Regulation Of Muscle Protein Synthesis Research Articles

Hydrogen Peroxide Does Not Potentiate Exercise-induced Mtorc1 In Rat Skeletal Muscle

Our previous study showed that administration of an antioxidant attenuated the phosphorylation of p70S6K and skeletal muscle hypertrophy induced by mechanical overload (Makanae et al. 2013), suggesting that reactive oxygen species (ROS) could be an activator of mammalian target of rapamycin complex 1(mTORC1), a key regulator of muscle protein synthesis. However, no study has investigated the effects of exogenous injection of ROS into skeletal muscle on the mTORC1 activity. PURPOSE: The purpose of this sturdy was to investigate the effect of ROS injection on mTORC1 activity at baseline and after a bout of exercise in rat skeletal muscle. METHODS: Experiment 1: The male Sprague-Dawley rats, at 11 weeks of age, were injected with hydrogen peroxide (1 mM, 0.1 mL) or placebo (PBS, 0.1 mL) into the right gastrocnemius muscle under isoflurane anesthesia, and killed by blood removal at 1 h after the injection. Experiment 2: The right gastrocnemius muscle of the rats was isometrically exercised (stimulating 3 sec × 10 contractions, with a 7 sec interval between contractions, per set for 5 sets, with 3 min rest intervals) via percutaneous electrical stimulation under isoflurane anesthesia. The hydrogen peroxide or placebo were injected into the right gastrocnemius muscle immediately after the resistance exercise. RESULTS: The injection of hydrogen peroxide increased significantly the phosphorylation of p70S6K (Thr389) and rspS6 (Ser235/Ser236) at 1 h after the injection (P < 0.05) as compared to the placebo injection. Resistance exercise increased the phosphorylation of p70S6K (Thr389) and prS6 (Ser235/Ser236) at 3 h after exercise in the both hydrogen peroxide and placebo injected groups. However, there was no difference in the level of either p70S6K (Thr389) or prS6 (Ser235/Ser236) phosphorylation between groups. CONCLUSIONS: These results suggest that ROS is one of the activator of mTORC1 signaling in the skeletal muscle, but the exogenous increase in ROS does not further stimulate resistance exercise-induced mTORC1 activation.

Mechanisms of Cachexia in Chronic Disease States

Sarcopenia and cachexia are muscle wasting syndromes associated with aging and with many chronic diseases, such as congestive heart failure (CHF), diabetes, cancer, chronic obstructive pulmonary disease and chronic kidney disease (CKD). While mechanisms are complex, these conditions are often accompanied by elevated angiotensin II (Ang II). Patients with advanced CHF or CKD often have increased Ang II levels and cachexia, and angiotensin-converting enzyme inhibitor treatment improves weight loss. It was found that Ang II infusion in rodents leads to skeletal muscle wasting. Ang II increases cytokines and circulating hormones, such as tumor necrosis factor-α, interleukin-6, serum amyloid-A and glucocorticoids, which regulate muscle protein synthesis and degradation. Ang II–induced muscle wasting is caused by alterations in insulin-like growth factor-1 signaling, enhanced muscle protein breakdown via the ubiquitin-proteasome system and decreased appetite resulting from the downregulation of hypothalamic orexigenic neuropeptides, such as Npy and orexin. Ang II also inhibits 5′ adenosine monophosphate–activated protein kinase activity and disrupts normal energy balance via the activation of 5′ adenosine monophosphate–activated protein kinase phosphatase PP2Cα. Furthermore, Ang II inhibits skeletal muscle stem (satellite) cell proliferation, leading to lowered muscle regenerative capacity. Distinct satellite cell angiotensin receptor subtypes have different effects on different stages of differentiation and are critical for the regulation of muscle regeneration. These data suggest that the renin-angiotensin system plays a critical role in mechanisms underlying cachexia in chronic disease states, and it is a promising target for the treatment of muscle atrophy in patients with diseases such as CHF and CKD.

