Assuntos
Insuficiência Cardíaca , Inibidores do Transportador 2 de Sódio-Glicose , Transportador 2 de Glucose-Sódio , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Animais , Camundongos , Transportador 2 de Glucose-Sódio/metabolismo , Transportador 2 de Glucose-Sódio/genética , Insuficiência Cardíaca/tratamento farmacológico , Volume Sistólico/efeitos dos fármacos , Camundongos Knockout , Camundongos Endogâmicos C57BL , MasculinoRESUMO
Cellular reprogramming of somatic cells to patient-specific induced pluripotent stem cells (iPSCs) enables in vitro modelling of human genetic disorders for pathogenic investigations and therapeutic screens. However, using iPSC-derived cardiomyocytes (iPSC-CMs) to model an adult-onset heart disease remains challenging owing to the uncertainty regarding the ability of relatively immature iPSC-CMs to fully recapitulate adult disease phenotypes. Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an inherited heart disease characterized by pathological fatty infiltration and cardiomyocyte loss predominantly in the right ventricle, which is associated with life-threatening ventricular arrhythmias. Over 50% of affected individuals have desmosome gene mutations, most commonly in PKP2, encoding plakophilin-2 (ref. 9). The median age at presentation of ARVD/C is 26 years. We used previously published methods to generate iPSC lines from fibroblasts of two patients with ARVD/C and PKP2 mutations. Mutant PKP2 iPSC-CMs demonstrate abnormal plakoglobin nuclear translocation and decreased ß-catenin activity in cardiogenic conditions; yet, these abnormal features are insufficient to reproduce the pathological phenotypes of ARVD/C in standard cardiogenic conditions. Here we show that induction of adult-like metabolic energetics from an embryonic/glycolytic state and abnormal peroxisome proliferator-activated receptor gamma (PPAR-γ) activation underlie the pathogenesis of ARVD/C. By co-activating normal PPAR-alpha-dependent metabolism and abnormal PPAR-γ pathway in beating embryoid bodies (EBs) with defined media, we established an efficient ARVD/C in vitro model within 2 months. This model manifests exaggerated lipogenesis and apoptosis in mutant PKP2 iPSC-CMs. iPSC-CMs with a homozygous PKP2 mutation also had calcium-handling deficits. Our study is the first to demonstrate that induction of adult-like metabolism has a critical role in establishing an adult-onset disease model using patient-specific iPSCs. Using this model, we revealed crucial pathogenic insights that metabolic derangement in adult-like metabolic milieu underlies ARVD/C pathologies, enabling us to propose novel disease-modifying therapeutic strategies.
Assuntos
Displasia Arritmogênica Ventricular Direita/metabolismo , Displasia Arritmogênica Ventricular Direita/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Modelos Biológicos , Transporte Ativo do Núcleo Celular , Idade de Início , Apoptose/genética , Displasia Arritmogênica Ventricular Direita/genética , Displasia Arritmogênica Ventricular Direita/fisiopatologia , Reprogramação Celular , Meios de Cultura/farmacologia , Corpos Embrioides/efeitos dos fármacos , Corpos Embrioides/fisiologia , Metabolismo Energético/genética , Ácidos Graxos/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Glucose/metabolismo , Glicólise , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lipogênese/genética , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/patologia , PPAR alfa/metabolismo , PPAR gama/metabolismo , Fenótipo , Placofilinas/genética , Fatores de Tempo , beta Catenina/metabolismoRESUMO
To identify new gene regulatory pathways controlling skeletal muscle energy metabolism, comparative studies were conducted on muscle-specific transgenic mouse lines expressing the nuclear receptors peroxisome proliferator-activated receptor α (PPARα; muscle creatine kinase [MCK]-PPARα) or PPARß/δ (MCK-PPARß/δ). MCK-PPARß/δ mice are known to have enhanced exercise performance, whereas MCK-PPARα mice perform at low levels. Transcriptional profiling revealed that the lactate dehydrogenase b (Ldhb)/Ldha gene expression ratio is increased in MCK-PPARß/δ muscle, an isoenzyme shift that diverts pyruvate into the mitochondrion for the final steps of glucose oxidation. PPARß/δ gain- and loss-of-function studies in skeletal myotubes demonstrated that PPARß/δ, but not PPARα, interacts with the exercise-inducible kinase AMP-activated protein kinase (AMPK) to synergistically activate Ldhb gene transcription by cooperating with myocyte enhancer factor 2A (MEF2A) in a PPARß/δ ligand-independent manner. MCK-PPARß/δ muscle was shown to have high glycogen stores, increased levels of GLUT4, and augmented capacity for mitochondrial pyruvate oxidation, suggesting a broad reprogramming of glucose utilization pathways. Lastly, exercise studies demonstrated that MCK-PPARß/δ mice persistently oxidized glucose compared with nontransgenic controls, while exhibiting supranormal performance. These results identify a transcriptional regulatory mechanism that increases capacity for muscle glucose utilization in a pattern that resembles the effects of exercise training.
