The mitochondrial rhomboid protease PSARL is a new candidate gene for type 2 diabetes

Aims/hypothesis This study aimed to identify genes that are expressed in skeletal muscle, encode proteins with functional significance in mitochondria, and are associated with type 2 diabetes.
Methods We screened for differentially expressed genes in skeletal muscle of Psammomys obesus (Israeli sand rats), and prioritised these on the basis of genomic localisation and bioinformatics analysis for proteins with likely mitochondrial functions.
Results We identified a mitochondrial intramembrane protease, known as presenilins-associated rhomboid-like protein (PSARL) that is associated with insulin resistance and type 2 diabetes. Expression of PSARL was reduced in skeletal muscle of diabetic Psammomys obesus, and restored after exercise training to successfully treat the diabetes. PSARL gene expression in human skeletal muscle was correlated with insulin sensitivity as assessed by glucose disposal during a hyperinsulinaemic–euglycaemic clamp. In 1,031 human subjects, an amino acid substitution (Leu262Val) in PSARL was associated with increased plasma insulin concentration, a key risk factor for diabetes. Furthermore, this variant interacted strongly with age to affect insulin levels, accounting for 5% of the variation in plasma insulin in elderly subjects.
Conclusions/interpretation Variation in PSARL sequence and/or expression may be an important new risk factor for type 2 diabetes and other components of the metabolic syndrome.

Identification of novel genes expressed during rhabdomyosarcoma differentiation using cDNA microarrays

Rhabdomyosarcomas (RMS) are highly aggressive tumors that are thought to arise as a consequence of the regulatory disruption of the growth and differentiation of skeletal muscle progenitor cells. Normal myogenesis is characterized by the expression of the myogenic regulatory factor gene family but, despite their expression in RMS, these tumor cells fail to complete the latter stages of myogenesis. The RMS cell line RD-A was treated with 12-O-tetradecanoylphorbol-13-acetate to induce differentiation and cultured for 10 days. RNA was extracted on days 1, 3, 6, 8 and 10. A human skeletal muscle cDNA microarray was developed and used to analyze the global gene expression of RMS tumors over the time-course of differentiation. As a comparison, the genes identified were subsequently examined during the differentiated primary human skeletal muscle cultures. Prothymosin alpha (PTMA), and translocase of inner mitochondrial membrane 10 (Tim10), two genes not previously implicated in RMS, showed reduced expression during differentiation. Marked differences in the expression of PTMA and Tim10 were observed during the differentiation of human primary skeletal muscle cells. These results identify several new genes with potential roles in the myogenic arrest present in rhabdomyosarcoma. PTMA expression in RMS biopsy samples might prove to be an effective diagnostic marker for this disease.

Regulation of metabolic transcriptional co-activators and transcription factors with acute exercise

Endurance exercise improves insulin sensitivity and increases fat oxidation, which are partly facilitated by the induction of metabolic transcription factors. Next to exercise, increased levels of FFA's also increase the gene expression of transcription factors, hence making it difficult to discern the effects from contractile signals produced during exercise, from those produced by increased circulatory FFA's. We aimed to investigate, in human skeletal muscle, whether acute exercise affects gene expression of metabolic transcriptional co-activators and transcription factors, including PGC-1α, PRC, PPARα, β/δ, and γ and RXR, SREBP-1c and FKHR, and to discern the effect of exercise per se from those of elevated levels of FFA. Two hours of endurance exercise was performed either in the fasted state, or following carbohydrate ingestion prior to and during exercise, thereby blunting the fasting-induced increase in FA availability and oxidation. Of the genes measured, PGC-1α and PRC mRNA increased immediately after, while PPARβ/δ and FKHR mRNA increased 1–4 h after exercise, irrespective of the increases in FFA's. Our results suggest that the induction in vivo of metabolic transcription factors implicated in mitochondrial biogenesis are under the control of inherent signals, (PGC-1α, PRC), while those implicated in substrate selection are under the control of associated signals (PPARβ/δ, FKHR) stimulated from the contracting skeletal muscle that are independent of circulating FFA levels.

