RESUMO
BACKGROUND AND AIMS: The mechanisms by which the I148M mutant variant of the patatin-like phospholipase domain-containing 3 (PNPLA3I148M ) drives development of nonalcoholic steatohepatitis (NASH) are not known. The aim of this study was to obtain insights on mechanisms underlying PNPLA3I148M -induced acceleration of NASH. APPROACH AND RESULTS: Hepatocyte-specific overexpression of empty vector (luciferase), human wild-type PNPLA3, or PNPLA3I148M was achieved using adeno-associated virus 8 in a diet-induced mouse model of nonalcoholic fatty liver disease followed by chow diet or high-fat Western diet with ad libitum administration of sugar in drinking water (WDSW) for 8 weeks. Under WDSW, PNPLA3I148M overexpression accelerated steatohepatitis with increased steatosis, inflammation ballooning, and fibrosis (P < 0.001 versus other groups for all). Silencing PNPLA3I148M after its initial overexpression abrogated these findings. PNPLA3I148M caused 22:6n3 docosahexanoic acid depletion and increased ceramides under WDSW in addition to increasing triglycerides and diglycerides, especially enriched with unsaturated fatty acids. It also increased oxidative stress and endoplasmic reticulum stress. Increased total ceramides was associated with signature of transducer and activator of transcription 3 (STAT3) activation with downstream activation of multiple immune-inflammatory pathways at a transcriptomic level by network analyses. Silencing PNPLA3I148M reversed STAT3 activation. Conditioned media from HepG2 cells overexpressing PNPLA3I148M increased procollagen mRNA expression in LX2 cells; this was abrogated by hepatocyte STAT3 inhibition. CONCLUSIONS: Under WDSW, PNPLA3I148M overexpression promotes steatosis and NASH by metabolic reprogramming characterized by increased triglycerides and diglycerides, n3 polyunsaturated fatty acid depletion, and increased ceramides with resultant STAT3 phosphorylation and downstream inflammatory pathway activation driving increased stellate cell fibrogenic activity.
Assuntos
Lipase , Cirrose Hepática , Proteínas de Membrana , Hepatopatia Gordurosa não Alcoólica , Animais , Dieta Hiperlipídica/efeitos adversos , Dieta Ocidental/efeitos adversos , Modelos Animais de Doenças , Progressão da Doença , Expressão Gênica , Células Hep G2 , Células Estreladas do Fígado/metabolismo , Hepatócitos/metabolismo , Humanos , Lipase/genética , Cirrose Hepática/etiologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Cirrose Hepática/fisiopatologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Polimorfismo Genético , TranscriptomaRESUMO
The histologic spectrum of nonalcoholic fatty liver disease (NAFLD) includes fatty liver (NAFL) and steatohepatitis (NASH), which can progress to cirrhosis in up to 20% of NASH patients. Bile acids (BA) are linked to the pathogenesis and therapy of NASH. We (1) characterized the plasma BA profile in biopsy-proven NAFL and NASH and compared to controls and (2) related the plasma BA profile to liver histologic features, disease activity, and fibrosis. Liquid chromatography/mass spectrometry quantified BAs. Descriptive statistics, paired and multiple group comparisons, and regression analyses were performed. Of 86 patients (24 controls, 25 NAFL, and 37 NASH; mean age 51.8 years and body mass index 31.9 kg/m2 ), 66% were women. Increased total primary BAs and decreased secondary BAs (both P < 0.05) characterized NASH. Total conjugated primary BAs were significantly higher in NASH versus NAFL (P = 0.047) and versus controls (P < 0.0001). NASH had higher conjugated to unconjugated chenodeoxycholate (P = 0.04), cholate (P = 0.0004), and total primary BAs (P < 0.0001). The total cholate to chenodeoxycholate ratio was significantly higher in NAFLD without (P = 0.005) and with (P = 0.02) diabetes. Increased key BAs were associated with higher grades of steatosis (taurocholate), lobular (glycocholate) and portal inflammation (taurolithocholate), and hepatocyte ballooning (taurocholate). Conjugated cholate and taurocholate directly and secondary to primary BA ratio inversely correlated to NAFLD activity score. A higher ratio of total secondary to primary BA decreased (odds ratio, 0.57; P = 0.004) and higher conjugated cholate increased the likelihood of significant fibrosis (F≥2) (P = 0.007). Conclusion: NAFLD is associated with significantly altered circulating BA composition, likely unaffected by type 2 diabetes, and correlated with histological features of NASH; these observations provide the foundation for future hypothesis-driven studies of specific effects of BAs on specific aspects of NASH. (Hepatology 2018;67:534-548).
