Intramuscular Signaling Research Articles

Association between myosin heavy chain protein isoforms and intramuscular anabolic signaling following resistance exercise in trained men.

Resistance exercise stimulates an increase in muscle protein synthesis regulated by intracellular anabolic signaling molecules in a mammalian/mechanistic target of rapamycin (mTOR)‐dependent pathway. The purpose of this study was to investigate acute anabolic signaling responses in experienced, resistance‐trained men, and to examine the association between myosin heavy chain (MHC) isoform composition and the magnitude of anabolic signaling. Eight resistance‐trained men (24.9 ± 4.3 years; 91.2 ± 12.4 kg; 176.7 ± 8.0 cm; 13.3 ± 3.9 body fat %) performed a whole body, high‐volume resistance exercise protocol (REX) and a control protocol (CTL) in a balanced, randomized order. Participants were provided a standardized breakfast, recovery drink, and meal during each protocol. Fine needle muscle biopsies were completed at baseline (BL), 2 h (2H) and 6 h post‐exercise (6H). BL biopsies were analyzed for MHC isoform composition. Phosphorylation of proteins specific to the Akt/mTOR signaling pathway and MHC mRNA expression was quantified. Phosphorylation of p70S6k was significantly greater in REX compared to CTL at 2H (P = 0.04). MHC mRNA expression and other targets in the Akt/mTOR pathway were not significantly influenced by REX. The percentage of type IIX isoform was inversely correlated (P < 0.05) with type I and type IIA MHC mRNA expression (r = −0.69 to −0.93). Maximal strength was also observed to be inversely correlated (P < 0.05) with Type I and Type IIA MHC mRNA expression (r = −0.75 to −0.77) and p70S6k phosphorylation (r = −0.75). Results indicate that activation of p70S6k occurs within 2‐h following REX in experienced, resistance‐trained men. Further, results also suggest that highly trained, stronger individuals have an attenuated acute anabolic response.

Phosphatidic acid feeding increases muscle protein synthesis and select mTORC1 pathway signaling mediators in rodent skeletal muscle

Background Human and cell culture studies have demonstrated that phosphatidic acid (PA) can increase muscle mass and anabolic signaling, respectively. However, no in vivo evidence to date has examined whether PA can increase intramuscular anabolic signaling in vivo. The purpose of this study was to examine – a) if PA feeding acutely increases post-prandial muscle protein synthesis (MPS) and anabolic signaling markers; and b) if PA can enhance the post-prandial anabolic effects of whey protein concentrate (WPC). Methods

Role of nitric oxide in skeletal muscle glucose uptake during exercise.

Nitric oxide is produced within skeletal muscle fibres and has various functions in skeletal muscle. There is evidence that NO may be essential for normal increases in skeletal muscle glucose uptake during contraction/exercise. Although there have been some discrepant results, it has been consistently demonstrated that inhibition of NO synthase (NOS) attenuates the increase in skeletal muscle glucose uptake during contraction in mouse and rat muscle ex vivo, during in situ contraction in rats and during exercise in humans. The NO-mediated increase in skeletal muscle glucose uptake during contraction/exercise is probably due to the modulation of intramuscular signalling that ultimately increases glucose transporter 4 (GLUT4) translocation and is, surprisingly, independent of blood flow. In this review, we discuss the evidence for and against a role of NO in regulating skeletal muscle glucose uptake during contraction/exercise and outline the possible mechanism(s) involved. Emerging findings regarding the role of neuronal NOS mu (nNOSμ) in this process are also discussed.