Assuntos
Glucose/metabolismo , Músculo Esquelético/metabolismo , Fatores de Regulação Miogênica/metabolismo , PPAR delta/metabolismo , Proteínas Quinases/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Animais , Células Cultivadas , Feminino , Lactato Desidrogenases/genética , Lactato Desidrogenases/metabolismo , Masculino , Camundongos , Músculo Esquelético/enzimologia , Oxirredução , PPAR alfa/metabolismo , Condicionamento Físico Animal , Ativação TranscricionalRESUMO
BACKGROUND: Significant evidence indicates that the failing heart is energy starved. During the development of heart failure, the capacity of the heart to utilize fatty acids, the chief fuel, is diminished. Identification of alternate pathways for myocardial fuel oxidation could unveil novel strategies to treat heart failure. METHODS AND RESULTS: Quantitative mitochondrial proteomics was used to identify energy metabolic derangements that occur during the development of cardiac hypertrophy and heart failure in well-defined mouse models. As expected, the amounts of proteins involved in fatty acid utilization were downregulated in myocardial samples from the failing heart. Conversely, expression of ß-hydroxybutyrate dehydrogenase 1, a key enzyme in the ketone oxidation pathway, was increased in the heart failure samples. Studies of relative oxidation in an isolated heart preparation using ex vivo nuclear magnetic resonance combined with targeted quantitative myocardial metabolomic profiling using mass spectrometry revealed that the hypertrophied and failing heart shifts to oxidizing ketone bodies as a fuel source in the context of reduced capacity to oxidize fatty acids. Distinct myocardial metabolomic signatures of ketone oxidation were identified. CONCLUSIONS: These results indicate that the hypertrophied and failing heart shifts to ketone bodies as a significant fuel source for oxidative ATP production. Specific metabolite biosignatures of in vivo cardiac ketone utilization were identified. Future studies aimed at determining whether this fuel shift is adaptive or maladaptive could unveil new therapeutic strategies for heart failure.
Assuntos
Dieta Cetogênica/métodos , Ácidos Graxos/metabolismo , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Corpos Cetônicos/metabolismo , Animais , Feminino , Perfilação da Expressão Gênica/métodos , Insuficiência Cardíaca/dietoterapia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α and -1ß serve as master transcriptional regulators of muscle mitochondrial functional capacity and are capable of enhancing muscle endurance when overexpressed in mice. We sought to determine whether muscle-specific transgenic overexpression of PGC-1ß affects the detraining response following endurance training. First, we established and validated a mouse exercise-training-detraining protocol. Second, using multiple physiological and gene expression end points, we found that PGC-1ß overexpression in skeletal muscle of sedentary mice fully recapitulated the training response. Lastly, PGC-1ß overexpression during the detraining period resulted in partial prevention of the detraining response. Specifically, an increase in the plateau at which O2 uptake (VÌo2) did not change from baseline with increasing treadmill speed [peak VÌo2 (ΔVÌo2max)] was maintained in trained mice with PGC-1ß overexpression in muscle 6 wk after cessation of training. However, other detraining responses, including changes in running performance and in situ half relaxation time (a measure of contractility), were not affected by PGC-1ß overexpression. We conclude that while activation of muscle PGC-1ß is sufficient to drive the complete endurance phenotype in sedentary mice, it only partially prevents the detraining response following exercise training, suggesting that the process of endurance detraining involves mechanisms beyond the reversal of muscle autonomous mechanisms involved in endurance fitness. In addition, the protocol described here should be useful for assessing early-stage proof-of-concept interventions in preclinical models of muscle disuse atrophy.