Assuntos
Ácidos e Sais Biliares/sangue , Hepatopatia Gordurosa não Alcoólica/sangue , Adulto , Idoso , Estudos Transversais , Feminino , Humanos , Cirrose Hepática/sangue , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Receptores Citoplasmáticos e Nucleares/fisiologia , Índice de Gravidade de DoençaRESUMO
BACKGROUND: Haptoglobin (Hp) genotype has been linked to oxidative stress and cardiovascular outcomes in response to vitamin E (VitE) among patients with diabetes mellitus. Its effect on histologic response to VitE in nonalcoholic steatohepatitis (NASH) is unknown. GOALS: Our objective was to determine if Hp genotype associates with response to VitE in patients with NASH. STUDY: A post hoc analysis of 228 patients receiving VitE or placebo in 2 clinical trials was performed. Regression analysis was used to assess the effect of VitE versus placebo, by Hp genotype (1-1, 2-1, or 2-2), on histologic features and laboratory markers of nonalcoholic fatty liver disease, comparing baseline to end of treatment values. An interaction term was included in the regression models to assess differential treatment effect across Hp genotype. RESULTS: Hp 2-2 patients treated with VitE versus placebo showed significant histologic improvement (51% vs. 20%; OR=4.2; P=0.006), resolution of steatohepatitis (44% vs. 12%; OR=6.2; P=0.009), decrease in nonalcoholic fatty liver disease Activity Score (NAS) (-2.2 vs. -0.6; P=0.001), and decrease in liver enzymes alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and γ-glutamyl transpeptidase. Hp 2-1 patients on VitE versus placebo showed improved resolution of steatohepatitis, NAS and liver enzymes. Hp 1-1 patients showed no significant improvement in histology or liver enzymes. VitE had no effect on fibrosis stage in any group. Regression analysis showed incremental benefit of having Hp 2-2 or 2-1 versus 1-1 for all liver enzyme. CONCLUSIONS: Hp 2 allele is associated with greater histologic and biological improvement in NASH with VitE treatment compared with the Hp 1 allele.
Assuntos
Haptoglobinas/genética , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Vitamina E/uso terapêutico , Adulto , Alelos , Feminino , Genótipo , Humanos , Masculino , Hepatopatia Gordurosa não Alcoólica/patologia , Ensaios Clínicos Controlados Aleatórios como Assunto , Resultado do Tratamento , Vitamina E/administração & dosagemRESUMO
BACKGROUND & AIMS: The lack of a preclinical model of progressive non-alcoholic steatohepatitis (NASH) that recapitulates human disease is a barrier to therapeutic development. METHODS: A stable isogenic cross between C57BL/6J (B6) and 129S1/SvImJ (S129) mice were fed a high fat diet with ad libitum consumption of glucose and fructose in physiologically relevant concentrations and compared to mice fed a chow diet and also to both parent strains. RESULTS: Following initiation of the obesogenic diet, B6/129 mice developed obesity, insulin resistance, hypertriglyceridemia and increased LDL-cholesterol. They sequentially also developed steatosis (4-8weeks), steatohepatitis (16-24weeks), progressive fibrosis (16weeks onwards) and spontaneous hepatocellular cancer (HCC). There was a strong concordance between the pattern of pathway activation at a transcriptomic level between humans and mice with similar histological phenotypes (FDR 0.02 for early and 0.08 for late time points). Lipogenic, inflammatory and apoptotic signaling pathways activated in human NASH were also activated in these mice. The HCC gene signature resembled the S1 and S2 human subclasses of HCC (FDR 0.01 for both). Only the B6/129 mouse but not the parent strains recapitulated all of these aspects of human NAFLD. CONCLUSIONS: We here describe a diet-induced animal model of non-alcoholic fatty liver disease (DIAMOND) that recapitulates the key physiological, metabolic, histologic, transcriptomic and cell-signaling changes seen in humans with progressive NASH. LAY SUMMARY: We have developed a diet-induced mouse model of non-alcoholic steatohepatitis (NASH) and hepatic cancers in a cross between two mouse strains (129S1/SvImJ and C57Bl/6J). This model mimics all the physiological, metabolic, histological, transcriptomic gene signature and clinical endpoints of human NASH and can facilitate preclinical development of therapeutic targets for NASH.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Animais , Carcinoma Hepatocelular , Dieta Hiperlipídica , Modelos Animais de Doenças , Humanos , Fígado , Neoplasias Hepáticas , Camundongos , Camundongos Endogâmicos C57BLRESUMO
UNLABELLED: Recent studies have identified a cholestatic variant of nonalcoholic fatty liver disease (NAFLD) with portal inflammation and ductular reaction. Based on reports of biliary damage, as well as increased circulating free fatty acids (FFAs) in NAFLD, we hypothesized the involvement of cholangiocyte lipoapoptosis as a mechanism of cellular injury. Here, we demonstrate that the saturated FFAs palmitate and stearate induced robust and rapid cell death in cholangiocytes. Palmitate and stearate induced cholangiocyte lipoapoptosis in a concentration-dependent manner in multiple cholangiocyte-derived cell lines. The mechanism of lipoapoptosis relied on the activation of caspase 3/7 activity. There was also a significant up-regulation of the proapoptotic BH3-containing protein, PUMA. In addition, palmitate-induced cholangiocyte lipoapoptosis involved a time-dependent increase in the nuclear localization of forkhead family of transcription factor 3 (FoxO3). We show evidence for posttranslational modification of FoxO3, including early (6 hours) deacetylation and dephosphorylation that coincide with localization of FoxO3 in the nuclear compartment. By 16 hours, nuclear FoxO3 is both phosphorylated and acetylated. Knockdown studies confirmed that FoxO3 and its downstream target, PUMA, were critical for palmitate- and stearate-induced cholangiocyte lipoapoptosis. Interestingly, cultured cholangiocyte-derived cells did not accumulate appreciable amounts of neutral lipid upon FFA treatment. CONCLUSION: Our data show that the saturated FFAs palmitate and stearate induced cholangiocyte lipoapoptosis by way of caspase activation, nuclear translocation of FoxO3, and increased proapoptotic PUMA expression. These results suggest that cholangiocyte injury may occur through lipoapoptosis in NAFLD and nonalcoholic steatohepatitis patients.
