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
We observed that exercise and calorie restriction reduced the body weight and blood glucose levels, concurrently improving insulin resistance and glucose tolerance in obese/diabetic model KKAy mice. Analysis of gene expression in the skeletal muscle showed enhanced mRNA levels of GLUT4 (glucose uptake), ATGL (lipolytic enzyme), and slow-twitch myosin heavy chain, which may contribute to the antiobesity and antidiabetic effects.
Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Animais , Camundongos , Restrição Calórica , Resistência à Insulina/fisiologia , Hipoglicemiantes/farmacologia , Músculo Esquelético/metabolismo , Obesidade/complicações , Obesidade/terapia , Obesidade/metabolismo , Glicemia/metabolismo , Insulina , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismoRESUMO
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
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
DNA methylation is an epigenetic mechanism regulating gene expression. In this study, we observed that DNA methyltransferase 3a (Dnmt3a) expression is decreased after muscle atrophy. We made skeletal muscle-specific Dnmt3a-knockout (Dnmt3a-KO) mice. The regeneration capacity after muscle injury was markedly decreased in Dnmt3a-KO mice. Diminished mRNA and protein expression of Dnmt3a were observed in skeletal muscles as well as in satellite cells, which are important for muscle regeneration, in Dnmt3a-KO mice. Dnmt3a-KO satellite cell showed smaller in size (length/area), suggesting suppressed myotube differentiation. Microarray analysis of satellite cells showed that expression of growth differentiation factor 5 (Gdf5) mRNA was markedly increased in Dnmt3a-KO mice. The DNA methylation level of the Gdf5 promoter was markedly decreased in Dnmt3a-KO satellite cells. In addition, DNA methylation inhibitor azacytidine treatment increased Gdf5 expression in wild-type satellite cells, suggesting Gdf5 expression is regulated by DNA methylation. Also, we observed increased inhibitor of differentiation (a target of Gdf5) mRNA expression in Dnmt3a-KO satellite cells. Thus, Dnmt3a appears to regulate satellite cell differentiation via DNA methylation. This mechanism may play a role in the decreased regeneration capacity during atrophy such as in aged sarcopenia.-Hatazawa, Y., Ono, Y., Hirose, Y., Kanai, S., Fujii, N. L., Machida, S., Nishino, I., Shimizu, T., Okano, M., Kamei, Y., Ogawa, Y. Reduced Dnmt3a increases Gdf5 expression with suppressed satellite cell differentiation and impaired skeletal muscle regeneration.
Assuntos
Diferenciação Celular , DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação da Expressão Gênica , Fator 5 de Diferenciação de Crescimento/biossíntese , Músculo Esquelético/fisiologia , Regeneração , Células Satélites de Músculo Esquelético/metabolismo , Animais , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Fator 5 de Diferenciação de Crescimento/genética , Camundongos Knockout , Músculo Esquelético/patologia , Células Satélites de Músculo Esquelético/patologiaRESUMO
We examined the effect of long-term exercise on the prevention of sarcopenia using a senescence-accelerated-prone mice (SAMP8) model. Mice were housed in a wheel cage for 25 weeks to increase voluntary exercise. At week 23, endurance running capacity was examined using a treadmill. In a treadmill running test, the wheel cage group had increased endurance running capacity, which suggests that aging-related loss of muscle function was recovered by long-term exercise. Mice were sacrificed and microarray analysis revealed that genes involved in protein synthesis and degradation were upregulated in the skeletal muscles of the wheel cage group, suggesting accelerated protein turnover. Total body and adipose tissue weights decreased following the use of the wheel cage. Thus, long-term, spontaneous physical exercise may assist in recovering from aging-related sarcopenia (loss of muscle function) and obesity.
