Testosterone represses ubiquitin ligases atrogin-1 and Murf-1 expression in an androgen-sensitive rat skeletal muscle in vivo.
Skeletal muscle atrophy induced by denervation and metabolic diseases has been associated with increased ubiquitin ligase expression. In the present study, we evaluate the influence of androgens on muscle ubiquitin ligases atrogin-1/MAFbx/FBXO32 and Murf-1/Trim63 expression and its correlation with maintenance of muscle mass by using the testosterone-dependent fast-twitch levator ani muscle (LA) from normal or castrated adult male Wistar rats. Gene expression was determined by qRT-PCR and/or immunoblotting. Castration induced progressive loss of LA mass (30% of control, 90 days) and an exponential decrease of LA cytoplasm-to-nucleus ratio (nuclear domain; 22% of control after 60 days). Testosterone deprivation induced a 31-fold increase in LA atrogin-1 mRNA and an 18-fold increase in Murf-1 mRNA detected after 2 and 7 days of castration, respectively. Acute (24 h) testosterone administration fully repressed atrogin-1 and Murf-1 mRNA expression to control levels. Atrogin-1 protein was also increased by castration up to 170% after 30 days. Testosterone administration for 7 days restored atrogin-1 protein to control levels. In addition to the well known stimulus of protein synthesis, our results show that testosterone maintains muscle mass by repressing ubiquitin ligases, indicating that inhibition of ubiquitin-proteasome catabolic system is critical for trophic action of androgens in skeletal muscle. Besides, since neither castration nor androgen treatment had any effect on weight or ubiquitin ligases mRNA levels of extensor digitorum longus muscle, a fast-twitch muscle with low androgen sensitivity, our study shows that perineal muscle LA is a suitable in vivo model to evaluate regulation of muscle proteolysis, closely resembling human muscle responsiveness to androgens.
Pires-Oliveira
Marcelo
M
Dept. of Pharmacology, Universidade Federal de São Paulo, Rua Três de Maio, São Paulo, SP, Brazil 04044-020.
Maragno
Ana Leticia G C
AL
Parreiras-e-Silva
Lucas T
LT
Chiavegatti
Tiago
T
Gomes
Marcelo D
MD
Godinho
Rosely O
RO
D000728:Androgens / Q000172:deficiency / Q000502:physiology*; D000818:Animals; D015153:Blotting, Western; D008297:Male; D051379:Mice; D018613:Microscopy, Confocal; D018656:Muscle Fibers, Fast-Twitch / Q000502:physiology / Q000648:ultrastructure; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D009124:Muscle Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*; D018482:Muscle, Skeletal / Q000033:anatomy & histology / Q000187:drug effects / Q000378:metabolism*; D009919:Orchiectomy; D009929:Organ Size / Q000187:drug effects; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis / Q000302:isolation & purification; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D044843:SKP Cullin F-Box Protein Ligases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*; D013739:Testosterone / Q000494:pharmacology*; D000070584:Tripartite Motif Proteins; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*
D000728:Androgens; D009124:Muscle Proteins; D012333:RNA, Messenger; D000070584:Tripartite Motif Proteins; D025801:Ubiquitin; D013739:Testosterone; C486692:Fbxo32 protein, rat; D044843:SKP Cullin F-Box Protein Ligases; C499086:Trim63 protein, rat; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex