RESUMO
StarD7 is a member of the START protein family required for phosphatidylcholine delivery to the mitochondria, thus key to maintain mitochondrial structure. Its deficiency has been associated with an impairment of cellular processes, such as proliferation and migration, and it has also been reported that it is needed in myogenic differentiation. Here, we show that StarD7 deficiency in C2C12 muscle cells results in the accumulation of abnormal mitochondria, a reduced number of mitochondria per cell area and increased glycolysis. In addition, StarD7-deficient cells undergo an increase in mitochondria-ER contact sites, reduced connexin 43 expression, and disturbances in lipid handling, evidenced by lipid droplet accumulation and decreased levels in phosphatidylserine synthase 1 and 2 expression. Interestingly, StarD7-deficient cells showed alterations in mitophagy markers. We observed accumulation of LC3B-II and BNIP3 proteins in mitochondria-enriched fractions and accumulation of autophagolysosomal and lysosomal vesicles in StarD7-deficient cells. Furthermore, live-cell imaging experiments of StarD7 knockdown cells expressing mitochondria-targeted mKeima indicated an enhanced mitochondria delivery into lysosomes. Importantly, StarD7 reconstitution in StarD7-deficient cells restores LC3B-II expression in mitochondria-enriched fractions at similar levels to those observed in control cells. Collectively, these findings suggest that StarD7-deficient C2C12 myoblasts are associated with altered cristae structure, disturbances in neutral lipid accumulation, glucose metabolism, and increased mitophagy flux. The alterations mentioned above allow for the maintenance of mitochondrial function.
Assuntos
Proteínas de Transporte , Mitofagia , Proteínas de Transporte/metabolismo , Glicólise/genética , Lipídeos , Mitofagia/genética , Mioblastos/metabolismo , Animais , CamundongosRESUMO
BACKGROUND AND AIM: The excessive accumulation of lipid droplets (LDs) is a defining characteristic of nonalcoholic fatty liver disease (NAFLD). The interaction between LDs and mitochondria is functionally important for lipid metabolism homeostasis. Exercise improves NAFLD, but it is not known if it has an effect on hepatic LD-mitochondria interactions. Here, we investigated the influence of exercise on LD-mitochondria interactions and its significance in the context of NAFLD. APPROACH AND RESULTS: Mice were fed high-fat diet (HFD) or HFD-0.1 % methionine and choline-deficient diet (MCD) to emulate simple hepatic steatosis or non-alcoholic steatohepatitis, respectively. In both models, aerobic exercise decreased the size of LDs bound to mitochondria and the number of LD-mitochondria contacts. Analysis showed that the effects of exercise on HOMA-IR and liver triglyceride levels were independent of changes in body weight, and a positive correlation was observed between the number of LD-mitochondria contacts and NAFLD severity and with the lipid droplet size bound to mitochondria. Cellular fractionation studies revealed that ATP-coupled respiration and fatty acid oxidation (FAO) were greater in hepatic peridroplet mitochondria (PDM) from HFD-fed exercised mice than from equivalent sedentary mice. Finally, exercise increased FAO and mitofusin-2 abundance exclusively in PDM through a mechanism involving the curvature of mitochondrial membranes and the abundance of saturated lipids. Accordingly, hepatic mitofusin-2 ablation prevented exercise-induced FAO in PDM. CONCLUSIONS: This study demonstrates that aerobic exercise has beneficial effects in murine NAFLD models by lessening the interactions between hepatic LDs and mitochondria, and by decreasing LD size, correlating with a reduced severity of NAFLD. Additionally, aerobic exercise increases FAO in PDM and this process is reliant on Mfn-2 enrichment, which modifies LD-mitochondria communication.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Animais , Masculino , Camundongos , Dieta Hiperlipídica , Ácidos Graxos/metabolismo , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismoRESUMO
Nek4 is a serine/threonine kinase which has been implicated in primary cilia stabilization, DNA damage response, autophagy and epithelial-to-mesenchymal transition. The role of Nek4 in cancer cell survival and chemotherapy resistance has also been shown. However, the precise mechanisms by which Nek4 operates remain to be elucidated. Here, we show that Nek4 overexpression activates mitochondrial respiration coupled to ATP production, which is paralleled by increased mitochondrial membrane potential, and resistance to mitochondrial DNA damage. Congruently, Nek4 depletion reduced mitochondrial respiration and mtDNA integrity. Nek4 deficiency caused mitochondrial elongation, probably via reduced activity of the fission protein DRP1. In Nek4 overexpressing cells, the increase in mitochondrial fission was concomitant to enhanced phosphorylation of DRP1 and Erk1/2 proteins, and the effects on mitochondrial respiration were abolished in the presence of a DRP1 inhibitor. This study shows Nek4 as a novel regulator of mitochondrial function that may explain the joint appearance of high mitochondrial respiration and mitochondrial fragmentation.
