RESUMO
Skeletal muscle consists of both fast- and slow-twitch fibers. Phospholipids are important structural components of cellular membranes, and the diversity of their fatty acid composition affects membrane characteristics. Although some studies have shown that acyl chain species in phospholipids differ among various muscle fiber types, the mechanisms underlying these differences are unclear. To investigate this, we analyzed phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecules in the murine extensor digitorum longus (EDL; fast-twitch) and soleus (slow-twitch) muscles. In the EDL muscle, the vast majority (93.6%) of PC molecules was palmitate-containing PC (16:0-PC), whereas in the soleus muscle, in addition to 16:0-PC, 27.9% of PC molecules was stearate-containing PC (18:0-PC). Most palmitate and stearate were bound at the sn-1 position of 16:0- and 18:0-PC, respectively, and 18:0-PC was found in type I and IIa fibers. The amount of 18:0-PE was higher in the soleus than in the EDL muscle. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) increased the amount of 18:0-PC in the EDL. Lysophosphatidylglycerol acyltransferase 1 (LPGAT1) was highly expressed in the soleus compared with that in the EDL muscle and was upregulated by PGC-1α. LPGAT1 knockout decreased the incorporation of stearate into PC and PE in vitro and ex vivo and the amount of 18:0-PC and 18:0-PE in murine skeletal muscle with an increase in the level of 16:0-PC and 16:0-PE. Moreover, knocking out LPGAT1 decreased the amount of stearate-containing phosphatidylserine (18:0-PS), suggesting that LPGAT1 regulated the acyl chain profiles of phospholipids, namely, PC, PE, and PS, in the skeletal muscle.
Assuntos
Fibras Musculares de Contração Rápida , Músculo Esquelético , Fosfolipídeos , Animais , Camundongos , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/metabolismo , Fosfatidilcolinas/metabolismo , Fosfolipídeos/química , Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Estearatos/metabolismo , Plasmalogênios , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fibras Musculares Esqueléticas/metabolismoRESUMO
Catabolic conditions, such as starvation, inactivity, and cancer cachexia, induce Forkhead box O (FOXO) transcription factor(s) expression and severe muscle atrophy via the induction of ubiquitin-proteasome system-mediated muscle proteolysis, resulting in frailty and poor quality of life. Although FOXOs are clearly essential for the induction of muscle atrophy, it is unclear whether there are other factors involved in the FOXO-mediated transcriptional regulation. As such, we identified FOXO-CCAAT/enhancer-binding protein δ (C/EBPδ) signaling pathway as a novel proteolytic pathway. By comparing the gene expression profiles of FOXO1-transgenic (gain-of-function model) and FOXO1,3a,4-/- (loss-of-function model) mice, we identified several novel FOXO1-target genes in skeletal muscle including Redd1, Sestrin1, Castor2, Chac1, Depp1, Lat3, as well as C/EBPδ. During starvation, C/EBPδ abundance was increased in a FOXOs-dependent manner. Notably, knockdown of C/EBPδ prevented the induction of the ubiquitin-proteasome system and decrease of myofibers in FOXO1-activated myotubes. Conversely, C/EBPδ overexpression in primary myotubes induced myotube atrophy. Furthermore, we demonstrated that FOXO1 enhances the promoter activity of target genes in cooperation with C/EBPδ and ATF4. This research comprehensively identifies novel FOXO1 target genes in skeletal muscle and clarifies the pathophysiological role of FOXO1, a master regulator of skeletal muscle atrophy.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Jejum/metabolismo , Proteína Forkhead Box O1/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Transcrição Gênica/fisiologia , Animais , Linhagem Celular , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Regiões Promotoras Genéticas/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Transdução de Sinais/fisiologia , Ubiquitina/metabolismoRESUMO
