Effect of sodium 4-phenylbutyrate on Clenbuterol-mediated muscle growth.

David M Brown,Madelaine C Brearley,Tim Parr,Francis J P Ebling,Zoe C T R Daniel,John M Brameld,Jo E Lewis,Sarah Jones,Ashok Kumar

doi:10.1371/journal.pone.0201481

David M Brown, Madelaine C Brearley + Show 7 more

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https://doi.org/10.1371/journal.pone.0201481

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Journal: PloS one	Publication Date: Jul 27, 2018
Citations: 12	License type: CC BY 4.0

Affiliation: University of Nottingham

Abstract

Previously, we highlighted induction of an integrated stress response (ISR) gene program in skeletal muscle of pigs treated with a beta-adrenergic agonist. Hence we tested the hypothesis that the ER-stress inhibitor, sodium 4-phenylbutyrate (PBA), would inhibit Clenbuterol-mediated muscle growth and reduce expression of genes that are known indicators of an ISR in mice. Clenbuterol (1mg/kg/day) administered to C57BL6/J mice for 21 days increased body weight (p<0.001), muscle weights (p<0.01), and muscle fibre diameters (p<0.05). Co-administration of PBA (100mg/kg/day) did not alter the Clenbuterol-mediated phenotype, nor did PBA alone have any effects compared to that of the vehicle treated mice. Clenbuterol increased skeletal muscle mRNA expression of phosphoserine amino transferase 1 (PSAT1, p<0.001) and cyclophillin A (p<0.01) at day 3, but not day 7. Clenbuterol decreased mRNA expression of activating transcription factor (ATF) 4 and ATF5 at day 3 (p<0.05) and day 7 (p<0.01), X-box binding protein 1 (XBP1) variant 2 mRNA at day 3 only (p<0.01) and DNA damage inducible transcript 3 (DDIT3/CHOP) mRNA at day 7 only (p<0.05). Co-administration of PBA had no effect on Clenbuterol-induced changes in skeletal muscle gene expression. In contrast, treatment of C2C12 myotubes with 5mM PBA (8hr) attenuated the thapsigargin-induced ISR gene program. Prolonged (24-48hr) treatment with PBA caused atrophy (p<0.01), reduced neoprotein synthesis (p<0.0001) and decreased expression of myogenin and fast myosin heavy chain genes (p<0.01), indicating an inhibition of myogenic differentiation. In summary, Clenbuterol did not induce an ISR gene program in mouse muscle. On the contrary, it reduced expression of a number of ISR genes, but it increased expression of PSAT1 mRNA. Co-administration of PBA had no effect on Clenbuterol-mediated muscle growth or gene expression in mice, whereas PBA did inhibit thapsigargin-induced ISR gene expression in cultured C2C12 cells and appeared to inhibit myogenic differentiation, independent of altering ISR gene expression.

Highlights

Retention of lean body mass is largely achieved by maintaining rates of skeletal muscle protein synthesis and degradation in equilibrium [1]
We recently demonstrated induction of an integrated stress response (ISR) gene program in skeletal muscle of pigs treated with a beta-adrenergic agonist (BA) [9], a potent class of muscle anabolic drug
Our previous work highlighted induction of an integrated stress response (ISR) gene program in skeletal muscle of pigs treated with a beta-adrenergic agonist [9]

Summary

Introduction

Retention of lean body mass is largely achieved by maintaining rates of skeletal muscle protein synthesis and degradation in equilibrium [1]. Depending on the severity of stress, the ISR leads to transcription of functionally related cohorts of ATF4 target genes, which attempt to restore cellular homeostasis or, if homeostasis cannot be resumed, initiate programmed cell death [8] Genes involved in this adaptive response regulate several aspects of protein metabolism, namely amino acid biosynthesis, protein translation, and amino acid transport. Treatment with a BA triggered a co-ordinated and temporal increase in expression of 20 + genes that are known transcriptional targets of ATF4, the best-characterized central effector of the ISR gene program The majority of these genes were involved in amino acid biosynthesis (Phgdh, Psat, Psph, Pck, Asns, Arg, Ass, Gpt2), tRNA charging (Sars, Tars, Gars, Wars, Iars, Aars), and amino acid transport (Slc3a2, Slc7a1, Slc5a1), as well as inhibition of cellular growth (Sesn and Cdkn1a). The identification of conserved mechanisms controlling skeletal muscle mass is required to develop novel therapeutic strategies against muscle wasting conditions, which remains a major unmet clinical need for several pathological conditions [29]

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