TNFAIP3 Interacting Protein 3 Overexpression Suppresses Nonalcoholic Steatohepatitis by Blocking TAK1 Activation.
Liu Dan,Zhang Peng,Zhou Junjie,Liao Rufang,Che Yan,Gao Mao-Mao,Sun Jiaqi,Cai Jingjing,Cheng Xu,Huang Yongping,Chen Guopeng,Nie Hongyu,Ji Yan-Xiao,Zhang Xiao-Jing,Huang Zan,Xu Haibo,She Zhi-Gang,Li Hongliang
Nonalcoholic steatohepatitis (NASH) is an unmet clinical challenge due to the rapid increase in its occurrence but the lack of approved drugs to treat it. Further unraveling of the molecular mechanisms underlying NASH may identify potential successful drug targets for this condition. Here, we identified TNFAIP3 interacting protein 3 (TNIP3) as a novel inhibitor of NASH. Hepatocyte-specific TNIP3 transgenic overexpression attenuates NASH in two dietary models in mice. Mechanistically, this inhibitory effect of TNIP3 is independent of its conventional role as an inhibitor of TNFAIP3. Rather, TNIP3 directly interacts with TAK1 and inhibits its ubiquitination and activation by the E3 ligase TRIM8 in hepatocytes in response to metabolic stress. Notably, adenovirus-mediated TNIP3 expression in the liver substantially blocks NASH progression in mice. These results suggest that TNIP3 may be a promising therapeutic target for NASH management.
The E3 ligase tripartite motif 8 targets TAK1 to promote insulin resistance and steatohepatitis.
Yan Feng-Juan,Zhang Xiao-Jing,Wang Wen-Xin,Ji Yan-Xiao,Wang Pi-Xiao,Yang Yang,Gong Jun,Shen Li-Jun,Zhu Xue-Yong,Huang Zan,Li Hongliang
Hepatology (Baltimore, Md.)
Tripartite motif 8 (TRIM8), an E3 ligase ubiquitously expressed in various cells, is closely involved in innate immunity. However, its role in nonalcoholic steatohepatitis is largely unknown. Here, we report evidence that TRIM8 is a robust enhancer of steatohepatitis and its complications induced by a high-fat diet or a genetic deficiency (ob/ob). Using gain-of-function and loss-of-function approaches, we observed dramatic exacerbation of insulin resistance, hepatic steatosis, inflammation, and fibrosis by hepatocyte-specific TRIM8 overexpression, whereas deletion or down-regulation of TRIM8 in hepatocytes led to a completely opposite phenotype. Furthermore, investigations of the underlying mechanisms revealed that TRIM8 directly binds to and ubiquitinates transforming growth factor-beta-activated kinase 1, thus promoting its phosphorylation and the activation of downstream c-Jun N-terminal kinase/p38 and nuclear factor κB signaling. Importantly, the participation of TRIM8 in human nonalcoholic fatty liver disease and nonalcoholic steatohepatitis was verified on the basis of its dramatically increased expression in the livers of these patients, suggesting a promising development of TRIM8 disturbance for the treatment of nonalcoholic steatohepatitis-related metabolic disorders. CONCLUSION:The E3 ligase TRIM8 is a potent regulator that exacerbates steatohepatitis and metabolic disorders dependent on its binding and ubiquitinating capacity on transforming growth factor-beta-activated kinase 1. (Hepatology 2017;65:1492-1511).
Hepatocyte TAZ/WWTR1 Promotes Inflammation and Fibrosis in Nonalcoholic Steatohepatitis.
Wang Xiaobo,Zheng Ze,Caviglia Jorge Matias,Corey Kathleen E,Herfel Tina M,Cai Bishuang,Masia Ricard,Chung Raymond T,Lefkowitch Jay H,Schwabe Robert F,Tabas Ira
Nonalcoholic steatohepatitis (NASH) is a leading cause of liver disease worldwide. However, the molecular basis of how benign steatosis progresses to NASH is incompletely understood, which has limited the identification of therapeutic targets. Here we show that the transcription regulator TAZ (WWTR1) is markedly higher in hepatocytes in human and murine NASH liver than in normal or steatotic liver. Most importantly, silencing of hepatocyte TAZ in murine models of NASH prevented or reversed hepatic inflammation, hepatocyte death, and fibrosis, but not steatosis. Moreover, hepatocyte-targeted expression of TAZ in a model of steatosis promoted NASH features, including fibrosis. In vitro and in vivo mechanistic studies revealed that a key mechanism linking hepatocyte TAZ to NASH fibrosis is TAZ/TEA domain (TEAD)-mediated induction of Indian hedgehog (Ihh), a secretory factor that activates fibrogenic genes in hepatic stellate cells. In summary, TAZ represents a previously unrecognized factor that contributes to the critical process of steatosis-to-NASH progression.
Targeting CASP8 and FADD-like apoptosis regulator ameliorates nonalcoholic steatohepatitis in mice and nonhuman primates.
Wang Pi-Xiao,Ji Yan-Xiao,Zhang Xiao-Jing,Zhao Ling-Ping,Yan Zhen-Zhen,Zhang Peng,Shen Li-Jun,Yang Xia,Fang Jing,Tian Song,Zhu Xue-Yong,Gong Jun,Zhang Xin,Wei Qiao-Fang,Wang Yong,Li Jing,Wan Lu,Xie Qingguo,She Zhi-Gang,Wang Zhihua,Huang Zan,Li Hongliang
Nonalcoholic steatohepatitis (NASH) is a progressive disease that is often accompanied by metabolic syndrome and poses a high risk of severe liver damage. However, no effective pharmacological treatment is currently available for NASH. Here we report that CASP8 and FADD-like apoptosis regulator (CFLAR) is a key suppressor of steatohepatitis and its metabolic disorders. We provide mechanistic evidence that CFLAR directly targets the kinase MAP3K5 (also known as ASK1) and interrupts its N-terminus-mediated dimerization, thereby blocking signaling involving ASK1 and the kinase MAPK8 (also known as JNK1). Furthermore, we identified a small peptide segment in CFLAR that effectively attenuates the progression of steatohepatitis and metabolic disorders in both mice and monkeys by disrupting the N-terminus-mediated dimerization of ASK1 when the peptide is expressed from an injected adenovirus-associated virus 8-based vector. Taken together, these findings establish CFLAR as a key suppressor of steatohepatitis and indicate that the development of CFLAR-peptide-mimicking drugs and the screening of small-molecular inhibitors that specifically block ASK1 dimerization are new and feasible approaches for NASH treatment.
USP18 protects against hepatic steatosis and insulin resistance through its deubiquitinating activity.
An Shimin,Zhao Ling-Ping,Shen Li-Jun,Wang Siyuan,Zhang Kuo,Qi Yu,Zheng Jilin,Zhang Xiao-Jing,Zhu Xue-Yong,Bao Rong,Yang Ling,Lu Yue-Xin,She Zhi-Gang,Tang Yi-Da
Hepatology (Baltimore, Md.)
Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis, impaired insulin sensitivity, and chronic low-grade inflammation. However, the pathogenic mechanism of NAFLD is poorly understood, which hinders the exploration of possible treatments. Here, we report that ubiquitin-specific protease 18 (USP18), a member of the deubiquitinating enzyme family, plays regulatory roles in NAFLD progression. Expression of USP18 was down-regulated in the livers of nonalcoholic steatohepatitis patients and high-fat diet (HFD)-induced or genetically obese mice. When challenged with HFD, hepatocyte-specific USP18 transgenic mice exhibited improved lipid metabolism and insulin sensitivity, whereas mice knocked out of USP18 expression showed adverse trends regarding hepatic steatosis and glucose metabolic disorders. Furthermore, the concomitant inflammatory response was suppressed in USP18-hepatocyte-specific transgenic mice and promoted in USP18-hepatocyte-specific knockout mice treated with HFD. Mechanistically, hepatocyte USP18 ameliorates hepatic steatosis by interacting with and deubiquitinating transforming growth factorβ-activated kinase 1 (TAK1), which inhibits TAK1 activation and subsequently suppresses the downstream c-Jun N-terminal kinase and nuclear factor kappa B signaling pathways. This is further validated by alleviated steatotic phenotypes and highly activated insulin signaling in HFD-fed USP18-hepatocyte-specific knockout mice administered a TAK1 inhibitor. The therapeutic effect of USP18 on NAFLD relies on its deubiquitinating activity because HFD-fed mice injected with active-site mutant USP18 failed to inhibit hepatic steatosis. CONCLUSION:USP18 associates with and deubiquitinates TAK1 to protect against hepatic steatosis, insulin resistance, and the inflammatory response. (Hepatology 2017;66:1866-1884).
Nonalcoholic Steatohepatitis (NASH) and Hepatic Fibrosis: Emerging Therapies.
