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

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

Autophagy, but Not Proteolysis, May Aid in Muscle Protein Synthesis

For muscle growth to occur, protein synthesis must be greater than protein degradation. However, up to this point, anabolic pathways have garnered the brunt of investigations examining anabolic capacity with little investigation into the connectedness of catabolic signaling on these anabolic targets. PURPOSE: The purpose of this study was to elucidate the contributions of proteasomal-dependent and autophagic-dependent catabolic pathways on anabolism via analysis of fractional synthetic rates (FSR) in L6 myotubes. METHODS: Differentiated, cultured L6 myoblasts were treated with media containing 4% deuterium oxide (stable isotope label) and a corresponding pharmacological treatment (NSC 185058 [autophagic inhibitor; 100 μM], MG-262 [proteasomal inhibitor; 0.01 μM] or DMSO control; n=3/group) during the final 24-hours of the differentiation period prior to harvest. The myofibrillar pellet of the processed samples was used to determine FSR via mass-spectrometry analysis. DMSO-treated myotubes served as controls, with a one-way analysis of variance and Tukey’s post-hoc test used to test for any differences among groups. RESULTS: Our results indicate that MG-262 had no impact on myofibrillar FSR when compared to DMSO control (MG-262 1.0993 %/day vs. control 1.239 %/day). However, NSC 185058 lowered myofibrillar FSR (NSC 185058 0.9009 %/day vs. control 1.239 %/day; P=0.0282). CONCLUSION: These data suggest that inhibition of autophagic machinery can impair anabolism. This may be due to autophagy’s role in increasing the amino acid pool within the cell. Further, the lack of inhibition seen from MG-262 suggests that there is a delineation of roles within the catabolic pathways in regard to their influence on anabolism in healthy, metabolically unchallenged myotubes

Insulin-induced Increase in Anabolic Capacity is Blunted by Autophagic Inhibition in L6 Myotubes

Insulin is an anabolic hormone that acts on skeletal muscle cells to stimulate protein synthesis, an effect that is enhanced by the availability of amino acids. While autophagy within the cell provides an intracellular source of amino acids to support anabolism, little is known about how this pathway impacts the insulin-induced increase in anabolic capacity within skeletal muscle cells. PURPOSE: The purpose of this study was to determine the impact of autophagic inhibition in cultured L6 myotubes in conjunction with insulin stimulation in vitro. METHODS: Differentiated, cultured L6 myotubes were treated for 24 hours with or without insulin (100 nM) and NSC 185058 (100 μM), a specialized inhibitor of the autophagic catabolic pathway, in media enriched with 4% deuterium. Cells were harvested from each treatment group (n=3/group) 24 hours post-deuterium enrichment and were processed for protein synthesis and western blot protein analysis. A one-way ANOVA was used to compare groups, and when significant F ratios were present, a Student’s Newman-Keuls post hoc procedure was used to test differences among group means. Alpha was set at p≤0.05 for all analyses. RESULTS: Cells treated with insulin (INS) had a higher ratio of phosphorylated to total P70S6K compared to untreated (CON) cells and those incubated with both insulin and NSC 185058 (INS+NSC; 1694% and 327%, respectively; p\u3c0.05). INS+NSC also decreased the ratio of phosphorylated to total 4EBP1 relative to CON (-51%) and INS (-49%), although these differences were not significant (p\u3e0.05). Myofibrillar protein synthesis was stimulated with INS compared to CON and INS+NSC (30.3% and 70.1% respectively; p\u3c0.05) but was lower in INS+NSC relative to CON (-23.4%; p\u3c0.05). CONCLUSION: Results from our study indicate that insulin (100 nM) stimulates anabolism in skeletal muscle cells, but that addition of the autophagic inhibitor NSC 185058 (100 μM) blunts this effect to a level similar to or less than control. Further, our data suggest that the reduction of protein synthesis is mediated through the downregulation of the mTORC1 signaling pathway. While it is widely recognized that insulin promotes anabolic activity through both the direct stimulation of mTOR signaling and extracellular amino acid uptake, our data strongly indicate that autophagic processes are necessary for full anabolic responses in muscle. This decrease in anabolic capacity supports previous literature indicating that the amino acid availability impacts the stimulatory impact of insulin on protein synthesis

