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Endoplasmic reticulum stress signalling and the pathogenesis of non-alcoholic fatty liver disease

  • Cynthia Lebeaupin
    Affiliations
    Université Côte d’Azur, INSERM, U1065, C3M, 06200 Nice, France
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  • Deborah Vallée
    Affiliations
    Université Côte d’Azur, INSERM, U1065, C3M, 06200 Nice, France
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  • Younis Hazari
    Affiliations
    Biomedical Neuroscience Institute (BNI), Faculty of Medicine, University of Chile, Santiago, Chile

    Center for Geroscience, Brain Health and Metabolism (GERO), Santiago, Chile

    Program of Cellular and Molecular Biology, Institute of Biomedical Sciences, University of Chile, Santiago, Chile
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  • Claudio Hetz
    Affiliations
    Biomedical Neuroscience Institute (BNI), Faculty of Medicine, University of Chile, Santiago, Chile

    Center for Geroscience, Brain Health and Metabolism (GERO), Santiago, Chile

    Program of Cellular and Molecular Biology, Institute of Biomedical Sciences, University of Chile, Santiago, Chile

    Buck Institute for Research on Aging, Novato, CA 94945, USA

    Department of Immunology and Infectious Diseases, Harvard School of Public Health, 02115 Boston, MA, USA
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  • Eric Chevet
    Affiliations
    “Chemistry, Oncogenesis, Stress, Signaling”, Inserm U1242, Université de Rennes, Rennes, France

    Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France
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  • Béatrice Bailly-Maitre
    Correspondence
    Corresponding author. Address: Bâtiment Universitaire ARCHIMED, INSERM, U1065, Team 8 “Hepatic complications in obesity”, 151 route Saint Antoine de Ginestière, BP 2 3194, 06204 Nice Cedex 03, France. Tel.:+33(0) 489064238; fax. +33(0)489064221.
    Affiliations
    Université Côte d’Azur, INSERM, U1065, C3M, 06200 Nice, France
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      Summary

      The global epidemic of obesity has been accompanied by a rising burden of non-alcoholic fatty liver disease (NAFLD), with manifestations ranging from simple steatosis to non-alcoholic steatohepatitis, potentially developing into hepatocellular carcinoma. Although much attention has focused on NAFLD, its pathogenesis remains largely obscure. The hallmark of NAFLD is the hepatic accumulation of lipids, which subsequently leads to cellular stress and hepatic injury, eventually resulting in chronic liver disease. Abnormal lipid accumulation often coincides with insulin resistance in steatotic livers and is associated with perturbed endoplasmic reticulum (ER) proteostasis in hepatocytes. In response to chronic ER stress, an adaptive signalling pathway known as the unfolded protein response is triggered to restore ER proteostasis. However, the unfolded protein response can cause inflammation, inflammasome activation and, in the case of non-resolvable ER stress, the death of hepatocytes. Experimental data suggest that the unfolded protein response influences hepatic tumour development, aggressiveness and response to treatment, offering novel therapeutic avenues. Herein, we provide an overview of the evidence linking ER stress to NAFLD and discuss possible points of intervention.

      Keywords

      Introduction

      The globally high prevalence of obesity is associated with the development of chronic liver diseases characterised by the build-up of extra fat in liver cells.
      The global epidemic of obesity poses a drastic threat to public health systems because of the increasing incidence of its related comorbidities. In developed countries, lifestyle changes have led to caloric excess, a lack of physical activity and increased life expectancy. Faced with these new challenges, physiological responses are no longer able to cope, leading to an imbalance between the homeostatic processes governing energy expenditure and energy uptake. Processes that largely affect liver function. Non-alcoholic fatty liver disease (NAFLD) is the most common aetiology of chronic liver disease, affecting 25% of the general population worldwide
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      NAFLD is often considered as the hepatic manifestation of the metabolic syndrome (insulin resistance, obesity and hyperlipidaemia), comprising hepatic steatosis and non-alcoholic steatohepatitis (NASH). NASH is a combination of lipid accumulation, hepatocyte death, inflammation and fibrosis, making this pathology a significant risk factor for hepatic cirrhosis, hepatocellular carcinoma (HCC) and ultimately liver-related mortality.
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      Dysfunction of the endoplasmic reticulum (ER), the main cellular compartment involved in secretory and transmembrane protein folding, calcium homeostasis and lipid biogenesis, is involved in metabolically-driven NAFLD pathologies
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      Endoplasmic reticulum stress in nonalcoholic fatty liver disease.
      through the activation of ER stress signalling. This review compiles the recent findings supporting the functional impact of ER stress signalling on chronic liver diseases, ranging from steatosis to NASH and HCC, and discusses possible therapeutic strategies.
      Chronic liver diseases range from steatosis to NASH featuring inflammation, hepatocyte death and various degrees of fibrosis. NASH may lead to liver cancer and end-stage liver disease.

      The physiology of the liver and the adaptive unfolded protein response

      The liver performs vital metabolic, secretory and excretory functions to preserve whole-body homeostasis. Hepatocytes represent up to 70% of total liver cells.
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      They are highly secretory cells responsible for the assembly and secretion of very low-density lipoprotein (VLDL) and for the synthesis of plasma proteins including albumin, α-1 antitrypsin, apolipoproteins and coagulation factors. Hepatocytes are responsible for lipogenesis, cholesterol biosynthesis, glucose and xenobiotic metabolism. To fulfil their myriad of metabolic functions, hepatocytes are enriched with both smooth and rough ER.
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      The ER in hepatocytes has a remarkable capacity to adapt to extracellular and intracellular changes, ensuring that vital hepatic metabolic functions are preserved. However, in humans, numerous disturbances (e.g. hyperlipidaemia, inflammation, viruses, drugs) can perturb hepatocyte ER homeostasis, contributing to the dysregulation of hepatic lipid metabolism and to liver disease. Consequently, the ER engages an evolutionarily conserved pathway termed the unfolded protein response (UPR)
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      The unfolded protein response: from stress pathway to homeostatic regulation.
      to control hepatic protein and lipid homeostasis. The UPR reduces the secretory protein load, enhances ER protein folding (transcription of chaperones and foldases) and increases clearance capacity by promoting autophagy and ER-associated degradation (ERAD) (Fig. 1).
      In liver cells, the ER is a critical site of protein synthesis, detoxification, lipid and glucose metabolism and Ca2+ regulation. Obesity leads to perturbed functions of the ER in liver cells.
      Figure thumbnail gr1
      Fig. 1The UPR signalling cascade. Multiple endogenous and exogenous stimuli can create ER stress leading to the accumulation of unfolded proteins and subsequent activation of the UPR. GRP78 dissociates from the three ER transmembrane sensors PERK, IRE1α and ATF6 that activate their respective signalling cascades to first re-establish proteostasis and promote cell survival through the transcription of UPR target genes of the mechanisms listed at the bottom. Oligomerised PERK phosphorylates eIF2α that inhibits translation to reduce the protein load on the ER, but concomitantly increases the expression of the transcription factor ATF4. In a negative feedback mechanism, ATF4 induces CHOP that induces GADD34, a regulatory subunit of PP1C, thus inhibiting eIF2α phosphorylation and reinitiating translation. PERK phosphorylation of NRF2 is involved in the regulation of redox homeostasis. Oligomerised IRE1α possesses both RNase and kinase activities. HSP47, a selective regulator of IRE1α, displaces GRP78 from IRE1α to facilitate its oligomerisation. Activation of the RNase function of IRE1α leads to RIDD-mediated degradation of mRNAs and microRNAs, such as miR17 leading to uninhibited TXNIP expression and NLRP3 inflammasome activation, and the splicing of XBP1 resulting in the activation of the potent transcription factor XBP1s. Activation of IRE1α can also lead to the recruitment of stress kinases involved in NFkB-mediated inflammation or JNK-mediated apoptosis and insulin resistance. ATF6 (p90) is transported from the ER to the Golgi to be cleaved by the proteases S1P and S2P, thereby releasing the cytosolic transcription factor ATF6 (p50). Similarly, SREBP1 is also proteolytically cleaved to activate the transcription of genes involved in lipid synthesis and uptake.
      Induction of the UPR involves the activation of three transmembrane ER-resident stress sensors: (i) inositol-requiring enzyme 1 (IRE1), (ii) PKR-like ER kinase (PERK) and (iii) activating transcription factor (ATF6). Under unstressed conditions, IRE1, PERK and ATF6 are maintained inactive upon binding to GRP78/BiP. Above a critical threshold of misfolded protein accumulation, GRP78 dissociates from the ER stress sensors, thereby priming all branches for activation. Protein disulfide isomerases (PDIs) may also regulate UPR stress sensors,
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      (Fig. 2)).
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      Figure thumbnail gr2
      Fig. 2Irremediable ER stress promotes hepatocyte apoptosis. Terminal ER stress and Ca2+ dysregulation leads to the mitochondrial release of apoptogenic factors, such as cytochrome c, that activate the caspase cascade of apoptosis. Regulating Ca2+ balance in the ER, SERCA actively pumps Ca2+ into the ER, while IP3-gated channels (IP3R) passively release Ca2+ into the cytosol. Massive efflux of Ca2+ by IP3R overcomes the mitochondria, resulting in the opening of the mitochondrial PTP leading to ionic imbalances, mitochondrial transmembrane potential disruption, matrix swelling and permeabilisation of the mitochondrial outer membrane (MOMP), resulting in a liberation of reactive oxygen species and cytochrome c. While anti-apoptotic proteins BCL2 and BI-1 aim to inhibit abnormal Ca2+ release, pro-apoptotic BAX/BAK promote Ca2+ release at the ER and mitochondria. When hyperactivated, PERK- and IRE1α-mediated signalling pathways detailed in promote proteotoxicity, inflammatory responses with associated pyropotosis and apoptosis. Finally, terminal ER stress signalling converges onto the pro-apoptotic transcription factor CHOP that upregulates pro-apoptotic BH3-only proteins (e.g. PUMA, BIM, BID) while suppressing anti-apoptotic BCL2/BCLXL expression. BH3-only proteins directly activate BAX and BAK to form a pore, release cytochrome c and induce apoptosis.

      The UPR in NAFLD: two faces of janus

      The UPR initially aims to maintain liver physiology by protecting hepatocytes from nutrient and xenobiotic exposure, or from cellular stress that arises from increased secretory demand or cellular differentiation. Concerning the physiological regulation of hepatic metabolism, transient UPR activation has been reported in the postprandial switch from glucose production to glucose storage.
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      Although the liver experiences transient ER stress in physiological conditions, this stress can become chronic in NAFLD, contributing to its progression towards more severe disease stages by inducing inflammatory responses and cell death (Fig. 3).
      Figure thumbnail gr3
      Fig. 3ER stress drives NAFLD progression. The three main actors of the UPR play a key role in chronic liver disease progression. Chronic UPR can drive steatosis through its involvement in hepatic insulin resistance and fat accumulation. The transition from a chronic to a terminal UPR may be responsible for steatohepatitis development through its involvement in cell death, inflammation and oxidative stress. With HCC development, a resistant UPR promotes cancerous cell survival and proliferation and stimulates angiogenesis.
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      A direct activation of the IRE1α-XBP1 branch was observed in vivo during the development of insulin resistance in the liver.
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      • Kaneto H.
      • Matsuoka T.A.
      • Kohno K.
      • Iwawaki T.
      • Nakatani Y.
      • et al.
      Direct monitoring of in vivo ER stress during the development of insulin resistance with ER stress-activated indicator transgenic mice.
      Furthermore, hepatic overexpression of the ER-resident chaperones, such as ORP150/HYOU1 and GRP78/BiP improved insulin sensitivity and resolved steatosis in obese mice,
      • Kammoun H.L.
      • Chabanon H.
      • Hainault I.
      • Luquet S.
      • Magnan C.
      • Koike T.
      • et al.
      GRP78 expression inhibits insulin and ER stress-induced SREBP-1c activation and reduces hepatic steatosis in mice.
      • Nakatani Y.
      • Kaneto H.
      • Kawamori D.
      • Yoshiuchi K.
      • Hatazaki M.
      • Matsuoka T.A.
      • et al.
      Involvement of endoplasmic reticulum stress in insulin resistance and diabetes.
      providing direct evidence that altered ER protein folding in the liver contributes to steatosis associated with obesity.

      The role of ER stress in the initiation of NAFLD

      Adding to its processing of secreted and membrane proteins; the ER is the major site of lipid synthesis in hepatocytes. Hepatocytes are involved in all of the major lipid metabolic pathways, including lipogenesis, triglyceride synthesis and storage, apolipoprotein assembly and secretion, and fatty acid oxidation. Firstly, de novo lipogenesis is regulated by ER membrane-localised transcription factors, the sterol regulatory element-binding proteins (SREBP1c (SREBF1) for fatty acid synthesis and SREBP2 (SREBF2) for sterol synthesis). Secondly, hepatocytes store lipids in the form of triglycerides that are synthesised from fatty acids and glycerol by ER-localised acyltransferase enzymes including diacylglycerol acyltransferase (DGAT). Thirdly, hepatocytes can secrete VLDLs, which are assembled in the ER prior to trafficking to the Golgi. Hepatic ER homeostasis is crucial for maintaining membrane lipid composition and controlling both intrahepatic and plasma lipid homeostasis.
      Steatosis is the first stage of NAFLD, characterised by ectopic triglyceride accumulation in hepatocytes. In patients with steatosis, 59% of triglycerides found in the liver were derived from serum non-esterified fatty acids stored in adipose tissue and 26% from increased de novo lipogenesis, while only 15% came from diet.
      • Donnelly K.L.
      • Smith C.I.
      • Schwarzenberg S.J.
      • Jessurun J.
      • Boldt M.D.
      • Parks E.J.
      Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease.
      Since the bulk of lipid synthesis takes place in the smooth ER, an important role for ER stress responses has emerged in the pathogenesis of steatosis. Studies using transgenic mice with liver-specific or whole-body deletions in specific UPR mediators have been performed to understand how the UPR regulates hepatic lipid homeostasis. In this section, we will describe how chronic ER stress could have a causative role in steatosis. Chronic ER stress can affect hepatic lipid metabolism directly by inducing de novo lipogenesis and indirectly through the alteration of VLDL secretion, insulin signalling and autophagy. Conversely, increased hepatocyte lipid content can initiate chronic ER stress. Herein, we highlight the intrinsic relationship between lipid and ER homeostasis.
      In obesity, the UPR is chronically engaged. ER stress may be the cause or consequence of dysregulated lipid and glucose signaling, inflammatory and cell death pathways in the evolution of the stages from steatosis, to NASH and to HCC.

