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Metabolites derived from the intestinal microbiota play an important role in maintaining skeletal muscle growth, function, and metabolism. Here, we found that D-malate (DMA) is produced by mouse intestinal microorganisms and its levels increase during aging. Moreover, we observed that dietary supplementation of 2% DMA inhibits metabolism in mice, resulting in reduced muscle mass, strength, and the number of blood vessels, as well as the skeletal muscle fiber type I/IIb ratio. In vitro assays demonstrate that DMA decreases the proliferation of vascular endothelial cells and suppresses the formation of blood vessels. In vivo, we further demonstrated that boosting angiogenesis by muscular VEGFB injection rescues the inhibitory effects of D-malate on muscle mass and fiber area. By transcriptomics analysis, we identified that the mechanism underlying the effects of DMA depends on the elevated intracellular acetyl-CoA content and increased Cyclin A acetylation rather than redox balance. This study reveals a novel mechanism by which gut microbes impair muscle angiogenesis and may provide a therapeutic target for skeletal muscle dysfunction in cancer or aging.
Assuntos
Células Endoteliais , Microbiota , Camundongos , Animais , Células Endoteliais/metabolismo , Acetilação , Ciclina A/metabolismo , Angiogênese , Malatos/metabolismo , Músculo Esquelético/metabolismo , EnvelhecimentoRESUMO
Beneficial effects of resistance exercise on metabolic health and particularly muscle hypertrophy and fat loss are well established, but the underlying chemical and physiological mechanisms are not fully understood. Here, we identified a myometabolite-mediated metabolic pathway that is essential for the beneficial metabolic effects of resistance exercise in mice. We showed that substantial accumulation of the tricarboxylic acid cycle intermediate α-ketoglutaric acid (AKG) is a metabolic signature of resistance exercise performance. Interestingly, human plasma AKG level is also negatively correlated with BMI. Pharmacological elevation of circulating AKG induces muscle hypertrophy, brown adipose tissue (BAT) thermogenesis, and white adipose tissue (WAT) lipolysis in vivo. We further found that AKG stimulates the adrenal release of adrenaline through 2-oxoglutarate receptor 1 (OXGR1) expressed in adrenal glands. Finally, by using both loss-of-function and gain-of-function mouse models, we showed that OXGR1 is essential for AKG-mediated exercise-induced beneficial metabolic effects. These findings reveal an unappreciated mechanism for the salutary effects of resistance exercise, using AKG as a systemically derived molecule for adrenal stimulation of muscle hypertrophy and fat loss.
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Ácidos Cetoglutáricos/sangue , Atrofia Muscular/genética , Receptores Purinérgicos P2/genética , Treinamento Resistido/métodos , Adulto , Idoso , Animais , Linhagem Celular , Feminino , Técnicas de Inativação de Genes , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Modelos Animais , Atrofia Muscular/metabolismo , Receptores Purinérgicos P2/metabolismoRESUMO
BACKGROUND/AIMS: Brown adipose tissue (BAT) is critical for mammals' survival in the cold environment. BAT-dependent non-shivering thermogenesis is attributed to uncoupling protein 1 (UCP1)'s disengagement of oxidative phosphorylation from ATP synthesis and dissipates energy as heat. Thus individuals with a substantial amount of BAT are better equipped during cold stress and less likely to become obese. Recently, our laboratory has shown pig adipocytes have no UCP1 protein. The inability of newborn piglets to generate heat contributed to its high death rate. Repairing the genetic defect of UCP1 in pig adipocytes has implications in defending against cold for piglets and developing an alternative treatment for human obesity. METHODS: Q-PCR, western blotting (WB) and oxygen consumption measurement were used to enable functional UCP1 protein in preadipocytes. Immunoprecipitation (IP), chromatin immunoprecipitation (CHIP), and dual-luciferase reporter assay system were used to clarify the thermogenesis mechanism of functional UCP1. RESULTS: Only co-overexpressing mice UCP1 and pig PGC-1α increased not only the mitochondrial number but also the uncoupled respiration rate in the transfected pig adipocytes. The functional mice UCP1 increased the pig PGC-1α activity through the AMPK-SIRT1 pathway. The active form PGC-1α interacted with transcription factors Lhx8, Zic1, ERRα, and PPARα to regulate the expression of mitochondrial energy metabolism and adipocytes browning-related genes. CONCLUSION: Our data suggest a model in which pig PGC-1α and mice UCP1 work collaboratively to restore uncoupling respiration in pig preadipocytes. These results have great implications for piglet survival and developing an alternative treatment for human obesity in the future.
