Luiz C. C. Navegantes

Adrenergic control of protein metabolism in skeletal muscle.

This review summarizes evidence indicating that the sympathetic nervous system, through hormonal and neurotransmitter actions, produces anabolic, protein-sparing effects on skeletal muscle protein metabolism. Studies are reviewed which indicate that catecholamines secreted by the adrenal medulla have an inhibitory effect on muscle Ca2+-dependent protein degradation independently of other hormones. In addition, norepinephrine released from adrenergic terminals may increase the rate of protein synthesis in oxidative muscles, leading to increased protein accretion. Evidence is also presented that these effects seem to be mediated by β2-adrenoceptors and cyclic adenosine monophosphate-dependent pathways. The understanding of the precise mechanisms by which endogenous catecholamines promote muscle anabolic effects may bring new perspectives for efficient treatment of muscle-wasting conditions and enhancement of growth efficacy in farm species.

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.

Involvement of cAMP/Epac/PI3K-dependent pathway in the antiproteolytic effect of epinephrine on rat skeletal muscle.

Very little is known about the signaling pathways by which catecholamines exert anabolic effects on muscle protein metabolism, stimulating protein synthesis and suppressing proteolysis. The present work tested the hypothesis that epinephrine-induced inhibition of muscle proteolysis is mediated through the cAMP/Epac/PI3K-dependent pathway with the involvement of AKT and Foxo. The incubation of extensor digitorum longus (EDL) muscles from rats with epinephrine and/or insulin increased the phosphorylation of AKT and its downstream target Foxo3a, a well-known effect that prevents Foxo translocation to the nucleus and the activation of proteolysis. Similar effects on AKT/Foxo signaling were observed in muscles incubated with DBcAMP (cAMP analog). The stimulatory effect of epinephrine on AKT phosphorylation was completely blocked by wortmannin (selective PI3K inhibitor), suggesting that the epinephrine-induced activation of AKT is mediated through PI3K. As for epinephrine and DBcAMP, the incubation of muscles with 8CPT-2Me-cAMP (selective Epac agonist) reduced rates of proteolysis and increased phosphorylation levels of AKT and Foxo3a. The specific PKA agonist (N6BZ-cAMP) inhibited proteolysis and abolished the epinephrine-induced AKT and Foxo3a phosphorylation. On the other hand, inhibition of PKA by H89 further increased the phosphorylation levels of AKT and Foxo3a induced by epinephrine, DBcAMP or 8CPT-2Me-cAMP. These findings suggest that the antiproteolytic effect of the epinephrine on isolated skeletal muscle may occur through a cAMP/Epac/PI3K-dependent pathway, which leads to the phosphorylation of AKT and Foxo3a. The parallel activation of PKA-dependent pathway also inhibits proteolysis and seems to limit the stimulatory effect of cAMP on AKT/Foxo3a signaling.

Low protein diet changes the energetic balance and sympathetic activity in brown adipose tissue of growing rats.

OBJECTIVE The aim of this study was to assess the effects of protein restriction in growing rats. METHODS Rats (approximate weight, 100g) were maintained with low-protein (LP; 6%) or normoproteic (control; 17%) diets, and at the end of the 15th day, hormonal and biochemistry parameters and energetic balance were evaluated. Data were analyzed using Students t test (with statistical significance set at P < or = .05). RESULTS LP animals were hyperphagic and showed increased energetic gain (24%) and energy expenditure (EE) compared with controls. The increase in EE was followed by increased sympathetic activity in brown adipose tissue, evidenced by increased norepinephrine turnover, suggesting increased thermogenesis. In spite of hyperphagia, protein ingestion in LP animals was lower than that of controls (P<0.01). The LP diet impaired body growth and caused deep alterations in body chemical composition, with an increase in carcass lipid content (64%) and reductions of protein and water. In LP animals, postprandial glycemia was unchanged, and insulinemia was lower than in controls (P < or = .01). Reduction in fasting glycemia without changes in insulinemia also was detected (P < .01), suggesting increased insulin sensitivity. The LP diet caused a 100% increase in serum leptin (P < .01). CONCLUSIONS Protein restriction led to an increase in EE, with probable activation of thermogenesis in brown adipose tissue, evidenced by an increase in catecholamines levels. Despite the higher EE, energetic gain and lipids increased. The high level of leptin associated with hyperphagia led to the supposition that these animals are leptin resistant, and the increase in insulin sensitivity, suggested by the relation between insulin and glycemia in fasting and fed animals, might contribute to lipid accumulation.

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.

Effect of guanethidine-induced adrenergic blockade on the different proteolytic systems in rat skeletal muscle

Overall proteolysis and the activity of skeletal muscle proteolytic systems were investigated in rats submitted to guanethidine-induced adrenergic blockade for 4 days. In soleus, overall proteolysis increased by 15-20% during the first 2 days of guanethidine treatment but decreased to levels below control values after 4 days. Extensor digitorum longus (EDL) did not show the initial increase in total proteolysis, which was already reduced after 2 days of guanethidine treatment. The initial rise in the rate of protein degradation in soleus was accompanied by an increased activity of the Ca(2+)-dependent proteolytic pathway. In both soleus and EDL, the reduction in overall proteolysis was paralleled by decreased activities of the Ca(2+)-dependent and ATP-dependent proteolytic processes. No change was observed in the activity of the lysosomal proteolytic system. Overall proteolysis in soleus and EDL from nontreated rats was partially inhibited by isoproterenol, in vitro. The data suggest an acute inhibitory control of skeletal muscle proteolysis by the adrenergic system, well evident in the oxidative muscle, with an important participation of the Ca(2+)-dependent pathway.

