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Muscle fiber size and oxidative metabolism are inversely related, suggesting that a glycolytic metabolism may offer a growth advantage in muscle fibers. However, the mechanisms underlying this advantage remains unknown. Nearly 100 years ago, Warburg reported that cancer cells take up more glucose to produce glycolytic intermediates for anabolic reactions such as amino acid-protein synthesis. The aim of this study was to test whether glycolysis contributes to anabolic signalling responses and hypertrophy in post-mitotic muscle cells. Skeletal muscle hypertrophy was induced in vitro by treating mouse C2C12 myotubes with IGF-1.14C glucose was added to differentiation medium and radioactivity in isolated protein was measured. We exposed differentiated C2C12 and primary mouse myotubes, to 2-deoxyglucose (2DG) and PHGDH siRNA upon which we assessed myotube diameter and signaling pathways involved in the regulation of muscle fiber size. Here, we present evidence that, hypertrophying C2C12 myotubes undergo a cancer-like metabolic reprogramming. First, IGF-1-induced C2C12 myotube hypertrophy increases shunting of carbon from glucose into protein. Second, reduction of glycolysis through 2-deoxy-D-glucose (2DG) lowers C2C12 and primary myotube size 16-40%. Third, reducing the cancer metabolism-associated enzyme PHGDH decreases C2C12 and primary myotube size 25-52%, whereas PHGDH overexpression increases C2C12 myotube size ≈20%. Fourth, the muscle hypertrophy-promoting kinase AKT regulates PHGDH expression. Together these results suggest that glycolysis is important for hypertrophying C2C12 myotubes by reprograming their metabolism similar to cancer cells.

Background: Exercise changes the concentrations of many metabolites, which are small molecules (< 1.5 kDa) metabolized by the reactions ofhuman metabolism. In recent years, especially mass spectrometry-based metabolomics methods have allowed researchers to measure up to hundreds of metabolites in a single sample in a non-biased fashion. To summarize human exercise metabolomics studies to date, we conducted a systematic review that reports the results of experiments that foundmetabolite concentrations changes after a bout of human endurance or resistance exercise. Methods: We carried out a systematic review following PRISMA guidelines and searched for human metabolomics studies that report metabolite concentrations before and within 24 h after endurance or resistance exercise in blood, urine, or sweat. We then displayedmetabolites that significantly changed their concentration in at least two experiments. Results: Twenty-seven studies and 57 experiments matched our search criteria and were analyzed. Within these studies, 196 metabolites changed their concentration significantly within 24 h after exercise in at least two experiments. Human biofluids contain mainly unphosphorylated metabolites as the phosphorylation of metabolites such as ATP, glycolytic intermediates, or nucleotides traps these metabolites within cells. Lactate, pyruvate, TCA cycle intermediates, fatty acids, acylcarnitines, and ketone bodies all typically increase after exercise, whereas bile acids decrease. In contrast, the concentrations of proteinogenic and non-proteinogenic amino acids change in different directions. Conclusion: Across different exercise modes and in different subjects, exercise often consistently changes the average concentrations of metabolites that belong to energy metabolism and other branches of metabolism. This dataset is a useful resource for those that wish to study human exercise metabolism.

One of the most striking adaptations to exercise is the skeletal muscle hypertrophy that occurs in response to resistance exercise. A large body of work shows that a mammalian target of rapamycin complex 1 (mTORC1)-mediated increase of muscle protein synthesis is the key, but not sole, mechanism by which resistance exercise causes muscle hypertrophy. While much of the hypertrophy signaling cascade has been identified, the initiating, resistance exercise-induced and hypertrophy-stimulating stimuli have remained elusive. For the purpose of this review, we define an initiating, resistance exercise-induced and hypertrophy-stimulating signal as “hypertrophy stimulus,” and the sensor of such a signal as “hypertrophy sensor.” In this review we discuss our current knowledge of specific mechanical stimuli, damage/injury-associated and metabolic stress-associated triggers, as potential hypertrophy stimuli. Mechanical signals are the prime hypertrophy stimuli candidates, and a filamin-C-BAG3-dependent regulation of mTORC1, Hippo, and autophagy signaling is a plausible albeit still incompletely characterized hypertrophy sensor. Other candidate mechanosensing mechanisms are nuclear deformation-initiated signaling or several mechanisms related to costameres, which are the functional equivalents of focal adhesions in other cells. While exercise-induced muscle damage is probably not essential for hypertrophy, it is still unclear whether and how such muscle damage could augment a hypertrophic response. Interventions that combine blood flow restriction and especially low load resistance exercise suggest that resistance exercise-regulated metabolites could be hypertrophy stimuli, but this is based on indirect evidence and metabolite candidates are poorly characterized.