Wattacheril Julia,Issa Danny,Sanyal Arun
Annual review of pharmacology and toxicology
Nonalcoholic fatty liver disease remains a major cause of liver-related morbidity and mortality worldwide. It is a complex disease associated with obesity, diabetes, and dyslipidemia but is increasingly recognized in normal-weight individuals. Its progressive inflammatory phenotype, nonalcoholic steatohepatitis (NASH), currently has no effective treatment apart from lifestyle interventions. Multiple pathogenic pathways are involved in disease progression, and targets for intervention have been identified. These targets mediate glucose, lipid, and bile acid metabolism; inflammation; apoptosis; and fibrosis. Novel therapeutic agents are being developed in each of these pathways, and several have shown promise in early phase testing. Given the complexity of the disease, intervention trials are large and long and require histologic confirmation as a primary endpoint for disease improvement or regression. We highlight active Phase 2 and 3 therapeutic trials for NASH as this field rapidly expands in development.
Hepatocyte DUSP14 maintains metabolic homeostasis and suppresses inflammation in the liver.
Wang Siyuan,Yan Zhen-Zhen,Yang Xia,An Shimin,Zhang Kuo,Qi Yu,Zheng Jilin,Ji Yan-Xiao,Wang Pi-Xiao,Fang Chun,Zhu Xue-Yong,Shen Li-Jun,Yan Feng-Juan,Bao Rong,Tian Song,She Zhi-Gang,Tang Yi-Da
Hepatology (Baltimore, Md.)
Nonalcoholic fatty liver disease (NAFLD) is a prevalent and complex disease that confers a high risk of severe liver disorders. Despite such public and clinical health importance, very few effective therapies are currently available for NAFLD. We report a protective function and the underlying mechanism of dual-specificity phosphatase 14 (DUSP14) in NAFLD and related metabolic disorders. Insulin resistance, hepatic lipid accumulation, and concomitant inflammatory responses, key pathological processes involved in NAFLD development, were significantly ameliorated by hepatocyte-specific DUSP14 overexpression (DUSP14-HTG) in high-fat diet (HFD)-induced or genetically obese mouse models. By contrast, specific DUSP14 deficiency in hepatocytes (DUSP14-HKO) aggravated these pathological alterations. We provided mechanistic evidence that DUSP14 directly binds to and dephosphorylates transforming growth factor β-activated kinase 1 (TAK1), resulting in the reduced activation of TAK1 and its downstream signaling molecules c-Jun N-terminal kinase 1 (JNK), p38, and nuclear factor kappa B NF-κB. This effect was further evidenced by the finding that inhibiting TAK1 activity effectively attenuated the deterioration of glucolipid metabolic phenotype in DUSP14-HKO mice challenged by HFD administration. Furthermore, we identified that both the binding domain and the phosphatase activity of DUSP14 are required for its protective role against hepatic steatosis, because interruption of the DUSP14-TAK1 interaction abolished the mitigative effects of DUSP14. CONCLUSION:Hepatocyte DUSP14 is required for maintaining hepatic metabolic homeostasis and for suppressing inflammation, a novel function that relies on constraining TAK1 hyperactivation. (Hepatology 2018;67:1320-1338).
Hepatic neuregulin 4 signaling defines an endocrine checkpoint for steatosis-to-NASH progression.
Guo Liang,Zhang Peng,Chen Zhimin,Xia Houjun,Li Siming,Zhang Yanqiao,Kobberup Sune,Zou Weiping,Lin Jiandie D
The Journal of clinical investigation
Nonalcoholic steatohepatitis (NASH) is characterized by progressive liver injury, inflammation, and fibrosis; however, the mechanisms that govern the transition from hepatic steatosis, which is relatively benign, to NASH remain poorly defined. Neuregulin 4 (Nrg4) is an adipose tissue-enriched endocrine factor that elicits beneficial metabolic effects in obesity. Here, we show that Nrg4 is a key component of an endocrine checkpoint that preserves hepatocyte health and counters diet-induced NASH in mice. Nrg4 deficiency accelerated liver injury, fibrosis, inflammation, and cell death in a mouse model of NASH. In contrast, transgenic expression of Nrg4 in adipose tissue alleviated diet-induced NASH. Nrg4 attenuated hepatocyte death in a cell-autonomous manner by blocking ubiquitination and proteasomal degradation of c-FLIPL, a negative regulator of cell death. Adeno-associated virus-mediated (AAV-mediated) rescue of hepatic c-FLIPL expression in Nrg4-deficent mice functionally restored the brake for steatosis to NASH transition. Thus, hepatic Nrg4 signaling serves as an endocrine checkpoint for steatosis-to-NASH progression by activating a cytoprotective pathway to counter stress-induced liver injury.
Preclinical models of non-alcoholic fatty liver disease.
Santhekadur Prasanna K,Kumar Divya P,Sanyal Arun J
Journal of hepatology
Non-alcoholic fatty liver disease (NAFLD) can manifest as non-alcoholic fatty liver (NAFL) or non-alcoholic steatohepatitis (NASH). NASH is often associated with progressive fibrosis which can lead to cirrhosis and hepatocellular carcinoma (HCC). NASH is increasing as an aetiology for end-stage liver disease as well as HCC. There are currently no approved therapies for NASH. A major barrier to development of therapeutics for NASH is the lack of preclinical models of disease that are appropriately validated to represent the biology and outcomes of human disease. Many in vitro and animal models have been developed. In vitro models do not fully capture the hepatic and extrahepatic milieu of human NASH and large animal models are expensive and logistically difficult to use. Therefore, there is considerable interest in the development and validation of mouse models for NAFLD, including NASH. Several models based on varying genetic or dietary manipulations have been developed. However, the majority do not recreate steatohepatitis, strictly defined as the presence of hepatocellular ballooning with or without Mallory-Denk bodies, accompanied by inflammation in the presence of macrovesicular steatosis. Others lack validation against human disease. Herein, we describe the best practices in development of mouse models of NASH. We further review existing models and the literature supporting their use as a surrogate for human disease. Finally, data on models to evaluate protective genes are discussed. It is hoped that this review will provide guidance for the interpretation of data derived from mouse models and also for the development and validation of newer models.
The deubiquitinating enzyme TNFAIP3 mediates inactivation of hepatic ASK1 and ameliorates nonalcoholic steatohepatitis.
Zhang Peng,Wang Pi-Xiao,Zhao Ling-Ping,Zhang Xin,Ji Yan-Xiao,Zhang Xiao-Jing,Fang Chun,Lu Yue-Xin,Yang Xia,Gao Mao-Mao,Zhang Yan,Tian Song,Zhu Xue-Yong,Gong Jun,Ma Xin-Liang,Li Feng,Wang Zhihua,Huang Zan,She Zhi-Gang,Li Hongliang
Activation of apoptosis signal-regulating kinase 1 (ASK1) in hepatocytes is a key process in the progression of nonalcoholic steatohepatitis (NASH) and a promising target for treatment of the condition. However, the mechanism underlying ASK1 activation is still unclear, and thus the endogenous regulators of this kinase remain open to be exploited as potential therapeutic targets. In screening for proteins that interact with ASK1 in the context of NASH, we identified the deubiquitinase tumor necrosis factor alpha-induced protein 3 (TNFAIP3) as a key endogenous suppressor of ASK1 activation, and we found that TNFAIP3 directly interacts with and deubiquitinates ASK1 in hepatocytes. Hepatocyte-specific ablation of Tnfaip3 exacerbated nonalcoholic fatty liver disease- and NASH-related phenotypes in mice, including glucose metabolism disorders, lipid accumulation and enhanced inflammation, in an ASK1-dependent manner. In contrast, transgenic or adeno-associated virus-mediated TNFAIP3 gene delivery in the liver in both mouse and nonhuman primate models of NASH substantially blocked the onset and progression of the disease. These results implicate TNFAIP3 as a functionally important endogenous suppressor of ASK1 hyperactivation in the pathogenesis of NASH and identify it as a potential new molecular target for NASH therapy.
Gasdermin D plays a key role as a pyroptosis executor of non-alcoholic steatohepatitis in humans and mice.