      ER stress regulates lipostasis

      The UPR has been extensively studied in terms of proteostatic control but mostly ignored in the context of lipostatic control. The UPR regulates hepatic lipid homeostasis. While each master regulator pathway of the UPR is required to protect from steatosis under pharmacologically-induced severe ER stress,
      • Rutkowski D.T.
      • Wu J.
      • Back S.H.
      • Callaghan M.U.
      • Ferris S.P.
      • Iqbal J.
      • et al.
      UPR pathways combine to prevent hepatic steatosis caused by ER stress-mediated suppression of transcriptional master regulators.
      each may contribute to the development of hepatic steatosis by promoting de novo lipogenesis and lipolysis, reducing fatty acid oxidation and disturbing lipoprotein and VLDL secretion.
      Firstly, the IRE1α-XBP1 arm was shown to be a crucial player in hepatic lipid metabolism through regulation of VLDL secretion and lipogenesis.
      • Reimold A.M.
      • Etkin A.
      • Clauss I.
      • Perkins A.
      • Friend D.S.
      • Zhang J.
      • et al.
      An essential role in liver development for transcription factor XBP-1.
      • Zhang K.
      • Wong H.N.
      • Song B.
      • Miller C.N.
      • Scheuner D.
      • Kaufman R.J.
      The unfolded protein response sensor IRE1α is required at 2 distinct steps in B cell lymphopoiesis.
      • Wang S.
      • Chen Z.
      • Lam V.
      • Han J.
      • Hassler J.
      • Finck B.N.
      • et al.
      IRE1alpha-XBP1s induces PDI expression to increase MTP activity for hepatic VLDL assembly and lipid homeostasis.
      Mice with a specific hepatocyte deletion of IRE1α and challenged with tunicamycin displayed increased steatosis due to the inhibition of lipid synthesis regulators (C/EBPβ, C/EBPβγ, PPARγ) and halted VLDL secretion.
      • Zhang K.
      • Wang S.
      • Malhotra J.
      • Hassler J.R.
      • Back S.H.
      • Wang G.
      • et al.
      The unfolded protein response transducer IRE1alpha prevents ER stress-induced hepatic steatosis.
      Similarly, the overexpression of BI-1 leading to the inhibition of the IRE1α pathway resulted in the downregulation of key enzymes of lipid homeostasis pathways (C/EBPα, SREBF1, PGC1α [PPARGC1A]).
      • Bailly-Maitre B.
      • Belgardt B.F.
      • Jordan S.D.
      • Coornaert B.
      • von Freyend M.J.
      • Kleinridders A.
      • et al.
      Hepatic Bax inhibitor-1 inhibits IRE1alpha and protects from obesity-associated insulin resistance and glucose intolerance.
      Unlike hepatocyte-specific IRE1α deletion, livers of mice with conditional XBP1 deletion had limited hepatic de novo lipogenesis, lower levels of serum triglycerides and cholesterol, and decreased steatosis in response to lipogenic diets.
      • Lee A.-H.
      • Scapa E.F.
      • Cohen D.E.
      • Glimcher L.H.
      Regulation of hepatic lipogenesis by the transcription factor XBP1.
      In hepatocytes, XBP1s binds to promoters of lipogenic genes (DGAT2, SCD, ACC2 [ACACB]).
      • Lee A.-H.
      • Scapa E.F.
      • Cohen D.E.
      • Glimcher L.H.
      Regulation of hepatic lipogenesis by the transcription factor XBP1.
      The discrepancy in the metabolic phenotype between IRE1α-deficient and XBP1-deficient mice is still under investigation. Furthermore, XBP1 deficiency results in feedback activation of IRE1α, inducing the mRNA degradation of genes involved in lipid metabolism and catabolism.
      • So J.S.
      • Hur K.Y.
      • Tarrio M.
      • Ruda V.
      • Frank-Kamenetsky M.
      • Fitzgerald K.
      • et al.
      Silencing of lipid metabolism genes through IRE1alpha-mediated mRNA decay lowers plasma lipids in mice.
      Secondly, the PERK-eIF2α-ATF4 arm was reported to regulate lipogenesis and steatosis. Sustained dephosphorylation of eIF2α due to the hepatic overexpression of GADD34 in the liver protects mice from HFD-induced obesity and NAFLD.
      • Oyadomari S.
      • Harding H.P.
      • Zhang Y.
      • Oyadomari M.
      • Ron D.
      Dephosphorylation of translation initiation factor 2alpha enhances glucose tolerance and attenuates hepatosteatosis in mice.
      However, a basal level of eIF2α phosphorylation prevents lipid accumulation in response to the direct challenge of ER stress, as inactive eIF2α in mice led to tunicamycin-induced fatty liver.
      • Rutkowski D.T.
      • Wu J.
      • Back S.H.
      • Callaghan M.U.
      • Ferris S.P.
      • Iqbal J.
      • et al.
      UPR pathways combine to prevent hepatic steatosis caused by ER stress-mediated suppression of transcriptional master regulators.
      ATF4 deficiency protected mice from steatosis in response to high-fructose feeding through attenuated de novo lipogenesis.
      • Xiao G.
      • Zhang T.
      • Yu S.
      • Lee S.
      • Calabuig-Navarro V.
      • Yamauchi J.
      • et al.
      ATF4 protein deficiency protects against high fructose-induced hypertriglyceridemia in mice.
      ATF4-null mice were protected from age-related and diet-induced obesity and steatosis.
      • Seo J.
      • Fortuno E.S.
      • Suh J.M.
      • Stenesen D.
      • Tang W.
      • Parks E.J.
      • et al.
      Atf4 regulates obesity, glucose homeostasis, and energy expenditure.
      In addition, CD154, a ligand of CD40 and a key mediator of inflammation, may play a protective role in hepatic steatosis via a connection to UPR signalling. Livers of CD154 (CD40LG) knockout mice fed an olive-oil rich diet presented reduced ApoB100 expression and increased lipogenic enzyme gene expression, associated with reduced p-eIF2α.
      • Villeneuve J.
      • Lepreux S.
      • Mulot A.
      • Berard A.M.
      • Higa-Nishiyama A.
      • Costet P.
      • et al.
      A protective role for CD154 in hepatic steatosis in mice.
      Thirdly, the ATF6α arm may be protective in steatosis. ATF6α knockout mice presented sustained CHOP expression, C/EBPα inhibition and hepatic steatosis when challenged with tunicamycin.
      • Rutkowski D.T.
      • Wu J.
      • Back S.H.
      • Callaghan M.U.
      • Ferris S.P.
      • Iqbal J.
      • et al.
      UPR pathways combine to prevent hepatic steatosis caused by ER stress-mediated suppression of transcriptional master regulators.
      • Yamamoto K.
      • Takahara K.
      • Oyadomari S.
      • Okada T.
      • Sato T.
      • Harada A.
      • et al.
      Induction of liver steatosis and lipid droplet formation in ATF6α-knockout mice burdened with pharmacological endoplasmic reticulum stress.
      • DeZwaan-McCabe D.
      • Sheldon R.D.
      • Gorecki M.C.
      • Guo D.F.
      • Gansemer E.R.
      • Kaufman R.J.
      • et al.
      ER stress inhibits liver fatty acid oxidation while unmitigated stress leads to anorexia-induced lipolysis and both liver and kidney steatosis.
      In hepatocytes, ATF6α interacted with PPARα to enhance its transcriptional activity, upregulating hepatic fatty acid oxidation.
      • Chen X.
      • Zhang F.
      • Gong Q.
      • Cui A.
      • Zhuo S.
      • Hu Z.
      • et al.
      Hepatic ATF6 increases fatty acid oxidation to attenuate hepatic steatosis in mice through peroxisome proliferator-activated receptor α.
      ATF6α suppresses SREBP2 transactivation in hepatocytes, downregulating lipogenesis.
      • Zeng L.
      • Lu M.
      • Mori K.
      • Luo S.
      • Lee A.S.
      • Zhu Y.
      • et al.
      ATF6 modulates SREBP2-mediated lipogenesis.
      Overall, studies suggest that the UPR has a relevant role in both lipostatic and proteostatic control. The lipid gene expression networks are directly responsive to chronic ER stress. While a transient UPR protects from steatosis, chronic ER stress in NAFLD aggravates hepatic steatosis, with each arm contributing to detrimental effects on hepatic lipid pathways. The crosstalk between these pathways remains to be explored. Identifying which UPR mediators contribute to adaptation and which induce metabolic dysfunction remains the challenge.

      ER modulates hepatic insulin sensitivity

      ER stress can trigger insulin resistance through excessive fat accumulation. In addition, ER stress can disrupt insulin action through the activation of each arm of the UPR.
      • Özcan U.
      • Cao Q.
      • Yilmaz E.
      • Lee A.H.
      • Iwakoshi N.N.
      • Ozdelen E.
      • et al.
      Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes.
      • Gregor M.F.
      • Hotamisligil G.S.
      Inflammatory mechanisms in obesity.
      • Hummasti S.
      • Hotamisligil G.S.
      Endoplasmic reticulum stress and inflammation in obesity and diabetes.
      • Özcan U.
      • Yilmaz E.
      • Özcan L.
      • Furuhashi M.
      • Vaillancourt E.
      • Smith R.O.
      • et al.
      Chemical chaperones reduce er stress and restore glucose homeostasis in a mouse model of type 2 diabetes.
      Through its kinase function, IRE1α phosphorylates JNKs and IkB kinase, inhibiting insulin signalling. However, alone, IRE1α-mediated JNK activation was found to be insufficient to induce hepatic insulin resistance, thus suggesting a causal relationship in which hepatic insulin resistance is secondary to ER stress-activated lipogenesis.
      • Jurczak M.J.
      • Lee A.H.
      • Jornayvaz F.R.
      • Lee H.Y.
      • Birkenfeld A.L.
      • Guigni B.A.
      • et al.
      Dissociation of inositol-requiring enzyme (IRE1alpha)-mediated c-Jun N-terminal kinase activation from hepatic insulin resistance in conditional X-box-binding protein-1 (XBP1) knock-out mice.
      Similarly, the PERK arm contributes to insulin resistance. The selective dephosphorylation of eIF2α through enforced expression of GADD34 led to enhanced insulin sensitivity.
      • Oyadomari S.
      • Harding H.P.
      • Zhang Y.
      • Oyadomari M.
      • Ron D.
      Dephosphorylation of translation initiation factor 2alpha enhances glucose tolerance and attenuates hepatosteatosis in mice.
      Interestingly, hepatic levels of p-eIF2α increased with worsening insulin resistance in obese, nondiabetic patients.
      • Kumashiro N.
      • Erion D.M.
      • Zhang D.
      • Kahn M.
      • Beddow S.A.
      • Chu X.
      • et al.
      Cellular mechanism of insulin resistance in nonalcoholic fatty liver disease.
      The role of ATF6α in hepatic insulin signalling has not been fully explored. The knockdown of ATF6α led to upregulated hepatic glucose output.
      • Wang Y.
      • Vera L.
      • Fischer W.H.
      • Montminy M.
      The CREB coactivator CRTC2 links hepatic ER stress and fasting gluconeogenesis.
      In support of a beneficial role for ATF6α, genetic overexpression of hepatic ATF6α in mice with diet-induced obesity led to improved insulin signalling and metabolic health.
      • Ozcan L.
      • Ghorpade D.S.
      • Zheng Z.
      • de Souza J.C.
      • Chen K.
      • Bessler M.
      • et al.
      Hepatocyte DACH1 is increased in obesity via nuclear exclusion of HDAC4 and promotes hepatic insulin resistance.

      ER regulates hepatic autophagic flux

      ER stress and autophagy are delicately balanced. Impaired autophagy in livers of lean mice augments ER stress, while the rescuing of autophagy alleviates obesity-induced hepatic ER stress as seen from the downregulation of LC3, Beclin, Atg5 and Atg7 in ob/ob and HFD murine models of obesity.
      • Yang L.
      • Li P.
      • Fu S.
      • Calay E.S.
      • Hotamisligil G.S.
      Defective hepatic autophagy in obesity promotes ER stress and causes insulin resistance.
      Interestingly, PERK and IRE1α were upregulated in livers of ob/ob models.
      • Yang L.
      • Li P.
      • Fu S.
      • Calay E.S.
      • Hotamisligil G.S.
      Defective hepatic autophagy in obesity promotes ER stress and causes insulin resistance.
      Furthermore, increased expression of LC3 in NASH murine models
      • Adkins Y.
      • Schie I.W.
      • Fedor D.
      • Reddy A.
      • Nguyen S.
      • Zhou P.
      • et al.
      A novel mouse model of nonalcoholic steatohepatitis with significant insulin resistance.
      • Sinha R.A.
      • You S.-H.
      • Zhou J.
      • Siddique M.M.
      • Bay B.-H.
      • Zhu X.
      • et al.
      Thyroid hormone stimulates hepatic lipid catabolism via activation of autophagy.
      and p62 in patients with NASH has been shown.
      • Gonzalez-Rodriguez A.
      • Mayoral R.
      • Agra N.
      • Valdecantos M.P.
      • Pardo V.
      • Miquilena-Colina M.E.
      • et al.
      Impaired autophagic flux is associated with increased endoplasmic reticulum stress during the development of NAFLD.
      In addition, ER stress increased parallel to elevated p62, LC3-II and autophagosomes in livers of murine models of NASH.
      • Gonzalez-Rodriguez A.
      • Mayoral R.
      • Agra N.
      • Valdecantos M.P.
      • Pardo V.
      • Miquilena-Colina M.E.
      • et al.
      Impaired autophagic flux is associated with increased endoplasmic reticulum stress during the development of NAFLD.
      BI-1 expression was also shown to modulate IRE1 signalling and autophagy in the liver of animals undergoing experimental ER stress.
      • Castillo K.
      • Rojas-Rivera D.
      • Lisbona F.
      • Caballero B.
      • Nassif M.
      • Court F.A.
      • et al.
      BAX inhibitor-1 regulates autophagy by controlling the IRE1alpha branch of the unfolded protein response.
      Although it is clear that ER stress-driven autophagy leads to cell death in NASH, it may still be too speculative to advocate a mitigative or augmentative role of autophagy in NAFLD.

