RESUMO
Triple-negative breast cancer (TNBC) poses significant challenges for treatment given the lack of targeted therapies and increased probability of relapse. It is pertinent to identify vulnerabilities in TNBC and develop newer treatments. Our prior research demonstrated that transcription factor EB (TFEB) is necessary for TNBC survival by regulating DNA repair, apoptosis signaling, and the cell cycle. However, specific mechanisms by which TFEB targets DNA repair and cell cycle pathways are unclear, and whether these effects dictate TNBC survival is yet to be determined. Here, we show that TFEB knockdown decreased the expression of genes and proteins involved in DNA replication and cell cycle progression in MDA-MB-231 TNBC cells. DNA replication was decreased in cells lacking TFEB, as measured by EdU incorporation. TFEB silencing in MDA-MB-231 and noncancerous MCF10A cells impaired progression through the S-phase following G1/S synchronization; however, this proliferation defect could not be rescued by co-knockdown of suppressor RB1. Instead, TFEB knockdown reduced origin licensing in G1 and early S-phase MDA-MB-231 cells. TFEB silencing was associated with replication stress in MCF10A but not in TNBC cells. Lastly, we identified that TFEB knockdown renders TNBC cells more sensitive to inhibitors of Aurora Kinase A, a protein facilitating mitosis. Thus, inhibition of TFEB impairs cell cycle progress by decreasing origin licensing, leading to delayed entry into the S-phase, while rendering TNBC cells sensitive to Aurora kinase A inhibitors and decreasing cell viability. In contrast, TFEB silencing in noncancerous cells is associated with replication stress and leads to G1/S arrest.
Assuntos
Aurora Quinase A , Ciclo Celular , Células Epiteliais , Fatores de Transcrição , Neoplasias de Mama Triplo Negativas , Humanos , Apoptose/genética , Aurora Quinase A/antagonistas & inibidores , Aurora Quinase A/metabolismo , Linhagem Celular Tumoral , Replicação do DNA/genética , Células Epiteliais/metabolismo , Fatores de Transcrição/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Técnicas de Silenciamento de Genes , Transdução de Sinais/genética , Ciclo Celular/genéticaRESUMO
Branched-chain α-keto acids (BCKAs) are catabolites of branched-chain amino acids (BCAAs). Intracellular BCKAs are cleared by branched-chain ketoacid dehydrogenase (BCKDH), which is sensitive to inhibitory phosphorylation by BCKD kinase (BCKDK). Accumulation of BCKAs is an indicator of defective BCAA catabolism and has been correlated with glucose intolerance and cardiac dysfunction. However, it is unclear whether BCKAs directly alter insulin signaling and function in the skeletal and cardiac muscle cell. Furthermore, the role of excess fatty acids (FAs) in perturbing BCAA catabolism and BCKA availability merits investigation. By using immunoblotting and ultra-performance liquid chromatography MS/MS to analyze the hearts of fasted mice, we observed decreased BCAA-catabolizing enzyme expression and increased circulating BCKAs, but not BCAAs. In mice subjected to diet-induced obesity (DIO), we observed similar increases in circulating BCKAs with concomitant changes in BCAA-catabolizing enzyme expression only in the skeletal muscle. Effects of DIO were recapitulated by simulating lipotoxicity in skeletal muscle cells treated with saturated FA, palmitate. Exposure of muscle cells to high concentrations of BCKAs resulted in inhibition of insulin-induced AKT phosphorylation, decreased glucose uptake, and mitochondrial oxygen consumption. Altering intracellular clearance of BCKAs by genetic modulation of BCKDK and BCKDHA expression showed similar effects on AKT phosphorylation. BCKAs increased protein translation and mTORC1 activation. Pretreating cells with mTORC1 inhibitor rapamycin restored BCKA's effect on insulin-induced AKT phosphorylation. This study provides evidence for FA-mediated regulation of BCAA-catabolizing enzymes and BCKA content and highlights the biological role of BCKAs in regulating muscle insulin signaling and function.