Assuntos
Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Proteína Desacopladora 1/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Tecido Adiposo Marrom/metabolismo , Animais , Metabolismo Energético/genética , Proteínas de Transporte de Ácido Graxo/genética , Proteínas de Transporte de Ácido Graxo/metabolismo , Camundongos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Regiões Promotoras Genéticas , Transdução de Sinais , Sirtuína 1/metabolismo , Suínos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Desacopladora 1/genéticaRESUMO
Brown adipose tissue (BAT) is critical for mammal's survival in the cold environment. Uncoupling protein 1 (UCP1) is responsible for the non-shivering thermogenesis in the BAT. Pig is important economically as a meat-producing livestock. However, whether BAT or more precisely UCP1 protein exists in pig remains a controversy. The objective of this study was to ascertain whether pig has UCP1 protein. In this study, we used rapid amplification of cDNA ends (RACE) technique to obtain the UCP1 mRNA 3' end sequence, confirmed only exons 1 and 2 of the UCP1 gene are transcribed in the pig. Then we cloned the pig UCP1 gene exons 1 and 2, and expressed the UCP1 protein from the truncated pig gene using E. coli BL21. We used the expressed pig UCP1 protein as antigen for antibody production in a rabbit. We could not detect any UCP1 protein expression in different pig adipose tissues by the specific pig UCP1 antibody, while our antibody can detect the cloned pig UCP1 as well as the mice adipose UCP1 protein. This result shows although exons 1 and 2 of the pig UCP1 gene were transcribed but not translated in the pig adipose tissue. Furthermore, we detected no uncoupled respiration in the isolated pig adipocytes. Thus, these results unequivocally demonstrate that pig has no UCP1 protein. Our results have resolved the controversy of whether pigs have the brown adipose tissue.
Assuntos
Proteína Desacopladora 1/genética , Animais , Masculino , SuínosRESUMO
BACKGROUND: Gut microbiota and their metabolites play a regulatory role in skeletal muscle growth and development, which be known as gut-muscle axis. 3-phenylpropionic acid (3-PPA), a metabolite produced by colonic microorganisms from phenylalanine in the gut, presents in large quantities in the blood circulation. But few study revealed its function in skeletal muscle development. RESULTS: Here, we demonstrated the beneficial effects of 3-PPA on muscle mass increase and myotubes hypertrophy both in vivo and vitro. Further, we discovered the 3-PPA effectively inhibited protein degradation and promoted protein acetylation in C2C12 and chick embryo primary skeletal muscle myotubes. Mechanistically, we supported that 3-PPA reduced NAD+ synthesis and subsequently suppressed tricarboxylic acid cycle and the mRNA expression of SIRT1/3, thus promoting the acetylation of total protein and Foxo3. Moreover, 3-PPA may inhibit Foxo3 activity by directly binding. CONCLUSIONS: This study firstly revealed the effect of 3-PPA on skeletal muscle growth and development, and newly discovered the interaction between 3-PPA and Foxo3/NAD+ which mechanically promote myotubes hypertrophy. These results expand new understanding for the regulation of gut microbiota metabolites on skeletal muscle growth and development.
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The beneficial effect of probiotics on host health is impaired due to the substantial loss of survivability during gastric transit caused by small intestinal enzymes and bile acids. Encapsulation helps to preserve the probiotics species from severe environmental factors. Lactobacillus paracasei, highly sensitive probiotic species to gastric acid, was encapsulated with polyacrylate resin. C57BL/6 male mice were equally divided into three groups; control group was fed with basal diet without any additives, the un-encapsulated group was fed with 0.1% of a mixture of encapsulating material and L. paracasei, and encapsulated group was fed with 0.1% encapsulated L. paracasei (microcapsule) for 4 weeks. The result showed elevated fecal moisture percentage in the encapsulated group, but not in the un-encapsulated group. Further study showed that the ratio of villus height to crypt depth in the small intestine was significantly higher compared to un-encapsulated and the control group. Microencapsulated probiotics also remarkably increased intestinal mucin and secretory immunoglobulin A (sIgA) concentration, intestinal MUC-2, and tight junction protein mRNA expression levels improving the intestinal barrier function of mice. In addition, microcapsules also reduced proinflammatory factor mRNA expression, while considerably increasing anti-inflammatory factor mRNA expression. Microbiota metabolites, fecal LPS (Lipopolysaccharide) were downregulated, and acetate and lactate were upraised compared to control. Furthermore, glutathione peroxidase (GSH-Px) and TAOC levels were increased and Malondialdehyde (MDA) was decreased improving antioxidant capacity. Microflora and bioinformatic predictive analysis of feces showed that encapsulated probiotics remarkably increased Lactobacillus proportions. Mice's intestinal health can thus be improved by using microencapsulated probiotics.
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Previously, we found that α-ketoglutaric acid (AKG) stimulates muscle hypertrophy and fat loss through 2-oxoglutarate receptor 1 (OXGR1). Here, we demonstrated the beneficial effects of AKG on glucose homeostasis in a diet-induced obesity (DIO) mouse model, which are independent of OXGR1. We also showed that AKG effectively decreased blood glucose and hepatic gluconeogenesis in DIO mice. By using transcriptomic and liver-specific serpina1e deletion mouse model, we further demonstrated that liver serpina1e is required for the inhibitory effects of AKG on hepatic gluconeogenesis. Mechanistically, we supported that extracellular AKG binds with a purinergic receptor, P2RX4, to initiate the solute carrier family 25 member 11 (SLC25A11)-dependent nucleus translocation of intracellular AKG and subsequently induces demethylation of lysine 27 on histone 3 (H3K27) in the seprina1e promoter region to decrease hepatic gluconeogenesis. Collectively, these findings reveal an unexpected mechanism for control of hepatic gluconeogenesis using circulating AKG as a signal molecule.