      The role of lipids and Ca2+ homeostasis in triggering chronic ER stress

      Although triglycerides are the most abundant lipid category, lipidomic analyses from human liver biopsies revealed diglycerides and ceramides in steatotic livers.
      • Gorden D.L.
      • Myers D.S.
      • Ivanova P.T.
      • Fahy E.
      • Maurya M.R.
      • Gupta S.
      • et al.
      Biomarkers of NAFLD progression: a lipidomics approach to an epidemic.
      These lipids inhibit hepatic insulin signalling pathways, contributing to the onset of hepatic insulin resistance and the genesis of ER stress. Interestingly, the expression of their transporter – fatty acid translocase CD36 – was upregulated in patients with NAFLD.
      • Miquilena-Colina M.E.
      • Lima-Cabello E.
      • Sanchez-Campos S.
      • Garcia-Mediavilla M.V.
      • Fernandez-Bermejo M.
      • Lozano-Rodriguez T.
      • et al.
      Hepatic fatty acid translocase CD36 upregulation is associated with insulin resistance, hyperinsulinaemia and increased steatosis in non-alcoholic steatohepatitis and chronic hepatitis C.
      While it remains unclear whether hepatic insulin resistance or steatosis develops first, it is well established that insulin resistance is a risk factor in NAFLD progression.
      • Farese Jr., R.V.
      • Zechner R.
      • Newgard C.B.
      • Walther T.C.
      The problem of establishing relationships between hepatic steatosis and hepatic insulin resistance.
      • Nagle C.A.
      • Klett E.L.
      • Coleman R.A.
      Hepatic triacylglycerol accumulation and insulin resistance.
      • Capeau J.
      Insulin resistance and steatosis in humans.
      Nevertheless, the common denominator between hepatic insulin resistance and steatosis is the unusually elevated amount of circulating and intracellular lipids.
      The aberrant lipid changes in hepatocytes could directly trigger chronic ER stress in the liver by disrupting Ca2+ homeostasis. In obese mice, the hepatic lipid burden leads to an imbalance in ER membrane lipid composition with a higher ratio of phosphatidylcholine vs. phosphatidylethanolamine compared to lean mice, thus altering membrane fluidity. Accumulation of phosphatidylcholine leads to the inhibition of SERCA (ATP2) activity and reduction of ER [Ca2+], which induces ER stress as most ER chaperones are Ca2+-dependent.
      • Fu S.
      • Yang L.
      • Li P.
      • Hofmann O.
      • Dicker L.
      • Hide W.
      • et al.
      Aberrant lipid metabolism disrupts calcium homeostasis causing liver endoplasmic reticulum stress in obesity.
      SERCA overexpression or ER phospholipid composition correction reduced ER stress and steatosis in obese animals,
      • Fu S.
      • Yang L.
      • Li P.
      • Hofmann O.
      • Dicker L.
      • Hide W.
      • et al.
      Aberrant lipid metabolism disrupts calcium homeostasis causing liver endoplasmic reticulum stress in obesity.
      suggesting that lipid perturbations and Ca2+ homeostasis (and protein-misfolding due to Ca2+ homeostasis disturbance in ER) are the main proponents of hepatic ER stress in obesity.
      • Fu S.
      • Yang L.
      • Li P.
      • Hofmann O.
      • Dicker L.
      • Hide W.
      • et al.
      Aberrant lipid metabolism disrupts calcium homeostasis causing liver endoplasmic reticulum stress in obesity.
      It remains unclear how obesity alters SERCA expression. The disruption of Ca2+ homeostasis may trigger ER stress and affect lipid metabolism as well. The ER makes close physical contacts with the mitochondria via mitochondrial-associated-membranes (MAMs) allowing Ca2+ and lipid transfer between these two organelles. During obesity, the number of MAMs increases,
      • Arruda A.P.
      • Pers B.M.
      • Parlakgul G.
      • Guney E.
      • Inouye K.
      • Hotamisligil G.S.
      Chronic enrichment of hepatic endoplasmic reticulum-mitochondria contact leads to mitochondrial dysfunction in obesity.
      which could overload mitochondria with Ca2+ and alter oxidative phosphorylation capacity. Suboptimal β-oxidation in NAFLD accompanied by dysfunctional tricarboxylic acid cycle activity results in the accumulation of toxic lipid intermediates, like ceramides and diacylglycerols, inevitably leading to chronic levels of ROS with inflammation and fibrosis during NASH.
      • Patterson R.E.
      • Kalavalapalli S.
      • Williams C.M.
      • Nautiyal M.
      • Mathew J.T.
      • Martinez J.
      • et al.
      Lipotoxicity in steatohepatitis occurs despite an increase in tricarboxylic acid cycle activity.
      Recent evidence indicates that steatosis is concurrent with fastened accretion of triglycerides in the liver, upregulating mitochondrial oxidative function and suggesting that mitochondrial remodelling is present in NAFLD.
      • Iozzo P.
      • Bucci M.
      • Roivainen A.
      • Nagren K.
      • Jarvisalo M.J.
      • Kiss J.
      • et al.
      Fatty acid metabolism in the liver, measured by positron emission tomography, is increased in obese individuals.
      • Sunny N.E.
      • Parks E.J.
      • Browning J.D.
      • Burgess S.C.
      Excessive hepatic mitochondrial TCA cycle and gluconeogenesis in humans with nonalcoholic fatty liver disease.
      Higher diacylglyceride, phospholipid, free cholesterol and free fatty acid (FFA) levels activate cellular ER stress. Hager et al. showed that a 2% cholesterol-rich diet leads to its accumulation in hepatic ER, resulting in the perturbed free cholesterol/phospholipid ratio of ER membranes.
      • Hager L.
      • Li L.
      • Pun H.
      • Liu L.
      • Hossain M.A.
      • Maguire G.F.
      • et al.
      Lecithin:cholesterol acyltransferase deficiency protects against cholesterol-induced hepatic endoplasmic reticulum stress in mice.
      Also, higher free cholesterol in the ER results in enhanced esterification due to acyl-CoA cholesterol acyl transferase (ACAT), leading to elevated free cholesterol/esterified cholesterol levels, further aggravating ER stress.
      • Gregor M.F.
      • Hotamisligil G.S.
      Thematic review series: adipocyte biology. Adipocyte stress: the endoplasmic reticulum and metabolic disease.
      Lipids were suggested to directly induce ER stress through its sensors, particularly IRE1α and PERK. A study using yeast genetics established that the luminal unfolded protein stress-sensing domain of IRE1α is dispensable for ER stress caused by lipid-induced membrane aberrancy, suggesting an additional sensing mechanism specific to ER stress sensors.
      • Promlek T.
      • Ishiwata-Kimata Y.
      • Shido M.
      • Sakuramoto M.
      • Kohno K.
      • Kimata Y.
      Membrane aberrancy and unfolded proteins activate the endoplasmic reticulum stress sensor Ire1 in different ways.
      Furthermore, WT and luminal domain-mutated IRE1α proteins could equally sense ER stress caused by lipid- or membrane-related aberrations resulting from inositol depletion. Myriocin, a sphingolipid biosynthesis inhibitor, prevented IRE1α activation induced by inositol depletion, but not by DTT exposure. Saturated fatty acid overload via palmitate or pharmacological inhibition of stearoyl CoA desaturase 1 increases XBP1 mRNA splicing eight- and fourfold, respectively, both in IRE1α WT and IRE1α luminal domain-deleted cells.
      • Volmer R.
      • van der Ploeg K.
      • Ron D.
      Membrane lipid saturation activates endoplasmic reticulum unfolded protein response transducers through their transmembrane domains.
      Moreover, the sensitivity of PERK activation towards membrane lipid perturbation was also revealed.
      • Promlek T.
      • Ishiwata-Kimata Y.
      • Shido M.
      • Sakuramoto M.
      • Kohno K.
      • Kimata Y.
      Membrane aberrancy and unfolded proteins activate the endoplasmic reticulum stress sensor Ire1 in different ways.
      • Volmer R.
      • van der Ploeg K.
      • Ron D.
      Membrane lipid saturation activates endoplasmic reticulum unfolded protein response transducers through their transmembrane domains.
      • Fu S.
      • Watkins S.M.
      • Hotamisligil G.S.
      The role of endoplasmic reticulum in hepatic lipid homeostasis and stress signaling.
      This clearly suggests that the transmembrane domains of IRE1α and PERK could act as lipid sensors, discerning the biophysical properties of lipid membranes, dependent on the ratio of unsaturated/saturated acyl chains that affect membrane fluidity and thickness.
      • Volmer R.
      • van der Ploeg K.
      • Ron D.
      Membrane lipid saturation activates endoplasmic reticulum unfolded protein response transducers through their transmembrane domains.
      Halbleib et al., utilising tools like in vitro reconstitution and molecular dynamics, elaborated on the molecular mechanism of UPR activation by lipid bilayer stress. Changing the amphipathic character of the amphipathic helix of IRE1α reduced cellular resistance to DTT, eventually imposing functional defects. This amphipathic helix region was required to sense aberrant physical membrane properties and for normal IRE1α functionality.
      • Halbleib K.
      • Pesek K.
      • Covino R.
      • Hofbauer H.F.
      • Wunnicke D.
      • Hanelt I.
      • et al.
      Activation of the unfolded protein response by lipid bilayer stress.
      Overall these observations suggest a novel sensing mechanism of ER stress sensors in response to lipid bilayer stress, which forms a vicious circle that disturbs lipid homeostasis and cell fate (see Section “ER stress-induced cell death in NASH”). Several mechanistic questions arise from these observations: What is the role of each class of lipids in activating the UPR sensors? Which class of lipids will activate cell death, due to an unresolved ER stress, driving steatosis to NASH transition?

      The role of chronic ER stress in NASH

      Under chronic conditions of ER stress, rather than alleviate the current stress the UPR can become counterproductive and drive key features of progressive NASH, including inflammation and cell death. It is well established that patients with NASH exhibit increased inflammation that correlates with their histological severity.
      • Patouraux S.
      • Rousseau D.
      • Bonnafous S.
      • Lebeaupin C.
      • Luci C.
      • Canivet C.M.
      • et al.
      CD44 is a key player in non-alcoholic steatohepatitis.
      Liver biopsies from patients with NASH also present a significantly greater number of TUNEL-positive hepatocytes, correlating with active caspases-3 and -7, activation of pro-apoptotic BCL2-family members, and death receptor Fas when compared to livers from normal or steatotic patients.
      • Feldstein A.E.
      • Canbay A.
      • Angulo P.
      • Taniai M.
      • Burgart L.J.
      • Lindor K.D.
      • et al.
      Hepatocyte apoptosis and fas expression are prominent features of human nonalcoholic steatohepatitis.

      ER stress-dependent inflammatory responses in NASH

      Consequences of ER stress-driven NF-kB and JNK in NASH

      Upon ER stress, activated IRE1α activates protein IkB and JNKs, which are implicated in the transcriptional activation of pro-inflammatory and pro-apoptotic pathways. The activation of NF-kB is pivotal in the induction of inflammatory responses that primarily promote survival. In NASH, NF-kB has been identified as a double-edged sword, acting as a central link between hepatic injury, fibrosis and even favouring progression to HCC. Activation of the PERK branch reduces translation of IkB, increasing NF-kB activity.
      • Deng J.
      • Lu P.D.
      • Zhang Y.
      • Scheuner D.
      • Kaufman R.J.
      • Sonenberg N.
      • et al.
      Translational repression mediates activation of nuclear factor kappa B by phosphorylated translation initiation factor 2.
      • Jiang H.Y.
      • Wek S.A.
      • McGrath B.C.
      • Scheuner D.
      • Kaufman R.J.
      • Cavener D.R.
      • et al.
      Phosphorylation of the subunit of eukaryotic initiation factor 2 is required for activation of NF- B in response to diverse cellular stresses.
      Although ATF6α can also trigger NF-kB activation via phosphorylation of Akt,
      • Yamazaki H.
      • Hiramatsu N.
      • Hayakawa K.
      • Tagawa Y.
      • Okamura M.
      • Ogata R.
      • et al.
      Activation of the Akt-NF-kappaB pathway by subtilase cytotoxin through the ATF6 branch of the unfolded protein response.
      the IRE1α and PERK arms appear crucial for ER stress-induced NF-kB activation. A vicious cycle of injury and inflammation exists between JNK-dependent hepatocyte death and NF-kB activation in Kupffer cells releasing mediators like IL-1β and TNFα.
      • Luedde T.
      • Schwabe R.F.
      NF-kappaB in the liver–linking injury, fibrosis and hepatocellular carcinoma.
      Gut-derived pathogens such as lipopolysaccharide (LPS) and subsequent LPS-induced cytokines may be toxic to hepatocytes, initially activating an NF-kB-mediated response that possess a pro-inflammatory yet anti-apoptotic dual function in hepatocytes. This suggests that normal or slightly upregulated NF-kB activity in hepatocytes protects against NASH by preventing hepatocyte death, but above a certain threshold, the fact that NF-kB promotes the secretion of inflammatory and chemotactic factors in hepatocytes leads to a worsening of hepatic inflammation and initiates fibrosis.
      • Luedde T.
      • Schwabe R.F.
      NF-kappaB in the liver–linking injury, fibrosis and hepatocellular carcinoma.
      In the livers of mice challenged with MCDD, NF-kB was a pro-inflammatory mediator of lesion development in NASH.
      • Dela Pena A.
      • Leclercq I.
      • Field J.
      • George J.
      • Jones B.
      • Farrell G.
      NF-kappaB activation, rather than TNF, mediates hepatic inflammation in a murine dietary model of steatohepatitis.
      A potential mechanism involved in steatosis progression to NASH was proposed through CHOP-mediated activation of NF-kB signalling in human primary hepatocytes exposed to saturated fatty acids.
      • Willy J.A.
      • Young S.K.
      • Stevens J.L.
      • Masuoka H.C.
      • Wek R.C.
      CHOP links endoplasmic reticulum stress to NF-κB activation in the pathogenesis of nonalcoholic steatohepatitis.

      Consequences of ER stress on ROS production in NASH

      Oxidative stress due to overproduction of ROS and free radicals in NAFLD contributes to its pathogenesis.
      • Browning Jeffrey D.
      • Szczepaniak Lidia S.
      • Dobbins R.
      • Nuremberg P.
      • Horton Jay D.
      • Cohen Jonathan C.
      • et al.
      Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity.
      • Rolo A.P.
      • Teodoro J.S.
      • Palmeira C.M.
      Role of oxidative stress in the pathogenesis of nonalcoholic steatohepatitis.
      During steatosis, the fatty acid and Ca2+ overload alters mitochondrial function (oxidation and respiration) leading to increased ROS production (as stated previously). Alternative oxidative pathways are then activated in the peroxisomes (β-oxidation) and microsomes (ω-oxidation), which results in more and more free radical production and a reduced cellular state.
      • Serviddio G.
      • Bellanti F.
      • Tamborra R.
      • Rollo T.
      • Romano A.D.
      • Giudetti A.M.
      • et al.
      Alterations of hepatic ATP homeostasis and respiratory chain during development of non-alcoholic steatohepatitis in a rodent model.
      • Serviddio G.
      • Bellanti F.
      • Tamborra R.
      • Rollo T.
      • Capitanio N.
      • Romano A.D.
      • et al.
      Uncoupling protein-2 (UCP2) induces mitochondrial proton leak and increases susceptibility of non-alcoholic steatohepatitis (NASH) liver to ischaemia-reperfusion injury.
      • Mannaerts G.P.
      • Debeer L.J.
      • Thomas J.
      • De Schepper P.J.
      Mitochondrial and peroxisomal fatty acid oxidation in liver homogenates and isolated hepatocytes from control and clofibrate-treated rats.
      • Hrycay E.G.
      • Bandiera S.M.
      Involvement of cytochrome P450 in reactive oxygen species formation and cancer.
      • De Craemer D.
      • Pauwels M.
      • Van den Branden C.
      Alterations of peroxisomes in steatosis of the human liver: a quantitative study.
      • Natarajan Sathish K.
      • Eapen Chundamannil E.
      • Pullimood Anna B.
      • Balasubramanian Kunissery A.
      Oxidative stress in experimental liver microvesicular steatosis: role of mitochondria and peroxisomes.
      • Zmijewski J.W.
      • Banerjee S.
      • Bae H.
      • Friggeri A.
      • Lazarowski E.R.
      • Abraham E.
      Exposure to hydrogen peroxide induces oxidation and activation of AMP-activated protein kinase.
      This reduced cellular state impairs fatty acid oxidation, enhances glycerol-3-phosphate production and lipogenesis, and aggravates steatosis.
      • Zmijewski J.W.
      • Banerjee S.
      • Bae H.
      • Friggeri A.
      • Lazarowski E.R.
      • Abraham E.
      Exposure to hydrogen peroxide induces oxidation and activation of AMP-activated protein kinase.
      • Begriche K.
      • Igoudjil A.
      • Pessayre D.
      • Fromenty B.
      Mitochondrial dysfunction in NASH: causes, consequences and possible means to prevent it.
      Similarly, saturated fatty acid and cholesterol accumulation also contribute to the altered cellular redox state and subsequent sequential changes in lipid metabolism.
      • Puri P.
      • Baillie Rebecca A.
      • Wiest Michelle M.
      • Mirshahi F.
      • Choudhury J.
      • Cheung O.
      • et al.
      A lipidomic analysis of nonalcoholic fatty liver disease.
      • Caballero F.
      • Fernández A.
      • De Lacy A.M.
      • Fernández-Checa J.C.
      • Caballería J.
      • García-Ruiz C.
      Enhanced free cholesterol, SREBP-2 and StAR expression in human NASH.
      Excessive toxic lipid peroxidation/oxidative stress can then act as a trigger in the steatosis to NASH transition.
      Studies have reported connections between the UPR and oxidative stress. Among them, NRF2 plays a central role in orchestrating the antioxidant response. NRF2 is highly expressed in the liver. In response to an increased protein-folding load in the ER, PERK can phosphorylate and stabilise NRF2 to compensate for high ROS levels, which are important mediators of inflammation.
      • Cullinan S.B.
      • Zhang D.
      • Hannink M.
      • Arvisais E.
      • Kaufman R.J.
      • Diehl J.A.
      Nrf2 Is a direct PERK substrate and effector of PERK-dependent cell survival.
      • Cullinan S.B.
      • Diehl J.A.
      PERK-dependent activation of Nrf2 contributes to redox homeostasis and cell survival following endoplasmic reticulum stress.
      • Cullinan S.B.
      • Diehl J.A.
      Coordination of ER and oxidative stress signaling: the PERK/Nrf2 signaling pathway.
      NRF2 therefore plays a cytoprotective role in response to ER stress-dependent inflammation in animal models of NASH. Nrf2 (Nfe2l2)-deficient mice fed an MCDD presented aggravated NASH features compared with control mice due to significantly increased oxidative stress and iron accumulation. Conversely, sustained NRF2 activation protects mice against NASH progression by inhibiting oxidative stress and the release of inflammatory cytokines and fibrosis stimulation factors.
      • Okada K.
      • Warabi E.
      • Sugimoto H.
      • Horie M.
      • Tokushige K.
      • Ueda T.
      • et al.
      Nrf2 inhibits hepatic iron accumulation and counteracts oxidative stress-induced liver injury in nutritional steatohepatitis.
      Pharmacological activators of NRF2 signalling significantly reduced fibrosis in rats with diet-induced NASH, demonstrating a potential strategy to treat patients with NASH and hepatic fibrosis.
      • Shimozono R.
      • Asaoka Y.
      • Yoshizawa Y.
      • Aoki T.
      • Noda H.
      • Yamada M.
      • et al.
      Nrf2 activators attenuate the progression of nonalcoholic steatohepatitis-related fibrosis in a dietary rat model.
      NRF2 can also repress the transcription of genes encoding pro-oxidant machinery, such as TXNIP,
      • Ma Q.
      Role of nrf2 in oxidative stress and toxicity.
      a protein known to link oxidative stress to inflammasome activation in a ROS-dependent manner.
      • Zhou R.
      • Tardivel A.
      • Thorens B.
      • Choi I.
      • Tschopp J.
      Thioredoxin-interacting protein links oxidative stress to inflammasome activation.
      IRE1α and PERK branch activation can increase TXNIP, ROS production, inflammasome activation and β-cell death.
      • Lerner A.G.
      • Upton J.P.
      • Praveen P.V.
      • Ghosh R.
      • Nakagawa Y.
      • Igbaria A.
      • et al.
      IRE1alpha induces thioredoxin-interacting protein to activate the NLRP3 inflammasome and promote programmed cell death under irremediable ER stress.
      • Oslowski C.M.
      • Hara T.
      • O'Sullivan-Murphy B.
      • Kanekura K.
      • Lu S.
      • Hara M.
      • et al.
      Thioredoxin-interacting protein mediates ER stress-induced beta cell death through initiation of the inflammasome.
      Overwhelmed IRE1a RNase activity in BI-1 deficient mice with NASH is associated with enhanced hepatic TXNIP expression.
      • Lebeaupin C.
      • Vallée D.
      • Rousseau D.
      • Patouraux S.
      • Bonnafous S.
      • Adam G.
      • et al.
      Bax Inhibitor-1 protects from non-alcoholic steatohepatitis by limiting IRE1α signaling.
      Collectively, ER stress activates oxidative stress markers such as TXNIP that exacerbate inflammation and cell death. ER stress tightly cooperates with oxidative stress in NASH. Further studies are needed to address the role of this crosstalk.