To examine effects of PP6 gene (Ppp6c) deficiency on pancreatic tumor development, we developed pancreas-specific, tamoxifen-inducible Cre-mediated KP (KRAS(G12D) plus Trp53-deficient) mice (cKP mice) and crossed them with Ppp6cflox / flox mice. cKP mice with the homozygous Ppp6c deletion developed pancreatic tumors, became emaciated and required euthanasia within 150 days of mutation induction, phenotypes that were not seen in heterozygous or wild-type (WT) mice. At 30 days, a comparative analysis of genes commonly altered in homozygous versus WT Ppp6c cKP mice revealed enhanced activation of Erk and NFκB pathways in homozygotes. By 80 days, the number and size of tumors and number of precancerous lesions had significantly increased in the pancreas of Ppp6c homozygous relative to heterozygous or WT cKP mice. Ppp6c-/- tumors were pathologically diagnosed as pancreatic ductal adenocarcinoma (PDAC) undergoing the epithelial-mesenchymal transition (EMT), and cancer cells had invaded surrounding tissues in three out of six cases. Transcriptome and metabolome analyses indicated an enhanced cancer-specific glycolytic metabolism in Ppp6c-deficient cKP mice and the increased expression of inflammatory cytokines. Individual Ppp6c-/- cKP mice showed weight loss, decreased skeletal muscle and adipose tissue, and increased circulating tumor necrosis factor (TNF)-α and IL-6 levels, suggestive of systemic inflammation. Overall, Ppp6c deficiency in the presence of K-ras mutations and Trp53 gene deficiency promoted pancreatic tumorigenesis with generalized cachexia and early death. This study provided the first evidence that Ppp6c suppresses mouse pancreatic carcinogenesis and supports the use of Ppp6c-deficient cKP mice as a model for developing treatments for cachexia associated with pancreatic cancer.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Fosfoproteínas Fosfatases/metabolismo , Animais , Caquexia/genética , Carcinogênese/genética , Carcinoma Ductal Pancreático/patologia , Humanos , Camundongos , Mutação , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Neoplasias PancreáticasRESUMO
Calorie restriction (CR) reportedly prevents atherosclerotic diseases. Furthermore, CR induces forkhead box protein-O1 (FOXO-1) expression in the skeletal muscle, altering the character of the skeletal muscle. We previously reported that the change in skeletal muscle character, induced by the overexpression of peroxisome proliferator-activated receptor γ coactivator-1α, suppresses atherosclerotic progression in an atherosclerotic apolipoprotein E-knockout (ApoE-KO) mouse model. Thus, we hypothesized that skeletal muscle alternation induced by FOXO-1 may also have an anti-atherosclerotic effect in ApoE-KO mice. In this study, we investigated whether skeletal muscle-specific FOXO-1 overexpression suppresses the progression of atherosclerosis in ApoE-KO mice. We generated ApoE-KO/FOXO-1 mice, in which an ApoE-KO mouse was crossbred with a mouse presenting skeletal muscle-specific FOXO-1 overexpression (FOXO-1Tg). The mice were sacrificed at 20 weeks of age, and atherosclerotic plaque area and protein expression in the plaque were measured. Additionally, we measured the tumor necrosis factor α (TNFα)- induced mRNA expression in human umbilical vein endothelial cells (HUVECs), using serum collected from the FOXO-1Tg mice. Accordingly, ApoE-KO/FOXO-1 mice showed a 65% reduced atherosclerotic plaque area when compared with the ApoE-KO mice, with concomitantly reduced vascular cell adhesion molecule-1 (VCAM-1) and macrophage infiltration. As compared to serum from wild-type mice, the serum collected from the FOXO-1Tg mice significantly suppressed the mRNA expression of VCAM-1, an atherosclerosis initiation factor, in TNFα-treated HUVECs. Therefore, these data suggest that skeletal muscle-specific FOXO-1 overexpression suppresses the progression of atherosclerosis in ApoE-KO mice. In part, the CR-induced anti-atherosclerotic effect could be attributed to FOXO-1 upregulation in the skeletal muscle.
Assuntos
Apolipoproteínas E/deficiência , Aterosclerose/genética , Aterosclerose/patologia , Progressão da Doença , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Músculo Esquelético/metabolismo , Animais , Apolipoproteínas E/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Phosphatidylcholine (PC) is an essential component of the plasma membrane. Its profile varies with species and tissues. However, the PC profiles in meat have not been explored in depth. This study aimed to investigate the differences in PC profiles between various meat animal species and meat cut sites, along with the identification of characteristic PC molecules. The results demonstrated that the PC profiles of chicken meat differed from those of other species. Significant differences were also observed between the PC profiles of pork meat and the meat obtained from other species. The amount of PCs containing ether bonds was high in pork meat. PCs containing an odd number of carbon atoms were characteristic of beef and lamb meats. Furthermore, PC profiles differed based on the muscle location in chicken and pork. These results suggest that the PC profiles of skeletal muscles are indicators of animal species and muscle location.
