Glucocorticoid action on protein synthesis and protein breakdown in isolated skeletal muscles.
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ABSTRACT:
SUBMITTER: McGrath JA
PROVIDER: S-EPMC1158634 | biostudies-other | 1982 Sep
REPOSITORIES: biostudies-other
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Project description:In the presence of stress, the hypothalamic-pituitary-adrenal (HPA) axis activity can be enhanced to promote the secretion of a large amount of glucocorticoids (GCs), which play an important role in the anabolism and catabolism of skeletal muscle. When the endogenous and exogenous glucocorticoids are deficient or excessive, the body will produce stress-related resistance and change the protein metabolism. In this study, we investigated the effect of GC receptor GRα on protein breakdown and synthesis in porcine skeletal muscle cells (PSCs). Overexpression of GRα was shown to increase the expression of protein degradation-related genes, while knockdown of GRα decreased the expression of these genes. Additionally, we found a relationship between GRα and solute carrier family 2 member 4 (SLC2A4), SLC2A4 expression level increases when stress occurs, suggesting that increasing SLC2A4 expression can partially alleviate stress-induced damage, and we found that there is a combination between them via luciferase reporter assays, which still needs to be confirmed in further studies.
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Project description:Spinal cord injury reduces the rate of skeletal muscle protein synthesis and increases protein breakdown, resulting in rapid muscle loss. The purpose of this study was to determine whether long-term paraplegia would eventually result in a downregulation of muscle mRNA and protein expression associated with both protein synthesis and breakdown. After 10 weeks of spinal cord transection, soleus muscle from 12 rats (6 sham-control, 6 paraplegic) was studied for mRNAs and proteins associated with protein synthesis and breakdown using real-time polymerase chain reaction and immunoblotting techniques. Protein kinase B (PKB/Akt), ribosomal S6 kinase 1 (S6K1), and myogenin mRNA were downregulated, whereas muscle ring finger 1 (MuRF1) and phospho-forkhead transcription factor 4 (FoxO4) protein were increased in paraplegic rats. We conclude that gene and protein expression of pathways associated with protein synthesis are reduced, whereas some markers of protein breakdown remain elevated following chronic paraplegia. Clinical interventions designed to increase muscle protein synthesis may be helpful in preventing excessive muscle loss during long-term paraplegia.
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Project description:BackgroundSkeletal muscle mass is controlled by myostatin and Akt-dependent signaling on mammalian target of rapamycin (mTOR), glycogen synthase kinase 3? (GSK3?) and forkhead box O (FoxO) pathways, but it is unknown how these pathways are regulated in critically ill human muscle. To describe factors involved in muscle mass regulation, we investigated the phosphorylation and expression of key factors in these protein synthesis and breakdown signaling pathways in thigh skeletal muscle of critically ill intensive care unit (ICU) patients compared with healthy controls.Methodology/principal findingsICU patients were systemically inflamed, moderately hyperglycemic, received insulin therapy, and showed a tendency to lower plasma branched chain amino acids compared with controls. Using Western blotting we measured Akt, GSK3?, mTOR, ribosomal protein S6 kinase (S6k), eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), and muscle ring finger protein 1 (MuRF1); and by RT-PCR we determined mRNA expression of, among others, insulin-like growth factor 1 (IGF-1), FoxO 1, 3 and 4, atrogin1, MuRF1, interleukin-6 (IL-6), tumor necrosis factor ? (TNF-?) and myostatin. Unexpectedly, in critically ill ICU patients Akt-mTOR-S6k signaling was substantially higher compared with controls. FoxO1 mRNA was higher in patients, whereas FoxO3, atrogin1 and myostatin mRNAs and MuRF1 protein were lower compared with controls. A moderate correlation (r2=0.36, p<0.05) between insulin infusion dose and phosphorylated Akt was demonstrated.Conclusions/significanceWe present for the first time muscle protein turnover signaling in critically ill ICU patients, and we show signaling pathway activity towards a stimulation of muscle protein synthesis and a somewhat inhibited proteolysis.
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Project description:Intracellular signaling exhibits circadian variation in the suprachiasmatic nucleus and liver. However, it is unclear whether circadian regulation also extends to intracellular signaling pathways in the cardiac and skeletal muscles. Here, we examined circadian variation in the intracellular mammalian target of rapamycin (mTOR)/70 kDa ribosomal protein S6 kinase 1 (p70S6K) and extracellular signal-regulated kinase (ERK) pathways, which regulate protein synthesis in rat cardiac and skeletal muscles. Seven-week-old male Wistar rats were assigned to six groups: Zeitgeber time (ZT) 2, ZT6, ZT10, ZT14, ZT18, and ZT22 (ZT0, lights on; ZT12, lights off). The cardiac, plantaris, and soleus muscles were removed after a 12-h fasting period, and signal transducers involved in protein synthesis (mTOR, p70S6K, and ERK) were analyzed by western blotting. Circadian rhythms of signal transducers were observed in both cardiac (mTOR, p70S6K, and ERK) and plantaris (p70S6K and ERK) muscles (p<0.05), but not in the soleus muscle. In the cardiac muscle, the phosphorylation rate of mTOR was significantly higher at ZT6 (peak) than at ZT18 (bottom), and the phosphorylation rate of p70S6K was significantly higher at ZT2 (peak) than at ZT18 (bottom). In contrast, in the plantaris muscle, the phosphorylation rate of ERK was significantly lower at ZT2 (bottom) than at ZT18 (peak). Our data suggested that protein synthesis via mTOR/p70S6K and ERK signaling molecules exhibits circadian variation in rat cardiac and fast-type plantaris muscles.