Assuntos
Aminoácidos de Cadeia Ramificada/metabolismo , Insulina/metabolismo , Músculo Esquelético/metabolismo , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/antagonistas & inibidores , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/genética , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/metabolismo , Aminoácidos de Cadeia Ramificada/sangue , Animais , Linhagem Celular , Dieta Hiperlipídica , Regulação para Baixo/efeitos dos fármacos , Insulina/farmacologia , Cetoácidos/sangue , Cetoácidos/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/citologia , Miocárdio/metabolismo , Palmitatos/farmacologia , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and autophagy with critical roles in several cancers. Lysosomal autophagy promotes cancer survival through the degradation of toxic molecules and the maintenance of adequate nutrient supply. Doxorubicin (DOX) is the standard of care treatment for triple-negative breast cancer (TNBC); however, chemoresistance at lower doses and toxicity at higher doses limit its usefulness. By targeting pathways of survival, DOX can become an effective antitumor agent. In this study, we examined the role of TFEB in TNBC and its relationship with autophagy and DNA damage induced by DOX. In TNBC cells, TFEB was hypo-phosphorylated and localized to the nucleus upon DOX treatment. TFEB knockdown decreased the viability of TNBC cells while increasing caspase-3 dependent apoptosis. Additionally, inhibition of the TFEB-phosphatase calcineurin sensitized cells to DOX-induced apoptosis in a TFEB dependent fashion. Regulation of apoptosis by TFEB was not a consequence of altered lysosomal function, as TFEB continued to protect against apoptosis in the presence of lysosomal inhibitors. RNA-Seq analysis of MDA-MB-231 cells with TFEB silencing identified a down-regulation in cell cycle and homologous recombination genes while interferon-γ and death receptor signaling genes were up-regulated. In consequence, TFEB knockdown disrupted DNA repair following DOX, as evidenced by persistent γH2A.X detection. Together, these findings describe in TNBC a novel lysosomal independent function for TFEB in responding to DNA damage.
Assuntos
Apoptose , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/fisiologia , Reparo do DNA , Lisossomos/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Doxorrubicina/farmacologia , Técnicas de Silenciamento de Genes , HumanosRESUMO
Besides serving as a structural membrane component and intermediate of the glycerolipid metabolism, lysophosphatidic acid (LPA) has a prominent role as a signaling molecule through its binding to LPA receptors at the cell surface. Extracellular LPA is primarily produced from lysophosphatidylcholine (LPC) through the activity of secreted lysophospholipase D, autotaxin (ATX). The degradation of extracellular LPA to monoacylglycerol is mediated by lipid phosphate phosphatases (LPPs) at the cell membrane. This review summarizes and interprets current literature on the role of the ATX-LPA-LPP3 axis in the regulation of energy homeostasis, insulin function, and adiposity at baseline and under conditions of obesity. We also discuss how the ATX-LPA-LPP3 axis influences obesity-related metabolic complications, including insulin resistance, fatty liver disease, and cardiomyopathy.
Assuntos
Metabolismo Energético , Lisofosfolipídeos/metabolismo , Doenças Metabólicas/metabolismo , Fosfatidato Fosfatase/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Transdução de Sinais , Animais , HumanosRESUMO
BACKGROUND: The objectives of the study were to characterize and quantify cellular inflammation and structural remodeling of human atria and correlate findings with molecular markers of inflammation and patient surrogate outcome. METHODS: Voluntary participants undergoing heart surgery were enrolled in the study and blood samples were collected prior to surgery, and right atrium samples were harvested intraoperatively. Blood samples were analyzed by flow cytometry and complete blood counts. Atrial samples were divided for fixed fibrosis analysis, homogenized for cytokine analysis and digested for single cell suspension flow cytometry. RESULTS: A total of 18 patients were enrolled and samples assessed. Isolated cells from the atria revealed a CD45+ population of ~ 20%, confirming a large number of leukocytes. Further characterization revealed this population as 57% lymphocytes and 26% monocyte/macrophages (MoΦ), with the majority of the latter cells being classical (CD14++/CD16-). Interstitial fibrosis was present in 87% of samples and correlated significantly with patient age. Older patients (> 65) had significantly more atrial fibrosis and cellular inflammation. AFib patients had no distinguishing feature of atrial fibrosis and had significantly greater CD45+ MoΦ, increased expression of MMP9 and presented with a significant correlation in length of stay to CCL-2/MCP-1 and NLR (neutrophil-to-lymphocyte ratio). CONCLUSION: Atrial fibrosis is correlated with age and not determinate to AFib. However, severity of atrial leukocyte infiltration and markers of matrix degradation are determinant to AFib. This also correlated with CCL2 (or MCP-1) and NLR-indicative of marked inflammation. These data show the potential importance of diagnostic and prognostic assessments that could inform clinical decision making in regard to the intensity of AFib patient management.
