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1.
J Biol Chem ; 294(45): 16831-16845, 2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31562244

RESUMO

The healthy heart has a dynamic capacity to respond and adapt to changes in nutrient availability. Metabolic inflexibility, such as occurs with diabetes, increases cardiac reliance on fatty acids to meet energetic demands, and this results in deleterious effects, including mitochondrial dysfunction, that contribute to pathophysiology. Enhancing glucose usage may mitigate metabolic inflexibility and be advantageous under such conditions. Here, we sought to identify how mitochondrial function and cardiac metabolism are affected in a transgenic mouse model of enhanced cardiac glycolysis (GlycoHi) basally and following a short-term (7-day) high-fat diet (HFD). GlycoHi mice constitutively express an active form of phosphofructokinase-2, resulting in elevated levels of the PFK-1 allosteric activator fructose 2,6-bisphosphate. We report that basally GlycoHi mitochondria exhibit augmented pyruvate-supported respiration relative to fatty acids. Nevertheless, both WT and GlycoHi mitochondria had a similar shift toward increased rates of fatty acid-supported respiration following HFD. Metabolic profiling by GC-MS revealed distinct features based on both genotype and diet, with a unique increase in branched-chain amino acids in the GlycoHi HFD group. Targeted quantitative proteomics analysis also supported both genotype- and diet-dependent changes in protein expression and uncovered an enhanced expression of pyruvate dehydrogenase kinase 4 (PDK4) in the GlycoHi HFD group. These results support a newly identified mechanism whereby the levels of fructose 2,6-bisphosphate promote mitochondrial PDK4 levels and identify a secondary adaptive response that prevents excessive mitochondrial pyruvate oxidation when glycolysis is sustained after a high-fat dietary challenge.

2.
Science ; 364(6436): 184-188, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30846611

RESUMO

Tissue regenerative potential displays striking divergence across phylogeny and ontogeny, but the underlying mechanisms remain enigmatic. Loss of mammalian cardiac regenerative potential correlates with cardiomyocyte cell-cycle arrest and polyploidization as well as the development of postnatal endothermy. We reveal that diploid cardiomyocyte abundance across 41 species conforms to Kleiber's law-the ¾-power law scaling of metabolism with bodyweight-and inversely correlates with standard metabolic rate, body temperature, and serum thyroxine level. Inactivation of thyroid hormone signaling reduces mouse cardiomyocyte polyploidization, delays cell-cycle exit, and retains cardiac regenerative potential in adults. Conversely, exogenous thyroid hormones inhibit zebrafish heart regeneration. Thus, our findings suggest that loss of heart regenerative capacity in adult mammals is triggered by increasing thyroid hormones and may be a trade-off for the acquisition of endothermy.


Assuntos
Coração/fisiologia , Miócitos Cardíacos/fisiologia , Poliploidia , Regeneração/fisiologia , Hormônios Tireóideos/fisiologia , Animais , Regulação da Temperatura Corporal , Pontos de Checagem do Ciclo Celular , Proliferação de Células , Diploide , Camundongos , Miócitos Cardíacos/classificação , Filogenia , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/fisiologia , Regeneração/efeitos dos fármacos , Regeneração/genética , Transdução de Sinais , Hormônios Tireóideos/farmacologia , Peixe-Zebra
3.
Circulation ; 139(11): 1422-1434, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30612451

RESUMO

BACKGROUND: Inorganic phosphate (Pi) is used extensively as a preservative and a flavor enhancer in the Western diet. Physical inactivity, a common feature of Western societies, is associated with increased cardiovascular morbidity and mortality. It is unknown whether dietary Pi excess contributes to exercise intolerance and physical inactivity. METHODS: To determine an association between Pi excess and physical activity in humans, we assessed the relationship between serum Pi and actigraphy-determined physical activity level, as well as left ventricular function by cardiac magnetic resonance imaging, in DHS-2 (Dallas Heart Study phase 2) participants after adjusting for relevant variables. To determine direct effects of dietary Pi on exercise capacity, oxygen uptake, serum nonesterified fatty acid, and glucose were measured during exercise treadmill test in C57/BL6 mice fed either a high-Pi (2%) or normal-Pi (0.6%) diet for 12 weeks. To determine the direct effect of Pi on muscle metabolism and expression of genes involved in fatty acid metabolism, additional studies in differentiated C2C12 myotubes were conducted after subjecting to media containing 1 to 3 mmol/L Pi (pH 7.0) to simulate in vivo phosphate conditions. RESULTS: In participants of the DHS-2 (n=1603), higher serum Pi was independently associated with reduced time spent in moderate to vigorous physical activity ( P=0.01) and increased sedentary time ( P=0.004). There was no association between serum Pi and left ventricular ejection fraction or volumes. In animal studies, compared with the control diet, consumption of high-Pi diet for 12 weeks did not alter body weight or left ventricular function but reduced maximal oxygen uptake, treadmill duration, spontaneous locomotor activity, fat oxidation, and fatty acid levels and led to downregulation of genes involved in fatty acid synthesis, release, and oxidation, including Fabp4, Hsl, Fasn, and Pparγ, in muscle. Similar results were recapitulated in vitro by incubating C2C12 myotubes with high-Pi media. CONCLUSIONS: Our data demonstrate a detrimental effect of dietary Pi excess on skeletal muscle fatty acid metabolism and exercise capacity that is independent of obesity and cardiac contractile function. Dietary Pi may represent a novel and modifiable target to reduce physical inactivity associated with the Western diet.


Assuntos
Metabolismo Energético/efeitos dos fármacos , Tolerância ao Exercício/efeitos dos fármacos , Ácidos Graxos/metabolismo , Músculo Esquelético/efeitos dos fármacos , Fosfatos/efeitos adversos , Fósforo na Dieta/efeitos adversos , Animais , Linhagem Celular , Metabolismo Energético/genética , Exercício , Tolerância ao Exercício/genética , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias Musculares/efeitos dos fármacos , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Consumo de Oxigênio , Fosfatos/administração & dosagem , Fosfatos/metabolismo , Fósforo na Dieta/administração & dosagem , Fósforo na Dieta/metabolismo , Comportamento Sedentário
4.
Cell Rep ; 23(13): 3701-3709, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29949755

RESUMO

Micropeptide regulator of ß-oxidation (MOXI) is a conserved muscle-enriched protein encoded by an RNA transcript misannotated as non-coding. MOXI localizes to the inner mitochondrial membrane where it associates with the mitochondrial trifunctional protein, an enzyme complex that plays a critical role in fatty acid ß-oxidation. Isolated heart and skeletal muscle mitochondria from MOXI knockout mice exhibit a diminished ability to metabolize fatty acids, while transgenic MOXI overexpression leads to enhanced ß-oxidation. Additionally, hearts from MOXI knockout mice preferentially oxidize carbohydrates over fatty acids in an isolated perfused heart system compared to wild-type (WT) animals. MOXI knockout mice also exhibit a profound reduction in exercise capacity, highlighting the role of MOXI in metabolic control. The functional characterization of MOXI provides insight into the regulation of mitochondrial metabolism and energy homeostasis and underscores the regulatory potential of additional micropeptides that have yet to be identified.


Assuntos
Ácidos Graxos/metabolismo , Mitocôndrias Musculares/metabolismo , Proteínas Mitocondriais/genética , Sequência de Aminoácidos , Animais , Ácidos Graxos/química , Humanos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredução , Alinhamento de Sequência
5.
J Biol Chem ; 293(18): 6915-6924, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29540486

RESUMO

Cardiac energy is produced primarily by oxidation of fatty acids and glucose, with the relative contributions of each nutrient being sensitive to changes in substrate availability and energetic demand. A major contributor to cardiac metabolic flexibility is pyruvate dehydrogenase (PDH), which converts glucose-derived pyruvate to acetyl-CoA within the mitochondria. PDH is inhibited by phosphorylation dependent on the competing activities of pyruvate dehydrogenase kinases (PDK1-4) and phosphatases (PDP1-2). A single high-fat meal increases cardiac PDK4 content and subsequently inhibits PDH activity, reducing pyruvate utilization when abundant fatty acids are available. In this study, we demonstrate that diet-induced increases in PDK4 are reversible and characterize a novel pathway that regulates PDK4 degradation in response to the cardiac metabolic environment. We found that PDK4 degradation is promoted by CoA (CoASH), the levels of which declined in mice fed a high-fat diet and normalized following transition to a control diet. We conclude that CoASH functions as a metabolic sensor linking the rate of PDK4 degradation to fatty acid availability in the heart. However, prolonged high-fat feeding followed by return to a low-fat diet resulted in persistent in vitro sensitivity of PDH to fatty acid-induced inhibition despite reductions in PDK4 content. Moreover, increases in the levels of proteins responsible for ß-oxidation and rates of palmitate oxidation by isolated cardiac mitochondria following long-term consumption of high dietary fat persisted after transition to the control diet. We propose that these changes prime PDH for inhibition upon reintroduction of fatty acids.


Assuntos
Coenzima A/metabolismo , Dieta Hiperlipídica , Miocárdio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Dieta com Restrição de Gorduras , Ácidos Graxos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/metabolismo , Oxirredução , Proteínas Serina-Treonina Quinases/genética , Proteólise , RNA Mensageiro/metabolismo
6.
Mol Metab ; 9: 141-155, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29398615

RESUMO

OBJECTIVE: A decline in mitochondrial function and biogenesis as well as increased reactive oxygen species (ROS) are important determinants of aging. With advancing age, there is a concomitant reduction in circulating levels of insulin-like growth factor-1 (IGF-1) that is closely associated with neuronal aging and neurodegeneration. In this study, we investigated the effect of the decline in IGF-1 signaling with age on astrocyte mitochondrial metabolism and astrocyte function and its association with learning and memory. METHODS: Learning and memory was assessed using the radial arm water maze in young and old mice as well as tamoxifen-inducible astrocyte-specific knockout of IGFR (GFAP-CreTAM/igfrf/f). The impact of IGF-1 signaling on mitochondrial function was evaluated using primary astrocyte cultures from igfrf/f mice using AAV-Cre mediated knockdown using Oroboros respirometry and Seahorse assays. RESULTS: Our results indicate that a reduction in IGF-1 receptor (IGFR) expression with age is associated with decline in hippocampal-dependent learning and increased gliosis. Astrocyte-specific knockout of IGFR also induced impairments in working memory. Using primary astrocyte cultures, we show that reducing IGF-1 signaling via a 30-50% reduction IGFR expression, comparable to the physiological changes in IGF-1 that occur with age, significantly impaired ATP synthesis. IGFR deficient astrocytes also displayed altered mitochondrial structure and function and increased mitochondrial ROS production associated with the induction of an antioxidant response. However, IGFR deficient astrocytes were more sensitive to H2O2-induced cytotoxicity. Moreover, IGFR deficient astrocytes also showed significantly impaired glucose and Aß uptake, both critical functions of astrocytes in the brain. CONCLUSIONS: Regulation of astrocytic mitochondrial function and redox status by IGF-1 is essential to maintain astrocytic function and coordinate hippocampal-dependent spatial learning. Age-related astrocytic dysfunction caused by diminished IGF-1 signaling may contribute to the pathogenesis of Alzheimer's disease and other age-associated cognitive pathologies.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Astrócitos/metabolismo , Memória de Curto Prazo , Mitocôndrias/metabolismo , Receptor IGF Tipo 1/genética , Envelhecimento/metabolismo , Animais , Células Cultivadas , Glucose/metabolismo , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismo , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais
7.
BMC Biol ; 15(1): 113, 2017 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-29183319

RESUMO

BACKGROUND: Peroxisome proliferator activated receptor-alpha (PPARα) is a ubiquitously expressed nuclear receptor. The role of endogenous PPARα in retinal neuronal homeostasis is unknown. Retinal photoreceptors are the highest energy-consuming cells in the body, requiring abundant energy substrates. PPARα is a known regulator of lipid metabolism, and we hypothesized that it may regulate lipid use for oxidative phosphorylation in energetically demanding retinal neurons. RESULTS: We found that endogenous PPARα is essential for the maintenance and survival of retinal neurons, with Pparα -/- mice developing retinal degeneration first detected at 8 weeks of age. Using extracellular flux analysis, we identified that PPARα mediates retinal utilization of lipids as an energy substrate, and that ablation of PPARα ultimately results in retinal bioenergetic deficiency and neurodegeneration. This may be due to PPARα regulation of lipid transporters, which facilitate the internalization of fatty acids into cell membranes and mitochondria for oxidation and ATP production. CONCLUSION: We identify an endogenous role for PPARα in retinal neuronal survival and lipid metabolism, and furthermore underscore the importance of fatty acid oxidation in photoreceptor survival. We also suggest PPARα as a putative therapeutic target for age-related macular degeneration, which may be due in part to decreased mitochondrial efficiency and subsequent energetic deficits.