Substantial skeletal muscle loss occurs during only 5 days of disuse

The impact of disuse on the loss of skeletal muscle mass and strength has been well documented. Given that most studies have investigated muscle atrophy after more than 2 weeks of disuse, few data are available on the impact of shorter periods of disuse. We assessed the impact of 5 and 14 days of disuse on skeletal muscle mass, strength and associated intramuscular molecular signalling responses. Twenty-four healthy, young (23 ± 1 year) males were subjected to either 5 (n = 12) or 14 (n = 12) days of one-legged knee immobilization using a full leg cast. Before and immediately after the immobilization period, quadriceps muscle cross-sectional area (CSA), leg lean mass and muscle strength were assessed, and biopsies were collected from the vastus lateralis. Quadriceps muscle CSA declined from baseline by 3.5 ± 0.5 (P < 0.0001) and 8.4 ± 2.8% (P < 0.001), leg lean mass was reduced by 1.4 ± 0.7 (P = 0.07) and 3.1 ± 0.7% (P < 0.01) and strength was decreased by 9.0 ± 2.3 (P < 0.0001) and 22.9 ± 2.6% (P < 0.001) following 5 and 14 days of immobilization respectively. Muscle myostatin mRNA expression doubled following immobilization (P < 0.05) in both groups, while the myostatin precursor isoform protein content decreased after 14 days only (P < 0.05). Muscle MAFBx mRNA expression increased from baseline by a similar magnitude following either 5 or 14 days of disuse, whereas MuRF1 mRNA expression had increased significantly only after 5 days. We conclude that even short periods of muscle disuse can cause substantial loss of skeletal muscle mass and strength and are accompanied by an early catabolic molecular signalling response.

Knockdown of the C. elegans kinome identifies kinases required for normal protein homeostasis, mitochondrial network structure, and sarcomere structure in muscle.

BackgroundKinases are important signalling molecules for modulating cellular processes and major targets of drug discovery programs. However, functional information for roughly half the human kinome is lacking. We conducted three kinome wide, >90%, RNAi screens and epistasis testing of some identified kinases against known intramuscular signalling systems to increase the functional annotation of the C. elegans kinome and expand our understanding of kinome influence upon muscle protein degradation.Results96 kinases were identified as required for normal protein homeostasis, 74 for normal mitochondrial networks and 50 for normal sarcomere structure. Knockdown of kinases required only for normal protein homeostasis and/or mitochondrial structure was significantly less likely to produce a developmental or behavioural phenotype than knockdown of kinases required for normal sarcomere structure and/or other sub-cellular processes. Lastly, assessment of kinases for which knockdown produced muscle protein degradation against the known regulatory pathways in C. elegans muscle revealed that close to half of kinase knockdowns activated autophagy in a MAPK dependent fashion.ConclusionsRoughly 40% of kinases studied, 159 of 397, are important in establishing or maintaining muscle cell health, with most required for both. For kinases where decreased expression triggers protein degradation, autophagy is most commonly activated. These results increase the annotation of the C. elegans kinome to roughly 75% and enable future kinome research. As 33% of kinases identified have orthologues expressed in human muscle, our results also enable testing of whether identified kinases function similarly in maintaining human muscle homeostasis.

Refining dietary protein recommendations for the athlete

Refining dietary protein recommendations for the athlete

Variable resistance training promotes greater fatigue resistance but not hypertrophy versus constant resistance training

Loading using variable resistance devices, where the external resistance changes in line with the force:angle relationship, has been shown to cause greater acute neuromuscular fatigue and larger serum hormone responses. This may indicate a greater potential for adaptation during long-term training. Twelve (constant resistance group) and 11 (variable resistance group) men completed 20 weeks of resistance training with 10 men as non-training controls. Training-induced adaptations were assessed by bilateral leg press one repetition maximum, a repetition to failure test using 75 % 1RM, lower limb lean mass and vastus lateralis cross-sectional area. Only the variable resistance training group improved the total number of repetitions (41 ± 46 %) and volume load (52 ± 37 %) during the repetition to failure test (P < 0.05). Similar improvements in maximum strength and hypertrophy of the lower limbs were observed in both training groups. Also, constant and variable resistance 5 × 10RM leg press loadings were performed before and after training in a crossover design. Acute loading-induced responses were assessed by concentric and isometric force, serum hormone concentrations and phosphorylation of intramuscular signalling proteins (0-30 min post-loading). Greater acute decreases in force (P < 0.05-0.01), and greater increases in serum testosterone and cortisol concentration (P < 0.05) and ERK 1/2 phosphorylation (P < 0.05) were observed following variable resistance loadings before and after training. Greater training-induced improvements in fatigue resistance occurred in the variable resistance training group, which may be due to greater acute fatigue and physiological responses during variable versus constant resistance loadings.