Protein supplementation does not alter intramuscular anabolic signaling or endocrine response after resistance exercise in trained men

The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway appears to be the primary regulator of muscle protein synthesis. A variety of stimuli including resistance exercise, amino acids, and hormonal signals activate mTORC1 signaling. The purpose of this study was to investigate the effect of a protein supplement on mTORC1 signaling following a resistance exercise protocol designed to promote elevations in circulating hormone concentrations. We hypothesized that the protein supplement would augment the intramuscular anabolic signaling response. Ten resistance-trained men (age, 24.7 ± 3.4 years; weight, 90.1 ± 11.3 kg; height, 176.0 ± 4.9 cm) received either a placebo or a supplement containing 20 g protein, 6 g carbohydrates, and 1 g fat after high-volume, short-rest lower-body resistance exercise. Blood samples were obtained at baseline, immediately, 30 minutes, 1 hour, 2 hours, and 5 hours after exercise. Fine-needle muscle biopsies were completed at baseline, 1 hour, and 5 hours after exercise. Myoglobin, lactate dehydrogenase, and lactate concentrations were significantly elevated after resistance exercise (P < .0001); however, no differences were observed between trials. Resistance exercise also elicited a significant insulin, growth hormone, and cortisol response (P < .01); however, no differences were observed between trials for insulin-like growth factor-1, insulin, testosterone, growth hormone, or cortisol. Intramuscular anabolic signaling analysis revealed significant elevations in RPS6 phosphorylation after resistance exercise (P = .001); however, no differences were observed between trials for signaling proteins including Akt, mTOR, p70S6k, and RPS6. The endocrine response and phosphorylation status of signaling proteins within the mTORC1 pathway did not appear to be altered by ingestion of supplement after resistance exercise in resistance-trained men.

Leucine is a major regulator of muscle protein synthesis in neonates.

Approximately 10% of infants born in the United States are of low birth weight. Growth failure during the neonatal period is a common occurrence in low birth weight infants due to their inability to tolerate full feeds, concerns about advancing protein supply, and high nutrient requirements for growth. An improved understanding of the nutritional regulation of growth during this critical period of postnatal growth is vital for the development of strategies to improve lean gain. Past studies with animal models have demonstrated that muscle protein synthesis is increased substantially following a meal and that this increase is due to the postprandial rise in amino acids as well as insulin. Both amino acids and insulin act independently to stimulate protein synthesis in a mammalian target of rapamycin-dependent manner. Further studies have elucidated that leucine, in particular, and its metabolites, α-ketoisocaproic acid and β-hydroxy-β-methylbutyrate, have unique anabolic properties. Supplementation with leucine, provided either parenterally or enterally, has been shown to enhance muscle protein synthesis in neonatal pigs, making it an ideal candidate for stimulating growth of low birth weight infants.

Muscle p70S6K phosphorylation in response to soy and dairy rich meals in middle aged men with metabolic syndrome: a randomised crossover trial.

BackgroundThe mammalian target of rapamycin (mTOR) pathway is the primary regulator of muscle protein synthesis. Metabolic syndrome (MetS) is characterized by central obesity and insulin resistance; little is known about how MetS affects the sensitivity of the mTOR pathway to feeding.MethodsThe responsiveness of mTOR pathway targets such as p706Sk to a high protein meal containing either dairy or soy foods was investigated in healthy insulin sensitive middle-aged men and those presenting with metabolic syndrome (MetS). Twenty male subjects (10 healthy controls, 10 MetS) participated in a single-blinded randomized cross-over study. In a random sequence, subjects ingested energy-matched breakfasts composed predominately of either dairy-protein or soy-protein foods. Skeletal muscle biopsies were collected in the fasted state and at 2 and 4 h post-meal ingestion for the analysis of mTOR- and insulin-signalling kinase activation.ResultsPhosphorylated Akt and Insulin receptor substrate 1 (IRS1) increased during the postabsorptive period with no difference between groups. mTOR (Ser448) and ribosomal protein S6 phosphorylation increased 2 h following dairy meal consumption only. p70S6K (Thr389) phosphorylation was increased after feeding only in the control subjects and not in the MetS group.ConclusionsThese data demonstrate that the consumption of a dairy-protein rich but not a soy-protein rich breakfast activates the phosphorylation of mTOR and ribosomal protein S6, required for protein synthesis in human skeletal muscle. Unlike healthy controls, subjects with MetS did not increase muscle p70S6K(Thr389) phosphorylation in response to a mixed meal.Trial registrationThis trial was registered with the Australian New Zealand Clinical Trials Registry (ANZCTR) as ACTRN12610000562077.

