RESUMO
During myoblast differentiation, mitochondria undergo numerous changes that are necessary for the progression of the myogenic program. Notably, we previously showed that alteration in mitochondrial activity was able to control the expression of keys regulator of cell cycle withdrawal and terminal differentiation. Here, we assessed whether inhibition of one of the respiratory complexes was a key factor in the regulation of myogenic differentiation in C2C12 cells, and was associated with alteration in reactive oxygen species (ROS) production. C2C12 cells were treated from proliferation to differentiation with specific inhibitors of mitochondrial complexes at a concentration that were inhibiting respiration but not altering cell morphology. Proliferation was significantly repressed with inhibition of complexes I, II, and III, or mitochondrial protein synthesis (using Chloramphenicol treatment), while complex IV inhibition did not alter myoblast proliferation compared to control cells. Moreover, inhibition of complexes I and II altered cell cycle regulators, with p21 protein expression upregulated since proliferation and p27 protein expression reduced at differentiation. Myotubes formation and myogenin expression were blunted with complexes I and II inhibitors while MyoD protein expression was maintained, suggesting an alteration in its transcriptional activity. Finally, a decrease in overall ROS production was observed with continuous inhibition of mitochondrial complexes I-IV. In summary, our data provide evidence that complexes I and II may be the primary regulators of C2C12 myogenic differentiation. This occurs through specific regulation of myogenic rather than cell cycle regulators expression and ROS production at mitochondrial rather than cell level.
Assuntos
Diferenciação Celular/fisiologia , Complexo II de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Mioblastos/metabolismo , Animais , Linhagem Celular , Transporte de Elétrons/fisiologia , Camundongos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Thyroid hormone is a major regulator of skeletal muscle development and repair, and also a key regulator of mitochondrial activity. We have previously identified a 43 kDa truncated form of the nuclear T3 receptor TRα1 (p43) which stimulates mitochondrial activity and regulates skeletal muscle features. However, its role in skeletal muscle regeneration remains to be addressed. To this end, we performed acute muscle injury induced by cardiotoxin in mouse tibialis in two mouse models where p43 is overexpressed in or depleted from skeletal muscle. The measurement of muscle fiber size distribution at different time point (up to 70 days) upon injury lead us to unravel requirement of the p43 signaling pathway for satellite cells dependent muscle regeneration; strongly delayed in the absence of p43; whereas the overexpression of the receptor enhances of the regeneration process. In addition, we found that satellite cells derived from p43-Tg mice display higher proliferation rates when cultured in vitro when compared to control myoblasts, whereas p43-/- satellites shows reduced proliferation capacity. These finding strongly support that p43 plays an important role in vivo by controling the duration of skeletal muscle regeneration after acute injury, possibly through the regulation of mitochondrial activity and myoblasts proliferation.
Assuntos
Mitocôndrias/metabolismo , Músculo Esquelético/fisiopatologia , Receptores alfa dos Hormônios Tireóideos/metabolismo , Animais , Proliferação de Células , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , Músculo Esquelético/lesões , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Regeneração , Células Satélites de Músculo Esquelético/metabolismo , Receptores alfa dos Hormônios Tireóideos/genéticaRESUMO
BACKGROUND: Skeletal muscle atrophy is a common feature of numerous chronic pathologies and is correlated with patient mortality. The REDD1 protein is currently recognized as a negative regulator of muscle mass through inhibition of the Akt/mTORC1 signaling pathway. REDD1 expression is notably induced following glucocorticoid secretion, which is a component of energy stress responses. RESULTS: Unexpectedly, we show here that REDD1 instead limits muscle loss during energetic stresses such as hypoxia and fasting by reducing glycogen depletion and AMPK activation. Indeed, we demonstrate that REDD1 is required to decrease O2 and ATP consumption in skeletal muscle via reduction of the extent of mitochondrial-associated endoplasmic reticulum membranes (MAMs), a central hub connecting energy production by mitochondria and anabolic processes. In fact, REDD1 inhibits ATP-demanding processes such as glycogen storage and protein synthesis through disruption of the Akt/Hexokinase II and PRAS40/mTORC1 signaling pathways in MAMs. Our results uncover a new REDD1-dependent mechanism coupling mitochondrial respiration and anabolic processes during hypoxia, fasting, and exercise. CONCLUSIONS: Therefore, REDD1 is a crucial negative regulator of energy expenditure that is necessary for muscle adaptation during energetic stresses. This present study could shed new light on the role of REDD1 in several pathologies associated with energetic metabolism alteration, such as cancer, diabetes, and Parkinson's disease.