Assuntos
Dinaminas , Dinâmica Mitocondrial , DNA Mitocondrial/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Fosforilação , RespiraçãoRESUMO
Platelet activation plays a key role in cardiovascular diseases. The generation of mitochondrial reactive oxygen species (ROS) has been described as a critical step required for platelet activation. For this reason, it is necessary to find new molecules with antiplatelet activity and identify their mechanisms of action. Mitoquinone (MitoQ) is a mitochondria-targeted antioxidant that reduces mitochondrial overproduction of ROS. In this work, the antiplatelet effect of MitoQ through platelet adhesion and spreading, secretion, and aggregation was evaluated. Thus MitoQ, in a non-toxic effect, decreased platelet adhesion and spreading on collagen surface, and expression of P-selectin and CD63, and inhibited platelet aggregation induced by collagen, convulxin, thrombin receptor activator peptide-6 (TRAP-6), and phorbol 12-myristate 13-acetate (PMA). As an antiplatelet mechanism, we showed that MitoQ produced mitochondrial depolarization and decreased ATP secretion. Additionally, in platelets stimulated with antimycin A and collagen MitoQ significantly decreased ROS production. Our findings showed, for the first time, an antiplatelet effect of MitoQ that is probably associated with its mitochondrial antioxidant effect.
Assuntos
Antioxidantes/farmacologia , Plaquetas/metabolismo , Compostos Organofosforados/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Ubiquinona/análogos & derivados , Trifosfato de Adenosina/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Células Cultivadas , Colágeno/metabolismo , Humanos , Camundongos , Mitocôndrias/metabolismo , Oligopeptídeos/farmacologia , Selectina-P/metabolismo , Ésteres de Forbol/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Tetraspanina 30/metabolismo , Ubiquinona/farmacologiaRESUMO
Cardiomyocyte hypertrophy has been associated with diminished mitochondrial metabolism. Mitochondria are crucial organelles for the production of ATP, and their morphology and function are regulated by the dynamic processes of fusion and fission. The relationship between mitochondrial dynamics and cardiomyocyte hypertrophy is still poorly understood. Here, we show that treatment of cultured neonatal rat cardiomyocytes with the hypertrophic agonist norepinephrine promotes mitochondrial fission (characterized by a decrease in mitochondrial mean volume and an increase in the relative number of mitochondria per cell) and a decrease in mitochondrial function. We demonstrate that norepinephrine acts through α1-adrenergic receptors to increase cytoplasmic Ca(2+), activating calcineurin and promoting migration of the fission protein Drp1 (encoded by Dnml1) to mitochondria. Dominant-negative Drp1 (K38A) not only prevented mitochondrial fission, it also blocked hypertrophic growth of cardiomyocytes in response to norepinephrine. Remarkably, an antisense adenovirus against the fusion protein Mfn2 (AsMfn2) was sufficient to increase mitochondrial fission and stimulate a hypertrophic response without agonist treatment. Collectively, these results demonstrate the importance of mitochondrial dynamics in the development of cardiomyocyte hypertrophy and metabolic remodeling.
Assuntos
Calcineurina/metabolismo , Mitocôndrias Cardíacas/fisiologia , Dinâmica Mitocondrial , Miócitos Cardíacos/fisiologia , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Cardiomegalia/metabolismo , Células Cultivadas , Dinaminas/genética , Dinaminas/metabolismo , GTP Fosfo-Hidrolases , Hipertrofia/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Norepinefrina/farmacologia , Transporte Proteico , Ratos Sprague-Dawley , Receptores Adrenérgicos alfa 1/metabolismoRESUMO
Insulin regulates heart metabolism through the regulation of insulin-stimulated glucose uptake. Studies have indicated that insulin can also regulate mitochondrial function. Relevant to this idea, mitochondrial function is impaired in diabetic individuals. Furthermore, the expression of Opa-1 and mitofusins, proteins of the mitochondrial fusion machinery, is dramatically altered in obese and insulin-resistant patients. Given the role of insulin in the control of cardiac energetics, the goal of this study was to investigate whether insulin affects mitochondrial dynamics in cardiomyocytes. Confocal microscopy and the mitochondrial dye MitoTracker Green were used to obtain three-dimensional images of the mitochondrial network in cardiomyocytes and L6 skeletal muscle cells in culture. Three hours of insulin treatment increased Opa-1 protein levels, promoted mitochondrial fusion, increased mitochondrial membrane potential, and elevated both intracellular ATP levels and oxygen consumption in cardiomyocytes in vitro and in vivo. Consequently, the silencing of Opa-1 or Mfn2 prevented all the metabolic effects triggered by insulin. We also provide evidence indicating that insulin increases mitochondrial function in cardiomyocytes through the Akt-mTOR-NFκB signaling pathway. These data demonstrate for the first time in our knowledge that insulin acutely regulates mitochondrial metabolism in cardiomyocytes through a mechanism that depends on increased mitochondrial fusion, Opa-1, and the Akt-mTOR-NFκB pathway.