Assuntos
Músculo Esquelético/fisiologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Condicionamento Físico Animal/métodos , Resistência Física/fisiologia , Aptidão Física/fisiologia , Corrida/fisiologia , Animais , Masculino , Camundongos , Camundongos Transgênicos , Transtornos Musculares Atróficos/fisiopatologia , Transtornos Musculares Atróficos/prevenção & controle , FenótipoRESUMO
RATIONALE: Increasing evidence has shown that proper control of mitochondrial dynamics (fusion and fission) is required for high-capacity ATP production in the heart. Transcriptional coactivators, peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1) α and PGC-1ß, have been shown to regulate mitochondrial biogenesis in the heart at the time of birth. The function of PGC-1 coactivators in the heart after birth has been incompletely understood. OBJECTIVE: Our aim was to assess the role of PGC-1 coactivators during postnatal cardiac development and in adult hearts in mice. METHODS AND RESULTS: Conditional gene targeting was used in mice to explore the role of PGC-1 coactivators during postnatal cardiac development and in adult hearts. Marked mitochondrial structural derangements were observed in hearts of PGC-1α/ß-deficient mice during postnatal growth, including fragmentation and elongation, associated with the development of a lethal cardiomyopathy. The expression of genes involved in mitochondrial fusion (Mfn1, Opa1) and fission (Drp1, Fis1) was altered in the hearts of PGC-1α/ß-deficient mice. PGC-lα was shown to directly regulate Mfn1 gene transcription by coactivating the estrogen-related receptor α on a conserved DNA element. Surprisingly, PGC-1α/ß deficiency in the adult heart did not result in evidence of abnormal mitochondrial dynamics or heart failure. However, transcriptional profiling demonstrated that PGC-1 coactivators are required for high-level expression of nuclear- and mitochondrial-encoded genes involved in mitochondrial dynamics and energy transduction in the adult heart. CONCLUSIONS: These results reveal distinct developmental stage-specific programs involved in cardiac mitochondrial dynamics.
Assuntos
Cardiomiopatias/metabolismo , Coração/crescimento & desenvolvimento , Mitocôndrias Cardíacas/metabolismo , Fatores de Transcrição/metabolismo , Fatores Etários , Animais , Cardiomiopatias/genética , Progressão da Doença , Metabolismo Energético/fisiologia , Receptor alfa de Estrogênio/metabolismo , Feminino , GTP Fosfo-Hidrolases/genética , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Knockout , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fatores de Transcrição/genéticaRESUMO
The energy demands of the adult mammalian heart are met largely by ATP generated via oxidation of fatty acids in a high capacity mitochondrial system. Peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1)-α and -ß serve as inducible transcriptional coregulators of genes involved in mitochondrial biogenesis and metabolism. Whether PGC-1 plays a role in the regulation of mitochondrial structure is unknown. In this study, mice with combined deficiency of PGC-1α and PGC-1ß (PGC-1αß(-/-)) in adult heart were analyzed. PGC-1αß(-/-) hearts exhibited a distinctive mitochondrial cristae-stacking abnormality suggestive of a phospholipid abnormality as has been described in humans with genetic defects in cardiolipin (CL) synthesis (Barth syndrome). A subset of molecular species, containing n-3 polyunsaturated species in the CL, phosphatidylcholine, and phosphatidylethanolamine profiles, was reduced in PGC-1αß-deficient hearts. Gene expression profiling of PGC-1αß(-/-) hearts revealed reduced expression of the gene encoding CDP-diacylglycerol synthase 1 (Cds1), an enzyme that catalyzes the proximal step in CL biosynthesis. Cds1 gene promoter-reporter cotransfection experiments and chromatin immunoprecipitation studies demonstrated that PGC-1α coregulates estrogen-related receptors to activate the transcription of the Cds1 gene. We conclude that the PGC-1/estrogen-related receptor axis coordinately regulates metabolic and membrane structural programs relevant to the maintenance of high capacity mitochondrial function in heart.
Assuntos
Diacilglicerol Colinofosfotransferase/biossíntese , Regulação Enzimológica da Expressão Gênica/fisiologia , Fosfatidilcolinas/biossíntese , Fosfatidiletanolaminas/biossíntese , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Animais , Síndrome de Barth/genética , Síndrome de Barth/metabolismo , Síndrome de Barth/patologia , Linhagem Celular , Diacilglicerol Colinofosfotransferase/genética , Feminino , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fosfatidilcolinas/genética , Fosfatidiletanolaminas/genética , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Fatores de Transcrição/genéticaRESUMO
Sodium-glucose co-transporter 2 inhibitors (SGLT2i) are novel, potent heart failure medications with an unknown mechanism of action. We sought to determine if the beneficial actions of SGLT2i in heart failure were on- or off-target, and related to metabolic reprogramming, including increased lipolysis and ketogenesis. The phenotype of mice treated with empagliflozin and genetically engineered mice constitutively lacking SGLT2 mirrored metabolic changes seen in human clinical trials (including reduced blood glucose, increased ketogenesis, and profound glucosuria). In a mouse heart failure model, SGLT2i treatment, but not generalized SGLT2 knockout, resulted in improved systolic function and reduced pathologic cardiac remodeling. SGLT2i treatment of the SGLT2 knockout mice sustained the cardiac benefits, demonstrating an off-target role for these drugs. This benefit is independent of metabolic changes, including ketosis. The mechanism of action and target of SGLT2i in HF remain elusive.