Xu Bing,Jiang Mingzuo,Chu Yi,Wang Weijie,Chen Di,Li Xiaowei,Zhang Zhao,Zhang Di,Fan Daiming,Nie Yongzhan,Shao Feng,Wu Kaichun,Liang Jie
Journal of hepatology
BACKGROUND & AIMS:Gasdermin D (GSDMD)-executed programmed necrosis is involved in inflammation and controls interleukin (IL)-1β release. However, the role of GSDMD in non-alcoholic steatohepatitis (NASH) remains unclear. We investigated the role of GSDMD in the pathogenesis of steatohepatitis. METHODS:Human liver tissues from patients with non-alcoholic fatty liver disease (NAFLD) and control individuals were obtained to evaluate GSDMD expression. Gsdmd knockout (Gsdmd) mice, obese db/db mice and their wild-type (WT) littermates were fed with methionine-choline deficient (MCD) or control diet to induce steatohepatitis. The Gsdmd and WT mice were also used in a high-fat diet (HFD)-induced NAFLD model. In addition, Alb-Cre mice were administered an adeno-associated virus (AAV) vector that expressed the gasdermin-N domain (AAV9-FLEX-GSDMD-N) and were fed with either MCD or control diet for 10 days. RESULTS:GSDMD and its pyroptosis-inducing fragment GSDMD-N were upregulated in liver tissues of human NAFLD/NASH. Importantly, hepatic GSDMD-N protein levels were significantly higher in human NASH and correlated with the NAFLD activity score and fibrosis. GSDMD-N remained a potential biomarker for the diagnosis of NASH. MCD-fed Gsdmd mice exhibit decreased severity of steatosis and inflammation compared with WT littermates. GSDMD was associated with the secretion of pro-inflammatory cytokines (IL-1β, TNF-α, and MCP-1 [CCL2]) and persistent activation of the NF-ĸB signaling pathway. Gsdmd mice showed lower steatosis, mainly because of reduced expression of the lipogenic gene Srebp1c (Srebf1) and upregulated expression of lipolytic genes, including Pparα, Aco [Klk15], Lcad [Acadl], Cyp4a10 and Cyp4a14. Alb-Cre mice administered with AAV9-FLEX-GSDMD-N showed significantly aggravated steatohepatitis when fed with MCD diet. CONCLUSION:As an executor of pyroptosis, GSDMD plays a key role in the pathogenesis of steatohepatitis, by controlling cytokine secretion, NF-ĸB activation, and lipogenesis. LAY SUMMARY:Non-alcoholic fatty liver disease has become one of the most feared chronic liver diseases, because it is the most rapidly growing indication for adult liver transplantation and a major cause of hepatocellular carcinoma. However, the mechanisms involved in the transformation of simple steatosis to steatohepatitis remain unclear. Herein, we show that gasdermin D driven pyroptosis is prominent in patients with non-alcoholic steatohepatitis (NASH), and gasdermin-N domain remains a potential biomarker for the diagnosis of NASH. Gasdermin D plays a key role in the pathogenesis of NASH by regulating lipogenesis, the inflammatory response, and the NF-ĸB signaling pathway, revealing potential treatment targets for NASH in humans.
The deubiquitinating enzyme cylindromatosis mitigates nonalcoholic steatohepatitis.
Ji Yan-Xiao,Huang Zan,Yang Xia,Wang Xiaozhan,Zhao Ling-Ping,Wang Pi-Xiao,Zhang Xiao-Jing,Alves-Bezerra Michele,Cai Lin,Zhang Peng,Lu Yue-Xin,Bai Lan,Gao Mao-Mao,Zhao Huan,Tian Song,Wang Yong,Huang Zhi-Xiang,Zhu Xue-Yong,Zhang Yan,Gong Jun,She Zhi-Gang,Li Feng,Cohen David E,Li Hongliang
Nonalcoholic steatohepatitis (NASH) is a common clinical condition that can lead to advanced liver diseases. Lack of effective pharmacotherapies for NASH is largely attributable to an incomplete understanding of its pathogenesis. The deubiquitinase cylindromatosis (CYLD) plays key roles in inflammation and cancer. Here we identified CYLD as a suppressor of NASH in mice and in monkeys. CYLD is progressively degraded upon interaction with the E3 ligase TRIM47 in proportion to NASH severity. We observed that overexpression of Cyld in hepatocytes concomitantly inhibits lipid accumulation, insulin resistance, inflammation and fibrosis in mice with NASH induced in an experimental setting. Mechanistically, CYLD interacts directly with the kinase TAK1 and removes its K63-linked polyubiquitin chain, which blocks downstream activation of the JNK-p38 cascades. Notably, reconstitution of hepatic CYLD expression effectively reverses disease progression in mice with dietary or genetically induced NASH and in high-fat diet-fed monkeys predisposed to metabolic syndrome. Collectively, our findings demonstrate that CYLD mitigates NASH severity and identify the CYLD-TAK1 axis as a promising therapeutic target for management of the disease.
Hsp72 protects against liver injury via attenuation of hepatocellular death, oxidative stress, and JNK signaling.
Levada Kateryna,Guldiken Nurdan,Zhang Xiaoji,Vella Giovanna,Mo Fa-Rong,James Laura P,Haybaeck Johannes,Kessler Sonja M,Kiemer Alexandra K,Ott Thomas,Hartmann Daniel,Hüser Norbert,Ziol Marianne,Trautwein Christian,Strnad Pavel
Journal of hepatology
BACKGROUND & AIMS:Heat shock protein (Hsp) 72 is a molecular chaperone that has broad cytoprotective functions and is upregulated in response to stress. To determine its hepatic functions, we studied its expression in human liver disorders and its biological significance in newly generated transgenic animals. METHODS:Double transgenic mice overexpressing Hsp72 (gene Hspa1a) under the control of a tissue-specific tetracycline-inducible system (Hsp72-LAP mice) were produced. Acute liver injury was induced by a single injection of acetaminophen (APAP). Feeding with either a methionine choline-deficient (MCD; 8 weeks) or a 3,5-diethoxycarbonyl-1,4-dihydrocollidine-supplemented diet (DDC; 12 weeks) was used to induce lipotoxic injury and Mallory-Denk body (MDB) formation, respectively. Primary hepatocytes were treated with palmitic acid. RESULTS:Patients with non-alcoholic steatohepatitis and chronic hepatitis C infection displayed elevated HSP72 levels. These levels increased with the extent of hepatic inflammation and HSP72 expression was induced after treatment with either interleukin (IL)-1β or IL-6. Hsp72-LAP mice exhibited robust, hepatocyte-specific Hsp72 overexpression. Primary hepatocytes from these animals were more resistant to isolation-induced stress and Hsp72-LAP mice displayed lower levels of hepatic injury in vivo. Mice overexpressing Hsp72 had fewer APAP protein adducts and were protected from oxidative stress and APAP-/MCD-induced cell death. Hsp72-LAP mice and/or hepatocytes displayed significantly attenuated Jnk activation. Overexpression of Hsp72 did not affect steatosis or the extent of MDB formation. CONCLUSIONS:Our results demonstrate that HSP72 induction occurs in human liver disease, thus, HSP72 represents an attractive therapeutic target owing to its broad hepatoprotective functions. LAY SUMMARY:HSP72 constitutes a stress-inducible, protective protein. Our data demonstrate that it is upregulated in patients with chronic hepatitis C and non-alcoholic steatohepatitis. Moreover, Hsp72-overexpressing mice are protected from various forms of liver stress.
Bax inhibitor-1 protects from nonalcoholic steatohepatitis by limiting inositol-requiring enzyme 1 alpha signaling in mice.
Lebeaupin Cynthia,Vallée Déborah,Rousseau Déborah,Patouraux Stéphanie,Bonnafous Stéphanie,Adam Gilbert,Luciano Frederic,Luci Carmelo,Anty Rodolphe,Iannelli Antonio,Marchetti Sandrine,Kroemer Guido,Lacas-Gervais Sandra,Tran Albert,Gual Philippe,Bailly-Maitre Béatrice
Hepatology (Baltimore, Md.)
Endoplasmic reticulum (ER) stress is activated in nonalcoholic fatty liver disease (NAFLD), raising the possibility that ER stress-dependent metabolic dysfunction, inflammation, and cell death underlie the transition from steatosis to steatohepatitis (nonalcoholic steatohepatitis; NASH). B-cell lymphoma 2 (BCL2)-associated X protein (Bax) inhibitor-1 (BI-1), a negative regulator of the ER stress sensor, inositol-requiring enzyme 1 alpha (IRE1α), has yet to be explored in NAFLD as a hepatoprotective agent. We hypothesized that the genetic ablation of BI-1 would render the liver vulnerable to NASH because of unrestrained IRE1α signaling. ER stress was induced in wild-type and BI-1 mice acutely by tunicamycin (TM) injection (1 mg/kg) or chronically by high-fat diet (HFD) feeding to determine NAFLD phenotype. Livers of TM-treated BI-1 mice showed IRE1α-dependent NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation, hepatocyte death, fibrosis, and dysregulated lipid homeostasis that led to liver failure within a week. The analysis of human NAFLD liver biopsies revealed BI-1 down-regulation parallel to the up-regulation of IRE1α endoribonuclease (RNase) signaling. In HFD-fed BI-1 mice that presented NASH and type 2 diabetes, exaggerated hepatic IRE1α, X-box binding protein 1 (XBP1), and C/EBP homologous protein (CHOP) expression was linked to activated NLRP3 inflammasome and caspase-1/-11. Rises in interleukin (IL)-1β, IL-6, monocyte chemoattractant protein 1 (MCP1), chemokine (C-X-C motif) ligand 1 (CXCL1), and alanine transaminase (ALT)/aspartate transaminase (AST) levels revealed significant inflammation and injury, respectively. Pharmacological inhibition of IRE1α RNase activity with the small molecules, STF-083010 or 4μ8c, was evaluated in HFD-induced NAFLD. In BI-1 mice, either treatment effectively counteracted IRE1α RNase activity, improving glucose tolerance and rescuing from NASH. The hepatocyte-specific role of IRE1α RNase activity in mediating NLRP3 inflammasome activation and cell death was confirmed in primary mouse hepatocytes by IRE1α axis knockdown or its inhibition with STF-083010 or 4μ8c. CONCLUSION:Targeting IRE1α-dependent NLRP3 inflammasome signaling with pharmacological agents or by BI-1 may represent a tangible therapeutic strategy for NASH. (Hepatology 2018).
