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Congenital Myasthenic Syndromes (CMS) are a set of genetic diseases that affect the neuromuscular transmission causing muscular weakness. The standard pharmacological treatment aims at ameliorating the myasthenic symptom by acetylcholinesterase inhibitors. Most patients respond well in the short and medium term, however, over time the beneficial effects rapidly fade, and the efficacy of the treatment diminishes. Increasing evidence shows that ß2-adrenergic agonists can be a suitable choice for the treatment of neuromuscular disorders, including CMS, as they promote beneficial effects in the neuromuscular system. The exact mechanism on which they rely is not completely understood, although patients and animal models respond well to the treatment, especially over extended periods. Here, we report the use of the long-lasting specific ß2-adrenergic agonist formoterol in a myasthenic mouse model (mnVAChT-KD), featuring deletion of VAChT (Vesicular Acetylcholine Transporter) specifically in the α-motoneurons. Our findings demonstrate that formoterol treatment (300 µg/kg/day; sc) for 30 days increased the neuromuscular junction area, induced skeletal muscle hypertrophy and altered fibre type composition in myasthenic mice. Interestingly, ß2-adrenergic agonists have shown efficacy even in the absence of ACh (acetylcholine). Our data provide important evidence supporting the potential of ß2-adrenergic agonists in treating neuromuscular disorders of pre-synaptic origin and characterized by disruptions in nerve-muscle communication, through a direct and beneficial action within the motor unit.
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BACKGROUND AND AIM: Although it is well established that hormones like glucagon stimulates gluconeogenesis via the PKA-mediated phosphorylation of CREB and dephosphorylation of the cAMP-regulated CREB coactivators CRTC2, the role of neural signals in the regulation of gluconeogenesis remains uncertain. METHODS AND RESULTS: Here, we characterize the noradrenergic bundle architecture in mouse liver; we show that the sympathoexcitation induced by acute cold exposure promotes hyperglycemia and upregulation of gluconeogenesis via triggering of the CREB/CRTC2 pathway. Following its induction by dephosphorylation, CRTC2 translocates to the nucleus and drives the transcription of key gluconeogenic genes. Rodents submitted to different models of sympathectomy or knockout of CRTC2 do not activate gluconeogenesis in response to cold. Norepinephrine directly acts in hepatocytes mainly through a Ca2+-dependent pathway that stimulates CREB/CRTC2, leading to activation of the gluconeogenic program. CONCLUSION: Our data demonstrate the importance of the CREB/CRTC2 pathway in mediating effects of hepatic sympathetic inputs on glucose homeostasis, providing new insights into the role of norepinephrine in health and disease.
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Temperatura Baixa , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Gluconeogênese , Fígado , Norepinefrina , Fatores de Transcrição , Animais , Gluconeogênese/fisiologia , Fígado/metabolismo , Camundongos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Masculino , Norepinefrina/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Neurônios Adrenérgicos/metabolismo , Neurônios Adrenérgicos/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/fisiologia , Hepatócitos/metabolismoRESUMO
Recent studies have shown that maternal vitamin D deficiency (VDD) causes long-term metabolic changes in offspring. However, little is known about the impact of maternal VDD on offspring endocrine pancreas development and insulin secretion in the adult life of male and female animals. Female rats (Wistar Hannover) were fed either control (1000 IU Vitamin D3/kg), VDD (0 IU Vitamin D3/kg), or a Ca2+-enriched VDD diet (0 IU Vitamin D3/kg + Ca2+ and P/kg) for 6 weeks and during gestation and lactation. At weaning, VDD status was confirmed based on low serum calcidiol levels in dams and pups. Next, male and female offspring were randomly separated and fed a standard diet for up to 90 days. At this age, serum calcidiol levels were restored to normal levels in all groups, but serum insulin levels were decreased in VDD males without affecting glucagon levels, glycemia, or glucose tolerance. Islets isolated from VDD males showed lower insulin secretion in response to different glucose concentrations, but this effect was not observed in VDD females. Furthermore, VDD males, but not females, showed a smaller total pancreatic islet area and lower ß cell mass, an effect that was accompanied by reduced gene expression of Ins1, Ins2, Pdx1, and SLC2A2. The decrease in Pdx1 expression was not related to the methylation profile of the promoter region of this gene. Most of these effects were observed in the male VDD+Ca2+ group, indicating that the effects were not due to alterations in Ca2+ metabolism. These data show that maternal VDD selectively impairs the morphology and function of ß cells in adult male offspring rats and that female offspring are fully protected from these deleterious effects.
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Células Secretoras de Insulina , Insulina , Ratos Wistar , Deficiência de Vitamina D , Animais , Feminino , Células Secretoras de Insulina/metabolismo , Masculino , Deficiência de Vitamina D/metabolismo , Ratos , Gravidez , Insulina/sangue , Insulina/metabolismo , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Efeitos Tardios da Exposição Pré-Natal/etiologia , Fatores Sexuais , Secreção de InsulinaRESUMO
Resistance exercise (RE) training and pharmacological stimulation of ß2-Adrenoceptors (ß2-ARs) alone can promote muscle hypertrophy and prevent muscle atrophy. Although the activation of the sympathetic nervous system (SNS) is a well-established response during RE, the physiological contribution of the endogenous catecholamines and ß2-ARs to the RE-induced changes on skeletal muscle protein metabolism remains unclear. This study investigated the effects of the ß2-ARs blockade on the acute molecular responses induced by a single bout of RE in rodent skeletal muscles. Male C57BL6/J mice were subjected to a single bout of progressive RE (until exhaustion) on a vertical ladder under ß2-AR blockade with ICI 118,551 (ICI; 10 mg kg-1, i. p.), or vehicle (sterile saline; 0.9%, i. p.), and the gene expression was analyzed in gastrocnemius (GAS) muscles by qPCR. We demonstrated that a single bout of RE acutely increased the circulating levels of stress-associated hormones norepinephrine (NE) and corticosterone (CORT), as well as the muscle phosphorylation levels of AMPK, p38 MAPK and CREB, immediately after the session. The acute increase in the phosphorylation levels of CREB was followed by the upregulation of CREB-target genes Sik1, Ppargc1a and Nr4a3 (a central regulator of the acute RE response), 3 h after the RE session. Conversely, ß2-AR blockade reduced significantly the Sik1 and Nr4a3 mRNA levels in muscles of exercised mice. Furthermore, a single bout of RE stimulated the mRNA levels of the atrophic genes Map1lc3b and Gabarapl1 (autophagy-related genes) and Mstn (a well-known negative regulator of muscle growth). Unexpectedly, the gene expression of Igf-1 or Il-6 were not affected by RE, while the atrophic genes Murf1/Trim63 and Atrogin-1/Mafbx32 (ubiquitin-ligases) were increased only in muscles of exercised mice under ß2-AR blockade. Interestingly, performing a single bout of RE under ß2-AR blockade increased the mRNA levels of Mstn in muscles of exercised mice. These data suggest that ß2-ARs stimulation during acute RE stimulates the hypertrophic gene Nr4a3 and prevents the overexpression of atrophic genes such as Mstn, Murf1/Trim63, and Atrogin-1/Mafbx32 in the first hours of postexercise recovery, indicating that he SNS may be physiologically important to muscle adaptations in response to resistance training.