Assuntos
Metabolismo Energético/genética , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Estresse Fisiológico/genética , Fatores de Transcrição/fisiologia , Adaptação Fisiológica/genética , Animais , Hipóxia Celular , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Atrofia Muscular/genética , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
Cardiolipin (CL) is a phospholipid at the heart of mitochondrial metabolism, which plays a key role in mitochondrial function and bioenergetics. Among mitochondrial activity regulators, SIRT3 plays a crucial role in controlling the acetylation status of many enzymes participating in the energy metabolism in particular concerning lipid metabolism and fatty acid oxidation. Data suggest that possible connection may exist between SIRT3 and CL status that has not been evaluated in skeletal muscle. In the present study, we have characterized skeletal muscle lipids as well as mitochondrial lipids composition in mice overexpressing long (SIRT3-M1) and short (SIRT3-M3) isoforms of SIRT3. Particular attention has been paid for CL. We reported no alteration in muscle lipids content and fatty acids composition between the two mice SIRT3 strains and the control mice. However, mitochondrial CL content was significantly decreased in SIRT3-M3 mice and associated to an upregulation of tafazzin gene expression. In addition, mitochondrial phospholipids and fatty acids composition was altered with an increase in the PC/PE ratio and arachidonic acid content and a reduction in the MUFA/SFA ratio. These modifications in mitochondrial membrane composition are associated with a reduction in the enzymatic activities of mitochondrial respiratory chain complexes I and IV. In spite of these mitochondrial enzymatic alterations, skeletal muscle mitochondrial respiration remained similar in SIRT3-M3 and control mice. Surprisingly, none of those metabolic alterations were detected in mitochondria from SIRT3-M1 mice. In conclusion, our data indicate a specific action of the shorter SIRT3 isoform on lipid mitochondrial membrane biosynthesis and functioning.
Assuntos
Cardiolipinas/metabolismo , Ácidos Graxos/metabolismo , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Sirtuína 3/fisiologia , Animais , Transporte de Elétrons , Camundongos , Membranas Mitocondriais/química , Membranas Mitocondriais/metabolismo , Fosfolipídeos/metabolismo , Isoformas de ProteínasRESUMO
Thyroid hormone is a major regulator of metabolism and mitochondrial function. Thyroid hormone also affects reactions in almost all pathways of lipids metabolism and as such is considered as the main hormonal regulator of lipid biogenesis. The aim of this study was to explore the possible involvement of p43, a 43 Kda truncated form of the nuclear thyroid hormone receptor TRα1 which stimulates mitochondrial activity. Therefore, using mouse models overexpressing p43 in skeletal muscle (p43-Tg) or lacking p43 (p43-/-), we have investigated the lipid composition in quadriceps muscle and in mitochondria. Here, we reported in the quadriceps muscle of p43-/- mice, a fall in triglycerides, an inhibition of monounsaturated fatty acids (MUFA) synthesis, an increase in elongase index and an decrease in desaturase index. However, in mitochondria from p43-/- mice, fatty acid profile was barely modified. In the quadriceps muscle of p43-Tg mice, MUFA content was decreased whereas the unsaturation index was increased. In addition, in quadriceps mitochondria of p43-Tg mice, we found an increase of linoleic acid level and unsaturation index. Last, we showed that cardiolipin content, a key phospholipid for mitochondrial function, remained unchanged both in quadriceps muscle and in its mitochondria whatever the mice genotype. In conclusion, this study shows that muscle lipid content and fatty acid profile are strongly affected in skeletal muscle by p43 levels. We also demonstrate that regulation of cardiolipin biosynthesis by the thyroid hormone does not imply p43.