Assuntos
Apoptose , Ductos Biliares Intra-Hepáticos/enzimologia , Ácidos Graxos não Esterificados/efeitos adversos , Fígado Gorduroso/etiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Fígado Gorduroso/metabolismo , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Humanos , Palmitatos/metabolismo , Proteínas Proto-Oncogênicas/metabolismoRESUMO
Hepatocyte apoptosis is a hallmark of nonalcoholic steatohepatitis. We have previously observed that the saturated free fatty acids (FFAs) induce hepatocyte apoptosis in part via a death receptor 5 (DR5)-mediated signaling pathway. Cellular inhibitor of apoptosis protein 1 and 2 (cIAP-1 and cIAP-2) proteins are potent inhibitors of death receptor-mediated apoptosis. However, the role of the cIAPs in FFA-mediated hepatocyte apoptosis is unexplored. Our aim was to determine whether cIAPs are dysregulated during hepatocyte lipoapoptosis. cIAP proteins underwent rapid cellular elimination following treatment with the saturated FFAs palmitate (PA) and stearate. In contrast, PA did not decrease cIAP-1 and cIAP-2 mRNA expression in the cells. Degradation of cIAPs was dependent on their E3-ligase activity, suggesting that cIAPs undergo autoubiquitination that leads to proteasomal degradation. Huh-7 cells stably expressing shRNA targeting cIAP-1, but not cIAP-2, displayed enhanced sensitivity to PA-mediated apoptosis. Incubation with the SMAC mimetic JP1584, which induces rapid degradation of cIAPs, also enhanced PA-mediated apoptosis. Hepatocytes isolated from DR5 knockout mice exhibited reduced apoptosis following treatment with PA plus JP1584, implying that degradation of cIAPs sensitizes to DR5-mediated cell death pathways. A decrease of cIAP-1 was also observed in tissue from patients with nonalcoholic steatohepatitis compared with normal obese subjects. Collectively, these results implicate proteasomal degradation of cIAPs by FFA as a mechanism contributing to hepatocyte lipoapoptosis.
Assuntos
Apoptose/fisiologia , Hepatócitos/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Hepatócitos/citologia , Humanos , Proteínas Inibidoras de Apoptose/genética , Camundongos , Hepatopatia Gordurosa não Alcoólica , Palmitatos/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Nonalcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA), endoplasmic reticulum (ER) stress, and hepatocyte lipoapoptosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 5 (DR5) is significantly elevated in patients with nonalcoholic steatohepatitis, and steatotic hepatocytes demonstrate increased sensitivity to TRAIL-mediated cell death. Nonetheless, a role for TRAIL and/or DR5 in mediating lipoapoptotic pathways is unexplored. Here, we examined the contribution of DR5 death signaling to lipoapoptosis by free fatty acids. The toxic saturated free fatty acid palmitate induces an increase in DR5 mRNA and protein expression in Huh-7 human hepatoma cells leading to DR5 localization into lipid rafts, cell surface receptor clustering with subsequent recruitment of the initiator caspase-8, and ultimately cellular demise. Lipoapoptosis by palmitate was not inhibited by a soluble human recombinant DR5-Fc chimera protein suggesting that DR5 cytotoxic signaling is ligand-independent. Hepatocytes from murine TRAIL receptor knock-out mice (DR(-/-)) displayed reduced palmitate-mediated lipotoxicity. Likewise, knockdown of DR5 or caspase-8 expression by shRNA technology attenuated palmitate-induced Bax activation and apoptosis in Huh-7 cells, without altering induction of ER stress markers. Similar observations were verified in other cell models. Finally, knockdown of CHOP, an ER stress-mediated transcription factor, reduced DR5 up-regulation and DR5-mediated caspase-8 activation upon palmitate treatment. Collectively, these results suggest that ER stress-induced CHOP activation by palmitate transcriptionally up-regulates DR5, likely resulting in ligand-independent cytotoxic signaling by this death receptor.