The Hippo effector YAP has recently been identified as a potent driver of embryonal rhabdomyosarcoma (ERMS). Most reports suggest that the YAP paralogue TAZ (gene symbol WWTR1) functions as YAP but, in skeletal muscle, TAZ has been reported to promote myogenic differentiation, whereas YAP inhibits it. Here, we investigated whether TAZ is also a rhabdomyosarcoma oncogene or whether TAZ acts as a YAP antagonist. Immunostaining of rhabdomyosarcoma tissue microarrays revealed that TAZ is significantly associated with poor survival in ERMS. In 12% of fusion gene‐negative rhabdomyosarcomas, the TAZ locus is gained, which is correlated with increased expression. Constitutively active TAZ S89A significantly increased proliferation of C2C12 myoblasts and, importantly, colony formation on soft agar, suggesting transformation. However, TAZ then switches to enhance myogenic differentiation in C2C12 myoblasts, unlike YAP. Conversely, lentiviral shRNA‐mediated TAZ knockdown in human ERMS cells reduced proliferation and anchorage‐independent growth. While TAZ S89A or YAP1 S127A similarly activated the 8XGTIIC–Luc Hippo reporter, only YAP1 S127A activated the Brachyury (T‐box) reporter. Consistent with its oncogene function, TAZ S89A induced expression of the ERMS cancer stem cell gene Myf5 and the serine biosynthesis pathway (Phgdh, Psat1, Psph) in C2C12 myoblasts. Thus, TAZ is associated with poor survival in ERMS and could act as an oncogene in rhabdomyosarcoma. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Persistent hyperactivity of the Hippo effector YAP in activated satellite cells is sufficient to cause embryonal rhabdomyosarcoma (ERMS) in mice. In humans, YAP is abundant and nuclear in the majority of ERMS cases, and high YAP expression is associated with poor survival. However, YAP1 is rarely mutated in human ERMS. Instead, the most common mutations in ERMS are oncogenic RAS mutations. First, to compare YAP1S127A and KRASG12V-driven rhabdomyosarcomas, we re-analysed gene expression microarray datasets from mouse rhabdomyosarcomas caused by these genes. This revealed that only 20% of the up or downregulated genes are identical, suggesting substantial differences in gene expression between YAP and KRAS-driven rhabdomyosarcomas. As oncogenic RAS has been linked to YAP in other types of cancer, we also tested whether KRAS G12V alone or in combination with loss of p53 and p16 activates YAP in myoblasts. We found that neither KRAS G12V alone nor KRAS G12V combined with loss of p53 and p16 activated Yap or Yap/Taz-Tead1–4 transcriptional activity in C2C12 myoblasts or U57810 cells. In conclusion, whilst oncogenic KRAS mutation might activate Yap in other cell types, we could find no evidence for this in myoblasts because the expression of KRAS G12V expression did not change Yap/Taz activity in myoblasts and there was a limited overlap in gene expression between KRAS G12V and YAP1 S127A-driven tumours.

Polyglutamine expansions in the huntingtin gene cause Huntington’s disease (HD). Huntingtin is ubiquitously expressed, leading to pathological alterations also in peripheral organs. Variations in the length of the polyglutamine tract explain up to 70% of the age-at-onset variance, with the rest of the variance attributed to genetic and environmental modifiers. To identify novel disease modifiers, we performed an unbiased mutagenesis screen on an HD mouse model, identifying a mutation in the skeletal muscle voltage-gated sodium channel (Scn4a, termed ‘draggen’ mutation) as a novel disease enhancer. Double mutant mice (HD; Scn4aDgn/+) had decreased survival, weight loss and muscle atrophy. Expression patterns show that the main tissue affected is skeletal muscle. Intriguingly, muscles from HD; Scn4aDgn/+ mice showed adaptive changes similar to those found in endurance exercise, including AMPK activation, fibre type switching and upregulation of mitochondrial biogenesis. Therefore, we evaluated the effects of endurance training on HD mice. Crucially, this training regime also led to detrimental effects on HD mice. Overall, these results reveal a novel role for skeletal muscle in modulating systemic HD pathogenesis, suggesting that some forms of physical exercise could be deleterious in neurodegeneration., Human Molecular Genetics, 27 (10), ISSN:0964-6906, ISSN:1460-2083

Sarcomas are rare cancers (≈1% of all solid tumours) usually of mesenchymal origin. Here, we review evidence implicating the Hippo pathway in soft tissue sarcomas. Several transgenic mouse models of Hippo pathway members (Nf2, Mob1, LATS1 and YAP1 mutants) develop various types of sarcoma. Despite that, Hippo member genes are rarely point mutated in human sarcomas. Instead, WWTR1-CAMTA1 and YAP1-TFE3 fusion genes are found in almost all cases of epithelioid haemangioendothelioma. Also copy number gains of YAP1 and other Hippo members occur at low frequencies but the most likely cause of perturbed Hippo signalling in sarcoma is the cross-talk with commonly mutated cancer genes such as KRAS, PIK3CA, CTNNB1 or FBXW7. Current Hippo pathway-targeting drugs include compounds that target the interaction between YAP and TEAD G protein-coupled receptors (GPCR) and the mevalonate pathway (e.g. statins). Given that many Hippo pathway-modulating drugs are already used in patients, this could lead to early clinical trials testing their efficacy in different types of sarcoma.

Magnetic resonance imaging (MRI) was used to investigate muscle rehabilitation following cast immobilization. The aim was to explore MRI as an imaging biomarker of muscle function. Sixteen patients completed an eight-week rehabilitation programme following six weeks of cast immobilization for an ankle fracture. MRI of the lower leg was performed at two-week intervals for 14 weeks. Total volume and anatomical cross-sectional areas at 70% of the distance from lateral malleolus to tibial tuberosity (ACSA) were measured for tibialis anterior (TA), medial and lateral gastrocnemius (GM and GL) and soleus (SOL). Pennation angle of muscle fascicules was measured at the same position in GM. Fractional fat/water contents and T2 relaxation times before and after exercise were calculated. Strength was measured as maximum isometric torque developed in plantar- and dorsi-flexion. Torque increased by (mean [SD]) 1.10 (0.32) N m day−1 in males, 0.74 (0.43) N m day−1 in females in plantar-flexion (0.9% of final strength p...

Ziel des Krafttrainings ist es, Muskelmasse und Kraft zu steigern. Um dies zu erreichen, fuhren Sportler ein progressives Krafttraining durch, um mit hohen Widerstanden die Innervation der trainierten Muskeln, die Muskelmasse und die Kraft per Muskelquerschnitt zu erhohen. Die Trainierbarkeit der Kraft und die absolute Kraft hangen zudem von dem Talent ab, welches von Variationen der DNA-Sequenz abhangt. Krafttraining aktiviert die mTOR-Kaskade und diese Aktivierung ist fur die Erhohung der Muskelmasse und damit die Hypertrophie notwendig. Der Myostatin-Smad-Signalweg reguliert die Muskelmasse auch, doch die Rolle bei der Anpassung an Krafttraining ist unklar. Zum Schluss dieses Kapitels wird auf die Satellitenzellen eingegangen, die bei der Regeneration und Anpassung nach einem Kraftreiz eine Sonderstellung einnehmen.

Molecular exercise physiology is the study of exercise physiology using molecular biology methods. The development of differentiated cell types is regulated by transcription factors like the muscle-making MyoD that specifies cell type, while others regulate the development of muscle, tendons, and bones. Maternal nutrition and exercise commonly affect embryonic development through epigenetic mechanisms. Adaptation to exercise involves sensor proteins detecting exercise-related signals, the processing of signals by signalling proteins and networks, and the regulation of the actual adaptations by effector proteins. Many sport- and exercise-related traits depend on both common and rare DNA sequence variations, including the muscle mass-increasing myostatin (GDF8) loss-of-function and the haematocrit-increasing EPOR gain-of-function mutations. Additionally, common DNA sequence variations contribute to the inherited variability of development, body height, strength, and endurance. Finally, in addition to ethical concerns, current genetic performance tests only explain a fraction of the variation of sport and exercise-related traits.

SummaryThe role of the Hippo pathway effector YAP1 in soft tissue sarcomas is poorly defined. Here we report that YAP1 activity is elevated in human embryonal rhabdomyosarcoma (ERMS). In mice, sustained YAP1 hyperactivity in activated, but not quiescent, satellite cells induces ERMS with high penetrance and short latency. Via its transcriptional program with TEAD1, YAP1 directly regulates several major hallmarks of ERMS. YAP1-TEAD1 upregulate pro-proliferative and oncogenic genes and maintain the ERMS differentiation block by interfering with MYOD1 and MEF2 pro-differentiation activities. Normalization of YAP1 expression reduces tumor burden in human ERMS xenografts and allows YAP1-driven ERMS to differentiate in situ. Collectively, our results identify YAP1 as a potent ERMS oncogenic driver and a promising target for differentiation therapy.

The use of human urine as a diagnostic tool has many advantages, such as ease of sample acquisition and noninvasiveness. However, the discovery of novel biomarkers, as well as biomarker patterns, in urine is hindered mainly by a lack of comparable datasets. To fill this gap, we assembled a new urinary fingerprint database. Here, we report the establishment of a human urinary proteomic fingerprint database using urine from 200 individuals analysed by SELDI-TOF (surface enhanced laser desorption ionisation-time of flight) mass spectrometry (MS) on several chip surfaces (SEND, HP50, NP20, Q10, CM10, and IMAC30). The database currently lists 2490 unique peaks/ion species from 1172 nonredundant SELDI analyses in the mass range of 1500 to 150000. All unprocessed mass spectrometric scans are available as “.xml” data files. Additionally, 1384 peaks were included from external studies using CE (capillary electrophoresis)-MS, MALDI (matrix assisted laser desorption/ionisation), and CE-MALDI hybrids. We propose to use this platform as a global resource to share and exchange primary data derived from MS analyses in urinary research.

This paper addresses practical and ethical considerations regarding genetic tests to predict performance and/or risk of exercise-related injury or illness. Various people might wish to conduct sport-related genetic tests for a variety of reasons. For example, an individual might seek personal genetic information to help guide their own sport participation. A sports coach might wish to test young athletes to aid team selection or individualize training. A physician might want to predict the risk of injury or illness in athletes and advise regarding selection or preventative measures. An insurance company might seek to estimate the risk of career-threatening injury for athletes based partly on genetic information. Whilst this information is, in part, encoded in our DNA sequence, the available tests allow generally only a poor prediction of the aforementioned variables. In other words, the current genetic tests and analysis methods are not powerful enough to inform important decisions in sport to a substantial degree. It is particularly disappointing that more than half of the commercially available genetic tests related to exercise and sport do not appear to identify publicly the genetic variants they assess, making scrutiny by academic scholars and consumers (or their representatives) impossible. There are also challenging ethical issues to consider. For example, the imposition of genetic tests on individuals (especially young people) by third parties is potentially susceptible to abuse. Scientists and practitioners should understand the limitations of the tests currently available, the ethical concerns and the importance of counselling before and after testing so that they are only used in a responsible manner.

