plasma glucose was abolished (0 minutes: 5.7 +/- 0.3; 20 minutes: 5.7 +/- 0.1 mmol. L(-1)). Glucose kinetics were measured using a primed, continuous infusion of [6,6-(2)H] glucose. Glucose production was not different between trials; on average these values were 25.3 +/- 3.9 and 30.9 +/- 4.4 micromol. kg(-1). min(-1) in CON and BLK, respectively. Glucose uptake during exercise was greater (P <.05) in BLK (30.6 +/- 4.6 micromol. kg(-1). min(-1)) compared with CON (18.4 +/- 2.5 micromol. kg(-1). min(-1)). In BLK, plasma insulin and catecholamines were higher (P <.05), while plasma glucagon was unchanged from CON. Free fatty acids (FFA) and glycerol were lower (P <.05) in BLK. These findings demonstrate that adrenergic blockade during intense exercise results in a blunted plasma glucose response that is due to enhanced glucose uptake, with no significant change in glucose production.

At the onset of exercise, signals from inside and outside the muscle cell increase the availability of carbohydrate (CHO) and fat to provide the fuel required for ATP production. CHO and fat oxidation are the dominant sources of aerobic ATP production and both pathways must be heavily upregulated during exercise to meet the increased energy demand. Within this paradigm, there is room for shifts between the proportion of energy that is provided from CHO and fat. It has long been known that increasing the availability of endogenous or exogenous CHO can increase the oxidation of CHO and decrease the oxidation of fat. The opposite is also true. While descriptive studies documenting these changes are numerous, the mechanisms regulating these shifts in fuel use in the face of constant energy demand have not been thoroughly elucidated. It would be expected, for example, that any fat-induced shift in CHO metabolism would target the enzymes that play key roles in regulating CHO metabolism and oxidation. Inside the muscle these could include glucose uptake (GLUT4) and phosphorylation (hexokinase), glycogenolysis (glycogen phosphorylase), glycolysis (phosphofructokinase) and conversion to acetyl CoA (pyruvate dehydrogenase). The same would be expected for a CHO-induced down regulation of fat metabolism and oxidation and might target transport of long chain fatty acids into the cell (fatty acid translocase CD36), release of fatty acids from intramuscular triacylglycerol (hormone sensitive lipase) and transport into the mitochondria (carnitine palmitoyl transferase complex). This review summarizes the work describing the interaction between CHO and fat metabolism in human skeletal muscle during exercise and presents the theories that may account for CHO/fat interaction during 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.

We investigated the effect of increased plasma adrenaline on hormone-sensitive lipase (HSL) activity and extracellular regulated kinase (ERK) 1/2 phosphorylation during exercise. Seven untrained men rested for 20 min and exercised for 10 min at 60 % peak pulmonary oxygen uptake on three occasions: with adrenaline infusion throughout rest and exercise (ADR), with no adrenaline infusion (CON) and with adrenaline infusion commencing after 3 min of exercise (EX+ADR). Muscle samples were obtained at rest before (Pre, -20 min) and after (0 min) infusion, and at 3 and 10 min of cycling. Exogenous adrenaline infusion increased (P < 0.05) plasma adrenaline at rest during ADR, which resulted in greater HSL activity (Pre, 2.14 +/- 0.10 mmol min-1 (kg dry matter (dm))-1; 0 min, 2.74 +/- 0.20 mmol min-1 (kg dm)-1). Subsequent exercise had no effect on HSL activity. During exercise in CON, HSL activity was increased (P < 0.05) above rest at 3 min but was not increased further by 10 min. The infusion of exogenous adrenaline at 3 min of exercise in EX+ADR resulted in a marked elevation in plasma adrenaline levels (3 min, 0.57 +/- 0.12 nM; 10 min, 10.08 +/- 0.84 nM) and increased HSL activity by 25 %. HSL activity at 10 min was greater (P < 0.05) in EX+ADR compared with CON. There were no changes between trials in the plasma concentrations of insulin and free fatty acids (FFA) and the muscle contents of free AMP, all putative regulators of HSL activity. ERK1/2 phosphorylation increased at 3 min in CON and EX+ADR. Because HSL activity did not increase during exercise when adrenaline was infused prior to exercise (ADR) and because HSL activity increased when adrenaline was infused during exercise (EX+ADR), we conclude that (1) high adrenaline levels can stimulate HSL activity regardless of the metabolic milieu and (2) large increases in adrenaline during exercise, independent of changes in other putative regulators, are able to further stimulate the contraction-induced increase in HSL activity. The results also demonstrate that increased ERK 1/2 phosphorylation coincides with elevated HSL activity, indicating that ERK 1/2 may mediate the contraction-induced increase in HSL activity early in exercise.

This study investigated the effect of reduced free fatty acid (FFA) availability on pyruvate dehydrogenase activation (PDHa) and carbohydrate metabolism during moderate aerobic exercise. Eight active male subjects cycled for 40 min at 55% Vo(2 peak) on two occasions. During one trial, subjects ingested 20 mg/kg body mass of the antilipolytic drug nicotinic acid (NA) during the hour before exercise to reduce FFA. Nothing was ingested in the control trial (CON). Blood and expired gas measurements were obtained throughout the trials, and muscle biopsy samples were obtained immediately before exercise and at 5, 20, and 40 min of exercise. Plasma FFA were lower in the NA trial (0.13 +/- 0.01 vs. 0.48 +/- 0.03 mM, P < 0.05), and the respiratory exchange ratio (RER) was increased with NA (0.93 +/- 0.01 vs. 0.89 +/- 0.01, P < 0.05), resulting in a 14.5 +/- 1.8% increase in carbohydrate oxidation compared with CON. PDHa increased rapidly in both trials at exercise onset but was approximately 15% higher (P < 0.05) throughout exercise in the NA trial (2.44 +/- 0.19 and 2.07 +/- 0.12 mmol x kg wet muscle(-1) x min(-1) for NA and CON at 40 min). Muscle glycogenolysis was 15.3 +/- 9.6% greater in the NA trial vs. the CON trial but did not reach statistical significance. Glucose 6-phosphate contents were elevated (P < 0.05) in the NA trial at 30 and 40 min of exercise, but pyruvate and lactate contents were unaffected. These data demonstrate that the reduction of exogenous FFA availability increased the activation of PDH and carbohydrate oxidation during moderate aerobic exercise in men. The increased activation of PDH was not explained by changes in muscle pyruvate or the ATP/ADP ratio but may be related to a decrease in the NADH/NAD(+) ratio or an epinephrine-induced increase in calcium concentration.

It has been proposed that hormone-sensitive lipase (HSL) regulates intramuscular triacylglycerol hydrolysis in skeletal muscle. The primary purpose of this study was to examine the early activation of HSL and the changes in the putative intramuscular and hormonal regulators of HSL activity at various aerobic exercise intensities. Eight male subjects cycled for 10 min at power outputs corresponding to 30, 60 and 90 % peak oxygen uptake (VO(2,peak)). Muscle samples were obtained at rest and following 1 and 10 min of exercise. Intramuscular triacylglycerol (mean +/- S.E.M.: 24.3 +/- 2.3 mmol (kg dry mass (DM))(-1)), long-chain fatty acyl CoA (13.9 +/- 1.4 micromol (kg DM)(-1)) and HSL activity (1.87 +/- 0.07 mmol min(-1) (kg DM)(-1))) were not different between trials at rest. HSL activity increased at 1 min of exercise at 30 and 60 % VO(2,peak), and to a greater extent at 90 % VO(2,peak). HSL activity remained elevated after 10 min of exercise at 30 and 60 % VO(2,peak), and decreased at 90 % VO(2,peak) from the rates observed at 1 min (1 min: 3.41 +/- 0.3 mmol min(-1) (kg DM)-1; 10 min: 2.92 +/- 0.26 mmol min(-1) (kg DM)(-1)), P < 0.05). There were no effects of exercise power output or time on long-chain fatty acyl CoA content. At 90 % VO(2,peak), skeletal muscle contents of ATP and phosphocreatine were decreased (P < 0.05), and free ADP and free AMP were increased (P < 0.05) during exercise. No changes in these metabolites occurred at 30 % VO(2,peak) and only modest changes were observed at 60 % VO(2,peak). Plasma adrenaline increased (P < 0.05) during exercise at 90 % VO(2,peak) only. These data suggest that a factor related to the onset of exercise (e.g. Ca2+) activates HSL early in exercise. Given the activation of HSL early in exercise, at a time when intramuscular triacylglycerol hydrolysis and fat oxidation are considered to be negligible, we propose that the control of intramuscular triacylglycerol hydrolysis is not solely related to the level of HSL activation, but must also be regulated by postactivational factors.