Assuntos
Músculo Esquelético/metabolismo , Condicionamento Físico Animal , Envelhecimento , Animais , Peso Corporal , Perfilação da Expressão Gênica , Masculino , Camundongos , Músculo Esquelético/patologia , Músculo Esquelético/fisiologia , Sarcopenia/prevenção & controleRESUMO
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
Unloading stress, such as bed rest, inhibits the regenerative potential of skeletal muscles; however, the underlying mechanisms remain largely unknown. FOXO1 expression, which induces the upregulated expression of the cell cycle inhibitors p57 and Gadd45α, is known to be increased in the skeletal muscle under unloading conditions. However, there is no report addressing FOXO1-induced inhibition of myoblast proliferation. Therefore, we induced muscle injury by cardiotoxin in transgenic mice overexpressing FOXO1 in the skeletal muscle (FOXO1-Tg mice) and observed regeneration delay in skeletal muscle mass and cross-sectional area in FOXO1-Tg mice. Increased p57 and Gadd45α mRNA levels, and decreased proliferation capacity were observed in C2C12 myoblasts expressing a tamoxifen-inducible active form of FOXO1. These results suggest that decreased proliferation capacity of myoblasts by FOXO1 disrupts skeletal muscle regeneration under FOXO1-increased conditions, such as unloading.
Assuntos
Proteínas de Ciclo Celular/genética , Inibidor de Quinase Dependente de Ciclina p57/genética , Proteína Forkhead Box O1/genética , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Proteínas Nucleares/genética , Regeneração/genética , Animais , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Proteínas Cardiotóxicas de Elapídeos/toxicidade , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Proteína Forkhead Box O1/metabolismo , Regulação da Expressão Gênica , Elevação dos Membros Posteriores , Mecanotransdução Celular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Esquelético/lesões , Músculo Esquelético/patologia , Mioblastos/efeitos dos fármacos , Mioblastos/patologia , Proteínas Nucleares/metabolismoRESUMO
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
Skeletal muscle is a reservoir of energy in the form of protein, which is degraded under catabolic conditions, resulting in the formation of amino acids and ammonia as a byproduct. The expression of FOXO1, a forkhead-type transcription factor, increases during starvation and exercise. In agreement, transgenic FOXO1-Tg mice that overexpress FOXO1 in skeletal muscle exhibit muscle atrophy. The aim of this study was to examine the role of FOXO1 in amino acid metabolism. The mRNA and protein expressions of glutamine synthetase (GS) were increased in skeletal muscle of FOXO1-Tg mice. Fasting induced FOXO1 and GS expression in wild-type mice but hardly increased GS expression in muscle-specific FOXO1 knockout (FOXO1-KO) mice. Activation of FOXO1 also increased GS mRNA and protein expression in C2C12 myoblasts. Using a transient transfection reporter assay, we observed that FOXO1 activated the GS reporter construct. Mutation of a putative FOXO1-binding consensus sequence in the downstream genomic region of GS decreased basal and FOXO1-dependent reporter activity significantly. A chromatin immunoprecipitation assay showed that FOXO1 was recruited to the 3' region of GS in C2C12 myoblasts. These results suggest that FOXO1 directly upregulates GS expression. GS is considered to mediate ammonia clearance in skeletal muscle. In agreement, an intravenous ammonia challenge increased blood ammonia concentrations to a twofold higher level in FOXO1-KO than in wild-type mice, demonstrating that the capacity for ammonia disposal correlated inversely with the expression of GS in muscle. These data indicate that FOXO1 plays a role in amino acid metabolism during protein degradation in skeletal muscle.
Assuntos
Regiões 3' não Traduzidas/fisiologia , Amônia/metabolismo , Fatores de Transcrição Forkhead/fisiologia , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/fisiologia , Músculo Esquelético/enzimologia , Regiões 3' não Traduzidas/genética , Aminoácidos/metabolismo , Amônia/toxicidade , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Proteína Forkhead Box O1 , Regulação da Expressão Gênica/fisiologia , Glutamina/metabolismo , Camundongos , Camundongos Transgênicos , Plasmídeos/genética , Reação em Cadeia da Polimerase em Tempo Real , TransfecçãoRESUMO
Previous studies indicate that muscle Pgc-1α expression governs the proportion of muscle fibre types. As a first step in using diet to manipulate the proportion of muscle fibre types by using Pgc-1α expression, the present study investigates the modulation of Pgc-1α expression by feedstuffs. A luciferase-based Pgc-1α reporter construct (Pgc-1α(-2553)-luc) that contains the mouse Pgc-1α promoter (-2553 to +78 bp) was prepared. A screen of ethanol extracts from 33 feedstuffs indicated that oolong tea and roasted green tea extracts decreased Pgc-1α(-2553)-luc expression in C2C12 myoblasts. The transcriptional repression of Pgc-1α by tea leaf extracts was reproduced in hepatic HepG2 cells. We further examined the effects of the alcohol extracts of tea waste and its silage on Pgc-1α transcription; the tea waste silage extract inhibited Pgc-1α transcription. Treatment with the extracts of raw tea leaves, tea waste and tea waste silage effectively decreased Pgc-1α mRNA levels during myogenesis of myosatellite cells. The present results suggest that tea leaves and their by-products could be used to modulate proportions of muscle fibre types.