This paper addresses practical and ethical considerations regarding genetic tests to predict performance and/or risk of exercise-related injury or illness. Various people might wish to conduct sport-related genetic tests for a variety of reasons. For example, an individual might seek personal genetic information to help guide their own sport participation. A sports coach might wish to test young athletes to aid team selection or individualize training. A physician might want to predict the risk of injury or illness in athletes and advise regarding selection or preventative measures. An insurance company might seek to estimate the risk of career-threatening injury for athletes based partly on genetic information. Whilst this information is, in part, encoded in our DNA sequence, the available tests allow generally only a poor prediction of the aforementioned variables. In other words, the current genetic tests and analysis methods are not powerful enough to inform important decisions in sport to a substantial degree. It is particularly disappointing that more than half of the commercially available genetic tests related to exercise and sport do not appear to identify publicly the genetic variants they assess, making scrutiny by academic scholars and consumers (or their representatives) impossible. There are also challenging ethical issues to consider. For example, the imposition of genetic tests on individuals (especially young people) by third parties is potentially susceptible to abuse. Scientists and practitioners should understand the limitations of the tests currently available, the ethical concerns and the importance of counselling before and after testing so that they are only used in a responsible manner.

Purpose: To investigate MRI biomarkers of muscle atrophy during cast immobilization of the lower leg. Materials and Methods: Eighteen patients (8 male, 10 female), who had one lower leg immobilized in a cast, underwent 3.0 Tesla (T) MR imaging 5, 8, 15, 29, and 43 days after casting. Measurements were made on both lower legs of total muscle volume. Cross-sectional area (CSA), fractional water content, and T2 were measured in tibialis anterior (TA), gastrocnemius medialis (GM) and lateralis (GL) and soleus (SOL). Fiber pennation angle was measured in GM. Results: Total muscle volume decreased by 17% (P < 0.001) over the 6 weeks of immobilization. The greatest loss in CSA (mean[SD]) was seen in GM (−23.3(8.7)%), followed by SOL (−19.0(9.8)%), GL (−17.1(6.5)%), and TA (−10.7(5.9)%). Significant reductions of CSA were also detectable in the contra-lateral leg. T2 increased in all muscles: TA 27.0(2.5) ms to 29.6(2.8) ms (P < 0.001), GM 34.6(2.9) ms to 39.8(5.4) ms (P < 0.001) and SOL 34.4 (2.9) ms to 44.9(5.9) ms (P < 0.001). Small reductions were found in fractional water content. Pennation angle decreased in the cast leg (P < 0.001). Conclusion: Quantitative MR imaging can detect and monitor progressive biochemical and biophysical changes in muscle during immobilization. J. Magn. Reson. Imaging 2012;35:686-695. © 2011 Wiley Periodicals, Inc.

Citrate synthase (CS) is an enzyme of the Krebs cycle that plays a key role in mitochondrial metabolism. The aim of this study was to investigate the mechanisms underlying low activity of citrate synthase (CS) in A/J mice compared with other inbred strains of mice. Enzyme activity, protein content, and mRNA levels of CS were studied in the quadriceps muscles of A/J, BALB/cByJ, C57BL/6J, C3H/HeJ, DBA/2J, and PWD/PhJ strains of mice. Cytochrome c protein content was also measured. The results of the study indicate that A/J mice have a 50–65% reduction in CS activity compared with other strains despite similar levels of Cs mRNA and lack of differences in CS and cytochrome c protein content. CS from A/J mice also showed lower Michaelis constant ( Km) for both acetyl CoA and oxaloacetate compared with the other strains of mice. In silico analysis of the genomic sequence identified a nonsynonymous single nucleotide polymorphism (SNP) (rs29358506, H55N) in Cs gene occurring near the site of the protein interacting with acetyl CoA. Allelic variants of the polymorphism segregated with the catalytic properties of CS enzyme among the strains. In summary, H55N polymorphism in Cs could be the underlying cause of low CS activity and its high affinity for substrates in A/J mice compared with other strains. This SNP might also play a role in resistance to obesity of A/J mice.

The expression, regulation and function of mammalian Hippo pathway members in skeletal muscle is largely unknown. The aim of this study was thus to test the hypothesis that core members of the mammalian Hippo pathway are expressed in skeletal muscle and that the transcriptional co-factor Yap, a core member of the Hippo pathway, regulates C2C12 myogenesis. We found that the major components of the mammalian Hippo pathway including Yap are all expressed in skeletal muscles, C2C12 myoblasts and myotubes. In C2C12 myoblasts, Yap Ser127 phosphorylation is low and Yap localises to nuclei. Upon differentiation, Yap Ser127 phosphorylation increases approximately 20-fold and Yap translocates from the nucleus to the cytosol. To test whether the observed increase of Yap Ser127 phosphorylation is required for differentiation we overexpressed hYAP1 S127A, a mutant that can not be phosphorylated at Ser127, in C2C12 myoblasts. We found that overexpression of hYAP S127A prevented myotube formation, whereas the overexpression of wildtype hYAP1 or empty vector had no effect. In addition, more hYAP1 S127A overexpressing cells progressed through the S phase of the cell cycle and the expression of MRFs (myogenin, Myf5), Mef2c and cell cycle regulators (p21, cyclin D1) was significantly changed when compared to wildtype hYAP1 and empty vector overexpressing cells. This data suggests that the phosphorylation of Yap at Ser127 leads to a changed expression of MRFs and cell cycle regulators and is required for C2C12 myoblasts to differentiate into myotubes.

