Comparison of protein synthesis and degradation in incubated and perfused muscle.
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ABSTRACT:
SUBMITTER: Clark AS
PROVIDER: S-EPMC1153139 | biostudies-other | 1983 Jun
REPOSITORIES: biostudies-other
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Project description:1. A method is described for perfusing the rat diaphragm muscle. 2. The following parameters were compared in both perfused and non-perfused incubated preparations: water content, sorbitol space, rate of lactate production, and the concentrations of tissue glucose, pyruvate, lactate, hexose phosphate intermediates, ATP and AMP. No significant differences were found. 3. Significant differences, however, were found on comparison of the tissue kept in vitro with the tissue in vivo. Immediately after removal of the tissue from the animal, the concentrations of the hexose phosphates and ATP were found to be much higher than after incubation or perfusion, and the concentrations of free glucose and of AMP were much lower, possibly indicating that the capacity for oxidative phosphorylation of glucose is impaired in vitro because of hypoxia.
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Project description:Several reports have shown that amino acid utilization via oxidation and gluconeogenesis is increased during exercise. The purpose of this study was to investigate whether these changes are accompanied by alterations in protein synthesis and degradation in the muscle of exercising rats. One group of rats was made in swim for 1h and then protein synthesis and protein degradation were measured in a perfused hemicorpus preparation. Protein synthesis was decreased and protein degradation was increased in exercised rats compared with sedentary control rats. Exercise also decreased amino acid incorporation by isolated polyribosomes from muscle. Measurement of several muscle proteinase activities demonstrated that exercise had no effect on alkaline proteinase or Ca2+-activated proteinase. However, the free (unbound) cathepsin D activity was elevated in muscle of exercised rats, whereas the total activity of catepsin D was unchanged. This increase in the proportion of free cathepsin D activity suggests that lysosomal enzymes may be involved in the increased protein degradation that was observed.
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Project description:Critical illness myopathy (CIM) is a debilitating common consequence of modern intensive care, characterized by severe muscle wasting, weakness and a decreased myosin/actin (M/A) ratio. Limb/trunk muscles are primarily affected by this myopathy while cranial nerve innervated muscles are spared or less affected, but the mechanisms underlying these muscle-specific differences remain unknown. In this time-resolved study, the cranial nerve innervated masseter muscle was studied in a unique experimental rat intensive care unit (ICU) model, where animals were exposed to sedation, neuromuscular blockade (NMB), mechanical ventilation, and immobilization for durations varying between 6 h and 14d. Gel electrophoresis, immunoblotting, RT-PCR and morphological staining techniques were used to analyze M/A ratios, myofiber size, synthesis and degradation of myofibrillar proteins, and levels of heat shock proteins (HSPs). Results obtained in the masseter muscle were compared with previous observations in experimental and clinical studies of limb muscles. Significant muscle-specific differences were observed, i.e., in the masseter, the decline in M/A ratio and muscle fiber size was small and delayed. Furthermore, transcriptional regulation of myosin and actin synthesis was maintained, and Akt phosphorylation was only briefly reduced. In studied degradation pathways, only mRNA, but not protein levels of MuRF1, atrogin-1 and the autophagy marker LC3b were activated by the ICU condition. The matrix metalloproteinase MMP-2 was inhibited and protective HSPs were up-regulated early. These results confirm that the cranial nerve innervated masticatory muscles is less affected by the ICU-stress response than limb muscles, in accordance with clinical observation in ICU patients with CIM, supporting the model' credibility as a valid CIM model.
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Project description:Increasing the extracellular pH over the range pH 7.4-8.9 stimulated protein synthesis by about 60% in the rat heart preparation anterogradely perfused in vitro. Protein degradation was inhibited by this pH increase. The magnitudes of the effects at pH 8.9 on protein synthesis and degradation were similar to those of high concentrations of insulin. Cardiac outputs were increased, as were cardiac phosphocreatine contents, indicating that the alterations in extracellular pH did not adversely affect the physiological viability of the preparation. ATP contents were unaltered. The creatine kinase equilibrium was used to assess the magnitude of the change in intracellular pH induced by these treatments. The increase in intracellular pH was about 0.2 for a 1-unit increase in extracellular pH. Thus small changes in intracellular pH have dramatic effects on cardiac protein turnover.
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Project description:BackgroundAnimal studies and clinical data support the interest of citrulline as a promising therapeutic for sarcopenia. Citrulline is known to stimulate muscle protein synthesis, but how it affects energy metabolism to support the highly energy-dependent protein synthesis machinery is poorly understood.MethodsHere, we used myotubes derived from primary culture of mouse myoblasts to study the effect of citrulline on both energy metabolism and protein synthesis under different limiting conditions.ResultsWhen serum/amino acid deficiency or energy stress (mild uncoupling) were applied, citrulline stimulated muscle protein synthesis by +22% and +11%, respectively. Importantly, this increase was not associated with enhanced energy status (ATP/ADP ratio) or mitochondrial respiration. We further analysed the share of mitochondrial respiration and thus of generated ATP allocated to different metabolic pathways by using specific inhibitors. Our results indicate that addition of citrulline allocated an increased share of mitochondrially generated ATP to the protein synthesis machinery under conditions of both serum/amino acid deficiency (+28%) and energy stress (+21%). This reallocation was not because of reduced ATP supply to DNA synthesis or activities of sodium and calcium cycling ion pumps.ConclusionsUnder certain stress conditions, citrulline increases muscle protein synthesis by specifically reallocating mitochondrial fuel to the protein synthesis machinery. Because ATP/ADP ratios and thus Gibbs free energy of ATP hydrolysis remained globally constant, this reallocation may be linked to decreased activation energies of one or several ATP (and GTP)-consuming reactions involved in muscle protein synthesis.