Assuntos
Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos , PPAR alfa/genética , Retina/metabolismo , Neurônios Retinianos/fisiologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , PPAR alfa/metabolismo , Ratos , Ratos Sprague-Dawley
8.
J Biol Chem ; 292(45): 18556-18564, 2017 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-28916720

RESUMO

Expansion of adipose tissue in response to a positive energy balance underlies obesity and occurs through both hypertrophy of existing cells and increased differentiation of adipocyte precursors (hyperplasia). To better understand the nutrient signals that promote adipocyte differentiation, we investigated the role of glucose availability in regulating adipocyte differentiation and maturation. 3T3-L1 preadipocytes were grown and differentiated in medium containing a standard differentiation hormone mixture and either 4 or 25 mm glucose. Adipocyte maturation at day 9 post-differentiation was determined by key adipocyte markers, including glucose transporter 4 (GLUT4) and adiponectin expression and Oil Red O staining of neutral lipids. We found that adipocyte differentiation and maturation required a pulse of 25 mm glucose only during the first 3 days of differentiation. Importantly, fatty acids were unable to substitute for the 25 mm glucose pulse during this period. The 25 mm glucose pulse increased adiponectin and GLUT4 expression and accumulation of neutral lipids via distinct mechanisms. Adiponectin expression and other early markers of differentiation required an increase in the intracellular pool of total NAD/P. In contrast, GLUT4 protein expression was only partially restored by increased NAD/P levels. Furthermore, GLUT4 mRNA expression was mediated by glucose-dependent activation of GLUT4 gene transcription through the cis-acting GLUT4-liver X receptor element (LXRE) promoter element. In summary, this study supports the conclusion that high glucose promotes adipocyte differentiation via distinct metabolic pathways and independently of fatty acids. This may partly explain the mechanism underlying adipocyte hyperplasia that occurs much later than adipocyte hypertrophy in the development of obesity.


Assuntos
Adipócitos Brancos/metabolismo , Adipogenia , Regulação da Expressão Gênica , Transportador de Glucose Tipo 4/metabolismo , Glucose/metabolismo , NADP/metabolismo , NAD/metabolismo , Células 3T3-L1 , Adipócitos Brancos/citologia , Adipócitos Brancos/patologia , Adiponectina/genética , Adiponectina/metabolismo , Tecido Adiposo Branco/citologia , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/patologia , Animais , Biomarcadores/metabolismo , Células Cultivadas , Transportador de Glucose Tipo 4/genética , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Hipoglicemia/metabolismo , Hipoglicemia/patologia , Lipogênese , Receptores X do Fígado/genética , Receptores X do Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Células Estromais/citologia , Células Estromais/metabolismo , Células Estromais/patologia , Regulação para Cima
9.
Radiat Res ; 187(6): 743-754, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28437190

RESUMO

Ketogenic diets are low in carbohydrates and high in fat, which forces cells to rely more heavily upon mitochondrial oxidation of fatty acids for energy. Relative to normal cells, cancer cells are believed to exist under a condition of chronic mitochondrial oxidative stress that is compensated for by increases in glucose metabolism to generate reducing equivalents. In this study we tested the hypothesis that a ketogenic diet concurrent with radiation and chemotherapy would be clinically tolerable in locally advanced non-small cell lung cancer (NSCLC) and pancreatic cancer and could potentially exploit cancer cell oxidative metabolism to improve therapeutic outcomes. Mice bearing MIA PaCa-2 pancreatic cancer xenografts were fed either a ketogenic diet or standard rodent chow, treated with conventionally fractionated radiation (2 Gy/fraction), and tumor growth rates were assessed daily. Tumors were assessed for immunoreactive 4-hydroxy-2-nonenal-(4HNE)-modfied proteins as a marker of oxidative stress. Based on this and another previously published preclinical study, phase 1 clinical trials in locally advanced NSCLC and pancreatic cancer were initiated, combining standard radiation and chemotherapy with a ketogenic diet for six weeks (NSCLC) or five weeks (pancreatic cancer). The xenograft experiments demonstrated prolonged survival and increased 4HNE-modfied proteins in animals consuming a ketogenic diet combined with radiation compared to radiation alone. In the phase 1 clinical trial, over a period of three years, seven NSCLC patients enrolled in the study. Of these, four were unable to comply with the diet and withdrew, two completed the study and one was withdrawn due to a dose-limiting toxicity. Over the same time period, two pancreatic cancer patients enrolled in the trial. Of these, one completed the study and the other was withdrawn due to a dose-limiting toxicity. The preclinical experiments demonstrate that a ketogenic diet increases radiation sensitivity in a pancreatic cancer xenograft model. However, patients with locally advanced NSCLC and pancreatic cancer receiving concurrent radiotherapy and chemotherapy had suboptimal compliance to the oral ketogenic diet and thus, poor tolerance.