Assuntos
Análise de Alimentos/métodos , Carne/análise , Músculo Esquelético/química , Fosfatidilcolinas/química , Animais , Bovinos , Galinhas , Cromatografia Líquida , Lipidômica/métodos , Músculo Esquelético/metabolismo , Especificidade de Órgãos , Fosfatidilcolinas/classificação , Fosfatidilcolinas/isolamento & purificação , Fosfatidilcolinas/metabolismo , Análise de Componente Principal , Ovinos , Especificidade da Espécie , Espectrometria de Massas por Ionização por Electrospray , SuínosRESUMO
PGC-1α expression increases in skeletal muscles during exercise and regulates the transcription of many target genes. In this study, we conducted a metabolomic analysis on the blood of transgenic mice overexpressing PGC-1α in its skeletal muscle (PGC-1α-Tg mice) using CE-TOFMS. The blood level of homovanillic acid (dopamine metabolite) and the gene expression of dopamine metabolic enzyme in the skeletal muscle of PGC-1α-Tg mice were high. The blood level of 5-methoxyindoleacetic acid was also high in PGC-1α-Tg mice. The blood levels of branched-chain α-keto acids and ß-alanine were low in PGC-1α-Tg mice. These metabolites in the skeletal muscle were present in low concentration. The changes in these metabolites may reflect the skeletal muscle condition with increasing PGC-1α, such as exercise.
Assuntos
Metabolômica/métodos , Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Animais , Eletroforese Capilar/métodos , Ácido Homovanílico/sangue , Ácido Hidroxi-Indolacético/análogos & derivados , Ácido Hidroxi-Indolacético/sangue , Espectrometria de Massas/métodos , Camundongos , Camundongos TransgênicosRESUMO
INTRODUCTION: Phospholipids are essential components of cellular membranes and are closely associated with cellular functions, but relationships involving skeletal muscle phospholipid profiles and their physiological phenotypes have remained unclear. METHODS: We carried out comprehensive phospholipid analyses using liquid chromatography-tandem mass spectrometry to determine the phospholipid profiles of skeletal muscles derived from muscle-wasting mouse models, including denervated and Duchenne muscular dystrophy mouse models (mdx) as well as rescued mdx mice expressing truncated dystrophin. RESULTS: Consistent phosphatidylcholine and phosphatidylethanolamine alterations in skeletal muscles isolated from denervated and mdx mice were observed. Notably, the levels of these phospholipids binding polyunsaturated fatty acids were reduced in denervated and mdx muscles. Moreover, rescuing the mdx pathology by expressing truncated dystrophin led to the restoration of phospholipid profiles. DISCUSSION: Our findings support the hypothesis that phospholipid profiles of the skeletal muscle may be associated with skeletal muscle function.
Assuntos
Glicerofosfolipídeos/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Animais , Cromatografia Líquida , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos mdx , Fenótipo , Espectrometria de Massas em TandemRESUMO
Endurance exercise training has been shown to induce peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) expression in skeletal muscle. We recently reported that skeletal muscle-specific PGC-1α overexpression suppressed atherosclerosis in apolipoprotein E-knockout (ApoE-/-) mice. ß-Aminoisobutyric acid (BAIBA) is a PGC-1α-dependent myokine secreted from myocytes that affects multiple organs. We have also reported that BAIBA suppresses tumor necrosis factor-alpha-induced vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) gene expression in endothelial cells. In the present study, we hypothesized that BAIBA suppresses atherosclerosis progression, and tested that hypothesis with ApoE-/- mice. The mice were administered water containing BAIBA for 14 weeks, and were then sacrificed at 20 weeks of age. Atherosclerotic plaque area, plasma BAIBA concentration, and plasma lipoprotein profiles were assessed. Immunohistochemical analyses of the plaque were performed to assess VCAM-1 and MCP-1 protein expression levels and macrophage infiltration. The results showed that BAIBA administration decreased atherosclerosis plaque area by 30%, concomitant with the elevation of plasma BAIBA levels. On the other hand, plasma lipoprotein profiles were not changed by the administration. Immunohistochemical analyses indicated reductions in VCAM-1, MCP-1, and Mac-2 protein expression levels in the plaque. These results suggest that BAIBA administration suppresses atherosclerosis progression without changing plasma lipoprotein profiles. We propose that the mechanisms of this suppression are reductions in both VCAM-1 and MCP-1 expression as well as macrophage infiltration into the plaque.