Assuntos
Apoptose , Fígado Gorduroso/metabolismo , Hepatócitos/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Técnicas de Silenciamento de Genes , Hepatócitos/patologia , Humanos , Microdomínios da Membrana/genética , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/patologia , Camundongos , Camundongos Knockout , Ácido Palmítico/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
BACKGROUND & AIMS: Ballooned hepatocytes in non-alcoholic steatohepatitis (NASH) generate sonic hedgehog (SHH). This observation is consistent with a cellular phenotype in which the cell death program has been initiated but cannot be executed. Our aim was to determine whether ballooned hepatocytes have potentially disabled the cell death execution machinery, and if so, can their functional biology be modeled in vitro. METHODS: Immunohistochemistry was performed on human NASH specimens. In vitro studies were performed using HuH-7 cells with shRNA targeted knockdown of caspase 9 (shC9 cells) or primary hepatocytes from caspase 3(-/-) mice. RESULTS: Ballooned hepatocytes in NASH display diminished expression of caspase 9. This phenotype was modeled using shC9 cells; these cells were resistant to lipoapoptosis by palmitate (PA) or lysophosphatidylcholine (LPC) despite lipid droplet formation. During lipid loading by either PA or LPC, shC9 cells activate JNK which induces SHH expression via AP-1. An autocrine hedgehog survival signaling pathway was further delineated in both shC9 and caspase 3(-/-) cells during lipotoxic stress. CONCLUSIONS: Ballooned hepatocytes in NASH downregulate caspase 9, a pivotal caspase executing the mitochondrial pathway of apoptosis. Hepatocytes engineered to reduce caspase 9 expression are resistant to lipoapoptosis, in part, due to a hedgehog autocrine survival signaling pathway.
Assuntos
Apoptose , Caspase 9/metabolismo , Fígado Gorduroso/metabolismo , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Clorometilcetonas de Aminoácidos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 1/genética , Caspase 9/genética , Linhagem Celular Tumoral , Regulação para Baixo , Técnicas de Silenciamento de Genes , Proteínas Hedgehog/genética , Hepatócitos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lisofosfatidilcolinas/farmacologia , Vírus Miúdo do Camundongo , Hepatopatia Gordurosa não Alcoólica , Palmitatos/farmacologia , Fosforilação/efeitos dos fármacos , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Fator de Transcrição AP-1/metabolismoRESUMO
Isolated hepatocytes undergo lipoapoptosis, a feature of hepatic lipotoxicity, on treatment with saturated free fatty acids (FFA) such as palmitate (PA). However, it is unknown if palmitate is directly toxic to hepatocytes or if its toxicity is indirect via the generation of lipid metabolites such as lysophosphatidylcholine (LPC). PA-mediated hepatocyte lipoapoptosis is associated with endoplasmic reticulum (ER) stress, c-Jun NH(2)-terminal kinase (JNK) activation, and a JNK-dependent upregulation of the potent proapoptotic BH3-only protein PUMA (p53 upregulated modulator of apoptosis). Our aim was to determine which of these mechanisms of lipotoxicity are activated by PA-derived LPC. We employed Huh-7 cells and isolated murine and human primary hepatocytes. Intracellular LPC concentrations increase linearly as a function of the exogenous, extracellular PA, stearate, or LPC concentration. Incubation of Huh-7 cells or primary hepatocytes with LPC induced cell death by apoptosis in a concentration-dependent manner. Substituting LPC for PA resulted in caspase-dependent cell death that was accompanied by activating phosphorylation of JNK with c-Jun phosphorylation and an increase in PUMA expression. LPC also induced ER stress as manifest by eIF2α phosphorylation and CAAT/enhancer binding homologous protein (CHOP) induction. LPC cytotoxicity was attenuated by pharmacological inhibition of JNK or glycogen synthase kinase-3 (GSK-3). Similarly, short-hairpin RNA (shRNA)-targeted knockdown of CHOP protected Huh-7 cells against LPC-induced toxicity. The LPC-induced PUMA upregulation was prevented by JNK inhibition or shRNA-targeted knockdown of CHOP. Finally, genetic deficiency of PUMA rendered murine hepatocytes resistant to LPC-induced apoptosis. We concluded that LPC-induced lipoapoptosis is dependent on mechanisms largely indistinguishable from PA. These data suggest that FFA-mediated cytotoxicity is indirect via the generation of the toxic metabolite, LPC.