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Although there are reports that polyphenol resveratrol (Rsv) may cause muscle hypertrophy in basal conditions and attenuate muscle wasting in catabolic situations, its mechanism of action is still unclear. Our study evaluated the ex vivo effects of Rsv on protein metabolism and intracellular signaling in innervated (sham-operated; Sham) and 3-day sciatic denervated (Den) rat skeletal muscles. Rsv (10-4 M) reduced total proteolysis (40%) in sham muscles. Den increased total proteolysis (~40%) in muscle, which was accompanied by an increase in the activities of ubiquitin-proteasome (~3-fold) and lysosomal (100%) proteolytic systems. Rsv reduced total proteolysis (59%) in Den muscles by inhibiting the hyperactivation of ubiquitin-proteasome (50%) and lysosomal (~70%) systems. Neither Rsv nor Den altered calcium-dependent proteolysis in muscles. Mechanistically, Rsv stimulated PKA/CREB signaling in Den muscles, and PKA blockage by H89 (50µM) abolished the antiproteolytic action of the polyphenol. Rsv reduced FoxO4 phosphorylation (~60%) in both Sham and Den muscles and Akt phosphorylation (36%) in Den muscles. Rsv also caused a homeostatic effect in Den muscles by returning their protein synthesis rates to levels similar to Sham muscles. These data indicate that Rsv directly inhibits the proteolytic activity of lysosomal and ubiquitin-proteasome systems, mainly in Den muscles through, at least in part, the activation of PKA/CREB signaling.
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Músculo Esquelético , Complexo de Endopeptidases do Proteassoma , Ratos , Animais , Proteólise , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/farmacologia , Resveratrol/farmacologia , Músculo Esquelético/metabolismo , Ratos Wistar , Ubiquitinas/metabolismo , Ubiquitinas/farmacologiaRESUMO
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.
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Hormônio Liberador da Corticotropina/metabolismo , Proteínas Proto-Oncogênicas c-akt , Urocortinas/metabolismo , Hormônio Adrenocorticotrópico/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Animais , Hipertrofia/metabolismo , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Urocortinas/farmacologiaRESUMO
Recent evidence has suggested that the carotid bodies might act as immunological sensors, detecting pro-inflammatory mediators and signalling to the central nervous system, which, in turn, orchestrates autonomic responses. Here, we confirmed that the TNF-α receptor type I is expressed in the carotid bodies of rats. The systemic administration of TNF-α increased carotid body afferent discharge and activated glutamatergic neurons in the nucleus tractus solitarius (NTS) that project to the rostral ventrolateral medulla (RVLM), where many pre-sympathetic neurons reside. The activation of these neurons was accompanied by an increase in splanchnic sympathetic nerve activity. Carotid body ablation blunted the TNF-α-induced activation of RVLM-projecting NTS neurons and the increase in splanchnic sympathetic nerve activity. Finally, plasma and spleen levels of cytokines after TNF-α administration were higher in rats subjected to either carotid body ablation or splanchnic sympathetic denervation. Collectively, our findings indicate that the carotid body detects circulating TNF-α to activate a counteracting sympathetic anti-inflammatory mechanism.
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Corpo Carotídeo , Animais , Anti-Inflamatórios , Bulbo/fisiologia , Ratos , Ratos Sprague-Dawley , Reflexo , Núcleo Solitário/fisiologia , Sistema Nervoso Simpático/fisiologia , Fator de Necrose Tumoral alfaRESUMO
Scorpion envenomation is a leading cause of morbidity and mortality among accidents caused by venomous animals. Major clinical manifestations that precede death after scorpion envenomation include heart failure and pulmonary edema. Here, we demonstrate that cardiac dysfunction and fatal outcomes caused by lethal scorpion envenomation in mice are mediated by a neuro-immune interaction linking IL-1 receptor signaling, prostaglandin E2, and acetylcholine release. IL-1R deficiency, the treatment with a high dose of dexamethasone or blockage of parasympathetic signaling using atropine or vagotomy, abolished heart failure and mortality of envenomed mice. Therefore, we propose the use of dexamethasone administration very early after envenomation, even before antiserum, to inhibit the production of inflammatory mediators and acetylcholine release, and to reduce the risk of death.
