Selective Inhibition of Ribosomal Biogenesis Over Cap-Dependent Translation by Rapamycin in L6 Myotubes

The mechanistic target of rapamycin, or mTOR, lies at the center of skeletal muscle anabolic regulation. The mTOR kinase has at least two complexes that aide in carrying out responses to anabolic signals. Complex 1 (mTORC1) activation leads to cap-dependent translation and ribosomal biogenesis via phosphorylation of its two downstream targets 4EBP1 and P70S6K respectively. mTORC1 is inhibited by rapamycin while mTORC2 is not; however, cap-dependent translation via mTORC1 may still occur under rapamycin inhibition. PURPOSE: To identify timing and mechanistic pathway by which selective skeletal muscle anabolism continues in the presence of rapamycin inhibition. METHODS: Murine L6 myoblasts were cultured in 10cm3 plates in standard culture medium supplemented with 1% pen/strep and 10% FBS. Differentiation was induced at 70% confluence by switching to a low serum medium. Myoblasts were differentiated until myotubes were visible at which point plates were treated with 100nM of rapamycin for 1 hour or 12 hours. Control plates were treated in the same way with vehicle control, DMSO. Myotubes were harvested immediately after treatment and protein content of key anabolic markers was measured using Western blotting techniques. RESULTS: Treatment with rapamycin significantly decreased P70S6K phosphorylation, and phosphorylated to total ratio after 12 hours of treatment compared to its respective DMSO control (pCONCLUSION: Treatment with rapamycin ablates P70S6K phosphorylation and subsequently inhibits mTORC1-dependent ribosomal biogenesis. Although there was no apparent reduction of the p4EBP1/4EBP1 ratio, acute cap-dependent translation is likely uninhibited by 1 or 12 hours of treatment with rapamycin. Total 4EBP1 protein content was decreased while its binding partner, eIF4E remained unchanged. Thus, the ratio of eIF4E to 4EBP1 was elevated after treatment with rapamycin making it likely that unbound eIF4E was free to initiate cap-dependent translation even in the presence of rapamycin

Acylated and Unacylated Ghrelin Impact on Protein synthesis and Signaling Pathways of L6 Myotubes

Ghrelin has recently become a hormone of interest in the fight against type two diabetes. Ghrelin is found in two forms (acylated vs. unacylated), with the acylated form of ghrelin being cited as diabetogenic purportedly due to its interaction with growth hormone secretion. Both the Acylated Ghrelin (AG) and Unacylated Ghrelin (UAG) forms are reported to have metabolic functions within skeletal muscle, despite having little expression of the only known ghrelin receptor known as the growth hormone secretagogue receptor 1a (ghsr-1a). The investigation into AG and UAG on skeletal muscle in type 2 diabetes (T2D) shows promise; however, the measures and dosing used to establish the impact of AG vs. UAG has varied widely, resulting in varied and sometimes contradictory results. PURPOSE: The purpose of this study was to establish the impact of AG or UAG on cultured myotubes in-vitro (100nM). METHODS: Differentiated, cultured L6 myotubes were treated for 48 hours with or without either AG or UAG. In the second 24-hour window, media was changed and enriched with 4% deuterium. Cells were harvested from each treatment group at 24 hours post deuterium enrichment and processed for protein synthesis and western blot protein analyses. RESULTS: Cells incubated with either AG or UAG doubled the content of phosphorylated AKT at Ser 473 (109% and 97% , respectively; p\u3c0.05), implicating an increase in mTORC2 activity. 100nM AG or UAG also increased phosphorylation of GSK3β (83% and 54%, respectively; p\u3c0.05). However, AG had increased Phosphorylation of 4EBP1 when compared to control (80%) while UAG did not, suggesting that mTORC1 was the predominate complex under that condition. Physiologically there were no differences of fractional synthesis rates among Control, 100nM AG or 100nM UAG. Interestingly, while protein synthesis data were similar among groups, the differences between mTORC1 vs. mTORC2 signaling may implicate that the impact of ghrelin might direct the types of proteins being manufactured. CONCLUSION: Results from this study indicate that 100nM of Ghrelin is sufficient to impact healthy skeletal muscle albeit by a yet to be defined mechanism. Further, our data suggest that both AG and UAG upregulate both mTOR pathways but that AG appears to favor mTORC1 as indicated by the hyperphosphorylation of 4EBP1. This increase in mTORC1 activity supports previous literature indicating that AG ay be a diabetogenic hormone, and furthers the understanding of Ghrelin’s impact on skeletal muscle metabolism and its involvement in development of diabetes