Assuntos
Apoptose/efeitos dos fármacos , Hepatócitos/metabolismo , Lisofosfatidilcolinas/farmacologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Hepatócitos/efeitos dos fármacos , Humanos , Lisofosfatidilcolinas/metabolismo , MAP Quinase Quinase 4/antagonistas & inibidores , MAP Quinase Quinase 4/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Palmitatos/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Estearatos/metabolismo , Fator de Transcrição CHOP/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
UNLABELLED: Cholangiocarcinoma (CCA) cells paradoxically express the death ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and, therefore, are dependent upon potent survival signals to circumvent TRAIL cytotoxicity. CCAs are also highly desmoplastic cancers with a tumor microenvironment rich in myofibroblasts (MFBs). Herein, we examine a role for MFB-derived CCA survival signals. We employed human KMCH-1, KMBC, HuCCT-1, TFK-1, and Mz-ChA-1 CCA cells, as well as human primary hepatic stellate and myofibroblastic LX-2 cells, for these studies. In vivo experiments were conducted using a syngeneic rat orthotopic CCA model. Coculturing CCA cells with myofibroblastic human primary hepatic stellate cells or LX-2 cells significantly decreased TRAIL-induced apoptosis in CCA cells, a cytoprotective effect abrogated by neutralizing platelet-derived growth factor (PDGF)-BB antiserum. Cytoprotection by PDGF-BB was dependent upon Hedgehog (Hh) signaling, because it was abolished by the smoothened (SMO; the transducer of Hh signaling) inhibitor, cyclopamine. PDGF-BB induced cyclic adenosine monophosphate-dependent protein kinase-dependent trafficking of SMO to the plasma membrane, resulting in glioma-associated oncogene (GLI)2 nuclear translocation and activation of a consensus GLI reporter gene-based luciferase assay. A genome-wide messenger RNA expression analysis identified 67 target genes to be commonly up- (50 genes) or down-regulated (17 genes) by both Sonic hedgehog and PDGF-BB in a cyclopamine-dependent manner in CCA cells. Finally, in a rodent CCA in vivo model, cyclopamine administration increased apoptosis in CCA cells, resulting in tumor suppression. CONCLUSIONS: MFB-derived PDGF-BB protects CCA cells from TRAIL cytotoxicity by a Hh-signaling-dependent process. These results have therapeutical implications for the treatment of human CCA.
Assuntos
Neoplasias dos Ductos Biliares/fisiopatologia , Ductos Biliares Intra-Hepáticos/fisiopatologia , Colangiocarcinoma/fisiopatologia , Proteínas Hedgehog/fisiologia , Proteínas Proto-Oncogênicas c-sis/fisiologia , Animais , Apoptose/efeitos dos fármacos , Becaplermina , Neoplasias dos Ductos Biliares/genética , Linhagem Celular Tumoral , Colangiocarcinoma/genética , Técnicas de Cocultura , Células Estreladas do Fígado/metabolismo , Humanos , Masculino , Ratos , Ratos Endogâmicos F344 , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptor Smoothened , Fatores de Transcrição/metabolismo , Alcaloides de Veratrum/farmacologia , Proteína GLI1 em Dedos de ZincoRESUMO
Saturated free fatty acids (FFA) induce hepatocyte lipoapoptosis, a key mediator of liver injury in nonalcoholic fatty liver disease (NAFLD). Lipoapoptosis involves the upregulation of the BH3-only protein PUMA, a potent pro-apoptotic protein. Given that dysregulation of hepatic microRNA expression has been observed in NAFLD, we examined the role of miRNA in regulating PUMA expression during lipotoxicity. By in silico analysis, we identified two putative binding sites for miR-296-5p within the 3' untranslated region (UTR) of PUMA mRNA. Enforced miR-296-5p levels efficiently reduced PUMA protein expression in Huh-7 cells, while antagonism of miR-296-5p function increased PUMA cellular levels. Reporter gene assays identified PUMA 3'UTR as a direct target of miR-296-5p. The saturated FFA, palmitate, repressed miR-296-5p expression; and Huh-7 cells were sensitized to palmitate-induced lipotoxicity by antagonism of miR-296-5p function using a targeted locked nucleic acid (LNA). Finally, miR-296-5p was reduced in liver samples from nonalcoholic steatohepatitis (NASH) patients compared with patients with simple steatosis (SS) or controls. Also miR-296-5p levels inversely varied with PUMA mRNA levels in human liver specimens. Our results implicate miR-296-5p in the regulation of PUMA expression during hepatic lipoapoptosis. We speculate that enhancement of miR-296-5p expression may represent a novel approach to minimize apoptotic damage in human fatty liver diseases.