Disruption of genes encoding eIF4E binding proteins-1 and -2 does not alter basal or sepsis-induced changes in skeletal muscle protein synthesis in male or female mice.

Sepsis decreases skeletal muscle protein synthesis in part by impairing mTOR activity and the subsequent phosphorylation of 4E-BP1 and S6K1 thereby controlling translation initiation; however, the relative importance of changes in these two downstream substrates is unknown. The role of 4E-BP1 (and -BP2) in regulating muscle protein synthesis was assessed in wild-type (WT) and 4E-BP1/BP2 double knockout (DKO) male mice under basal conditions and in response to sepsis. At 12 months of age, body weight, lean body mass and energy expenditure did not differ between WT and DKO mice. Moreover, in vivo rates of protein synthesis in gastrocnemius, heart and liver did not differ between DKO and WT mice. Sepsis decreased skeletal muscle protein synthesis and S6K1 phosphorylation in WT and DKO male mice to a similar extent. Sepsis only decreased 4E-BP1 phosphorylation in WT mice as no 4E-BP1/BP2 protein was detected in muscle from DKO mice. Sepsis decreased the binding of eIF4G to eIF4E in WT mice; however, eIF4E•eIF4G binding was not altered in DKO mice under either basal or septic conditions. A comparable sepsis-induced increase in eIF4B phosphorylation was seen in both WT and DKO mice. eEF2 phosphorylation was similarly increased in muscle from WT septic mice and both control and septic DKO mice, compared to WT control values. The sepsis-induced increase in muscle MuRF1 and atrogin-1 (markers of proteolysis) as well as TNFα and IL-6 (inflammatory cytokines) mRNA was greater in DKO than WT mice. The sepsis-induced decrease in myocardial and hepatic protein synthesis did not differ between WT and DKO mice. These data suggest overall basal protein balance and synthesis is maintained in muscle of mice lacking both 4E-BP1/BP2 and that sepsis-induced changes in mTOR signaling may be mediated by a down-stream mechanism independent of 4E-BP1 phosphorylation and eIF4E•eIF4G binding.

Differential effects of PDCD4 depletion on protein synthesis in myoblast and myotubes

BackgroundReduced muscle mass is a hallmark of metabolic diseases like diabetes and cancer. The mammalian (mechanistic) target of rapamycin complex 1/S6 kinase 1 (mTORC1/S6K1) pathway is critical to the regulation of muscle protein synthesis and mass but its mechanism of action is not completely understood.ResultsUsing L6 myotubes, we characterized the regulation of programmed cell death 4 (PDCD4), a recently described substrate of S6K1. The abundance, but not Ser67 phosphorylation, of PDCD4 was sensitive to amino acid and serum deprivation: values in starved cells were 4.5X of control (P < 0.001). Refeeding had opposite effects. Growth factors, compared to amino acids, appeared more critical in regulating PDCD4 abundance. Furthermore, inhibition of mTORC1 or the proteasome prevented the refeeding-associated decrease in PDCD4 abundance. Amino acid and serum deprivation significantly increased PDCD4 binding to eIF4A (P < 0.05); this was reversed during refeeding. PDCD4 depletion by RNA interference had no significant effect on phenylalanine incorporation into myotube mixed proteins in control cells but further suppressed (30%) this measure in nutrient-deprived cells (P < 0.0005). This was not observed in myoblasts. In starved myotubes, PDCD4 depletion further reduced the association of eIF4G with eIF4E.ConclusionOur data suggest that in myotubes, PDCD4 abundance is sensitive to nutritional manipulation in an mTORC1 and proteasome depended manner. Furthermore, the role of PDCD4 in regulating protein synthesis appears dependent on the developmental state of the cell.