Aortic carboxypeptidase-like protein, a WNT ligand, exacerbates nonalcoholic steatohepatitis.
Teratani Toshiaki,Tomita Kengo,Suzuki Takahiro,Furuhashi Hirotaka,Irie Rie,Nishikawa Makoto,Yamamoto Junji,Hibi Toshifumi,Miura Soichiro,Minamino Tohru,Oike Yuichi,Hokari Ryota,Kanai Takanori
The Journal of clinical investigation
Incidence of nonalcoholic steatohepatitis (NASH), which is considered a hepatic manifestation of metabolic syndrome, has been increasing worldwide with the rise in obesity; however, its pathological mechanism is poorly understood. Here, we demonstrate that the hepatic expression of aortic carboxypeptidase-like protein (ACLP), a glycosylated, secreted protein, increases in NASH in humans and mice. Furthermore, we elucidate that ACLP is a ligand, unrelated to WNT proteins, that activates the canonical WNT pathway and exacerbates NASH pathology. In the liver, ACLP is specifically expressed in hepatic stellate cells (HSCs). As fatty liver disease progresses, ACLP expression is enhanced via activation of STAT3 signaling by obesity-related factors in serum. ACLP specifically binds to frizzled-8 and low-density lipoprotein-related receptor 6 to form a ternary complex that activates canonical WNT signaling. Consequently, ACLP activates HSCs by inhibiting PPARγ signals. HSC-specific ACLP deficiency inhibits fibrosis progression in NASH by inhibiting canonical WNT signaling in HSCs. The present study elucidates the role of canonical WNT pathway activation by ACLP in NASH pathology, indicating that NASH can be treated by targeting ACLP-induced canonical WNT pathway activation in HSCs.
Ubiquitin-Specific Protease 4 Is an Endogenous Negative Regulator of Metabolic Dysfunctions in Nonalcoholic Fatty Liver Disease in Mice.
Zhao Yichao,Wang Fang,Gao Lingchen,Xu Longwei,Tong Renyang,Lin Nan,Su Yuanyuan,Yan Yang,Gao Yu,He Jie,Kong Lingcong,Yuan Ancai,Zhuge Ying,Pu Jun
Hepatology (Baltimore, Md.)
Nonalcoholic fatty liver disease (NAFLD), characterized by hepatic steatosis (HS), insulin resistance (IR), and inflammation, poses a high risk of cardiometabolic disorders. Ubiquitin specific protease 4 (USP4), a deubiquitinating enzyme, is pivotally involved in regulating multiple inflammatory pathways; however, the role of USP4 in NAFLD is unknown. Here, we report that USP4 expression was dramatically down-regulated in livers from NAFLD patients and different NAFLD mouse models induced by high-fat diet (HFD) or genetic deficiency (ob/ob) as well as in palmitate-treated hepatocytes. Hepatocyte-specific USP4 depletion exacerbated HS, IR, and inflammatory response in HFD-induced NAFLD mice. Conversely, hepatic USP4 overexpression notably alleviated the pathological alterations in two different NAFLD models. Mechanistically, hepatocyte USP4 directly bound to and deubiquitinated transforming growth factor-β activated kinase 1 (TAK1), leading to a suppression of the activation of downstream nuclear factor kappa B (NF-κB) and c-Jun N-terminal kinase (JNK) cascades, which, in turn, reversed the disruption of insulin receptor substrate/protein kinase B/glycogen synthase kinase 3 beta (IRS-AKT-GSK3β) signaling. In addition, USP4-TAK1 interaction and subsequent TAK1 deubiquitination were required for amelioration of metabolic dysfunctions. Conclusion: Collectively, the present study provides evidence that USP4 functions as a pivotal suppressor in NAFLD and related metabolic disorders. (Hepatology 2018; 00:000-000).
Ubiquitin-Specific Peptidase 10 (USP10) Inhibits Hepatic Steatosis, Insulin Resistance, and Inflammation Through Sirt6.
Luo Pengcheng,Qin Cong,Zhu Lihua,Fang Chun,Zhang Yan,Zhang Hai,Pei Fei,Tian Song,Zhu Xue-Yong,Gong Jun,Mao Qing,Xiao Chengcheng,Su Yang,Zheng Haizhou,Xu Tao,Lu Jingxiao,Zhang Jie
Hepatology (Baltimore, Md.)
Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis, insulin resistance and inflammation, and the pathogenic mechanism of NAFLD is poorly understood. Ubiquitin-specific peptidase 10 (USP10), a member of the ubiquitin-specific protease family, is involved in environmental stress responses, tumor growth, inflammation, and cellular metabolism. However, the role of USP10 in hepatic steatosis, insulin resistance, and inflammation remains largely unexplored. USP10 expression was detected in livers of patients with NAFLD, mice with high-fat diet (HFD)-induced obesity, and genetically obese (ob/ob) mice, as well as in palmitate-induced hepatocytes. The function of USP10 in hepatic steatosis, insulin resistance, and inflammation was investigated using hepatocyte-specific USP10 deficiency or overexpression in mice induced by HFD treatment or genetic defect. The molecular mechanisms underlying USP10-regulated hepatic steatosis were further investigated in HFD-treated mice. USP10 expression was significantly decreased in the fatty livers of NAFLD patients and obese mice and in palmitate-treated hepatocytes. USP10 deficiency exacerbated the metabolic dysfunction induced by HFD treatment for 12 weeks. Conversely, USP10 overexpression significantly suppressed metabolic dysfunction in mice after HFD treatment and inhibited the development of NAFLD in ob/ob mice. Further investigation indicated that USP10 regulates hepatic steatosis by interacting with Sirt6 and inhibiting its ubiquitination and degradation. Sirt6 overexpression markedly ameliorated the effects of USP10 deficiency in hepatic steatosis, insulin resistance, and inflammation. Conversely, Sirt6 deficiency decreased the ameliorative effects of USP10 overexpression in response to HFD treatment. Conclusion: USP10 inhibits hepatic steatosis, insulin resistance, and inflammation through Sirt6.
TRUSS Exacerbates NAFLD Development by Promoting IκBα Degradation in Mice.
Yu Chang-Jiang,Wang Qiu-Shi,Wu Ming-Ming,Song Bin-Lin,Liang Chen,Lou Jie,Tang Liang-Liang,Yu Xiao-Di,Niu Na,Yang Xu,Zhang Bao-Long,Qu Yao,Liu Yang,Dong Zhi-Chao,Zhang Zhi-Ren
Hepatology (Baltimore, Md.)
There is no effective treatment method for nonalcoholic fatty liver disease (NAFLD), the most common liver disease. The exact mechanism underlying the pathogenesis of NAFLD remains to be elucidated. Here, we report that tumor necrosis factor receptor-associated ubiquitous scaffolding and signaling protein (TRUSS) acts as a positive regulator of NAFLD and in a variety of metabolic disorders. TRUSS expression was increased in the human liver specimens with NAFLD or nonalcoholic steatohepatitis, and in the livers of high-fat diet (HFD)-induced and genetically obese mice. Conditional knockout of TRUSS in hepatocytes significantly ameliorated hepatic steatosis, insulin resistance, glucose intolerance, and inflammatory responses in mice after HFD challenge or in spontaneous obese mice with normal chow feeding. All of these HFD-induced pathological phenotypes were exacerbated in mice overexpressing TRUSS in hepatocytes. We show that TRUSS physically interacts with the inhibitor of nuclear factor κB α (IκBα) and promotes the ubiquitination and degradation of IκBα, which leads to aberrant activation of nuclear factor κB (NF-κB). Overexpressing IκBα , a phosphorylation-resistant mutant of IκBα, in the hepatocyte-specific TRUSS overexpressing mice almost abolished HFD-induced NAFLD and metabolic disorders. Conclusion: Hepatocyte TRUSS promotes pathological stimuli-induced NAFLD and metabolic disorders, through activation of NF-κB by promoting ubiquitination and degradation of IκBα. Our findings may provide a strategy for the prevention and treatment of NAFLD by targeting TRUSS.
CD36 palmitoylation disrupts free fatty acid metabolism and promotes tissue inflammation in non-alcoholic steatohepatitis.