      ER stress and acute-phase proteins in NASH

      Acute-phase proteins secreted by hepatocytes are indicators of hepatic inflammation and can be associated with ER stress. CREBH is a UPR-mediated transcription factor expressed only in the liver and implicated in the acute-phase response. Upon ER stress in hepatocytes, full-length and latent CREBH on the ER membrane undergoes regulated intramembrane proteolysis that transactivates genes of the hepatic acute-phase response including C-reactive protein (CRP) and serum amyloid P component.
      • Zhang K.
      • Shen X.
      • Wu J.
      • Sakaki K.
      • Saunders T.
      • Rutkowski D.T.
      • et al.
      Endoplasmic reticulum stress activates cleavage of CREBH to induce a systemic inflammatory response.
      Compared to patients with steatosis, patients with NASH displayed significantly elevated serum CRP levels.
      • Yoneda M.
      • Mawatari H.
      • Fujita K.
      • Iida H.
      • Yonemitsu K.
      • Kato S.
      • et al.
      High-sensitivity C-reactive protein is an independent clinical feature of nonalcoholic steatohepatitis (NASH) and also of the severity of fibrosis in NASH.
      However, while plasma CRP levels were elevated in another cohort of severely obese patients, they were not predictive of the diagnosis of NASH.
      • Anty R.
      • Bekri S.
      • Luciani N.
      • Saint-Paul M.C.
      • Dahman M.
      • Iannelli A.
      • et al.
      The inflammatory C-reactive protein is increased in both liver and adipose tissue in severely obese patients independently from metabolic syndrome, Type 2 diabetes, and NASH.
      An additional acute-phase protein induced by CREBH is hepcidin, a peptide hormone that regulates iron homeostasis and is secreted from the liver in response to ER stress and inflammation.
      • Vecchi C.
      • Montosi G.
      • Zhang K.
      • Lamberti I.
      • Duncan S.A.
      • Kaufman R.J.
      • et al.
      ER stress controls iron metabolism through induction of hepcidin.
      Iron overload was identified in patients with NASH who had an increased risk of worsened histological severity and advanced fibrosis.
      • Kowdley K.V.
      • Belt P.
      • Wilson L.A.
      • Yeh M.M.
      • Neuschwander-Tetri B.A.
      • Chalasani N.
      • et al.
      Serum ferritin is an independent predictor of histologic severity and advanced fibrosis in patients with nonalcoholic fatty liver disease.
      Iron overload was also found to modulate ER stress-associated pathways in a murine model of iron- and HFD-induced liver injury.
      • Tan T.C.
      • Crawford D.H.
      • Jaskowski L.A.
      • Subramaniam V.N.
      • Clouston A.D.
      • Crane D.I.
      • et al.
      Excess iron modulates endoplasmic reticulum stress-associated pathways in a mouse model of alcohol and high-fat diet-induced liver injury.

      ER stress-dependent NLRP3 inflammasome activation in NASH

      Much evidence has linked ER stress to the NLRP3 inflammasome, found to be particularly active in livers of mice and humans with NASH.
      • Csak T.
      • Ganz M.
      • Pespisa J.
      • Kodys K.
      • Dolganiuc A.
      • Szabo G.
      Fatty acid and endotoxin activate inflammasomes in mouse hepatocytes that release danger signals to stimulate immune cells.
      • Lebeaupin C.
      • Proics E.
      • de Bieville C.H.
      • Rousseau D.
      • Bonnafous S.
      • Patouraux S.
      • et al.
      ER stress induces NLRP3 inflammasome activation and hepatocyte death.
      • Szabo G.
      • Csak T.
      Inflammasomes in liver diseases.
      Mice expressing a constitutively active form of global-specific vs. myeloid-cell-derived NLRP3 developed more extensive liver inflammation, hepatocyte pyroptosis, stellate cell activation and liver fibrosis.
      • Wree A.
      • Eguchi A.
      • McGeough M.D.
      • Pena C.A.
      • Johnson C.D.
      • Canbay A.
      • et al.
      NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice.
      A stronger NASH phenotype would need to be directly confirmed in hepatocyte-specific NLRP3 active mice. Livers from caspase-1-deficient mice were protected from ER stress-induced inflammasome activation.
      • Zhang J.
      • Zhang K.
      • Li Z.
      • Guo B.
      ER stress-induced inflammasome activation contributes to hepatic inflammation and steatosis.
      More specifically, endotoxaemia provoked in genetically obese mice overwhelmed hepatic IRE1α and PERK activities. This led to the overexpression of CHOP found to regulate caspase-1, -11 and PUMA expression, triggering hepatocyte pyroptosis and apoptosis.
      • Lebeaupin C.
      • Proics E.
      • de Bieville C.H.
      • Rousseau D.
      • Bonnafous S.
      • Patouraux S.
      • et al.
      ER stress induces NLRP3 inflammasome activation and hepatocyte death.
      Thus, inflammation and cell death (pyroptosis, apoptosis) in NASH may drive a feed forward cycle in liver disease progression.

      ER stress-induced cell death in NASH

      Apoptosis induced by ER stress

      The notion that enhanced ER stress-induced apoptosis within liver cells may be relevant in the progression from steatosis to NASH in humans was supported by the display of elevated ER stress markers, namely CHOP (DDIT3) and GRP78 (HSPA5), in liver biopsies from patients with NASH.
      • Gonzalez-Rodriguez A.
      • Mayoral R.
      • Agra N.
      • Valdecantos M.P.
      • Pardo V.
      • Miquilena-Colina M.E.
      • et al.
      Impaired autophagic flux is associated with increased endoplasmic reticulum stress during the development of NAFLD.
      The deleterious CHOP/GRP78 ratio was positively correlated with both NAFLD activity score and liver injury in humans.
      • Lebeaupin C.
      • Proics E.
      • de Bieville C.H.
      • Rousseau D.
      • Bonnafous S.
      • Patouraux S.
      • et al.
      ER stress induces NLRP3 inflammasome activation and hepatocyte death.
      This study suggested that both ER-apoptosis and pyroptosis contributed to NASH development in humans. Since this study, a central role for IRE1α-RNase activity has been reported in NASH patient liver biopsies and NASH animal models.
      • Lebeaupin C.
      • Vallée D.
      • Rousseau D.
      • Patouraux S.
      • Bonnafous S.
      • Adam G.
      • et al.
      Bax Inhibitor-1 protects from non-alcoholic steatohepatitis by limiting IRE1α signaling.
      A previous report on liver biopsies from patients with NASH found inconsistencies in the downstream pathways of PERK, displaying activated eIF2α but unchanged ATF4-GADD34-CHOP signalling, and IRE1α, presenting activated JNK but unmodified XBP1.
      • Puri P.
      • Mirshahi F.
      • Cheung O.
      • Natarajan R.
      • Maher J.W.
      • Kellum J.M.
      • et al.
      Activation and dysregulation of the unfolded protein response in nonalcoholic fatty liver disease.
      Supporting a pro-apoptotic role for JNK in NASH pathogenesis, Jnk1 (Mapk8)-deficient mice fed an MCDD were protected from steatohepatitis development due to the reduction in apoptosis.
      • Schattenberg J.M.
      • Singh R.
      • Wang Y.
      • Lefkowitch J.H.
      • Rigoli R.M.
      • Scherer P.E.
      • et al.
      JNK1 but not JNK2 promotes the development of steatohepatitis in mice.

      Lipotoxicity and cell death

      The dangerous build-up of lipids in steatotic livers increases hepatic exposure to lipotoxicity that can aggravate the symptoms leading to NASH. Lipidomic analyses revealed significantly elevated serum FFA levels in patients with NASH.
      • Tavares De Almeida I.
      • Cortez-Pinto H.
      • Fidalgo G.
      • Rodrigues D.
      • Camilo M.E.
      Plasma total and free fatty acids composition in human non-alcoholic steatohepatitis.
      Data indicate that FFAs may contribute more to lipotoxicity-induced liver damage than their esterified product (triglycerides). The inhibition of triglyceride synthesis at DGAT2 in MCDD-fed mice led to an FFA build-up accompanied by higher levels of cytochrome P4502E1 (CYP2E1), markers of lipid peroxidation/oxidant stress, lobular necroinflammation and fibrosis than in MCDD-fed mice with uninhibited DGAT2.
      • Yamaguchi K.
      • Yang L.
      • McCall S.
      • Huang J.
      • Yu X.X.
      • Pandey S.K.
      • et al.
      Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis.
      Saturated FFA, such as palmitic acid or stearic acid, as opposed to unsaturated FFA, such as oleic acid, can become toxic to hepatocytes, activating ER stress-induced apoptosis present in NASH pathogenesis.
      • Hirsova P.
      • Ibrabim S.H.
      • Gores G.J.
      • Malhi H.
      Lipotoxic lethal and sublethal stress signaling in hepatocytes: relevance to NASH pathogenesis.
      Rats fed an HFD rich in saturated fats showed increased expression of CHOP and active caspase-3.
      • Wang D.
      • Wei Y.
      • Pagliassotti M.J.
      Saturated fatty acids promote endoplasmic reticulum stress and liver injury in rats with hepatic steatosis.
      In hepatocyte cell lines, palmitic acid activated PERK and IRE1α, increasing CHOP expression,
      • Pfaffenbach K.T.
      • Gentile C.L.
      • Nivala A.M.
      • Wang D.
      • Wei Y.
      • Pagliassotti M.J.
      Linking endoplasmic reticulum stress to cell death in hepatocytes: roles of C/EBP homologous protein and chemical chaperones in palmitate-mediated cell death.
      which induces expression of DR5 leading to generation of truncated BID (tBID) by caspase-8.
      • Cazanave S.C.
      • Mott J.L.
      • Bronk S.F.
      • Werneburg N.W.
      • Fingas C.D.
      • Meng X.W.
      • et al.
      Death receptor 5 signaling promotes hepatocyte lipoapoptosis.
      Importantly, deletion of BAX and BAK in the liver completely blocks apoptosis induction under ER stress, correlating with modified UPR signalling.
      • Hetz C.
      • Bernasconi P.
      • Fisher J.
      • Lee A.H.
      • Bassik M.C.
      • Antonsson B.
      • et al.
      Proapoptotic BAX and BAK modulate the unfolded protein response by a direct interaction with IRE1alpha.
      Palmitic acid-induced activation of the IRE1α pathway through sustained JNK and/or CHOP activities promotes the expression of BH3-only proteins, PUMA and BIM, that directly activate BAX
      • Akazawa Y.
      • Cazanave S.
      • Mott J.L.
      • Elmi N.
      • Bronk S.F.
      • Kohno S.
      • et al.
      Palmitoleate attenuates palmitate-induced bim and PUMA up-regulation and hepatocyte lipoapoptosis.
      and hepatocyte death. Thus, lipotoxicity triggers PERK- and IRE1α-mediated cell death via the mitochondrial pathway of apoptosis.
      • Chipuk J.E.
      • Green D.R.
      How do BCL-2 proteins induce mitochondrial outer membrane permeabilization?.
      • Deniaud A.
      • Sharaf el dein O.
      • Maillier E.
      • Poncet D.
      • Kroemer G.
      • Lemaire C.
      • Brenner C.
      Endoplasmic reticulum stress induces calcium-dependent permeability transition, mitochondrial outer membrane permeabilization and apoptosis.
      However, in models of liver damage induced by CCl4, deletion of CHOP did not protect animals against tissue damage despite there being clear signs of ER stress.
      • Campos G.
      • Schmidt-Heck W.
      • Ghallab A.
      • Rochlitz K.
      • Pütter L.
      • Medinas D.B.
      • et al.
      The transcription factor CHOP, a central component of the transcriptional regulatory network induced upon CCl4 intoxication in mouse liver, is not a critical mediator of hepatotoxicity.