Assuntos
Ácidos Aminoisobutíricos/uso terapêutico , Aterosclerose/tratamento farmacológico , Ácidos Aminoisobutíricos/sangue , Ácidos Aminoisobutíricos/farmacocinética , Ácidos Aminoisobutíricos/farmacologia , Animais , Valva Aórtica/efeitos dos fármacos , Valva Aórtica/patologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Quimiocina CCL2/metabolismo , Galectina 3/metabolismo , Lipídeos/sangue , Camundongos Knockout para ApoE , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
We investigated whether the deletion of glycerol-3-phosphate dehydrogenase (GPD) 1 would affect carbohydrate oxidation, fat oxidation, and body weight by using the GPD1 null mice (BALB/cHeA (HeA)). We found that fat oxidation in HeA mice was significantly high during the early active phase than in BALB/cBy (By) mice used as a control under ad libitum conditions. Metabolic tracer experiment revealed that fatty acid oxidation in the skeletal muscle of HeA mice tended to be high. The energy expenditure and fat oxidation in HeA mice under fasting conditions were significantly higher than that in the By mice. Moreover, we monitored body weight gain in HeA mice under ad libitum feeding and found lower body weight gain. These data indicate that GPD1 deficiency induces enhancement of fat oxidation with suppression of weight gain. We propose that GPD1 deletion contributes to the reduction of body weight gain via enhancement of fat oxidation.
Assuntos
Tecido Adiposo/metabolismo , Peso Corporal , Glicerolfosfato Desidrogenase/deficiência , Animais , Metabolismo dos Carboidratos , Camundongos , OxirreduçãoRESUMO
Most fatty acids in phospholipids and other lipid species carry an even number of carbon atoms. Also odd-chain fatty acids (OCFAs), such as C15:0 and C17:0, are widespread throughout the living organism. However, the qualitative and quantitative profiles of OCFAs-containing lipids in living organisms remain unclear. Here, we show that OCFAs are present in Drosophila phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and that their level increases in accordance with progression of growth. Furthermore, we found that food-derived propionic acid/propanoic acid (C3:0) is utilized for production of OCFA-containing PC and PE. This study provides the basis for understanding in vivo function of OCFA-containing phospholipids in development and lipid homeostasis.
Assuntos
Drosophila/química , Ácidos Graxos/química , Fosfolipídeos/química , Animais , Drosophila/metabolismo , Ácidos Graxos/biossíntese , Propionatos/metabolismoRESUMO
Chemokines are critical mediators of angiogenesis in several physiological and pathological conditions; however, a potential role for muscle-derived chemokines in exercise-stimulated angiogenesis in skeletal muscle remains poorly understood. Here, we postulated that the chemokine stromal cell-derived factor-1 (SDF-1α/C-X-C motif chemokine ligand 12: CXCL12), shown to promote neovascularization in several organs, contributes to angiogenesis in skeletal muscle. We found that CXCL12 is abundantly expressed in capillary-rich oxidative soleus and exercise-trained plantaris muscles. CXCL12 mRNA and protein were also abundantly expressed in muscle-specific peroxisome proliferator-activated receptor γ coactivator 1α transgenic mice, which have a high proportion of oxidative muscle fibers and capillaries when compared with wild-type littermates. We then generated CXCL12 muscle-specific knockout mice but observed normal baseline capillary density and normal angiogenesis in these mice when they were exercise trained. To get further insight into a potential CXCL12 role in a myofiber-endothelial cell crosstalk, we first mechanically stretched C2C12 myotubes, a model known to induce stretch-related chemokine release, and observed increased CXCL12 mRNA and protein. Human umbilical vein endothelial cells (HUVECs) exposed to conditioned medium from cyclically stretched C2C12 myotubes displayed increased proliferation, which was dependent on CXCL12-mediated signaling through the CXCR4 receptor. However, HUVEC migration and tube formation were unaltered under these conditions. Collectively, our findings indicate that increased muscle contractile activity enhances CXCL12 production and release from muscle, potentially contributing to endothelial cell proliferation. However, redundant signals from other angiogenic factors are likely sufficient to sustain normal endothelial cell migration and tube formation activity, thereby preserving baseline capillary density and exercise training-mediated angiogenesis in muscles lacking CXCL12.