Exercise-induced activation of STAT3 signaling is increased with age

Activation of the transcription factor signal transducers and activators of transcription (STAT) 3 is common to many inflammatory cytokines and growth factors, with recent evidence of involvement in skeletal muscle regeneration. The purpose of this study was to determine whether STAT3 signaling activation is regulated differentially, at rest and following intense resistance exercise, in aged human skeletal muscle. Skeletal muscle biopsies were harvested from healthy younger (n = 11, 20.4 ± 0.8 years) and older men (n = 10, 67.4 ± 1.3 years) under resting conditions and 2 h after the completion of resistance exercise. No differences were evident at rest, whereas the phosphorylation of STAT3 was significantly increased in old (23-fold) compared to young (5-fold) subjects after exercise. This correlated with significantly higher induction of the STAT3 target genes including; interleukin-6 (IL-6), JUNB, c-MYC, and suppressor of cytokine signaling (SOCS) 3 mRNA in older subjects following exercise. Despite increased SOCS3 mRNA, cellular protein abundance was suppressed. SOCS3 protein is an important negative regulator of STAT3 activation and cytokine signaling. Thus, in aged human muscle, elevated responsiveness of the STAT3 signaling pathway and suppressed SOCS3 protein are evident following resistance exercise. These data suggest that enhanced STAT3 signaling responsiveness to proinflammatory factors may impact on mechanisms of muscle repair and regeneration.

Whey protein ingestion activates mTOR-dependent signalling after resistance exercise in young men : a double-blinded randomized controlled trial

The effect of resistance exercise with the ingestion of supplementary protein on the activation of the mTOR cascade, in human skeletal muscle has not been fully elucidated. In this study, the impact of a single bout of resistance exercise, immediately followed by a single dose of whey protein isolate (WPI) or placebo supplement, on the activation of mTOR signalling was analyzed. Young untrained men completed a maximal single-legged knee extension exercise bout and were randomized to ingest either WPI supplement (n = 7) or the placebo (n = 7). Muscle biopsies were taken from the vastus lateralis before, and 2, 4 and 24 hr post-exercise. WPI or placebo ingestion consumed immediately post-exercise had no impact on the phosphorylation of Akt (Ser⁴⁷³). However, WPI significantly enhanced phosphorylation of mTOR (Ser²⁴⁴⁸), 4E-BP1 (Thr^37/46) and p70S6K (Thr³⁸⁹) at 2 hr post-exercise. This study demonstrates that a single dose of WPI, when consumed in modest quantities, taken immediately after resistance exercise elicits an acute and transient activation of translation initiation within the exercised skeletal muscle.

Exercise as an intervention to improve metabolic outcomes after intrauterine growth restriction

Individuals born after intrauterine growth restriction (IUGR) are at an increased risk of developing diabetes in their adult life. IUGR impairs β-cell function and reduces β-cell mass, thereby diminishing insulin secretion. IUGR also induces insulin resistance, with impaired insulin signaling in muscle in adult humans who were small for gestational age (SGA) and in rodent models of IUGR. There is epidemiological evidence in humans that exercise in adults can reduce the risk of metabolic disease following IUGR. However, it is not clear whether adult IUGR individuals benefit to the same extent from exercise as do normal-birth-weight individuals, as our rat studies suggest less of a benefit in those born IUGR. Importantly, however, there is some evidence from studies in rats that exercise in early life might be able to reverse or reprogram the long-term metabolic effects of IUGR. Studies are needed to address gaps in current knowledge, including determining the mechanisms involved in the reprogramming effects of early exercise in rats, whether exercise early in life or in adulthood has similar beneficial metabolic effects in larger animal models in which insulin resistance develops after IUGR. Human studies are also needed to determine whether exercise training improves insulin secretion and insulin sensitivity to the same extent in IUGR adults as in control populations. Such investigations will have implications for customizing the recommended level and timing of exercise to improve metabolic health after IUGR.

Validation of a multiplex reverse transcription and pre-amplification method using TaqMan(®) MicroRNA assays.

Since the discovery of microRNAs (miRNAs), different approaches have been developed to label, amplify and quantify miRNAs. The TaqMan(®) technology, provided by Applied Biosystems (ABIs), uses a stem-loop reverse transcription primer system to reverse transcribe the RNA and amplify the cDNA. This method is widely used to identify global differences between the expression of 100s of miRNAs across comparative samples. This technique also allows the quantification of the expression of targeted miRNAs to validate observations determined by whole-genome screening or to analyze few specific miRNAs on a large number of samples. Here, we describe the validation of a method published by ABIs on their web site allowing to reverse transcribe and pre-amplify multiple miRNAs and snoRNAs simultaneously. The validation of this protocol was performed on human muscle and plasma samples. Fast and cost efficient, this method achieves an easy and convenient way to screen a relatively large number of miRNAs in parallel.

Changes in mitochondrial function and mitochondria associated protein expression in response to 2-weeks of high intensity interval training

PURPOSE: High-intensity short-duration interval training (HIT) stimulates functional and metabolic adaptation in skeletal muscle, but the influence of HIT on mitochondrial function remains poorly studied in humans. Mitochondrial metabolism as well as mitochondrial-associated protein expression were tested in untrained participants performing HIT over a 2-week period. METHODS: Eight males performed a single-leg cycling protocol (12 × 1 min intervals at 120% peak power output, 90 s recovery, 4 days/week). Muscle biopsies (vastus lateralis) were taken pre- and post-HIT. Mitochondrial respiration in permeabilized fibers, citrate synthase (CS) activity and protein expression of peroxisome proliferator-activated receptor gamma coactivator (PGC-1α) and respiratory complex components were measured. RESULTS: HIT training improved peak power and time to fatigue. Increases in absolute oxidative phosphorylation (OXPHOS) capacities and CS activity were observed, but not in the ratio of CCO to the electron transport system (CCO/ETS), the respiratory control ratios (RCR-1 and RCR-2) or mitochondrial-associated protein expression. Specific increases in OXPHOS flux were not apparent after normalization to CS, indicating that gross changes mainly resulted from increased mitochondrial mass. CONCLUSION: Over only 2 weeks HIT significantly increased mitochondrial function in skeletal muscle independently of detectable changes in mitochondrial-associated and mitogenic protein expression.

Effect of resistance exercise contraction mode and protein supplementation on members of the STARS signalling pathway

The striated muscle activator of Rho signalling (STARS) pathway is suggested to provide a link between external stress responses and transcriptional regulation in muscle. However, the sensitivity of STARS signalling to different mechanical stresses has not been investigated. In a comparative study, we examined the regulation of the STARS signalling pathway in response to unilateral resistance exercise performed as either eccentric (ECC) or concentric (CONC) contractions as well as prolonged training; with and without whey protein supplementation. Skeletal muscle STARS, myocardian-related transcription factor-A (MRTF-A) and serum response factor (SRF) mRNA and protein, as well as muscle cross-sectional area and maximal voluntary contraction, were measured. A single-bout of exercise produced increases in STARS and SRF mRNA and decreases in MRTF-A mRNA with both ECC and CONC exercise, but with an enhanced response occurring following ECC exercise. A 31% increase in STARS protein was observed exclusively after CONC exercise (P < 0.001), while pSRF protein levels increased similarly by 48% with both CONC and ECC exercise (P < 0.001). Prolonged ECC and CONC training equally stimulated muscle hypertrophy and produced increases in MRTF-A protein of 125% and 99%, respectively (P < 0.001). No changes occurred for total SRF protein. There was no effect of whey protein supplementation. These results show that resistance exercise provides an acute stimulation of the STARS pathway that is contraction mode dependent. The responses to acute exercise were more pronounced than responses to accumulated training, suggesting that STARS signalling is primarily involved in the initial phase of exercise-induced muscle adaptations.

Regulation of the STARS signaling pathway in response to endurance and resistance exercise and training

The striated muscle activator of Rho signaling (STARS) protein and members of its downstream signaling pathway, including myocardin-related transcription factor-A (MRTF-A) and SRF, are increased in response to prolonged resistance exercise training but also following a single bout of endurance cycling. The aim of the present study was to measure and compare the regulation of STARS, MRTF-A and SRF mRNA and protein following 10 weeks of endurance training (ET) versus resistance training (RT), as well as before and following a single bout of endurance (EE) versus resistance exercise (RE). Following prolonged training, STARS, MRTF-A and SRF mRNA levels were all increased by similar magnitude, irrespective of training type. In the training-habituated state, STARS mRNA increased following a single-bout RE when measured 2.5 and 5 h post-exercise and had returned to resting level by 22 h following exercise. MRTF-A and SRF mRNA levels were decreased by 2.5, 5, and 22 h following a single bout of RE and EE exercise when compared to their respective basal levels, with no significant difference seen between the groups at any of the time points. No changes in protein levels were observed following the two modes of exercise training or a single bout of exercise. This study demonstrates that the stress signals elicited by ET and RT result in a comparable regulation of members of the STARS pathway. In contrast, a single bout of EE and RE, performed in the trained state, elicit different responses. These observations suggest that in the trained state, the acute regulation of the STARS pathway following EE or RE may be responsible for exercise-specific muscle adaptations.

Modulating exercise-induced hormesis: does less equal more?