Assuntos
Proteínas Reguladoras de Apoptose , Fígado Gorduroso/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , MicroRNAs , Palmitatos/farmacologia , Proteínas Proto-Oncogênicas , Regiões 3' não Traduzidas/genética , Idoso , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Sítios de Ligação/genética , Linhagem Celular Tumoral , Fígado Gorduroso/patologia , Fígado Gorduroso/terapia , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , Luciferases/análise , Masculino , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , MicroRNAs/metabolismo , MicroRNAs/farmacologia , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Hepatopatia Gordurosa não Alcoólica , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismoRESUMO
BACKGROUNDS & AIMS: Saturated free fatty acids induce hepatocyte lipoapoptosis, a key pathologic feature of non-alcoholic steatohepatitis. The saturated free fatty acid palmitate induces hepatocyte lipoapoptosis via an endoplasmic reticulum stress pathway resulting in c-Jun-N-terminal (JNK) activation. Glycogen synthase kinase (GSK)-3 is a serine/threonine kinase which may also promote JNK activation. Thus, our aim was to determine if GSK-3 inhibition suppresses palmitate induced JNK activation and lipoapoptosis. METHODS: For these studies, we employed mouse primary hepatocytes, Huh-7 and Hep3B cell lines. RESULTS: Palmitate-induced GSK-3 activation was identified by phosphorylation of its substrate glycogen synthase. GSK-3 pharmacologic inhibition, by GSK-3 inhibitor IX and enzastaurin, significantly reduced PA-mediated lipoapoptosis. More importantly, Huh-7 cells, in which either GSK-3α or GSK-3ß isoforms were stably and selectively knocked down by shRNA, displayed resistance to palmitate-induced cytotoxicity. GSK-3 pharmacological inhibitors and shRNA-targeted knockdown of GSK-3α or GSK-3ß also suppressed JNK activation by palmitate. JNK activation, in part, promotes lipoapotosis by inducing expression of the pro-apoptotic effector p53-upregulated modulator of apoptosis (PUMA). Consistent with this concept, GSK-3 pharmacologic inhibition also reduced PUMA cellular protein levels during exposure to palmitate. On the other hand, the GSK-3 inhibitors did not prevent PA induction of ER stress. CONCLUSIONS: Our results suggest that GSK-3 activation promotes a JNK-dependent cytotoxic signaling cascade culminating in lipoapoptosis.
Assuntos
Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Sequência de Bases , Linhagem Celular , Células Cultivadas , Primers do DNA/genética , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Técnicas de Silenciamento de Genes , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Hepatócitos/citologia , Humanos , Indóis/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Hepatopatia Gordurosa não Alcoólica , Palmitatos/farmacologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Proteínas Supressoras de Tumor/metabolismoRESUMO
Although there are small animal platforms that recapitulate some of the histological features of nonalcoholic fatty liver disease, there are no small animal models of nonalcoholic steatohepatitis (NASH) with consistent hepatocellular ballooning and progressive fibrosis that also exhibit fidelity to the human condition physiologically. We examined the metabolic and histological effects of a diet on the basis of the composition of "fast food" (high saturated fats, cholesterol, and fructose). Mice (n = 8 in each group) were assigned to diets as follows: 1) standard chow (SC), i.e., 13% energy as fat [1% saturated fatty acids (SFA)], 2) high fat (HF), i.e., 60% energy as fat (1% SFA), and 3) fast food (FF), i.e., 40% energy as fat (12% SFA, 2% cholesterol). All three diets were supplemented with high fructose. All diets produced obesity. The HF and FF diets produced insulin resistance. Liver histology was normal in animals fed the SC diet. Steatohepatitis with pronounced ballooning and progressive fibrosis (stage 2) was observed in mice fed the FF diet. Although the HF diet produced obesity, insulin resistance, and some steatosis; inflammation was minimal, and there was no increase in fibrosis. The FF diet produced a gene expression signature of increased fibrosis, inflammation, and endoplasmic reticulum stress and lipoapoptosis. A diet based on high cholesterol, high saturated fat, and high fructose recapitulates features of the metabolic syndrome and NASH with progressive fibrosis. This represents a novel small animal model of fibrosing NASH with high fidelity to the human condition. These results highlight the contribution of dietary composition to the development of nonalcoholic fatty liver disease and NASH.