Assuntos
Quimiorradioterapia/métodos , Dietoterapia/métodos , Dieta Cetogênica/métodos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/terapia , Neoplasias Pancreáticas/terapia , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Iowa , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Neoplasias Pancreáticas/diagnóstico , Resultado do Tratamento
10.
J Biol Chem ; 292(1): 305-312, 2017 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-27856638

RESUMO

Cardiac metabolic inflexibility is driven by robust up-regulation of pyruvate dehydrogenase kinase 4 (PDK4) and phosphorylation-dependent inhibition of pyruvate dehydrogenase (PDH) within a single day of feeding mice a high fat diet. In the current study, we have discovered that PDK4 is a short lived protein (t½ ∼ 1 h) and is specifically degraded by the mitochondrial protease Lon. Lon does not rapidly degrade PDK1 and -2, indicating specificity toward the PDK isoform that is a potent modulator of metabolic flexibility. Moreover, PDK4 degradation appears regulated by dissociation from the PDH complex dependent on the respiratory state and energetic substrate availability of mouse heart mitochondria. Finally, we demonstrate that pharmacologic inhibition of PDK4 promotes PDK4 degradation in vitro and in vivo These findings reveal a novel strategy to manipulate PDH activity by selectively targeting PDK4 content through dissociation and proteolysis.


Assuntos
Regulação Enzimológica da Expressão Gênica , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Protease La/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Animais , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/citologia , Fosforilação , Protease La/genética , Proteínas Serina-Treonina Quinases/genética , Complexo Piruvato Desidrogenase/genética
11.
Nature ; 541(7636): 222-227, 2017 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-27798600

RESUMO

The adult mammalian heart is incapable of regeneration following cardiomyocyte loss, which underpins the lasting and severe effects of cardiomyopathy. Recently, it has become clear that the mammalian heart is not a post-mitotic organ. For example, the neonatal heart is capable of regenerating lost myocardium, and the adult heart is capable of modest self-renewal. In both of these scenarios, cardiomyocyte renewal occurs via the proliferation of pre-existing cardiomyocytes, and is regulated by aerobic-respiration-mediated oxidative DNA damage. Therefore, we reasoned that inhibiting aerobic respiration by inducing systemic hypoxaemia would alleviate oxidative DNA damage, thereby inducing cardiomyocyte proliferation in adult mammals. Here we report that, in mice, gradual exposure to severe systemic hypoxaemia, in which inspired oxygen is gradually decreased by 1% and maintained at 7% for 2 weeks, results in inhibition of oxidative metabolism, decreased reactive oxygen species production and oxidative DNA damage, and reactivation of cardiomyocyte mitosis. Notably, we find that exposure to hypoxaemia 1 week after induction of myocardial infarction induces a robust regenerative response with decreased myocardial fibrosis and improvement of left ventricular systolic function. Genetic fate-mapping analysis confirms that the newly formed myocardium is derived from pre-existing cardiomyocytes. These results demonstrate that the endogenous regenerative properties of the adult mammalian heart can be reactivated by exposure to gradual systemic hypoxaemia, and highlight the potential therapeutic role of hypoxia in regenerative medicine.


Assuntos
Coração/crescimento & desenvolvimento , Hipóxia/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Regeneração , Medicina Regenerativa/métodos , Animais , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Proliferação de Células , Respiração Celular , Dano ao DNA , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitose , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/patologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Função Ventricular Esquerda
12.
Am J Physiol Heart Circ Physiol ; 311(5): H1091-H1096, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27614223

RESUMO

We have recently demonstrated that catalase content in mouse cardiac mitochondria is selectively elevated in response to high dietary fat, a nutritional state associated with oxidative stress and loss in insulin signaling. Catalase and various isoforms of glutathione peroxidase and peroxiredoxin each catalyze the consumption of H2O2 Catalase, located primarily within peroxisomes and to a lesser extent mitochondria, has a low binding affinity for H2O2 relative to glutathione peroxidase and peroxiredoxin. As such, the contribution of catalase to mitochondrial H2O2 consumption is not well understood. In the current study, using highly purified cardiac mitochondria challenged with micromolar concentrations of H2O2, we found that catalase contributes significantly to mitochondrial H2O2 consumption. In addition, catalase is solely responsible for removal of H2O2 in nonrespiring or structurally disrupted mitochondria. Finally, in mice fed a high-fat diet, mitochondrial-derived H2O2 is responsible for diminished insulin signaling in the heart as evidenced by reduced insulin-stimulated Akt phosphorylation. While elevated mitochondrial catalase content (∼50%) enhanced the capacity of mitochondria to consume H2O2 in response to high dietary fat, the selective increase in catalase did not prevent H2O2-induced loss in cardiac insulin signaling. Taken together, our results indicate that mitochondrial catalase likely functions to preclude the formation of high levels of H2O2 without perturbing redox-dependent signaling.


