Brendan Egan

Exercise intensity‐dependent regulation of peroxisome proliferator‐activated receptor γ coactivator‐1α mRNA abundance is associated with differential activation of upstream signalling kinases in human skeletal muscle

Skeletal muscle contraction increases intracellular ATP turnover, calcium flux, and mechanical stress, initiating signal transduction pathways that modulate peroxisome proliferator‐activated receptor γ coactivator‐1α (PGC‐1α)‐dependent transcriptional programmes. The purpose of this study was to determine if the intensity of exercise regulates PGC‐1α expression in human skeletal muscle, coincident with activation of signalling cascades known to regulate PGC‐1α transcription. Eight sedentary males expended 400 kcal (1674 kj) during a single bout of cycle ergometer exercise on two separate occasions at either 40% (LO) or 80% (HI) of . Skeletal muscle biopsies from the m. vastus lateralis were taken at rest and at +0, +3 and +19 h after exercise. Energy expenditure during exercise was similar between trials, but the high intensity bout was shorter in duration (LO, 69.9 ± 4.0 min; HI, 36.0 ± 2.2 min, P < 0.05) and had a higher rate of glycogen utilization (P < 0.05). PGC‐1α mRNA abundance increased in an intensity‐dependent manner +3 h after exercise (LO, 3.8‐fold; HI, 10.2‐fold, P < 0.05). AMP‐activated protein kinase (AMPK) (2.8‐fold, P < 0.05) and calcium/calmodulin‐dependent protein kinase II (CaMKII) phosphorylation (84%, P < 0.05) increased immediately after HI but not LO. p38 mitogen‐activated protein kinase (MAPK) phosphorylation increased after both trials (∼2.0‐fold, P < 0.05), but phosphorylation of the downstream transcription factor, activating transcription factor‐2 (ATF‐2), increased only after HI (2.4‐fold, P < 0.05). Cyclic‐AMP response element binding protein (CREB) phosphorylation was elevated at +3 h after both trials (∼80%, P < 0.05) and class IIa histone deacetylase (HDAC) phosphorylation increased only after HI (2.0‐fold, P < 0.05). In conclusion, exercise intensity regulates PGC‐1α mRNA abundance in human skeletal muscle in response to a single bout of exercise. This effect is mediated by differential activation of multiple signalling pathways, with ATF‐2 and HDAC phosphorylation proposed as key intensity‐dependent mediators.

Time course analysis reveals gene-specific transcript and protein kinetics of adaptation to short-term aerobic exercise training in human skeletal muscle

Repeated bouts of episodic myofibrillar contraction associated with exercise training are potent stimuli for physiological adaptation. However, the time course of adaptation and the continuity between alterations in mRNA expression and protein content are not well described in human skeletal muscle. Eight healthy, sedentary males cycled for 60 min at 80% of peak oxygen consumption (VO2peak) each day for fourteen consecutive days, resulting in an increase in VO2peak of 17.5±3.8%. Skeletal muscle biopsies were taken at baseline, and on the morning following (+16 h after exercise) the first, third, seventh, tenth and fourteenth training sessions. Markers of mitochondrial adaptation (Cyt c and COXIV expression, and citrate synthase activity) were increased within the first week of training, but the mtDNA/nDNA ratio was unchanged by two weeks of training. Accumulation of PGC-1α and ERRα protein during training suggests a regulatory role for these factors in adaptations of mitochondrial and metabolic gene expression. A subset of genes were transiently increased after one training session, but returned to baseline levels thereafter, which is supportive of the concept of transcriptional capacity being particularly sensitive to the onset of a new level of contractile activity. Thus, gene-specific temporal patterns of induction of mRNA expression and protein content are described. Our results illustrate the phenomenology of skeletal muscle plasticity and support the notion that transcript level adjustments, coupled to accumulation of encoded protein, underlie the modulation of skeletal muscle metabolism and phenotype by regular exercise.

Autocrine role of interleukin-13 on skeletal muscle glucose metabolism in type 2 diabetic patients involves microRNA let-7

Low-grade inflammation associated with type 2 diabetes (T2DM) is postulated to exacerbate insulin resistance. We report that serum levels, as well as IL-13 secreted from cultured skeletal muscle, are reduced in T2DM vs. normal glucose-tolerant (NGT) subjects. IL-13 exposure increases skeletal muscle glucose uptake, oxidation, and glycogen synthesis via an Akt-dependent mechanism. Expression of microRNA let-7a and let-7d, which are direct translational repressors of the IL-13 gene, was increased in skeletal muscle from T2DM patients. Overexpression of let-7a and let-7d in cultured myotubes reduced IL-13 secretion. Furthermore, basal glycogen synthesis was reduced in cultured myotubes exposed to an IL-13-neutralizing antibody. Thus, IL-13 is synthesized and released by skeletal muscle through a mechanism involving let-7, and this effect is attenuated in skeletal muscle from insulin-resistant T2DM patients. In conclusion, IL-13 plays an autocrine role in skeletal muscle to increase glucose uptake and metabolism, suggesting a role in glucose homeostasis in metabolic disease.

2-D DIGE analysis of the mitochondrial proteome from human skeletal muscle reveals time course-dependent remodelling in response to 14 consecutive days of endurance exercise training.

Adaptation of skeletal muscle to repeated bouts of endurance exercise increases aerobic capacity and improves mitochondrial function. However, the adaptation of human skeletal muscle mitochondrial proteome to short‐term endurance exercise training has not been investigated. Eight sedentary males cycled for 60 min at 80% of peak oxygen consumption (VO2peak) each day for 14 consecutive days, resulting in an increase in VO2peak of 17.5±3.8% (p<0.01). Mitochondria‐enriched protein fractions from skeletal muscle biopsies taken from m. vastus lateralis at baseline, and on the morning following the 7th and 14th training sessions were subjected to 2‐D DIGE analysis with subsequent MS followed by database interrogation to identify the proteins of interest. Thirty‐one protein spots were differentially expressed after either 7 or 14 days of training (ANOVA, p<0.05). These proteins included subunits of the electron transport chain, enzymes of the tricarboxylic acid cycle, phosphotransfer enzymes, and regulatory factors in mitochondrial protein synthesis, oxygen transport, and antioxidant capacity. Several proteins demonstrated a time course‐dependent induction during training. Our results illustrate the phenomenon of skeletal muscle plasticity with the extensive remodelling of the mitochondrial proteome occurring after just 7 days of exercise training suggestive of enhanced capacity for adenosine triphosphate generation at a cellular level.

Altered miR-29 Expression in Type 2 Diabetes Influences Glucose and Lipid Metabolism in Skeletal Muscle

MicroRNAs have emerged as important regulators of glucose and lipid metabolism in several tissues; however, their role in skeletal muscle remains poorly characterized. We determined the effects of the miR-29 family on glucose metabolism, lipid metabolism, and insulin responsiveness in skeletal muscle. We provide evidence that miR-29a and miR-29c are increased in skeletal muscle from patients with type 2 diabetes and are decreased following endurance training in healthy young men and in rats. In primary human skeletal muscle cells, inhibition and overexpression strategies demonstrate that miR-29a and miR-29c regulate glucose uptake and insulin-stimulated glucose metabolism. We identified that miR-29 overexpression attenuates insulin signaling and expression of insulin receptor substrate 1 and phosphoinositide 3-kinase. Moreover, miR-29 overexpression reduces hexokinase 2 expression and activity. Conversely, overexpression of miR-29 by electroporation of mouse tibialis anterior muscle decreased glucose uptake and glycogen content in vivo, concomitant with decreased abundance of GLUT4. We also provide evidence that fatty acid oxidation is negatively regulated by miR-29 overexpression, potentially through the regulation of peroxisome proliferator–activated receptor γ coactivator-1α expression. Collectively, we reveal that miR-29 acts as an important regulator of insulin-stimulated glucose metabolism and lipid oxidation, with relevance to human physiology and type 2 diabetes.

Metabolism of ketone bodies during exercise and training: physiological basis for exogenous supplementation

Optimising training and performance through nutrition strategies is central to supporting elite sportspeople, much of which has focused on manipulating the relative intake of carbohydrate and fat and their contributions as fuels for energy provision. The ketone bodies, namely acetoacetate, acetone and β‐hydroxybutyrate (βHB), are produced in the liver during conditions of reduced carbohydrate availability and serve as an alternative fuel source for peripheral tissues including brain, heart and skeletal muscle. Ketone bodies are oxidised as a fuel source during exercise, are markedly elevated during the post‐exercise recovery period, and the ability to utilise ketone bodies is higher in exercise‐trained skeletal muscle. The metabolic actions of ketone bodies can alter fuel selection through attenuating glucose utilisation in peripheral tissues, anti‐lipolytic effects on adipose tissue, and attenuation of proteolysis in skeletal muscle. Moreover, ketone bodies can act as signalling metabolites, with βHB acting as an inhibitor of histone deacetylases, an important regulator of the adaptive response to exercise in skeletal muscle. Recent development of ketone esters facilitates acute ingestion of βHB that results in nutritional ketosis without necessitating restrictive dietary practices. Initial reports suggest this strategy alters the metabolic response to exercise and improves exercise performance, while other lines of evidence suggest roles in recovery from exercise. The present review focuses on the physiology of ketone bodies during and after exercise and in response to training, with specific interest in exploring the physiological basis for exogenous ketone supplementation and potential benefits for performance and recovery in athletes.