Effects of AMP-activated protein kinase (AMPK) signaling and essential amino acids on mammalian target of rapamycin (mTOR) signaling and protein synthesis rates in mammary cells

Regulation of mammary protein synthesis potentially changes the relationships between AA supply and milk protein output represented in current nutrient requirement models. Glucose and AA regulate muscle protein synthesis via cellular signaling pathways involving mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK). The objective of this study was to investigate the effects of essential AA (EAA) and acetate or glucose on mTOR and AMPK signaling pathways and milk protein synthesis rates. A bovine mammary epithelial cell line, MAC-T, was subjected to different media containing 0 or 3.5mmol/L EAA concentrations with 0 or 5mmol/L acetate or 0 or 17.5mmol/L glucose in 2 separate 2×2 factorial studies. In a separate set of experiments, lactogenic bovine mammary tissue slices were subjected to the same treatments except that the low EAA treatment contained a low level of EAA (0.18mmol/L). Supplementation of EAA enhanced phosphorylation of mTOR (Ser2448) and eukaryotic initiation factor 4E binding protein 1 (4EBP1, Thr37/46), and reduced phosphorylation of eukaryotic elongation factor 2 (eEF2, Thr56) in MAC-T cells. Concentration of ATP and phosphorylation of AMPK increased and decreased, respectively, in the presence of EAA in MAC-T cells. Acetate, EAA, or glucose numerically reduced AMPK phosphorylation by about 16% in mammary tissue slices. Provision of EAA increased phosphorylation of mTOR and 4EBP1, intracellular total EAA concentration, and casein synthesis rates in mammary tissue slices, irrespective of the presence of acetate or glucose in the medium. Phosphorylation of mTOR had a marginally negative association with AMPK phosphorylation, which was positively related to eEF2 phosphorylation. Casein synthesis rates were positively and more strongly linked to mTOR phosphorylation than the negative link between eEF2 phosphorylation and casein synthesis rates. A 100% increase in mTOR phosphorylation was associated with an increase in the casein synthesis rate of 0.74%·h−1, whereas a 100% increase in eEF2 phosphorylation was related to a decline in the casein synthesis rate of 0.33%·h−1. Although AMPK phosphorylation was responsive to cellular energy status and had a negative effect on mTOR-mediated signals in bovine mammary epithelial cells, its effect on milk protein synthesis rates appeared to be marginal compared with the mTOR-mediated regulation of milk protein synthesis by EAA.

Exercise, nutrition, and aging in the regulation of muscle protein synthesis

Exercise, nutrition, and aging in the regulation of muscle protein synthesis

Muscle protein kinetics in cancer cachexia

Skeletal muscle loss appears to be the most significant event in cancer cachexia and is associated with a poor outcome. The balance between mechanisms that control synthesis and degradation is fundamental when designing new therapies. This review aims to highlight the molecular mechanisms that are associated with protein kinetics. The mechanisms that promote muscle synthesis have been explored in detail recently but moreover they have been the mechanisms behind degradation. Specific advances in cellular signalling molecules related to autophagy pathways including signal transducer and activators of transcription-3, activin type-2 receptor, TRAF6, and transcriptomic research have been given special attention in this review to highlight their roles in degradation and as potential targets for therapeutics. Ways to quantify muscle loss are badly needed for outcome measures; recent research using radiolabelled amino acids has also been discussed in this review. Only by having an appreciation of the complex regulation of muscle protein synthesis and degradation, will potential new therapeutics be able to be developed. This review identifies known targets in molecular pathways of current interest, explores methods used to find novel genes which may be involved in muscle kinetics and also highlights ways in which muscle kinetics may be measured to assess the efficacy of such interventions.

SREBP-1 Transcription Factors Regulate Skeletal Muscle Cell Size by Controlling Protein Synthesis through Myogenic Regulatory Factors

SREBP-1 are ubiquitously expressed transcription factors, strongly expressed in lipogenic tissues where they regulate several metabolic processes like fatty acid synthesis. In skeletal muscle, SREBP-1 proteins regulate the expression of hundreds of genes, and we previously showed that their overexpression induced muscle atrophy together with a combined lack of expression of myogenic regulatory factors. Here we present evidences that SREBP-1 regulate muscle protein synthesis through the downregulation of the expression of MYOD1, MYOG and MEF2C factors. In myotubes overexpressing SREBP-1, restoring the expression of myogenic factors prevented atrophy and rescued protein synthesis, without affecting SREBP-1 action on atrogenes and proteolysis. Our results point out the roles of MRFs in the maintenance of the protein content and cell size in adult muscle fibre, and contribute to decipher the mechanisms by which SREBP-1 regulate muscle mass.