RESUMO
Background: Two general phenotypes of heart failure (HF) are recognized: HF with reduced ejection fraction (HFrEF) and with preserved EF (HFpEF). To develop HF disease phenotype-specific approaches to define and guide treatment, distinguishing biomarkers are needed. The goal of this study was to utilize quantitative metabolomics on a large, diverse population to replicate and extend existing knowledge of the plasma metabolic signatures in human HF. Methods: Quantitative, targeted LC/MS plasma metabolomics was conducted on 787 samples collected by the Penn Medicine BioBank from subjects with HFrEF (n=219), HFpEF (n=357), and matched non-failing Controls (n=211). A total of 90 metabolites were analyzed, comprising 28 amino acids, 8 organic acids, and 54 acylcarnitines. 733 of these samples were also processed via an OLINK protein panel for proteomic profiling. Results: Consistent with previous studies, unsaturated forms of medium/long chain acylcarnitines were elevated in the HFrEF group to a greater extent than the HFpEF group compared to Controls. A number of amino acid derivatives, including 1- and 3-methylhistidine, homocitrulline, and symmetric (SDMA) and asymmetric (ADMA) dimethylarginine were elevated in HF, with ADMA elevated uniquely in HFpEF. Plasma branched-chain amino acids (BCAA) were not different across the groups; however, short-chain acylcarnitine species indicative of BCAA catabolism were significantly elevated in both HF groups. The ketone body 3-hydroxybutyrate (3-HBA) and its metabolite C4-OH carnitine were uniquely elevated in the HFrEF group. Linear regression models demonstrated a significant correlation between plasma 3-HBA and NT-proBNP in both forms of HF, stronger in HFrEF. Conclusions: These results identify plasma signatures that are shared as well as potentially distinguish between HFrEF and HFpEF. Metabolite markers for ketogenic metabolic reprogramming in extra-cardiac tissues were identified as unique signatures in the HFrEF group, possibly related to the lipolytic action of increased levels of BNP. Future studies will be necessary to further validate these metabolites as HF biosignatures that may guide phenotype-specific therapeutics and provide insight into the systemic metabolic responses to HFpEF and HFrEF.
RESUMO
During the development of heart failure (HF), the capacity for cardiomyocyte (CM) fatty acid oxidation (FAO) and ATP production is progressively diminished, contributing to pathologic cardiac hypertrophy and contractile dysfunction. Receptor-interacting protein 140 (RIP140, encoded by Nrip1) has been shown to function as a transcriptional corepressor of oxidative metabolism. We found that mice with striated muscle deficiency of RIP140 (strNrip1-/-) exhibited increased expression of a broad array of genes involved in mitochondrial energy metabolism and contractile function in heart and skeletal muscle. strNrip1-/- mice were resistant to the development of pressure overload-induced cardiac hypertrophy, and CM-specific RIP140-deficient (csNrip1-/-) mice were protected against the development of HF caused by pressure overload combined with myocardial infarction. Genomic enhancers activated by RIP140 deficiency in CMs were enriched in binding motifs for transcriptional regulators of mitochondrial function (estrogen-related receptor) and cardiac contractile proteins (myocyte enhancer factor 2). Consistent with a role in the control of cardiac fatty acid oxidation, loss of RIP140 in heart resulted in augmented triacylglyceride turnover and fatty acid utilization. We conclude that RIP140 functions as a suppressor of a transcriptional regulatory network that controls cardiac fuel metabolism and contractile function, representing a potential therapeutic target for the treatment of HF.
Assuntos
Insuficiência Cardíaca , Proteína 1 de Interação com Receptor Nuclear , Animais , Camundongos , Cardiomegalia/metabolismo , Metabolismo Energético/genética , Ácidos Graxos/metabolismo , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Miócitos Cardíacos/metabolismo , Proteína 1 de Interação com Receptor Nuclear/genética , Proteína 1 de Interação com Receptor Nuclear/metabolismoRESUMO
Diabetic cardiac dysfunction is associated with decreased rates of myocardial glucose oxidation (GO) and increased fatty acid oxidation (FAO), a fuel shift that has been shown to sensitize the heart to ischemic insult and ventricular dysfunction. We sought to evaluate the metabolic and functional consequences of chronic suppression of GO in heart as modeled by transgenic mice with cardiac-specific overexpression of pyruvate dehydrogenase kinase 4 (myosin heavy chain (MHC)-PDK4 mice), an inhibitor of pyruvate dehydrogenase. Hearts of MHC-PDK4 mice were shown to exhibit an insulin-resistant substrate utilization profile, characterized by low GO rates and high FAO flux. Surprisingly, MHC-PDK4 mice were not sensitized to cardiac ischemia-reperfusion injury despite a fuel utilization pattern that phenocopied the diabetic heart. In addition, MHC-PDK4 mice were protected against high fat diet-induced myocyte lipid accumulation, likely related to increased capacity for FAO. The high rates of mitochondrial FAO in the MHC-PDK4 heart were related to heightened activity of the AMP-activated protein kinase, reduced levels of malonyl-CoA, and increased capacity for mitochondrial uncoupled respiration. The expression of the known AMP-activated protein kinase target, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a master regulator of mitochondrial function and biogenesis, was also activated in the MHC-PDK4 heart. These results demonstrate that chronic activation of PDK4 triggers transcriptional and post-transcriptional mechanisms that re-program the heart for chronic high rates of FAO without the expected deleterious functional or metabolic consequences.