Autophagy is Required for the Anabolic Response to Muscle Contraction

Exercise is a key stimulus in regulating the behavior and metabolism of skeletal muscle, with exercise inducing muscular growth through activation of the anabolic mechanistic target of rapamycin kinase (mTOR). Separately, there is mounting evidence that exercise increases autophagy (one of the main routes by which intracellular proteins are degraded) and that the autophagic process may indeed be required for adaptations to exercise training. PURPOSE: To investigate the effects of autophagy inhibition on mTOR signaling and cellular anabolism after muscular contraction. METHODS: Cultured L6 myotubes were to exposed to electrical pulse stimulation using a stimulator set to deliver bipolar pulses of 30V at 100 Hz for 200 ms every fifth second for 60 minutes. Subsequently, cells received either vehicle control, or 100 μM NSC-185058, an antagonist of the key autophagy protein ATG4B and known inhibitor of autophagy. All groups were also exposed to 4% deuterium oxide, a stable isotopic tracer for measurements of protein synthesis. 24 hours post “exercise” bout, cells were lysed in ice-cold Norris buffer, and prepared for Western immunoblot of protein expression, or determination of protein fractional synthesis rate (FSR) of the myofibrillar fraction via mass-spectrometry analysis. Non-stimulated cells receiving vehicle control treatment served as controls, with a one-way analysis of variance and Tukey’s post-hoc test used to test for any differences between groups. RESULTS: We found that phosphorylation of a key downstream target of mTOR, P70S6 kinase, was roughly seven times greater in cells subjected to EPS and vehicle control (710.3%) relative to control (p0.05). While there was a trend for EPS treatment to increase expression of ATG4B, along with a reduction of ATG4B content as a result of NSC-185058 treatment, this finding did not rise to the level of statistical significance. There were no differences in FSR between cells exposed to EPS; however, NSC-185058 treatment significantly reduced FSR in EPS treated cells relative to controls (0.8712 %/hr vs 1.193 %/hr). CONCLUSION: These findings present two conclusions: high-intensity EPS as an in vitro model of exercise elevates mTOR signaling through P70S6K 24 hours post exercise, and mTOR activation as a result of muscular contraction is reliant upon autophagy in skeletal muscle. Further work will be required to elucidate the dynamics of this relationship, and the interplay between skeletal muscle autophagy and anabolism

TRY plant trait database – enhanced coverage and open access

Plant traits - the morphological, anatomical, physiological, biochemical and phenological characteristics of plants - determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait‐based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits - almost complete coverage for ‘plant growth form’. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait–environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives

Stages of condom use in a high HIV-risk sample

The goals were to (1) clarify high risk individuals into one of five stages of readiness for consistent condom use based on the Transtheoretical Model of behavior change (e.g., Prochaska, Norcross and DiClemente, 1994); (2) investigate whether frequency of condom use was different for vaginal vs. anal sex, and for steady vs. other partners; and (3) explore whether condom use was related to several demographic variables. Street interviews were conducted in six major geographic areas across the United States on a diverse sample of 345 individuals engaging in high HIV-risk behaviors. Percentages of individuals found in each of five stages of condom use were: 36% Precontemplators, 15% Contemplators, 7% in Preparation, 7% in Action, and 35% in Maintenance. Individuals used condoms more with other, casual partners than with a steady partner, and slightly more when engaging in anal sex rather than vaginal sex. Condoms were also used more by: men, Caucasians, non-heterosexuals, and those who were not in a steady relationship than by others not in these categories. Still, the majority of high-risk individuals were not using condoms and were not prepared to start. This presents an urgent need for effective interventions to encourage condom use