Assuntos
Dieta Hiperlipídica , Fígado Gorduroso/patologia , Cirrose Hepática/patologia , Fígado/patologia , Animais , Apoptose/fisiologia , Modelos Animais de Doenças , Progressão da Doença , Fígado Gorduroso/metabolismo , Fígado/metabolismo , Cirrose Hepática/metabolismo , Camundongos , Hepatopatia Gordurosa não Alcoólica , Resposta a Proteínas não Dobradas/fisiologiaRESUMO
Noninvasive detection of nonalcoholic steatohepatitis (NASH), the progressive form of nonalcoholic fatty liver disease, promises to improve patient screening, accelerate drug trials, and reduce health care costs. On the basis of protease dysregulation of the biological pathways of fibrotic NASH, we developed the Glympse Bio Test System (GBTS) for multiplexed quantification of liver protease activity. GBTS-NASH comprises a mixture of 19 mass-barcoded PEGylated peptides that is administered intravenously and senses liver protease activity by releasing mass-barcoded reporters into urine for analysis by mass spectrometry. To identify a protease signature of NASH, transcriptomic analysis of 355 human liver biopsies identified a 13-protease panel that discriminated clinically relevant NASH ≥F2 fibrosis from F0-F1 with high classification accuracy across two independent patient datasets. We screened 159 candidate substrates to identify a panel of 19 peptides that exhibited high activity for our 13-protease panel. In the choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) mouse model, binary classifiers trained on urine samples discriminated fibrotic NASH from simple steatosis and healthy controls across a range of nondisease conditions and indicated disease regression upon diet change [area under receiver operating characteristics (AUROCs) > 0.97]. Using a hepatoprotective triple combination treatment (FXR agonist, ACC and ASK1 inhibitors) in a rat model of NASH, urinary classification distinguished F0-F1 from ≥F2 animals and indicated therapeutic response as early as 1 week on treatment (AUROCs >0.91). Our results support GBTS-NASH to diagnose fibrotic NASH via an infusion of peptides, monitor changes in disease severity, and indicate early treatment response.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Fibrose , Humanos , PeptídeosRESUMO
Free fatty acids (FFA) induce hepatocyte lipoapoptosis by a c-Jun N-terminal kinase (JNK)-dependent mechanism. However, the cellular processes by which JNK engages the core apoptotic machinery during lipotoxicity, especially activation of BH3-only proteins, remain incompletely understood. Thus, our aim was to determine whether JNK mediates induction of BH3-only proteins during hepatocyte lipoapoptosis. The saturated FFA palmitate, but not the monounsaturated FFA oleate, induces an increase in PUMA mRNA and protein levels. Palmitate induction of PUMA was JNK1-dependent in primary murine hepatocytes. Palmitate-mediated PUMA expression was inhibited by a dominant negative c-Jun, and direct binding of a phosphorylated c-Jun containing the activator protein 1 complex to the PUMA promoter was identified by electrophoretic mobility shift assay and a chromatin immunoprecipitation assay. Short hairpin RNA-targeted knockdown of PUMA attenuated Bax activation, caspase 3/7 activity, and cell death. Similarly, the genetic deficiency of Puma rendered murine hepatocytes resistant to lipoapoptosis. PUMA expression was also increased in liver biopsy specimens from patients with non-alcoholic steatohepatitis as compared with patients with simple steatosis or controls. Collectively, the data implicate JNK1-dependent PUMA expression as a mechanism contributing to hepatocyte lipoapoptosis.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Hepatócitos/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Antracenos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Expressão Gênica/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 8 Ativada por Mitógeno/genética , Palmitatos/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
MicroRNAs regulate pathways contributing to oncogenesis, and thus the mechanisms causing dysregulation of microRNA expression in cancer are of significant interest. Mature mir-29b levels are decreased in malignant cells, and this alteration promotes the malignant phenotype, including apoptosis resistance. However, the mechanism responsible for mir-29b suppression is unknown. Here, we examined mir-29 expression from chromosome 7q32 using cholangiocarcinoma cells as a model for mir-29b downregulation. Using 5' rapid amplification of cDNA ends, the transcriptional start site was identified for this microRNA locus. Computational analysis revealed the presence of two putative E-box (Myc-binding) sites, a Gli-binding site, and four NF-kappaB-binding sites in the region flanking the transcriptional start site. Promoter activity in cholangiocarcinoma cells was repressed by transfection with c-Myc, consistent with reports in other cell types. Treatment with the hedgehog inhibitor cyclopamine, which blocks smoothened signaling, increased the activity of the promoter and expression of mature mir-29b. Mutagenesis analysis and gel shift data are consistent with a direct binding of Gli to the mir-29 promoter. Finally, activation of NF-kappaB signaling, via ligation of Toll-like receptors, also repressed mir-29b expression and promoter function. Of note, activation of hedgehog, Toll-like receptor, and c-Myc signaling protected cholangiocytes from TRAIL-induced apoptosis. Thus, in addition to c-Myc, mir-29 expression can be suppressed by hedgehog signaling and inflammatory pathways, both commonly activated in the genesis of human malignancies.