The impact of a single dose of resveratrol on intramuscular signaling and whole body fat oxidation in humans

Resveratrol's (RSV) mechanism of action in skeletal muscle has been characterized in cellular and murine models, but RSV's in vivo signaling pathways remain unconfirmed in human skeletal muscle. Overweight males (n=8; BMI, 30.5±3.6; VO2peak, 34.0±7.3 ml/kg) reported to the lab on 2 occasions following a standardized dinner and 12 h fast. Respiratory exchange ratio (RER) and a muscle biopsy were taken while fasted, and 2 hours after breakfast supplemented with 0.3g of RSV or a placebo (PL) pill. Experiments were performed in random order using a double blind crossover design. RER increased from 0.81±0.04 to 0.84±0.06 with PL but decreased from 0.85±0.07 to 0.81±0.05 following RSV. The interaction for RER approached significance (p=0.09). A significant effect of RSV treatment was observed for p‐p38 MAPK (interaction, p &lt; 0.05) with the decrease observed after PL (−31±28%) being reversed following RSV (+7±13%). p‐AKT increased (p &lt; 0.05) in a time dependent fashion following both RSV (+111±68%) and PL (+79±19%). While p‐ACC tended to increase with time this change did not reach significance in either group. Significant interactions were not observed for p‐AKT (p=0.12) or p‐ACC (p=0.47). These results suggest that a single dose of RSV can influence intramuscular signaling; specifically pathways involved in the control of mitochondrial biogenesis, and may increase whole body fat oxidation. This study was funded by NSERC

Divergent intramuscular mTORC1 signaling responses to habitual consumption of milk‐and soy‐based energy restricted diets

The effects of milk‐or soy‐based, high‐protein, energy restricted (ER) diets on intracellular regulators of muscle mass are not well described. Eighty male Sprague‐Dawley rats were randomly divided into groups and fed energy adequate (EA) and ER (60% of average ad libitum EA feed intake) diets containing two protein sources (milk protein concentrate: MPC; or isoflavone‐free soy isolate: SOY) and levels (control protein: CP, 10%; high protein: HP, 35%) for 16 weeks. Basal protein expression and phosphorylation of mammalian target of rapamycin complex 1 (mTORC1) associated proteins were assessed using multiplex. Phosphorylation of mTOR was lower (P &lt; 0.05) for ER compared to EA. Independent of protein level, p70S6K1 expression was lower (P &lt; 0.05) for ER MPC compared to EA MPC. However, mTOR and rpS6 protein expression were higher (P &lt; 0.05) for ER SOY than ER MPC, and TSC2, mTOR, and rpS6 were higher (P &lt; 0.05) for ER SOY compared to EA SOY. Phosphorylation of rpS6 was also higher (P &lt; 0.05) for EA SOY compared to EA MPC, and p70S6K1 phosphorylation was higher (P &lt; 0.05) for SOY versus MPC, regardless of energy status and protein level. These data demonstrate divergent mTORC1 responses to soy‐and milk‐based energy restricted diets but suggest that habitual consumption of soy‐based diets may enhance protein synthetic responses to feeding by upregulating the expression of key mTORC1 signaling proteins. Supported by Dairy Research Institute and USAMRMC

Energy intake modulates intramuscular mTORC1 signaling and ubiquitin‐mediated proteolysis during energy deficit independent of dietary protein variations

Effects of variations in habitual and acute dietary protein consumption on intracellular regulators of human skeletal muscle mass during energy deficit (ED) were investigated. Using a randomized‐block design, 32 men and 7 women consumed diets providing protein at 0.8, 1.6, or 2.4 g·kg−1·d−1 for 31 days. A 10‐day weight maintenance (WM) period preceded 21 days of ED, during which energy intake was reduced to 60% of daily requirements. Phosphorylation of mTORC1 associated signaling proteins, protein ubiquitylation (Ub), and NFκB expression were assessed in vastus lateralis muscle samples obtained after an overnight fast (FAST) and again 120 min after consuming a mixed meal (480 kcal, 20 g protein; FED) on days 10 and 31. Independent of dietary protein level and feeding state, Ub was 25% higher (P&lt; 0.05) for ED versus WM. Energy state and feeding altered p70S6K1 phosphorylation, as FED‐ED levels were 26% lower than FED‐WM (energy‐by‐feeding, P&lt; 0.05). Overall, Akt and rpS6 phosphorylation were higher (P&lt; 0.05), and NFκB and Ub were lower (P&lt; 0.05), for FED versus FAST. These data suggest that ED may upregulate intramuscular ubiquitin‐mediated proteolysis, and attenuate p70S6K1 sensitivity to feeding, although energy consumption, independent of habitual protein intake, may confer skeletal muscle protection through concomitant stimulation of mTORC1 signaling and inhibition of Ub during ED.Supported by USAMRMC, EMU CHHS SSSA, and USDA ARS