Nutritional regulation of muscle protein synthesis with resistance exercise: strategies to enhance anabolism.

Provision of dietary amino acids increases skeletal muscle protein synthesis (MPS), an effect that is enhanced by prior resistance exercise. As a fundamentally necessary process in the enhancement of muscle mass, strategies to enhance rates of MPS would be beneficial in the development of interventions aimed at increasing skeletal muscle mass particularly when combined with chronic resistance exercise. The purpose of this review article is to provide an update on current findings regarding the nutritional regulation of MPS and highlight nutrition based strategies that may serve to maximize skeletal muscle protein anabolism with resistance exercise. Such factors include timing of protein intake, dietary protein type, the role of leucine as a key anabolic amino acid, and the impact of other macronutrients (i.e. carbohydrate) on the regulation of MPS after resistance exercise. We contend that nutritional strategies that serve to maximally stimulate MPS may be useful in the development of nutrition and exercise based interventions aimed at enhancing skeletal muscle mass which may be of interest to elderly populations and to athletes.

Supplementation of a suboptimal protein dose with leucine or essential amino acids: effects on myofibrillar protein synthesis at rest and following resistance exercise in men

Leucine is a nutrient regulator of muscle protein synthesis by activating mTOR and possibly other proteins in this pathway. The purpose of this study was to examine the role of leucine in the regulation of human myofibrillar protein synthesis (MPS). Twenty-four males completed an acute bout of unilateral resistance exercise prior to consuming either: a dose (25 g) of whey protein (WHEY); 6.25 g whey protein with total leucine equivalent to WHEY (LEU); or 6.25 g whey protein with total essential amino acids (EAAs) equivalent to WHEY for all EAAs except leucine (EAA-LEU). Measures of MPS, signalling through mTOR, and amino acid transporter (AAT) mRNA abundance were made while fasted (FAST), and following feeding under rested (FED) and post-exercise (EX-FED) conditions. Leucinaemia was equivalent between WHEY and LEU and elevated compared to EAA-LEU (P=0.001). MPS was increased above FAST at 1–3 h post-exercise in both FED (P <0.001) and EX-FED (P <0.001) conditions with no treatment effect.At 3–5 h, only WHEY remained significantly elevated above FAST in EX-FED(WHEY 184% vs. LEU 55% and EAA-LEU 35%; P =0.036). AAT mRNA abundance was increased above FAST after feeding and exercise with no effect of leucinaemia. In summary, a low dose of whey protein supplemented with leucine or all other essential amino acids was as effective as a complete protein (WHEY) in stimulating postprandial MPS; however only WHEY was able to sustain increased rates of MPS post-exercise and may therefore be most suited to increase exercise-induced muscle protein accretion.

The abundance of the mRNA translation initiation inhibitor PDCD4 in L6 myotubes is regulated by mTORC1 and the proteasome

The mammalian target of rapamycin complex 1/ S6 kinase 1 (mTORC1/S6K1) pathway is a critical regulator of muscle protein synthesis and mass. Using L6 myotubes, we characterized the regulation of the mRNA translation inhibitor programmed cell death 4 (PDCD4), a recently described substrate of the mTORCI/S6K1 pathway. Myotubes were cultured in amino acid‐free, serum‐free medium for 12 h, and then refed for 1 or 3 h. Starvation decreased the phosphorylation of S6K1, the PDCD4 kinase (P&lt;0.01). Starvation increased the abundance, but not the phosphorylation, of PDCD4 &gt;4 fold (P&lt;0.001); refeeding reversed the effects of starvation on S6K1 and PDCD4. Rapamycin or MG132 (an inhibitor of the proteasome) prevented the refeeding‐associated decrease in PDCD4 abundance. Because PDCD4 inhibits translation by binding to the eukaryotic initiation factor 4A (eIF4A), we assessed this interaction. Starvation increased the % of PDCD4 bound to eIF4A (P&lt;0.05); this was reversed during refeeding. Rapamycin prevented the effect of refeeding. Thus, we showed that in skeletal muscle cells, the abundance of PDCD4 and its interaction with the initiation factor eIF4A were reversibly modified by nutrients, and that the activities of mTORC1 and of the ubiquitin dependent proteolytic system were required for this regulation. Manipulation of PDCD4 abundance may offer an approach to stimulate mass. Funded by NSERC.

