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

Rodent herpesvirus Peru encodes a secreted chemokine decoy receptor

Viruses have long been studied not only for their pathology and associated disease but also as model systems for understanding cellular and immunological processes. Rodent herpesvirus Peru (RHVP) is a recently characterized rhadinovirus related to murine gammaherpesvirus 68 (MHV68) and Kaposi's sarcoma-associated herpesvirus (KSHV) that establishes acute and latent infection in laboratory mice. RHVP encodes numerous unique proteins that we hypothesize might facilitate host immune evasion during infection. We report here that open reading frame (ORF) R17 encodes a high-affinity chemokine binding protein that broadly recognizes human and murine CC and C chemokines. The interaction of R17 with chemokines is generally characterized by rapid association kinetics, and in the case of CCL3, CCL4, CCL5, CCL24, and XCL1, extremely stable complexes are formed. Functionally, R17 potently inhibited CCL2-driven chemotaxis of the human monocytic cell line THP-1, CCL3-driven chemotaxis of peripheral blood mononuclear cells, and CCL2-mediated calcium flux. Our studies also reveal that R17 binds to glycosaminoglycans (GAGs) in a process dependent upon two BBXB motifs and that chemokine and GAG binding can occur simultaneously at distinct sites. Collectively, these studies suggest that R17 may play a role in RHVP immune evasion through the targeted sabotage of chemokine-mediated immune surveillance

Analysing the impact of rescheduling time in hybrid manufacturing control

Hybrid manufacturing control architectures merge the benefits of hierarchical and heterarchical approaches. Disturbances can be handled at upper or lower decision levels, depending on the type of disturbance, its impact and the time the control system has to react. This paper focuses particularly on a disturbance handling mechanism at upper decision levels using a rescheduling manufacturing method. Such rescheduling is more complex that the offline scheduling since the control system must take into account the current system status, obtain a satisfactory performance under the new conditions, and also come up with a new schedule in a restricted amount of time. Then, this paper proposes a simple and generic rescheduling method which, based on the satisfying principle, analyses the trade-off between the rescheduling time and the performance achieved after a perturbation. The proposed approach is validated on a simulation model of a realistic assembly cell and results demonstrate that adaptation of the rescheduling time might be beneficial in terms of overall performance and reactivity.info:eu-repo/semantics/publishedVersio

Noninvasive optical inhibition with a red-shifted microbial rhodopsin

Optogenetic inhibition of the electrical activity of neurons enables the causal assessment of their contributions to brain functions. Red light penetrates deeper into tissue than other visible wavelengths. We present a red-shifted cruxhalorhodopsin, Jaws, derived from Haloarcula (Halobacterium) salinarum (strain Shark) and engineered to result in red light–induced photocurrents three times those of earlier silencers. Jaws exhibits robust inhibition of sensory-evoked neural activity in the cortex and results in strong light responses when used in retinas of retinitis pigmentosa model mice. We also demonstrate that Jaws can noninvasively mediate transcranial optical inhibition of neurons deep in the brains of awake mice. The noninvasive optogenetic inhibition opened up by Jaws enables a variety of important neuroscience experiments and offers a powerful general-use chloride pump for basic and applied neuroscience.McGovern Institute for Brain Research at MIT (Razin Fellowship)United States. Defense Advanced Research Projects Agency. Living Foundries Program (HR0011-12-C-0068)Harvard-MIT Joint Research Grants Program in Basic NeuroscienceHuman Frontier Science Program (Strasbourg, France)Institution of Engineering and Technology (A. F. Harvey Prize)McGovern Institute for Brain Research at MIT. Neurotechnology (MINT) ProgramNew York Stem Cell Foundation (Robertson Investigator Award)National Institutes of Health (U.S.) (New Innovator Award 1DP2OD002002)National Institute of General Medical Sciences (U.S.) (EUREKA Award 1R01NS075421)National Institutes of Health (U.S.) (Grant 1R01DA029639)National Institutes of Health (U.S.) (Grant 1RC1MH088182)National Institutes of Health (U.S.) (Grant 1R01NS067199)National Science Foundation (U.S.) (Career Award CBET 1053233)National Science Foundation (U.S.) (Grant EFRI0835878)National Science Foundation (U.S.) (Grant DMS0848804)Society for Neuroscience (Research Award for Innovation in Neuroscience)Wallace H. Coulter FoundationNational Institutes of Health (U.S.) (RO1 MH091220-01)Whitehall FoundationEsther A. & Joseph Klingenstein Fund, Inc.JPB FoundationPIIF FundingNational Institute of Mental Health (U.S.) (R01-MH102441-01)National Institutes of Health (U.S.) (DP2-OD-017366-01)Massachusetts Institute of Technology. Simons Center for the Social Brai

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

Heparan sulfate proteoglycans: structure, protein interactions and cell signaling

Heparan sulfate proteoglycans are ubiquitously found at the cell surface and extracellular matrix in all the animal species. This review will focus on the structural characteristics of the heparan sulfate proteoglycans related to protein interactions leading to cell signaling. The heparan sulfate chains due to their vast structural diversity are able to bind and interact with a wide variety of proteins, such as growth factors, chemokines, morphogens, extracellular matrix components, enzymes, among others. There is a specificity directing the interactions of heparan sulfates and target proteins, regarding both the fine structure of the polysaccharide chain as well precise protein motifs. Heparan sulfates play a role in cellular signaling either as receptor or co-receptor for different ligands, and the activation of downstream pathways is related to phosphorylation of different cytosolic proteins either directly or involving cytoskeleton interactions leading to gene regulation. The role of the heparan sulfate proteoglycans in cellular signaling and endocytic uptake pathways is also discussed.Proteoglicanos de heparam sulfato são encontrados tanto superfície celular quanto na matriz extracelular em todas as espécies animais. Esta revisão tem enfoque nas características estruturais dos proteoglicanos de heparam sulfato e nas interações destes proteoglicanos com proteínas que levam à sinalização celular. As cadeias de heparam sulfato, devido a sua variedade estrutural, são capazes de se ligar e interagir com ampla gama de proteínas, como fatores de crescimento, quimiocinas, morfógenos, componentes da matriz extracelular, enzimas, entreoutros. Existe uma especificidade estrutural que direciona as interações dos heparam sulfatos e proteínas alvo. Esta especificidade está relacionada com a estrutura da cadeia do polissacarídeo e os motivos conservados da cadeia polipeptídica das proteínas envolvidas nesta interação. Os heparam sulfatos possuem papel na sinalização celular como receptores ou coreceptores para diferentes ligantes. Esta ligação dispara vias de sinalização celular levam à fosforilação de diversas proteínas citosólicas ou com ou sem interações diretas com o citoesqueleto, culminando na regulação gênica. O papel dos proteoglicanos de heparam sulfato na sinalização celular e vias de captação endocítica também são discutidas nesta revisão.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo (UNIFESP) Departamento de BioquímicaUniversidade Federal de São Paulo (UNIFESP) Departamento de OftalmologiaUNIFESP, Depto. de BioquímicaUNIFESP, Depto. de OftalmologiaSciEL

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