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[Figure: see text].
Assuntos
Antioxidantes/metabolismo , Citocinas/metabolismo , Cardiomiopatias Diabéticas/enzimologia , Miócitos Cardíacos/enzimologia , Nicotinamida Fosforribosiltransferase/metabolismo , Estresse Oxidativo , Animais , Apoptose , Autofagia , Células Cultivadas , Citocinas/genética , Cardiomiopatias Diabéticas/genética , Cardiomiopatias Diabéticas/patologia , Dieta Hiperlipídica , Modelos Animais de Doenças , Fibrose , Glutationa/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/patologia , Mitofagia , Miócitos Cardíacos/patologia , NAD/metabolismo , NADP/metabolismo , Nicotinamida Fosforribosiltransferase/genética , Ratos Wistar , Sirtuínas/genética , Sirtuínas/metabolismo , Tiorredoxinas/metabolismoRESUMO
Obesity is closely associated with low-grade chronic and systemic inflammation and dyslipidemia, and the consumption of omega-3 polyunsaturated fatty acids (n-3 PUFAs) may modulate obesity-related disorders, such as inflammation and dyslipidemia. An emerging research question is to understand the dietary intervention strategy that is more important regarding n-3 PUFA consumption: (1) a lower ratio of n-6/n-3 PUFAs or (2) a higher amount of n-3 PUFAs consumption. To understand the desirable dietary intervention method of n-3 PUFAs consumption, we replaced lard from the experimental diets with either perilla oil (PO) or corn oil (CO) to have identical n-3 amounts in the experimental diets. PO had a lower n-6/n-3 ratio, whereas CO contained higher amounts of PUFAs; it inherently contained relatively lower n-3 but higher n-6 PUFAs than PO. After the 12-week dietary intervention in ob/ob mice, dyslipidemia was observed in the normal chow and CO-fed ob/ob mice; however, PO feeding increased the high density lipoprotein-cholesterol (HDL-C) level; further, not only did the HDL-C level increase, the low density lipoprotein-cholesterol (LDL-C) and triglyceride (TG) levels also decreased significantly after lipopolysaccharide (LPS) injection. Consequently, extra TG accumulated in the liver and white adipose tissue (WAT) of normal chow- or CO-fed ob/ob mice after LPS injection; however, PO consumption decreased serum TG accumulation in the liver and WAT. PUFAs replacement attenuated systemic inflammation induced by LPS injection by increasing anti-inflammatory cytokines but inhibiting pro-inflammatory cytokine production in the serum and WAT. PO further decreased hepatic inflammation and fibrosis in comparison with the ND and CO. Hepatic functional biomarkers (aspartate aminotransferase (AST) and alanine transaminase (ALT) levels) were also remarkably decreased in the PO group. In LPS-challenged ob/ob mice, PO and CO decreased adipocyte size and adipokine secretion, with a reduction in phosphorylation of MAPKs compared to the ND group. In addition, LPS-inducible endoplasmic reticulum (ER) and oxidative stress decreased with consumption of PUFAs. Taken together, PUFAs from PO and CO play a role in regulating obesity-related disorders. Moreover, PO, which possesses a lower ratio of n-6/n-3 PUFAs, remarkably alleviated metabolic dysfunction in LPS-induced ob/ob mice. Therefore, an interventional trial considering the ratio of n-6/n-3 PUFAs may be desirable for modulating metabolic complications, such as inflammatory responses and ER stress in the circulation, liver, and/or WAT.
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Dislipidemias , Ácidos Graxos Ômega-3 , Animais , LDL-Colesterol/metabolismo , Dislipidemias/metabolismo , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-3/farmacologia , Inflamação/metabolismo , Lipopolissacarídeos/metabolismo , Fígado/metabolismo , Camundongos , Obesidade/metabolismoRESUMO
Cardiovascular disease (CVD) remains the leading cause of death globally, and heart failure is a major component of CVD-related morbidity and mortality. The development of cardiac hypertrophy in response to hemodynamic overload is initially considered to be beneficial; however, this adaptive response is limited and, in the presence of prolonged stress, will transition to heart failure. Yes-associated protein (YAP), the central downstream effector of the Hippo signaling pathway, regulates proliferation and survival in mammalian cells. Our previous work demonstrated that cardiac-specific loss of YAP leads to increased cardiomyocyte (CM) apoptosis and impaired CM hypertrophy during chronic myocardial infarction (MI) in the mouse heart. Because of its documented cardioprotective effects, we sought to determine the importance of YAP in response to acute pressure overload (PO). Our results indicate that endogenous YAP is activated in the heart during acute PO. YAP activation that depended upon RhoA was also observed in CMs subjected to cyclic stretch. To examine the function of endogenous YAP during acute PO, Yap+/flox;Creα-MHC (YAP-CHKO) and Yap+/flox mice were subjected to transverse aortic constriction (TAC). We found that YAP-CHKO mice had attenuated cardiac hypertrophy and significant increases in CM apoptosis and fibrosis that correlated with worsened cardiac function after 1 week of TAC. Loss of CM YAP also impaired activation of the cardioprotective kinase Akt, which may underlie the YAP-CHKO phenotype. Together, these data indicate a prohypertrophic, prosurvival function of endogenous YAP and suggest a critical role for CM YAP in the adaptive response to acute PO.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cardiomegalia/metabolismo , Fosfoproteínas/metabolismo , Pressão , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose , Cardiomegalia/etiologia , Cardiomegalia/patologia , Ciclo Celular , Proteínas de Ciclo Celular , Regulação para Baixo/genética , Fibrose , Técnicas de Inativação de Genes , Heterozigoto , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , PTEN Fosfo-Hidrolase/metabolismo , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Sinalização YAP , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Inflammation is a central feature of cardiovascular disease, including myocardial infarction and heart failure. Reperfusion of the ischemic myocardium triggers a complex inflammatory response that can exacerbate injury and worsen heart function, as well as prevent myocardial rupture and mediate wound healing. Therefore, a more complete understanding of this process could contribute to interventions that properly balance inflammatory responses for improved outcomes. In this study, we leveraged several approaches, including global and regional ischemia/reperfusion (I/R), genetically modified mice, and primary cell culture, to investigate the cell type-specific function of the tumor suppressor Ras association domain family member 1 isoform A (RASSF1A) in cardiac inflammation. Our results revealed that genetic inhibition of RASSF1A in cardiomyocytes affords cardioprotection, whereas myeloid-specific deletion of RASSF1A exacerbates inflammation and injury caused by I/R in mice. Cell-based studies revealed that RASSF1A negatively regulates NF-κB and thereby attenuates inflammatory cytokine expression. These findings indicate that myeloid RASSF1A antagonizes I/R-induced myocardial inflammation and suggest that RASSF1A may be a promising target in immunomodulatory therapy for the management of acute heart injury.
Assuntos
Inflamação/metabolismo , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Células Cultivadas , Masculino , Camundongos , Camundongos Knockout , Infarto do Miocárdio/patologia , Miocárdio/patologia , Células RAW 264.7 , Proteínas Supressoras de Tumor/deficiênciaRESUMO
The heart requires high-energy production, but metabolic ability declines in the failing heart. Nicotinamide phosphoribosyl-transferase (Nampt) is a rate-limiting enzyme in the salvage pathway of nicotinamide adenine dinucleotide (NAD) synthesis. NAD is directly involved in various metabolic processes and may indirectly regulate metabolic gene expression through sirtuin 1 (Sirt1), an NAD-dependent protein deacetylase. However, how Nampt regulates cardiac function and metabolism in the failing heart is poorly understood. Here we show that pressure-overload (PO)-induced heart failure is exacerbated in both systemic Nampt heterozygous knockout (Nampt+/-) mice and mice with cardiac-specific Nampt overexpression (Tg-Nampt). The NAD level declined in Nampt+/- mice under PO (wild: 377 pmol/mg tissue; Nampt+/-: 119 pmol/mg tissue; P = 0.028). In cultured cardiomyocytes, Nampt knockdown diminished mitochondrial NAD content and ATP production (relative ATP production: wild: 1; Nampt knockdown: 0.56; P = 0.0068), suggesting that downregulation of Nampt induces mitochondrial dysfunction. On the other hand, the NAD level was increased in Tg-Nampt mice at baseline but not during PO, possibly due to increased consumption of NAD by Sirt1. The expression of Sirt1 was increased in Tg-Nampt mice, in association with reduced overall protein acetylation. PO-induced downregulation of metabolic genes was exacerbated in Tg-Nampt mice. In cultured cardiomyocytes, Nampt and Sirt1 cooperatively suppressed mitochondrial proteins and ATP production, thereby promoting mitochondrial dysfunction. In addition, Nampt overexpression upregulated inflammatory cytokines, including TNF-α and monocyte chemoattractant protein-1. Thus endogenous Nampt maintains cardiac function and metabolism in the failing heart, whereas Nampt overexpression is detrimental during PO, possibly due to excessive activation of Sirt1, suppression of mitochondrial function, and upregulation of proinflammatory mechanisms.NEW & NOTEWORTHY Nicotinamide phosphoribosyl-transferase (Nampt) is a rate-limiting enzyme in the salvage pathway of nicotinamide adenine dinucleotide synthesis. We demonstrate that pressure overload-induced heart failure is exacerbated in both systemic Nampt heterozygous knockout mice and mice with cardiac-specific Nampt overexpression. Both loss- and gain-of-function models exhibited reduced protein acetylation, suppression of metabolic genes, and mitochondrial energetic dysfunction. Thus endogenous Nampt maintains cardiac function and metabolism in the failing heart, but cardiac-specific Nampt overexpression is detrimental rather than therapeutic.
