Maternal vitamin D deficiency affects the morphology and function of glycolytic muscle in adult offspring rats.

BACKGROUND: Fetal stage is a critical developmental window for the skeletal muscle, but little information is available about the impact of maternal vitamin D (Vit. D) deficiency (VDD) on offspring lean mass development in the adult life of male and female animals. METHODS: Female rats (Wistar Hannover) were fed either a control (1000 IU Vit. D3/kg) or a VDD diet (0 IU Vit. D3/kg) for 6 weeks and during gestation and lactation. At weaning, male and female offspring were randomly separated and received a standard diet up to 180 days old. RESULTS: Vitamin D deficiency induced muscle atrophy in the male (M-VDD) offspring at the end of weaning, an effect that was reverted along the time. Following 180 days, fast-twitch skeletal muscles [extensor digitorum longus (EDL)] from the M-VDD showed a decrease (20%; P < 0.05) in the number of total fibres but an increase in the cross-sectional area of IIB (17%; P < 0.05), IIA (19%; P < 0.05) and IIAX (21%; P < 0.05) fibres. The fibre hypertrophy was associated with the higher protein levels of MyoD (73%; P < 0.05) and myogenin (55% %; P < 0.05) and in the number of satellite cells (128.8 ± 14 vs. 91 ± 7.6 nuclei Pax7 + in the M-CTRL; P < 0.05). M-VDD increased time to fatigue during ex vivo contractions of EDL muscles and showed an increase in the phosphorylation levels of IGF-1/insulin receptor and their downstream targets related to anabolic processes and myogenic activation, including Ser 473 Akt and Ser 21/9 GSK-3ß. In such muscles, maternal VDD induced a compensatory increase in the content of calcitriol (two-fold; P < 0.05) and CYP27B1 (58%; P < 0.05), a metabolizing enzyme that converts calcidiol to calcitriol. Interestingly, most morphological and biochemical changes found in EDL were not observed in slow-twitch skeletal muscles (soleus) from the M-VDD group as well as in both EDL and soleus muscles from the female offspring. CONCLUSIONS: These data show that maternal VDD selectively affects the development of type-II muscle fibres in male offspring rats but not in female offspring rats and suggest that the enhancement of their size and fatigue resistance in fast-twitch skeletal muscle (EDL) is probably due to a compensatory increase in the muscle content of Vit. D in the adult age.

Urocortin 2 promotes hypertrophy and enhances skeletal muscle function through cAMP and insulin/IGF-1 signaling pathways.

OBJECTIVE: Although it is well established that urocortin 2 (Ucn2), a peptide member of the corticotrophin releasing factor (CRF) family, and its specific corticotrophin-releasing factor 2 receptor (CRF2R) are highly expressed in skeletal muscle, the role of this peptide in the regulation of skeletal muscle mass and protein metabolism remains elusive. METHODS: To elucidate the mechanisms how Ucn2 directly controls protein metabolism in skeletal muscles of normal mice, we carried out genetic tools, physiological and molecular analyses of muscles in vivo and in vitro. RESULTS: Here, we demonstrated that Ucn2 overexpression activated cAMP signaling and promoted an expressive muscle hypertrophy associated with higher rates of protein synthesis and activation of Akt/mTOR and ERK1/2 signaling pathways. Furthermore, Ucn2 induced a decrease in mRNA levels of atrogin-1 and in autophagic flux inferred by an increase in the protein content of LC3-I, LC3-II and p62. Accordingly, Ucn2 reduced both the transcriptional activity of FoxO in vivo and the overall protein degradation in vitro through an inhibition of lysosomal proteolytic activity. In addition, we demonstrated that Ucn2 induced a fast-to-slow fiber type shift and improved fatigue muscle resistance, an effect that was completely blocked in muscles co-transfected with mitogen-activated protein kinase phosphatase 1 (MKP-1), but not with dominant-negative Akt mutant (Aktmt). CONCLUSIONS: These data suggest that Ucn2 triggers an anabolic and anti-catabolic response in skeletal muscle of normal mice probably through the activation of cAMP cascade and participation of Akt and ERK1/2 signaling. These findings open new perspectives in the development of therapeutic strategies to cope with the loss of muscle mass.