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Project description:Background/aimsMethodological challenges have been associated with the dynamic measurement of muscle protein breakdown (MPB), as have the measurement of both muscle protein synthesis (MPS) and MPB within the same experiment. Our aim was to use the transmethylation properties of methionine as proof-of-concept to measure rates of MPB via its methylation of histidine within skeletal muscle myofibrillar proteins, whilst simultaneously utilising methionine incorporation into bound protein to measure MPS.ResultsDuring the synthesis measurement period, incorporation of methyl[D3]-13C-methionine into cellular protein in C2C12 myotubes was observed (representative of MPS), alongside an increase in the appearance of methyl[D3]-methylhistidine into the media following methylation of histidine (representative of MPB). For further validation of this approach, fractional synthetic rates (FSR) of muscle protein were increased following treatment of the cells with the anabolic factors insulin-like growth factor-1 (IGF-1) and insulin, while dexamethasone expectedly reduced MPS. Conversely, rates of MPB were reduced with IGF-1 and insulin treatments, whereas dexamethasone accelerated MPB.ConclusionsThis is a novel stable isotope tracer approach that permits the dual assessment of muscle cellular protein synthesis and breakdown rates, through the provision of a single methionine amino acid tracer that could be utilised in a wide range of biological settings.
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Project description:IntroductionFuel sources for skeletal muscle tissue include carbohydrates and fatty acids, and utilization depends upon fiber type, workload, and substrate availability. The use of isotopically labeled substrate tracers combined with nuclear magnetic resonance (NMR) enables a deeper examination of not only utilization of substrates by a given tissue, but also their contribution to tricarboxylic acid (TCA) cycle intermediates.ObjectivesThe goal of this study was to determine the differential utilization of substrates in isolated murine skeletal muscle, and to evaluate how isopotomer anlaysis provided insight into skeletal muscle metabolism.MethodsIsolated C57BL/6 mouse hind limb muscles were incubated in oxygenated solution containing uniformly labeled 13C6 glucose, 13C3 pyruvate, or 13C2 acetate at room temperature. Isotopomer analysis of 13C labeled glutamate was performed on pooled extracts of isolated soleus and extensor digitorum longus (EDL) muscles.ResultsPyruvate and acetate were more avidly consumed than glucose with resultant increases in glutamate labeling in both muscle groups. Glucose incubation resulted in glutamate labeling, but with high anaplerotic flux in contrast to the labeling by pyruvate. Muscle fiber type distinctions were evident by differences in lactate enrichment and extent of substrate oxidation.ConclusionIsotope tracing experiments in isolated muscles reveal that pyruvate and acetate are avidly oxidized by isolated soleus and EDL muscles, whereas glucose labeling of glutamate is accompanied by high anaplerotic flux. We believe our results may set the stage for future examination of metabolic signatures of skeletal muscles from pre-clinical models of aging, type-2 diabetes and neuromuscular disease.
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Project description:Glucocorticoids are frequently used as anti-inflammatory and immunosuppressive agents. However, high doses and/or prolonged use induce undesired secondary effects such as muscular atrophy. Recently, de novo expression of connexin43 and connexin45 hemichannels (Cx43 HCs and Cx45 HCs, respectively) has been proposed to play a critical role in the mechanism underlying myofiber atrophy induced by dexamethasone (Dex: a synthetic glucocorticoid), but their involvement in specific muscle changes promoted by Dex remains poorly understood. Moreover, treatments that could prevent the undesired effects of glucocorticoids on skeletal muscles remain unknown. In the present work, a 7-day Dex treatment in adult mice was found to induce weight loss and skeletal muscle changes including expression of functional Cx43/Cx45 HCs, elevated atrogin immunoreactivity, atrophy, oxidative stress and mitochondrial dysfunction. All these undesired effects were absent in muscles of mice simultaneously treated with Dex and vitamin E (VitE). Moreover, VitE was found to rapidly inhibit the activity of Cx HCs in freshly isolated myofibers of Dex treated mice. Exposure to alkaline pH induced free radical generation only in HeLa cells expressing Cx43 or Cx45 where Ca2+ was present in the extracellular milieu, response that was prevented by VitE. Besides, VitE and two other anti-oxidant compounds, Tempol and Resveratrol, were found to inhibit Cx43 HCs in HeLa cells transfectants. Thus, we propose that in addition to their intrinsic anti-oxidant potency, some antioxidants could be used to reduce expression and/or opening of Cx HCs and consequently reduce the undesired effect of glucocorticoids on skeletal muscles.
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