Assuntos
Citocinas/metabolismo , Metabolismo Energético , Insuficiência Cardíaca/enzimologia , Mitocôndrias Cardíacas/enzimologia , Miócitos Cardíacos/enzimologia , Nicotinamida Fosforribosiltransferase/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Aorta Torácica/fisiopatologia , Aorta Torácica/cirurgia , Células Cultivadas , Citocinas/deficiência , Citocinas/genética , Modelos Animais de Doenças , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/fisiopatologia , Mediadores da Inflamação/metabolismo , Ligadura , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/patologia , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/deficiência , Nicotinamida Fosforribosiltransferase/genética , Sirtuína 1/genética , Sirtuína 1/metabolismoRESUMO
BACKGROUND: Human pluripotent stem cell (hPSC)-derived endothelial cells (ECs) have limited clinical utility because of undefined components in the differentiation system and poor cell survival in vivo. Here, we aimed to develop a fully defined and clinically compatible system to differentiate hPSCs into ECs. Furthermore, we aimed to enhance cell survival, vessel formation, and therapeutic potential by encapsulating hPSC-ECs with a peptide amphiphile (PA) nanomatrix gel. METHODS: We induced differentiation of hPSCs into the mesodermal lineage by culturing on collagen-coated plates with a glycogen synthase kinase 3ß inhibitor. Next, vascular endothelial growth factor, endothelial growth factor, and basic fibroblast growth factor were added for endothelial lineage differentiation, followed by sorting for CDH5 (VE-cadherin). We constructed an extracellular matrix-mimicking PA nanomatrix gel (PA-RGDS) by incorporating the cell adhesive ligand Arg-Gly-Asp-Ser (RGDS) and a matrix metalloproteinase-2-degradable sequence. We then evaluated whether the encapsulation of hPSC-CDH5+ cells in PA-RGDS could enhance long-term cell survival and vascular regenerative effects in a hind-limb ischemia model with laser Doppler perfusion imaging, bioluminescence imaging, real-time reverse transcription-polymerase chain reaction, and histological analysis. RESULTS: The resultant hPSC-derived CDH5+ cells (hPSC-ECs) showed highly enriched and genuine EC characteristics and proangiogenic activities. When injected into ischemic hind limbs, hPSC-ECs showed better perfusion recovery and higher vessel-forming capacity compared with media-, PA-RGDS-, or human umbilical vein EC-injected groups. However, the group receiving the PA-RGDS-encapsulated hPSC-ECs showed better perfusion recovery, more robust and longer cell survival (> 10 months), and higher and prolonged angiogenic and vascular incorporation capabilities than the bare hPSC-EC-injected group. Surprisingly, the engrafted hPSC-ECs demonstrated previously unknown sustained and dynamic vessel-forming behavior: initial perivascular concentration, a guiding role for new vessel formation, and progressive incorporation into the vessels over 10 months. CONCLUSIONS: We generated highly enriched hPSC-ECs via a clinically compatible system. Furthermore, this study demonstrated that a biocompatible PA-RGDS nanomatrix gel substantially improved long-term survival of hPSC-ECs in an ischemic environment and improved neovascularization effects of hPSC-ECs via prolonged and unique angiogenic and vessel-forming properties. This PA-RGDS-mediated transplantation of hPSC-ECs can serve as a novel platform for cell-based therapy and investigation of long-term behavior of hPSC-ECs.
Assuntos
Células Endoteliais da Veia Umbilical Humana/transplante , Isquemia/terapia , Metaloproteinase 2 da Matriz/administração & dosagem , Nanoestruturas/administração & dosagem , Oligopeptídeos/administração & dosagem , Células-Tronco Pluripotentes/transplante , Animais , Diferenciação Celular/fisiologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Células Endoteliais/fisiologia , Células Endoteliais/transplante , Membro Posterior/irrigação sanguínea , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Isquemia/fisiopatologia , Masculino , Camundongos , Camundongos Nus , Células-Tronco Pluripotentes/fisiologia , Distribuição Aleatória , Resultado do TratamentoRESUMO
BACKGROUND: Although methods for generating cardiomyocytes from pluripotent stem cells have been reported, current methods produce heterogeneous mixtures of cardiomyocytes and noncardiomyocyte cells. Here, we report an entirely novel system in which pluripotent stem cell-derived cardiomyocytes are purified by cardiomyocyte-specific molecular beacons (MBs). MBs are nanoscale probes that emit a fluorescence signal when hybridized to target mRNAs. METHOD AND RESULTS: Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6/7 were generated. Among 5 MBs, an MB that targeted myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 cardiomyocytes, a mouse cardiomyocyte cell line, but <3% of 4 noncardiomyocyte cell types in flow cytometry analysis, which indicates that MHC1-MB is specific for identifying cardiomyocytes. We delivered MHC1-MB into cardiomyogenically differentiated pluripotent stem cells through nucleofection. The detection rate of cardiomyocytes was similar to the percentages of cardiac troponin T- or cardiac troponin I-positive cardiomyocytes, which supports the specificity of MBs. Finally, MHC1-MB-positive cells were sorted by fluorescence-activated cell sorter from mouse and human pluripotent stem cell differentiating cultures, and ≈97% cells expressed cardiac troponin T or cardiac troponin I as determined by flow cytometry. These MB-based sorted cells maintained their cardiomyocyte characteristics, which was verified by spontaneous beating, electrophysiological studies, and expression of cardiac proteins. When transplanted in a myocardial infarction model, MB-based purified cardiomyocytes improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors. CONCLUSIONS: We developed a novel cardiomyocyte selection system that allows production of highly purified cardiomyocytes. These purified cardiomyocytes and this system can be valuable for cell therapy and drug discovery.