α-Calcitonin gene-related peptide inhibits autophagy and calpain systems and maintains the stability of neuromuscular junction in denervated muscles.

OBJECTIVE: Although it is well established that a-calcitonin gene-related peptide (CGRP) stabilizes muscle-type cholinergic receptors nicotinic subunits (AChR), the underlying mechanism by which this neuropeptide regulates muscle protein metabolism and neuromuscular junction (NMJ) morphology is unclear. METHODS: To elucidate the mechanisms how CGRP controls NMJ stability in denervated mice skeletal muscles, we carried out physiological, pharmacological, and molecular analyses of atrophic muscles induced by sciatic nerve transection. RESULTS: Here, we report that CGRP treatment in vivo abrogated the deleterious effects on NMJ upon denervation (DEN), an effect that was associated with suppression of skeletal muscle proteolysis, but not stimulation of protein synthesis. CGRP also blocked the DEN-induced increase in endocytic AChR vesicles and the elevation of autophagosomes per NMJ area. The treatment of denervated animals with rapamycin blocked the stimulatory effects of CGRP on mTORC1 and its inhibitory actions on autophagic flux and NMJ degeneration. Furthermore, CGRP inhibited the DEN-induced hyperactivation of Ca2+-dependent proteolysis, a degradative system that has been shown to destabilize NMJ. Consistently, calpain was found to be activated by cholinergic stimulation in myotubes leading to the dispersal of AChR clusters, an effect that was abolished by CGRP. CONCLUSION: Taken together, these data suggest that the inhibitory effect of CGRP on autophagy and calpain may represent an important mechanism for the preservation of synapse morphology when degradative machinery is exacerbated upon denervation conditions.

Acute intermittent hypoxia in rats activates muscle proteolytic pathways through a gluccorticoid-dependent mechanism.

Although it is well known that chronic hypoxia induces muscle wasting, the effects of intermittent hypoxia on skeletal muscle protein metabolism remain unclear. We hypothesized that acute intermittent hypoxia (AIH), a challenge that activates the hypothalamic-pituitary-adrenal axis, would alter muscle protein homeostasis through a glucocorticoid-dependent mechanism. Three-week-old rats were submitted to adrenalectomy (ADX) and exposed to 8 h of AIH (6% O2 for 40 s at 9-min intervals). Animals were euthanized, and the soleus and extensor digitorum longus (EDL) muscles were harvested and incubated in vitro for measurements of protein turnover. AIH increased plasma levels of corticosterone and induced insulin resistance as estimated by the insulin tolerance test and lower rates of muscle glucose oxidation and the HOMA index. In both soleus and EDL muscles, rates of overall proteolysis increased after AIH. This rise was accompanied by an increased proteolytic activities of the ubiquitin(Ub)-proteasome system (UPS) and lysosomal and Ca2+-dependent pathways. Furthermore, AIH increased Ub-protein conjugates and gene expression of atrogin-1 and MuRF-1, two key Ub-protein ligases involved in muscle atrophy. In parallel, AIH increased the mRNA expression of the autophagy-related genes LC3b and GABARAPl1. In vitro rates of protein synthesis in skeletal muscles did not differ between AIH and control rats. ADX completely blocked the insulin resistance in hypoxic rats and the AIH-induced activation of proteolytic pathways and atrogene expression in both soleus and EDL muscles. These results demonstrate that AIH induces insulin resistance in association with activation of the UPS, the autophagic-lysosomal process, and Ca2+-dependent proteolysis through a glucocorticoid-dependent mechanism.NEW & NOTEWORTHY Since hypoxia is a condition in which the body is deprived of adequate oxygen supply and muscle wasting is induced, the present work provides evidence linking hypoxia to proteolysis through a glucocorticoid-dependent mechanism. We show that the activation of proteolytic pathways, atrophy-related genes, and insulin resistance in rats exposed to acute intermittent hypoxia was abolished by surgical removal of adrenal gland. This finding will be helpful for understanding of the muscle wasting in hypoxemic conditions.

Lipolytic response of adipose tissue and metabolic adaptations to long periods of fasting in red tilapia (Oreochromis sp., Teleostei: Cichlidae).