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Project description:We have previously shown that incubation for 1h with excess glucose or leucine causes insulin resistance in rat extensor digitorum longus (EDL) muscle by inhibiting AMP-activated protein kinase (AMPK). To examine the events that precede and follow these changes, studies were performed in rat EDL incubated with elevated levels of glucose or leucine for 30min-2h. Incubation in high glucose (25mM) or leucine (100?M) significantly diminished AMPK activity by 50% within 30min, with further decreases occurring at 1 and 2h. The initial decrease in activity at 30min coincided with a significant increase in muscle glycogen. The subsequent decreases at 1h were accompanied by phosphorylation of ?AMPK at Ser485/491, and at 2h by decreased SIRT1 expression and increased PP2A activity, all of which have previously been shown to diminish AMPK activity. Glucose infusion in vivo, which caused several fold increases in plasma glucose and insulin, produced similar changes but with different timing. Thus, the initial decrease in AMPK activity observed at 3h was associated with changes in Ser485/491 phosphorylation and SIRT1 expression and increased PP2A activity was a later event. These findings suggest that both ex vivo and in vivo, multiple factors contribute to fuel-induced decreases in AMPK activity in skeletal muscle and the insulin resistance that accompanies it.
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Project description:Disuse is a potent inducer of muscle atrophy, but the molecular mechanisms driving this loss of muscle mass are highly debated. In particular, the extent to which disuse triggers decreases in protein synthesis or increases in protein degradation, and whether these changes are uniform across muscles or influenced by age, is unclear. We aimed to determine the impact of disuse on protein synthesis and protein degradation in lower limb muscles of varied function and fiber type in adult and old rats. Alterations in protein synthesis and degradation were measured in the soleus, medial gastrocnemius, and tibialis anterior (TA) muscles of adult and old rats subjected to hindlimb unloading (HU) for 3, 7, or 14 days. Loss of muscle mass was progressive during the unloading period, but highly variable (-9 to -38%) across muscle types and between ages. Protein synthesis decreased significantly in all muscles, except for the old TA. Atrophy-associated gene expression was only loosely associated with protein degradation as muscle RING finger-1, muscle atrophy F-box (MAFbx), and Forkhead box O1 expression significantly increased in all muscles, but an increase in proteasome activity was only observed in the adult soleus. MAFbx protein levels were significantly higher in the old muscles compared with adult muscles, despite the old having higher expression of microRNA-23a. These results indicate that adult and old muscles respond similarly to HU, and the greatest loss in muscle mass occurs in predominantly slow-twitch extensor muscles due to a concomitant decrease in protein synthesis and increase in protein degradation.NEW & NOTEWORTHY In this study, we showed that age did not intensify the atrophy response to unloading in rats, but rather that the degree of atrophy was highly variable across muscles, indicating that changes in protein synthesis and protein degradation occur in a muscle-specific manner. Our data emphasize the importance of studying muscles of varying fiber-type and physiological function at multiple time points to fully understand the molecular mechanisms responsible for disuse atrophy.
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Project description:In eight anaesthetized post-absorptive dogs we measured the concentration and specific radioactivity of phenylalanine and leucine in arterial and femoral-venous plasma, together with hindlimb flow during a continuous infusion of L-[ring-2,6-3H]phenylalanine and [1-14C]leucine. The femoral-venous plasma concentration was greater than arterial for both phenylalanine and leucine (P less than 0.05 for each). Despite net amino acid release there was a significant removal of both labelled phenylalanine and labelled leucine. Consequently, a significant dilution of specific radioactivity was observed between artery and vein for both radio-tracers. The uptake of leucine from the arterial circulation by the hindlimb exceeded by 2.6-fold that of phenylalanine; the measured molar ratio of leucine to phenylalanine in hindlimb muscle protein averaged 2.4 +/- 0.1. Since phenylalanine is neither synthesized nor degraded by muscle tissue, the measured removal of tracer and the dilution of tracer specific radioactivity across the hindlimb can be used to estimate rates of phenylalanine incorporation into, and release from, tissue protein. The estimated rate of protein synthesis by hindlimb averaged 644 +/- 250 nmol of phenylalanine/min. This was exceeded by the rate of tissue protein degradation (987 +/- 285 nmol of phenylalanine/min). The present results demonstrate that the dilution of the specific radioactivity of labelled phenylalanine can be readily measured across dog hindlimb. This measurement, coupled with an estimate of tissue blood flow, can provide a readily measured, non-destructive, method for estimation of protein turnover in specific muscle beds in vivo. Measurements can be made repeatedly over time in a single experiment, allowing the study of factors which regulate protein turnover. The method developed here in dogs can be readily extended to clinical studies.
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