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Acetilcolina/metabolismo , Dinoprostona/biossíntese , Insuficiência Cardíaca/etiologia , Receptores Tipo I de Interleucina-1/metabolismo , Venenos de Escorpião/toxicidade , Animais , Antivenenos/administração & dosagem , Atropina/farmacologia , Dexametasona/administração & dosagem , Modelos Animais de Doenças , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/fisiopatologia , Humanos , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Cardiovasculares , Neuroimunomodulação/efeitos dos fármacos , Receptores Tipo I de Interleucina-1/deficiência , Receptores Tipo I de Interleucina-1/genética , Picadas de Escorpião/complicações , Escorpiões , Transdução de Sinais , VagotomiaRESUMO
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.
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Glucocorticoides/metabolismo , Hipóxia/fisiopatologia , Músculo Esquelético/fisiopatologia , Animais , Cálcio/metabolismo , Sistema Hipotálamo-Hipofisário/metabolismo , Sistema Hipotálamo-Hipofisário/fisiopatologia , Hipóxia/metabolismo , Resistência à Insulina/fisiologia , Lisossomos/metabolismo , Lisossomos/fisiologia , Masculino , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Atrofia Muscular/fisiopatologia , Sistema Hipófise-Suprarrenal/metabolismo , Sistema Hipófise-Suprarrenal/fisiopatologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Proteólise , Ratos , Ratos Wistar , Ubiquitina/metabolismoRESUMO
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 ß2-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.
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Saúde , Homeostase , Doenças do Sistema Nervoso/patologia , Junção Neuromuscular/patologia , Sistema Nervoso Simpático/patologia , Transporte Ativo do Núcleo Celular , Animais , Técnicas Biossensoriais , Núcleo Celular/metabolismo , AMP Cíclico/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Músculo Esquelético/inervação , Junção Neuromuscular/metabolismo , Neurônios/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fenótipo , Transdução de Sinais , Simpatectomia , Sistema Nervoso Simpático/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The our objective was to investigate the adaptations induced by a low-protein, high-carbohydrate (LPHC) diet in growing rats, which by comparison with the rats fed a control (C) diet at displayed lower fasting glycemia and similar fasting insulinemia, despite impairment in insulin signaling in adipose tissues. In the insulin tolerance test the LPHC rats showed higher rates of glucose disappearance (30%) and higher tolerance to overload of glucose than C rats. The glucose uptake by the soleus muscle, evaluated in vivo by administration of 2-deoxy-[(14)C]glucose, increased by 81%. The phosphoenolpyruvate carboxykinase content and the incorporation of [1-(14)C]pyruvate into glucose was also higher in the slices of liver from the LPHC rats than in those from C rats. The LPHC rats showed increases in l-lactate as well as in other gluconeogenic precursors in the blood. These rats also had a higher hepatic production of glucose, evaluated by in situ perfusion. The data obtained indicate that the main substrates for gluconeogenesis in the LPHC rats are l-lactate and glycerol. Thus, we concluded that the fasting glycemia in the LPHC animals was maintained mainly by increases in the hepatic gluconeogenesis from glycerol and l-lactate, compensating, at least in part, for the higher glucose uptake by the tissues.
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Glicemia/metabolismo , Dieta com Restrição de Proteínas , Carboidratos da Dieta/administração & dosagem , Jejum/sangue , Gluconeogênese , Glucose/biossíntese , Fígado/metabolismo , Tecido Adiposo/metabolismo , Animais , Teste de Tolerância a Glucose , Glicerol/sangue , Insulina/sangue , Ácido Láctico/sangue , Masculino , Músculo Esquelético/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , RatosRESUMO
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.