      Ca2+ and cell death

      The inability of cells to restore normal ER proteostasis because of aberrant Ca2+ signalling can lead to Ca2+ efflux from the ER to the mitochondria through MAMs, which triggers ER stress-induced apoptosis, contributing to the development of NAFLD.
      • Arruda A.P.
      • Pers B.M.
      • Parlakgul G.
      • Guney E.
      • Inouye K.
      • Hotamisligil G.S.
      Chronic enrichment of hepatic endoplasmic reticulum-mitochondria contact leads to mitochondrial dysfunction in obesity.
      • Rieusset J.
      Endoplasmic reticulum-mitochondria calcium signaling in hepatic metabolic diseases.
      A myriad of downstream effectors of Ca2+ release from the ER include the opening of the permeability transition pore, leading to mitochondrial swelling and outer membrane permeabilisation, and the activation of cathepsins, proteases implicated in the pathological hepatocyte death seen in humans with NASH.
      • Walenbergh S.M.
      • Houben T.
      • Rensen S.S.
      • Bieghs V.
      • Hendrikx T.
      • van Gorp P.J.
      • et al.
      Plasma cathepsin D correlates with histological classifications of fatty liver disease in adults and responds to intervention.
      Finally, ER stress is an important trigger of apoptotic, necrotic and other forms of hepatocyte death and hence a potential accelerator of inflammation, leading to NASH, fibrosis and HCC.

      The role of chronic ER stress in HCC

      The high worldwide prevalence of NAFLD may contribute to the rising incidence of HCC. HCC occurs within an established background of chronic liver disease and cirrhosis, with risk factors including NASH, excessive alcoholic intake or viral hepatitis.
      • El-Serag H.B.
      • Rudolph K.L.
      Hepatocellular carcinoma: epidemiology and molecular carcinogenesis.
      In the United States, the increasing incidence of HCC coupled with an overall five-year survival rate of less than 12% makes HCC the fastest growing cause of cancer-related mortality.
      • Mittal S.
      • Kanwal F.
      • Ying J.
      • Chung R.
      • Sada Y.H.
      • Temple S.
      • et al.
      Effectiveness of surveillance for hepatocellular carcinoma in clinical practice: a United States cohort.
      The role of ER stress signalling in the development of HCC remains to be defined. The tumour microenvironment in HCC may activate ER stress via oxygen and nutrient deprivation and acidic waste accumulation. While chronic UPR initiates steatosis development and activates hepatocyte death in steatohepatitis, the HCC microenvironment adapts to alter the integrated signalling of the UPR and prevent ER stress-induced apoptosis, thereby safe-guarding the cancerous cells and increasing their aggressiveness and resistance to chemotherapeutic agents.

      The roles of ER stress proximal sensors IRE1a, PERK and ATF6 in HCC

      The monitoring of UPR kinetics in HCC revealed that the IRE1α pathway is activated during tumour initiation, the PERK pathway during tumour progression and the ATF6α pathway is modestly activated in developed tumours.
      • Vandewynckel Y.-P.
      • Laukens D.
      • Bogaerts E.
      • Paridaens A.
      • Van den Bussche A.
      • Verhelst X.
      • et al.
      Modulation of the unfolded protein response impedes tumor cell adaptation to proteotoxic stress: a PERK for hepatocellular carcinoma therapy.
      IRE1α exhibits one somatic mutation in human HCC tumours,
      • Guichard C.
      • Amaddeo G.
      • Imbeaud S.
      • Ladeiro Y.
      • Pelletier L.
      • Maad I.B.
      • et al.
      Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma.
      • Greenman C.
      • Stephens P.
      • Smith R.
      • Dalgliesh G.L.
      • Hunter C.
      • Bignell G.
      • et al.
      Patterns of somatic mutation in human cancer genomes.
      which could account for a modulation of its kinase and/or RNase function. HCC biopsies from human patients revealed elevated XBP1s expression levels.
      • Shuda M.
      • Kondoh N.
      • Imazeki N.
      • Tanaka K.
      • Okada T.
      • Mori K.
      • et al.
      Activation of the ATF6, XBP1 and grp78 genes in human hepatocellular carcinoma: a possible involvement of the ER stress pathway in hepatocarcinogenesis.
      • Kishimoto T.
      • Kokura K.
      • Ohkawa N.
      • Makino Y.
      • Yoshida M.
      • Hirohashi S.
      • et al.
      Enhanced expression of a new class of liver-enriched b-Zip transcription factors, hepatocarcinogenesis-related transcription factor, in hepatocellular carcinomas of rats and humans.
      A specific target gene of XBP1 in the liver is alpha-fetoprotein,
      • Reimold A.M.
      • Etkin A.
      • Clauss I.
      • Perkins A.
      • Friend D.S.
      • Zhang J.
      • et al.
      An essential role in liver development for transcription factor XBP-1.
      the most widely used biomarker for HCC surveillance.
      • Mittal S.
      • Kanwal F.
      • Ying J.
      • Chung R.
      • Sada Y.H.
      • Temple S.
      • et al.
      Effectiveness of surveillance for hepatocellular carcinoma in clinical practice: a United States cohort.
      Cancerous cells deficient for XBP1 are less prone to developing solid tumours in nude mice.
      • Spiotto M.T.
      • Banh A.
      • Papandreou I.
      • Cao H.
      • Galvez M.G.
      • Gurtner G.C.
      • et al.
      Imaging the unfolded protein response in primary tumors reveals microenvironments with metabolic variations that predict tumor growth.
      Similarly, IRE1α or XBP1 deficiency compromises the mircoenvironment support for cancerous cell growth.
      • Xu G.
      • Liu K.
      • Anderson J.
      • Patrene K.
      • Lentzsch S.
      • Roodman G.D.
      • et al.
      Expression of XBP1s in bone marrow stromal cells is critical for myeloma cell growth and osteoclast formation.
      Among the 37 RIDD substrates of IRE1α identified in metazoans, 68% are associated with cancer.
      • Maurel M.
      • Chevet E.
      • Tavernier J.
      • Gerlo S.
      Getting RIDD of RNA: IRE1 in cell fate regulation.
      The RIDD function targets cancer-relevant mRNAs like SPARC and microRNAs such as miR-17 in the liver. SPARC inhibits the tumorigenic capacity of HCC cells and improves survival of HepG2-injected nude mice.
      • Atorrasagasti C.
      • Malvicini M.
      • Aquino J.B.
      • Alaniz L.
      • Garcia M.
      • Bolontrade M.
      • et al.
      Overexpression of SPARC obliterates the in vivo tumorigenicity of human hepatocellular carcinoma cells.
      PERK signalling contributes to adaptive rather than terminal pathways, promoting tumour growth and angiogenesis. Under hypoxic stress, PERK regulates proangiogenic genes involved in cell-cell adhesion, matrix remodelling and extracellular matrix proteolysis.
      • Blais J.D.
      • Addison C.L.
      • Edge R.
      • Falls T.
      • Zhao H.
      • Wary K.
      • et al.
      Perk-dependent translational regulation promotes tumor cell adaptation and angiogenesis in response to hypoxic stress.
      The role of ATF6α in cancer still remains unclear. Data suggested a role for ATF6α in hepatocarcinogenesis
      • Arai M.
      • Kondoh N.
      • Imazeki N.
      • Hada A.
      • Hatsuse K.
      • Kimura F.
      • et al.
      Transformation-associated gene regulation by ATF6alpha during hepatocarcinogenesis.
      without clear mechanistic information. However, ATF6α is essential for the adaptation of dormant cells to chemotherapy, nutrient starvation and in vivo microenvironmental challenges among other environmental factors.
      • Schewe D.M.
      • Aguirre-Ghiso J.A.
      ATF6alpha-Rheb-mTOR signaling promotes survival of dormant tumor cells in vivo.

      The roles of UPR targets GRP78 and CHOP in HCC

      GRP78 is constitutively overexpressed in HCCs.
      • Shuda M.
      • Kondoh N.
      • Imazeki N.
      • Tanaka K.
      • Okada T.
      • Mori K.
      • et al.
      Activation of the ATF6, XBP1 and grp78 genes in human hepatocellular carcinoma: a possible involvement of the ER stress pathway in hepatocarcinogenesis.
      Both ATF6α- and IRE1α-XBP1-dependent UPR systems are essential for the transformation-associated expression of the GRP78 gene in HCC.
      • Shuda M.
      • Kondoh N.
      • Imazeki N.
      • Tanaka K.
      • Okada T.
      • Mori K.
      • et al.
      Activation of the ATF6, XBP1 and grp78 genes in human hepatocellular carcinoma: a possible involvement of the ER stress pathway in hepatocarcinogenesis.
      Cleaved GRP78 has been reported in the sera of patients with HCC.
      • Chignard N.
      • Shang S.
      • Wang H.
      • Marrero J.
      • Brechot C.
      • Hanash S.
      • et al.
      Cleavage of endoplasmic reticulum proteins in hepatocellular carcinoma: detection of generated fragments in patient sera.
      Elevated GRP78 levels correlate with higher pathologic grade, recurrence, and poor patient survival.
      • Luo B.
      • Lee A.S.
      The critical roles of endoplasmic reticulum chaperones and unfolded protein response in tumorigenesis and anticancer therapies.
      Autoantibodies against GRP78 were identified as promising biomarkers for HCC.
      • Shao Q.
      • Ren P.
      • Li Y.
      • Peng B.
      • Dai L.
      • Lei N.
      • et al.
      Autoantibodies against glucose-regulated protein 78 as serological diagnostic biomarkers in hepatocellular carcinoma.
      Because of the pro-apoptotic function of CHOP under ER stress, a strategy aimed at inducing CHOP to kill cancerous cells would appear logical; however, CHOP may contribute to HCC pathogenesis. Chop-deficient mice exhibit both reduced apoptosis and cellular proliferation, and are resistant to hepatocarcinogenesis.
      • DeZwaan-McCabe D.
      • Riordan J.D.
      • Arensdorf A.M.
      • Icardi M.S.
      • Dupuy A.J.
      • Rutkowski D.T.
      The stress-regulated transcription factor CHOP promotes hepatic inflammatory gene expression, fibrosis, and oncogenesis.
      Nevertheless, we can question the cell autonomy of CHOP’s oncogenic effects in the liver. The pro-inflammatory genes differentially expressed in Chop-deficient mice were suggested to arise from a hepatocyte-independent function. In that respect, Chop-deficient mice with carcinogen-induced HCC had less inflammation.
      • Scaiewicz V.
      • Nahmias A.
      • Chung R.T.
      • Mueller T.
      • Tirosh B.
      • Shibolet O.
      CCAAT/enhancer-binding protein homologous (CHOP) protein promotes carcinogenesis in the DEN-induced hepatocellular carcinoma model.
      Despite robust expression of CHOP in HCC tumours, no evidence of cell death was observed, suggesting a potential switch of CHOP function from pro-apoptotic to pro-tumourigenic.
      • Scaiewicz V.
      • Nahmias A.
      • Chung R.T.
      • Mueller T.
      • Tirosh B.
      • Shibolet O.
      CCAAT/enhancer-binding protein homologous (CHOP) protein promotes carcinogenesis in the DEN-induced hepatocellular carcinoma model.
      Finally, the ATF6α branch was specifically tumour-activated in DEN-induced HCC and described as a putative inducer of CHOP.
      • Scaiewicz V.
      • Nahmias A.
      • Chung R.T.
      • Mueller T.
      • Tirosh B.
      • Shibolet O.
      CCAAT/enhancer-binding protein homologous (CHOP) protein promotes carcinogenesis in the DEN-induced hepatocellular carcinoma model.
      In liver biopsies from human patients, CHOP protein levels increased parallel to the evolution of the stages from steatosis, to NASH, to liver cirrhosis and to HCC.
      • Toriguchi K.
      • Hatano E.
      • Tanabe K.
      • Takemoto K.
      • Nakamura K.
      • Koyama Y.
      • et al.
      Attenuation of steatohepatitis, fibrosis, and carcinogenesis in mice fed a methionine-choline deficient diet by CCAAT/enhancer-binding protein homologous protein deficiency.
      Lastly, ER stress and HFD feeding were found to synergistically induce HCC through an inflammatory mechanism dependent on the TNF-TNFR1-IKKβ-NF-kB pathway, and independent of CHOP, perpetuating the pathogenesis of NASH to HCC.
      • Nakagawa H.
      • Umemura A.
      • Taniguchi K.
      • Font-Burgada J.
      • Dhar D.
      • Ogata H.
      • et al.
      ER stress cooperates with hypernutrition to trigger TNF-dependent spontaneous HCC development.
      This study demonstrated that ER stress and steatosis trigger oxidative stress leading to genomic instability, oncogenic mutations and/or gene-copy-number variations.

      Current and potential therapies for the treatment of NAFL/NASH

      Despite this major public health concern, no effective treatment yet exists. A consensus of confirmed experimental models of NAFLD is necessary to test the potential of pharmacological strategies.
      In the early stages, NAFLD can be successfully managed with lifestyle changes. Only an improved diet and/or increased physical activity can slow the progression of NAFLD.
      • Zelber-Sagi S.
      • Godos J.
      • Salomone F.
      Lifestyle changes for the treatment of nonalcoholic fatty liver disease: a review of observational studies and intervention trials.
      Nevertheless, because of the long-term challenge, many patients are unable maintain their weight loss. A more drastic option includes bariatric surgery for the morbidly obese, but a recent retrospective study showed that 41% of patients that underwent a Roux-en-Y gastric bypass had regained weight after 10 years.
      • Monaco-Ferreira D.V.
      • Leandro-Merhi V.A.
      Weight regain 10 years after Roux-en-Y gastric bypass.
      As no established treatment yet exists for NAFLD, it is urgent to develop effective therapies.
      The development of animal models of NAFLD that meet the pathophysiological criteria of the disease and its evolution is necessary to test promising compounds before initiating clinical trials. Experimental models of NAFLD reported in the literature mainly cover dietary, genetic and/or chemical interventions in mice (Table 1). An international consensus on the ideal murine model is needed to improve the translatability into human NAFLD pathogenesis and treatment.
      Table 1Overview of mouse models of NAFLD with ER stress markers.
      InterventionModelCharacteristicsObesitySteatosisNASHFibrosisHCCIncreased ER stress markers?
      Dietary*

      (*primary models used to study NAFLD, but composition usually exceeds western culture diets in fat and cholesterol content)
      High-fatUsually 40–75% fat to bring about triglyceride accumulation in the liver.YesYesYes (mild)Yes (mild)NoYes

      (IRE1α, PERK and ATF6 signalling pathways, GRP78)
      High-fat high-sugarUsually 30–55% fructose and/or sucrose.YesYesYes (mild)Yes (mild)NoYes

      (IRE1α-XBP1, PERK, CHOP, GRP78)
      High-cholesterolUsually 1–2% cholesterol.YesYesYesYesNo?
      High-cholesterol high-cholateAtherogenic diet.

      1.25% cholesterol and 0.5% cholate added to diet, promoting cholesterol and fat absorption and suppressing the conversion of cholesterol to bile acids, thus promoting atherosclerosis.
      NoYesYesYesNoYes

      (CHOP)
      High-fat high-sugar high-cholesterolFast-food diet.YesYesYesYesYesYes

      (p-PERK, p-ATF2, p-ELF2)
      Methionine- and choline-deficientDeficiency of essential nutrients, resulting in impaired β-oxidation and production/secretion of very low-density lipoprotein (VLDL) particles. Usually contains high sucrose (40%) and moderate fat (10%).No

      (significant weight loss)
      YesYesYesYes

      (if animals survive)
      Yes

      (p-PERK, p-eIF2α, XBP1s, p-JNK, p-NF-kB, CHOP, GRP78)
      Choline-deficient l-amino acid-definedDiet proteins are substituted with an equivalent and corresponding mixture of L-amino acids.