Assuntos
Fibrilação Atrial/patologia , Fibrilação Atrial/cirurgia , Procedimentos Cirúrgicos Cardíacos , Leucócitos/patologia , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Fibrilação Atrial/sangue , Plaquetas/patologia , Contagem de Células , Estudos de Coortes , Feminino , Fibrose , Átrios do Coração/patologia , Humanos , Tempo de Internação , Antígenos Comuns de Leucócito/metabolismo , Linfócitos/patologia , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Neutrófilos/patologia , Prognóstico , Nó Sinoatrial/patologiaRESUMO
Autotaxin (ATX) is an adipokine that generates the bioactive lipid, lysophosphatidic acid (LPA). ATX-LPA signaling has been implicated in diet-induced obesity and systemic insulin resistance. However, it remains unclear whether the ATX-LPA pathway influences insulin function and energy metabolism in target tissues, particularly skeletal muscle, the major site of insulin-stimulated glucose disposal. The objective of this study was to test whether the ATX-LPA pathway impacts tissue insulin signaling and mitochondrial metabolism in skeletal muscle during obesity. Male mice with heterozygous ATX deficiency (ATX+/-) were protected from obesity, systemic insulin resistance, and cardiomyocyte dysfunction following high-fat high-sucrose (HFHS) feeding. HFHS-fed ATX+/- mice also had improved insulin-stimulated AKT phosphorylation in white adipose tissue, liver, heart, and skeletal muscle. Preserved insulin-stimulated glucose transport in muscle from HFHS-fed ATX+/- mice was associated with improved mitochondrial pyruvate oxidation in the absence of changes in fat oxidation and ectopic lipid accumulation. Similarly, incubation with LPA decreased insulin-stimulated AKT phosphorylation and mitochondrial energy metabolism in C2C12 myotubes at baseline and following palmitate-induced insulin resistance. Taken together, our results suggest that the ATX-LPA pathway contributes to obesity-induced insulin resistance in metabolically relevant tissues. Our data also suggest that LPA directly impairs skeletal muscle insulin signaling and mitochondrial function.
Assuntos
Resistência à Insulina , Lisofosfolipídeos/metabolismo , Mitocôndrias/patologia , Obesidade/metabolismo , Obesidade/patologia , Diester Fosfórico Hidrolases/metabolismo , Transdução de Sinais , Animais , Glucose/metabolismo , Homeostase , Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Especificidade de ÓrgãosRESUMO
The heart balances uptake, metabolism and oxidation of fatty acids (FAs) to maintain ATP production, membrane biosynthesis and lipid signaling. Under conditions where FA uptake outpaces FA oxidation and FA sequestration as triacylglycerols in lipid droplets, toxic FA metabolites such as ceramides, diacylglycerols, long-chain acyl-CoAs, and acylcarnitines can accumulate in cardiomyocytes and cause cardiomyopathy. Moreover, studies using mutant mice have shown that dysregulation of enzymes involved in triacylglycerol, phospholipid, and sphingolipid metabolism in the heart can lead to the excess deposition of toxic lipid species that adversely affect cardiomyocyte function. This review summarizes our current understanding of lipid uptake, metabolism and signaling pathways that have been implicated in the development of lipotoxic cardiomyopathy under conditions including obesity, diabetes, aging, and myocardial ischemia-reperfusion. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk.
Assuntos
Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/toxicidade , Miocárdio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos BiológicosRESUMO
Impaired cardiac metabolism in the obese and diabetic heart leads to glucolipotoxicity and ensuing cardiomyopathy. Glucolipotoxicity causes cardiomyocyte injury by increasing energy insufficiency, impairing proteasomal-mediated protein degradation and inducing apoptosis. Proteasome-evading proteins are degraded by autophagy in the lysosome, whose metabolism and function are regulated by master regulator transcription factor EB (TFEB). Limited studies have examined the impact of glucolipotoxicity on intra-lysosomal signaling proteins and their regulators. By utilizing a mouse model of diet-induced obesity, type-1 diabetes (Akita) and ex-vivo model of glucolipotoxicity (H9C2 cells and NRCM, neonatal rat cardiomyocyte), we examined whether glucolipotoxicity negatively targets TFEB and lysosomal proteins to dysregulate autophagy and cause cardiac injury. Despite differential effects of obesity and diabetes on LC3B-II, expression of proteins facilitating autophagosomal clearance such as TFEB, LAMP-2A, Hsc70 and Hsp90 were decreased in the obese and diabetic heart. In-vivo data was recapitulated in H9C2 and NRCM cells, which exhibited impaired autophagic flux and reduced TFEB content when exposed to a glucolipotoxic milieu. Notably, overloading myocytes with a saturated fatty acid (palmitate) but not an unsaturated fatty acid (oleate) depleted cellular TFEB and suppressed autophagy, suggesting a fatty acid specific regulation of TFEB and autophagy in the cardiomyocyte. The effect of glucolipotoxicity to reduce TFEB content was also confirmed in heart tissue from patients with Class-I obesity. Therefore, during glucolipotoxicity, suppression of lysosomal autophagy was associated with reduced lysosomal content, decreased cathepsin-B activity and diminished cellular TFEB content likely rendering myocytes susceptible to cardiac injury.