Assuntos
Cardiomiopatias Diabéticas/enzimologia , Glucose/metabolismo , Mitocôndrias Cardíacas/enzimologia , Miocárdio/enzimologia , Proteínas Serina-Treonina Quinases/biossíntese , Animais , Cardiomiopatias Diabéticas/genética , Modelos Animais de Doenças , Glucose/genética , Frequência Cardíaca/genética , Resistência à Insulina/genética , Camundongos , Camundongos Transgênicos , Mitocôndrias Cardíacas/genética , Isquemia Miocárdica/enzimologia , Isquemia Miocárdica/genética , Miocárdio/patologia , Cadeias Pesadas de Miosina/genética , Oxirredução , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Proteínas Serina-Treonina Quinases/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição , Disfunção Ventricular/enzimologia , Disfunção Ventricular/genéticaRESUMO
RATIONALE: Long chain fatty acids (LCFAs) are the preferred substrate for energy provision in hearts. However, the contribution of endogenous triacylglyceride (TAG) turnover to LCFA oxidation and the overall dependence of mitochondrial oxidation on endogenous lipid is largely unstudied. OBJECTIVE: We sought to determine the role of TAG turnover in supporting LCFA oxidation and the influence of the lipid-activated nuclear receptor, proliferator-activated receptor (PPAR)alpha, on this balance. METHODS AND RESULTS: Palmitoyl turnover within TAG and palmitate oxidation rates were quantified in isolated hearts, from normal mice (nontransgenic) and mice with cardiac-specific overexpression of PPARalpha (MHC-PPARalpha). Turnover of palmitoyl units within TAG, and thus palmitoyl-coenzyme A recycling, in nontransgenic (4.5+/-2.3 micromol/min per gram dry weight) was 3.75-fold faster than palmitate oxidation (1.2+/-0.4). This high rate of palmitoyl unit turnover indicates preferential oxidation of palmitoyl units derived from TAG in normal hearts. PPARalpha overexpression augmented TAG turnover 3-fold over nontransgenic hearts, despite similar fractions of acetyl-coenzyme A synthesis from palmitate and oxygen use at the same workload. Palmitoyl turnover within TAG of MHC-PPARalpha hearts (16.2+/-2.9, P<0.05) was 12.5-fold faster than oxidation (1.3+/-0.2). Elevated TAG turnover in MHC-PPARalpha correlated with increased mRNA for enzymes involved in both TAG synthesis, Gpam (glycerol-3-phosphate acyltransferase, mitochondrial), Dgat1 (diacylglycerol acetyltransferase 1), and Agpat3 (1-acylglycerol-3-phospate O-acyltransferase 3), and lipolysis, Pnliprp1 (pancreatic lipase related protein 1). CONCLUSIONS: The role of endogenous TAG in supporting beta-oxidation in the normal heart is much more dynamic than previously thought, and lipolysis provides the bulk of LCFA for oxidation. Accelerated palmitoyl turnover in TAG, attributable to chronic PPARalpha activation, results in near requisite oxidation of LCFAs from TAG.