The application of 2H2O to measure skeletal muscle protein synthesis

Skeletal muscle protein synthesis has generally been determined by the precursor:product labeling approach using labeled amino acids (e.g., [13C]leucine or [13C]-, [15N]-, or [2H]phenylalanine) as the tracers. Although reliable for determining rates of protein synthesis, this methodological approach requires experiments to be conducted in a controlled environment, and as a result, has limited our understanding of muscle protein renewal under free-living conditions over extended periods of time (i.e., integrative/cumulative assessments). An alternative tracer, 2H2O, has been successfully used to measure rates of muscle protein synthesis in mice, rats, fish and humans. Moreover, perturbations such as feeding and exercise have been included in these measurements without exclusion of common environmental and biological factors. In this review, we discuss the principle behind using 2H2O to measure muscle protein synthesis and highlight recent investigations that have examined the effects of feeding and exercise. The framework provided in this review should assist muscle biologists in designing experiments that advance our understanding of conditions in which anabolism is altered (e.g., exercise, feeding, growth, debilitating and metabolic pathologies)

Pharmacological Inhibition of mTOR and ERK1/2 Resulted in Attenuated Protein Synthesis Rates in Differentiated C2C12 Myoblasts in a Similar Fashion to in vivo Rodent Studies.

Fractional protein synthesis rates have long been used as in indicator of acute alterations in the anabolic state of various tissues. Through the use of a number of stable and isotopic tracer methodologies, the measurement of fractional synthesis rates (FSR) in vivo has become a staple of skeletal muscle physiology. Through the application of a deuterium oxide tracer, this project sought to measure pharmacological perturbations in fractional synthesis rates in culture in differentiated C2C12 murine myotubes. PURPOSE: To assess myofibrillar protein FSR in differentiated C2C12 murine myotubes following pharmacological inhibition of rapamycin-sensitive (mTOR) or -insensitive (ERK1/2) pathways, and how signal transduction through these pathways impact FSR as compared to previous in vivo studies of pharmacological inhibition studies in skeletal muscle. METHODS: C2C12 murine myoblasts were cultured in collagen coated 6 well culture dishes, and grown to 60-70% confluency using a high glucose DMEM growth media (GM). Cultures were transitioned to a differentiation media (DM) upon reaching target confluency. DM was changed daily for 4 days to allow for complete differentiation to myotubes. Cultures were randomly assigned treatment conditions of cell control (CC), rapamycin inhibition (RAPA), ERK1/2 inhibition (ERK), and electrical stimulation (ESTIM). Cultures underwent treatment conditions for 24 hours with a 4% deuterium oxide GM supplement. Analysis was carried out using a gas chromatography mass spectrometer. RESULTS: Fractional rates of protein synthesis were significantly lower in the RAPA (p=0.028) and ERK (p=0.029) groups as compared to CC, with no differences between RAPA and ERK groups (p\u3e0.05). Although statistics were not applied to the ESTIM group due to low sample size, electrical pulse stimulation shows promise for the stimulation of FSR in cultured myotubes. CONCLUSION: Diminished FSR in both RAPA and ERK groups are consistent with previous findings from in vivo rodent studies. These results may indicate comparable alterations in skeletal muscle anabolic signaling in cell culture as well as in vivo rodent models. Further investigations into anabolic signaling mechanisms related to the control of protein synthesis are needed

Characterization of Protein Metabolism in Undifferentiated and Differentiated Murine Muscle Tissue