Assuntos
Ácidos Graxos/análise , Músculo Esquelético/metabolismo , Receptores alfa dos Hormônios Tireóideos/genética , Animais , Cardiolipinas/biossíntese , Ácidos Graxos/metabolismo , Lipídeos/análise , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/química , Mitocôndrias/metabolismo , Músculo Esquelético/química , Músculo Quadríceps/química , Músculo Quadríceps/metabolismoRESUMO
Sirtuin 3 (SIRT3), one of the seven mammalian sirtuins, is a mitochondrial NAD+-dependent deacetylase known to control key metabolic pathways. SIRT3 deacetylases and activates a large number of mitochondrial enzymes involved in the respiratory chain, in ATP production, and in both the citric acid and urea cycles. We have previously shown that the regulation of myoblast differentiation is tightly linked to mitochondrial activity. Since SIRT3 modulates mitochondrial activity, we decide to address its role during myoblast differentiation. For this purpose, we first investigated the expression of endogenous SIRT3 during C2C12 myoblast differentiation. We further studied the impact of SIRT3 silencing on both the myogenic potential and the mitochondrial activity of C2C12 cells. We showed that SIRT3 protein expression peaked at the onset of myoblast differentiation. The inhibition of SIRT3 expression mediated by the stable integration of SIRT3 short inhibitory RNA (SIRT3shRNA) in C2C12 myoblasts, resulted in: 1) abrogation of terminal differentiation - as evidenced by a marked decrease in the myoblast fusion index and a significant reduction of Myogenin, MyoD, Sirtuin 1 and Troponin T protein expression - restored upon MyoD overexpression; 2) a decrease in peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and citrate synthase protein expression reflecting an alteration of mitochondrial density; and 3) an increased production of reactive oxygen species (ROS) mirrored by the decreased activity of manganese superoxide dismutase (MnSOD). Altogether our data demonstrate that SIRT3 mainly regulates myoblast differentiation via its influence on mitochondrial activity.
Assuntos
Diferenciação Celular , Mitocôndrias/enzimologia , Mioblastos/citologia , NAD/metabolismo , Sirtuína 3/metabolismo , Animais , Linhagem Celular , Regulação para Baixo , Técnicas de Silenciamento de Genes , Camundongos , Mitocôndrias/metabolismo , Proteína MyoD/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Sirtuína 3/deficiência , Sirtuína 3/genética , Fatores de Transcrição/metabolismoRESUMO
Chicoric acid (CA) is a caffeoyl derivative previously described as having potential anti-diabetic properties. As similarities in cellular mechanism similarities between diabetes and aging have been shown, we explored on L6 myotubes the effect of CA on the modulation of intracellular pathways involved in diabetes and aging. We also determined its influence on lifespan of Caenorhabditis elegans worm (C. elegans). In L6 myotubes, CA was a potent reactive oxygen species (ROS) scavenger, reducing ROS accumulation under basal as well as oxidative stress conditions. CA also stimulated the AMP-activated kinase (AMPK) pathway and displayed various features associated with AMPK activation: CA (a) enhanced oxidative enzymatic defences through increase in glutathion peroxidase (GPx) and superoxide dismutase (SOD) activities, (b) favoured mitochondria protection against oxidative damage through up-regulation of MnSOD protein expression, (c) increased mitochondrial biogenesis as suggested by increases in complex II and citrate synthase activities, along with up-regulation of PGC-1α mRNA expression and (d) inhibited the insulin/Akt/mTOR pathway. As AMPK stimulators (e.g. the anti-diabetic agent meformin or polyphenols such as epigallocatechingallate or quercetin) were shown to extend lifespan in C. elegans, we also determined the effect of CA on the same model. A concentration-dependant lifespan extension was observed with CA (5-100 µM). These data indicate that CA is a potent antioxidant compound activating the AMPK pathway in L6 myotubes. Similarly to other AMPK stimulators, CA is able to extend C. elegans lifespan, an effect measurable even at the micromolar range. Future studies will explore CA molecular targets and give new insights about its possible effects on metabolic and aging-related diseases.