Assuntos
Transplante de Células/métodos , Infarto do Miocárdio/terapia , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , RNA Mensageiro/isolamento & purificação , Potenciais de Ação/fisiologia , Animais , Biomarcadores , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Citometria de Fluxo/métodos , Humanos , Camundongos , Miócitos Cardíacos/fisiologia , Cadeias Pesadas de Miosina/genética , Nanotecnologia , Conformação de Ácido Nucleico , Células-Tronco Pluripotentes/fisiologia , Sondas RNA/química , Sondas RNA/isolamento & purificação , RNA Mensageiro/química , Troponina I/genética , Troponina T/genéticaRESUMO
The prevalence of obesity has been recognized as a major public health issue with rapid increase globally. Obesity triggers other metabolic complications, such as diabetes, dyslipidemia, liver diseases, and cardiovascular diseases. Helianthus tuberosus L. (the Jerusalem artichoke) is an important edible plant that may provide health benefits in treating metabolic diseases. In this study, we investigated potential antiobesity effects of saccharified H. tuberosus L. (SH) and its fermented vinegar (fermented H. tuberosus L. [FH]) in a high-fat diet (HFD)-induced obesity murine model. FH exhibited significantly lower pH, Brix, and total sugar content compared with the SH, along with higher radical-scavenging activity. The body weight and adipose tissue weights were significantly decreased with the administration of SH and FH compared with the HFD group. SH and FH groups significantly attenuated hepatomegaly and lipid accumulation. The increased triglyceride (TG) content in obese mice was remarkably lower in the SH and FH groups. SH and FH alleviated serum dyslipidemia and atherogenic risk. Furthermore, expression of adipogenic genes was significantly downregulated after SH and FH supplementation compared with the HFD group. The TG and total cholesterol (TC) content of serum and adipose tissues significantly decreased by SH and FH administration in comparison with the HFD group. Reduced adiposity with SH and FH administration was confirmed by reduced adipocyte size and weight with inhibition of lipoprotein lipase expression. Our study showed that SH and FH, indeed FH was superior to SH, had antiobesity effects by decreasing adiposity, regulating dyslipidemia in systemic tissues, and inhibiting adipogenic gene expression.
Assuntos
Dislipidemias , Helianthus , Animais , Camundongos , Bebidas , Dieta Hiperlipídica , Dislipidemias/tratamento farmacológico , Dislipidemias/metabolismo , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Obesidade/genética , TriglicerídeosRESUMO
Introduction: Metabolic syndrome (MetS) is considered as a complex, intertwined multiple risk factors that directly increase the risk of various metabolic diseases, especially cardiovascular atherosclerotic diseases and diabetes mellitus type 2. While lifestyle changes, including dietary intervention are effective in mitigating or preventing MetS, there are no specific therapies against MetS. Typical western diets comprise of high saturated fatty acid, cholesterol, and simple sugar; consequently their consumption may increase the potential pathological developmental risk of MetS. Partial replacement of dietary fatty acids with polyunsaturated fatty acids (PUFAs) is widely recommended measure to manage MetS-related disorders. Methods: In the present study, we used rat model to investigate the role of n-3 PUFA enriched beef tallows (BT) on MetS and tunicamycin (TM)-induced endoplasmic reticulum (ER) stress, by partially replacing dietary fat (lard) with equal amounts of two different BTs; regular BT or n-3 PUFA-enriched BT. The experimental rats were randomly assigned to three different dietary groups (n = 16 per group): (1) high-fat and high-cholesterol diet (HFCD); (2) HFCD partially replaced with regular BT (HFCD + BT1); (3) HFCD partially replaced with n-3 enhanced BT (w/w) (HFCD + BT2). After 10 weeks of dietary intervention, each experimental rodent was intraperitoneally injected with either phosphate-buffered saline or 1 mg/kg body weight of TM. Results: HFCD + BT2 showed improved dyslipidemia before TM injection, and increased serum high-density lipoprotein cholesterol (HDL-C) levels after TM injection. BT replacement groups had significantly reduced hepatic triglyceride (TG) levels, and decreased total cholesterol (TC) and TG levels in epididymal adipose tissue (EAT). Furthermore, BT replacement remarkably attenuated TM-induced unfolded protein responses (UPRs) in liver, showing reduced ER stress, with BT2 being more effective in the EAT. Discussion: Therefore, our findings suggest that partially replacing dietary fats with n-3 PUFA to lower the ratio of n-6/n-3 PUFAs is beneficial in preventing pathological features of MetS by alleviating HFCD- and/or TM-induced dyslipidemia and ER stress.
