Mechanisms Involved in 3′,5′-Cyclic Adenosine Monophosphate-Mediated Inhibition of the Ubiquitin-Proteasome System in Skeletal Muscle

Abstract

Although it is well known that catecholamines inhibit skeletal muscle protein degradation, the molecular underlying mechanism remains unclear. This study was undertaken to investigate the role of beta(2)-adrenoceptors (AR) and cAMP in regulating the ubiquitin-proteasome system (UPS) in skeletal muscle. We report that increased levels of cAMP in isolated muscles, promoted by the cAMP phosphodiesterase inhibitor isobutylmethylxanthine was accompanied by decreased activity of the UPS, levels of ubiquitin-protein conjugates, and expression of atrogin-1, a key ubiquitin-protein ligase involved in muscle atrophy. In cultured myotubes, atrogin-1 induction after dexamethasone treatment was completely prevented by isobutylmethylxanthine. Furthermore, administration of clenbuterol, a selective beta(2)-agonist, to mice increased muscle cAMP levels and suppressed the fasting-induced expression of atrogin-1 and MuRF-1, atrogin-1 mRNA being much more responsive to clenbuterol. Moreover, clenbuterol increased the phosphorylation of muscle Akt and Foxo3a in fasted rats. Similar responses were observed in muscles exposed to dibutyryl-cAMP. The stimulatory effect of clenbuterol on cAMP and Akt was abolished in muscles from beta(2)-AR knockout mice. The suppressive effect of beta(2)-agonist on atrogin-1 was not mediated by PGC-1alpha (peroxisome proliferator-activated receptor-gamma coactivator 1alpha known to be induced by beta(2)-agonists and previously shown to inhibit atrogin-1 expression), because food-deprived PGC-1alpha knockout mice were still sensitive to clenbuterol. These findings suggest that the cAMP increase induced by stimulation of beta(2)-AR in skeletal muscles from fasted mice is possibly the mechanism by which catecholamines suppress atrogin-1 and the UPS, this effect being mediated via phosphorylation of Akt and thus inactivation of Foxo3.

Clenbuterol, a β2-adrenergic agonist, reciprocally alters PGC-1 alpha and RIP140 and reduces fatty acid and pyruvate oxidation in rat skeletal muscle

“…In contrast to our observations, a recent report indicated that clenbuterol increased PGC-1␣ mRNA in skeletal muscles (19,47). Given that the long-term effect of clenbuterol is to transform skeletal muscle to a more glycolytic phenotype (14,35,46,53,58), these PGC-1␣ mRNA responses to ␤2-adrenergic agonist treatment are perplexing and may reflect an acute stress response that activates AMP-activated protein kinase (33), which can upregulate PGC-1␣ (24,39,63).…”

“…Given that the long-term effect of clenbuterol is to transform skeletal muscle to a more glycolytic phenotype (14,35,46,53,58), these PGC-1␣ mRNA responses to ␤2-adrenergic agonist treatment are perplexing and may reflect an acute stress response that activates AMP-activated protein kinase (33), which can upregulate PGC-1␣ (24,39,63). In contrast to these unusual responses of clenbuterol treatment with respect to changes in PGC-1␣ mRNA (19,47), our present results are consistent with studies showing that in skeletal muscle, clenbuterol decreases mitochondrial enzyme activity and contents and increases glycolytic enzyme activity (present study; 35,49,53), and our observed reduction in PGC-1␣ protein and mRNA is fully consistent with such changes. Also, long-term clenbuterol administration decreased ␤-adrenergic receptor concentration in skeletal muscle (64,65), effects that may inhibit the transcription of PGC-1␣, thereby contributing to reduced mitochondrial content and capacity in skeletal muscle.…”

cAMP signaling in skeletal muscle adaptation: hypertrophy, metabolism, and regeneration

“…Consistently, clenbuterol potently suppresses both MuRF1 and MAFbx/Atrogin-1 transcription in normal muscle and attenuates expression of these genes in atrophying muscle in rats (127). In addition, cAMP signaling activates Akt and represses ubiquitin-proteasome pathway activity in C 2 C 12 cells and rat skeletal muscle (73). It is tempting to speculate that FoxO transcription factors might mediate the activity of ␤-AR on Murf1 and Mafbx/Atrogin-1; indeed, FoxO3A phosphorylation correlates with clenbuterol-induced Akt phosphorylation in vivo in muscle from fasted animals (73).…”

“…3). Additionally, ␤-adrenergic agonists blunt muscle atrophy after denervation (253), muscular dystrophy (192), or nutritional deficiency (73). At the cellular level, clenbuterol has been shown to stimulate total protein synthesis (57,154) and translational efficiency (155) as well as reduce proteolysis in rabbit, rat, and chick skeletal muscles (18,63,190).…”

“…In addition, cAMP signaling activates Akt and represses ubiquitin-proteasome pathway activity in C 2 C 12 cells and rat skeletal muscle (73). It is tempting to speculate that FoxO transcription factors might mediate the activity of ␤-AR on Murf1 and Mafbx/Atrogin-1; indeed, FoxO3A phosphorylation correlates with clenbuterol-induced Akt phosphorylation in vivo in muscle from fasted animals (73). FoxO1 phosphorylation has not been explored in this model, and much uncertainty remains regarding the mechanisms by which ␤ 2 -AR agonists modulate ubiquitin-mediated protein degradation in skeletal muscle.…”