Muscle Kinetics and Exercise: Any Advantages of Dietary Proteins

Muscle Kinetics and Exercise: Any Advantages of Dietary Proteins

Contributions to the understanding of the anabolic properties of different dietary proteins

Contributions to the understanding of the anabolic properties of different dietary proteins

Leucine: a nutrient 'trigger' for muscle anabolism, but what more?

Leucine: a nutrient 'trigger' for muscle anabolism, but what more?

Concurrent resistance and aerobic exercise stimulates both myofibrillar and mitochondrial protein synthesis in sedentary middle-aged men

We determined myofibrillar and mitochondrial protein fractional synthesis rates (FSR), intramuscular signaling protein phosphorylation, and mRNA expression responses after isolated bouts of resistance exercise (RE), aerobic exercise (AE), or in combination [termed concurrent exercise (CE)] in sedentary middle-aged men. Eight subjects (age = 53.3 ± 1.8 yr; body mass index = 29.4 ± 1.4 kg·m(2)) randomly completed 8 × 8 leg extension repetitions at 70% of one repetition-maximum, 40 min of cycling at 55% peak aerobic power output (AE), or (consecutively) 50% of the RE and AE trials (CE). Biopsies were obtained (during a primed, constant infusion of l-[ring-(13)C(6)]phenylalanine) while fasted, and at 1 and 4 h following postexercise ingestion of 20 g of protein. All trials increased mitochondrial FSR above fasted rates (RE = 1.3-fold; AE = 1.5; CE = 1.4; P < 0.05), although only CE (2.2) and RE (1.8) increased myofibrillar FSR (P < 0.05). At 1 h postexercise, phosphorylation of Akt on Ser(473) (CE = 7.7; RE = 4.6) and Thr(308) (CE = 4.4; RE = 2.9), and PRAS40 on Thr(246) (CE = 3.8; AE = 2.5) increased (P < 0.05), with CE greater than AE for Akt Ser(473)-Thr(308) and greater than RE for PRAS40 (P < 0.05). Despite increased phosphorylation of Akt-PRAS40, phosphorylation of mammalian target of rapamycin (Ser(2448)) remained unchanged (P > 0.05), while rpS6 (Ser(235/236)) increased only in RE (10.4) (P < 0.05). CE and AE both resulted in increased peroxisome proliferator receptor-γ coactivator 1-α (PGC1α) expression at 1 h (CE = 2.9; AE = 2.8; P < 0.05) and 4 h (CE = 2.6; AE = 2.4) and PGC1β expression at 4 h (CE = 2.1; AE = 2.6; P < 0.05). These data suggest that CE-induced acute stimulation of myofibrillar and mitochondrial FSR, protein signaling, and mRNA expression are equivalent to either isolate mode (RE or AE). These results occurred without an interference effect on muscle protein subfractional synthesis rates, protein signaling, or mRNA expression.

Muscle protein synthesis in response to nutrition and exercise

Muscle protein synthesis (MPS) is the driving force behind adaptive responses to exercise and represents a widely adopted proxy for gauging chronic efficacy of acute interventions, (i.e. exercise/nutrition). Recent findings in this arena have been progressive. Nutrient-driven increases in MPS are of finite duration (∼1.5 h), switching off thereafter despite sustained amino acid availability and intramuscular anabolic signalling. Intriguingly, this 'muscle-full set-point' is delayed by resistance exercise (RE) (i.e. the feeding × exercise combination is 'more anabolic' than nutrition alone) even 24 h beyond a single exercise bout, casting doubt on the importance of nutrient timing vs. sufficiency per se. Studies manipulating exercise intensity/workload have shown that increases in MPS are negligible with RE at 20-40% but maximal at 70-90% of one-repetition maximum when workload is matched (according to load × repetition number). However, low-intensity exercise performed to failure equalises this response. Analysing distinct subcellular fractions (e.g. myofibrillar, sarcoplasmic, mitochondrial) may provide a readout of chronic exercise efficacy in addition to effect size in MPS per se, i.e. while 'mixed' MPS increases similarly with endurance and RE, increases in myofibrillar MPS are specific to RE, prophetic of adaptation (i.e. hypertrophy). Finally, the molecular regulation of MPS by exercise and its regulation via 'anabolic' hormones (e.g. IGF-1) has been questioned, leading to discovery of alternative mechanosensing-signalling to MPS.