The small molecule inhibitor SB431542 inhibits activin type I receptors. The muscle growth-inhibitor myostatin binds to and signals via these receptors. The aim of this study was to test the hypothesis that SB431542 can inhibit myostatin-related Smad signaling and induce muscle growth in cultured C2C12 myotubes and increase growth and specific force in cultured Xenopus muscle fibers. The effect of SB431542 was assessed in vitro on C2C12 myotubes and ex vivo using mature Xenopus muscle fibers. SB431542 treatment reduced myostatin-induced C-terminal Smad2 phosphorylation and resulted in the formation of enlarged myotubes. However myogenin expression was unchanged, while p70 S6k phosphorylation at Thr389, total myosin heavy chain, and the rate of protein synthesis were all reduced. Mature Xenopus muscle fibers that were treated with SB431542 had a higher fiber cross-sectional area but decreased specific force production than control. SB431542 can initially antagonize myostatin signaling, but long-term unexpected signaling effects occur. Muscle fibers hypertrophy, but their specific force decreases compared to control.

Exercise results in an increase in interleukin-6 (IL-6), its receptor (IL-6R) and skeletal muscle glucose transport. Interleukin-6 has been found to have equivocal effects on glucose transport, with no studies, to our knowledge, investigating any potential role of IL-6R. In the present study, we hypothesized that a combined preparation of IL-6 and soluble IL-6R (sIL-6R) would stimulate glucose transport. Mouse soleus muscles were incubated with physiological and supraphysiological concentrations of IL-6 and a combination of IL-6 and sIL-6R. Total and phosphorylated AMP-activated protein kinase (AMPK) and Protein Kinase B (PKB/Akt) were also measured by Western blotting. Exposure to both physiological (80 pg ml(-1)) and supraphysiological IL-6 (120 ng ml(-1)) had no effect on glucose transport. At physiological levels, exposure to a combination of IL-6 and sIL-6R (32 ng ml(-1)) resulted in a 1.4-fold increase (P < 0.05) in basal glucose transport with no change to the phosphorylation of AMPK. Exposure to supraphysiological levels of IL-6 and sIL-6R (120 ng ml(-1)) resulted in an approximately twofold increase (P < 0.05) in basal glucose transport and an increase (P < 0.05) in AMPK phosphorylation. No effect of IL-6 or sIL-6R was observed on insulin-stimulated glucose transport. These findings demonstrate that, while IL-6 alone does not stimulate glucose transport in mouse soleus muscle, when sIL-6R is introduced glucose transport is directly stimulated, partly through AMPK-dependent signalling.

Progressive high-resistance exercise with 8–12 repetitions per set to near failure for beginners and 1–12 repetitions for athletes will increase muscle protein synthesis for up to 72 h; approx. 20 g of protein, especially when ingested directly after exercise, will promote high growth by elevating protein synthesis above breakdown. Muscle growth is regulated by signal transduction pathways that sense and compute local and systemic signals and regulate various cellular functions. The main signalling mechanisms are the phosphorylation of serine, threonine and tyrosine residues by kinases and their dephosphorylation by phosphatases. Muscle growth is stimulated by the mTOR (mammalian target of rapamycin) system, which senses (i) IGF-1 (insulin-like growth factor 1)/MGF (mechano-growth factor)/insulin and/or (ii) mechanical signals, (iii) amino acids and (iv) the energetic state of the muscle, and regulates protein synthesis accordingly. The action of the mTOR system is opposed by myostatin-Smad signalling which inhibits muscle growth via gene transcription.

Activation of AMP-activated protein kinase (AMPK) in rodent muscle by exercise, metformin, 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside (AICAR), and adiponectin increases glucose uptake. The aim of this study was to determine whether AICAR stimulates muscle glucose uptake in humans. We studied 29 healthy men (aged 26 +/- 8 years, BMI 25 +/- 4 kg/m(2) [mean +/- SD]). Rates of muscle 2-deoxyglucose (2DG) uptake were determined by measuring accumulation of total muscle 2DG (2DG and 2DG-6-phosphate) during a primed, continuous 2DG infusion. The effects of AICAR and exercise on muscle AMPK activity/phosphorylation and 2DG uptake were determined. Whole-body glucose disposal was compared before and during AICAR with the euglycemic-hyperinsulinemic clamp. Muscle 2DG uptake was linear over 9 h (R(2) = 0.88 +/- 0.09). After 3 h, 2DG uptake increased 2.1 +/- 0.8- and 4.7 +/- 1.7-fold in response to AICAR or bicycle exercise, respectively. AMPK alpha(1) and alpha(2) activity or AMPK phosphorylation was unchanged after 20 min or 3 h of AICAR, but AMPK phosphorylation significantly increased immediately and 3 h after bicycle exercise. AICAR significantly increased phosphorylation of extracellular signal-regulated kinase 1/2, but phosphorylation of beta-acetyl-CoA carboxylase, glycogen synthase, and protein kinase B or insulin receptor substrate-1 level was unchanged. Mean whole-body glucose disposal increased by 7% with AICAR from 9.3 +/- 0.6 to 10 +/- 0.6 mg x kg(-1) x min(-1) (P < 0.05). In healthy people, AICAR acutely stimulates muscle 2DG uptake with a minor effect on whole-body glucose disposal.

The ubiquitous transcriptional coactivators Yap (gene symbol Yap1) and Taz (gene symbol Wwtr1) regulate gene expression mainly by coactivating the Tead transcription factors. Being at the center of the Hippo signaling network, Yap and Taz are regulated by the Hippo kinase cassette and additionally by a plethora of exercise-associated signals and signaling modules. These include mechanotransduction, the AKT-mTORC1 network, the SMAD transcription factors, hypoxia, glucose homeostasis, AMPK, adrenaline/epinephrine and angiotensin II through G protein-coupled receptors, and IL-6. Consequently, exercise should alter Hippo signaling in several organs to mediate at least some aspects of the organ-specific adaptations to exercise. Indeed, Tead1 overexpression in muscle fibers has been shown to promote a fast-to-slow fiber type switch, whereas Yap in muscle fibers and cardiomyocytes promotes skeletal muscle hypertrophy and cardiomyocyte adaptations, respectively. Finally, genome-wide association studies in humans have linked the Hippo pathway members LATS2, TEAD1, YAP1, VGLL2, VGLL3, and VGLL4 to body height, which is a key factor in sports.