Assuntos
Metabolismo Energético , Lipase/metabolismo , Miocárdio/metabolismo , PPAR alfa/metabolismo , Ácido Palmítico/metabolismo , Triglicerídeos/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , 1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Acetilcoenzima A/metabolismo , Animais , Cardiotônicos/farmacologia , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/metabolismo , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Regulação Enzimológica da Expressão Gênica , Glicerol-3-Fosfato O-Aciltransferase/genética , Glicerol-3-Fosfato O-Aciltransferase/metabolismo , Hemodinâmica , Isoproterenol/farmacologia , Lipase/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oxirredução , Consumo de Oxigênio , PPAR alfa/genética , Palmitoil Coenzima A/metabolismo , Perfusão , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
Perturbations in hepatic lipid homeostasis are linked to the development of obesity-related steatohepatitis. Mutations in the gene encoding lipin 1 cause hepatic steatosis in fld mice, a genetic model of lipodystrophy. However, the molecular function of lipin 1 is unclear. Herein, we demonstrate that the expression of lipin 1 is induced by peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator 1alpha (PGC-1alpha), a transcriptional coactivator controlling several key hepatic metabolic pathways. Gain-of-function and loss-of-function strategies demonstrated that lipin selectively activates a subset of PGC-1alpha target pathways, including fatty acid oxidation and mitochondrial oxidative phosphorylation, while suppressing the lipogenic program and lowering circulating lipid levels. Lipin activates mitochondrial fatty acid oxidative metabolism by inducing expression of the nuclear receptor PPARalpha, a known PGC-1alpha target, and via direct physical interactions with PPARalpha and PGC-1alpha. These results identify lipin 1 as a selective physiological amplifier of the PGC-1alpha/PPARalpha-mediated control of hepatic lipid metabolism.
Assuntos
Metabolismo dos Lipídeos/fisiologia , Fígado/metabolismo , Proteínas Nucleares/metabolismo , PPAR alfa/metabolismo , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Animais , Linhagem Celular , Ácidos Graxos/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/fisiopatologia , Regulação da Expressão Gênica/fisiologia , Hepatócitos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Nucleares/genética , Fosforilação Oxidativa , PPAR alfa/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fosfatidato Fosfatase , Transativadores/genética , Fatores de Transcrição , Ativação Transcricional/fisiologiaRESUMO
PGC-1α is a potent, inducible transcriptional coactivator that exerts control on mitochondrial biogenesis and multiple cellular energy metabolic pathways. PGC-1α levels are controlled in a highly dynamic manner reflecting regulation at both transcriptional and post-transcriptional levels. Here, we demonstrate that PGC-1α is rapidly degraded in the nucleus (t(½ 0.3 h) via the ubiquitin proteasome system. An N-terminal deletion mutant of 182 residues, PGC182, as well as a lysine-less mutant form, are nuclear and rapidly degraded (t(½) 0.5 h), consistent with degradation via the N terminus-dependent ubiquitin subpathway. Both PGC-1α and PGC182 degradation rates are increased in cells under low serum conditions. However, a naturally occurring N-terminal splice variant of 270 residues, NT-PGC-1α is cytoplasmic and stable (t(½>7 h), providing additional evidence that PGC-1α is degraded in the nucleus. These results strongly suggest that the nuclear N terminus-dependent ubiquitin proteasome pathway governs PGC-1α cellular degradation. In contrast, the cellular localization of NT-PCG-1α results in a longer-half-life and possible distinct temporal and potentially biological actions.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Choque Térmico/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Sequência de Aminoácidos , Animais , Núcleo Celular/genética , Citoplasma/genética , Células HeLa , Proteínas de Choque Térmico/genética , Células Hep G2 , Humanos , Camundongos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Complexo de Endopeptidases do Proteassoma/genética , Estabilidade Proteica , Estrutura Terciária de Proteína , Deleção de Sequência , Transativadores/genética , Fatores de Transcrição/genética , Ubiquitina/genéticaRESUMO
Hypertension and diabetes are common side effects of glucocorticoid treatment. To determine whether peroxisome proliferator-activated receptor-alpha (PPAR-alpha) mediates these sequelae, mice deficient in low-density lipoprotein receptor (Ldlr-/-), with (Ppara+/+) or without (Ppara-/-) PPAR-alpha, were treated chronically with dexamethasone. Ppara+/+, but not Ppara-/-, mice developed hyperglycemia, hyperinsulinemia and hypertension. Similar effects on glucose metabolism were seen in a different model using C57BL/6 mice. Hepatic gluconeogenic gene expression was increased and insulin-mediated suppression of endogenous glucose production was less effective in dexamethasone-treated Ppara+/+ mice. Adenoviral reconstitution of PPAR-alpha in the livers of nondiabetic, normotensive, dexamethasone-treated Ppara-/- mice induced hyperglycemia, hyperinsulinemia and increased gluconeogenic gene expression. It also increased blood pressure, renin activity, sympathetic nervous activity and renal sodium retention. Human hepatocytes treated with dexamethasone and the PPAR-alpha agonist Wy14,643 induced PPARA and gluconeogenic gene expression. These results identify hepatic activation of PPAR-alpha as a mechanism underlying glucocorticoid-induced insulin resistance.