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Aberrant epigenetic transcriptional regulation is linked to metastasis, a primary cause of cancer-related death. Dissecting the epigenetic mechanisms controlling metastatic progression may uncover important insights to tumor biology and potential therapeutic targets. Here, we investigated the role of the SIN3A histone deacetylase 1 and 2 (SIN3A-HDAC1/2) complex in cancer metastasis. Using a mouse model of melanoma metastasis, we found that the SIN3A-HDAC1/2 transcription repressor complex silences BMP6 expression, causing increased metastatic dissemination and tumor growth via suppression of BMP6-activated SMAD5 signaling. We further discovered that FAM83G/PAWS1, a downstream effector of BMP6-SMAD5 signaling, contributes critically to metastatic progression by promoting actin-dependent cytoskeletal dynamics and cell migration. Pharmacologic inhibition of the SIN3A-HDAC1/2 complex reduced the numbers of melanoma cells in the circulation and inhibited metastatic tumor growth by inducing disseminated cell dormancy, highlighting the SIN3A-HDAC1/2 repressor complex as a potential therapeutic target for blocking cancer metastasis. IMPLICATIONS: This study identifies the novel molecular links in the metastatic progression to target cytoskeletal dynamics in melanoma and identifies the SIN3A-HDAC1/2 complex and FAM83G/PAWS1 as potential targets for melanoma adjuvant therapy.
Assuntos
Proteína Morfogenética Óssea 6/metabolismo , Epigênese Genética/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Proteínas/metabolismo , Animais , Humanos , Melanoma , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Metástase NeoplásicaRESUMO
Aims: PERM1 is a striated muscle-specific regulator of mitochondrial bioenergetics. We previously demonstrated that PERM1 is downregulated in the failing heart and that PERM1 positively regulates metabolic genes known as targets of the transcription factor ERRα and its coactivator PGC-1α in cultured cardiomyocytes. The aims of this study were to determine the effect of loss of PERM1 on cardiac function and energetics using newly generated Perm1-knockout (Perm1 -/-) mice and to investigate the molecular mechanisms of its transcriptional control. Methods and results: Echocardiography showed that ejection fraction and fractional shortening were lower in Perm1 -/- mice than in wild-type mice (both p < 0.05), and the phosphocreatine-to-ATP ratio was decreased in Perm1 -/- hearts (p < 0.05), indicating reduced contractile function and energy reserves of the heart. Integrated proteomic and metabolomic analyses revealed downregulation of oxidative phosphorylation and upregulation of glycolysis and polyol pathways in Perm1 -/- hearts. To examine whether PERM1 regulates energy metabolism through ERRα, we performed co-immunoprecipitation assays, which showed that PERM1 bound to ERRα in cardiomyocytes and the mouse heart. DNA binding and reporter gene assays showed that PERM1 was localized to and activated the ERR target promoters partially through ERRα. Mass spectrometry-based screening in cardiomyocytes identified BAG6 and KANK2 as potential PERM1's binding partners in transcriptional regulation. Mammalian one-hybrid assay, in which PERM1 was fused to Gal4 DNA binding domain, showed that the recruitment of PERM1 to a gene promoter was sufficient to activate transcription, which was blunted by silencing of either PGC-1α, BAG6, or KANK2. Conclusion: This study demonstrates that PERM1 is an essential regulator of cardiac energetics and function and that PERM1 is a novel transcriptional coactivator in the ERRα/PGC-1α axis that functionally interacts with BAG6 and KANK2.
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Obesity has recently risen and become a serious health concern in Korea according to the westernized diet and altered lifestyle. Hence, there is a growing interest in the supplementation of phytochemicals to find a safe and effective functional ingredient to treat obesity. Spergularia marina Griseb (SM) has traditionally been used as a natural herb against chronic diseases in Korea. In this study, we investigated the antiobesity effects of SM in vitro and in vivo. SM ethanol extract (SME) inhibited proliferation and differentiation in murine adipocytes and primary porcine pre-adipocytes in a dose-dependent manner. In the in vivo study, supplementation of SM powder (SMP) remarkably attenuated fat accumulation in HFD-induced obese rats. In addition, SMP supplementation improved lipid profiles in the serum and tissues of high-fat induced obese rats. Collectively, these data indicated that SME exhibited antiobesity effects by modulating adipogenesis and lipolysis. Furthermore, SMP could be developed as an obesity-induced metabolic syndrome treatment.