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Epinefrina/deficiência , Jejum/metabolismo , Fibras Musculares de Contração Rápida/metabolismo , Proteólise , Tecido Adiposo/anatomia & histologia , Tecido Adiposo/efeitos dos fármacos , Medula Suprarrenal/fisiologia , Medula Suprarrenal/cirurgia , Animais , Composição Corporal/efeitos dos fármacos , Composição Corporal/fisiologia , Catecolaminas/sangue , Epinefrina/farmacologia , Masculino , Norepinefrina/sangue , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Autonomic regulation processes in striated muscles are largely mediated by cAMP/PKA-signaling. In order to achieve specificity of signaling its spatial-temporal compartmentation plays a critical role. We discuss here how specificity of cAMP/PKA-signaling can be achieved in skeletal muscle by spatio-temporal compartmentation. While a microdomain containing PKA type I in the region of the neuromuscular junction (NMJ) is important for postsynaptic, activity-dependent stabilization of the nicotinic acetylcholine receptor (AChR), PKA type I and II microdomains in the sarcomeric part of skeletal muscle are likely to play different roles, including the regulation of muscle homeostasis. These microdomains are due to specific A-kinase anchoring proteins, like rapsyn and myospryn. Importantly, recent evidence indicates that compartmentation of the cAMP/PKA-dependent signaling pathway and pharmacological activation of cAMP production are aberrant in different skeletal muscles disorders. Thus, we discuss here their potential as targets for palliative treatment of certain forms of dystrophy and myasthenia. Under physiological conditions, the neuropeptide, α-calcitonin-related peptide, as well as catecholamines are the most-mentioned natural triggers for activating cAMP/PKA signaling in skeletal muscle. While the precise domains and functions of these first messengers are still under investigation, agonists of ß2-adrenoceptors clearly exhibit anabolic activity under normal conditions and reduce protein degradation during atrophic periods. Past and recent studies suggest direct sympathetic innervation of skeletal muscle fibers. In summary, the organization and roles of cAMP-dependent signaling in skeletal muscle are increasingly understood, revealing crucial functions in processes like nerve-muscle interaction and muscle trophicity.
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We have previously shown that a high-protein, carbohydrate-free diet can decrease the production of glycerol-3-phosphate (G3P) from glucose and increase glyceroneogenesis in both brown (BAT) and epididymal (EAT) adipose tissue. Here, we utilized an in-vivo approach to examine the hypothesis that there is reciprocal regulation in the G3P synthesis from glucose (via glycolysis) and glyceroneogenesis in BAT, EAT and liver of fasted rats and cafeteria diet-fed rats. Glyceroneogenesis played a prominent role in the generation of G3P in the liver (~70 %) as well as in BAT and EAT (~80 %) in controls rats. The cafeteria diet induced an increase in the total glyceride-glycerol synthesis and G3P synthesis from glucose and a decrease in glyceroneogenesis in BAT; this diet did not affect either the total glyceride-glycerol synthesis or G3P generation from glyceroneogenesis or glycolysis in the liver or EAT. Fasting induced an increase in total glyceride-glycerol synthesis and glyceroneogenesis and a decrease in G3P synthesis from glucose in the liver but did not affect either the total glyceride-glycerol synthesis or G3P synthesis from glyceroneogenesis in BAT and EAT, despite a reduction in glycolysis in these tissues. These data demonstrate that reciprocal changes in the G3P generation from glucose and from glyceroneogenesis in the rat liver and BAT occur only when the synthesis of glycerides-glycerol is increased. Further, our data suggest that this increase may be essential for the systemic recycling of fatty acids by the liver from fasted rats and for the maintenance of the thermogenic capacity of BAT from cafeteria diet-fed rats.
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Tecido Adiposo Marrom/metabolismo , Glicerídeos/biossíntese , Glicerol/metabolismo , Glicólise , Fígado/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Jejum , Glicerofosfatos/biossíntese , Masculino , Ratos , Ratos WistarRESUMO
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.