Assuntos
Catalase/genética , Peróxido de Hidrogênio/metabolismo , Mitocôndrias Cardíacas/metabolismo , Estresse Oxidativo , Animais , Western Blotting , Catalase/metabolismo , Dieta com Restrição de Gorduras , Dieta Hiperlipídica , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Camundongos , Camundongos Knockout , NADP/metabolismo , Oxirredução , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
13.
mBio ; 7(4)2016 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-27531911

RESUMO

UNLABELLED: Syntrophus aciditrophicus is a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation by S. aciditrophicus However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome of S. aciditrophicus leaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show that S. aciditrophicus uses AMP-forming, acetyl-CoA synthetase (Acs1) for ATP synthesis from acetyl-CoA. acs1 mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, of S. aciditrophicus grown in pure culture and coculture. Cell extracts of S. aciditrophicus had low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified from S. aciditrophicus and recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) in S. aciditrophicus cells support the operation of Acs1 in the acetate-forming direction. Thus, S. aciditrophicus has a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase. IMPORTANCE: Bacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA. Syntrophus aciditrophicus apparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as its genome does not have homologs to the genes for phosphate acetyltransferase and acetate kinase. Here, we show that S. aciditrophicus uses an alternative approach, an AMP-forming, acetyl-CoA synthetase, to make ATP from acetyl-CoA. AMP-forming, acetyl-CoA synthetases were previously thought to function only in the activation of acetate to acetyl-CoA.


Assuntos
Acetilcoenzima A/metabolismo , Trifosfato de Adenosina/metabolismo , Coenzima A Ligases/metabolismo , Deltaproteobacteria/enzimologia , Deltaproteobacteria/metabolismo , Difosfatos/metabolismo , Acetatos/metabolismo , Perfilação da Expressão Gênica , Metaboloma , Proteoma/análise
14.
Biochimie ; 124: 74-83, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26476002

RESUMO

A central feature of obesity-related cardiometabolic diseases is the impaired ability to transition between fatty acid and glucose metabolism. This impairment, referred to as "metabolic inflexibility", occurs in a number of tissues, including the heart. Although the heart normally prefers to metabolize fatty acids over glucose, the inability to upregulate glucose metabolism under energetically demanding conditions contributes to a pathological state involving energy imbalance, impaired contractility, and post-translational protein modifications. This review discusses pathophysiologic processes that contribute to cardiac metabolic inflexibility and speculates on the potential physiologic origins that lead to the current state of cardiometabolic disease in an obesogenic environment.


Assuntos
Metabolismo Energético , Glucose/metabolismo , Cardiopatias , Contração Miocárdica , Miocárdio/metabolismo , Obesidade , Processamento de Proteína Pós-Traducional , Animais , Cardiopatias/etiologia , Cardiopatias/metabolismo , Cardiopatias/patologia , Cardiopatias/fisiopatologia , Humanos , Obesidade/complicações , Obesidade/metabolismo , Obesidade/patologia , Obesidade/fisiopatologia
15.
Biochemistry ; 54(25): 4008-18, 2015 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-26061789

RESUMO

High-throughput proteomics studies have identified several thousand acetylation sites on more than 1000 proteins. Mitochondrial aconitase, the Krebs cycle enzyme that converts citrate to isocitrate, has been identified in many of these reports. Acetylated mitochondrial aconitase has also been identified as a target for sirtuin 3 (SIRT3)-catalyzed deacetylation. However, the functional significance of mitochondrial aconitase acetylation has not been determined. Using in vitro strategies, mass spectrometric analyses, and an in vivo mouse model of obesity, we found a significant acetylation-dependent activation of aconitase. Isolated heart mitochondria subjected to in vitro chemical acetylation with either acetic anhydride or acetyl-coenzyme A resulted in increased aconitase activity that was reversed with SIRT3 treatment. Quantitative mass spectrometry was used to measure acetylation at 21 lysine residues and revealed significant increases with both in vitro treatments. A high-fat diet (60% of kilocalories from fat) was used as an in vivo model and also showed significantly increased mitochondrial aconitase activity without changes in protein level. The high-fat diet also produced an increased level of aconitase acetylation at multiple sites as measured by the quantitative mass spectrometry assays. Treatment of isolated mitochondria from these mice with SIRT3 abolished the high-fat diet-induced activation of aconitase and reduced acetylation. Finally, kinetic analyses found that the increase in activity was a result of increased maximal velocity, and molecular modeling suggests the potential for acetylation at K144 to perturb the tertiary structure of the enzyme. The results of this study reveal a novel activation of mitochondrial aconitase by acetylation.