Assuntos
Autofagia/fisiologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Diabetes Mellitus/metabolismo , Lisossomos/metabolismo , Miócitos Cardíacos/metabolismo , Obesidade/metabolismo , Animais , Apoptose/fisiologia , Autofagossomos/metabolismo , Linhagem Celular , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ácido Oleico/metabolismo , Palmitatos/metabolismo , Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologiaRESUMO
Dieldrin is a legacy organochlorine pesticide that is persistent in the environment, despite being discontinued from use in North America since the 1970s. Some epidemiologic studies suggest that exposure to dieldrin is associated with increased risks of neurodegenerative disease and breast cancer by inducing inflammatory responses in tissues as well as oxidative stress. However, the direct effects of organochlorine pesticides on the heart have not been adequately addressed to date given that these chemicals are detectable in human serum and are environmentally persistent; thus, individuals may show latent adverse effects in the cardiovascular system due to long-term, low-dose exposure over time. Our objective was to determine whether low-level exposure to dieldrin at an environmentally relevant dose results in aberrant molecular signaling in the vertebrate heart. Using transcriptomic profiling and immunoblotting, we determined the global gene and targeted protein expression response to dieldrin treatment and show that dieldrin affects gene networks in the heart that are associated with processes related to cardiovascular disease, specifically cardiac arrest and ventricular fibrillation. We report that genes regulating inflammatory responses, a significant risk factor for cardiovascular disease, are upregulated by dieldrin whereas transcripts related to lysosomal function are significantly downregulated. To verify these findings, proteins in these pathways were examined with immunoblotting, and our results demonstrate that dieldrin constitutively activates Akt/mTOR signaling and downregulates lysosomal genes, participating in autophagy. Our data demonstrate that dieldrin induces genes associated with cardiovascular dysfunction and compromised lysosomal physiology, thereby identifying a novel mechanism for pesticide-induced cardiotoxicity.
Assuntos
Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Dieldrin/farmacologia , Coração/efeitos dos fármacos , Inseticidas/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Relação Dose-Resposta a Droga , Feminino , Redes Reguladoras de Genes/efeitos dos fármacos , Redes Reguladoras de Genes/genética , Masculino , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Peixe-ZebraRESUMO
Doxorubicin (DOX) is an effective anti-cancer agent. However, DOX treatment increases patient susceptibility to dilated cardiomyopathy. DOX predisposes cardiomyocytes to insult by suppressing mitochondrial energy metabolism, altering calcium flux, and disrupting proteolysis and proteostasis. Prior studies have assessed the role of macroautophagy in DOX cardiotoxicity; however, limited studies have examined whether DOX mediates cardiac injury through dysfunctions in inter- and/or intra-lysosomal signaling events. Lysosomal signaling and function is governed by transcription factor EB (TFEB). In the present study, we hypothesized that DOX caused myocyte injury by impairing lysosomal function and signaling through negative regulation of TFEB. Indeed, we found that DOX repressed cellular TFEB expression, which was associated with impaired cathepsin proteolytic activity across in vivo, ex vivo, and in vitro models of DOX cardiotoxicity. Furthermore, we observed that loss of TFEB was associated with reduction in macroautophagy protein expression, inhibition of autophagic flux, impairments in lysosomal cathepsin B activity, and activation of cell death. Restoration and/or activation of TFEB in DOX-treated cardiomyocytes prevented DOX-induced suppression of cathepsin B activity, reduced DOX-mediated reactive oxygen species (ROS) overproduction, attenuated activation of caspase-3, and improved cellular viability. Collectively, loss of TFEB inhibits lysosomal autophagy, rendering cardiomyocytes susceptible to DOX-induced proteotoxicity and injury. Our data reveal a novel mechanism wherein DOX primes cardiomyocytes for cell death by depleting cellular TFEB.