Zhao Lei,Zhang Chang,Luo Xiaoxiao,Wang Pei,Zhou Wei,Zhong Shan,Xie Yunxia,Jiang Yibo,Yang Ping,Tang Renkuang,Pan Qin,Hall Andrew R,Luong Tu Vinh,Fan Jiangao,Varghese Zac,Moorhead John F,Pinzani Massimo,Chen Yaxi,Ruan Xiong Z
Journal of hepatology
BACKGROUND AND AIMS:Fatty acid translocase CD36 (CD36) is a membrane protein with multiple immuno-metabolic functions. Palmitoylation has been suggested to regulate the distribution and functions of CD36, but little is known about its significance in non-alcoholic steatohepatitis (NASH). METHODS:Human liver tissue samples were obtained from patients undergoing liver biopsy for diagnostic purposes. CD36 knockout mice were injected with lentiviral vectors expressing wild-type CD36 or CD36 with mutated palmitoylation sites. Liver histology, immunofluorescence, mRNA expression profile, subcellular distributions and functions of CD36 protein were assessed. RESULTS:The localization of CD36 on the plasma membrane of hepatocytes was markedly increased in patients with NASH compared to patients with normal liver and those with simple steatosis. Increased CD36 palmitoylation and increased localization of CD36 on the plasma membrane of hepatocytes were also observed in livers of mice with NASH. Furthermore, inhibition of CD36 palmitoylation protected mice from developing NASH. The absence of palmitoylation decreased CD36 protein hydrophobicity reducing its localization on the plasma membrane as well as in lipid raft of hepatocytes. Consequently, a lack of palmitoylation decreased fatty acid uptake and CD36/Fyn/Lyn complex in HepG2 cells. Inhibition of CD36 palmitoylation not only ameliorated intracellular lipid accumulation via activation of the AMPK pathway, but also inhibited the inflammatory response through the inhibition of the JNK signaling pathway. CONCLUSIONS:Our findings demonstrate the key role of palmitoylation in regulating CD36 distributions and its functions in NASH. Inhibition of CD36 palmitoylation may represent an effective therapeutic strategy in patients with NASH. LAY SUMMARY:Fatty acid translocase CD36 (CD36) is a multifunctional membrane protein which contributes to the development of liver steatosis. In the present study, we demonstrated that the localization of CD36 on the plasma membrane of hepatocytes is increased in patients with non-alcoholic steatohepatitis. Blocking the palmitoylation of CD36 reduces CD36 distribution in hepatocyte plasma membranes and protects mice from non-alcoholic steatohepatitis. The inhibition of CD36 palmitoylation not only improved fatty acid metabolic disorders but also reduced the inflammatory response in vitro and in vivo. The present study suggests that CD36 palmitoylation is important for non-alcoholic steatohepatitis development and inhibition of CD36 palmitoylation could be used to cure non-alcoholic steatohepatitis.
Caspase Recruitment Domain Protein 6 Protects Against Hepatic Steatosis and Insulin Resistance by Suppressing Apoptosis Signal-Regulating Kinase 1.
Sun Peng,Zeng Qiang,Cheng Daqing,Zhang Kuo,Zheng Jilin,Liu Yupeng,Yuan Yu-Feng,Tang Yi-Da
Hepatology (Baltimore, Md.)
The rapidly increasing prevalence of metabolic disorders associated with nonalcoholic fatty liver disease (NAFLD) warrants further study of the underlying mechanisms to identify key regulators as targets for the development of therapeutic interventions. Caspase recruitment domain protein 6 (Card6), as a member of the CARD family that regulates cell death and immunity, may potentially control this process. Indeed, Card6 down-regulation was found to be closely associated with the fatty livers observed in NAFLD patients, obese mice, and a palmitate-treated hepatocyte model. Gain-of-function and loss-of-function Card6 mouse models demonstrated that Card6 protected mice from insulin resistance, hepatic steatosis, and inflammatory responses upon high-fat diet administration. Mechanistically, Card6 interacted with and inhibited apoptosis signal-regulating kinase 1 (Ask1) and its subsequent downstream c-Jun N-terminal kinase/p38 signaling. Furthermore, Ask1 was sufficient to mediate Card6 function, and the interaction between Ask1 and Card6 was absolutely required for Card6 function in vivo. Adenovirus-mediated Card6 overexpression in the liver effectively ameliorated insulin resistance and hepatic steatosis in ob/ob mice. Therefore, we identified Card6 as an important negative regulator in NAFLD. Conclusion: Targeting Ask1 by Card6 may be a good strategy to develop a therapeutic method against NAFLD.
Mechanisms of NAFLD development and therapeutic strategies.
Friedman Scott L,Neuschwander-Tetri Brent A,Rinella Mary,Sanyal Arun J
There has been a rise in the prevalence of nonalcoholic fatty liver disease (NAFLD), paralleling a worldwide increase in diabetes and metabolic syndrome. NAFLD, a continuum of liver abnormalities from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH), has a variable course but can lead to cirrhosis and liver cancer. Here we review the pathogenic and clinical features of NAFLD, its major comorbidities, clinical progression and risk of complications and in vitro and animal models of NAFLD enabling refinement of therapeutic targets that can accelerate drug development. We also discuss evolving principles of clinical trial design to evaluate drug efficacy and the emerging targets for drug development that involve either single agents or combination therapies intended to arrest or reverse disease progression.
Insulin/Snail1 axis ameliorates fatty liver disease by epigenetically suppressing lipogenesis.
Liu Yan,Jiang Lin,Sun Chengxin,Ireland Nicole,Shah Yatrik M,Liu Yong,Rui Liangyou
Insulin stimulates lipogenesis but insulin resistance is also associated with increased hepatic lipogenesis in obesity. However, the underlying mechanism remains poorly characterized. Here, we show a noncanonical insulin-Snail1 pathway that suppresses lipogenesis. Insulin robustly upregulates zinc-finger protein Snail1 in a PI 3-kinase-dependent manner. In obesity, the hepatic insulin-Snail1 cascade is impaired due to insulin resistance. Hepatocyte-specific deletion of Snail1 enhances insulin-stimulated lipogenesis in hepatocytes, exacerbates dietary NAFLD in mice, and attenuates NAFLD-associated insulin resistance. Liver-specific overexpression of Snail1 has the opposite effect. Mechanistically, Snail1 binds to the fatty acid synthase promoter and recruits HDAC1/2 to induce deacetylation of H3K9 and H3K27, thereby repressing fatty acid synthase promoter activity. Our data suggest that insulin pathways bifurcate into canonical (lipogenic) and noncanonical (anti-lipogenesis by Snail1) two arms. The noncanonical arm counterbalances the canonical arm through Snail1-elicited epigenetic suppression of lipogenic genes. Impairment in the insulin-Snail1 arm may contribute to NAFLD in obesity.
Mitochondrial Stasis Reveals p62-Mediated Ubiquitination in Parkin-Independent Mitophagy and Mitigates Nonalcoholic Fatty Liver Disease.
Yamada Tatsuya,Murata Daisuke,Adachi Yoshihiro,Itoh Kie,Kameoka Shoichiro,Igarashi Atsushi,Kato Takashi,Araki Yoichi,Huganir Richard L,Dawson Ted M,Yanagawa Toru,Okamoto Koji,Iijima Miho,Sesaki Hiromi
It is unknown what occurs if both mitochondrial division and fusion are completely blocked. Here, we introduced mitochondrial stasis by deleting two dynamin-related GTPases for division (Drp1) and fusion (Opa1) in livers. Mitochondrial stasis rescues liver damage and hypotrophy caused by the single knockout (KO). At the cellular level, mitochondrial stasis re-establishes mitochondrial size and rescues mitophagy defects caused by division deficiency. Using Drp1KO livers, we found that the autophagy adaptor protein p62/sequestosome-1-which is thought to function downstream of ubiquitination-promotes mitochondrial ubiquitination. p62 recruits two subunits of a cullin-RING ubiquitin E3 ligase complex, Keap1 and Rbx1, to mitochondria. Resembling Drp1KO, diet-induced nonalcoholic fatty livers enlarge mitochondria and accumulate mitophagy intermediates. Resembling Drp1Opa1KO, Opa1KO rescues liver damage in this disease model. Our data provide a new concept that mitochondrial stasis leads the spatial dimension of mitochondria to a stationary equilibrium and a new mechanism for mitochondrial ubiquitination in mitophagy.
Long noncoding RNA licensing of obesity-linked hepatic lipogenesis and NAFLD pathogenesis.