Assuntos
Aconitato Hidratase/metabolismo , Lisina/metabolismo , Mitocôndrias/enzimologia , Miocárdio/enzimologia , Acetilação , Aconitato Hidratase/química , Aconitato Hidratase/genética , Motivos de Aminoácidos , Animais , Lisina/química , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/química , Miocárdio/química , Miocárdio/metabolismo , Sirtuína 3/genética , Sirtuína 3/metabolismo
16.
FASEB J ; 28(10): 4534-50, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25016030

RESUMO

The chemokine receptor CXCR4 is expressed on adipocytes and macrophages in adipose tissue, but its role in this tissue remains unknown. We evaluated whether deficiency in either adipocyte or myeloid leukocyte CXCR4 affects body weight (BW) and adiposity in a mouse model of high-fat-diet (HFD)-induced obesity. We found that ablation of adipocyte, but not myeloid leukocyte, CXCR4 exacerbated obesity. The HFD-fed adipocyte-specific CXCR4-knockout (AdCXCR4ko) mice, compared to wild-type C57BL/6 control mice, had increased BW (average: 52.0 g vs. 35.5 g), adiposity (average: 49.3 vs. 21.0% of total BW), and inflammatory leukocyte content in white adipose tissue (WAT), despite comparable food intake. As previously reported, HFD feeding increased uncoupling protein 1 (UCP1) expression (fold increase: 3.5) in brown adipose tissue (BAT) of the C57BL/6 control mice. However, no HFD-induced increase in UCP1 expression was observed in the AdCXCR4ko mice, which were cold sensitive. Thus, our study suggests that adipocyte CXCR4 limits development of obesity by preventing excessive inflammatory cell recruitment into WAT and by supporting thermogenic activity of BAT. Since CXCR4 is conserved between mouse and human, the newfound role of CXCR4 in mouse adipose tissue may parallel the role of this chemokine receptor in human adipose tissue.


Assuntos
Adipócitos/metabolismo , Tecido Adiposo Marrom/metabolismo , Obesidade/metabolismo , Receptores CXCR4/metabolismo , Termogênese , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/fisiologia , Tecido Adiposo Branco/citologia , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/fisiologia , Animais , Dieta Hiperlipídica/efeitos adversos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Leucócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Obesidade/etiologia , Receptores CXCR4/genética , Proteína Desacopladora 1
17.
Cell ; 157(3): 565-79, 2014 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-24766806

RESUMO

The mammalian heart has a remarkable regenerative capacity for a short period of time after birth, after which the majority of cardiomyocytes permanently exit cell cycle. We sought to determine the primary postnatal event that results in cardiomyocyte cell-cycle arrest. We hypothesized that transition to the oxygen-rich postnatal environment is the upstream signal that results in cell-cycle arrest of cardiomyocytes. Here, we show that reactive oxygen species (ROS), oxidative DNA damage, and DNA damage response (DDR) markers significantly increase in the heart during the first postnatal week. Intriguingly, postnatal hypoxemia, ROS scavenging, or inhibition of DDR all prolong the postnatal proliferative window of cardiomyocytes, whereas hyperoxemia and ROS generators shorten it. These findings uncover a protective mechanism that mediates cardiomyocyte cell-cycle arrest in exchange for utilization of oxygen-dependent aerobic metabolism. Reduction of mitochondrial-dependent oxidative stress should be an important component of cardiomyocyte proliferation-based therapeutic approaches.


Assuntos
Pontos de Checagem do Ciclo Celular , Miócitos Cardíacos/citologia , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Dano ao DNA , Depuradores de Radicais Livres/farmacologia , Camundongos , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Peixe-Zebra
18.
PLoS One ; 8(10): e77280, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24116221