Assuntos
Insulina/farmacologia , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Miócitos Cardíacos/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Células Cultivadas , GTP Fosfo-Hidrolases/metabolismo , Camundongos , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
The endoplasmic reticulum (ER) is a dynamic intracellular organelle with multiple functions essential for cellular homeostasis, development, and stress responsiveness. In response to cellular stress, a well-established signaling cascade, the unfolded protein response (UPR), is activated. This intricate mechanism is an important means of re-establishing cellular homeostasis and alleviating the inciting stress. Now, emerging evidence has demonstrated that the UPR influences cellular metabolism through diverse mechanisms, including calcium and lipid transfer, raising the prospect of involvement of these processes in the pathogenesis of disease, including neurodegeneration, cancer, diabetes mellitus and cardiovascular disease. Here, we review the distinct functions of the ER and UPR from a metabolic point of view, highlighting their association with prevalent pathologies.
Assuntos
Retículo Endoplasmático/metabolismo , Resposta a Proteínas não Dobradas , Animais , Doença , Retículo Endoplasmático/ultraestrutura , Estresse do Retículo Endoplasmático , Humanos , ProteóliseRESUMO
Increasing evidence indicates that endoplasmic reticulum (ER) stress activates the adaptive unfolded protein response (UPR), but that beyond a certain degree of ER damage, this response triggers apoptotic pathways. The general mechanisms of the UPR and its apoptotic pathways are well characterized. However, the metabolic events that occur during the adaptive phase of ER stress, before the cell death response, remain unknown. Here, we show that, during the onset of ER stress, the reticular and mitochondrial networks are redistributed towards the perinuclear area and their points of connection are increased in a microtubule-dependent fashion. A localized increase in mitochondrial transmembrane potential is observed only in redistributed mitochondria, whereas mitochondria that remain in other subcellular zones display no significant changes. Spatial re-organization of these organelles correlates with an increase in ATP levels, oxygen consumption, reductive power and increased mitochondrial Ca²âº uptake. Accordingly, uncoupling of the organelles or blocking Ca²âº transfer impaired the metabolic response, rendering cells more vulnerable to ER stress. Overall, these data indicate that ER stress induces an early increase in mitochondrial metabolism that depends crucially upon organelle coupling and Ca²âº transfer, which, by enhancing cellular bioenergetics, establishes the metabolic basis for the adaptation to this response.
Assuntos
Retículo Endoplasmático/metabolismo , Metabolismo Energético , Mitocôndrias/metabolismo , Estresse Fisiológico , Antibacterianos/farmacologia , Apoptose/fisiologia , Cálcio/metabolismo , Respiração Celular , Inibidores Enzimáticos/farmacologia , Células HeLa , Agonistas dos Receptores Histamínicos/farmacologia , Humanos , Potencial da Membrana Mitocondrial , Consumo de Oxigênio/efeitos dos fármacos , Fosfatos de Fosfatidilinositol/metabolismo , Transdução de Sinais/fisiologiaRESUMO
AIMS: In cells, mitochondria are organized as a network of interconnected organelles that fluctuate between fission and fusion events (mitochondrial dynamics). This process is associated with cell death. We investigated whether activation of apoptosis with ceramides affects mitochondrial dynamics and promotes mitochondrial fission in cardiomyocytes. METHODS AND RESULTS: Neonatal rat cardiomyocytes were incubated with C(2)-ceramide or the inactive analog dihydro-C(2)-ceramide for up to 6 h. Three-dimensional images of cells loaded with mitotracker green were obtained by confocal microscopy. Dynamin-related protein-1 (Drp-1) and mitochondrial fission protein 1 (Fis1) distribution and levels were studied by immunofluorescence and western blot. Mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c (cyt c) distribution were used as indexes of early activation of apoptosis. Cell viability and DNA fragmentation were determined by propidium iodide staining/flow cytometry, whereas cytotoxicity was evaluated by lactic dehydrogenase activity. To decrease the levels of the mitochondrial fusion protein mitofusin 2, we used an antisense adenovirus (AsMfn2). C(2)-ceramide, but not dihydro-C(2)-ceramide, promoted rapid fragmentation of the mitochondrial network in a concentration- and time-dependent manner. C(2)-ceramide also increased mitochondrial Drp-1 and Fis1 content, Drp-1 colocalization with Fis1, and caused early activation of apoptosis. AsMfn2 accentuated the decrease in DeltaPsi(m) and cyt c redistribution induced by C(2)-ceramide. Doxorubicin, which induces cardiomyopathy and apoptosis through ceramide generation, also stimulated mitochondrial fragmentation. CONCLUSION: Ceramides stimulate mitochondrial fission and this event is associated with early activation of cardiomyocyte apoptosis.