Eduardo C. Lira

Clenbuterol suppresses proteasomal and lysosomal proteolysis and atrophy-related genes in denervated rat soleus muscles independently of Akt

Although it is well known that administration of the selective β(2)-adrenergic agonist clenbuterol (CB) protects muscle following denervation (DEN), the underlying molecular mechanism remains unclear. We report that in vivo treatment with CB (3 mg/kg sc) for 3 days induces antiproteolytic effects in normal and denervated rat soleus muscle via distinct mechanisms. In normal soleus muscle, CB treatment stimulates protein synthesis, inhibits Ca(2+)-dependent proteolysis, and increases the levels of calpastatin protein. On the other hand, the administration of CB to DEN rats ameliorates the loss of muscle mass, enhances the rate of protein synthesis, attenuates hyperactivation of proteasomal and lysosomal proteolysis, and suppresses the transcription of the lysosomal protease cathepsin L and of atrogin-1/MAFbx and MuRF1, two ubiquitin (Ub) ligases involved in muscle atrophy. These effects were not associated with alterations in either IGF-I content or Akt phosphorylation levels. In isolated muscles, CB (10(-6) M) treatment significantly attenuated DEN-induced overall proteolysis and upregulation in the mRNA levels of the Ub ligases. Similar responses were observed in denervated muscles exposed to 6-BNZ-cAMP (500 μM), a PKA activator. The in vitro addition of triciribine (10 μM), a selective Akt inhibitor, did not block the inhibitory effects of CB on proteolysis and Ub ligase mRNA levels. These data indicate that short-term treatment with CB mitigates DEN-induced atrophy of the soleus muscle through the stimulation of protein synthesis, downregulation of cathepsin L and Ub ligases, and consequent inhibition of lysosomal and proteasomal activities and that these effects are independent of Akt and possibly mediated by the cAMP/PKA signaling pathway.

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

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.

Cyclic adenosine monophosphate-phosphodiesterase inhibitors reduce skeletal muscle protein catabolism in septic rats

ABSTRACT We have previously shown that catecholamines exert an inhibitory effect on muscle protein degradation through a pathway involving the cyclic adenosine monophosphate (cAMP) cascade in normal rats. In the present work, we investigated in vivo and in vitro effects of cAMP-phosphodiesterase inhibitors on protein metabolism in skeletal muscle from rats submitted to a model of acute sepsis. The in vivo muscle protein metabolism was evaluated indirectly by measurements of the tyrosine interstitial concentration using microdialysis. Muscle blood flow (MBF) was monitored by ethanol perfusion technique. Sepsis was induced by cecal ligation and puncture and resulted in lactate acidosis, hypotension, and reduction in MBF (−30%; P < 0.05). Three-hour septic rats showed an increase in muscle interstitial tyrosine concentration (˜150%), in arterial plasma tyrosine levels (˜50%), and in interstitial-arterial tyrosine concentration difference (˜200%; P < 0.05). Pentoxifylline (50 mg/kg of body weight, i.v.) infusion during 1 h after cecal ligation and puncture prevented the tumor necrosis factor &agr; increase and significantly reduced by 50% (P < 0.05) the interstitial-arterial tyrosine difference concentration. In situ perfusion with isobutylmethylxanthine (IBMX; 10−3 M) reduced by 40% (P< 0.05) the muscle interstitial tyrosine in both sham-operated and septic rats. Neither pentoxifylline nor IBMX altered MBF. The addition of IBMX (10−3 M) to the incubation medium increased (P < 0.05) muscle cAMP levels and reduced proteolysis in both groups. The in vitro addition of H89, a protein kinase A inhibitor, completely blocked the antiproteolytic effect of IBMX. The data show that activation of cAMP-dependent pathways and protein kinase A reduces muscle protein catabolism during basal and septic state.

Phosphodiesterase-4 inhibition reduces proteolysis and atrogenes expression in rat skeletal muscles.

Phosphodiesterase (PDE) inhibition reduces skeletal muscle atrophy, but the underlying molecular mechanism remains unclear. We used microdialysis to investigate the effects of different PDE inhibitors on interstitial tyrosine concentration as well as proteolytic activity and atrogenes expression in isolated rat muscle. Rolipram, a PDE‐4–selective inhibitor, reduced the interstitial tyrosine concentration and rates of muscle protein degradation. The rolipram‐induced muscle cAMP increase was accompanied by a decrease in ubiquitin–proteasome system (UPS) activity and atrogin‐1 mRNA, a ubiquitin‐ligase involved in muscle atrophy. This effect was not associated with Akt phosphorylation but was partially blocked by a protein kinase A inhibitor. Fasting increased atrogin‐1, MuRF‐1 and LC3b expression, and these effects were markedly suppressed by rolipram. Our data suggest that activation of cAMP signaling by PDE‐4 blockade leads to inhibition of UPS activity and atrogenes expression independently of Akt. These findings are important for identifying novel approaches to attenuate muscle atrophy. Muscle Nerve 44: 371–381, 2011

Epinephrine depletion exacerbates the fasting-induced protein breakdown in fast-twitch skeletal muscles

