Bertrand Léger

Mitofusins 1/2 and ERRα expression are increased in human skeletal muscle after physical exercise

Mitochondrial impairment is hypothesized to contribute to the pathogenesis of insulin resistance. Mitofusin (Mfn) proteins regulate the biogenesis and maintenance of the mitochondrial network, and when inactivated, cause a failure in the mitochondrial architecture and decreases in oxidative capacity and glucose oxidation. Exercise increases muscle mitochondrial content, size, oxidative capacity and aerobic glucose oxidation. To address if Mfn proteins are implicated in these exercise‐induced responses, we measured Mfn1 and Mfn2 mRNA levels, pre‐, post‐, 2 and 24 h post‐exercise. Additionally, we measured the expression levels of transcriptional regulators that control mitochondrial biogenesis and functions, including PGC‐1α, NRF‐1, NRF‐2 and the recently implicated ERRα. We show that Mfn1, Mfn2, NRF‐2 and COX IV mRNA were increased 24 h post‐exercise, while PGC‐1α and ERRα mRNA increased 2 h post‐exercise. Finally, using in vitro cellular assays, we demonstrate that Mfn2 gene expression is driven by a PGC‐1α programme dependent on ERRα. The PGC‐1α/ERRα‐mediated induction of Mfn2 suggests a role of these two factors in mitochondrial fusion. Our results provide evidence that PGC‐1α not only mediates the increased expression of oxidative phosphorylation genes but also mediates alterations in mitochondrial architecture in response to aerobic exercise in humans.

Akt signalling through GSK-3β, mTOR and Foxo1 is involved in human skeletal muscle hypertrophy and atrophy

Skeletal muscle size is tightly regulated by the synergy between anabolic and catabolic signalling pathways which, in humans, have not been well characterized. Akt has been suggested to play a pivotal role in the regulation of skeletal muscle hypertrophy and atrophy in rodents and cells. Here we measured the amount of phospho‐Akt and several of its downstream anabolic targets (glycogen synthase kinase‐3β (GSK‐3β), mTOR, p70s6k and 4E‐BP1) and catabolic targets (Foxo1, Foxo3, atrogin‐1 and MuRF1). All measurements were performed in human quadriceps muscle biopsies taken after 8 weeks of both hypertrophy‐stimulating resistance training and atrophy‐stimulating de‐training. Following resistance training a muscle hypertrophy (∼10%) and an increase in phospho‐Akt, phospho‐GSK‐3β and phospho‐mTOR protein content were observed. This was paralleled by a decrease in Foxo1 nuclear protein content. Following the de‐training period a muscle atrophy (5%), relative to the post‐training muscle size, a decrease in phospho‐Akt and GSK‐3β and an increase in Foxo1 were observed. Atrogin‐1 and MuRF1 increased after the hypertrophy and decreased after the atrophy phases. We demonstrate, for the first time in human skeletal muscle, that the regulation of Akt and its downstream signalling pathways GSK‐3β, mTOR and Foxo1 are associated with both the skeletal muscle hypertrophy and atrophy processes.

The role and regulation of MAFbx/atrogin-1 and MuRF1 in skeletal muscle atrophy

Skeletal muscle atrophy occurs in many chronic diseases and disuse conditions. Its severity reduces patient recovery, independence and quality of life. The discovery of two muscle-specific E3 ubiquitin ligases, MAFbx/atrogin-1 and Muscle RING Finger-1 (MuRF1), promoted an expectation of these molecules as targets for therapeutic development. While numerous studies have determined the conditions in which MAFbx/atrogin-1 and MuRF1 mRNA levels are regulated, few studies have investigated their functional role in skeletal muscle. Recently, studies identifying new target substrates for MAFbx/atrogin-1 and MuRF1, outside of their response to the initiation of muscle atrophy, suggest that there is more to these proteins than previously appreciated. This review will highlight our present knowledge of MAFbx/atrogin-1 and MuRF1 in skeletal muscle atrophy, the impact of potential therapeutics and their known regulators and substrates. Finally, we will comment on new approaches that may expand our knowledge of these two molecules in their control of skeletal muscle function.

A Reservoir of Brown Adipocyte Progenitors in Human Skeletal Muscle

Brown adipose tissue uncoupling protein‐1 (UCP1) plays a major role in the control of energy balance in rodents. It has long been thought, however, that there is no physiologically relevant UCP1 expression in adult humans. In this study we show, using an original approach consisting of sorting cells from various tissues and differentiating them in an adipogenic medium, that a stationary population of skeletal muscle cells expressing the CD34 surface protein can differentiate in vitro into genuine brown adipocytes with a high level of UCP1 expression and uncoupled respiration. These cells can be expanded in culture, and their UCP1 mRNA expression is strongly increased by cell‐permeating cAMP derivatives and a peroxisome‐proliferator‐activated receptor‐γ (PPARγ) agonist. Furthermore, UCP1 mRNA was detected in the skeletal muscle of adult humans, and its expression was increased in vivo by PPARγ agonist treatment. All the studies concerning UCP1 expression in adult humans have until now been focused on the white adipose tissue. Here we show for the first time the existence in human skeletal muscle and the prospective isolation of progenitor cells with a high potential for UCP1 expression. The discovery of this reservoir generates a new hope of treating obesity by acting on energy dissipation.