Diabetes mellitus (DM) is associated with pancreatic atrophy and compromised digestion of carbohydrates as a result of exocrine pancreatic insufficiency and lower alpha-amylase synthesis and secretion. The reduced production of digestive enzymes is likely to be caused by deregulated protein metabolism. The relative concentrations and phosphorylation of signaling proteins associated with protein translation, such as PKB, p70S6K1, 4E-BP1, ERK1/2, and also some of those implicated in protein breakdown, such as ubiquitin and NF-kappaB, in the pancreas of streptozotocin (STZ)-induced type I diabetic pancreas were measured using Western blotting. There were significant decreases in the levels of total PKB, p70S6K, 4E-BP1, ERK1/2, and NF-kappaB in the diabetic pancreas compared to control. In contrast, the phosphorylation of p70S6K1, 4E-BP1, ERK1/2, and protein ubiquitination increased significantly compared to controls. Together, these results indicate that STZ-induced DM leads to reduced levels of enzymes mediating protein synthesis while their phosphorylation is actually increased, perhaps in an attempt to maintain protein homeostasis, which is further compromised by heightened ubiquitin-dependent protein breakdown. It is likely that these factors are responsible for pancreatic atrophy, enzyme synthesis, and net protein loss in DM.

Previous research has pointed to the efficacy of physical activity in individuals suffering from type 2 diabetes mellitus (type 2 DM). However, as with other populations, adherence to exercise programs is often problematic. This study assessed the effectiveness of a combination of exercise and psychological interventions in type 2 diabetics in terms of disease management and exercise adherence. Forty newly diagnosed type 2 diabetic subjects (54 +/- 6.5 years) took part in the study. Subjects were allocated to an exercise-only intervention (EO) or a combined exercise and psychological adherence intervention (EP) group. Adherence to the program was also monitored at a 6-month follow-up. The results confirmed a significant improvement in physiological parameters (total mass, fat mass, grip strength, peak flow, flexibility, and VO2 max) after the 12-week program in both groups (P < 0.001). The EP group had significant changes in body fat, grip strength, and peak flow (P < 0.05) in comparison to the EO group. Components of the visual analog mood scale (VAMS) were positively influenced in both groups from the therapy program (P < 0.001). The directed psychological intervention had a significant influence on attendance to the 12-week program (P < 0.001). This also resulted in significantly better adherence 6 months later (P < 0.05). In conclusion, the results have demonstrated that psychological intervention is of paramount importance for ensuring high adherence rates during exercise therapy for type 2 diabetic subjects.

Myostatin (Mstn) is a potent negative regulator of skeletal development shown to inhibit myoblast proliferation by impinging on cell cycle and suppressing the synthesis of MyoD. Moreover, Mstn causes muscle wasting and its expression is linked with several conditions of muscle loss, mainly dystrophy and cachexia. NF-κB is a transcription factor that is constitutively active in proliferating myoblasts and also plays a role in cell growth control and skeletal muscle differentiation. NF-κB inhibits myogenesis by promoting myoblast growth and inducing loss of MyoD, and NF-κB activity is required in states of muscle wasting. However, the extracellular factors that regulate NF-κB activity to modulate myogenesis are currently not known. Given the similarities in Mstn and NF-κB activities in muscle cells, we investigated the possibility that Mstn-induced regulation of myogenesis may signal via NF-κB. Using a variety of assays to monitor for NF-κB activity, we found that Mstn signaling does not activate NF-κB in differentiating C2C12 myoblasts, nor is the constitutive activity of NF-κB required for Mstn-mediated inhibition of myogenesis. Likewise, in pre-differentiated myotubes, Mstn signaling induces only a modest activation of NF-κB DNA binding activity. We also investigated whether NF-κB inhibition of myogenesis may occur through the regulation of Mstn. However, activation of NF-κB by TNFα or IL-1β failed to induce Mstn expression. These results thus highlight the distinctive differences by which Mstn and NF-κB signal to regulate myogenesis, a finding which broadens our understanding of how these pathways function in both development and disease.

Endurance training induces a partial fast-to-slow muscle phenotype transformation and mitochondrial biogenesis but no growth. In contrast, resistance training mainly stimulates muscle protein synthesis resulting in hypertrophy. The aim of this study was to identify signaling events that may mediate the specific adaptations to these types of exercise. Isolated rat muscles were electrically stimulated with either high frequency (HFS; 6x10 repetitions of 3 s-bursts at 100 Hz to mimic resistance training) or low frequency (LFS; 3 h at 10 Hz to mimic endurance training). HFS significantly increased myofibrillar and sarcoplasmic protein synthesis 3 h after stimulation 5.3- and 2.7-fold, respectively. LFS had no significant effect on protein synthesis 3 h after stimulation but increased UCP3 mRNA 11.7-fold, whereas HFS had no significant effect on UCP3 mRNA. Only LFS increased AMPK phosphorylation significantly at Thr172 by approximately 2-fold and increased PGC-1alpha protein to 1.3 times of control. LFS had no effect on PKB phosphorylation but reduced TSC2 phosphorylation at Thr1462 and deactivated translational regulators. In contrast, HFS acutely increased phosphorylation of PKB at Ser473 5.3-fold and the phosphorylation of TSC2, mTOR, GSK-3beta at PKB-sensitive sites. HFS also caused a prolonged activation of the translational regulators p70 S6k, 4E-BP1, eIF-2B, and eEF2. These data suggest that a specific signaling response to LFS is a specific activation of the AMPK-PGC-1alpha signaling pathway which may explain some endurance training adaptations. HFS selectively activates the PKB-TSC2-mTOR cascade causing a prolonged activation of translational regulators, which is consistent with increased protein synthesis and muscle growth. We term this behavior the "AMPK-PKB switch." We hypothesize that the AMPK-PKB switch is a mechanism that partially mediates specific adaptations to endurance and resistance training, respectively.