The physiological role of epinephrine in the regulation of skeletal muscle protein metabolism under fasting is unknown. We examined the effects of plasma epinephrine depletion, induced by adrenodemedullation (ADMX), on muscle protein metabolism in fed and 2-day-fasted rats. In fed rats, ADMX for 10 days reduced muscle mass, the cross-sectional area of extensor digitorum longus (EDL) muscle fibers, and the phosphorylation levels of Akt. In addition, ADMX led to a compensatory increase in muscle sympathetic activity, as estimated by the rate of norepinephrine turnover; this increase was accompanied by high rates of muscle protein synthesis. In fasted rats, ADMX exacerbated fasting-induced proteolysis in EDL but did not affect the low rates of protein synthesis. Accordingly, ADMX activated lysosomal proteolysis and further increased the activity of the ubiquitin (Ub)-proteasome system (UPS). Moreover, expression of the atrophy-related Ub ligases atrogin-1 and MuRF1 and the autophagy-related genes LC3b and GABARAPl1 were upregulated in EDL muscles from ADMX-fasted rats compared with sham-fasted rats, and ADMX reduced cAMP levels and increased fasting-induced Akt dephosphorylation. Unlike that observed for EDL muscles, soleus muscle proteolysis and Akt phosphorylation levels were not affected by ADMX. In isolated EDL, epinephrine reduced the basal UPS activity and suppressed overall proteolysis and atrogin-1 and MuRF1 induction following fasting. These data suggest that epinephrine released from the adrenal medulla inhibits fasting-induced protein breakdown in fast-twitch skeletal muscles, and these antiproteolytic effects on the UPS and lysosomal system are apparently mediated through a cAMP-Akt-dependent pathway, which suppresses ubiquitination and autophagy.

Tyrosine: A Possible Marker of Severe Intestinal Injury During Ischemia

BACKGROUND Long periods of ischemia can cause organ injury and dysfunction. The protein degradation occurring in the muscular layer and in the mucosa of the intestinal wall during ischemia may release amino acids into the intestinal lumen or into the circulation. The small intestine, like skeletal muscle, cannot synthesize or degrade tyrosine. Thus, the tyrosine concentration released from the gut mucosa reflects the balance between protein synthesis and degradation. We aimed to determine whether tyrosine can be used as a marker of intestinal injury during ischemia. METHODS In 19 anesthetized rabbits, an ultrasonic flow probe was placed around the superior mesenteric artery to estimate blood flow. A segment from the ileum was isolated using two multilumen catheters with inflated balloons to create a closed segment for perfusion. Animals were allocated into three groups: a sham group without intervention (group I); a group submitted to superior mesenteric artery ligation only (group II); and a group submitted to 1 h of SMA clamping followed by 1 h of reperfusion (group III). Concentrations of lactate and tyrosine (fluorometry) were determined in the serum and the gut luminal perfusate. RESULTS Gut luminal perfusate tyrosine concentrations increased significantly in group II (from 10 +/- 8 to 93 +/- 63 mm/mL at 2 h) and were significantly higher than in group I (26 +/- 24 mm/mL) and group III (11 +/- 13 mm/mL) (P < 0.05 for all). CONCLUSION Tyrosine is released from cells into the lumen during severe intestinal ischemia. Regional measurements of tyrosine levels may be a useful indicator of severe intestinal villus compromise.

Effect of Cyclosporine A on Glucose Interstitial Concentration in Renal Cortex and Medulla from Rats

Aim: To standardize microdialysis in rat kidneys and address cyclosporine A (CsA) effects on renal cortex and medulla interstitial glucose. Methods: Munich-Wistar rats were treated with vehicle or CsA (15 mg/kg/day) for 3 weeks. Glucose was assessed by spectrophotometry in dialysate samples from cortex, medulla and arterial plasma. Plasma insulin was measured by radioimmunoassay. Renal blood flow (RBF) was measured by Doppler ultrasound. Creatinine and urea were measured by spectrophotometry. Results: CsA significantly increased the plasma levels of urea and creatinine (1.5 ± 0.20 vs. 0.73 ± 0.03 mg/dl in controls, p < 0.05). Medullary glucose in control was 44% lower than arterial glucose (56 ± 6 vs. 101 ± 8 mg/dl, p < 0.05). At the same time, CsA increased arterial (163 ± 35 vs. 101 ± 8 mg/dl in controls, p < 0.05) and medullary interstitial glucose (100 ± 18 vs. 56 ± 6 mg/dl in controls, p < 0.05), but did not affect cortical glucose (114 ± 21 vs. 90 ± 11 mg/dl in controls). These changes occurred in the presence of a decreased plasma insulin level (2.7 ± 0.2 vs. 9.3 ± 0.4 µU/ml in controls, p < 0.05). The increment in medullary glucose in CsA group occurred despite a reduction in RBF (4.6 ± 0.8 vs. 6.5 ± 1.0 ml/min/kidney in controls, p < 0.05). Conclusions: Microdialysis was an adequate tool to investigate in vivo regulation of renal glucose metabolism. Renal glucose uptake was dependent on medullary cells and CsA treatment induced diabetogenic effects on renal medulla in situ.