Assuntos
Adipócitos/efeitos dos fármacos , Caryophyllaceae/química , Obesidade/induzido quimicamente , Obesidade/tratamento farmacológico , Extratos Vegetais/farmacologia , Células 3T3-L1 , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Lipídeos/sangue , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Camundongos , Extratos Vegetais/química , Ratos , Ratos Sprague-DawleyRESUMO
AIMS: Thioredoxin 1 (Trx1) is an evolutionarily conserved oxidoreductase that cleaves disulphide bonds in oxidized substrate proteins such as mechanistic target of rapamycin (mTOR) and maintains nuclear-encoded mitochondrial gene expression. The cardioprotective effect of Trx1 has been demonstrated via cardiac-specific overexpression of Trx1 and dominant negative Trx1. However, the pathophysiological role of endogenous Trx1 has not been defined with a loss-of-function model. To address this, we have generated cardiac-specific Trx1 knockout (Trx1cKO) mice. METHODS AND RESULTS: Trx1cKO mice were viable but died with a median survival age of 25.5 days. They developed heart failure, evidenced by contractile dysfunction, hypertrophy, and increased fibrosis and apoptotic cell death. Multiple markers consistently indicated increased oxidative stress and RNA-sequencing revealed downregulation of genes involved in energy production in Trx1cKO mice. Mitochondrial morphological abnormality was evident in these mice. Although heterozygous Trx1cKO mice did not show any significant baseline phenotype, pressure-overload-induced cardiac dysfunction, and downregulation of metabolic genes were exacerbated in these mice. mTOR was more oxidized and phosphorylation of mTOR substrates such as S6K and 4EBP1 was impaired in Trx1cKO mice. In cultured cardiomyocytes, Trx1 knockdown inhibited mitochondrial respiration and metabolic gene promoter activity, suggesting that Trx1 maintains mitochondrial function in a cell autonomous manner. Importantly, mTOR-C1483F, an oxidation-resistant mutation, prevented Trx1 knockdown-induced mTOR oxidation and inhibition and attenuated suppression of metabolic gene promoter activity. CONCLUSION: Endogenous Trx1 is essential for maintaining cardiac function and metabolism, partly through mTOR regulation via Cys1483.
Assuntos
Metabolismo Energético , Insuficiência Cardíaca/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Tiorredoxinas/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Metabolismo Energético/genética , Regulação da Expressão Gênica , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/patologia , Estresse Oxidativo , Ratos Wistar , Transdução de Sinais , Tiorredoxinas/genéticaRESUMO
The transcriptional regulatory machinery in mitochondrial bioenergetics is complex and is still not completely understood. We previously demonstrated that the histone methyltransferase Smyd1 regulates mitochondrial energetics. Here, we identified Perm1 (PPARGC-1 and ESRR-induced regulator, muscle specific 1) as a downstream target of Smyd1 through RNA-seq. Chromatin immunoprecipitation assay showed that Smyd1 directly interacts with the promoter of Perm1 in the mouse heart, and this interaction was significantly reduced in mouse hearts failing due to pressure overload for 4 weeks, where Perm1 was downregulated (24.4 ± 5.9% of sham, p<0.05). Similarly, the Perm1 protein level was significantly decreased in patients with advanced heart failure (55.2 ± 13.1% of donors, p<0.05). Phenylephrine (PE)-induced hypertrophic stress in cardiomyocytes also led to downregulation of Perm1 (55.7 ± 5.7% of control, p<0.05), and adenovirus-mediated overexpression of Perm1 rescued PE-induced downregulation of estrogen-related receptor alpha (ERRα), a key transcriptional regulator of mitochondrial energetics, and its target gene, Ndufv1 (Complex I). Pathway enrichment analysis of cardiomyocytes in which Perm1 was knocked-down by siRNA (siPerm1), revealed that the most downregulated pathway was metabolism. Cell stress tests using the Seahorse XF analyzer showed that basal respiration and ATP production were significantly reduced in siPerm1 cardiomyocytes (40.7% and 23.6% of scrambled-siRNA, respectively, both p<0.05). Luciferase reporter gene assay further revealed that Perm1 dose-dependently increased the promoter activity of the ERRα gene and known target of ERRα, Ndufv1 (Complex I). Overall, our study demonstrates that Perm1 is an essential regulator of cardiac energetics through ERRα, as part of the Smyd1 regulatory network.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Fatores de Transcrição/metabolismo , Adulto , Idoso , Animais , Metilação de DNA , Modelos Animais de Doenças , Regulação para Baixo , Complexo I de Transporte de Elétrons/genética , Metabolismo Energético/genética , Feminino , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/cirurgia , Transplante de Coração , Histonas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Musculares/genética , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fosforilação Oxidativa , Fenilefrina/farmacologia , Cultura Primária de Células , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/metabolismo , RNA-Seq , Ratos , Receptores de Estrogênio/genética , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
BACKGROUND: Proper dynamics of RNA polymerase II, such as promoter recruitment and elongation, are essential for transcription. PGC-1α (peroxisome proliferator-activated receptor [PPAR]-γ coactivator-1α), also termed PPARGC1a, is a transcriptional coactivator that stimulates energy metabolism, and PGC-1α target genes are downregulated in the failing heart. However, whether the dysregulation of polymerase II dynamics occurs in PGC-1α target genes in heart failure has not been defined. METHODS AND RESULTS: Chromatin immunoprecipitation-sequencing revealed that reduced promoter occupancy was a major form of polymerase II dysregulation on PGC-1α target metabolic gene promoters in the pressure-overload-induced heart failure model. PGC-1α-cKO (cardiac-specific PGC-1α knockout) mice showed phenotypic similarity to the pressure-overload-induced heart failure model in wild-type mice, such as contractile dysfunction and downregulation of PGC-1α target genes, even under basal conditions. However, the protein levels of PGC-1α were neither changed in the pressure-overload model nor in human failing hearts. Chromatin immunoprecipitation assays revealed that the promoter occupancy of polymerase II and PGC-1α was consistently reduced both in the pressure-overload model and PGC-1α-cKO mice. In vitro DNA binding assays using an endogenous PGC-1α target gene promoter sequence confirmed that PGC-1α recruits polymerase II to the promoter. CONCLUSIONS: These results suggest that PGC-1α promotes the recruitment of polymerase II to the PGC-1α target gene promoters. Downregulation of PGC-1α target genes in the failing heart is attributed, in part, to a reduction of the PGC-1α occupancy and the polymerase II recruitment to the promoters, which might be a novel mechanism of metabolic perturbations in the failing heart.