Hormesis encompasses the notion that low levels of stress stimulate or upregulate existing cellular and molecular pathways that improve the capacity of cells and organisms to withstand greater stress. This notion underlies much of what we know about how exercise conditions the body and induces long-term adaptations. During exercise, the body is exposed to various forms of stress, including thermal, metabolic, hypoxic, oxidative, and mechanical stress. These stressors activate biochemical messengers, which in turn activate various signaling pathways that regulate gene expression and adaptive responses. Historically, antioxidant supplements, nonsteroidal anti-inflammatory drugs, and cryotherapy have been favored to attenuate or counteract exercise-induced oxidative stress and inflammation. However, reactive oxygen species and inflammatory mediators are key signaling molecules in muscle, and such strategies may mitigate adaptations to exercise. Conversely, withholding dietary carbohydrate and restricting muscle blood flow during exercise may augment adaptations to exercise. In this review article, we combine, integrate, and apply knowledge about the fundamental mechanisms of exercise adaptation. We also critically evaluate the rationale for using interventions that target these mechanisms under the overarching concept of hormesis. There is currently insufficient evidence to establish whether these treatments exert dose-dependent effects on muscle adaptation. However, there appears to be some dissociation between the biochemical/molecular effects and functional/performance outcomes of some of these treatments. Although several of these treatments influence common kinases, transcription factors, and proteins, it remains to be determined if these interventions complement or negate each other, and whether such effects are strong enough to influence adaptations to exercise.

Heterogeneous atrophy occurs within individual lower limb muscles during 60 days of bed rest

To better understand disuse muscle atrophy, via magnetic resonance imaging, we sequentially measured muscle cross-sectional area along the entire length of all individual muscles from the hip to ankle in nine male subjects participating in 60-day head-down tilt bed rest (2nd Berlin BedRest Study; BBR2-2). We hypothesized that individual muscles would not atrophy uniformly along their length such that different regions of an individual muscle would atrophy to different extents. This hypothesis was confirmed for the adductor magnus, vasti, lateral hamstrings, medial hamstrings, rectus femoris, medial gastrocnemius, lateral gastrocnemius, tibialis posterior, flexor hallucis longus, flexor digitorum longus, peroneals, and tibialis anterior muscles (P ≤ 0.004). In contrast, the hypothesis was not confirmed in the soleus, adductor brevis, gracilis, pectineus, and extensor digitorum longus muscles (P ≥ 0.20). The extent of atrophy only weakly correlated (r = -0.30, P < 0.001) with the location of greatest cross-sectional area. The rate of atrophy during bed rest also differed between muscles (P < 0.0001) and between some synergists. Most muscles recovered to their baseline size between 14 and 90 days after bed rest, but flexor hallucis longus, flexor digitorum longus, and lateral gastrocnemius required longer than 90 days before recovery occurred. On the basis of findings of differential atrophy between muscles and evidence in the literature, we interpret our findings of intramuscular atrophy to reflect differential disuse of functionally different muscle regions. The current work represents the first lower-limb wide survey of intramuscular differences in disuse atrophy. We conclude that intramuscular differential atrophy occurs in most, but not all, of the muscles of the lower limb during prolonged bed rest.

Effect of soccer training on the running speed and the blood lactate concentration at the lactate minimum test

Tegtbur et al. [23] devised a new method able to estimate the intensity at maximal lactate steady state termed lactate minimum test. According to Billat et al. [7], no studies have yet been published on the affect of training on highest blood lactate concentration that can be maintained over time without continual blood lactate accumulation. Therefore, the aim of the present study was to verify the effect of soccer training on the running speed and the blood lactate concentration (BLC) at the lactate minimum test (Lac(min)). Thirteen Brazilian male professional soccer players, all members of the same team playing at National level, volunteered for this study. Measurements were carried out before (pre) and after (post) eight weeks of soccer training. The Lac(min) test was adapted to the procedures reported by Tegtbur et al. [23]. The running speed at the Lac(min) test was taken when the gradient of the line was zero. Differences in running speed and blood lactate concentration at the Lac(min) test before (pre) and after (post) the training program were evaluated by Student's paired t-test. The training program increased the running speed at the Lac(min) test (14.94 +/- 0.21 vs. 15.44 +/- 0.42* km(.)h(-1)) and the blood lactate concentration (5.11 +/- 2.31 vs. 6.93 +/- 1.33* mmol(.)L(-1)). The enhance in the blood lactate concentration may be explained by an increase in the lactate/H+ transport capacity of human skeletal muscle verified by other authors.