The emergence of cell culture experiments have greatly expanded the understanding of skeletal muscle physiology. However, there is a paucity of data regarding the behaviors of cells grown in culture at various stages versus in vivo. This preliminary set of studies was designed to assess alterations of anabolic responses between undifferentiated and differentiated muscle tissue in [high] and [low] glucose media along with varying dosages of insulin. Purpose: Determine if there is a disparity in fractional synthesis rates (FSR) between C2C12 myoblasts and myotubes with varying levels of insulin and in [high] (4.5g/L) and [low] glucose (2.75 g/L) media. Methods: All cells that were going to be differentiated were started on a [high] glucose differentiation media for 48 hours. The [high] glucose differentiation media was continually applied for the [high] glucose group until harvest of the cells. The [low] glucose media group had the [high] glucose differentiation media removed and [low] glucose differentiation media was applied for 48 hours until the cells were harvested. Both [low] and [high] glucose groups received three different levels of insulin. T-25’s received either 75 µL, 150 µL, or 300 µL. T-75’s 195 µL, 390 µL, and 780 µL. Deuterium oxide was applied 24 hours prior to harvest of the cells at a level of 4%. Results: Preliminary data demonstrates that differentiated murine myotubes have slightly elevated FSR than undifferentiated myoblasts (p\u3c0.013). When insulin was added to the growth media, FSR was found to be elevated in undifferentiated cells compared to controls (p\u3c0.05). Within the differentiated myotubes, the [low] glucose myotubes had higher FSR than myotubes that were incubated in [high] glucose myotubes (p\u3c0.001). There was also no difference in FSR based on flask size for either the undifferentiated (p\u3e0.181) or differentiated (p\u3e0.464) C2C12’s. Conclusion: Future investigators must be aware of the ratio of undifferentiated cells and differentiated myotubes as this ratio could confound results as myoblasts are still present even at later stages of differentiation. Current protocols for differentiation media, regarding insulin addition, provide for optimal anabolic responses. Elevated FSR rates in the myotubes fed [low] glucose media could be explained by the cells having a higher turnover rate of cellular proteins

Effects of Voluntary Resistance Exercise Training During Recovery From Hindlimb Unloading on Rat Gastrocnemius Muscle

As research continues to examine the deleterious impact of long-duration spaceflight on human muscle mass and function, there remain gaps in our knowledge of muscle physiology, especially in examining how muscle’s ability to recover or rehabilitate from unloading may alter the results of multiple exposures to microgravity followed by 1g recovery. The purpose of this study was to analyze the effects of resistance exercise training of gastrocnemius muscle mass and anabolism during the initial recovery period immediately following a bout of unloading, as well as to examine the role that exercise may have on a subsequent period of weightlessness. This was achieved in rodent models of simulated spaceflight (0g), recovery (1g), and resistance training (\u3e1g) using male Sprague-Dawley (6 mo) rats randomly assigned to the following groups: 28d hindlimb unloading (HU), 28d HU followed by a 56d recovery period of normal cage ambulation at 1g (1HU+REC), 2 cycles of 28d HU with a 56d recovery period between unloading (2HU), 2HU followed by an additional 56d recovery at 1g (2HU+REC), or an age- and housing-matched control group (CON). In addition, following the initial 28d HU period, two groups of animals were given 7d recovery at 1g followed by a 7wk (3 sessions/wk) moderate-intensity, moderate-volume voluntary resistance exercise program (EX) in which the animals were trained to perform a squat-like motion with full extension of the lower limb and resistance was applied incrementally by weighted pouches over the scapula to ~65% bodyweight. At the conclusion of the experiments, gastrocnemius muscles were carefully excised, weighed, and evaluated for cumulative (24h) rates of protein synthesis (FSR). Values of both muscle mass and FSR were lower than control during periods of unloading (p\u3c0.05), but with recovery, control values were reached for mass and surpassed for FSR. Interestingly, there was no significant difference between the mass of 2HU and 2HU+EX (p\u3e0.05), and both were diminished in comparison to control animals, suggesting that benefits of exercise during periods of ambulatory reloading after disuse/microgravity may not be additive. In conclusion, our data suggest that given adequate recovery, microgravity-induced losses of muscle mass can be fully restored to control values, and this adaptational response persists even with multiple exposures. These findings may have important implications not only for career astronauts, but also for individuals who have been subjected to casting of a limb or a period of bed rest following severe injury or illness

Rider energy expenditure during high intensity horse activity and the potential for health benefits