Sprint exercise enhances skeletal muscle p70S6k phosphorylation and more so in women than in men

Sprint exercise is characterized by repeated sessions of brief intermittent exercise at a high relative workload. However, little is known about the effect on mTOR pathway, an important link in the regulation of muscle protein synthesis. An earlier training study showed a greater increase in muscle fibre cross-sectional area in women than men. Therefore, we tested the hypothesis that the activation of mTOR signalling is more pronounced in women than in men. Healthy men (n=9) and women (n=8) performed three bouts of 30-s sprint exercise with 20-min rest in between. Multiple blood samples were collected over time, and muscle biopsy specimens were obtained at rest and 140 min after the last sprint. Serum insulin increased by sprint exercise and more so in women than in men [gender (g) × time (t)]: P=0.04. In skeletal muscle, phosphorylation of Akt increased by 50% (t, P=0.001) and mTOR by 120% (t, P=0.002) independent of gender. The elevation in p70S6k phosphorylation was larger in women (g × t, P=0.03) and averaged 230% (P=0.006) as compared to 60% in men (P=0.04). Phosphorylation rpS6 increased by 660% over time independent of gender (t, P=0.003). Increase in the phosphorylation of p70S6k was directly related to increase in serum insulin (r=0.68, P=0.004). It is concluded that repeated 30-s all-out bouts of sprint exercise separated by 20 min of rest increases Akt/mTOR signalling in skeletal muscle. Secondly, signalling downstream of mTOR was stronger in women than in men after sprint exercise indicated by the increased phosphorylation of p70S6k.

Regulation of muscle protein synthesis in humans

Investigations into the regulation of muscle protein synthesis (MPS) are a cornerstone of understanding the control of muscle mass. Rates of MPS are finely tuned according to levels of activity, nutrient availability and health status. For instance, rates of MPS are positively regulated by exercise and nutrition, and negatively regulated by inactivity (e.g. disuse), ageing (i.e. sarcopenia) and in muscle-wasting related diseases (e.g. cancer). Skeletal muscles display a high degree of intrinsic regulation. Increases in MPS after exercise occur independently of the systemic milieu for example growth hormone/testosterone concentrations. In the absence of exercise, increases in MPS after feeding are of finite duration despite enduring precursor availability; that is muscles can sense they are 'full'. Intriguingly, exercise delays this 'muscle-full' response to allow for building and repair. In contrast, muscle-wasting conditions exhibit a premature 'muscle-full' response to nutrition and exercise (i.e. anabolic resistance), which may cause atrophy. Observations of 'dissociations' between MPS and anabolic signalling pathways have cast doubt on how much we understand of the molecular regulation of human MPS. Anabolic and anticatabolic interventions in health and disease should be aimed at manipulating the 'muscle-full' set point to maximize muscle maintenance/hypertrophy.

Leucine content of dietary proteins is a determinant of postprandial skeletal muscle protein synthesis in adult rats

BackgroundLeucine (Leu) regulates muscle protein synthesis (MPS) producing dose-dependent plasma Leu and MPS responses from free amino acid solutions. This study examined the role of Leu content from dietary proteins in regulation of MPS after complete meals.MethodsExperiment 1 examined 4 protein sources (wheat, soy, egg, and whey) with different Leu concentrations (6.8, 8.0, 8.8, and 10.9% (w/w), respectively) on the potential to increase plasma Leu, activate translation factors, and stimulate MPS. Male rats (~250 g) were trained for 14 day to eat 3 meals/day consisting of 16/54/30% of energy from protein, carbohydrates and fats. Rats were killed on d14 either before or 90 min after consuming a 4 g breakfast meal. Experiment 2 compared feeding wheat, whey, and wheat + Leu to determine if supplementing the Leu content of the wheat meal would yield similar anabolic responses as whey.ResultsIn Experiment 1, only whey and egg groups increased post-prandial plasma Leu and stimulated MPS above food-deprived controls. Likewise, greater phosphorylation of p70 S6 kinase 1 (S6K1) and 4E binding protein-1 (4E-BP1) occurred in whey and egg groups versus wheat and soy groups. Experiment 2 demonstrated that supplementing wheat with Leu to equalize the Leu content of the meal also equalized the rates of MPS.ConclusionThese findings demonstrate that Leu content is a critical factor for evaluating the quantity and quality of proteins necessary at a meal for stimulation of MPS.