Assuntos
Dexametasona/farmacologia , Diabetes Mellitus/metabolismo , Glucocorticoides/farmacologia , Hipertensão/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de LDL/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , HDL-Colesterol/metabolismo , Glucose/metabolismo , Hepatócitos/citologia , Hepatócitos/fisiologia , Humanos , Insulina/metabolismo , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de LDL/genética , Sódio/metabolismoRESUMO
BACKGROUND: Accumulating evidence suggests that the failing heart reprograms fuel metabolism toward increased utilization of ketone bodies and that increasing cardiac ketone delivery ameliorates cardiac dysfunction. As an initial step toward development of ketone therapies, we investigated the effect of chronic oral ketone ester (KE) supplementation as a prevention or treatment strategy in rodent heart failure models. METHODS: Two independent rodent heart failure models were used for the studies: transverse aortic constriction/myocardial infarction (MI) in mice and post-MI remodeling in rats. Seventy-five mice underwent a prevention treatment strategy with a KE comprised of hexanoyl-hexyl-3-hydroxybutyrate KE (KE-1) diet, and 77 rats were treated in either a prevention or treatment regimen using a commercially available ß-hydroxybutyrate-(R)-1,3-butanediol monoester (DeltaG; KE-2) diet. RESULTS: The KE-1 diet in mice elevated ß-hydroxybutyrate levels during nocturnal feeding, whereas the KE-2 diet in rats induced ketonemia throughout a 24-hour period. The KE-1 diet preventive strategy attenuated development of left ventricular dysfunction and remodeling post-transverse aortic constriction/MI (left ventricular ejection fraction±SD, 36±8 in vehicle versus 45±11 in KE-1; P=0.016). The KE-2 diet therapeutic approach also attenuated left ventricular dysfunction and remodeling post-MI (left ventricular ejection fraction, 41±11 in MI-vehicle versus 61±7 in MI-KE-2; P<0.001). In addition, ventricular weight, cardiomyocyte cross-sectional area, and the expression of ANP (atrial natriuretic peptide) were significantly attenuated in the KE-2-treated MI group. However, treatment with KE-2 did not influence cardiac fibrosis post-MI. The myocardial expression of the ketone transporter and 2 ketolytic enzymes was significantly increased in rats fed KE-2 diet along with normalization of myocardial ATP levels to sham values. CONCLUSIONS: Chronic oral supplementation with KE was effective in both prevention and treatment of heart failure in 2 preclinical animal models. In addition, our results indicate that treatment with KE reprogrammed the expression of genes involved in ketone body utilization and normalized myocardial ATP production following MI, consistent with provision of an auxiliary fuel. These findings provide rationale for the assessment of KEs as a treatment for patients with heart failure.
Assuntos
Suplementos Nutricionais , Insuficiência Cardíaca/fisiopatologia , Hidroxibutiratos , Infarto do Miocárdio/fisiopatologia , Miocárdio/metabolismo , Volume Sistólico/fisiologia , Disfunção Ventricular Esquerda/fisiopatologia , Trifosfato de Adenosina/metabolismo , Animais , Aorta/cirurgia , Fator Natriurético Atrial/metabolismo , Constrição Patológica , Fibrose , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Camundongos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/patologia , Miócitos Cardíacos/patologia , Tamanho do Órgão , Ratos , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/patologia , Função Ventricular EsquerdaRESUMO
OBJECTIVE: UCP2 -866G>A (rs659366) has been implicated in cardiometabolic disease and represents a novel candidate gene for beta-blocker response, particularly among patients with diabetes. We assessed the function of -866G>A and its role as a modifier of beta-blocker treatment outcomes by diabetes status in an acute coronary syndrome (ACS) cohort. METHODS: ACS patients with genetic samples and 12 months of follow-up for cardiac rehospitalizations or death (n=468) were assessed. The influence of -866G>A on beta-blocker treatment outcomes was evaluated in those with diabetes and without. To assess functional correlates of -866G>A, we compared uncoupling protein 2 (UCP2) expression in the skeletal muscle of obese participants by genotype and compared the activity of UCP2 luciferase promoters with -866G and -866A alleles. RESULTS: An interaction between -866G>A and beta-blocker treatment was found in individuals with diabetes (P=0.002) but not those without (P=0.79). Among G/G individuals with diabetes, discharge beta-blocker use was associated with an 80% reduction in cardiac rehospitalization (adjusted hazard ratio: 0.20; 95% confidence interval: 0.04-1.02). In contrast, among A-carrier patients with diabetes, there was an 11-fold increase in cardiac rehospitalizations with discharge beta-blocker therapy (adjusted hazard ratio: 11.75; 95% confidence interval: 1.28-108.2). Promoter activity assays showed that -866G had greater cyclic AMP response element binding protein-responsiveness compared with -866A, and compared with -866A carriers G/G individuals exhibited increased UCP2 expression in the skeletal muscle. CONCLUSION: We identified a significant interaction between -866G>A and beta-blocker response among ACS patients with diabetes. Furthermore, -866G conferred greater gene transcriptional activity than -866A in cell lines and in obese patients. These findings may help us gain insight into the mechanisms underlying the beneficial and detrimental effects of beta-blockers in those with diabetes.
