Effects of high doses of glucocorticoids on free amino acids, ribosomes and protein turnover in human muscle
Erland LöfbergA GutiérrezJan WernermanBjörn AnderstamWilliam E. MitchS. Russ PriceJonas BergströmAnders Alvestrand
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Abstract Background Treatment with glucocorticosteroids causes a negative nitrogen balance, but the kinetic mechanisms responsible for this catabolic effect are controversial. We investigated the effects of 60 mg day −1 prednisolone on protein synthesis and degradation in human skeletal muscle. Materials and methods Healthy adults ( n = 9) were studied in the postabsorptive state, before and after 3 days of prednisolone treatment. The L‐[ring 2,6 ‐3 H 5 ]‐phenylalanine tracer technique, concentration and size distribution of the ribosomes, mRNA content of the ubiquitin‐proteasome pathway components in muscle, phenylalanine flux across the leg, and the free amino acid concentrations in skeletal muscle were used to study muscle protein metabolism. Results The concentrations of most amino acids in arterial blood increased after prednisolone. There were also increased effluxes of phenylalanine, asparagine, arginine, alanine, methionine and isoleucine from the leg. The rate of protein degradation, as measured by the appearance rate (Ra) of phenylalanine, increased by 67% ( P = 0·023) which, together with a doubling of the net release of phenylalanine from the leg ( P = 0·007), indicated accelerated protein degradation. The pathway was not identified but there was no significant increase in mRNAs’ encoding components of the ubiquitin‐proteasome pathway. There was a 6% reduction in polyribosomes ( P = 0·007), suggesting a decrease in the capacity for protein synthesis, although there was no measured decrease in the rate of protein synthesis. Conclusions These findings indicate that high doses of prednisolone lead to a sharp increase in net protein catabolism, which depends more on enhanced protein breakdown, and an uncertain effect on protein synthesis. The mechanisms stimulating proteolysis and the pathway stimulated to increase muscle protein degradation should be explored.Keywords:
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The effects of thyroxine (T4) on protein turnover in skeletal muscle were studied using normal, thyroidectomized (thyrex), and hypophysectomized (hypox) rats. Thyrex rats had a depressed growth rate that was accompanied by 50% reductions in the level of RNA and the rate of protein synthesis in gastrocnemius muscle, as determined in the perfused hemicorpus. Protein synthetic efficiency (protein synthesis per unit RNA) was decreased by 18%. Daily treatment of thyrex rats with T4 at different dose levels for up to 16 days led to improved growth rates, elevated RNA concentrations, and increased protein synthesis rates. The primary effect of T4 was to increase the protein synthetic capacity of muscle. Protein degradation, determined in the perfused hemicorpus, and activity of a lysosomal protease, determined in unperfused muscle, were reduced in the thyrex condition. Treatment of thyrex rats with T4 increased protein degradative rates, but not protease activity. Hypox rats, which also exhibited depressed skeletal muscle protein synthesis, responded to T4 and combined T4 and growth hormone with marked improvements in protein synthesis.
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An activator as well as an inhibitor of protein synthesis, were isolated from rabbit reticulocyte ribosomes. The ribosomes of the erythrocytes are inactive in protein synthesis and lacked the activator. However, when the activator moiety was added to the erythrocyte ribosomes, there was a significant increase in their capacity for protein synthesis, provided these ribosomes were freed of their inhibitor by washing beforehand with a detergent.
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The inhibition of cell division and the ultimate loss of viability after removal of streptomycin from growing cultures of streptomycin-dependent bacteria are not the result of “unbalanced growth” or of the breakdown of ribosomes. The streptomycin-dependent strain of Escherichia coli K-12 studied continued to synthesize ribonucleic acid (RNA) and protein during streptomycin starvation. There was no evidence of a gross imbalance in the ratio of RNA to protein synthesized or of selective degradation of either protein or RNA. Using the sedimentation of subunits in sucrose as the criterion, normal ribosomes were synthesized even after 18 h of streptomycin deprivation, although the rates of appearance of mature 30 S and 50 S subunits decreased with time of deprivation. Once formed, these ribosomes appeared stable, as did those synthesized before the onset of starvation. Ribosomes isolated from starved dependent cells were as “functional” as ribosomes from cells grown with streptomycin in their capacity to bind aminoacyl-transfer RNA in response to polyuridylic acid or natural messenger RNA to interconvert between active and inactive transfer RNA binding states, and to synthesize proteins in cell-free systems. The effects are consistent with an impaired rate of synthesis of ribosomal components or assembly of ribosomes resulting in a continually diminishing rate of protein synthesis. The effect on cell division may be the result of a decreased rate of protein synthesis in general and the requirement for a specific protein(s) in particular.
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Abstract The level of protein synthetic activity in dark‐grown cultures of Verticillium agaricinum was significantly enhanced by light. As expected the enhancement of protein synthetic activity was accompanied by a transformation of cytoplasmic monoribosomes to polyribosomes. Amino acid incorporation studies utilizing the synthetic mRNA, poly (U), suggest that the transformation was preceded by an activation of pre‐existing ribosomes. The change in ribosome activity related, at least in part, to an increase in the level of peptidyl‐tRNA associated with the ribosomes. In this regard the response of V. agaricinum ribosomes was similar to ribosome activation in several higher plant systems. The initial response at the level of the ribosome remains to be elucidated.
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The function of protein L1 in protein biosynthesis has been examined using ribosomes from two independently derived mutants of Escherichia coli, both of which lacked this protein. In systems in vitro with phage MS2 RNA or poly(U) as message, the mutant ribosomes showed from 40% to 60% of the activity of wild-type ribosomes. The reduction in activity was apparent in the kinetics of [14C]phenylalanine incorporation throughout the incubation period. The activities were restored fully to the wild-type level by the addition of purified protein L1. These results show on the one hand that protein L1 is not essential for protein biosynthesis but, on the other, its presence can significantly increase the overall rate of this process. The data further indicate it as likely that protein L1 exerts its effect at the elongation step
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