Assuntos
Proteínas Hedgehog/metabolismo , MicroRNAs/genética , NF-kappa B/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sítios de Ligação/genética , Linhagem Celular , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Proteínas Hedgehog/antagonistas & inibidores , Humanos , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Transcrição Gênica , Alcaloides de Veratrum/farmacologia , Proteína GLI1 em Dedos de ZincoRESUMO
BACKGROUND & AIMS: Saturated free fatty acids induce hepatocyte lipoapoptosis. This lipotoxicity involves an endoplasmic reticulum stress response, activation of JNK, and altered expression and function of Bcl-2 proteins. The mono-unsaturated free fatty acid palmitoleate is an adipose-derived lipokine which suppresses free fatty acid-mediated lipotoxicity by unclear mechanisms. Herein we examined the mechanisms responsible for cytoprotection. METHODS: We employed isolated human and mouse primary hepatocytes, and the Huh-7 and Hep 3B cell lines for these studies. Cells were incubated in presence and absence of palmitate (16:0), stearate (18:0), and or palmitoleate (16:1, n-7). RESULTS: Palmitoleate significantly reduced lipoapoptosis by palmitate or stearate in both primary cells and cell lines. Palmitoleate accentuated palmitate-induced steatosis in Huh-7 cells excluding inhibition of steatosis as a mechanism for reduced apoptosis. Palmitoleate inhibited palmitate induction of the endoplasmic reticulum stress response as demonstrated by reductions in CHOP expression, eIF2-alpha phosphorylation, XBP-1 splicing, and JNK activation. Palmitate increased expression of the BH3-only proteins PUMA and Bim, which was attenuated by palmitoleate. Consistent with its inhibition of PUMA and Bim induction, palmitoleate prevented activation of the downstream death mediator Bax. CONCLUSIONS: These data suggest palmitoleate inhibits lipoapoptosis by blocking endoplasmic reticulum stress-associated increases of the BH3-only proteins Bim and PUMA.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Ácidos Graxos Monoinsaturados/metabolismo , Fígado Gorduroso/fisiopatologia , Hepatócitos/fisiologia , Proteínas de Membrana/genética , Palmitatos/metabolismo , Proteínas Proto-Oncogênicas/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Interações Medicamentosas , Retículo Endoplasmático/fisiologia , Ácidos Graxos Monoinsaturados/farmacologia , Fígado Gorduroso/patologia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Palmitatos/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição de Fator Regulador X , Estresse Fisiológico/fisiologia , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Proteína 1 de Ligação a X-Box , Proteína X Associada a bcl-2/metabolismoRESUMO
Endoplasmic reticulum (ER) stress-mediated apoptosis is a key feature of hepatocyte cytotoxicity by saturated free fatty acids (FFA). This lipoapoptosis is dependent, in part, on the transcriptional upregulation of the BH3-only protein PUMA (p53 upregulated modulator of apoptosis). Although the activator protein (AP)-1 complex facilitates PUMA expression by saturated FFA, the transcription factor CAAT/enhancer binding homologous protein (CHOP) is also induced by ER stress and promotes apoptosis. To integrate the role of these two transcription factors in ER stress-induced apoptosis, we examined the relative contribution of CHOP and AP-1 in mediating PUMA induction by saturated FFA. Our results demonstrate that short-hairpin RNA-targeted knockdown of CHOP attenuates palmitate-induced apoptosis in Huh-7 cells. Loss of CHOP induction also reduced the increase in PUMA mRNA and protein levels as well as Bax activation by palmitate. No functional CHOP binding sites were identified in the PUMA promoter sequence. Rather, we observed that CHOP physically interacts with the AP-1 complex protein c-Jun upon palmitate treatment, and a CHOP:phosphorylated c-Jun heteromeric complex binds to the AP-1 consensus binding sequence within the PUMA promoter region. Finally, loss of function studies suggest that both transcription factors are necessary for maximal PUMA induction. Collectively, these data suggest that CHOP and AP-1 cooperatively mediate PUMA induction during hepatocyte lipoapoptosis.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Fígado Gorduroso/metabolismo , Hepatócitos/metabolismo , Ácido Oleico/metabolismo , Ácido Palmítico/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição CHOP/metabolismo , Proteínas Reguladoras de Apoptose/genética , Sítios de Ligação , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Hepatócitos/patologia , Humanos , Fosforilação , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , Fator de Transcrição AP-1/genética , Fator de Transcrição CHOP/genética , Ativação Transcricional , Transfecção , Regulação para Cima , Proteína X Associada a bcl-2/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
BACKGROUND/AIMS: There are limited chemotherapy options for hepatocellular carcinoma (HCC). The heparin-degrading endosulfatase SULF1 functions as a liver tumor suppressor. We investigated the effects of the histone deacetylase inhibitor apicidin in combination with doxorubicin in SULF1-expressing HCC cells in vitro and in SULF1-expressing xenografts in nude mice. METHODS: We evaluated the effects of apicidin alone or combined with doxorubicin on apoptosis, caspase activity, and phosphorylation of Erk and Akt in SULF1-transfected Huh7 and Hep3B cells in vitro and in vivo. RESULTS: Apicidin induced HCC cell apoptosis and caspase activation in a dose- and time-dependent manner. Apicidin-induced caspase activation was significantly inhibited by the caspase inhibitor Z-Vad-fmk. Apicidin also decreased phosphorylation of both Erk and Akt. Expression of constitutively-active Mek1 and Akt significantly decreased apicidin-induced apoptosis. The combination of doxorubicin with apicidin significantly increased the anti-tumor effect in the SULF1-expressing Huh7 and Hep3B cells as compared to either apicidin or doxorubicin alone, both in vitro and in vivo. CONCLUSIONS: The combination of a histone deacetylase inhibitor with doxorubicin may be a novel and promising therapeutic modality for HCCs, particularly for SULF1-expressing HCCs.