Lipotoxicity induces hepatic protein inclusions through TANK binding kinase 1-mediated p62/sequestosome 1 phosphorylation.
Cho Chun-Seok,Park Hwan-Woo,Ho Allison,Semple Ian A,Kim Boyoung,Jang Insook,Park Haeli,Reilly Shannon,Saltiel Alan R,Lee Jun Hee
Hepatology (Baltimore, Md.)
Obesity commonly leads to hepatic steatosis, which often provokes lipotoxic injuries to hepatocytes that cause nonalcoholic steatohepatitis (NASH). NASH, in turn, is associated with the accumulation of insoluble protein aggregates that are composed of ubiquitinated proteins and ubiquitin adaptor p62/sequestosome 1 (SQSTM1). Formation of p62 inclusions in hepatocytes is the critical marker that distinguishes simple fatty liver from NASH and predicts a poor prognostic outcome for subsequent liver carcinogenesis. However, the molecular mechanism by which lipotoxicity induces protein aggregation is currently unknown. Here, we show that, upon saturated fatty acid-induced lipotoxicity, TANK binding kinase 1 (TBK1) is activated and phosphorylates p62. TBK1-mediated p62 phosphorylation is important for lipotoxicity-induced aggregation of ubiquitinated proteins and formation of large protein inclusions in hepatocytes. In addition, cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING), upstream regulators of TBK1, are involved in lipotoxic activation of TBK1 and subsequent p62 phosphorylation in hepatocytes. Furthermore, TBK1 inhibition prevented formation of ubiquitin-p62 aggregates not only in cultured hepatocytes, but also in mouse models of obesity and NASH. CONCLUSION:These results suggest that lipotoxic activation of TBK1 and subsequent p62 phosphorylation are critical steps in the NASH pathology of protein inclusion accumulation in hepatocytes. This mechanism can provide an explanation for how hypernutrition and obesity promote the development of severe liver pathologies, such as steatohepatitis and liver cancer, by facilitating the formation of p62 inclusions. (Hepatology 2018).
Hypoxia-inducible factor 2α drives nonalcoholic fatty liver progression by triggering hepatocyte release of histidine-rich glycoprotein.
Morello Elisabetta,Sutti Salvatore,Foglia Beatrice,Novo Erica,Cannito Stefania,Bocca Claudia,Rajsky Martina,Bruzzì Stefania,Abate Maria Lorena,Rosso Chiara,Bozzola Cristina,David Ezio,Bugianesi Elisabetta,Albano Emanuele,Parola Maurizio
Hepatology (Baltimore, Md.)
Mechanisms underlying progression of nonalcoholic fatty liver disease (NAFLD) are still incompletely characterized. Hypoxia and hypoxia-inducible factors (HIFs) have been implicated in the pathogenesis of chronic liver diseases, but the actual role of HIF-2α in the evolution of NAFLD has never been investigated in detail. In this study, we show that HIF-2α is selectively overexpressed in the cytosol and the nuclei of hepatocytes in a very high percentage (>90%) of liver biopsies from a cohort of NAFLD patients at different stages of the disease evolution. Similar features were also observed in mice with steatohepatitis induced by feeding a methionine/choline-deficient diet. Experiments performed in mice carrying hepatocyte-specific deletion of HIF-2α and related control littermates fed either a choline-deficient L-amino acid-defined or a methionine/choline-deficient diet showed that HIF-2α deletion ameliorated the evolution of NAFLD by decreasing parenchymal injury, fatty liver, lobular inflammation, and the development of liver fibrosis. The improvement in NAFLD progression in HIF-2α-deficient mice was related to a selective down-regulation in the hepatocyte production of histidine-rich glycoprotein (HRGP), recently proposed to sustain macrophage M1 polarization. In vitro experiments confirmed that the up-regulation of hepatocyte HRGP expression was hypoxia-dependent and HIF-2α-dependent. Finally, analyses performed on specimens from NAFLD patients indicated that HRGP was overexpressed in all patients showing hepatocyte nuclear staining for HIF-2α and revealed a significant positive correlation between HIF-2α and HRGP liver transcript levels in these patients. CONCLUSIONS:These results indicate that hepatocyte HIF-2α activation is a key feature in both human and experimental NAFLD and significantly contributes to the disease progression through the up-regulation of HRGP production. (Hepatology 2018;67:2196-2214).
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.
Disruption of adenosine 2A receptor exacerbates NAFLD through increasing inflammatory responses and SREBP1c activity.