Assuntos
Camellia sinensis/química , Extratos Vegetais/farmacologia , Folhas de Planta/química , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Chá , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Regulação para Baixo , Masculino , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Regiões Promotoras Genéticas , Ratos , Ratos Wistar , Células Satélites de Músculo Esquelético/metabolismo , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
The neuromuscular junction (NMJ)-formed between a motor nerve terminal and skeletal muscle fiber-plays an important role in muscle contraction and other muscle functions. Aging and neurodegeneration worsen NMJ formation and impair muscle function. Downstream of tyrosine kinase-7 (Dok-7), expressed in skeletal muscle fibers, is essential for the formation of NMJ. Exercise increases the expression of the transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) in skeletal muscles and restores NMJ formation. In this study, we used skeletal muscle-specific PGC1α knockout or overexpression mice to examine the role of PGC1α in regulating Dok-7 expression and NMJ formation. Our findings revealed that Dok-7 expression is regulated by PGC1α, and luciferase activity of the Dok-7 promoter is greatly increased by coexpressing PGC1α and estrogen receptor-related receptor α. Thus, we suggest PGC1α is involved in exercise-mediated restoration of NMJ formation.
Assuntos
Junção Neuromuscular , PPAR gama , Animais , Camundongos , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Junção Neuromuscular/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , PPAR gama/metabolismo , Proteínas Tirosina Quinases/metabolismoRESUMO
DNA methylation is a key epigenetic contributor to gene regulation in mammals. We have recently found that in the mouse liver, the promoter region of glycerol-3-phosphate acyltransferase 1, a rate-limiting enzyme of de novo lipogenesis, is regulated by DNA methylation, which is mediated by Dnmt3b, an enzyme required for the initiation of de novo methylation. In this study, using primary cultures of mouse hepatocytes with adenoviral overexpression of Dnmt3b, we characterized Dnmt3b-dependent DNA methylation on a genome-wide basis. A genome-wide DNA methylation analysis, called microarray-based integrated analysis of methylation by isoschizomers, identified 108 genes with Dnmt3b dependent DNA methylation. In DNA expression array analysis, expression of some genes with Dnmt3b-dependent DNA methylation was suppressed. Studies with primary mouse hepatocytes overexpressing Dnmt3b or Dnmt3a revealed that many genes with Dnmt3b-dependent methylation are not methylated by Dnmt3a, whereas those methylated by Dnmt3a are mostly methylated by Dnmt3b. Bioinformatic analysis showed that the CANAGCTG and CCGGWNCSC (N denotes A, T, G, or C; W denotes A or T; and S denotes C or G) sequences are enriched in genes methylated by overexpression of Dnmt3b and Dnmt3a, respectively. We also observed a large number of genes with Dnmt3b-dependent DNA methylation in primary cultures of mouse hepatocytes with adenoviral overexpression of Dnmt3, suggesting that Dnmt3b is an important DNA methyltransferase in primary mouse hepatocytes, targets specific genes, and potentially plays a role in vivo.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Animais , Sequência de Bases , Western Blotting , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Hepatócitos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Motivos de Nucleotídeos/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , DNA Metiltransferase 3BRESUMO