Assuntos
Autofagia/efeitos dos fármacos , Doxorrubicina/farmacologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Immunoblotting , Marcação In Situ das Extremidades Cortadas , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Proteólise/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Cardiomyocytes are vulnerable to hypoxia in the adult, but adapted to hypoxia in utero. Current understanding of endogenous cardiac oxygen sensing pathways is limited. Myocardial oxygen consumption is determined by regulation of energy metabolism, which shifts from glycolysis to lipid oxidation soon after birth, and is reversed in failing adult hearts, accompanying re-expression of several "fetal" genes whose role in disease phenotypes remains unknown. Here we show that hypoxia-controlled expression of the transcription factor Hand1 determines oxygen consumption by inhibition of lipid metabolism in the fetal and adult cardiomyocyte, leading to downregulation of mitochondrial energy generation. Hand1 is under direct transcriptional control by HIF1α. Transgenic mice prolonging cardiac Hand1 expression die immediately following birth, failing to activate the neonatal lipid metabolising gene expression programme. Deletion of Hand1 in embryonic cardiomyocytes results in premature expression of these genes. Using metabolic flux analysis, we show that Hand1 expression controls cardiomyocyte oxygen consumption by direct transcriptional repression of lipid metabolising genes. This leads, in turn, to increased production of lactate from glucose, decreased lipid oxidation, reduced inner mitochondrial membrane potential, and mitochondrial ATP generation. We found that this pathway is active in adult cardiomyocytes. Up-regulation of Hand1 is protective in a mouse model of myocardial ischaemia. We propose that Hand1 is part of a novel regulatory pathway linking cardiac oxygen levels with oxygen consumption. Understanding hypoxia adaptation in the fetal heart may allow development of strategies to protect cardiomyocytes vulnerable to ischaemia, for example during cardiac ischaemia or surgery.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Metabolismo Energético/genética , Metabolismo dos Lipídeos/genética , Miocárdio/metabolismo , Consumo de Oxigênio/genética , Trifosfato de Adenosina/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Hipóxia Celular/genética , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Coração/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Potencial da Membrana Mitocondrial/genética , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Isquemia Miocárdica/genética , Isquemia Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Oxigênio/metabolismo , Ativação TranscricionalRESUMO
RATIONALE: The energy sensor AMP-activated protein kinases (AMPK) is thought to play an important role in regulating myocardial fatty acid oxidation (FAO) via its phosphorylation and inactivation of acetyl coenzyme A carboxylase (ACC). However, studies supporting this have not directly assessed whether the maintenance of FAO rates and subsequent cardiac function requires AMPK-dependent inhibitory phosphorylation of ACC. OBJECTIVE: To determine whether preventing AMPK-mediated inactivation of ACC influences myocardial FAO or function. METHODS AND RESULTS: A double knock-in (DKI) mouse (ACC-DKI) model was generated in which the AMPK phosphorylation sites Ser79 on ACC1 and Ser221 (Ser212 mouse) on ACC2 were mutated to prevent AMPK-dependent inhibitory phosphorylation of ACC. Hearts from ACC-DKI mice displayed a complete loss of ACC phosphorylation at the AMPK phosphorylation sites. Despite the inability of AMPK to regulate ACC activity, hearts from ACC-DKI mice displayed normal basal AMPK activation and cardiac function at both standard and elevated workloads. In agreement with the inability of AMPK in hearts from ACC-DKI mice to phosphorylate and inhibit ACC, there was a significant increase in cardiac malonyl-CoA content compared with wild-type mice. However, cardiac FAO rates were comparable between wild-type and ACC-DKI mice at baseline, during elevated workloads, and after a more stressful condition of myocardial ischemia that is known to robustly activate AMPK. CONCLUSIONS: Our findings show AMPK-dependent inactivation of ACC is not essential for the control of myocardial FAO and subsequent cardiac function during a variety of conditions involving AMPK-independent and AMPK-dependent metabolic adaptations.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Acetil-CoA Carboxilase/metabolismo , Metabolismo Energético , Ácidos Graxos/metabolismo , Contração Miocárdica , Miocárdio/enzimologia , Acetil-CoA Carboxilase/genética , Animais , Feminino , Técnicas de Introdução de Genes , Masculino , Malonil Coenzima A/metabolismo , Camundongos Mutantes , Camundongos Transgênicos , Mutação , Oxirredução , Fosforilação , Serina , Fatores de Tempo , Função Ventricular EsquerdaRESUMO
Myocardial triacylglycerol (TAG) constitutes a highly dynamic fatty acid (FA) storage pool that can be used for an energy reserve in the cardiomyocyte. However, derangements in myocardial TAG metabolism and accumulation are commonly associated with cardiac disease, suggesting an important role of intramyocardial TAG turnover in the regulation of cardiac function. In cardiomyocytes, TAG is synthesized by acyltransferases and phosphatases at the sarcoplasmic reticulum and mitochondrial membrane and then packaged into cytosolic lipid droplets for temporary storage or into lipoproteins for secretion. A complex interplay among lipases, lipase regulatory proteins, and lipid droplet scaffold proteins leads to the controlled release of FAs from the cardiac TAG pool for subsequent mitochondrial ß-oxidation and energy production. With the identification and characterization of proteins involved in myocardial TAG metabolism as well as the identification of the importance of cardiac TAG turnover, it is now evident that adequate regulation of myocardial TAG metabolism is critical for both cardiac energy metabolism and function. In this article, we review the current understanding of myocardial TAG metabolism and discuss the potential role of myocardial TAG turnover in cardiac health and disease. This article is part of a Special Issue entitled "Focus on Cardiac Metabolism".