      Sometimes iron-supplemented.
      NoYesYesYesYesYes

      (CHOP)
      Dietary and lifestyleALIOS (American lifestyle-induced obesity syndrome)High-fat (45%, rich in trans fat (30% of fat content)) high-fructose diet and promoted sedentary lifestyle (removal of cage racks).YesYesYes (without hepatocyte ballooning)YesYesYes

      (CHOP)
      Genetic*

      (*dietary intervention is frequently required to induce NAFL to NASH transition)
      ob/obLeptin-deficient (spontaneous mutation). Hyperphagic and inactive.YesYesNo (not spontaneous)No (resistant)NoYes

      (IRE1α, PERK and ATF6 signalling pathways, GRP78)
      db/dbLeptin receptor-deficient (spontaneous mutation). Hyperleptinaemic, hyperphagic and inactive.YesYesNo (not spontaneous)NoNoYes

      (IRE1α, PERK and ATF6 signalling pathways, GRP78)
      foz/fozAlstrom syndrome 1 (Alms1)-deficient (more severe on C57BL6/J than BALB/c strain).YesYesNo (not spontaneous)NoNo?
      Dietary and geneticob/ob + MCDYesYesYesNoNo?
      db/db + MCDYesYesYesYes?Yes

      (IRE1α, PERK and ATF6 signalling pathways)
      foz/foz + atherogenic dietMice fed a diet composed of 23% fat, 45% carbohydrate, 20% protein, 0.2% cholesterol.YesYesYesYes??
      MUP-uPA + HFDUrokinase-type plasminogen activator (uPA) transgenic mice under the control of the mature hepatocyte-specific promoter for major urinary protein (MUP). Mice fed a diet composed of 60% fat.YesYesYesYesYesYes

      (p-eIF2α, p-JNK, CHOP)
      DIAMONDInbred isogenic strain of C57BL6/J and 129S1/SvImJ mice fed a high-fat, high-carbohydrate diet with sugar water.YesYesYesYesYesYes

      (p-JNK, CHOP)
      Chemical*

      (*not very physiological, but rapid disease evolution)
      TunicamycinSingle injection (1mg/kg).NoYes

      (transient)
      No

      (unless transgenic model vulnerable to ER stress)
      No

      (mild if transgenic model vulnerable to ER stress)
      NoYes

      (all markers significantly increased)
      CCl4Repeat dosing (0.2–0.5 ml/kg) induces oxidative stress leading to toxic lipid and protein peroxidation product accumulation and a strong necrotic response.No

      (weight loss)
      YesYes (without hepatocyte ballooning)YesYesYes

      (CHOP)
      DENSingle DEN injection (1mg/kg). Has been associated with repeat dosing of CCl4 (0.2 ml/kg).No??YesYes?
      Dietary and chemicalDEN + HFD or DEN + MCDHFD yes

      MCD no
      YesYesYesYesYes

      (CHOP)
      STAMNeonatal streptozotocin (200 µg) type 1 diabetes model + HFD feeding.NoYesYesYesYes?
      ER, endoplasmic reticulum; HCC, hepatocellular carcinoma; MCD, methionine-choline-deficient; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis.
      Over the past three years, 74% of published articles studying NASH pathogenesis have used dietary models.
      • Van Herck M.A.
      • Vonghia L.
      • Francque S.M.
      Animal models of nonalcoholic fatty liver disease-a starter's guide.
      MCDD represents the most commonly used diet to produce steatohepatitis with liver fibrosis. While this diet is adequate for intrahepatic study, it may be inadequate for multisystemic analysis as mice fed an MCDD lose considerable weight and do not develop systemic insulin resistance. Other diets have sought to mimic the development of NAFLD by diet-induced obesity, which is the major risk factor in humans. Obesogenic diets are characterised by high-fat and/or high-sucrose and/or high-cholesterol, which may often exceed the dietary amounts found in western diets. Genetically and diet-induced obesity, as well as MCDD, have been used extensively to study ER stress signalling in NAFLD pathogenesis (Table 1). C57BL/6J mice fed with MCD showed robust activation of eIF2α and CHOP, indicating preferential induction of the PERK arm.
      • Henkel A.S.
      • Dewey A.M.
      • Anderson K.A.
      • Olivares S.
      • Green R.M.
      Reducing endoplasmic reticulum stress does not improve steatohepatitis in mice fed a methionine- and choline-deficient diet.
      MCD-diet-induced steatohepatitis was reversibly reduced in Chop knockout mice. In a similar context, we demonstrated that ob/ob mice challenged with LPS mimicked the extent of steatohepatitis found in human NASH progression, with concurrent activation of IRE1α and PERK resulting in synergic CHOP activation.
      • Lebeaupin C.
      • Proics E.
      • de Bieville C.H.
      • Rousseau D.
      • Bonnafous S.
      • Patouraux S.
      • et al.
      ER stress induces NLRP3 inflammasome activation and hepatocyte death.
      In addition to this, C57BL/6J WT mice fed with a high-fat high-sucrose diet significantly perturbed the ER FC/PL ratio (free cholesterol to phospholipid), which precisely correlated with upregulated CHOP and XBP1s levels.
      • Bashiri A.
      • Nesan D.
      • Tavallaee G.
      • Sue-Chue-Lam I.
      • Chien K.
      • Maguire G.F.
      • et al.
      Cellular cholesterol accumulation modulates high fat high sucrose (HFHS) diet-induced ER stress and hepatic inflammasome activation in the development of non-alcoholic steatohepatitis.
      Similarly, a study confirmed the upregulation of CHOP in an atherogenic diet-induced mouse model of steatohepatitis.
      • Kandel-Kfir M.
      • Almog T.
      • Shaish A.
      • Shlomai G.
      • Anafi L.
      • Avivi C.
      • et al.
      Interleukin-1alpha deficiency attenuates endoplasmic reticulum stress-induced liver damage and CHOP expression in mice.
      Furthermore, a high-cholesterol-containing fast food diet (24 weeks) resulted in disrupted ER and significantly elevated p-PERK levels in C57BL/6J WT mice.
      • Abdelmegeed M.A.
      • Choi Y.
      • Godlewski G.
      • Ha S.-K.
      • Banerjee A.
      • Jang S.
      • et al.
      Cytochrome P450–2E1 promotes fast food-mediated hepatic fibrosis.
      Genetic models are frequently used in combination with modified diets to induce a robust steatosis to NASH transition. For example, foz/foz mice are an overnutrition model featuring obesity, diabetes and hypercholesterolaemia. When challenged with an atherogenic diet foz/foz mice developed NASH with fibrosis, which enabled the testing of an anti-inflammatory approach to NASH treatment.
      • Mridha A.R.
      • Wree A.
      • Robertson A.A.B.
      • Yeh M.M.
      • Johnson C.D.
      • Van Rooyen D.M.
      • et al.
      NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice.
      It was reported that ER stress does not contribute to NASH in HFD-foz/foz mice.
      • Legry V.
      • Van Rooyen D.M.
      • Lambert B.
      • Sempoux C.
      • Poekes L.
      • Espanol-Suner R.
      • et al.
      Endoplasmic reticulum stress does not contribute to steatohepatitis in obese and insulin-resistant high-fat-diet-fed foz/foz mice.
      The diet-induced animal model of NAFLD (DIAMOND) was recently developed to recapitulate human NAFLD pathophysiology by crossing two mouse strains (C57BL6/J and 129S1/SvImJ) and feeding the mice a high-fat and high-carbohydrate diet with a high fructose-glucose solution.
      • Asgharpour A.
      • Cazanave S.C.
      • Pacana T.
      • Seneshaw M.
      • Vincent R.
      • Banini B.A.
      • et al.
      A diet-induced animal model of non-alcoholic fatty liver disease and hepatocellular cancer.
      Moreover, DIAMOND-fed mice rapidly developed NASH, mild fibrosis (16–22 weeks), bridging fibrosis (around 52 weeks) and HCC (32–52 weeks). Interestingly, they showed similar transcriptional, metabolic, histological, physiological and cell-signalling patterns to progressive NASH in humans.
      • Asgharpour A.
      • Cazanave S.C.
      • Pacana T.
      • Seneshaw M.
      • Vincent R.
      • Banini B.A.
      • et al.
      A diet-induced animal model of non-alcoholic fatty liver disease and hepatocellular cancer.
      The pathological progression of steatosis to NASH with fibrosis and eventually HCC in STAM™ mice is very similar to the human disease.
      • Fujii M.
      • Shibazaki Y.
      • Wakamatsu K.
      • Honda Y.
      • Kawauchi Y.
      • Suzuki K.
      • et al.
      A murine model for non-alcoholic steatohepatitis showing evidence of association between diabetes and hepatocellular carcinoma.
      STAM mice recapitulated the lipidomic status of human NASH (8 weeks) and fibrosis (12 weeks).
      • Saito K.
      • Uebanso T.
      • Maekawa K.
      • Ishikawa M.
      • Taguchi R.
      • Nammo T.
      • et al.
      Characterization of hepatic lipid profiles in a mouse model with nonalcoholic steatohepatitis and subsequent fibrosis.
      The authors found elevated sphingolipids, including ceramides, in STAM mice, which at the stage of NASH could trigger inflammation, apoptosis and insulin resistance, eventually culminating in fibrosis.
      • Saito K.
      • Uebanso T.
      • Maekawa K.
      • Ishikawa M.
      • Taguchi R.
      • Nammo T.
      • et al.
      Characterization of hepatic lipid profiles in a mouse model with nonalcoholic steatohepatitis and subsequent fibrosis.
      Studies are needed to analyse the contribution of ER stress in DIAMOND and STAM mice. In any case, all animal models should be limited to clearly defined and liver-specific research goals for the development of therapeutics.
      Promising strategies using drugs that reduce ER stress levels, pharmacological inhibitors of the UPR, or genetic probes, are listed (Table 2). Some of these molecules have been tested in mice with NAFLD (summarised below).
      Table 2Notable compounds that target ER stress-related pathways in NAFLD.
      FamilyNameER TargetsEffectsTherapeutic potential in liver DiseaseReference
      Chemical chaperones4-PBAUnfolded proteinsPromotes protein folding capacity and ERAD efficiency.

      Stabilises the adaptive UPR.
      (NAFL/NASH)

      Urea-cycle disorders
      (Özcan et al., 2006)
      • Özcan U.
      • Yilmaz E.
      • Özcan L.
      • Furuhashi M.
      • Vaillancourt E.
      • Smith R.O.
      • et al.
      Chemical chaperones reduce er stress and restore glucose homeostasis in a mouse model of type 2 diabetes.


      (Ben Mosbah et al., 2010)
      • Ben Mosbah I.
      • Alfany-Fernandez I.
      • Martel C.
      • Zaouali M.A.
      • Bintanel-Morcillo M.
      • Rimola A.
      • et al.
      Endoplasmic reticulum stress inhibition protects steatotic and non-steatotic livers in partial hepatectomy under ischemia-reperfusion.


      (Lebeaupin et al., 2015)
      • Lebeaupin C.
      • Proics E.
      • de Bieville C.H.
      • Rousseau D.
      • Bonnafous S.
      • Patouraux S.
      • et al.
      ER stress induces NLRP3 inflammasome activation and hepatocyte death.


      (Nakagawa et al., 2014)
      • Nakagawa H.
      • Umemura A.
      • Taniguchi K.
      • Font-Burgada J.
      • Dhar D.
      • Ogata H.
      • et al.
      ER stress cooperates with hypernutrition to trigger TNF-dependent spontaneous HCC development.
      TUDCA(NAFL/NASH)

      Cholestatic liver disease
      Chemical inhibitors4μ8cIRE1α RNaseInhibits XBP1 mRNA splicing.

      Inhibits RIDD function.
      (NASH)

      (HCC)
      (Tufanli et al., 2017)
      • Tufanli O.
      • Telkoparan Akillilar P.
      • Acosta-Alvear D.
      • Kocaturk B.
      • Onat U.I.
      • Hamid S.M.
      • et al.
      Targeting IRE1 with small molecules counteracts progression of atherosclerosis.


      (Lebeaupin et al., 2018)
      • Lebeaupin C.
      • Vallée D.
      • Rousseau D.
      • Patouraux S.
      • Bonnafous S.
      • Adam G.
      • et al.
      Bax Inhibitor-1 protects from non-alcoholic steatohepatitis by limiting IRE1α signaling.
      STF-083010(Lerner et al., 2012)
      • Lerner A.G.
      • Upton J.P.
      • Praveen P.V.
      • Ghosh R.
      • Nakagawa Y.
      • Igbaria A.
      • et al.
      IRE1alpha induces thioredoxin-interacting protein to activate the NLRP3 inflammasome and promote programmed cell death under irremediable ER stress.


      (Kim et al., 2015)
      • Kim S.
      • Joe Y.
      • Kim H.J.
      • Kim Y.S.
      • Jeong S.O.
      • Pae H.O.
      • et al.
      Endoplasmic reticulum stress-induced IRE1alpha activation mediates cross-talk of GSK-3beta and XBP-1 to regulate inflammatory cytokine production.


      (Tufanli et al., 2017)
      • Tufanli O.
      • Telkoparan Akillilar P.
      • Acosta-Alvear D.
      • Kocaturk B.
      • Onat U.I.
      • Hamid S.M.
      • et al.
      Targeting IRE1 with small molecules counteracts progression of atherosclerosis.


      (Lebeaupin et al., 2018)
      • Lebeaupin C.
      • Vallée D.
      • Rousseau D.
      • Patouraux S.
      • Bonnafous S.
      • Adam G.
      • et al.
      Bax Inhibitor-1 protects from non-alcoholic steatohepatitis by limiting IRE1α signaling.
      MKC-3946(Mimura et al., 2012)
      • Mimura N.
      • Fulciniti M.
      • Gorgun G.
      • Tai Y.T.
      • Cirstea D.
      • Santo L.
      • et al.
      Blockade of XBP1 splicing by inhibition of IRE1alpha is a promising therapeutic option in multiple myeloma.
      KIRA6/KIRA8IRE1α kinase and RNasePromotes cell survival under ER stress.(Insulin resistance)

      (NAFL/NASH)
      (Wang et al., 2012)
      • Wang L.
      • Perera B.G.
      • Hari S.B.
      • Bhhatarai B.
      • Backes B.J.
      • Seeliger M.A.
      • et al.
      Divergent allosteric control of the IRE1alpha endoribonuclease using kinase inhibitors.


      (Ghosh et al., 2014)
      • Ghosh R.
      • Wang L.
      • Wang E.S.
      • Perera B.G.
      • Igbaria A.
      • Morita S.
      • et al.
      Allosteric inhibition of the IRE1alpha RNase preserves cell viability and function during endoplasmic reticulum stress.


      (Morita et al., 2017)
      • Morita S.
      • Villalta S.A.
      • Feldman H.C.
      • Register A.C.
      • Rosenthal W.
      • Hoffmann-Petersen I.T.
      • et al.
      Targeting ABL-IRE1alpha signaling spares ER-stressed pancreatic beta cells to reverse autoimmune diabetes.
      APY29IRE1α kinaseATP-competitive inhibitor that inhibits IRE1α kinase, but increases dimerisation/oligomerisation of IRE1α, enhancing RNase activity.?(Korennykh et al., 2009)
      • Korennykh A.V.
      • Egea P.F.
      • Korostelev A.A.
      • Finer-Moore J.
      • Zhang C.
      • Shokat K.M.
      • et al.
      The unfolded protein response signals through high-order assembly of Ire1.
      SelonsertibIRE1α-ASK1Reduces collagen content and inflammation.(NASH with liver fibrosis)(Loomba et al., 2017)
      • Loomba R.
      • Lawitz E.
      • Mantry P.S.
      • Jayakumar S.
      • Caldwell S.H.
      • Arnold H.
      • et al.
      The ASK1 inhibitor selonsertib in patients with nonalcoholic steatohepatitis: A randomized, phase 2 trial.
      SalubrinalPERK-eIF2αPrevents eIF2α dephosphorylation, maintaining protein translation inhibition to limit the ER protein load.(NAFL/NASH)(Vandewynckel et al., 2015)
      • Vandewynckel Y.-P.
      • Laukens D.
      • Bogaerts E.
      • Paridaens A.
      • Van den Bussche A.
      • Verhelst X.
      • et al.
      Modulation of the unfolded protein response impedes tumor cell adaptation to proteotoxic stress: a PERK for hepatocellular carcinoma therapy.
      Guanabenz(Tsaytler et al., 2011)
      • Tsaytler P.
      • Harding H.P.
      • Ron D.
      • Bertolotti A.
      Selective inhibition of a regulatory subunit of protein phosphatase 1 restores proteostasis.
      ISRIBPERK-p-eIF2αInhibits p-eIF2α to resume global protein translation.?(Sidrauski et al., 2015)
      • Sidrauski C.
      • McGeachy A.M.
      • Ingolia N.T.
      • Walter P.
      The small molecule ISRIB reverses the effects of eIF2alpha phosphorylation on translation and stress granule assembly.
      CeapinsATF6αPrevents ATF6α cleavage and activation.?(Gallagher et al., 2016)
      • Gallagher C.M.
      • Garri C.
      • Cain E.L.
      • Ang K.K.
      • Wilson C.G.
      • Chen S.
      • et al.
      Ceapins are a new class of unfolded protein response inhibitors, selectively targeting the ATF6alpha branch.