Significant Molecular and Systemic Adaptations after Repeated Sprint Training in Hypoxia

While intermittent hypoxic training (IHT) has been reported to evoke cellular responses via hypoxia inducible factors (HIFs) but without substantial performance benefits in endurance athletes, we hypothesized that repeated sprint training in hypoxia could enhance repeated sprint ability (RSA) performed in normoxia via improved glycolysis and O2 utilization. 40 trained subjects completed 8 cycling repeated sprint sessions in hypoxia (RSH, 3000 m) or normoxia (RSN, 485 m). Before (Pre-) and after (Post-) training, muscular levels of selected mRNAs were analyzed from resting muscle biopsies and RSA tested until exhaustion (10-s sprint, work-to-rest ratio 1∶2) with muscle perfusion assessed by near-infrared spectroscopy. From Pre- to Post-, the average power output of all sprints in RSA was increased (p<0.01) to the same extent (6% vs 7%, NS) in RSH and in RSN but the number of sprints to exhaustion was increased in RSH (9.4±4.8 vs. 13.0±6.2 sprints, p<0.01) but not in RSN (9.3±4.2 vs. 8.9±3.5). mRNA concentrations of HIF-1α (+55%), carbonic anhydrase III (+35%) and monocarboxylate transporter-4 (+20%) were augmented (p<0.05) whereas mitochondrial transcription factor A (−40%), peroxisome proliferator-activated receptor gamma coactivator 1α (−23%) and monocarboxylate transporter-1 (−36%) were decreased (p<0.01) in RSH only. Besides, the changes in total hemoglobin variations (Δ[tHb]) during sprints throughout RSA test increased to a greater extent (p<0.01) in RSH. Our findings show larger improvement in repeated sprint performance in RSH than in RSN with significant molecular adaptations and larger blood perfusion variations in active muscles.

Human skeletal muscle atrophy in amyotrophic lateral sclerosis reveals a reduction in Akt and an increase in atrogin-1

The molecular mechanisms influencing muscle atrophy in humans are poorly understood. Atrogin‐1 and MuRF1, two ubiquitin E3‐ligases, mediate rodent and cell muscle atrophy and are suggested to be regulated by an Akt/Forkhead (FKHR) signaling pathway. Here we investigated the expression of atrogin‐1, MuRF1, and the activity of Akt and its catabolic (FKHR and FKHRL1) and anabolic (p70s6k and GSK‐3?) targets in human skeletal muscle atrophy. The muscle atrophy model used was amyotrophic lateral sclerosis (ALS). All measurements were performed in biopsies from 22 ALS patients and 16 healthy controls as well as in G93A ALS mice. ALS patients had a significant increase in atrogin‐1 mRNA and protein content, which was associated with a decrease in Akt activity. There was no difference in the mRNA and protein content of FKHR, FKHRL1, p70s6k, and GSK‐3?. Similar observations were made in the G93A ALS mice. Human skeletal muscle atrophy, as seen in the ALS model, is associated with an increase in atrogin‐1 and a decrease in Akt. The transcriptional regulation of human atrogin‐1 may be controlled by an Akt‐mediated transcription factor other than FKHR or via another signaling pathway.

β1/β2/β3-adrenoceptor knockout mice are obese and cold-sensitive but have normal lipolytic responses to fasting

Catecholamines are viewed as major stimulants of diet‐ and cold‐induced thermogenesis and of fasting‐induced lipolysis, through the β‐adrenoceptors (β1/β2/β3). To test this hypothesis, we generated β1/β2/β3‐adrenoceptor triple knockout (TKO) mice and compared them to wild type animals. TKO mice exhibited normophagic obesity and cold‐intolerance. Their brown fat had impaired morphology and lacked responses to cold of uncoupling protein‐1 expression. In contrast, TKO mice had higher circulating levels of free fatty acids and glycerol at basal and fasted states, suggesting enhanced lipolysis. Hence, β‐adrenergic signalling is essential for the resistance to obesity and cold, but not for the lipolytic response to fasting.