Adaptive changes of carbohydrate and lipid metabolism induced by 7, 15, 30, 60, 90, 150 and 200 days of fasting were investigated in red tilapia (Oreochromis sp.). Plasma glucose, lactate and free fatty acids (FFA) levels, liver and muscle glycogen and total lipid contents and rates of FFA release from mesenteric adipose tissue (MAT) were measured. Plasma glucose levels showed significant differences only after 90 days of fasting, when glycemia was 34% lower (50±5mg.dL-1) than fed fish values (74±1mg.dL-1), remaining relatively constant until 200 days of fasting. The content of liver glycogen ("15%) in fed tilapia fell 40% in 7 days of food deprivation. In 60, 90 and 150 days of fasting, plasma FFA levels increased 49%, 64% and 90%, respectively, compared to fed fish values. In agreement with the increase in plasma FFA, fasting induced a clear increase in lipolytic activity of MAT incubated in vitro. Addition of isobutylmethylxanthine (cAMP-phosphodiesterase inhibitor) and isoproterenol (non selective beta adrenergic agonist) to the incubation medium induced a reduction of lipolysis in fasted fish, differently to what was observed in mammal adipose tissue. This study allowed a physiological assessment of red tilapia response to starvation.

Calcitonin gene-related peptide inhibits autophagic-lysosomal proteolysis through cAMP/PKA signaling in rat skeletal muscles.

Calcitonin gene-related peptide (CGRP) is a neuropeptide released by motor neuron in skeletal muscle and modulates the neuromuscular transmission by induction of synthesis and insertion of acetylcholine receptor on postsynaptic muscle membrane; however, its role in skeletal muscle protein metabolism remains unclear. We examined the in vitro and in vivo effects of CGRP on protein breakdown and signaling pathways in control skeletal muscles and muscles following denervation (DEN) in rats. In isolated muscles, CGRP (10(-10) to 10(-6)M) reduced basal and DEN-induced activation of overall proteolysis in a concentration-dependent manner. The in vitro anti-proteolytic effect of CGRP was completely abolished by CGRP8-37, a CGRP receptor antagonist. CGRP down-regulated the lysosomal proteolysis, the mRNA levels of LC3b, Gabarapl1 and cathepsin L and the protein content of LC3-II in control and denervated muscles. In parallel, CGRP elevated cAMP levels, stimulated PKA/CREB signaling and increased Foxo1 phosphorylation in both conditions. In denervated muscles and starved C2C12 cells, Rp-8-Br-cAMPs or PKI, two PKA inhibitors, completely abolished the inhibitory effect of CGRP on Foxo1, 3 and 4 and LC3 lipidation. A single injection of CGRP (100 µg kg(-1)) in denervated rats increased the phosphorylation levels of CREB and Akt, inhibited Foxo transcriptional activity, the LC3 lipidation as well as the mRNA levels of LC3b and cathepsin L, two bona fide targets of Foxo. This study shows for the first time that CGRP exerts a direct inhibitory action on autophagic-lysosomal proteolysis in control and denervated skeletal muscle by recruiting cAMP/PKA signaling, effects that are related to inhibition of Foxo activity and LC3 lipidation.

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.

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 ß(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.

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.

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.

In vivo effects of Bothrops jararaca venom on metabolic profile and on muscle protein metabolism in rats.

This study investigated the in vivo effects of the Bothrops jararaca venom (BjV) on general metabolic profile and, specifically, on muscle protein metabolism in rats. The crude venom (0.4 mg/kg body weight, IV) was infused in awake rats, and plasma activity of enzymes and metabolites levels were determined after 1, 2, 3, and 4 hours. BjV increased urea, lactate, and activities of creatine kinase, lactate dehydrogenase, and aspartate aminotransferase after 4 hours. The content of liver glycogen was reduced by BjV. Protein metabolism was evaluated by means of microdialysis technique and in isolated muscles. BjV induced increase in the muscle interstitial-arterial tyrosine concentration difference, indicating a high protein catabolism. The myotoxicity induced by this venom is associated with reduction of protein synthesis and increase in rates of overall proteolysis, which was accompanied by activation of lysosomal and ubiquitin-proteasome systems without changes in protein levels of cathepsins and ubiquitin-protein conjugates.

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

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 (approximately 150%), in arterial plasma tyrosine levels (approximately 50%), and in interstitial-arterial tyrosine concentration difference (approximately 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 alpha 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.