Assuntos
Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/farmacologia , Células Endoteliais/citologia , Neovascularização Fisiológica/fisiologia , Condicionamento Físico Animal/fisiologia , Animais , Proliferação de Células , Quimiocina CXCL12/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/metabolismo , Estresse OxidativoRESUMO
Muscle mitochondria are crucial for systemic metabolic function, yet their regulation remains unclear. The zinc finger MYND domain-containing protein 17 (Zmynd17) was recently identified as a muscle-specific gene in mammals. Here, we investigated the role of Zmynd17 in mice. We found Zmynd17 predominantly expressed in skeletal muscle, especially in fast glycolytic muscle. Genetic Zmynd17 inactivation led to morphologic and functional abnormalities in muscle mitochondria, resulting in decreased respiratory function. Metabolic stress induced by a high-fat diet upregulated Zmynd17 expression and further exacerbated muscle mitochondrial morphology in Zmynd17-deficient mice. Strikingly, Zmynd17 deficiency significantly aggravated metabolic stress-induced hepatic steatosis, glucose intolerance, and insulin resistance. Furthermore, middle-aged mice lacking Zmynd17 exhibited impaired aerobic exercise performance, glucose intolerance, and insulin resistance. Thus, our results indicate that Zmynd17 is a metabolic stress-inducible factor that maintains muscle mitochondrial integrity, with its deficiency profoundly affecting whole-body glucose metabolism.-Fujita, R., Yoshioka, K., Seko, D., Suematsu, T., Mitsuhashi, S., Senoo, N., Miura, S., Nishino, I., Ono, Y. Zmynd17 controls muscle mitochondrial quality and whole-body metabolism.
Assuntos
Peso Corporal/fisiologia , Proteínas de Ligação a DNA/metabolismo , Dieta Hiperlipídica , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Animais , Fígado Gorduroso/metabolismo , Intolerância à Glucose/metabolismo , Resistência à Insulina/fisiologia , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
RATIONALE: In skeletal muscles, there are four myofiber types, Types I, IIa, IIx, and IIb, which show different contraction characteristics and have different metabolic statuses. To understand muscle function, it is necessary to analyze myofiber-specific metabolic changes. However, these fibers are heterogeneous and are hard to discriminate by conventional analyses using tissue extracts. In this study, we found myofiber-specific molecules and molecular markers of other cells such as smooth muscle cells, fat cells, and motor neurons, and visualized them within muscle sections. METHODS: We used three different muscle tissues, namely extensor digitorum longus, soleus, and gastrocnemius tissues, from ICR mice. After the muscles had been harvested, cross-sections were prepared using a cryostat and analyzed using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI), and conventional immunofluorescence imaging. RESULTS: By comparing the MALDI MSI results with the immunofluorescence imaging results, we were able to identify each fiber and cell-specific ion. It was especially important that we could find Type IIa and IIb specific ions, because these were difficult to distinguish. CONCLUSIONS: Through MSI analyses, we performed a comprehensive survey to identify cell- and myofiber-specific molecular markers. In conclusion, we assigned muscle fiber Type I, IIa, and IIb-specific molecular ions at m/z 856.6, 872.6, and 683.8, respectively. These molecular markers might be useful for verifying changes that occur due to exercise and/or disease.