      GSK2656157
      PERKInhibits PERK autophosphorylation.

      Reduces viability and proliferation of HCC cells, reduces HCC tumour growth in vivo.
      (HCC)(Axten et al., 2013)
      • Axten J.M.
      • Romeril S.P.
      • Shu A.
      • Ralph J.
      • Medina J.R.
      • Feng Y.
      • et al.
      Discovery of GSK2656157: an optimized perk inhibitor selected for preclinical development.


      (Vandewynckel et al., 2015)
      • Vandewynckel Y.-P.
      • Laukens D.
      • Bogaerts E.
      • Paridaens A.
      • Van den Bussche A.
      • Verhelst X.
      • et al.
      Modulation of the unfolded protein response impedes tumor cell adaptation to proteotoxic stress: a PERK for hepatocellular carcinoma therapy.
      LBY135Agonistic DR5 antibodySensitises resistant HCC cells to apoptosis.(HCC)(Chen et al., 2010)
      • Chen K.F.
      • Tai W.T.
      • Liu T.H.
      • Huang H.P.
      • Lin Y.C.
      • Shiau C.W.
      • et al.
      Sorafenib overcomes TRAIL resistance of hepatocellular carcinoma cells through the inhibition of STAT3.
      PACMA 31PDI inhibitorIncreases ER stress to reduce cell viability.(HCC)(Won et al., 2017)
      • Won J.K.
      • Yu S.J.
      • Hwang C.Y.
      • Cho S.H.
      • Park S.M.
      • Kim K.
      • et al.
      Protein disulfide isomerase inhibition synergistically enhances the efficacy of sorafenib for hepatocellular carcinoma.
      BacitracinPDI inhibitorIncreases ER stress to reduce cell viability.(HCC)(Yu et al., 2012)
      • Yu S.J.
      • Yoon J.H.
      • Yang J.I.
      • Cho E.J.
      • Kwak M.S.
      • Jang E.S.
      • et al.
      Enhancement of hexokinase II inhibitor-induced apoptosis in hepatocellular carcinoma cells via augmenting ER stress and anti-angiogenesis by protein disulfide isomerase inhibition.
      Gene therapyAdenovirus-GRP78Overexpression of GRP78Reduces hepatic steatosis and improved insulin sensitivity in obese mice.(Insulin resistance)

      (NAFL/NASH)
      (Kammoun et al., 2009)
      • Kammoun H.L.
      • Chabanon H.
      • Hainault I.
      • Luquet S.
      • Magnan C.
      • Koike T.
      • et al.
      GRP78 expression inhibits insulin and ER stress-induced SREBP-1c activation and reduces hepatic steatosis in mice.


      (Nakagawa et al., 2014)
      • Nakagawa H.
      • Umemura A.
      • Taniguchi K.
      • Font-Burgada J.
      • Dhar D.
      • Ogata H.
      • et al.
      ER stress cooperates with hypernutrition to trigger TNF-dependent spontaneous HCC development.


      (Gomez and Rutkowski, 2016)
      • Gomez J.A.
      • Rutkowski D.T.
      Experimental reconstitution of chronic ER stress in the liver reveals feedback suppression of BiP mRNA expression.
      Adenovirus-BI-1Overexpression/reintroduction of BI-1Improves glucose metabolism and insulin sensitivity.

      Reduces steatosis.
      (Insulin resistance)

      (NAFL/NASH)
      (Bailly-Maitre et al., 2010)
      • Bailly-Maitre B.
      • Belgardt B.F.
      • Jordan S.D.
      • Coornaert B.
      • von Freyend M.J.
      • Kleinridders A.
      • et al.
      Hepatic Bax inhibitor-1 inhibits IRE1alpha and protects from obesity-associated insulin resistance and glucose intolerance.


      (Lee et al., 2016a)
      • Lee G.H.
      • Oh K.J.
      • Kim H.R.
      • Han H.S.
      • Lee H.Y.
      • Park K.G.
      • et al.
      Effect of BI-1 on insulin resistance through regulation of CYP2E1.


      (Lee et al., 2016b)
      • Lee H.Y.
      • Lee G.H.
      • Bhattarai K.R.
      • Park B.H.
      • Koo S.H.
      • Kim H.R.
      • et al.
      Bax Inhibitor-1 regulates hepatic lipid accumulation via ApoB secretion.
      Adenovirus-XBP1sOverexpression of XBP1sImproves glucose metabolism and insulin sensitivity.(Insulin resistance)

      (NAFL/NASH)
      (Zhou et al., 2011)
      • Zhou Y.
      • Lee J.
      • Reno C.M.
      • Sun C.
      • Park S.W.
      • Chung J.
      • et al.
      Regulation of glucose homeostasis through a XBP-1-FoxO1 interaction.
      Adenovirus-ASK1Overexpression/reintroduction of ASK1Restores the JNK-BIM and TNFα pro-apoptotic pathways.(HCC)(Nakagawa et al., 2011)
      • Nakagawa H.
      • Hirata Y.
      • Takeda K.
      • Hayakawa Y.
      • Sato T.
      • Kinoshita H.
      • et al.
      Apoptosis signal-regulating kinase 1 inhibits hepatocarcinogenesis by controlling the tumor-suppressing function of stress-activated mitogen-activated protein kinase.
      Oncolytic viro-therapyM1 virus with valosin-containing protein inhibitorsIRE1α-XBP1Suppresses adaptive and promotes terminal UPR pathway.(HCC)(Zhang et al., 2017)
      • Zhang H.
      • Li K.
      • Lin Y.
      • Xing F.
      • Xiao X.
      • Cai J.
      • et al.
      Targeting VCP enhances anticancer activity of oncolytic virus M1 in hepatocellular carcinoma.
      Natural compoundsMelatoninInduction of CHOPIncreases ER stress-induced apoptosis.(HCC)(Moreira et al., 2015)
      • Moreira A.J.
      • Ordonez R.
      • Cerski C.T.
      • Picada J.N.
      • Garcia-Palomo A.
      • Marroni N.P.
      • et al.
      Melatonin activates endoplasmic reticulum stress and apoptosis in rats with diethylnitrosamine-induced hepatocarcinogenesis.
      ResveratrolIRE1αReduces DNA-binding capacity of XBP1s to its pro-survival target genes.(HCC)(Rojas et al., 2014)
      • Rojas C.
      • Pan-Castillo B.
      • Valls C.
      • Pujadas G.
      • Garcia-Vallve S.
      • Arola L.
      • et al.
      Resveratrol enhances palmitate-induced ER stress and apoptosis in cancer cells.
      ER, endoplasmic reticulum; HCC, hepatocellular carcinoma; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis.

      Broad-spectrum ER stress-reducing drugs

      Compounds have been identified that alleviate ER stress by increasing protein-folding capacity. These include the 4-phenyl butyric acid (PBA), a short-chain fatty acid also described as a potent histone deacetylase inhibitor, and the taurine-conjugated ursodeoxycholic acid (TUDCA), a derivative of an endogenous bile acid that has been safely used as a hepatoprotective agent in humans.
      • Carey E.J.
      • Ali A.H.
      • Lindor K.D.
      Primary biliary cirrhosis.
      However, because of their poor selectivity, these chaperones usually require high concentrations to take effect, making them largely neglected as therapeutic agents. Treatment with either 4-PBA (1 g/kg/day; orally) or TUDCA (500 mg/kg/day; intraperitoneally [i.p.]) for up to 30 days resolved hepatic steatosis in ob/ob mice.
      • Özcan U.
      • Yilmaz E.
      • Özcan L.
      • Furuhashi M.
      • Vaillancourt E.
      • Smith R.O.
      • et al.
      Chemical chaperones reduce er stress and restore glucose homeostasis in a mouse model of type 2 diabetes.
      While 4-PBA (100–200 mg/kg) and TUDCA (50–100 mg/kg) administered in rats with both normal and steatotic livers before partial hepatectomy and ischaemia-reperfusion protected against liver injury and regeneration failure by reducing inflammation, apoptosis and necrosis.
      • Ben Mosbah I.
      • Alfany-Fernandez I.
      • Martel C.
      • Zaouali M.A.
      • Bintanel-Morcillo M.
      • Rimola A.
      • et al.
      Endoplasmic reticulum stress inhibition protects steatotic and non-steatotic livers in partial hepatectomy under ischemia-reperfusion.
      TUDCA (500 mg/kg/day; i.p.) administered as a protective five-day pretreatment or as a potential six-hour treatment protected against NASH by limiting ER stress-dependent NLRP3 inflammasome activation in ob/ob mice, with endotoxaemia induced by a single LPS injection (2 mg/kg; i.p.).
      • Lebeaupin C.
      • Proics E.
      • de Bieville C.H.
      • Rousseau D.
      • Bonnafous S.
      • Patouraux S.
      • et al.
      ER stress induces NLRP3 inflammasome activation and hepatocyte death.
      In HFD-fed MUP-uPA mice, daily TUDCA (250 mg/kg; i.p.) injections over four weeks limited hepatic steatosis and liver injury.
      • Nakagawa H.
      • Umemura A.
      • Taniguchi K.
      • Font-Burgada J.
      • Dhar D.
      • Ogata H.
      • et al.
      ER stress cooperates with hypernutrition to trigger TNF-dependent spontaneous HCC development.
      TUDCA and PBA have been approved by the FDA for the treatment of primary biliary cirrhosis and urea-cycle disorders respectively. TUDCA is currently in a phase II clinical trial for type 1 diabetes. While the direct mechanisms of action of 4-PBA and TUDCA have yet to be clearly defined, they have proven their potency in reducing ER stress in metabolic disorders and may represent a therapeutic option for the treatment of NAFLD.