The nature of the deficit underlying age-related muscle wasting remains controversial. To test whether it could be due to a poor anabolic response to dietary amino acids, we measured the rates of myofibrillar and sarcoplasmic muscle protein synthesis (MPS) in 44 healthy young and old men, of similar body build, after ingesting different amounts of essential amino acids (EAA). Basal rates of MPS were indistinguishable, but the elderly showed less anabolic sensitivity and responsiveness of MPS to EAA, possibly due to decreased intramuscular expression, and activation (phosphorylation) after EAA, of amino acid sensing/signaling proteins (mammalian target of rapamycin, mTOR; p70 S6 kinase, or p70(S6k); eukaryotic initiation factor [eIF]4BP-1; and eIF2B). The effects were independent of insulin signaling since plasma insulin was clamped at basal values. Associated with the anabolic deficits were marked increases in NFkappaB, the inflammation-associated transcription factor. These results demonstrate first, EAA stimulate MPS independently of increased insulin availability; second, in the elderly, a deficit in MPS in the basal state is unlikely; and third, the decreased sensitivity and responsiveness of MPS to EAA, associated with decrements in the expression and activation of components of anabolic signaling pathways, are probably major contributors to the failure of muscle maintenance in the elderly. Countermeasures to maximize muscle maintenance should target these deficits.

The fruit of Momordica charantia (family: Cucurbitacea) is used widely as a hypoglycaemic agent to treat diabetes mellitus (DM). The mechanism of the hypoglycaemic action of M. charantia in vitro is not fully understood. This study investigated the effect of M. charantia juice on either 3H-2-deoxyglucose or N-methyl-amino-a-isobutyric acid (14C-Me-AIB) uptake in L6 rat muscle cells cultured to the myotube stage. The fresh juice was centrifuged at 5000 rpm and the supernatant lyophilised. L6 myotubes were incubated with either insulin (100 nM), different concentrations (1-10 microg ml(-1)) of the juice or its chloroform extract or wortmannin (100 nM) over a period of 1- 6 h. The results were expressed as pmol min(-1) (mg cell protein)(-1), n = 6-8 for each value. Basal 3H-deoxyglucose and 14C-Me-AIB uptakes by L6 myotubes after 1 h of incubation were (means +/- S.E.M.) 32.14 +/- 1.34 and 13.48 +/- 1.86 pmol min(-1) (mg cell protein)(-1), respectively. Incubation of L6 myotubes with 100 nM insulin for 1 h resulted in significant (ANOVA, p < 0.05) increases in 3H-deoxyglucose and 14C-Me-AIB uptakes. Typically, 3H-deoxyglucose and 14C-Me-AIB uptakes in the presence of insulin were 58.57 +/- 4.49 and 29.52 +/- 3.41 pmol min(-1) (mg cell protein(-1)), respectively. Incubation of L6 myotubes with three different concentrations (1, 5 and 10 microg ml(-1)) of either the lyophilised juice or its chloroform extract resulted in time-dependent increases in 3H-deoxy-D-glucose and 14C-Me-AIB uptakes, with maximal uptakes occurring at a concentration of 5 microg ml(-1). Incubation of either insulin or the juice in the presence of wortmannin (a phosphatidylinositol 3-kinase inhibitor) resulted in a marked inhibition of 3H-deoxyglucose by L6 myotubes compared to the uptake obtained with either insulin or the juice alone. The results indicate that M. charantia fruit juice acts like insulin to exert its hypoglycaemic effect and moreover, it can stimulate amino acid uptake into skeletal muscle cells just like insulin.

Differences in the concentrations of signal transduction proteins often alter cellular function and phenotype, as is evident from numerous, heterozygous knockout mouse models for signal transduction proteins. Here, we measured signal transduction proteins involved in the adaptation to exercise and insulin signalling in fast rat extensor digitorum longus (EDL; 3% type I fibres) and the slow soleus muscles (84% type I fibres). The EDL and soleus were excised from four rats, the proteins extracted and subjected to Western blots for various signal transduction proteins. Our results show major differences in signal transduction protein concentrations between EDL and soleus. The EDL to soleus concentration ratios were: Calcineurin: 1.43 +/- 0.10; ERK1: 0.38 +/- 0.18; ERK2: 0.61 +/- 0.16; p38alpha, beta: 1.36 +/- 0.15; p38gamma/ERK6: 0.95 +/- 0.11; PKB/AKT: 1.44 +/- 0.08; p70S6k: 6.86 +/- 3.58; GSK3beta: 0.69 +/- 0.03; myostatin: 1.95 +/- 0.43; NF-kappaB: 0.32 +/- 0.10 (values1 indicate higher expression in the EDL, and values1 indicate higher expression in the soleus). With the exception of p38gamma/ERK6, the concentration of each signal transduction protein was uniformly higher in one muscle than in the other in all four animals. These experiments show that signal transduction protein concentrations vary between fast and slow muscles, presumably reflecting a concentration difference on a fibre level. Proteins that promote particular functions such as growth or slow phenotype are not necessarily higher in muscles with that particular trait (e.g. higher in larger fibres or slow muscle). Interindividual differences in fibre composition might explain variable responses to training and insulin.