Assuntos
Biomarcadores/análise , Fibras Musculares Esqueléticas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Biomarcadores/metabolismo , Cromatografia em Camada Fina , Diglicerídeos/análise , Diglicerídeos/metabolismo , Processamento de Imagem Assistida por Computador , Lipídeos/análise , Masculino , Camundongos Endogâmicos ICR , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Músculo Liso/química , Músculo Liso/metabolismo , Espectrometria de Massas em TandemRESUMO
A low-protein diet (LPD) protects against the progression of renal injury in patients with chronic kidney disease (CKD). However, LPD may accelerate muscle wasting in these patients. Both exercise and branched-chain amino acids (BCAA) are known to increase muscle protein synthesis by activating the mammalian target of rapamycin (mTOR) pathway. The aim of this study was to investigate whether endurance exercise and BCAA play a role for increasing muscle protein synthesis in LPD-fed CKD (5/6 nephrectomized) rats. Both CKD and sham rats were pair-fed on LPD or LPD fortified with a BCAA diet (BD), and approximately one-half of the animals in each group was subjected to treadmill exercise (15 m/min, 1 h/day, 5 days/wk). After 7 wk, renal function was measured, and soleus muscles were collected to evaluate muscle protein synthesis. Renal function did not differ between LPD- and BD-fed CKD rats, and the treadmill exercise did not accelerate renal damage in either group. The treadmill exercise slightly increased the phosphorylation of p70s6 kinase, a marker of mTOR activity, in the soleus muscle of LPD-fed CKD rats compared with the sham group. Furthermore, BCAA supplementation of the LPD-fed, exercise-trained CKD rats restored the phosphorylation of p70s6 kinase to the same level observed in the sham group; however, the corresponding induced increase in muscle protein synthesis and muscle mass was marginal. These results indicate that the combination of treadmill exercise and BCAA stimulates cell signaling to promote muscle protein synthesis; however, the implications of this effect for muscle growth remain to be clarified.
Assuntos
Aminoácidos de Cadeia Ramificada/administração & dosagem , Dieta com Restrição de Proteínas , Terapia por Exercício/métodos , Proteínas Musculares/biossíntese , Músculo Esquelético/metabolismo , Atrofia Muscular/prevenção & controle , Resistência Física , Insuficiência Renal Crônica/dietoterapia , Ração Animal , Animais , Dieta com Restrição de Proteínas/efeitos adversos , Modelos Animais de Doenças , Interleucina-6/biossíntese , Interleucina-6/genética , Rim/fisiopatologia , Masculino , Músculo Esquelético/fisiopatologia , Atrofia Muscular/etiologia , Atrofia Muscular/metabolismo , Atrofia Muscular/fisiopatologia , Fosforilação , Proteólise , Ratos Wistar , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/fisiopatologia , Proteínas Quinases S6 Ribossômicas 70-kDa/biossíntese , Transdução de Sinais , Serina-Treonina Quinases TOR/biossíntese , Fatores de TempoRESUMO
In skeletal muscle, resting intracellular Ca2+ concentration ([Ca2+]i) homeostasis is exquisitely regulated by Ca2+ transport across the sarcolemmal, mitochondrial, and sarcoplasmic reticulum (SR) membranes. Of these three systems, the relative importance of the mitochondria in [Ca2+]i regulation remains poorly understood in in vivo skeletal muscle. We tested the hypothesis that the capacity for Ca2+ uptake by mitochondria is a primary factor in determining [Ca2+]i regulation in muscle at rest and following contractions. Tibialis anterior muscle of anesthetized peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α)-overexpressing (OE, increased mitochondria model) and wild-type (WT) littermate mice was exteriorized in vivo and loaded with the fluorescent probe fura 2-AM, and Rhod 2-AM Ca2+ buffering and mitochondrial [Ca2+] were evaluated at rest and during recovery from fatiguing tetanic contractions induced by electrical stimulation (120 s, 100 Hz). In addition, the effects of pharmacological inhibition of SR (thapsigargin) and mitochondrial [carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP)] function were examined at rest. [Ca2+]i in WT remained elevated for the entire postcontraction recovery period (+6 ± 1% at 450 s), but in PGC-1α OE [Ca2+]i returned to resting baseline within 150 s. Thapsigargin immediately and substantially increased resting [Ca2+]i in WT, whereas in PGC-1α OE this effect was delayed and markedly diminished (WT, +12 ± 3; PGC-1α OE, +1 ± 2% at 600 s after thapsigargin treatment, P < 0.05). FCCP abolished this improvement of [Ca2+]i regulation in PGC-1α OE. Mitochondrial [Ca2+] accumulation was observed in PGC-1α OE following contractions and thapsigargin treatment. In the SR, PGC-1α OE downregulated SR Ca2+-ATPase 1 (Ca2+ uptake) and parvalbumin (Ca2+ buffering) protein levels, whereas mitochondrial Ca2+ uptake-related proteins (Mfn1, Mfn2, and mitochondrial Ca2+ uniporter) were upregulated. These data demonstrate a heretofore unappreciated role for skeletal muscle mitochondria in [Ca2+]i regulation in vivo following fatiguing tetanic contractions and at rest.