Zhao Xu-Yun,Xiong Xuelian,Liu Tongyu,Mi Lin,Peng Xiaoling,Rui Crystal,Guo Liang,Li Siming,Li Xiaoying,Lin Jiandie D
Hepatic lipogenesis is aberrantly induced in nonalcoholic fatty liver disease (NAFLD) via activation of the LXR-SREBP1c pathway. To date, a number of protein factors impinging on the transcriptional activity of LXR and SREBP1c have been elucidated. However, whether this regulatory axis interfaces with long noncoding RNAs (lncRNAs) remains largely unexplored. Here we show that hepatic expression of the lncRNA Blnc1 is strongly elevated in obesity and NAFLD in mice. Blnc1 is required for the induction of SREBP1c and hepatic lipogenic genes in response to LXR activation. Liver-specific inactivation of Blnc1 abrogates high-fat diet-induced hepatic steatosis and insulin resistance and protects mice from diet-induced nonalcoholic steatohepatitis. Proteomic analysis of the Blnc1 ribonucleoprotein complex identified EDF1 as a component of the LXR transcriptional complex that acts in concert with Blnc1 to activate the lipogenic gene program. These findings illustrate a lncRNA transcriptional checkpoint that licenses excess hepatic lipogenesis to exacerbate insulin resistance and NAFLD.
Loss of the E3 ubiquitin ligase MKRN1 represses diet-induced metabolic syndrome through AMPK activation.
Lee Min-Sik,Han Hyun-Ji,Han Su Yeon,Kim Il Young,Chae Sehyun,Lee Choong-Sil,Kim Sung Eun,Yoon Seul Gi,Park Jun-Won,Kim Jung-Hoon,Shin Soyeon,Jeong Manhyung,Ko Aram,Lee Ho-Young,Oh Kyoung-Jin,Lee Yun-Hee,Bae Kwang-Hee,Koo Seung-Hoi,Kim Jea-Woo,Seong Je Kyung,Hwang Daehee,Song Jaewhan
AMP-activated protein kinase (AMPK) plays a key role in controlling energy metabolism in response to physiological and nutritional status. Although AMPK activation has been proposed as a promising molecular target for treating obesity and its related comorbidities, the use of pharmacological AMPK activators has been met with contradictory therapeutic challenges. Here we show a regulatory mechanism for AMPK through its ubiquitination and degradation by the E3 ubiquitin ligase makorin ring finger protein 1 (MKRN1). MKRN1 depletion promotes glucose consumption and suppresses lipid accumulation due to AMPK stabilisation and activation. Accordingly, MKRN1-null mice show chronic AMPK activation in both liver and adipose tissue, resulting in significant suppression of diet-induced metabolic syndrome. We demonstrate also its therapeutic effect by administering shRNA targeting MKRN1 into obese mice that reverses non-alcoholic fatty liver disease. We suggest that ubiquitin-dependent AMPK degradation represents a target therapeutic strategy for metabolic disorders.
Expression of STING Is Increased in Liver Tissues From Patients With NAFLD and Promotes Macrophage-Mediated Hepatic Inflammation and Fibrosis in Mice.
Luo Xianjun,Li Honggui,Ma Linqiang,Zhou Jing,Guo Xin,Woo Shih-Lung,Pei Ya,Knight Linda R,Deveau Michael,Chen Yanming,Qian Xiaoxian,Xiao Xiaoqiu,Li Qifu,Chen Xiangbai,Huo Yuqing,McDaniel Kelly,Francis Heather,Glaser Shannon,Meng Fanyin,Alpini Gianfranco,Wu Chaodong
BACKGROUND & AIMS:Transmembrane protein 173 (TMEM173 or STING) signaling by macrophage activates the type I interferon-mediated innate immune response. The innate immune response contributes to hepatic steatosis and non-alcoholic fatty liver disease (NAFLD). We investigated whether STING regulates diet-induced in hepatic steatosis, inflammation, and liver fibrosis in mice. METHODS:Mice with disruption of Tmem173 (STING) on a C57BL/6J background, mice without disruption of this gene (controls), and mice with disruption of Tmem173 only in myeloid cells were fed a standard chow diet, a high-fat diet (HFD; 60% fat calories), or a methionine- and choline-deficient diet (MCD). Liver tissues were collected and analyzed by histology and immunohistochemistry. Bone marrow cells were isolated from mice, differentiated into macrophages, and incubated with 5,6-dimethylxanthenone-4-acetic acid (DMXAA; an activator of STING) or cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Macrophages or their media were applied to mouse hepatocytes or human hepatic stellate cells (LX2) cells, which were analyzed for cytokine expression, protein phosphorylation, and fat deposition (by oil red O staining after incubation with palmitate). We obtained liver tissues from patients with and without NAFLD and analyzed these by immunohistochemistry. RESULTS:Non-parenchymal cells of liver tissues from patients with NAFLD had higher levels of STING than cells of liver tissues from patients without NAFLD. STING mice and mice with disruption only in myeloid cells developed less severe hepatic steatosis, inflammation, and/or fibrosis after the HFD or MCD than control mice. Levels of phosphorylated c-Jun N-terminal kinase and p65 and mRNAs encoding tumor necrosis factor and interleukins 1B and 6 (markers of inflammation) were significantly lower in liver tissues from STING mice vs control mice after the HFD or MCD. Transplantation of bone marrow cells from control mice to STING mice restored the severity of steatosis and inflammation after the HFD. Macrophages from control, but not STING, mice increased markers of inflammation in response to lipopolysaccharide and cGAMP. Hepatocytes and stellate cells cocultured with STING macrophages in the presence of DMXAA or incubated with the medium collected from these macrophages had decreased fat deposition and markers of inflammation compared with hepatocytes or stellate cells incubated with control macrophages. CONCLUSIONS:Levels of STING were increased in liver tissues from patients with NAFLD and mice with HFD-induced steatosis. In mice, loss of STING from macrophages decreased the severity of liver fibrosis and the inflammatory response. STING might be a therapeutic target for NAFLD.
ER Stress Drives Lipogenesis and Steatohepatitis via Caspase-2 Activation of S1P.
Kim Ju Youn,Garcia-Carbonell Ricard,Yamachika Shinichiro,Zhao Peng,Dhar Debanjan,Loomba Rohit,Kaufman Randal J,Saltiel Alan R,Karin Michael
Nonalcoholic fatty liver disease (NAFLD) progresses to nonalcoholic steatohepatitis (NASH) in response to elevated endoplasmic reticulum (ER) stress. Whereas the onset of simple steatosis requires elevated de novo lipogenesis, progression to NASH is triggered by accumulation of hepatocyte-free cholesterol. We now show that caspase-2, whose expression is ER-stress inducible and elevated in human and mouse NASH, controls the buildup of hepatic-free cholesterol and triglycerides by activating sterol regulatory element-binding proteins (SREBP) in a manner refractory to feedback inhibition. Caspase-2 colocalizes with site 1 protease (S1P) and cleaves it to generate a soluble active fragment that initiates SCAP-independent SREBP1/2 activation in the ER. Caspase-2 ablation or pharmacological inhibition prevents diet-induced steatosis and NASH progression in ER-stress-prone mice. Caspase-2 inhibition offers a specific and effective strategy for preventing or treating stress-driven fatty liver diseases, whereas caspase-2-generated S1P proteolytic fragments, which enter the secretory pathway, are potential NASH biomarkers.
Rho-kinase/AMPK axis regulates hepatic lipogenesis during overnutrition.
Huang Hu,Lee Seung-Hwan,Sousa-Lima Inês,Kim Sang Soo,Hwang Won Min,Dagon Yossi,Yang Won-Mo,Cho Sungman,Kang Min-Cheol,Seo Ji A,Shibata Munehiko,Cho Hyunsoo,Belew Getachew Debas,Bhin Jinhyuk,Desai Bhavna N,Ryu Min Jeong,Shong Minho,Li Peixin,Meng Hua,Chung Byung-Hong,Hwang Daehee,Kim Min Seon,Park Kyong Soo,Macedo Maria Paula,White Morris,Jones John,Kim Young-Bum
The Journal of clinical investigation
Obesity is a major risk factor for developing nonalcoholic fatty liver disease (NAFLD). NAFLD is the most common form of chronic liver disease and is closely associated with insulin resistance, ultimately leading to cirrhosis and hepatocellular carcinoma. However, knowledge of the intracellular regulators of obesity-linked fatty liver disease remains incomplete. Here we showed that hepatic Rho-kinase 1 (ROCK1) drives obesity-induced steatosis in mice through stimulation of de novo lipogenesis. Mice lacking ROCK1 in the liver were resistant to diet-induced obesity owing to increased energy expenditure and thermogenic gene expression. Constitutive expression of hepatic ROCK1 was sufficient to promote adiposity, insulin resistance, and hepatic lipid accumulation in mice fed a high-fat diet. Correspondingly, liver-specific ROCK1 deletion prevented the development of severe hepatic steatosis and reduced hyperglycemia in obese diabetic (ob/ob) mice. Of pathophysiological significance, hepatic ROCK1 was markedly upregulated in humans with fatty liver disease and correlated with risk factors clustering around NAFLD and insulin resistance. Mechanistically, we found that hepatic ROCK1 suppresses AMPK activity and a ROCK1/AMPK pathway is necessary to mediate cannabinoid-induced lipogenesis in the liver. Furthermore, treatment with metformin, the most widely used antidiabetes drug, reduced hepatic lipid accumulation by inactivating ROCK1, resulting in activation of AMPK downstream signaling. Taken together, our findings establish a ROCK1/AMPK signaling axis that regulates de novo lipogenesis, providing a unique target for treating obesity-related metabolic disorders such as NAFLD.