Assuntos
Miocárdio/metabolismo , Triglicerídeos/metabolismo , Animais , Metabolismo Energético , Humanos , Metabolismo dos Lipídeos , Lipoproteínas/metabolismo , Miocárdio/enzimologia , Triglicerídeos/biossínteseRESUMO
Although pre-clinical evidence has suggested that partial inhibition of myocardial fatty acid oxidation (FAO) and subsequent switch to greater glucose oxidation for ATP production can prevent ischemia/reperfusion injury, controversy about this approach persists. For example, mice with germline deletion of the FA transporter CD36, exhibited either impaired or unchanged post-ischemic functional recovery despite a 40-60% reduction in FAO rates. Because there are limitations to cardiac studies utilizing whole body CD36 knockout (totalCD36KO) mice, we have now generated an inducible and cardiomyocyte-specific CD36 KO (icCD36KO) mouse to better address the role of cardiomyocyte CD36 and its regulation of FAO and post-ischemic functional recovery. Four to six weeks following CD36 ablation, hearts from icCD36KO mice had significantly decreased FA uptake compared to controls, which was paralleled by significant reductions in intramyocardial triacylglycerol content. Analysis of cardiac energy metabolism using ex vivo working heart perfusions showed that reduced FAO rates were compensated by enhanced glucose oxidation in the hearts from icCD36KO mice. In contrast to the totalCD36KO mice, hearts from icCD36KO mice exhibited significantly improved functional recovery following ischemia/reperfusion (18min of global no-flow ischemia followed by 40min of aerobic reperfusion). This improved recovery was associated with lower calculated proton production prior to and following ischemia compared to controls. Moreover, the amount of ATP generated relative to cardiac work was significantly lower in the hearts from icCD36KO mice compared to controls, indicating significantly increased cardiac efficiency in the hearts from icCD36KO mice. These data provide genetic evidence that reduced FAO as a result of diminished CD36-mediated FA uptake improves post-ischemic cardiac efficiency and functional recovery. As such, targeting cardiomyocyte FA uptake and FAO via inhibition of CD36 in the adult myocardium may provide therapeutic benefit during ischemia-reperfusion.
Assuntos
Antígenos CD36/genética , Isquemia Miocárdica/genética , Isquemia Miocárdica/fisiopatologia , Miócitos Cardíacos/metabolismo , Animais , Modelos Animais de Doenças , Metabolismo Energético , Ácidos Graxos/metabolismo , Ordem dos Genes , Marcação de Genes , Recombinação Homóloga , Masculino , Camundongos , Camundongos Knockout , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologiaRESUMO
Classical ischemia-reperfusion (IR) preconditioning relies on phosphatidylinositol 3-kinase (PI3K) for protective signaling. Surprisingly, inhibition of PI3Kα activity using a dominant negative (DN) strategy protected the murine heart from IR injury. It has been proposed that increased signaling through PI3Kγ may contribute to the improved recovery of PI3KαDN hearts following IR. To investigate the mechanism by which PI3KαDN hearts are protected from IR injury, we created a double mutant (PI3KDM) model by crossing p110γ(-/-) (PI3KγKO) with cardiac-specific PI3KαDN mice. The PI3KDM model has morphological and hemodynamic features that are characteristic of both PI3Kγ(-/-) and PI3KαDN mice. Interestingly, when subjected to IR using ex vivo Langendorff perfusion, PI3KDM hearts showed significantly enhanced functional recovery when compared to wildtype (WT) hearts. However, signaling downstream of PI3K through Akt and GSK3ß, which has been associated with IR protection, was reduced in PI3KDM hearts. Using ex vivo working heart perfusion, we found no difference in functional recovery after IR between PI3KDM and PI3KαDN; also, glucose oxidation rates were significantly increased in PI3KαDN hearts when compared to WT, and this metabolic shift has been associated with enhanced IR recovery. However, we found that PI3KαDN hearts still had enhanced recovery when perfused exclusively with fatty acids (FA). We then investigated parallel signaling pathways, and found that mitogen-activated protein kinase signaling was increased in PI3KαDN hearts, possibly through the inhibition of negative feedback loops downstream of PI3Kα.