Assuntos
Cálcio/metabolismo , Contração Muscular , Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Animais , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Genótipo , Homeostase , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias Musculares/efeitos dos fármacos , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/inervação , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Fenótipo , Ionóforos de Próton/farmacologia , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Tapsigargina/farmacologia , Fatores de Tempo , Regulação para CimaRESUMO
The expression of the transcriptional coactivator PGC1α is increased in skeletal muscles during exercise. Previously, we showed that increased PGC1α leads to prolonged exercise performance (the duration for which running can be continued) and, at the same time, increases the expression of branched-chain amino acid (BCAA) metabolism-related enzymes and genes that are involved in supplying substrates for the TCA cycle. We recently created mice with PGC1α knockout specifically in the skeletal muscles (PGC1α KO mice), which show decreased mitochondrial content. In this study, global gene expression (microarray) analysis was performed in the skeletal muscles of PGC1α KO mice compared with that of wild-type control mice. As a result, decreased expression of genes involved in the TCA cycle, oxidative phosphorylation, and BCAA metabolism were observed. Compared with previously obtained microarray data on PGC1α-overexpressing transgenic mice, each gene showed the completely opposite direction of expression change. Bioinformatic analysis of the promoter region of genes with decreased expression in PGC1α KO mice predicted the involvement of several transcription factors, including a nuclear receptor, ERR, in their regulation. As PGC1α KO microarray data in this study show opposing findings to the PGC1α transgenic data, a loss-of-function experiment, as well as a gain-of-function experiment, revealed PGC1α's function in the oxidative energy metabolism of skeletal muscles.
Assuntos
Deleção de Genes , Regulação da Expressão Gênica , Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Transativadores/metabolismo , Animais , Biologia Computacional , Regulação para Baixo/genética , Masculino , Redes e Vias Metabólicas/genética , Camundongos Knockout , Mitocôndrias/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Oxirredução , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/genética , Transativadores/genéticaRESUMO
Leucine is known to increase mTOR-mediated phosphorylation of 4EBP. In this study, leucine was administered to skeletal muscle-PGC-1α knockout mice. We observed attenuated 4EBP phosphorylation in the skeletal muscle, but not in the liver, of the PGC-1α knockout mice. These data suggest that skeletal muscle-PGC-1α is important for leucine-mediated mTOR activation and protein biosynthesis.
Assuntos
Proteínas de Transporte/genética , Leucina/administração & dosagem , Músculo Esquelético/efeitos dos fármacos , Fosfoproteínas/genética , RNA Mensageiro/genética , Serina-Treonina Quinases TOR/genética , Fatores de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal , Administração Oral , Animais , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Fatores de Iniciação em Eucariotos , Regulação da Expressão Gênica , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Especificidade de Órgãos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fosfoproteínas/metabolismo , Fosforilação , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/deficiênciaRESUMO
Exercise training influences phospholipid fatty acid composition in skeletal muscle and these changes are associated with physiological phenotypes; however, the molecular mechanism of this influence on compositional changes is poorly understood. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a nuclear receptor coactivator, promotes mitochondrial biogenesis, the fiber-type switch to oxidative fibers, and angiogenesis in skeletal muscle. Because exercise training induces these adaptations, together with increased PGC-1α, PGC-1α may contribute to the exercise-mediated change in phospholipid fatty acid composition. To determine the role of PGC-1α, we performed lipidomic analyses of skeletal muscle from genetically modified mice that overexpress PGC-1α in skeletal muscle or that carry KO alleles of PGC-1α. We found that PGC-1α affected lipid profiles in skeletal muscle and increased several phospholipid species in glycolytic muscle, namely phosphatidylcholine (PC) (18:0/22:6) and phosphatidylethanolamine (PE) (18:0/22:6). We also found that exercise training increased PC (18:0/22:6) and PE (18:0/22:6) in glycolytic muscle and that PGC-1α was required for these alterations. Because phospholipid fatty acid composition influences cell permeability and receptor stability at the cell membrane, these phospholipids may contribute to exercise training-mediated functional changes in the skeletal muscle.