Apoptosis and necroptosis in the liver: a matter of life and death.
Schwabe Robert F,Luedde Tom
Nature reviews. Gastroenterology & hepatology
Cell death represents a basic biological paradigm that governs outcomes and long-term sequelae in almost every hepatic disease condition. Acute liver failure is characterized by massive loss of parenchymal cells but is usually followed by restitution ad integrum. By contrast, cell death in chronic liver diseases often occurs at a lesser extent but leads to long-term alterations in organ architecture and function, contributing to chronic hepatocyte turnover, the recruitment of immune cells and activation of hepatic stellate cells. These chronic cell death responses contribute to the development of liver fibrosis, cirrhosis and cancer. It has become evident that, besides apoptosis, necroptosis is a highly relevant form of programmed cell death in the liver. Differential activation of specific forms of programmed cell death might not only affect outcomes in liver diseases but also offer novel opportunities for therapeutic intervention. Here, we summarize the underlying molecular mechanisms and open questions about disease-specific activation and roles of programmed cell death forms, their contribution to response signatures and their detection. We focus on the role of apoptosis and necroptosis in acute liver injury, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH) and liver cancer, and possible translations into clinical applications.
Gα12 ablation exacerbates liver steatosis and obesity by suppressing USP22/SIRT1-regulated mitochondrial respiration.
Kim Tae Hyun,Yang Yoon Mee,Han Chang Yeob,Koo Ja Hyun,Oh Hyunhee,Kim Su Sung,You Byoung Hoon,Choi Young Hee,Park Tae-Sik,Lee Chang Ho,Kurose Hitoshi,Noureddin Mazen,Seki Ekihiro,Wan Yu-Jui Yvonne,Choi Cheol Soo,Kim Sang Geon
The Journal of clinical investigation
Nonalcoholic fatty liver disease (NAFLD) arises from mitochondrial dysfunction under sustained imbalance between energy intake and expenditure, but the underlying mechanisms controlling mitochondrial respiration have not been entirely understood. Heterotrimeric G proteins converge with activated GPCRs to modulate cell-signaling pathways to maintain metabolic homeostasis. Here, we investigated the regulatory role of G protein α12 (Gα12) on hepatic lipid metabolism and whole-body energy expenditure in mice. Fasting increased Gα12 levels in mouse liver. Gα12 ablation markedly augmented fasting-induced hepatic fat accumulation. cDNA microarray analysis from Gna12-KO liver revealed that the Gα12-signaling pathway regulated sirtuin 1 (SIRT1) and PPARα, which are responsible for mitochondrial respiration. Defective induction of SIRT1 upon fasting was observed in the liver of Gna12-KO mice, which was reversed by lentivirus-mediated Gα12 overexpression in hepatocytes. Mechanistically, Gα12 stabilized SIRT1 protein through transcriptional induction of ubiquitin-specific peptidase 22 (USP22) via HIF-1α increase. Gα12 levels were markedly diminished in liver biopsies from NAFLD patients. Consistently, Gna12-KO mice fed a high-fat diet displayed greater susceptibility to diet-induced liver steatosis and obesity due to decrease in energy expenditure. Our results demonstrate that Gα12 regulates SIRT1-dependent mitochondrial respiration through HIF-1α-dependent USP22 induction, identifying Gα12 as an upstream molecule that contributes to the regulation of mitochondrial energy expenditure.
Serotonin signals through a gut-liver axis to regulate hepatic steatosis.
Choi Wonsuk,Namkung Jun,Hwang Inseon,Kim Hyeongseok,Lim Ajin,Park Hye Jung,Lee Hye Won,Han Kwang-Hyub,Park Seongyeol,Jeong Ji-Seon,Bang Geul,Kim Young Hwan,Yadav Vijay K,Karsenty Gerard,Ju Young Seok,Choi Chan,Suh Jae Myoung,Park Jun Yong,Park Sangkyu,Kim Hail
Nonalcoholic fatty liver disease (NAFLD) is increasing in worldwide prevalence, closely tracking the obesity epidemic, but specific pharmaceutical treatments for NAFLD are lacking. Defining the key molecular pathways underlying the pathogenesis of NAFLD is essential for developing new drugs. Here we demonstrate that inhibition of gut-derived serotonin synthesis ameliorates hepatic steatosis through a reduction in liver serotonin receptor 2A (HTR2A) signaling. Local serotonin concentrations in the portal blood, which can directly travel to and affect the liver, are selectively increased by high-fat diet (HFD) feeding in mice. Both gut-specific Tph1 knockout mice and liver-specific Htr2a knockout mice are resistant to HFD-induced hepatic steatosis, without affecting systemic energy homeostasis. Moreover, selective HTR2A antagonist treatment prevents HFD-induced hepatic steatosis. Thus, the gut TPH1-liver HTR2A axis shows promise as a drug target to ameliorate NAFLD with minimal systemic metabolic effects.
The role of macrophages in nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.
Kazankov Konstantin,Jørgensen Simon Mark Dahl,Thomsen Karen Louise,Møller Holger Jon,Vilstrup Hendrik,George Jacob,Schuppan Detlef,Grønbæk Henning
Nature reviews. Gastroenterology & hepatology
Nonalcoholic fatty liver disease (NAFLD) and its inflammatory and often progressive subtype nonalcoholic steatohepatitis (NASH) are becoming the leading cause of liver-related morbidity and mortality worldwide, and a primary indication for liver transplantation. The pathophysiology of NASH is multifactorial and not yet completely understood; however, innate immunity is a major contributing factor in which liver-resident macrophages (Kupffer cells) and recruited macrophages play a central part in disease progression. In this Review, we assess the evidence for macrophage involvement in the development of steatosis, inflammation and fibrosis in NASH. In this process, not only the polarization of liver macrophages towards a pro-inflammatory phenotype is important, but adipose tissue macrophages, especially in the visceral compartment, also contribute to disease severity and insulin resistance. Macrophage activation is mediated by factors such as endotoxins and translocated bacteria owing to increased intestinal permeability, factors released from damaged or lipoapoptotic hepatocytes, as well as alterations in gut microbiota and defined nutritional components, including certain free fatty acids, cholesterol and their metabolites. Reflecting the important role of macrophages in NASH, we also review studies investigating drugs that target macrophage recruitment to the liver, macrophage polarization and their inflammatory effects as potential treatment options for patients with NASH.
STING-mediated inflammation in Kupffer cells contributes to progression of nonalcoholic steatohepatitis.
Yu Yongsheng,Liu Yu,An Weishuai,Song Jingwen,Zhang Yuefan,Zhao Xianxian
The Journal of clinical investigation
Innate immune activation contributes to the transition from nonalcoholic fatty liver to nonalcoholic steatohepatitis (NASH). Stimulator of IFN genes (STING, also referred to Tmem173) is a universal receptor that recognizes released DNA and triggers innate immune activation. In this work, we investigated the role of STING in the progression of NASH in mice. Both methionine- and choline-deficient diet (MCD) and high-fat diet (HFD) were used to induce NASH in mice. Strikingly, STING deficiency attenuated steatosis, fibrosis, and inflammation in livers in both murine models of NASH. Additionally, STING deficiency increased fasting glucose levels in mice independently of insulin, but mitigated HFD-induced insulin resistance and weight gain and reduced levels of cholesterol, triglycerides, and LDL in serum; it also enhanced levels of HDL. The mitochondrial DNA (mtDNA) from hepatocytes of HFD-fed mice induced TNF-α and IL-6 expression in cultured Kupffer cells (KCs), which was attenuated by STING deficiency or pretreatment with BAY11-7082 (an NF-κB inhibitor). Finally, chronic exposure to 5,6-dimethylxanthenone-4-acetic acid (DMXAA, a STING agonist) led to hepatic steatosis and inflammation in WT mice, but not in STING-deficient mice. We proposed that STING functions as an mtDNA sensor in the KCs of liver under lipid overload and induces NF-κB-dependent inflammation in NASH.
Hepatocyte-specific loss of GPS2 in mice reduces non-alcoholic steatohepatitis via activation of PPARα.