Assuntos
Insuficiência Cardíaca/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Regiões Promotoras Genéticas/genética , RNA Polimerase II/genética , Animais , Modelos Animais de Doenças , Regulação para Baixo , Camundongos , Camundongos Knockout , RNA Polimerase II/metabolismoRESUMO
The purpose of this study was to examine the isomer-specific effect of conjugated linoleic acid (CLA) on inflammatory markers associated with fat accumulation in cultures of differentiating 3T3-L1 adipocytes. trans-10,cis-12 CLA (t10c12 CLA) reduced leptin secretion and fat accumulation. Linoleic acid (LA) and cis-9,trans-11 CLA (c9t11 CLA) increased them, but not significantly. t10c12 CLA and LA showed similar effects on mRNA expression of inflammatory markers. t10c12 CLA and LA tended to up-regulate the mRNA levels of inflammatory cytokines such as interleukin (IL)-6 (not significantly), tumor necrosis factor (TNF)-alpha, and C-reactive protein (CRP) with no significant change in the secretion of adiponectin, an anti-inflammatory adipokine. However, c9t11 CLA induced no significant change in the mRNA expression of IL-6, TNF-alpha, or CRP, but significantly increased adiponectin secretion. In conclusion, CLA exerted isomer-specific effects on fat accumulation and mRNA expression of inflammatory markers in 3T3-L1 adipocytes. t10c12 CLA up-regulated inflammatory markers in spite of the decreased fat accumulation, and TNF-alpha might be one of the causal factors.
Assuntos
Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Citocinas/genética , Ácidos Linoleicos Conjugados/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Células 3T3-L1 , Adiponectina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteína C-Reativa/genética , Expressão Gênica/efeitos dos fármacos , Interleucina-6/genética , Leptina/metabolismo , Camundongos , RNA Mensageiro/análise , Fator de Necrose Tumoral alfa/genéticaRESUMO
BACKGROUND: Heart failure is accompanied by changes in cardiac metabolism characterized by reduced fatty acid (FA) utilization. However, the underlying mechanism and its causative involvement in the progression of heart failure are poorly understood. The peroxisome proliferator activated receptor-α (PPARα)/retinoid X receptor (RXR) heterodimer promotes transcription of genes involved in FA metabolism through binding to the PPAR response element, called direct repeat 1 (DR1). Silent information regulator 1 (Sirt1) is a histone deacetylase, which interacts with PPARα. METHODS AND RESULTS: To investigate the role of PPARα in the impaired FA utilization observed during heart failure, genetically altered mice were subjected to pressure overload. The DNA binding of PPARα, RXRα, and Sirt1 to DR1 was evaluated with oligonucleotide pull-down and chromatin immunoprecipitation assays. Although the binding of PPARα to DR1 was enhanced in response to pressure overload, that of RXRα was attenuated. Sirt1 competes with RXRα to dimerize with PPARα, thereby suppressing FA utilization in the failing heart. DR1 sequence analysis indicated that the typical DR1 sequence favors PPARα/RXRα heterodimerization, whereas the switch from RXRα to Sirt1 takes place on degenerate DR1s. Sirt1 bound to PPARα through a region homologous to the PPARα binding domain in RXRα. A short peptide corresponding to the RXRα domain not only inhibited the interaction between PPARα and Sirt1 but also improved FA metabolism and ameliorated cardiac dysfunction. CONCLUSIONS: A change in the heterodimeric partner of PPARα from RXRα to Sirt1 is responsible for the impaired FA metabolism and cardiac dysfunction in the failing heart.