This study investigated the effect of reduced acetylcarnitine availability on oxidative metabolism during the transition from rest to steady-state exercise. Eight male subjects completed two randomised exercise trials at 68 % of the peak rate of O(2) uptake (V((O(2)),peak)). On one occasion subjects ingested 1 g (kg body mass)(-1) glucose 75 min prior to exercise (CHO), whereas the other trial acted as a control (CON). Muscle samples were obtained pre- and 75 min post-ingestion, and following 1 and 10 min of exercise. Plasma glucose and insulin were elevated (P < 0.05), and plasma free fatty acids (FFA) were lower at the onset of exercise in CHO. Acetylcarnitine (CON, 4.8 +/- 1.8; CHO, 1.5 +/- 0.9 mmol (kg dry mass (d.m.))(-1), P < 0.05) and acetyl CoA (CON, 13.2 +/- 2.3; CHO, 6.3 +/- 0.6 micromol (kg d.m.)(-1), P < 0.05) were lower at rest, whereas pyruvate dehydrogenase activation (PDHa) was greater in CHO compared with CON (CON, 0.78 +/- 0.07; CHO, 1.44 +/- 0.19 mmol min(-1) (kg wet mass (w.m.))(-1)). Respiratory exchange ratio (RER) was significantly elevated during exercise in CHO. The acetyl groups increased at similar rates at the onset of exercise (1 min) and there was no difference in substrate phosphorylation as determined from lactate accumulation and phosphocreatine degradation between trials. Subsequently, oxidative metabolism during the transition from rest to steady-state exercise was not affected by prior carbohydrate ingestion. Although exercise resulted in the rapid activation of PDH in both trials, PDHa was greater at 1 min in CHO (CON, 2.36 +/- 0.22; CHO, 2.91 +/- 0.18 mmol min(-1) (kg w.m.)(-1)). No differences in muscle metabolite levels and PDHa were observed after 10 min of moderate exercise between trials. In summary, at rest, carbohydrate ingestion induced multiple metabolic changes which included decreased acetylcarnitine availability and small increases in PDHa. The prior changes in PDHa and acetylcarnitine availability had no effect on substrate phosphorylation and oxidative metabolism at the onset of exercise. These data suggest that acetylcarnitine availability is unlikely to be the site of metabolic inertia during the transition from rest to steady-state moderate intensity exercise.

Intramuscular triacylglyerols (IMTGs) represent a potentially important energy source for contracting human skeletal muscle. Although the majority of evidence from isotope tracer and (1)H-magnetic resonance spectroscopy (MRS) studies demonstrate IMTG utilization during exercise, controversy regarding the importance of IMTG as a metabolic substrate persists. The controversy stems from studies that measure IMTG in skeletal muscle biopsy samples and report no significant net IMTG degradation during prolonged moderate-intensity (55-70% maximal O(2) consumption) exercise lasting 90-120 min. Although postexercise decrements in IMTG levels are often reported from direct muscle measurements, the marked between-biopsy variability (approximately 23%) that has been reported with this technique in untrained subjects is larger than the expected decrease in IMTG content, effectively precluding significant findings. In contrast, recent data obtained in endurance-trained subjects demonstrated reduced variability between duplicate biopsies (approximately 12%), and significant changes in IMTG were detected after 120 min of moderate-intensity exercise. Therefore, it is our contention that the muscle biopsy, isotope tracer, and (1)H-MRS techniques report significant and energetically important oxidation of free fatty acids derived from IMTGs during prolonged moderate exercise.