Liang Ning,Damdimopoulos Anastasius,Goñi Saioa,Huang Zhiqiang,Vedin Lise-Lotte,Jakobsson Tomas,Giudici Marco,Ahmed Osman,Pedrelli Matteo,Barilla Serena,Alzaid Fawaz,Mendoza Arturo,Schröder Tarja,Kuiper Raoul,Parini Paolo,Hollenberg Anthony,Lefebvre Philippe,Francque Sven,Van Gaal Luc,Staels Bart,Venteclef Nicolas,Treuter Eckardt,Fan Rongrong
Obesity triggers the development of non-alcoholic fatty liver disease (NAFLD), which involves alterations of regulatory transcription networks and epigenomes in hepatocytes. Here we demonstrate that G protein pathway suppressor 2 (GPS2), a subunit of the nuclear receptor corepressor (NCOR) and histone deacetylase 3 (HDAC3) complex, has a central role in these alterations and accelerates the progression of NAFLD towards non-alcoholic steatohepatitis (NASH). Hepatocyte-specific Gps2 knockout in mice alleviates the development of diet-induced steatosis and fibrosis and causes activation of lipid catabolic genes. Integrative cistrome, epigenome and transcriptome analysis identifies the lipid-sensing peroxisome proliferator-activated receptor α (PPARα, NR1C1) as a direct GPS2 target. Liver gene expression data from human patients reveal that Gps2 expression positively correlates with a NASH/fibrosis gene signature. Collectively, our data suggest that the GPS2-PPARα partnership in hepatocytes coordinates the progression of NAFLD in mice and in humans and thus might be of therapeutic interest.
Inhibiting Interleukin 11 Signaling Reduces Hepatocyte Death and Liver Fibrosis, Inflammation, and Steatosis in Mouse Models of Nonalcoholic Steatohepatitis.
Widjaja Anissa A,Singh Brijesh K,Adami Eleonora,Viswanathan Sivakumar,Dong Jinrui,D'Agostino Giuseppe A,Ng Benjamin,Lim Wei Wen,Tan Jessie,Paleja Bhairav S,Tripathi Madhulika,Lim Sze Yun,Shekeran Shamini Guna,Chothani Sonia P,Rabes Anne,Sombetzki Martina,Bruinstroop Eveline,Min Lio Pei,Sinha Rohit A,Albani Salvatore,Yen Paul M,Schafer Sebastian,Cook Stuart A
BACKGROUND & AIMS:We studied the role of interleukin 11 (IL11) signaling in the pathogenesis of nonalcoholic steatohepatitis (NASH) using hepatic stellate cells (HSCs), hepatocytes, and mouse models of NASH. METHODS:We stimulated mouse and human fibroblasts, HSCs, or hepatocytes with IL11 and other cytokines and analyzed them by imaging, immunoblot, and functional assays and enzyme-linked immunosorbent assays. Mice were given injections of IL11. Mice with disruption of the interleukin 11 receptor subunit alpha1 gene (Il11ra1) mice and Il11ra1 mice were fed a high-fat methionine- and choline-deficient diet (HFMCD) or a Western diet with liquid fructose (WDF) to induce steatohepatitis; control mice were fed normal chow. db/db mice were fed with methionine- and choline-deficient diet for 12 weeks and C57BL/6 NTac were fed with HFMCD for 10 weeks or WDF for 16 weeks. Some mice were given intraperitoneal injections of anti-IL11 (X203), anti-IL11RA (X209), or a control antibody at different timepoints on the diets. Livers and blood were collected; blood samples were analyzed by biochemistry and liver tissues were analyzed by histology, RNA sequencing, immunoblots, immunohistochemistry, hydroxyproline, and mass cytometry time of flight assays. RESULTS:HSCs incubated with cytokines produced IL11, resulting in activation (phosphorylation) of ERK and expression of markers of fibrosis. Livers of mice given injections of IL11 became damaged, with increased markers of fibrosis, hepatocyte cell death and inflammation. Following the HFMCD or WDF, livers from Il11ra1 mice had reduced steatosis, fibrosis, expression of markers of inflammation and steatohepatitis, compared to and Il11ra1 mice on the same diets. Depending on the time of administration of anti-IL11 or anti-IL11RA antibodies to wild-type mice on the HFMCD or WDF, or to db/db mice on the methionine and choline-deficient diet, the antibodies prevented, stopped, or reversed development of fibrosis and steatosis. Blood samples from Il11ra1 mice fed the WDF and given injections of anti-IL11 or anti-IL11RA, as well as from Il11ra1 mice fed WDF, had lower serum levels of lipids and glucose than mice not injected with antibody or with disruption of Il11ra1. CONCLUSIONS:Neutralizing antibodies that block IL11 signaling reduce fibrosis, steatosis, hepatocyte death, inflammation and hyperglycemia in mice with diet-induced steatohepatitis. These antibodies also improve the cardiometabolic profile of mice and might be developed for the treatment of NASH.
Brain leptin reduces liver lipids by increasing hepatic triglyceride secretion and lowering lipogenesis.
Hackl Martina Theresa,Fürnsinn Clemens,Schuh Christina Maria,Krssak Martin,Carli Fabrizia,Guerra Sara,Freudenthaler Angelika,Baumgartner-Parzer Sabina,Helbich Thomas H,Luger Anton,Zeyda Maximilian,Gastaldelli Amalia,Buettner Christoph,Scherer Thomas
Hepatic steatosis develops when lipid influx and production exceed the liver's ability to utilize/export triglycerides. Obesity promotes steatosis and is characterized by leptin resistance. A role of leptin in hepatic lipid handling is highlighted by the observation that recombinant leptin reverses steatosis of hypoleptinemic patients with lipodystrophy by an unknown mechanism. Since leptin mainly functions via CNS signaling, we here examine in rats whether leptin regulates hepatic lipid flux via the brain in a series of stereotaxic infusion experiments. We demonstrate that brain leptin protects from steatosis by promoting hepatic triglyceride export and decreasing de novo lipogenesis independently of caloric intake. Leptin's anti-steatotic effects are generated in the dorsal vagal complex, require hepatic vagal innervation, and are preserved in high-fat-diet-fed rats when the blood brain barrier is bypassed. Thus, CNS leptin protects from ectopic lipid accumulation via a brain-vagus-liver axis and may be a therapeutic strategy to ameliorate obesity-related steatosis.
ER-residential Nogo-B accelerates NAFLD-associated HCC mediated by metabolic reprogramming of oxLDL lipophagy.
Tian Yuan,Yang Bin,Qiu Weinan,Hao Yajing,Zhang Zhenxing,Yang Bo,Li Nan,Cheng Shuqun,Lin Zhangjun,Rui Yao-Cheng,Cheung Otto K W,Yang Weiqin,Wu William K K,Cheung Yue-Sun,Lai Paul B S,Luo Jianjun,Sung Joseph J Y,Chen Runsheng,Wang Hong-Yang,Cheng Alfred S L,Yang Pengyuan
Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome that elevates the risk of hepatocellular carcinoma (HCC). Although alteration of lipid metabolism has been increasingly recognized as a hallmark of cancer cells, the deregulated metabolic modulation of HCC cells in the NAFLD progression remains obscure. Here, we discovers an endoplasmic reticulum-residential protein, Nogo-B, as a highly expressed metabolic modulator in both murine and human NAFLD-associated HCCs, which accelerates high-fat, high-carbohydrate diet-induced metabolic dysfunction and tumorigenicity. Mechanistically, CD36-mediated oxLDL uptake triggers CEBPβ expression to directly upregulate Nogo-B, which interacts with ATG5 to promote lipophagy leading to lysophosphatidic acid-enhanced YAP oncogenic activity. This CD36-Nogo-B-YAP pathway consequently reprograms oxLDL metabolism and induces carcinogenetic signaling for NAFLD-associated HCCs. Targeting the Nogo-B pathway may represent a therapeutic strategy for HCC arising from the metabolic syndrome.
Landscape of Intercellular Crosstalk in Healthy and NASH Liver Revealed by Single-Cell Secretome Gene Analysis.
Xiong Xuelian,Kuang Henry,Ansari Sahar,Liu Tongyu,Gong Jianke,Wang Shuai,Zhao Xu-Yun,Ji Yewei,Li Chuan,Guo Liang,Zhou Linkang,Chen Zhimin,Leon-Mimila Paola,Chung Meng Ting,Kurabayashi Katsuo,Opp Judy,Campos-Pérez Francisco,Villamil-Ramírez Hugo,Canizales-Quinteros Samuel,Lyons Robert,Lumeng Carey N,Zhou Beiyan,Qi Ling,Huertas-Vazquez Adriana,Lusis Aldons J,Xu X Z Shawn,Li Siming,Yu Yonghao,Li Jun Z,Lin Jiandie D
Cell-cell communication via ligand-receptor signaling is a fundamental feature of complex organs. Despite this, the global landscape of intercellular signaling in mammalian liver has not been elucidated. Here we perform single-cell RNA sequencing on non-parenchymal cells isolated from healthy and NASH mouse livers. Secretome gene analysis revealed a highly connected network of intrahepatic signaling and disruption of vascular signaling in NASH. We uncovered the emergence of NASH-associated macrophages (NAMs), which are marked by high expression of triggering receptors expressed on myeloid cells 2 (Trem2), as a feature of mouse and human NASH that is linked to disease severity and highly responsive to pharmacological and dietary interventions. Finally, hepatic stellate cells (HSCs) serve as a hub of intrahepatic signaling via HSC-derived stellakines and their responsiveness to vasoactive hormones. These results provide unprecedented insights into the landscape of intercellular crosstalk and reprogramming of liver cells in health and disease.