Assuntos
Adenilato Quinase/metabolismo , Antioxidantes/farmacologia , Caenorhabditis elegans/enzimologia , Ácidos Cafeicos/farmacologia , Longevidade/efeitos dos fármacos , Fibras Musculares Esqueléticas/enzimologia , Succinatos/farmacologia , Adenilato Quinase/genética , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/biossíntese , Proteínas de Caenorhabditis elegans/genética , Citrato (si)-Sintase/biossíntese , Citrato (si)-Sintase/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Longevidade/fisiologia , Oxirredutases/biossíntese , Oxirredutases/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
The commonly used live attenuated vaccine in ovine brucellosis prophylaxis is Brucella melitensis Rev.1. This vaccine is known to induce antibody responses in vaccinated animals indistinguishable by the current conventional serological tests from those observed in challenged animals. Brucella BP26 and Omp31 proteins have shown an interesting potential as diagnostic antigens for ovine brucellosis. Accordingly, the bp26 gene and both bp26 and omp31 genes have been deleted from the vaccine strain Rev.1. Immunogenicity and vaccine efficacy of the parental Rev.1 strain and of both mutants in protecting sheep against B. melitensis strain H38 challenge was evaluated by clinical and bacteriological examination of ewes. They were conjunctivally or subcutaneously vaccinated when 4 months old and then challenged with B. melitensis H38 at the middle of the first pregnancy following vaccination. Deletion of bp26 and omp31 genes did not significantly affect the well recognised capacity of Rev.1 to protect sheep against B. melitensis challenge. However, the protection conferred by the CGV2631 mutant was significantly lower than that conferred by the CGV26 mutant or the Rev.1 strain. Vaccinated and challenged animals were detected positive in classical serological tests and in the IFN-gamma assay. A BP26-based ELISA was investigated to discriminate between ewes vaccinated by the mutants and ewes challenged with B. melitensis H38. The cut-off which was chosen in order to have 100% specificity resulted in a moderate sensitivity for the detection of challenged ewes. The use in the field of one of the mutants as vaccine against a B. melitensis infection, combined with classic diagnostic tests and a BP26 ELISA, could thus give an improvement in the differentiation between vaccinated and infected animals and contribute to the objective of eradication of brucellosis in small ruminants.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Vacina contra Brucelose/imunologia , Brucella melitensis/imunologia , Brucelose/prevenção & controle , Proteínas de Membrana/genética , Vacinas Sintéticas/imunologia , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Deleção de Genes , Interferon gama/biossíntese , Leite/microbiologia , Ovinos , Vacinação , Vagina/microbiologiaRESUMO
Brucella, the causative agent of brucellosis in animals and humans, can survive and proliferate within macrophages. Macrophages mediate mouse resistance to various pathogens through the expression of the Nramp1 gene. The role of this gene in the control of Brucella infection was investigated. When BALB/c mice (Nramp1(s)) and C.CB congenic mice (Nramp1(r)) were infected with Brucella melitensis, the number of Brucella organisms per spleen was significantly larger in the C.CB mice than in the BALB/c mice during the first week postinfection (p.i.). This Nramp1-linked susceptibility to Brucella was temporary, since similar numbers of Brucella were recovered from the two strains of mice 2 weeks p.i. The effect of Nramp1 expression occurred within splenocytes intracellularly infected by BRUCELLA: However, there was no difference between in vitro replication rates of Brucella in macrophages isolated from the two strains of mice infected in vivo or in Nramp1 RAW264 transfectants. In mice, infection with Brucella induced an inflammatory response, resulting in splenomegaly and recruitment of phagocytes in the spleen, which was amplified in C.CB mice. Reverse transcription-PCR (RT-PCR), performed 5 days p.i., showed that inducible nitric oxide synthase, tumor necrosis factor alpha (TNF-alpha), interleukin-12 p40 (IL-12p40), gamma interferon (IFN-gamma), and IL-10 mRNAs were similarly induced in spleens of the two strains. In contrast, the mRNA of KC, a C-X-C chemokine, was induced only in infected C.CB mice at this time. This pattern of mRNA expression was maintained at 14 days p.i., with IFN-gamma and IL-12p40 mRNAs being more intensively induced in the infected C.CB mice, but TNF-alpha mRNA was no longer induced. The higher recruitment of neutrophils observed in the spleens of infected C.CB mice could explain the temporary susceptibility of C.CB mice to B. melitensis infection. In contrast to infections with Salmonella, Leishmania, and Mycobacterium, the expression of the Nramp1 gene appears to be of limited importance for the natural resistance of mice to Brucella.