SnapShot: Exercise metabolism

Exercise represents a major challenge to whole-body homeostasis. To meet this challenge, myriad acute and adaptive responses take place at multiple cellular and systemic levels. The molecular bases of skeletal muscle adaptations to exercise are mediated by an array of signaling events, pre- and post-transcriptional processes, regulation of translation, and ultimately the increased abundance and/or maximal activity of key proteins with roles in energy provision.

Temporal analysis of reciprocal miRNA-mRNA expression patterns predicts regulatory networks during differentiation in human skeletal muscle cells

microRNAs (miRNAs) are short noncoding RNAs that regulate gene expression through posttranscriptional repression of target genes. miRNAs exert a fundamental level of control over many developmental processes, but their role in the differentiation and development of skeletal muscle from myogenic progenitor cells in humans remains incompletely understood. Using primary cultures established from human skeletal muscle satellite cells, we performed microarray profiling of miRNA expression during differentiation of myoblasts (day 0) into myotubes at 48 h intervals (day 2, 4, 6, 8, and 10). Based on a time-course analysis, we identified 44 miRNAs with altered expression [false discovery rate (FDR) < 5%, fold change > ±1.2] during differentiation, including the marked upregulation of the canonical myogenic miRNAs miR-1, miR-133a, miR-133b, and miR-206. Microarray profiling of mRNA expression at day 0, 4, and 10 identified 842 and 949 genes differentially expressed (FDR < 10%) at day 4 and 10, respectively. At day 10, 42% of altered transcripts demonstrated reciprocal expression patterns in relation to the directional change of their in silico predicted regulatory miRNAs based on analysis using Ingenuity Pathway Analysis microRNA Target Filter. Bioinformatic analysis predicted networks of regulation during differentiation including myomiRs miR-1/206 and miR-133a/b, miRNAs previously established in differentiation including miR-26 and miR-30, and novel miRNAs regulated during differentiation of human skeletal muscle cells such as miR-138-5p and miR-20a. These reciprocal expression patterns may represent new regulatory nodes in human skeletal muscle cell differentiation. This analysis serves as a reference point for future studies of human skeletal muscle differentiation and development in healthy and disease states.

Transient Increase in Homocysteine but Not Hyperhomocysteinemia during Acute Exercise at Different Intensities in Sedentary Individuals

Considering that hyperhomocysteinemia is an independent risk factor for cardiovascular disease, the purpose of this study was to determine the kinetics of serum homocysteine (tHcy) and the vitamins involved in its metabolism (folates, B12, and B6) in response to acute exercise at different intensities. Eight sedentary males (18–27 yr) took part in the study. Subjects were required to complete two isocaloric (400 kcal) acute exercise trials on separate occasions at 40% (low intensity, LI) and 80% VO2peak (high intensity, HI). Blood samples were drawn at different points before (pre4 and pre0 h), during (exer10, exer20, exer30, exer45, and exer60 min), and after exercise (post0, post3, and post19 h). Dietary, genetic, and lifestyle factors were controlled. Maximum tHcy occurred during exercise, both at LI (8.6 (8.0–10.1) µmol/L, 9.3% increase from pre0) and HI (9.4 (8.2–10.6) µmol/L, 25.7% increase from pre0), coinciding with an accumulated energy expenditure independent of the exercise intensity. From this point onwards tHcy declined until the cessation of exercise and continued descending. At post19, tHcy was not different from pre-exercise values. No values of hyperhomocysteinemia were observed at any sampling point and intensity. In conclusion, acute exercise in sedentary individuals, even at HI, shows no negative effect on tHcy when at least 400 kcal are spent during exercise and the nutritional status for folate, B12, and B6 is adequate, since no hyperhomocysteinemia has been observed and basal concentrations were recovered in less than 24 h. This could be relevant for further informing healthy exercise recommendations.

Hunting for the SNARK in metabolic disease

dysregulation of energy balance is a primary constituent in the etiology of obesity and type 2 diabetes mellitus, which is manifested by altered metabolic homeostasis and insulin resistance in a variety of tissues, including brain, liver, and skeletal muscle. The discovery of the AMP-activated