Assuntos
Glucose/metabolismo , Sistema de Sinalização das MAP Quinases , Traumatismo por Reperfusão Miocárdica/enzimologia , Fosfatidilinositol 3-Quinases/genética , Animais , Classe I de Fosfatidilinositol 3-Quinases , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , AMP Cíclico/metabolismo , Feminino , Genes Dominantes , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Frequência Cardíaca , Técnicas In Vitro , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/enzimologia , Oxirredução , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sistemas do Segundo Mensageiro , Pressão VentricularRESUMO
Obesity is a state of metabolic dysfunction that can lead to dyslipidemia and impaired glucose homeostasis. Apple polyphenols have been shown to ameliorate dyslipidemia/metabolic dysfunction in humans. The influence of apple (poly)phenols on energy metabolism in high-fat (HF) diet-induced obese mice remains controversial. This study examined the effect of dietary supplementation of (poly)phenol-rich 'Daux Belan' apple (DB; 6.2 mg gallic acid equivalence (GAE)/mouse/day; 0.15% (poly)phenol) in the form of freeze-dried powder on glucose and lipid metabolism in male HF-fed C57BL/6NCrl mice, in comparison to low-(poly)phenol-containing 'Zestar' apple (Z; 0.4 mg GAE/mouse/day). Obesity, glucose intolerance, hypertriglyceridemia, and hepatic lipid vacuolation were induced by HF feeding while circulating cholesterol levels remained unchanged. DB apple supplementation did not protect against HF-induced body weight gain, hyperglycemia, hepatic triglyceride level elevation, and hepatic lipid vacuolation at the tested dosage. Future studies should be conducted with increased DB dosage and employ apple (poly)phenols supplemented in the form of extracts or sugar-free powder.
Assuntos
Dislipidemias , Intolerância à Glucose , Humanos , Masculino , Camundongos , Animais , Intolerância à Glucose/etiologia , Intolerância à Glucose/prevenção & controle , Intolerância à Glucose/metabolismo , Fenol/metabolismo , Camundongos Endogâmicos C57BL , Pós/farmacologia , Obesidade/metabolismo , Fígado/metabolismo , Dieta Hiperlipídica/efeitos adversos , Glucose/metabolismo , Suplementos Nutricionais , Fenóis/farmacologia , Fenóis/metabolismo , Dislipidemias/etiologia , Dislipidemias/prevenção & controle , Dislipidemias/metabolismo , Lipídeos/farmacologiaRESUMO
Lipin-1 is the major phosphatidate phosphatase (PAP) in the heart and a transcriptional coactivator that regulates fatty acid (FA) oxidation in the liver. As the control of FA metabolism is essential for maintaining cardiac function, we investigated whether lipin-1 deficiency affects cardiac metabolism and performance. Cardiac PAP activity in lipin-1 deficient [fatty liver dystrophy (fld)] mice was decreased by >80% compared with controls. Surprisingly, oleate oxidation and incorporation in triacylglycerol (TG), as well as glucose oxidation, were not significantly different in perfused working fld hearts. Despite this, [³H]oleate accumulation in phosphatidate and phosphatidylinositol was increased in fld hearts, reflecting the decreased PAP activity. Phosphatidate accumulation was linked to increased cardiac mammalian target of rapamycin complex 1 (mTORC1) signaling and endoplasmic reticulum (ER) stress. Transthoracic echocardiography showed decreased cardiac function in fld mice; however, cardiac dysfunction was not observed in ex vivo perfused working fld hearts. This showed that changes in systemic factors due to the global absence of lipin-1 could contribute to the decreased cardiac function in vivo. Collectively, this study shows that fld hearts exhibit unchanged oleate esterification, as well as oleate and glucose oxidation, despite the absence of lipin-1. However, lipin-1 deficiency increases the accumulation of newly synthesized phosphatidate and induces aberrant cell signaling.
Assuntos
Glucose/metabolismo , Coração/fisiologia , Proteínas Nucleares/deficiência , Ácido Oleico/metabolismo , Fosfatidato Fosfatase/deficiência , Animais , Fígado Gorduroso/fisiopatologia , Técnicas In Vitro , Masculino , Camundongos , Miocárdio/metabolismo , Triglicerídeos/biossínteseRESUMO
Maintenance of circadian alignment between an organism and its environment is essential to ensure metabolic homeostasis. Synchrony is achieved by cell autonomous circadian clocks. Despite a growing appreciation of the integral relation between clocks and metabolism, little is known regarding the direct influence of a peripheral clock on cellular responses to fatty acids. To address this important issue, we utilized a genetic model of disrupted clock function specifically in cardiomyocytes in vivo (termed cardiomyocyte clock mutant (CCM)). CCM mice exhibited altered myocardial response to chronic high fat feeding at the levels of the transcriptome and lipidome as well as metabolic fluxes, providing evidence that the cardiomyocyte clock regulates myocardial triglyceride metabolism. Time-of-day-dependent oscillations in myocardial triglyceride levels, net triglyceride synthesis, and lipolysis were markedly attenuated in CCM hearts. Analysis of key proteins influencing triglyceride turnover suggest that the cardiomyocyte clock inactivates hormone-sensitive lipase during the active/awake phase both at transcriptional and post-translational (via AMP-activated protein kinase) levels. Consistent with increased net triglyceride synthesis during the end of the active/awake phase, high fat feeding at this time resulted in marked cardiac steatosis. These data provide evidence for direct regulation of triglyceride turnover by a peripheral clock and reveal a potential mechanistic explanation for accelerated metabolic pathologies after prevalent circadian misalignment in Western society.