Assuntos
Ácidos Graxos/metabolismo , Insuficiência Cardíaca/metabolismo , Metabolismo dos Lipídeos/fisiologia , PPAR alfa/metabolismo , Receptores X de Retinoides/metabolismo , Sirtuína 1/metabolismo , Animais , Estudos de Casos e Controles , Dimerização , Modelos Animais de Doenças , Insuficiência Cardíaca/genética , Humanos , Camundongos , PPAR alfa/genética , RNA Mensageiro/metabolismo , Receptores X de Retinoides/genética , Sirtuína 1/genéticaRESUMO
Various stem cells and their progeny have been used therapeutically for vascular regeneration. One of the major hurdles for cell-based therapy is low cell retention in vivo, and to improve cell survival several biomaterials have been used to encapsulate cells before transplantation. Vascular regeneration involves new blood vessel formation which consists of two processes, vasculogenesis and angiogenesis. While embryonic stem cell (ESC)-derived endothelial cells (ESC-ECs) have clearer vasculogenic potency, adult cells exert their effects mainly through paracrine angiogenic activities. While these two cells have seemingly complementary advantages, there have not been any studies to date combining these two cell types for vascular regeneration. We have developed a novel chitosan-based hydrogel construct that encapsulates both CD31-expressing BM-mononuclear cells (BM-CD31(+) cells) and ESC-ECs, and is loaded with VEGF-releasing microtubes. This cell construct showed high cell survival and minimal cytotoxicity in vitro. When implanted into a mouse model of hindlimb ischemia, it induced robust cell retention, neovascularization through vasculogenesis and angiogenesis, and efficiently induced recovery of blood flow in ischemic hindlimbs. This chitosan-based hydrogel encapsulating mixed adult and embryonic cell derivatives and containing VEGF can serve as a novel platform for treating various cardiovascular diseases.
Assuntos
Quitosana/química , Células-Tronco Embrionárias/transplante , Células Endoteliais/transplante , Membro Posterior/irrigação sanguínea , Isquemia/terapia , Alicerces Teciduais/química , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Animais , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células Endoteliais/citologia , Membro Posterior/patologia , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Isquemia/patologia , Masculino , Camundongos , Neovascularização Fisiológica , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Peroxisome proliferator-activated receptor-α (PPARα), a nuclear receptor, plays an important role in the transcription of genes involved in fatty acid metabolism through heterodimerization with the retinoid x receptor (RXR). The consensus sequence of the PPAR response element (PPRE) is composed of two AGGTCA-like sequences directionally aligned with a single nucleotide spacer. PPARα and RXR bind to the 5' and 3' hexad sequences, respectively. However, the precise sequence definition of the PPRE remains obscure, and thus, the consensus sequence currently available remains AGGTCANAGGTCA with unknown redundancy. The vague PPRE sequence definition poses an obstacle to understanding how PPARα regulates fatty acid metabolism. Here we show that, rather than the generally accepted 6-bp sequence, PPARα actually recognized a 12-bp DNA sequence, of which the preferred binding sequence was WAWVTRGGBBAH. Additionally, the optimized RXRα hexad binding sequence was RGKTYA. Thus, the optimal PPARα/RXRα heterodimer binding sequence was WAWVTRGGBBAHRGKTYA. The single nucleotide substitution, which reduces binding of RXRα to DNA, attenuated PPARα-induced transcriptional activation, but this is not always true for PPARα. Using the definition of the PPRE sequence, novel PPREs were successfully identified. Taken altogether, the provided PPRE sequence definition contributes to the understanding of PPARα signaling by identifying PPARα direct target genes with functional PPARα response elements.
Assuntos
DNA/metabolismo , PPAR alfa/metabolismo , Elementos de Resposta , Receptor X Retinoide alfa/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Células Cultivadas , Chlorocebus aethiops , Sequência Consenso , Dimerização , Ácidos Graxos/metabolismo , Genes Reporter , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Miócitos Cardíacos/metabolismo , PPAR alfa/deficiência , Ligação Proteica , Ratos , Organismos Livres de Patógenos Específicos , Transcrição Gênica , Ativação TranscricionalRESUMO
Nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme for nicotinamide adenine dinucleotide (NAD+) synthesis, and Sirt1, an NAD+-dependent histone deacetylase, protect the heart against ischemia/reperfusion (I/R). It remains unknown whether Nampt mediates the protective effect of ischemic preconditioning (IPC), whether nicotinamide mononucleotide (NMN, 500 mg/kg), a product of Nampt in the NAD+ salvage pathway, mimics the effect of IPC, or whether caloric restriction (CR) upregulates Nampt and protects the heart through a Sirt1-dependent mechanism. IPC upregulated Nampt protein, and the protective effect of IPC against ischemia (30 minutes) and reperfusion (24 hours) was attenuated at both early and late phases in Nampt +/- mice, suggesting that Nampt plays an essential role in mediating the protective effect of IPC. In order to mimic the effect of Nampt, NMN was administered by intraperitoneal injection. NMN significantly increased the level of NAD+ in the heart at baseline and prevented a decrease in NAD+ during ischemia. NMN protected the heart from I/R injury when it was applied once 30 minutes before ischemia or 4 times just before and during reperfusion, suggesting that exogenous NMN protects the heart from I/R injury in both ischemic and reperfusion phases. The protective effect of NMN was accompanied by decreases in acetylation of FoxO1, but it was not obvious in Sirt1 KO mice, suggesting that the effect of NMN is mediated through activation of Sirt1. Compared to control diet (90% calories), CR (60% calories for 6 weeks) in mice led to a significant reduction in I/R injury, accompanied by upregulation of Nampt. The protective effect of CR against I/R injury was not significant in cardiac-specific Sirt1 KO mice, suggesting that the protective effect of CR is in part mediated through the Nampt-Sirt1 pathway. In conclusion, exogenous application of NMN and CR protects the heart by both mimicking IPC and activating Sirt1.