Abstract The exposure of hepatocytes to high concentrations of lipids and carbohydrates and the ensuing hepatocellular injury are termed lipotoxicity and glucotoxicity, respectively. A common ...denominator is metabolic derangement, especially in regards to intracellular energy homeostasis, which is brought on by glucose intolerance and insulin resistance in tissues. In this review, we highlight the lipids and carbohydrates that provoke hepatocyte injury and the mechanisms involved in lipotoxicity and glucotoxicity, including endoplasmic reticulum stress, oxidative stress and mitochondrial impairment. Through upregulation of proteins involved in various pathways including PKR-like ER kinase (PERK), CCAAT/enhancer-binding homologous protein (CHOP), c-Jun NH2-terminal kinase-1 (JNK), Bcl-2 interacting mediator (BIM), p53 upregulated modulator of apoptosis (PUMA), and eventually caspases, hepatocytes in lipotoxic states ultimately undergo apoptosis. The protective role of certain lipids and possible targets for pharmacological therapy are explored. Finally, we discuss the role of high fructose and glucose diets in contributing to organelle impairment and poor glucose transport mechanisms, which perpetuate hyperglycemia and hyperlipidemia by shunting of excess carbohydrates into lipogenesis.
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The lack of a preclinical model of progressive non-alcoholic steatohepatitis (NASH) that recapitulates human disease is a barrier to therapeutic development.
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.
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.
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.
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.
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‐κB‐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‐κB 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. J. Cell. Biochem. 110: 1155–1164, 2010. Published 2010 Wiley‐Liss, Inc.
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.
CHOP and AP-1 cooperatively mediate PUMA expression during lipoapoptosis Cazanave, Sophie C; Elmi, Nafisa A; Akazawa, Yuko ...
American journal of physiology. Gastrointestinal and liver physiology/American journal of physiology: Gastrointestinal and liver physiology,
07/2010, Letnik:
299, Številka:
1
Journal Article
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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.
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. (Hepatology 2014;60:1941–1955)
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. (HEPATOLOGY 2011;)
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.
Death Receptor 5 Signaling Promotes Hepatocyte Lipoapoptosis Cazanave, Sophie C.; Mott, Justin L.; Bronk, Steven F. ...
Journal of biological chemistry/The Journal of biological chemistry,
11/2011, Letnik:
286, Številka:
45
Journal Article
Recenzirano
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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.
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.
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