LEUCINE ATTENUATES SKELETAL MUSCLE WASTING VIA INHIBITION OF UBIQUITIN LIGASES IGOR L. BAPTISTA, PhD(Stud), 1 MARCELO L. LEAL, PhD(Stud), 1 GUILHERME G. ARTIOLI, PhD(Stud), 1 MARCELO S. AOKI, PhD, 2 JARLEI FIAMONCINI, PhD, 3 ANTONIO O. TURRI, 1 RUI CURI, PhD, 3 ELEN H. MIYABARA, PhD, 4 and ANSELMO S. MORISCOT, PhD 1 1 Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of Sa ˜o Paulo, Avenida Prof. Lineu Prestes, 1524, Butanta ˜, CEP 05508-900, Sa ˜o Paulo, Brazil 2 School of Arts, Sciences, and Humanities, University of Sa ˜o Paulo, Sa ˜o Paulo, Brazil 3 Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sa ˜o Paulo, Sa ˜o Paulo, Brazil 4 Department of Anatomy, Institute of Biomedical Sciences, University of Sa ˜o Paulo, Brazil Accepted 5 October 2009 ABSTRACT: The aim of this study was to assess the effect of leucine supplementation on elements of the ubiquitin–protea- some system (UPS) in rat skeletal muscle during immobiliza- tion. This effect was evaluated by submitting the animals to a leucine supplementation protocol during hindlimb immobiliza- tion, after which different parameters were determined, includ- ing: muscle mass; cross-sectional area (CSA); gene expression of E3 ligases/deubiquitinating enzymes; content of ubiquitinated proteins; and rate of protein synthesis. Our results show that leucine supplementation attenuates soleus muscle mass loss driven by immobilization. In addition, the marked decrease in the CSA in soleus muscle type I fibers, but not type II fibers, induced by immobilization was minimized by leucine feeding. Interestingly, leucine supplementation severely minimized the early transient increase in E3 ligase [muscle ring finger 1 (MuRF1) and muscle atrophy F-box (MAFbx)/atrogin-1] gene expression observed during immobilization. The reduced peak of E3 ligase gene expression was paralleled by a decreased content of ubiquitinated proteins during leucine feeding. The protein synthesis rate decreased by immobilization and was not affected by leucine supplementation. Our results strongly sug- gest that leucine supplementation attenuates muscle wasting induced by immobilization via minimizing gene expression of E3 ligases, which consequently could downregulate UPS-driven protein degradation. It is notable that leucine supplementation does not restore decreased protein synthesis driven by immobi- lization. Muscle Nerve 41: 800–808, 2010 Prolonged periods of skeletal muscle inactivity due to bed rest, denervation, hindlimb unloading, microgravity, or immobilization can result in con- siderable muscle wasting. The well-known func- tional and morphological consequences of muscle inactivity include decreased muscle fiber cross- sectional area (CSA) and protein content, reduced force and power output, increased fatigability, and increased insulin resistance. 1,2 However, the molec- ular mechanisms responsible for muscle wasting induced by disuse are still poorly understood. 3–5 Much attention has been given to intracellular pathways associated with muscle mass control such as Akt–mammalian target of rapamycin (Akt-mTOR), myostatin, and the ubiquitin-proteasome system (UPS). 5–8 Evidence suggests that the enhancement of proteolysis in atrophying muscles results mainly from a general activation of the UPS protein deg- radation. 4,9,10 In various types of atrophy, includ- ing Cushing’s syndrome, 11 diabetes, 12 sepsis, 13 can- cer cachexia, 14 and renal failure, 15 muscles exhibit a common series of adaptations, including increased content of ubiquitin (Ub)–protein con- jugates 16 and mRNA encoding Ub, 17 certain ubiq- uitination enzymes, 12 and multiple proteasome subunits. 15,18 The UPS is an adenosine triphosphate (ATP)- requiring multienzyme process that mediates pro- tein degradation by the proteasome. Briefly, prior to degradation, a target protein undergoes a three- step process which covalently links a polyubiquitin chain to the substrate. Three enzyme components are involved in this process, E1 (Ub-activating enzyme), E2 (Ub-conjugating enzymes), and E3 (ubiquitin protein ligase), which present substrate recognition sites. 19,20 The ubiquitinated substrate can then be recognized and degraded by the pro- teasome. In atrophy models, the genes atrogin-1, also called muscle atrophy F-box (MAFbx), and muscle ring finger 1 (MuRF1), encoding E3 ubiq- uitin ligases, are upregulated. 9,21 In addition, UPS activity is also influenced by a family of enzymes known as deubiquitinating enzymes (DUBs). 22 These enzymes can destabilize the covalent bond formed between polyubiquitin chain and sub- strate. 22 The role of DUBs in skeletal muscle plas- ticity is elusive; to date, only one study has deter- mined gene expression of DUBs during longitudinal skeletal muscle growth. 23 In addition to mechanical stimuli, certain essential amino acids (leucine in particular) also modulate protein turnover in skeletal muscle. 24,25 It is well known that leucine, per se, can promote Abbreviations: ANOVA, analysis of variance; ATP, adenosine triphos- phate; BSA, bovine serum albumin; CSA, cross-sectional area; CSA/BM, cross-sectional area/body mass; Ct, cycle threshold; dNTP, deoxynucleo- tide phosphate; DTT, dithiothreitol; DUB, deubiquitinating enzyme; ECL, electrochemiluminescence; EDTA, ethylene-diamine tetraacetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid; Im, immobi- lized; KHB, Krebs–Henseleit buffer; Leu, leucine; MAFbx, muscle atrophy F-box; MuRF1, muscle ring finger 1; PBS, phosphate-buffered saline; Rec, recovery; soleus M/BM, soleus mass/body mass; RT, reverse tran- scription; TCA, trichloroacetic acid; Ub, ubiquitin; SDS-PAGE, sodium dodecylsulfate–polyacrylamide gel electrophoresis; UPS, ubiquitin–protea- some system Correspondence to: A.S. Moriscot; e-mail: moriscot@usp.br V C 2010 Wiley Periodicals, Inc. Published online 15 January 2010 in Wiley InterScience (www. interscience.wiley.com). DOI 10.1002/mus.21578 Key words: atrophy; immobilization; leucine; soleus; ubiquitin–proteasome system 800 Leucine Attenuates Skeletal Muscle Wasting MUSCLE & NERVE June 2010