Assuntos
Diabetes Mellitus , Hiperglicemia , Animais , Diabetes Mellitus/metabolismo , Gluconeogênese , Hiperglicemia/tratamento farmacológico , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/farmacologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Endurance training and explosive strength training, with different contraction protein and energy metabolism adaptation in skeletal muscle, are both beneficial for physical function and quality of life. Our previous study found that chronic succinate feeding enhanced the endurance exercise of mice by inducing skeletal muscle fiber-type transformation. The purpose of this study is to investigate the effect of acute succinate administration on skeletal muscle explosive strength and its potential mechanism. Succinate was injected to mature mice to explore the acute effect of succinate on skeletal muscle explosive strength. And C2C12 cells were used to verify the short-term effect of succinate on oxidative phosphorylation. Then the cells interfered with succinate receptor 1 (SUCNR1) siRNA, and the SUCNR1-GKO mouse model was used for verifying the role of SUCNR1 in succinate-induced muscle metabolism and expression and explosive strength. The results showed that acute injection of succinate remarkably improved the explosive strength in mice and also decreased the ratio of nicotinamide adenine dinucleotide (NADH) to NAD+ and increased the mitochondrial complex enzyme activity and creatine kinase (CK) activity in skeletal muscle tissue. Similarly, treatment of C2C12 cells with succinate revealed that succinate significantly enhanced oxidative phosphorylation with increased adenosine triphosphate (ATP) content, CK, and the activities of mitochondrial complex I and complex II, but with decreased lactate content, reactive oxygen species (ROS) content, and NADH/NAD+ ratio. Moreover, the succinate's effects on oxidative phosphorylation were blocked in SUCNR1-KD cells and SUCNR1-KO mice. In addition, succinate-induced explosive strength was also abolished by SUCNR1 knockout. All the results indicate that acute succinate administration increases oxidative phosphorylation and skeletal muscle explosive strength in a SUCNR1-dependent manner.
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Pig is a useful medical model for humans due to its similarity in size and physiology. Skeletal muscle plays an essential role in body movement. However, the skeletal muscle injuries are common. Skeletal muscle function maintenance largely depends on preserving the regenerative capacity of muscle. Muscle satellite cells proliferation plays an essential role in postnatal muscle growth and regeneration. Therefore, understanding the mechanisms associated with muscle satellite cells proliferation is essential for devising the alternative treatments for muscle injury. Previous studies showed JAG1-Notch1 signaling pathway and miRNAs regulate the skeletal muscle development. JAG1-Notch1 signal pathway regulates the transcription of certain types of miRNAs which further affects target gene expression. However, the specific relationship between JAG1-Notch1 signal pathway and miRNAs during muscle development has not been established. We found overexpression of intracellular domain of the Notch1 protein (N1ICD) in porcine muscle satellite cells (PSCs) decreased miR-199b level. We demonstrated that miR-199b inhibits PSCs proliferation using the overexpression and inhibition of miR-199b experiment. We also found JAG1, the miR-199b target gene, promotes PSCs proliferation through activating the Notch1 signal pathway. Furthermore, we demonstrated miR-199b forms a feedback loop with the JAG1-Notch1 signal pathway to maintain the PSCs niche homeostasis. Our results of miRNAs and genes work collaboratively in regulating PSCs proliferation expand our understanding in PSCs proliferation mechanism. Furthermore, this finding indicates miR-199b is a potential therapeutic target for muscle atrophy.
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Proteína Jagged-1/genética , MicroRNAs/genética , Células Satélites de Músculo Esquelético/citologia , Regiões 3' não Traduzidas , Animais , Animais Recém-Nascidos , Proliferação de Células , Células Cultivadas , Regulação da Expressão Gênica , Masculino , Receptor Notch1/metabolismo , Células Satélites de Músculo Esquelético/química , Transdução de Sinais , Sus scrofa , SuínosRESUMO
The positive regulatory domain containing 16 (PRDM16) gene is a dominant transcriptional regulator that favors the "browning" of white adipocytes in rodents. Since the "browning" of white fat is important in pig in terms of producing heat fighting against cold environment, avoiding obesity, and improving meat quality, understanding the critical role that PRDM16 gene played in pig adipose "browning" and energy metabolism is of great significance. However, the constitution of pig fat differs a lot from rodents and human as they do not have brown adipose tissue (BAT) even in the newborn piglets. In this study, we isolated porcine primary preadipocytes and investigated the function of PRDM16 during preadipocytes differentiation. Our results showed that overexpression of the PR domain of PRDM16 repressed the differentiation of porcine preadipocytes, indicated by oil red O staining and the deposition of the triglyceride. Overexpression of the PR domain significantly increased the level of lipolysis and mitochondrial oxidative capacity detected by Western blotting during differentiation. Furthermore, we purified the protein coded by the PR domain and demonstrated that this protein has the H3K9me1 methyltransferase activity. In conclusion, the PR domain of the porcine PRDM16 gene repressed the mature of the porcine preadipocytes by promoting its oxidative activity.