Insulin Suppresses Atrophy- and Autophagy-related Genes in Heart Tissue and Cardiomyocytes Through AKT/FOXO Signaling

Insulin is an important regulator of the ubiquitin-proteasome system (UPS) and of lysosomal proteolysis in cardiac muscle. However, the role of insulin in the regulation of the muscle atrophy-related Ub-ligases atrogin-1 and MuRF1 as well as in autophagy, a major adaptive response to nutritional stress, in the heart has not been characterized. We report here that acute insulin deficiency in the cardiac muscle of rats induced by streptozotocin increased the expression of atrogin-1 and MuRF1 as well as LC3 and Gabarapl1, 2 autophagy-related genes. These effects were associated with decreased phosphorylation levels of Akt and its downstream target Foxo3a; this phenomenon is a well-known effect that permits the maintenance of Foxo in the nucleus to activate protein degradation by proteasomal and autophagic processes. The administration of insulin increased Akt and Foxo3a phosphorylation and suppressed the diabetes-induced expression of Ub-ligases and autophagy-related genes. In cultured neonatal rat cardiomyocytes, nutritional stress induced by serum/glucose deprivation strongly increased the expression of Ub-ligases and autophagy-related genes; this effect was inhibited by insulin. Furthermore, the addition of insulin in vitro prevented the decrease in Akt/Foxo signaling induced by nutritional stress. These findings demonstrate that insulin suppresses atrophy- and autophagy-related genes in heart tissue and cardiomyocytes, most likely through the phosphorylation of Akt and the inactivation of Foxo3a.

Sympathetic innervation controls homeostasis of neuromuscular junctions in health and disease

Significance The sympathetic nervous system regulates basic body functions such as heartbeat, blood pressure, and gland activities. Whereas hormone secretion from the adrenal medulla modulates these processes systemically, local and fast responses can be mediated by direct sympathetic innervation. Although many effects of the sympathetic system on skeletal muscle physiology and disease are known, direct sympathetic innervation targets in skeletal muscle have been scarcely studied. We investigated this aspect and found that neuromuscular junctions, the contact points between motor neurons and muscle fibers, are innervated by sympathetic neurons, which is of crucial importance for the integrity and function of nerve–muscle contact. Our findings help to understand and refine treatment of neuromuscular diseases, including myasthenic syndromes. The distribution and function of sympathetic innervation in skeletal muscle have largely remained elusive. Here we demonstrate that sympathetic neurons make close contact with neuromuscular junctions and form a network in skeletal muscle that may functionally couple different targets including blood vessels, motor neurons, and muscle fibers. Direct stimulation of sympathetic neurons led to activation of muscle postsynaptic β2-adrenoreceptor (ADRB2), cAMP production, and import of the transcriptional coactivator peroxisome proliferator-activated receptor γ-coactivator 1α (PPARGC1A) into myonuclei. Electrophysiological and morphological deficits of neuromuscular junctions upon sympathectomy and in myasthenic mice were rescued by sympathicomimetic treatment. In conclusion, this study identifies the neuromuscular junction as a target of the sympathetic nervous system and shows that sympathetic input is crucial for synapse maintenance and function.

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.

A low-protein, high-carbohydrate diet increases the adipose lipid content without increasing the glycerol-3-phosphate or fatty acid content in growing rats

The amount of triacylglycerol (TAG) that accumulates in adipose tissue depends on 2 opposing processes: lipogenesis and lipolysis. We have previously shown that the weight and lipid content of epididymal (EPI) adipose tissue increases in growing rats fed a low-protein, high-carbohydrate (LPHC) diet for 15 days. The aim of this work was to study the pathways involved in lipogenesis and lipolysis, which ultimately regulate lipid accumulation in the tissue. De novo fatty acid synthesis was evaluated in vivo and was similar for rats fed an LPHC diet or a control diet; however, the LPHC-fed rats had decreased lipoprotein lipase activity in the EPI adipose tissue, which suggests that there was a decreased uptake of fatty acids from the circulating lipoproteins. The LPHC diet did not affect synthesis of glycerol-3-phosphate (G3P) via glycolysis or glyceroneogenesis. Glycerokinase activity - i.e., the phosphorylation of glycerol from the hydrolysis of endogenous TAG to form G3P - was also not affected in LPHC-fed rats. In contrast, adipocytes from LPHC animals had a reduced lipolytic response when stimulated by norepinephrine, even though the basal adipocyte lipolytic rate was similar for both of the groups. Thus, the results suggest that the reduction of lipolytic activity stimulated by norepinephrine seems essential for the TAG increase observed in the EPI adipose tissue of LPHC animals, probably by impairment of the process of activation of lipolysis by norepinephrine.