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.

This study investigated intramuscular triacylglycerol (IMTG) and glycogen utilisation, pyruvate dehydrogenase activation (PDHa) and acetyl group accumulation during prolonged moderate intensity exercise. Seven endurance-trained men cycled for 240 min at 57 % maximal oxygen consumption (V(O2,max)) and duplicate muscle samples were obtained at rest and at 10, 120 and 240 min of exercise. We hypothesised that IMTG utilisation would be augmented during 2-4 h of exercise, while PDHa would be decreased secondary to reduced glycogen metabolism. IMTG was measured on both muscle samples at each time point and the coefficient of variation was 12.3 +/- 9.4 %. Whole body respiratory exchange ratio (RER) decreased from 0.89 +/- 0.01 at 30 min to 0.83 +/- 0.01 at 150 min and remained low throughout exercise. Plasma glycerol and free fatty acids (FFAs) had increased compared with rest at 90 min and progressively increased until exercise cessation. Although plasma glucose tended to decrease with exercise, this was not significant. IMTG was reduced at 120 min compared with rest (0 min, 15.6 +/- 0.8 mmol kg(-1) d.m.; 120 min, 12.8 +/- 0.7 mmol kg(-1) d.m.) but no further reduction in IMTG was observed at 240 min. Muscle glycogen was 468 +/- 49 mmol kg(-1) d.m. at rest and decreased at 120 min and again at 240 min (217 +/- 48 and 144 + 47 mmol kg(-1) d.m.). PDHa increased above rest at 10 and 120 min, but decreased at 240 min, which coincided with reduced whole body carbohydrate oxidation. Muscle pyruvate and ATP were unchanged with exercise. Acetyl CoA increased at 10 min and remained elevated throughout exercise. Acetylcarnitine increased at exercise onset but returned to resting values by 240 min. Contrary to our first hypothesis, significant utilisation of IMTG occurred during the first 2 h of moderate exercise but not during hours 2-4. The reduced utilisation of intramuscular fuels during the last 120 min was offset by greater FFA delivery and oxidation. Consistent with the second hypothesis, PDHa decreased late in moderate exercise and closely matched the estimates of lower carbohydrate flux. Although the factor underlying the PDHa decrease was not apparent, reduced pyruvate provision secondary to diminished glycolytic flux is the most likely mechanism.

To investigate the influence of heat stress on the regulation of skeletal muscle carbohydrate metabolism, six active, but not specifically trained, men performed 5 min of cycling at a power output eliciting 70% maximal O2 uptake in either 20 degrees C (Con) or 40 degrees C (Heat) after 20 min of passive exposure to either environmental condition. Although muscle temperature (T(mu)) was similar at rest when comparing trials, 20 min of passive exposure and 5 min of exercise increased (P < 0.05) T(mu) in Heat compared with Con (37.5 +/- 0.1 vs. 36.9 +/- 0.1 degrees C at 5 min for Heat and Con, respectively). Rectal temperature and plasma epinephrine were not different at rest, preexercise, or 5 min of exercise between trials. Although intramuscular glycogen phosphorylase and pyruvate dehydrogenase activity increased (P < 0.05) at the onset of exercise, there were no differences in the activities of these regulatory enzymes when comparing Heat with Con. Accordingly, glycogen use in the first 5 min of exercise was not different when comparing Heat with Con. Similarly, no differences in intramuscular concentrations of glucose 6-phosphate, lactate, pyruvate, acetyl-CoA, creatine, phosphocreatine, or ATP were observed at any time point when comparing Heat with Con. These results demonstrate that, whereas mild heat stress results in a small difference in contracting T(mu), it does not alter the activities of the key regulatory enzymes for carbohydrate metabolism or glycogen use at the onset of exercise, when plasma epinephrine levels are unaltered.