RESUMO

Cardiac function depends on the ability to switch between fatty acid and glucose oxidation for energy production in response to changes in substrate availability and energetic stress. In obese and diabetic individuals, increased reliance on fatty acids and reduced metabolic flexibility are thought to contribute to the development of cardiovascular disease. Mechanisms by which cardiac mitochondria contribute to diet-induced metabolic inflexibility were investigated. Mice were fed a high fat or low fat diet for 1 d, 1 wk, and 20 wk. Cardiac mitochondria isolated from mice fed a high fat diet displayed a diminished ability to utilize the glycolytically derived substrate pyruvate. This response was rapid, occurring within the first day on the diet, and persisted for up to 20 wk. A selective increase in the expression of pyruvate dehydrogenase kinase 4 and inhibition of pyruvate dehydrogenase are responsible for the rapid suppression of pyruvate utilization. An important consequence is that pyruvate dehydrogenase is sensitized to inhibition when mitochondria respire in the presence of fatty acids. Additionally, increased expression of pyruvate dehydrogenase kinase 4 preceded any observed diet-induced reductions in the levels of glucose transporter type 4 and glycolytic enzymes and, as judged by Akt phosphorylation, insulin signaling. Importantly, diminished insulin signaling evident at 1 wk on the high fat diet did not occur in pyruvate dehydrogenase kinase 4 knockout mice. Dietary intervention leads to a rapid decline in pyruvate dehydrogenase kinase 4 levels and recovery of pyruvate dehydrogenase activity indicating an additional form of regulation. Finally, an overnight fast elicits a metabolic response similar to that induced by high dietary fat obscuring diet-induced metabolic changes. Thus, our data indicate that diet-induced inhibition of pyruvate dehydrogenase may be an initiating event in decreased oxidation of glucose and increased reliance of the heart on fatty acids for energy production.


Assuntos
Gorduras na Dieta/metabolismo , Miocárdio/metabolismo , Proteínas Quinases/metabolismo , Animais , Dieta com Restrição de Gorduras , Dieta Hiperlipídica/efeitos adversos , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Miocárdio/enzimologia , Proteínas Quinases/genética , Ácido Pirúvico/metabolismo
19.
Redox Biol ; 1: 80-5, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24024140

RESUMO

Alzheimer disease (AD) is an age-related neurodegenerative disease characterized by the presence of three pathological hallmarks: synapse loss, extracellular senile plaques (SP) and intracellular neurofibrillary tangles (NFTs). The major component of SP is amyloid ß-peptide (Aß), which has been shown to induce oxidative stress. The AD brain shows increased levels of lipid peroxidation products, including 4-hydroxy-2-nonenal (HNE). HNE can react covalently with Cys, His, or Lys residues on proteins, altering structure and function of the latter. In the present study we measured the levels of the HNE-modified lipoic acid in brain of subjects with AD and age-matched controls. Lipoic acid is a key co-factor for a number of proteins including pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, key complexes for cellular energetics. We observed a significant decrease in the levels of HNE-lipoic acid in the AD brain compared to that of age-matched controls. To investigate this phenomenon further, the levels and activity of lipoamide dehydrogenase (LADH) were measured in AD and control brains. Additionally, LADH activities were measured after in-vitro HNE-treatment to mice brains. Both LADH levels and activities were found to be significantly reduced in AD brain compared to age-matched control. HNE-treatment also reduced the LADH activity in mice brain. These data are consistent with a two-hit hypothesis of AD: oxidative stress leads to lipid peroxidation that, in turn, causes oxidative dysfunction of key energy-related complexes in mitochondria, triggering neurodegeneration. This study is consonant with the notion that lipoic acid supplementation could be a potential treatment for the observed loss of cellular energetics in AD and potentiate the antioxidant defense system to prevent or delay the oxidative stress in and progression of this devastating dementing disorder.


Assuntos
Aldeídos/metabolismo , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Di-Hidrolipoamida Desidrogenase/metabolismo , Ácido Tióctico/metabolismo , Animais , Estudos de Casos e Controles , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo
20.
Am J Physiol Heart Circ Physiol ; 305(5): H634-43, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23792672

RESUMO

Obesity enhances the risk for the development of type 2 diabetes and cardiovascular disease. Loss in insulin sensitivity and diminished ability of muscle to take up and use glucose are characteristics of type 2 diabetes. Paradoxically, regulatory mechanisms that promote utilization of fatty acids appear to initiate diet-induced insulin insensitivity. In this review, we discuss recent findings implicating increased mitochondrial production of the prooxidant H2O2 due to enhanced utilization of fatty acids, as a signal to diminish reliance on glucose and its metabolites for energy. In the short term, the ability to preferentially use fatty acids may be beneficial, promoting a metabolic shift that ensures use of available fat by skeletal muscle and heart while preventing intracellular glucose accumulation and toxicity. However, with prolonged consumption of high dietary fat and ensuing obesity, the near exclusive dependence on fatty acid oxidation for production of energy by the mitochondria drives insulin resistance, diabetes, and cardiovascular disease.


Assuntos
Metabolismo Energético/fisiologia , Ácidos Graxos/metabolismo , Resistência à Insulina/fisiologia , Mitocôndrias Musculares/metabolismo , Animais , Doenças Cardiovasculares/epidemiologia , Diabetes Mellitus/epidemiologia , Glucose/metabolismo , Humanos , Obesidade/complicações , Obesidade/metabolismo , Oxirredução , Fatores de Risco
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