Antibody-directed myostatin inhibition in 21-mo-old mice reveals novel roles for myostatin signaling in skeletal muscle structure and function

Sarcopenia is the progressive loss of skeletal muscle mass and function with advancing age, leading to reduced mobility and quality of life. We tested the hypothesis that antibody‐directed myostatin inhibition would attenuate the decline in mass and function of muscles of aged mice and that apoptosis would be reduced. Eighteen‐month‐old C57BL/6 mice were treated for 14 wk with a once‐weekly injection of saline (control, n=9) or a mouse chimera of anti‐human myostatin antibody (PF‐354, 10 mg/kg; n=12). PF‐354 prevented the age‐related reduction in body mass and increased soleus, gastrocnemius, and quadriceps muscle mass (P<0.05). PF‐354 increased fiber cross‐sectional area by 12% and enhanced maximum in situ force of tibialis anterior (TA) muscles by 35% (P<0.05). PF‐354 increased the proportion of type IIa fibers by 114% (P<0.01) and enhanced activity of oxidative enzymes (SDH) by 39% (P<0.01). PF‐354 reduced markers of apoptosis in TA muscle cross‐sections by 56% (P<0.03) and reduced caspase3 mRNA by 65% (P<0.04). Antibody‐directed myostatin inhibition attenuated the decline in mass and function of muscles of aging mice, in part, by reducing apoptosis. These observations identify novel roles for myostatin in regulation of muscle mass and highlight the therapeutic potential of antibody‐directed myostatin inhibition for sarcopenia.—Murphy, K. T., Koopman, R., Naim, T., Léger, B., Trieu, J., Ibebunjo, C. Lynch, G. S. Antibody‐directed myostatin inhibition in 21‐mo‐old mice reveals novel roles for myostatin signaling in skeletal muscle structure and function. FASEB J. 24, 4433–4442 (2010). www.fasebj.org

Exercise in the fasted state facilitates fibre type-specific intramyocellular lipid breakdown and stimulates glycogen resynthesis in humans

The effects were compared of exercise in the fasted state and exercise with a high rate of carbohydrate intake on intramyocellular triglyceride (IMTG) and glycogen content of human muscle. Using a randomized crossover study design, nine young healthy volunteers participated in two experimental sessions with an interval of 3 weeks. In each session subjects performed 2 h of constant‐load bicycle exercise (∼75%), followed by 4 h of controlled recovery. On one occasion they exercised after an overnight fast (F), and on the other (CHO) they received carbohydrates before (∼150 g) and during (1 g (kg bw)−1 h−1) exercise. In both conditions, subjects ingested 5 g carbohydrates per kg body weight during recovery. Fibre type‐specific relative IMTG content was determined by Oil red O staining in needle biopsies from m. vastus lateralis before, immediately after and 4 h after exercise. During F but not during CHO, the exercise bout decreased IMTG content in type I fibres from 18 ± 2% to 6 ± 2% (P= 0.007) area lipid staining. Conversely, during recovery, IMTG in type I fibres decreased from 15 ± 2% to 10 ± 2% in CHO, but did not change in F. Neither exercise nor recovery changed IMTG in type IIa fibres in any experimental condition. Exercise‐induced net glycogen breakdown was similar in F and CHO. However, compared with CHO (11.0 ± 7.8 mmol kg−1 h−1), mean rate of postexercise muscle glycogen resynthesis was 3‐fold greater in F (32.9 ± 2.7 mmol kg−1 h−1, P= 0.01). Furthermore, oral glucose loading during recovery increased plasma insulin markedly more in F (+46.80 μU ml−1) than in CHO (+14.63 μU ml−1, P= 0.02). We conclude that IMTG breakdown during prolonged submaximal exercise in the fasted state takes place predominantly in type I fibres and that this breakdown is prevented in the CHO‐fed state. Furthermore, facilitated glucose‐induced insulin secretion may contribute to enhanced muscle glycogen resynthesis following exercise in the fasted state.

Regulation of STARS and its downstream targets suggest a novel pathway involved in human skeletal muscle hypertrophy and atrophy

Skeletal muscle atrophy is a severe consequence of ageing, neurological disorders and chronic disease. Identifying the intracellular signalling pathways controlling changes in skeletal muscle size and function is vital for the future development of potential therapeutic interventions. Striated activator of Rho signalling (STARS), an actin‐binding protein, has been implicated in rodent cardiac hypertrophy; however its role in human skeletal muscle has not been determined. This study aimed to establish if STARS, as well as its downstream signalling targets, RhoA, myocardin‐related transcription factors A and B (MRTF‐A/B) and serum response factor (SRF), were increased and decreased respectively, in human quadriceps muscle biopsies taken after 8 weeks of both hypertrophy‐stimulating resistance training and atrophy‐stimulating de‐training. The mRNA levels of the SRF target genes involved in muscle structure, function and growth, such as α‐actin, myosin heavy chain IIa (MHCIIa) and insulin‐like growth factor‐1 (IGF‐1), were also measured. Following resistance training, STARS, MRTF‐A, MRTF‐B, SRF, α‐actin, MHCIIa and IGF‐1 mRNA, as well as RhoA and nuclear SRF protein levels were all significantly increased by between 1.25‐ and 3.6‐fold. Following the de‐training period all measured targets, except for RhoA, which remained elevated, returned to base‐line. Our results show that the STARS signalling pathway is responsive to changes in skeletal muscle loading and appears to play a role in both human skeletal muscle hypertrophy and atrophy.