Molecular Determinants of Skeletal Muscle Hypertrophy in Animals

Beyond skeletal muscle’s primary function as a force generator for locomotion, there is a growing recognition of the important role skeletal muscle plays in overall health through its impact on whole-body metabolism as well as directly influencing quality of life issues with chronic disease and aging. Over the last decade, extensive progress has been made with regard to our understanding of the molecules that regulate skeletal muscle mass. Various growth factors such as insulin-like growth factor-I (IGF-I), hepatocyte growth factor (HGF), and leukemia inhibitory factor (LIF) have been shown to stimulate the activation, proliferation, and differentiation of satellite cells, and then contribute to muscle hypertrophy as well as normal muscle growth. In contrast, myostatin inhibits these processes through forkhead box O (FOXO) and/or Smad 2/3-dependent signaling. Intramuscular signaling by PI3-K/Akt/mTOR, calcineurin, and serum response factor (SRF) activates protein synthesis but some signaling also inhibits protein degradation at the same time. Although various studies using in vitro cell cultures and in vivo rodent models have revealed candidates for proteins that modulate the hypertrophic process in muscle fibers after mechanical loading, these findings do not necessarily apply to the adaptations that occur in human muscle. In this review, we discuss several possible factors regulating muscle hypertrophy, and the adaptations of human muscle after resistance training, a model of mechanical loading.

A step towards underpinning the molecular signalling events regulating muscle protein loss in critically ill patients

A step towards underpinning the molecular signalling events regulating muscle protein loss in critically ill patients

The curious case of anabolic resistance: old wives' tales or new fables?

the progressive loss of muscle mass, commonly termed sarcopenia, accompanies the process of healthy aging ([27][1]). The underlying basis of this condition, in a very simplistic view, would be at a more advanced age skeletal muscle proteins are being lost because of an imbalance between muscle

Rapid aminoacidemia enhances myofibrillar protein synthesis and anabolic intramuscular signaling responses after resistance exercise

Ingestion of whey or casein yields divergent patterns of aminoacidemia that influence whole-body and skeletal muscle myofibrillar protein synthesis (MPS) after exercise. Direct comparisons of the effects of contrasting absorption rates exhibited by these proteins are confounded by their differing amino acid contents. Our objective was to determine the effect of divergent aminoacidemia by manipulating ingestion patterns of whey protein alone on MPS and anabolic signaling after resistance exercise. In separate trials, 8 healthy men consumed whey protein either as a single bolus (BOLUS; 25-g dose) or as repeated, small, "pulsed" drinks (PULSE; ten 2.5-g drinks every 20 min) to mimic a more slowly digested protein. MPS and phosphorylation of signaling proteins involved in protein synthesis were measured at rest and after resistance exercise. BOLUS increased blood essential amino acid (EAA) concentrations above those of PULSE (162% compared with 53%, P < 0.001) 60 min after exercise, whereas PULSE resulted in a smaller but sustained increase in aminoacidemia that remained elevated above BOLUS amounts later (180-220 min after exercise, P < 0.05). Despite an identical net area under the EAA curve, MPS was elevated to a greater extent after BOLUS than after PULSE early (1-3 h: 95% compared with 42%) and later (3-5 h: 193% compared with 121%) (both P < 0.05). There were greater changes in the phosphorylation of the Akt-mammalian target of rapamycin pathway after BOLUS than after PULSE. Rapid aminoacidemia in the postexercise period enhances MPS and anabolic signaling to a greater extent than an identical amount of protein fed in small pulses that mimic a more slowly digested protein. A pronounced peak aminoacidemia after exercise enhances protein synthesis. This trial was registered at clinicaltrials.gov as NCT01319513.

No role for early IGF-1 signalling in stimulating acute ‘muscle building’ responses

No role for early IGF-1 signalling in stimulating acute ‘muscle building’ responses