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Agonistas Adrenérgicos beta/uso terapêutico , Clembuterol/uso terapêutico , Lisossomos/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Inibidores de Proteassoma , Agonistas Adrenérgicos beta/farmacologia , Animais , Catepsina L/metabolismo , Clembuterol/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/química , Ativadores de Enzimas/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Lisossomos/enzimologia , Masculino , Denervação Muscular/efeitos adversos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/inervação , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/enzimologia , Atrofia Muscular/metabolismo , Atrofia Muscular/prevenção & controle , Complexo de Endopeptidases do Proteassoma/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
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.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Músculo Esquelético/metabolismo , Atrofia Muscular/genética , Inibidores da Fosfodiesterase 4/farmacologia , Proteólise/efeitos dos fármacos , Rolipram/farmacologia , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/fisiologia , Expressão Gênica/fisiologia , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Animais , Proteínas Musculares/metabolismo , Músculo Esquelético/efeitos dos fármacos , Atrofia Muscular/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas com Motivo Tripartido , Tirosina/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
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.
Assuntos
Tecido Adiposo/metabolismo , Carboidratos da Dieta/administração & dosagem , Proteínas Alimentares/administração & dosagem , Ácidos Graxos/metabolismo , Glicerofosfatos/metabolismo , Metabolismo dos Lipídeos , Adipócitos/metabolismo , Adiposidade , Animais , Dieta , Dieta com Restrição de Proteínas , Carboidratos da Dieta/metabolismo , Proteínas Alimentares/metabolismo , Ingestão de Alimentos , Glicerol Quinase/metabolismo , Glicólise , Lipogênese , Lipólise , Lipase Lipoproteica/metabolismo , Lipoproteínas/metabolismo , Masculino , Norepinefrina/metabolismo , Ratos , Ratos Wistar , Triglicerídeos/metabolismoRESUMO
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.
Assuntos
AMP Cíclico/metabolismo , Músculo Esquelético/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Agonistas de Receptores Adrenérgicos beta 2 , Animais , Western Blotting , Linhagem Celular , Clembuterol/farmacologia , Dexametasona/farmacologia , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Inibidores de Fosfodiesterase/farmacologia , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição , Proteínas com Motivo Tripartido , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
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.
Assuntos
Venenos de Crotalídeos/farmacologia , Proteínas Musculares/metabolismo , Músculo Esquelético/efeitos dos fármacos , Animais , Pressão Sanguínea , Bothrops , Hidrólise , Metabolismo dos Lipídeos , Glicogênio Hepático/metabolismo , Masculino , Microdiálise , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/metabolismo , Ratos , Ratos Wistar , Fluxo Sanguíneo RegionalRESUMO
In vivo fatty acid synthesis and the pathways of glycerol-3-phosphate (G3P) production were investigated in brown adipose tissue (BAT) from rats fed a cafeteria diet for 3 weeks. In spite of BAT activation, the diet promoted an increase in the carcass fatty acid content. Plasma insulin levels were markedly increased in cafeteria diet-fed rats. Two insulin-sensitive processes, in vivo fatty acid synthesis and in vivo glucose uptake (which was used to evaluate G3P generation via glycolysis) were increased in BAT from rats fed the cafeteria diet. Direct glycerol phosphorylation, evaluated by glycerokinase (GyK) activity and incorporation of [U-14C]glycerol into triacylglycerol (TAG)-glycerol, was also markedly increased in BAT from these rats. In contrast, the cafeteria diet induced a marked reduction of BAT glyceroneogenesis, evaluated by phosphoenolpyruvate carboxykinase-C activity and incorporation of [1-14C]pyruvate into TAG-glycerol. BAT denervation resulted in an approximately 50% reduction of GyK activity, but did not significantly affect BAT in vivo fatty acid synthesis, in vivo glucose uptake, or glyceroneogenesis. The data suggest that the supply of G3P for BAT TAG synthesis can be adjusted independently from the sympathetic nervous system and solely by reciprocal changes in the generation of G3P via glycolysis and via glyceroneogenesis, with no participation of direct phosphorylation of glycerol by GyK.