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

Effects of leucine and citrulline versus non-essential amino acids on muscle protein synthesis in fasted rat: a common activation pathway?

Leucine (LEU) is recognized as a major regulator of muscle protein synthesis (MPS). Citrulline (CIT) is emerging as a potent new regulator. The aim of our study was to compare MPS modulation by CIT and LEU in food-deprived rats and to determine whether their action was driven by similar mechanisms. Rats were either freely fed (F, n=10) or food deprived for 18h. Food-deprived rats were randomly assigned to one of four groups and received per os, i.e., gavage, saline (S, n=10), L: -leucine (1.35g/kg, LEU, n=10), L: -citrulline (1.80g/kg CIT, n=10) or isonitrogenous non-essential amino acids (NEAA, n=10). After gavage, the rats were injected with a flooding dose of [(13)C] valine to determine MPS. The rats were killed 50min after the injection of the flooding dose. Blood was collected for amino acid, glucose and insulin determinations. Tibialis anterior muscles were excised for determination of MPS and for Western blot analyses of the PI3K/Akt, mTORC1, ERK1/2/MAPK pathways and AMP kinase component. MPS was depressed by 61% in starved rats (Saline vs. Fed, P<0.05). Administration of amino acids (NEAA, LEU or CIT) completely abolished this decrease (NEAA, CIT, LEU vs. Fed, NS). Food deprivation affected the phosphorylation status of the mTORC1 pathway and AMP kinase (Saline vs. Fed, P<0.05). LEU and CIT administration differently stimulated the mTORC1 pathway (LEU>CIT). LEU but not CIT increased the phosphorylation of rpS6 at serine 235/236. Our findings clearly demonstrated that both CIT and LEU were able to stimulate MPS, but this effect was likely related to the nitrogen load. LEU, CIT and NEAA may have different actions on MPS in this model as they share different mTORC1 regulation capacities.

Sprint exercise enhances skeletal muscle p70S6k phosphorylation and more so in females than in males

Aim: Sprint exercise is characterized by repeated sessions of brief intermittent exercise at a high relative workload. However, little is known about the effect on mTOR pathway, an important link in regulation of muscle protein synthesis. An earlier training study showed a greater increase in muscle fibre cross sectional area in females than males. Therefore, we tested the hypothesis that activation of mTOR signalling is more pronounced in females than in males. Healthy males (n=9) and females (n=8) performed three bouts of 30-s sprint exercise with 20 min rest between. Methods: Multiple blood samples were collected over time and muscle biopsy specimens were obtained at rest and 140 min after the last sprint. Results: Serum insulin increased by sprint exercise and more so in females than in males (gender (g) x time (t):P=0.04. In skeletal muscle, phosphorylation of Akt increased by 50% (t, P=0.001) and mTOR by 120% (t, P=0.002) independent of gender. The elevation in p70S6k phosphorylation was larger in females (g x t, P=0.03) and averaged 230% (P=0.006) as compared to 60% in males (P=0.04). Phosphorylation rpS6 increased by 660% over time independent of gender (t, P=0.003). Increase in phosphorylation of p70S6k was directly related to increase in serum insulin (r=0.68, P=0.004). Conclusion: It is concluded that repeated 30-s all out bouts of sprint exercise separated by 20 min of rest, increases Akt/mTOR signalling in skeletal muscle. Secondly, signalling downstream of mTOR was stronger in females than in males after sprint exercise indicated by increased phosphorylation of p70S6k.