1. To evaluate the role of adrenaline in regulating carbohydrate metabolism during moderate exercise, 10 moderately trained men completed two 20 min exercise bouts at 58 +/- 2 % peak pulmonary oxygen uptake (V(O2,peak)). On one occasion saline was infused (CON), and on the other adrenaline was infused intravenously for 5 min prior to and throughout exercise (ADR). Glucose kinetics were measured by a primed, continuous infusion of 6,6-[(2)H]glucose and muscle samples were obtained prior to and at 1 and 20 min of exercise. 2. The infusion of adrenaline elevated (P < 0.01) plasma adrenaline concentrations at rest (pre-infusion, 0.28 +/- 0.09; post-infusion, 1.70 +/- 0.45 nmol l(-1); means +/- S.E.M.) and this effect was maintained throughout exercise. Total carbohydrate oxidation increased by 18 % and this effect was due to greater skeletal muscle glycogenolysis (P < 0.05) and pyruvate dehydrogenase (PDH) activation (P < 0.05, treatment effect). Glucose rate of appearance was not different between trials, but the infusion of adrenaline decreased (P < 0.05, treatment effect) skeletal muscle glucose uptake in ADR. 3. During exercise muscle glucose 6-phosphate (G-6-P) (P = 0.055, treatment effect) and lactate (P < 0.05) were elevated in ADR compared with CON and no changes were observed for pyruvate, creatine, phosphocreatine, ATP and the calculated free concentrations of ADP and AMP. 4. The data demonstrate that elevated plasma adrenaline levels during moderate exercise in untrained men increase skeletal muscle glycogen breakdown and PDH activation, which results in greater carbohydrate oxidation. The greater muscle glycogenolysis appears to be due to increased glycogen phosphorylase transformation whilst the increased PDH activity cannot be readily explained. Finally, the decreased glucose uptake observed during exercise in ADR is likely to be due to the increased intracellular G-6-P and a subsequent decrease in glucose phosphorylation.

Purpose: We investigated the effects of acute plasma volume expansion on exercise performance in the heat., Methods: Six moderately trained men cycled for 40 min at 64 +/- 2% peak pulmonary oxygen uptake (VO2peak) followed by an individual performance time trial, where subjects completed a set amount of work (267 +/- 15 kJ) in as little time as possible. Exercise trials were performed at 35 degrees C with a relative humidity of 40%. Subjects performed two exercise trials: one after 13.1 +/- 1% acute plasma volume expansion (PVE), which was achieved by the intravenous infusion of 8 mL x kg(-1) body weight of Hemaccel (35 g x L(-1) polygeline, 145 mmol x L(-1) Na+, and 145 mmol x L(-1) Cl-) and the other without prior treatment (CON)., Results: Core temperature, skin blood flow, and heart rate progressively increased (P < 0.05) during exercise, but no differences were observed between trials. Plasma glucose and lactate were similar at rest and during exercise, as was VO2 during exercise. Exercise performance was not influenced by plasma volume expansion (CON 17.5 +/- 0.4 min and PVE 17.1 +/- 0.2 min)., Conclusion: These data suggest that, in moderately trained men, plasma volume expansion alone does not enhance thermoregulatory function and exercise performance during moderate intensity exercise in the heat.

To examine the effect of acute plasma volume expansion (PVE) on substrate selection during exercise, seven untrained men cycled for 40 min at 72 +/- 2% peak oxygen uptake (VO(2 peak)) on two occasions. On one occasion, subjects had their plasma volume expanded by 12 +/- 2% via an intravenous infusion of the plasma substitute Haemaccel, whereas on the other occasion no such infusion took place. Muscle samples were obtained before and immediately after exercise. In addition, heart rate and pulmonary gas and venous blood samples were obtained throughout exercise. No differences in oxygen uptake or heart rate during exercise were observed between trials, whereas respiratory exchange ratio, blood glucose, and lactate were unaffected by PVE. Muscle glycogen and lactate concentrations were not different either before or after exercise. In addition, there was no difference in total carbohydrate oxidation between trials (control: 108 +/- 2 g; PVE group: 105 +/- 2 g). Plasma catecholamine levels were not affected by PVE. These data indicate that substrate metabolism during submaximal exercise in untrained men is unaltered by acute hypervolemia.