Assuntos
Músculo Esquelético/metabolismo , Fosfolipídeos/metabolismo , Condicionamento Físico Animal/fisiologia , Fatores de Transcrição/metabolismo , Animais , Humanos , Masculino , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fatores de Transcrição/genéticaRESUMO
A large percentage of energy produced during high-intensity exercise depends on the aerobic glycolytic pathway. Maintenance of a cytoplasmic redox balance ([NADH]/[NAD(+)] ratio) by the glycerophosphate shuttle involves sustained aerobic glycolysis. Glycerol 3-phosphate dehydrogenase 1 (GPD1) catalyzes an oxidation reaction in the glycerophosphate shuttle. In this study, we examined whether GPD1 deficiency decreases exercise capacity due to impairment of aerobic glycolysis by using the GPD1 null mouse model BALB/cHeA (HeA). Unexpectedly, we found that exercise endurance was significantly higher in HeA mice than in BALBc/By (By) mice used as controls. Furthermore, aerobic glycolysis in HeA mice was not impaired. During exercise, lipid oxidation was significantly higher in HeA mice than in By mice, concomitant with an increase in phosphorylation of AMP-activated protein kinase (AMPK). HeA mice also showed a delay in the onset of muscle glycogen usage and lactate production during exercise. These data suggest that contribution of lipid oxidation as a fuel source for exercise is increased in HeA mice, and GPD1 deficiency enhances exercise capacity by increasing lipid oxidation, probably due to activation of AMPK. We propose that GPD1 deficiency induces an adaptation that enhances lipid availability in the skeletal muscle during exercise.
Assuntos
Glicerolfosfato Desidrogenase/genética , Glicólise , Metabolismo dos Lipídeos , Condicionamento Físico Animal , Esforço Físico , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Deleção de Genes , Glicerolfosfato Desidrogenase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Músculo Esquelético/metabolismo , Oxirredução , Consumo de OxigênioRESUMO
Acute ethanol consumption leads to the accumulation of triglycerides (TGs) in hepatocytes. The increase in lipogenesis and reduction of fatty acid oxidation are implicated as the mechanisms underlying ethanol-induced hepatic TG accumulation. Although glycerol-3-phosphate (Gro3P), formed by glycerol kinase (GYK) or glycerol-3-phosphate dehydrogenase 1 (GPD1), is also required for TG synthesis, the roles of GYK and GPD1 have been the subject of some debate. In this study, we examine (1) the expression of genes involved in Gro3P production in the liver of C57BL/6J mice in the context of hepatic TG accumulation after acute ethanol intake, and (2) the role of GPD1 in the progression of ethanol-induced fatty liver using GPD1 null mice. As a result, in C57BL/6J mice, ethanol-induced hepatic TG accumulation began within 2h and was 1.7-fold greater than that observed in the control group after 6h. The up-regulation of GPD1 began 2h after administering ethanol, and significantly increased 6h later with the concomitant escalation in the glycolytic gene expression. The incorporation of (14)C-labelled glucose into TG glycerol moieties increased during the same period. On the other hand, in GPD1 null mice carrying normal GYK activity, no significant increase in hepatic TG level was observed after acute ethanol intake. In conclusion, GPD1 and glycolytic gene expression is up-regulated by ethanol, and GPD1-mediated incorporation of glucose into TG glycerol moieties together with increased lipogenesis, is suggested to play an important role in ethanol-induced hepatic TG accumulation.