Obesity and disease associated with sedentary lifestyles have become major concerns in the United States. Exercise has been described as a crucial part of disease prevention and overall health, with activities such as biking or running cited as examples. Horseback riding is a popular activity; however, little information on the exercise value of horseback riding exists. The objective of this experiment was to define the energy expenditure of participants in several common riding events. All procedures were approved by the Texas A&M Institutional Review Board and Institutional Animal Care and Use Committee; subjects gave written consent prior to participation. Twenty subjects completed each of three riding tests: a 45min walk-trot-canter ride (WTC), a reining pattern, and a cutting pattern while wearing a telemetric gas analyzer. Anthropometric data were obtained for each subject through DEXA scans. Total energy expenditure (tEE), as well as mean and peak energy expenditure per minute (EE/min), metabolic equivalents of task (MET), heart rate (HR), respiratory frequency (RF), pulmonary ventilation (VE), oxygen consumption (VO2), relative oxygen consumption (relVO2) and carbon dioxide production (­VCO2)were measured. Mean energy expenditure per minute, and HR responses were greater (P \u3c 0.05) for reining (6.96±0.23Kcal/min, 163.28±4.2bpm) and cutting (4.98±0.23Kcal/min, 146.9±4.2bpm) than for WTC (4.27±0.23Kcal/min, 131.5±4.2bpm). When WTC test was evaluated by gait component, mean EE/min and MET increased as gait speed increased. Mean EE/min and MET were higher (P \u3c 0.05) for riders at long trot (6.9±0.21Kcal/min, 6.19±0.21MET) and canter (6.93±0.21Kcal/min, 5.95±0.21MET) gaits than during the walk (2.34±0.21Kcal.min, 2.01±0.21MET) or trot (3.5±0.21Kcal/min, 3.2±0.21MET) gaits. Similar patterns were observed for RF, VE, VO2, VCO2 and RelVO2 across disciplines and by gait component. The tEE (194.7±3.84kcal/min) and mean MET (4.27±0.23MET) observed during the 45-min WTC ride were within current national recommendations of intensity (3-6MET), time (\u3e30min) and calories burned (~1000Kcal/wk). The results of this study provide novel information about exercise intensity values for horseback activities and differences among riding disciplines measured in real time using a portable system. Riders engaged in cutting and reining experienced more intense exercise in short durations, while WTC provided a greater total energy expenditure. These data suggest that it is possible, if riding at the more intense gaits such as long trot and canter, for health benefits to be achieved through accumulated weekly horseback riding exercise

Effects of Multiple Bouts of Long-duration Hindlimb Unloading and Recovery on Rat Plantaris Muscle

Exposure to microgravity results in a rapid reduction of muscle mass. However, few studies exist designed to examine the effects of multiple long-term exposures to microgravity with alternating recovery periods on skeletal muscle. To determine what happens to the recovery of skeletal muscle when faced with subsequent unloading and recovery periods. Male Sprague-Dawley (6 mo) were assigned to the following groups as shown in figure 1 below: 28d hindlimb unloading (1HU), 28d HU session followed by a 56d recovery bout of normal cage ambulation at 1g (1HU+REC), 2 cycles of 28d HU with a 56d recovery period between unloadings (2HU), 2 cycles of 28d HU as in the 2HU group, but followed by an additional 56d recovery at 1g (2HU+REC), and an age- and housing-matched control group (CON). On the final day of the experimental period, plantaris muscles were excised and weighed. The 1HU+REC (0.548 ± 0.012), 2HU+REC (0.562 ± 0.015), and CON (0.550 ± 0.013) showed no statistical difference (p\u3e0.05) between each other. The 1 HU (0.442 ± 0.020) and 2 HU (0.431 ± 0.011) groups were significantly less (p\u3c0.001) than recovery and aged control animals but were not significantly different from each other. The results show that the plantaris muscle presented reduction of muscle mass with initial and subsequent exposures to microgravity. However, with the recovery period, animals were able to regain lost muscle mass, similar to age-matched controls. These findings would be relevant for astronauts participating in multiple long-duration missions throughout their career