Assuntos
Síndrome Coronariana Aguda/etiologia , Síndrome Coronariana Aguda/genética , Antagonistas Adrenérgicos beta/efeitos adversos , Diabetes Mellitus/tratamento farmacológico , Canais Iônicos/genética , Proteínas Mitocondriais/genética , Polimorfismo de Nucleotídeo Único , Antagonistas Adrenérgicos beta/farmacocinética , Antagonistas Adrenérgicos beta/uso terapêutico , Idoso , Linhagem Celular , Estudos de Coortes , Complicações do Diabetes/etiologia , Complicações do Diabetes/genética , Feminino , Expressão Gênica , Estudos de Associação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/genética , Farmacogenética , Proteínas Recombinantes/genética , Transcrição Gênica , Transfecção , Proteína Desacopladora 2RESUMO
To define the necessity of calcineurin (Cn) signaling for cardiac maturation and function, the postnatal phenotype of mice with cardiac-specific targeted ablation of the Cn B1 regulatory subunit (Ppp3r1) gene (csCnb1(-/-) mice) was characterized. csCnb1(-/-) mice develop a lethal cardiomyopathy, characterized by impaired postnatal growth of the heart and combined systolic and diastolic relaxation abnormalities, despite a lack of structural derangements. Notably, the csCnb1(-/-) hearts did not exhibit diastolic dilatation, despite the severe functional phenotype. Myocytes isolated from the mutant mice exhibited reduced rates of contraction/relaxation and abnormalities in calcium transients, consistent with altered sarcoplasmic reticulum loading. Levels of sarco(endo) plasmic reticulum Ca-ATPase 2a (Atp2a2) and phospholamban were normal, but phospholamban phosphorylation was markedly reduced at Ser(16) and Thr(17). In addition, levels of the Na/Ca exchanger (Slc8a1) were modestly reduced. These results define a novel mouse model of cardiac-specific Cn deficiency and demonstrate novel links between Cn signaling, postnatal growth of the heart, pathological ventricular remodeling, and excitation-contraction coupling.
Assuntos
Calcineurina/deficiência , Sinalização do Cálcio , Cardiomiopatias/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Proteínas Musculares/deficiência , Contração Miocárdica , Miocárdio/metabolismo , Disfunção Ventricular Esquerda/metabolismo , Envelhecimento/metabolismo , Animais , Calcineurina/genética , Sinalização do Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Cardiomiopatias/genética , Cardiomiopatias/patologia , Cardiomiopatias/fisiopatologia , Cardiotônicos/administração & dosagem , Dobutamina/administração & dosagem , Ácidos Graxos/metabolismo , Genótipo , Ventrículos do Coração/metabolismo , Ventrículos do Coração/fisiopatologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas/metabolismo , Proteínas Musculares/genética , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/genética , Miocárdio/patologia , Oxirredução , Fenótipo , Fosforilação , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Serina , Trocador de Sódio e Cálcio/metabolismo , Treonina , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/fisiopatologia , Remodelação VentricularRESUMO
The gene encoding the transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) was targeted in mice. PGC-1alpha null (PGC-1alpha(-/-)) mice were viable. However, extensive phenotyping revealed multi-system abnormalities indicative of an abnormal energy metabolic phenotype. The postnatal growth of heart and slow-twitch skeletal muscle, organs with high mitochondrial energy demands, is blunted in PGC-1alpha(-/-) mice. With age, the PGC-1alpha(-/-) mice develop abnormally increased body fat, a phenotype that is more severe in females. Mitochondrial number and respiratory capacity is diminished in slow-twitch skeletal muscle of PGC-1alpha(-/-) mice, leading to reduced muscle performance and exercise capacity. PGC-1alpha(-/-) mice exhibit a modest diminution in cardiac function related largely to abnormal control of heart rate. The PGC-1alpha(-/-) mice were unable to maintain core body temperature following exposure to cold, consistent with an altered thermogenic response. Following short-term starvation, PGC-1alpha(-/-) mice develop hepatic steatosis due to a combination of reduced mitochondrial respiratory capacity and an increased expression of lipogenic genes. Surprisingly, PGC-1alpha(-/-) mice were less susceptible to diet-induced insulin resistance than wild-type controls. Lastly, vacuolar lesions were detected in the central nervous system of PGC-1alpha(-/-) mice. These results demonstrate that PGC-1alpha is necessary for appropriate adaptation to the metabolic and physiologic stressors of postnatal life.