Assuntos
Regulação da Expressão Gênica , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Triglicerídeos/metabolismo , Animais , Ritmo Circadiano , Ácidos Graxos , Perfilação da Expressão Gênica , Coração , Masculino , Camundongos , Perfusão , Processamento de Proteína Pós-Traducional , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
Consumption of milk-derived whey proteins has been demonstrated to have insulin-sensitizing effects in mice and humans, in part through the generation of bioactive whey peptides. While whey peptides can prevent insulin resistance in vitro, it is unclear whether consumption of whey peptides can prevent obesity-induced metabolic dysfunction in vivo. We sought to determine whether whey peptides consumption can protect from high fat (HF) diet-induced obesity and dysregulation of glucose homeostasis. Male C57BL/6J mice were fed either a low or HF diet for 13 weeks. HF diet fed mice were provided drinking water with no addition (control), undigested whey protein isolate (WPI, 1 mg ml-1) or whey protein hydrolysate (WPH, 1 mg ml-1) throughout the diet regimen. Mice consuming WPH gained more body weight and were more glucose intolerant compared to those consuming WPI or water only. Despite increased body weight gain, perigonadal adipose tissue weight and lipid accumulation were unchanged. However, excess lipids accumulated ectopically in the liver and skeletal muscle in mice consuming WPH, which was associated with elevated inflammatory markers systemically and in adipose tissue, liver, and skeletal muscle. In skeletal muscle, mitochondrial fat oxidation and electron transport chain proteins were decreased with WPH consumption, indicative of mitochondrial dysfunction. Taken together, our results demonstrate that WPH, but not WPI, exacerbates HF-induced body weight gain and impairs glucose homeostasis, which is accompanied by increased inflammation, ectopic fat accumulation and mitochondrial dysfunction. Thus, our results argue against the use of dietary whey peptide supplementation as a preventative option against HF diet-induced metabolic dysfunction.
Assuntos
Obesidade/metabolismo , Aumento de Peso/efeitos dos fármacos , Proteínas do Soro do Leite/farmacologia , Animais , Dieta Hiperlipídica , Modelos Animais de Doenças , Glucose/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Proteínas do Soro do Leite/administração & dosagemRESUMO
Triple-negative breast cancers (TNBCs) are characterized by poor survival, prognosis, and gradual resistance to cytotoxic chemotherapeutics, like doxorubicin (DOX). The clinical utility of DOX is limited by its cardiotoxic and chemoresistant effects that manifest over time. To induce chemoresistance, TNBC rewires oncogenic gene expression and cell signaling pathways. Recent studies have demonstrated that reprogramming of branched-chain amino acids (BCAAs) metabolism facilitates tumor growth and survival. Branched-chain ketoacid dehydrogenase kinase (BCKDK), a regulatory kinase of the rate-limiting enzyme of the BCAA catabolic pathway, is reported to activate RAS/RAF/MEK/ERK signaling to promote tumor cell proliferation. However, it remains unexplored if BCKDK action remodels TNBC proliferation and survival per se and influences susceptibility to DOX-induced genotoxic stress. TNBC cells treated with DOX exhibited reduced BCKDK expression and intracellular BCKAs. Genetic and pharmacological inhibition of BCKDK in TNBC cell lines also showed a similar reduction in intracellular and secreted BCKAs. BCKDK silencing in TNBC cells downregulated mitochondrial metabolism genes, reduced electron complex protein expression, oxygen consumption, and ATP production. Transcriptome analysis of BCKDK silenced cells confirmed dysregulation of mitochondrial metabolic networks and upregulation of the apoptotic signaling pathway. Furthermore, BCKDK inhibition with concurrent DOX treatment exacerbated apoptosis, caspase activity, and loss of TNBC proliferation. Inhibition of BCKDK in TNBC also upregulated sestrin 2 and concurrently decreased mTORC1 signaling and protein synthesis. Overall, loss of BCKDK action in TNBC remodels BCAA flux, reduces protein translation triggering cell death, ATP insufficiency, and susceptibility to genotoxic stress.