Significant Predictors of Nonalcoholic Fatty Liver Disease in Texas Firefighters

Risk factors for Nonalcoholic Fatty Liver Disease (NAFLD) include obesity, hypertension, dyslipidemia, and diabetes mellitus. Not only are these prevalent in the general US population, but they are also present at high rates in a specific subset responsible for public safety – firefighters. PURPOSE: The aim of the present study is to use logistic regression to predict the likelihood of occurrence of NAFLD in firefighters using a subset of health-related factors associated with common cardiometabolic risk factors. METHODS: Data were collected on 136 firefighters (128 males, 8 females; 36.3 ± 9.0 yrs; 95.7 ± 17.0 kg; 178.9 ± 7.4 cm; 29.8 ± 4.2 kg/m2) participating in FITLIFE, a university-based fitness program at Texas A&M University. Nominal logistic regression with stepwise removal was used to estimate the best model to predict fatty liver disease. Stepwise removal identified resting systolic blood pressure (RSBP, mm HG), Body Mass Index (BMI, kg/m2), visceral adipose tissue (VAT, cm2), whether or not has hypertension or is on medication (HTNMED; 0=No,1=Yes), and plasma triglyceride concentrations (TG, mg/dL) as independent predictors (p\u3c0.05). Odds ratios (OR) were calculated to determine the change in the odds of NAFLD per unit increase in each predictor. RESULTS: Logistic regression yielded the following equation to predict the probability of developing NAFLD: Logit = -22.5176 + 0.0918(RSBP) + 0.2154(BMI) + 0.0065(TG) + 0.0161(VAT) + 1.830(HTNMED) (R2 = 0.4655, p \u3c 0.001). Of the predictors, the ORs from largest to smallest were 6.235, 1.240, 1.096, 1.016, and 1.002 for HTNMED, BMI, RSBP, VAT, and TG, respectively. CONCLUSION: Using RSBP, BMI, VAT, TG, and HTNMED as predictors, this study demonstrates that the probability of developing NAFLD in Texas firefighters can be reasonably predicted. This regression model and individual predictors may be used by health practitioners as a cost-effective screening tool to identify those at higher risk for NAFLD

Autophagy is Required for mTOR-Mediated Anabolism in Skeletal Muscle

PURPOSE: While much has been discovered about the role autophagy in protein degradation, recent evidence suggests that autophagy is required for muscular adaptations to exercise, hinting at a hitherto unknown cross-talk between autophagic proteolysis and muscle protein anabolism. Here, we set out to further elucidate the metabolic mechanisms by which autophagy may influence protein anabolism. METHODS: L6 myoblasts received either electrical pulse stimulation (EPS) to induce muscle contraction or were unstimulated to serve as controls, and were then treated with an inhibitor of the ATG4 enzyme which catalyzes the initial step of autophagy NSC185058 (NSC, 100 μM) or DMSO as a vehicle control (VC). After 24 hours, cells were lysed and Western immunoblotted for P70S6K, DEPTOR, MAPK, AMPK, LC3, and P62. Differences between VC and NSC treated groups were assessed by a two-tailed t-test, while comparisons between VC, EPS, and EPS+NSC groups were made using one-way ANOVA and SNK post-hoc test, with α levels set at 0.05. RESULTS: EPS induced a 97% increase in P70S6K phosphorylation (p\u3c0.05), with NSC treatment blunting this effect, leading to a 22% increase (P\u3e0.05). EPS resulted in a 37% reduction in DEPTOR content (p\u3c0.05); however, NSC treatment alone produced a 166% decrease in DEPTOR level (p\u3c0.05), with EPS+NSC leading to an even larger reduction (-766%) in DEPTOR than EPS alone. NSC treatment led to a decrease (-85%, p\u3e0.05) LC3II/I ratio relative to VC, which was reduced in both the EPS (-68%, p\u3c0.05) and EPS+NSC (-87%, p\u3c0.05) conditions. P62 content increased by 749% with EPS (p\u3c0.05), with no significant difference in P62 level between VC and EPS+NSC, and NSC treatment alone led to a 61% decrease in P62 (p\u3c0.05). MAPK phosphorylation was elevated in both EPS (99.9%, p\u3e0.05) and EPS+NSC (149.13, p\u3c0.05). Neither NSC nor EPS+NSC altered phosphorylation status of AMPK. CONCLUSION: Despite reductions in DEPTOR, mTOR activity was blunted in EPS+NSC cells, indicating that mTOR mediated anabolic signaling requires autophagy post muscle contraction. This is particular to the mTOR pathway, as an increase in MAPK phosphorylation was still observed in EPS+NSC. While the decrease in LC3II/I ratio and accumulation of P62 seen after EPS are likely due to inhibition of autophagy due to mTOR activity, our data indicate that inhibition of ATG4 by NSC185058 blunts mTOR activity after muscle contraction. This effect is not due to activation of the cellular energy sensor AMPK, as we found no increase in AMPK phosphorylation in any condition. Further work will be required to fully elucidate the mechanism by which NSC185058 inhibits mTOR-mediated anabolism