Nutritional deprivation or malnutrition suppresses immune function in humans and animals, thereby conferring higher susceptibility to infectious diseases. Indeed, nutritional deprivation induces atrophy of lymphoid tissues such as thymus and spleen and decreases the number of circulating lymphocytes. Leptin, a major adipocytokine, is exclusively produced in the adipose tissue in response to the nutritional status and acts on the hypothalamus, thereby regulating energy homeostasis. Although leptin plays a critical role in the starvation-induced T-cell-mediated immunosuppression, little is known about its role in B-cell homeostasis under starvation conditions. Here we show the alteration of B-cell development in the bone marrow of fasted mice, characterized by decrease in pro-B, pre-B, and immature B cells and increase in mature B cells. Interestingly, intracerebroventricular leptin injection was sufficient to prevent the alteration of B-cell development of fasted mice. The alteration of B lineage cells in the bone marrow of fasted mice was markedly prevented by oral administration of glucocorticoid receptor antagonist RU486 (11ß-[p-(dimethylamino)phenyl]-17ß-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one). It was also effectively prevented by intracerebroventricular injection of neuropeptide Y Y(1) receptor antagonist BIBP3226 [(2R)-5-(diaminomethylideneamino)-2-[(2,2-diphenylacetyl)amino]-N-[(4-hydroxyphenyl)methyl]pentanamide], along with suppression of the otherwise increased serum corticosterone concentrations. This study provides the first in vivo evidence for the role of central leptin signaling in the starvation-induced alteration of B-cell development. The data of this study suggest that the CNS, which is inherent to integrate information from throughout the organism, is able to control immune function.
Assuntos
Linfócitos B/metabolismo , Diferenciação Celular/fisiologia , Hipotálamo/metabolismo , Leptina/metabolismo , Transdução de Sinais/fisiologia , Inanição/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/imunologia , Tecido Adiposo/metabolismo , Adrenalectomia , Animais , Linfócitos B/imunologia , Glicemia , Diferenciação Celular/efeitos dos fármacos , Citometria de Fluxo , Hipotálamo/efeitos dos fármacos , Hipotálamo/imunologia , Leptina/farmacologia , Masculino , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Inanição/imunologiaRESUMO
Obesity may be viewed as a state of chronic low-grade inflammation that participates in the development of the metabolic syndrome. Nonalcoholic steatohepatitis (NASH) is considered a hepatic phenotype of the metabolic syndrome and a high risk for progression to cirrhosis and hepatocellular carcinoma. Although the "two hit" hypothesis suggests involvement of excessive hepatic lipid accumulation and chronic inflammation, the molecular mechanisms underlying the development of NASH remain unclear, in part because of lack of appropriate animal models. Herein we report that melanocortin 4 receptor-deficient mice (MC4R-KO) develop steatohepatitis when fed a high-fat diet, which is associated with obesity, insulin resistance, and dyslipidemia. Histologic analysis reveals inflammatory cell infiltration, hepatocyte ballooning, and pericellular fibrosis in the liver in MC4R-KO mice. Of note, all of the MC4R-KO mice examined developed well-differentiated hepatocellular carcinoma after being fed a high-fat diet for 1 year. They also demonstrated enhanced adipose tissue inflammation, ie, increased macrophage infiltration and fibrotic changes, which may contribute to excessive lipid accumulation and enhanced fibrosis in the liver. Thus, MC4R-KO mice provide a novel mouse model of NASH with which to investigate the sequence of events that make up diet-induced hepatic steatosis, liver fibrosis, and hepatocellular carcinoma and to aid in understanding the pathogenesis of NASH, pursuing specific biomarkers, and evaluating potential therapeutic strategies.