      Pharmacological inhibitors of the UPR

      Screening studies have identified multiple compounds that target distinct UPR signalling members (reviewed in
      • Hetz C.
      • Chevet E.
      • Harding H.P.
      Targeting the unfolded protein response in disease.
      ). However, the efficacy and safety of most of these compounds remain to be investigated in murine models of NAFLD to confirm their therapeutic potential.
      While it remains to be further investigated in the context of hepatic disorders, the therapeutic targeting of the UPR pathways provides a promising approach to prevent or treat different stages of NAFLD.
      The dual functions of IRE1α, which possesses a catalytic core on its RNase domain and an ATP-binding pocket on its kinase domain, have the potential to be manipulated therapeutically. Small molecules inhibit IRE1α RNase activity, specifically blocking RIDD and XBP1 splicing, without affecting its kinase activity or ability to dimerise/oligomerise. Such compounds include salicylaldehyde-based RNase inhibitors exemplified by 4μ8c,
      • Lebeaupin C.
      • Vallée D.
      • Rousseau D.
      • Patouraux S.
      • Bonnafous S.
      • Adam G.
      • et al.
      Bax Inhibitor-1 protects from non-alcoholic steatohepatitis by limiting IRE1α signaling.
      • Tufanli O.
      • Telkoparan Akillilar P.
      • Acosta-Alvear D.
      • Kocaturk B.
      • Onat U.I.
      • Hamid S.M.
      • et al.
      Targeting IRE1 with small molecules counteracts progression of atherosclerosis.
      STF-083010
      • Lebeaupin C.
      • Vallée D.
      • Rousseau D.
      • Patouraux S.
      • Bonnafous S.
      • Adam G.
      • et al.
      Bax Inhibitor-1 protects from non-alcoholic steatohepatitis by limiting IRE1α signaling.
      • Lerner A.G.
      • Upton J.P.
      • Praveen P.V.
      • Ghosh R.
      • Nakagawa Y.
      • Igbaria A.
      • et al.
      IRE1alpha induces thioredoxin-interacting protein to activate the NLRP3 inflammasome and promote programmed cell death under irremediable ER stress.
      • Tufanli O.
      • Telkoparan Akillilar P.
      • Acosta-Alvear D.
      • Kocaturk B.
      • Onat U.I.
      • Hamid S.M.
      • et al.
      Targeting IRE1 with small molecules counteracts progression of atherosclerosis.
      • Kim S.
      • Joe Y.
      • Kim H.J.
      • Kim Y.S.
      • Jeong S.O.
      • Pae H.O.
      • et al.
      Endoplasmic reticulum stress-induced IRE1alpha activation mediates cross-talk of GSK-3beta and XBP-1 to regulate inflammatory cytokine production.
      and MKC-6688.
      • Mimura N.
      • Fulciniti M.
      • Gorgun G.
      • Tai Y.T.
      • Cirstea D.
      • Santo L.
      • et al.
      Blockade of XBP1 splicing by inhibition of IRE1alpha is a promising therapeutic option in multiple myeloma.
      Treatment of hepatic mouse macrophages with STF-083010 (30 µM) resulted in specific inhibition of tunicamycin (10 µg/ml)-induced XBP1 splicing and TNFα transcription.
      • Kim S.
      • Joe Y.
      • Kim H.J.
      • Kim Y.S.
      • Jeong S.O.
      • Pae H.O.
      • et al.
      Endoplasmic reticulum stress-induced IRE1alpha activation mediates cross-talk of GSK-3beta and XBP-1 to regulate inflammatory cytokine production.
      This study emphasised the importance of targeting ER stress-induced IRE1α RNase activation in the production of inflammatory cytokines, which could be applied to NASH. Pretreatment of both mouse and human macrophages with either STF-083010 (150 µM) or 4μ8c (100 µM) prevented the synergistic effects of palmitate-induced ER stress coupled with LPS-induced inflammasome activation.
      • Tufanli O.
      • Telkoparan Akillilar P.
      • Acosta-Alvear D.
      • Kocaturk B.
      • Onat U.I.
      • Hamid S.M.
      • et al.
      Targeting IRE1 with small molecules counteracts progression of atherosclerosis.
      In vivo administration of either IRE1α RNase inhibitors STF-083010 or 4µ8c (10 mg/kg/day; i.p.; 4–6 weeks) in foz/foz mice fed a western-type diet counteracted atherosclerotic plaque formation and metaflammation without inducing hepatic toxicity.
      • Tufanli O.
      • Telkoparan Akillilar P.
      • Acosta-Alvear D.
      • Kocaturk B.
      • Onat U.I.
      • Hamid S.M.
      • et al.
      Targeting IRE1 with small molecules counteracts progression of atherosclerosis.
      Similarly, administration of STF-083010 or 4µ8c (30 mg/kg/biweekly; i.p.) in the last two weeks of a three-month HFD rescued BI-1-deficient mice, vulnerable to IRE1α RNase hyperactivation, from NASH development.
      • Lebeaupin C.
      • Vallée D.
      • Rousseau D.
      • Patouraux S.
      • Bonnafous S.
      • Adam G.
      • et al.
      Bax Inhibitor-1 protects from non-alcoholic steatohepatitis by limiting IRE1α signaling.
      It was further shown that IRE1α RNase function could be modulated through its kinase function by ATP-competitive inhibitors, such as APY29.
      • Korennykh A.V.
      • Egea P.F.
      • Korostelev A.A.
      • Finer-Moore J.
      • Zhang C.
      • Shokat K.M.
      • et al.
      The unfolded protein response signals through high-order assembly of Ire1.
      or the new pharmacological class of inhibitors called kinase-inhibiting RNase attenuators
      • Wang L.
      • Perera B.G.
      • Hari S.B.
      • Bhhatarai B.
      • Backes B.J.
      • Seeliger M.A.
      • et al.
      Divergent allosteric control of the IRE1alpha endoribonuclease using kinase inhibitors.
      with therapeutic potential in diabetes.
      • Ghosh R.
      • Wang L.
      • Wang E.S.
      • Perera B.G.
      • Igbaria A.
      • Morita S.
      • et al.
      Allosteric inhibition of the IRE1alpha RNase preserves cell viability and function during endoplasmic reticulum stress.
      • Morita S.
      • Villalta S.A.
      • Feldman H.C.
      • Register A.C.
      • Rosenthal W.
      • Hoffmann-Petersen I.T.
      • et al.
      Targeting ABL-IRE1alpha signaling spares ER-stressed pancreatic beta cells to reverse autoimmune diabetes.
      ASK1 is an intermediary of the IRE1α-JNK1 signalling pathway that is associated with hepatic inflammation, apoptosis and fibrosis, but can be targeted by the selective inhibitor selonsertib. In a multicentre phase II trial, 72 patients with NASH and liver fibrosis were randomised to receive 24 weeks of treatment with either selonsertib (GS-4997; 6 or 18 mg/day; orally), with or without simtuzumab (125 mg/week; subcutaneous injection) a monoclonal antibody directed against collagen and elastin cross-linking enzyme, or simtuzumab alone.
      • Loomba R.
      • Lawitz E.
      • Mantry P.S.
      • Jayakumar S.
      • Caldwell S.H.
      • Arnold H.
      • et al.
      The ASK1 inhibitor selonsertib in patients with nonalcoholic steatohepatitis: A randomized, phase 2 trial.
      Patients with NASH treated with a combination of selonsertib and simtuzumab exhibited improved collagen content and lobular inflammation as well as reduced serum biomarkers of apoptosis and necrosis.
      • Loomba R.
      • Lawitz E.
      • Mantry P.S.
      • Jayakumar S.
      • Caldwell S.H.
      • Arnold H.
      • et al.
      The ASK1 inhibitor selonsertib in patients with nonalcoholic steatohepatitis: A randomized, phase 2 trial.
      Compounds that modulate the PERK pathway primarily through eIF2α have also been tested. Salubrinal prevents eIF2α dephosphorylation thereby maintaining inhibition of protein translation and resulting in a reduced protein load on the ER. In HepG2 cells incubated with tunicamcyin (1.25 µg/ml) for 48 h, co-treatment with salubrinal (50 µM) significantly improved hepatocyte viability.
      • Vandewynckel Y.-P.
      • Laukens D.
      • Bogaerts E.
      • Paridaens A.
      • Van den Bussche A.
      • Verhelst X.
      • et al.
      Modulation of the unfolded protein response impedes tumor cell adaptation to proteotoxic stress: a PERK for hepatocellular carcinoma therapy.
      Similarly, guanabenz promotes persistent eIF2α phosphorylation in ER stress conditions by selectively binding to GADD34.
      • Tsaytler P.
      • Harding H.P.
      • Ron D.
      • Bertolotti A.
      Selective inhibition of a regulatory subunit of protein phosphatase 1 restores proteostasis.
      However, excessive inhibition of global translation may have undesirable effects in the long-term. Conversely, integrated stress response inhibitor was shown to inhibit the effects of eIF2α phosphorylation.
      • Sidrauski C.
      • McGeachy A.M.
      • Ingolia N.T.
      • Walter P.
      The small molecule ISRIB reverses the effects of eIF2alpha phosphorylation on translation and stress granule assembly.
      Compounds that directly modulate ATF6α expression or activity are scarce. Recently, high-throughput screening has identified small molecule inhibitors of ATF6α called Ceapins that trap the full-length protein in ER-resident foci, preventing ATF6α from ER stress-induced trafficking to the Golgi and subsequent proteolytic activation.
      • Gallagher C.M.
      • Garri C.
      • Cain E.L.
      • Ang K.K.
      • Wilson C.G.
      • Chen S.
      • et al.
      Ceapins are a new class of unfolded protein response inhibitors, selectively targeting the ATF6alpha branch.
      The testing of these compounds that inhibit IRE1α, PERK and/or ATF6 is still limited and has yet to be explored in the context of NAFL/NASH.

      Gene therapy

      The UPR has a central role in maintaining the physiology of various organs and specialised secretory cells.
      • Dufey E.
      • Sepulveda D.
      • Rojas-Rivera D.
      • Hetz C.
      Cellular mechanisms of endoplasmic reticulum stress signaling in health and disease. 1. An overview.
      This is why the long-term administration of UPR targeting drugs is predicted to cause serious side effects.
      • Hetz C.
      • Chevet E.
      • Harding H.P.
      Targeting the unfolded protein response in disease.
      Gene therapy is emerging as an alternative approach to target the UPR in a tissue specific manner.
      • Valenzuela V.
      • Jackson K.L.
      • Sardi S.P.
      • Hetz C.
      Gene therapy strategies to restore er proteostasis in disease.
      Promising results have featured gene therapy as a novel approach to traditional therapies in order to combat chronic liver disease. Hepatic overexpression of GRP78 improves the UPR dynamics, reducing steatosis associated with obesity.
      • Kammoun H.L.
      • Chabanon H.
      • Hainault I.
      • Luquet S.
      • Magnan C.
      • Koike T.
      • et al.
      GRP78 expression inhibits insulin and ER stress-induced SREBP-1c activation and reduces hepatic steatosis in mice.
      • Nakagawa H.
      • Umemura A.
      • Taniguchi K.
      • Font-Burgada J.
      • Dhar D.
      • Ogata H.
      • et al.
      ER stress cooperates with hypernutrition to trigger TNF-dependent spontaneous HCC development.
      Recently, the hepatoprotective effects of GRP78 in obese mice were proposed to act through a negative feedback loop that suppresses UPR activity (by suppressing ATF6α) but stimulates the degradation of its own mRNA (by perpetuating IRE1α’s RIDD function).
      • Gomez J.A.
      • Rutkowski D.T.
      Experimental reconstitution of chronic ER stress in the liver reveals feedback suppression of BiP mRNA expression.
      Interestingly, chronic ER stress may be marked by cycles of activation and deactivation that suppress UPR-dependent transcription, but potentially maintain RIDD. The inhibition of IRE1α by adenoviral gene transfer of BI-1 in genetically and diet-induced obese mice significantly improved hepatic glucose metabolism and insulin sensitivity.
      • Bailly-Maitre B.
      • Belgardt B.F.
      • Jordan S.D.
      • Coornaert B.
      • von Freyend M.J.
      • Kleinridders A.
      • et al.
      Hepatic Bax inhibitor-1 inhibits IRE1alpha and protects from obesity-associated insulin resistance and glucose intolerance.
      BI-1 could prevent ER stress-associated CYP2E1 activation which leads to oxidative stress.
      • Lee G.H.
      • Oh K.J.
      • Kim H.R.
      • Han H.S.
      • Lee H.Y.
      • Park K.G.
      • et al.
      Effect of BI-1 on insulin resistance through regulation of CYP2E1.
      BI-1 could also inhibit hepatic lipid accumulation through the regulation of ApoB secretion,
      • Lee H.Y.
      • Lee G.H.
      • Bhattarai K.R.
      • Park B.H.
      • Koo S.H.
      • Kim H.R.
      • et al.
      Bax Inhibitor-1 regulates hepatic lipid accumulation via ApoB secretion.
      although this remains controversial. In livers of ob/ob mice, ectopic overexpression of XBP1s improved glucose metabolism and insulin sensitivity by binding Foxo1.
      • Zhou Y.
      • Lee J.
      • Reno C.M.
      • Sun C.
      • Park S.W.
      • Chung J.
      • et al.
      Regulation of glucose homeostasis through a XBP-1-FoxO1 interaction.
      While XBP1s overexpression may protect against hepatic insulin resistance, it cannot be excluded that it may increase hepatic steatosis due to its function in activating the lipogenic machinery.

      Current and potential therapies for the treatment of HCC

      The oral multitargeted tyrosine kinase inhibitor sorafenib is the only FDA-approved medication for patients with advanced HCC. Because of genetic heterogeneity, some HCC cells are initially resistant to sorafenib, increasing the interest in a combinatorial treatment strategy with the small molecules mentioned previously. Of course, the intention of such treatment in this case would be to override the adaptive response of cancerous cells, leading to their eradication. In that sense, the use of chemical chaperones (TUDCA, 4-PBA) in HCC may be questionable as the targeted increase in the adaptive vs. terminal UPR may actually promote cancer cell survival.
      Specific targeting of the UPR branches is still under investigation.
      • Vandewynckel Y.-P.
      • Laukens D.
      • Bogaerts E.
      • Paridaens A.
      • Van den Bussche A.
      • Verhelst X.
      • et al.
      Modulation of the unfolded protein response impedes tumor cell adaptation to proteotoxic stress: a PERK for hepatocellular carcinoma therapy.
      • Chen K.F.
      • Tai W.T.
      • Liu T.H.
      • Huang H.P.
      • Lin Y.C.
      • Shiau C.W.
      • et al.
      Sorafenib overcomes TRAIL resistance of hepatocellular carcinoma cells through the inhibition of STAT3.
      • Won J.K.
      • Yu S.J.
      • Hwang C.Y.
      • Cho S.H.
      • Park S.M.
      • Kim K.
      • et al.
      Protein disulfide isomerase inhibition synergistically enhances the efficacy of sorafenib for hepatocellular carcinoma.
      • Yu S.J.
      • Yoon J.H.
      • Yang J.I.
      • Cho E.J.
      • Kwak M.S.
      • Jang E.S.
      • et al.
      Enhancement of hexokinase II inhibitor-induced apoptosis in hepatocellular carcinoma cells via augmenting ER stress and anti-angiogenesis by protein disulfide isomerase inhibition.
      • Axten J.M.
      • Romeril S.P.
      • Shu A.
      • Ralph J.
      • Medina J.R.
      • Feng Y.
      • et al.
      Discovery of GSK2656157: an optimized perk inhibitor selected for preclinical development.
      The IRE1α-ASK1-JNK pathway is at the crossroads of ER stress and cell death. ASK1-deficient mice are more susceptible to DEN-induced HCC, and the reintroduction of ASK1 by adenoviral vector restored the JNK-BIM pathways of hepatocyte death.
      • Nakagawa H.
      • Hirata Y.
      • Takeda K.
      • Hayakawa Y.
      • Sato T.
      • Kinoshita H.
      • et al.
      Apoptosis signal-regulating kinase 1 inhibits hepatocarcinogenesis by controlling the tumor-suppressing function of stress-activated mitogen-activated protein kinase.
      ASK1 overexpression was further shown to eliminate the tumorigenicity of subcutaneous HCC xenografts in nude mice.
      • Jiang C.-F.
      • Wen L.-Z.
      • Yin C.
      • Xu W.-P.
      • Shi B.
      • Zhang X.
      • et al.
      Apoptosis signal-regulating kinase 1 mediates the inhibitory effect of hepatocyte nuclear factor-4α on hepatocellular carcinoma.
      Concerning a potential strategy aimed at PERK inhibition in the liver, 25 weeks following DEN injection, mice treated with GSK2656157 (100 mg/kg/bidaily; i.p.) for four weeks exhibited greater ER-stress-driven cell death and HCC regression via proteotoxicity.
      • Vandewynckel Y.-P.
      • Laukens D.
      • Bogaerts E.
      • Paridaens A.
      • Van den Bussche A.
      • Verhelst X.
      • et al.
      Modulation of the unfolded protein response impedes tumor cell adaptation to proteotoxic stress: a PERK for hepatocellular carcinoma therapy.
      However, the selectivity profile of GSK2656157 has been questioned because of its potency as a RIPK1 inhibitor. No reported studies have targeted the ATF6 branch in HCC, probably because of the lack of availability of specific inhibitors working in vivo.
      Another promising therapeutic target involves the ER protein PDI. Aggressive pathological features of HCC and poor clinical outcome are associated with upregulated PDI expression, as seen in HCC tissues from patients.
      • Won J.K.
      • Yu S.J.
      • Hwang C.Y.
      • Cho S.H.
      • Park S.M.
      • Kim K.
      • et al.
      Protein disulfide isomerase inhibition synergistically enhances the efficacy of sorafenib for hepatocellular carcinoma.
      • Yu S.J.
      • Won J.K.
      • Ryu H.S.
      • Choi W.M.
      • Cho H.
      • Cho E.J.
      • et al.
      A novel prognostic factor for hepatocellular carcinoma: protein disulfide isomerase.
      A combinatorial daily treatment of sorafenib (30 mg/kg; i.p.) and a PDI inhibitor (PACMA 31; 20 mg/kg; i.p.) in a Hep3B xenograft model reduced HCC tumour volume by improving the efficiency of sorafenib in inducing proteotoxic stress, leading to apoptosis through JNK and CHOP induction.
      • Won J.K.
      • Yu S.J.
      • Hwang C.Y.
      • Cho S.H.
      • Park S.M.
      • Kim K.
      • et al.
      Protein disulfide isomerase inhibition synergistically enhances the efficacy of sorafenib for hepatocellular carcinoma.
      Another PDI inhibitor, bacitracin (10, 50 or 100 mg/kg/day; i.m.; 12 days, enhanced HK II inhibitor-induced anti-tumour efficacy synergistically by activating JNK, leading to apoptosis and limiting angiogenesis in mouse models of HCC.
      • Yu S.J.
      • Yoon J.H.
      • Yang J.I.
      • Cho E.J.
      • Kwak M.S.
      • Jang E.S.
      • et al.
      Enhancement of hexokinase II inhibitor-induced apoptosis in hepatocellular carcinoma cells via augmenting ER stress and anti-angiogenesis by protein disulfide isomerase inhibition.
      Pharmacological agents may conditionally enhance oncolytic viral efficacy. Small-molecule valosin-containing protein inhibitors cooperate with M1 virus by suppressing the IRE1α-XBP1 pathway and promoting irresolvable ER stress which kills HCC cells.
      • Zhang H.
      • Li K.
      • Lin Y.
      • Xing F.
      • Xiao X.
      • Cai J.
      • et al.
      Targeting VCP enhances anticancer activity of oncolytic virus M1 in hepatocellular carcinoma.
      This study also presented the favourable outcomes of this combinatorial therapeutic strategy in nonhuman primates, encouraging the rapid clinical translation of such therapies.
      Certain natural compounds may modulate the ER stress-related pro-apoptotic pathway in HCC. Melatonin and resveratrol have been shown to act as pro-apoptotic agents by initiating a terminal ER stress response in HCC cells through CHOP.
      • Moreira A.J.
      • Ordonez R.
      • Cerski C.T.
      • Picada J.N.
      • Garcia-Palomo A.
      • Marroni N.P.
      • et al.
      Melatonin activates endoplasmic reticulum stress and apoptosis in rats with diethylnitrosamine-induced hepatocarcinogenesis.