▪ Abstract This review is divided into two parts, the first dealing with the cell and molecular biology of muscle in terms of growth and wasting and the second being an account of current knowledge of physiological mechanisms involved in the alteration of size of the human muscle mass. Wherever possible, attempts have been made to interrelate the information in each part and to provide the most likely explanation for phenomena that are currently only partially understood. The review should be of interest to cell and molecular biologists who know little of human muscle physiology and to physicians, physiotherapists, and kinesiologists who may be familiar with the gross behavior of human muscle but wish to understand more about the underlying mechanisms of change.

Skeletal muscle adapts to various forms of exercise depending on the force, speed and duration characteristics of the contraction pattern. The stresses and signals associated with each contraction pattern are likely to specifically activate a network of signal transduction pathways that integrate this information. These pathways include the calcineurin, Calcium/calmodulin-dependent protein kinase (CaMK), mitogen-activated protein kinase (MAPK), protein kinase C (PKC), nuclear factor kappa B (NF-B), AMP-dependent protein kinase (AMPK), insulin signalling and developmental pathways. Activated signal transduction pathways activate or increase the expression of transcription factors via various mechanisms. Skeletal muscle genes are usually regulated by combinatorial control exerted by several transcription factors and possibly other mechanisms. In addition, adaptations such as an increase in mitochondrial biogenesis or the activation of satellite cell proliferation involve distinct regulatory mechanisms

Background We previously measured the rate of regaining muscle strength during rehabilitation of lower leg muscles in patients following lower leg casting. Our primary aim in this study was to measure the rate of gain of strength in healthy individuals undergoing a similar training regime. Our secondary aim was to test the ability of MRI to provide a biomarker for muscle function. Methods Men and women were recruited in three age groups: 20–30, 50–65 and over 70 years. Their response to resistance training of the right lower leg twice a week for 8 weeks was monitored using a dynamometer and MRI of tibialis anterior, soleus and gastrocnemius muscles at 2 weekly intervals to measure muscle size (anatomical cross-sectional area (ACSA)) and quality (T2 relaxation). Forty-four volunteers completed the study. Results Baseline strength declined with age. Training had no effect in middle-aged females or in elderly men in dorsiflexion. Other groups significantly increased both plantarflexion and dorsiflexion strength at rates up to 5.5 N m week-1 in young females in plantarflexion and 1.25 N m week-1 in young males in dorsiflexion. No changes were observed in ACSA or T2 in any age group in any muscle. Conclusion Exercise training improves muscle strength in males at all ages except the elderly in dorsiflexion. Responses in females were less clear with variation across age and muscle groups. These results were not reflected in simple MRI measures that do not, therefore, provide a good biomarker for muscle atrophy or the efficacy of rehabilitation.

Purpose Cancer of the upper digestive tract (uGI) is a major contributor to cancer-related death worldwide. Due to a rise in occurrence, together with poor survival rates and a lack of diagnostic or prognostic clinical assays, there is a clear need to establish molecular biomarkers. Experimental design Initial assessment was performed on urine samples from 60 control and 60 uGI cancer patients using MS to establish a peak pattern or fingerprint model, which was validated by a further set of 59 samples. Results We detected 86 cluster peaks by MS above frequency and detection thresholds. Statistical testing and model building resulted in a peak profiling model of five relevant peaks with 88% overall sensitivity and 91% specificity, and overall correctness of 90%. High-resolution MS of 40 samples in the 2–10 kDa range resulted in 646 identified proteins, and pattern matching identified four of the five model peaks within significant parameters, namely programmed cell death 6 interacting protein (PDCD6IP/Alix/AIP1), Rabenosyn-5 (ZFYVE20), protein S100A8, and protein S100A9, of which the first two were validated by Western blotting. Conclusions and clinical relevance We demonstrate that MS analysis of human urine can identify lead biomarker candidates in uGI cancers, which makes this technique potentially useful in defining and consolidating biomarker patterns for uGI cancer screening.

The discovery of the Hippo pathway can be traced back to two areas of research. Genetic screens in fruit flies led to the identification of the Hippo pathway kinases and scaffolding proteins that function together to suppress cell proliferation and tumor growth. Independent research, often in the context of muscle biology, described Tead (TEA domain) transcription factors, which bind CATTCC DNA motifs to regulate gene expression. These two research areas were joined by the finding that the Hippo pathway regulates the activity of Tead transcription factors mainly through phosphorylation of the transcriptional coactivators Yap and Taz, which bind to and activate Teads. Additionally, many other signal transduction proteins crosstalk to members of the Hippo pathway forming a Hippo signal transduction network. We discuss evidence that the Hippo signal transduction network plays important roles in myogenesis, regeneration, muscular dystrophy, and rhabdomyosarcoma in skeletal muscle, as well as in myogenesis, organ size control, and regeneration of the heart. Understanding the role of Hippo kinases in skeletal and heart muscle physiology could have important implications for translational research.