Assuntos
Fígado Gorduroso/etiologia , Receptor Tipo 4 de Melanocortina/deficiência , Animais , Carcinoma Hepatocelular/etiologia , Modelos Animais de Doenças , Metabolismo dos Lipídeos/fisiologia , Peróxidos Lipídicos/metabolismo , Cirrose Hepática/etiologia , Neoplasias Hepáticas/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica , Estresse Oxidativo/fisiologia , Fenótipo , Triglicerídeos/metabolismoRESUMO
Amino acids are compounds that contain an amino group (-NH2) and a carboxyl group (-COOH) and are components of proteins and materials for various bioactive molecules. The skeletal muscle, which is the largest organ in the human body, representing ~40% of the total body weight, plays important roles in exercise, energy expenditure, and glucose/amino acid usage-processes that are modulated by various amino acids and their metabolites. In this review, we address the metabolism and function of amino acids, especially non-proteinogenic amino acids, in the skeletal muscle. Leucine, a BCAA, and its metabolite, ß-hydroxy-ß-methylbutyrate (HMB), both activate mammalian target of rapamycin complex 1 (mTORC1) and increase protein synthesis, but the mechanisms of activation appear to be different. The metabolite of valine (another BCAA), ß-aminoisobutyric acid (BAIBA), is increased by exercise, is secreted by the skeletal muscle, and acts on other tissues, such as white adipose tissue, to increase energy expenditure. In addition, several amino acid-related molecules reportedly activate skeletal muscle function. Oral 5-aminolevulinic acid (ALA) supplementation can protect against mild hyperglycemia and help prevent type 2 diabetes. ß-alanine levels are decreased in the skeletal muscles of aged mice. ß-alanine supplementation increased the physical performance and improved the executive function induced by endurance exercise in middle-aged individuals. Further studies focusing on the effects of amino acids and their metabolites on skeletal muscle function will provide data essential for the production of food supplements for older adults, athletes, and individuals with metabolic diseases.
Assuntos
Aminoácidos , Diabetes Mellitus Tipo 2 , Pessoa de Meia-Idade , Humanos , Animais , Camundongos , Idoso , Músculo Esquelético , beta-Alanina/farmacologia , Leucina/farmacologia , MamíferosRESUMO
Sarcopenia and frailty are urgent socio-economic problems worldwide. Here we demonstrate a functional connection between the lateral hypothalamus (LH) and skeletal muscle through Slc12a8, a recently identified nicotinamide mononucleotide transporter, and its relationship to sarcopenia and frailty. Slc12a8-expressing cells are mainly localized in the LH. LH-specific knockdown of Slc12a8 in young mice decreases activity-dependent energy and carbohydrate expenditure and skeletal muscle functions, including muscle mass, muscle force, intramuscular glycolysis, and protein synthesis. LH-specific Slc12a8 knockdown also decreases sympathetic nerve signals at neuromuscular junctions and ß2-adrenergic receptors in skeletal muscle, indicating the importance of the LH-sympathetic nerve-ß2-adrenergic receptor axis. LH-specific overexpression of Slc12a8 in aged mice significantly ameliorates age-associated decreases in energy expenditure and skeletal muscle functions. Our results highlight an important role of Slc12a8 in the LH for regulation of whole-body metabolism and skeletal muscle functions and provide insights into the pathogenesis of sarcopenia and frailty during aging.
Assuntos
Fragilidade , Sarcopenia , Envelhecimento/fisiologia , Animais , Metabolismo Energético , Fragilidade/metabolismo , Fragilidade/patologia , Região Hipotalâmica Lateral , Camundongos , Músculo Esquelético/metabolismo , Sarcopenia/metabolismoRESUMO
Vitamin D is a fat-soluble molecule, well known for its role in regulating calcium homeostasis in bone. It has become increasingly clear that it also has important effects in many other organs, including the skeletal muscle. In order to gain insight into the role of vitamin D in the skeletal muscle, we performed microarray analysis using C2C12 myoblasts treated with 1,25-dihydroxyvitamin D (1,25(OH)2D), active form of vitamin D. We found multiple genes upregulated by 1,25(OH)2D. Some of them, i.e., vitamin D receptor (Vdr), diacylglycerol O-acyltransferase (Dgat1 and Dgat2, the rate limiting steps of triacylglycerol acylation), and vascular endothelial growth factor A (Vegfa), were previously reported to be upregulated by 1,25(OH)2D in C2C12 cells. RT-qPCR analysis confirmed increased mRNA levels of Rarres2, Dio2, Tgm2, Lpl, Mdfi, Igfbp3, Dgat1, Crabp2, Gadd45a, Vagfa, Dgat2, C3, Ldhb, Cebpa, Igfbp5, Mrc2, Vdr. Thus, many genes, including lipid metabolism genes as well as genes related to muscle functions, appear to be upregulated by 1,25(OH)2D in muscle cells.