Cai Yuli,Li Honggui,Liu Mengyang,Pei Ya,Zheng Juan,Zhou Jing,Luo Xianjun,Huang Wenya,Ma Linqiang,Yang Qiuhua,Guo Shaodong,Xiao Xiaoqiu,Li Qifu,Zeng Tianshu,Meng Fanyin,Francis Heather,Glaser Shannon,Chen Lulu,Huo Yuqing,Alpini Gianfranco,Wu Chaodong
Hepatology (Baltimore, Md.)
Adenosine 2A receptor (A R) exerts protective roles in endotoxin- and/or ischemia-induced tissue damage. However, the role for A R in nonalcoholic fatty liver disease (NAFLD) remains largely unknown. We sought to examine the effects of global and/or myeloid cell-specific A R disruption on the aspects of obesity-associated NAFLD and to elucidate the underlying mechanisms. Global and/or myeloid cell-specific A R-disrupted mice and control mice were fed a high-fat diet (HFD) to induce NAFLD. In addition, bone marrow-derived macrophages and primary mouse hepatocytes were examined for inflammatory and metabolic responses. Upon feeding an HFD, both global A R-disrupted mice and myeloid cell-specific A R-defcient mice revealed increased severity of HFD-induced hepatic steatosis and inflammation compared with their respective control mice. In in vitro experiments, A R-deficient macrophages exhibited increased proinflammatory responses, and enhanced fat deposition of wild-type primary hepatocytes in macrophage-hepatocyte cocultures. In primary hepatocytes, A R deficiency increased the proinflammatory responses and enhanced the effect of palmitate on stimulating fat deposition. Moreover, A R deficiency significantly increased the abundance of sterol regulatory element-binding protein 1c (SREBP1c) in livers of fasted mice and in hepatocytes upon nutrient deprivation. In the absence of A R, SREBP1c transcription activity was significantly increased in mouse hepatocytes. CONCLUSION:Taken together, our results demonstrate that disruption of A R in both macrophage and hepatocytes accounts for increased severity of NAFLD, likely through increasing inflammation and through elevating lipogenic events due to stimulation of SREBP1c expression and transcription activity. (Hepatology 2018;68:48-61).
Hsp72 protects against liver injury via attenuation of hepatocellular death, oxidative stress, and JNK signaling.
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.
Non-alcoholic steatohepatitis pathogenesis: sublethal hepatocyte injury as a driver of liver inflammation.
Ibrahim Samar H,Hirsova Petra,Gores Gregory J
A subset of patients with non-alcoholic fatty liver disease develop an inflammatory condition, termed non-alcoholic steatohepatitis (NASH). NASH is characterised by hepatocellular injury, innate immune cell-mediated inflammation and progressive liver fibrosis. The mechanisms whereby hepatic inflammation occurs in NASH remain incompletely understood, but appear to be linked to the proinflammatory microenvironment created by toxic lipid-induced hepatocyte injury, termed lipotoxicity. In this review, we discuss the signalling pathways induced by sublethal hepatocyte lipid overload that contribute to the pathogenesis of NASH. Furthermore, we will review the role of proinflammatory, proangiogenic and profibrotic hepatocyte-derived extracellular vesicles as disease biomarkers and pathogenic mediators during lipotoxicity. We also review the potential therapeutic strategies to block the feed-forward loop between sublethal hepatocyte injury and liver inflammation.
MicroRNAs in the pathogenesis and treatment of progressive liver injury in NAFLD and liver fibrosis.
Su Qiaozhu,Kumar Virender,Sud Neetu,Mahato Ram I
Advanced drug delivery reviews
Non-alcoholic fatty liver disease (NAFLD) increases the risk of various liver injuries, ranging from simple steatosis to non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis, and ultimately hepatocellular carcinoma (HCC). Ample evidence has suggested that aberrant expression of microRNAs (miRNAs) is functionally involved in the activation of cellular stress, inflammation and fibrogenesis in hepatic cells, including hepatocytes, Kupffer and hepatic stellate cells (HSCs), at different pathological stages of NAFLD and liver fibrosis. Here, we overview recent findings on the potential role of miRNAs in the pathogenesis of NAFLD, including lipotoxicity, oxidative stress, metabolic inflammation and fibrogenesis. We critically assess the literatures on both human subjects and animal models of NAFLD and liver fibrosis with miRNA dysregulation and their mechanisms of actions in liver damage. We further highlight the potential use of miRNA mimics or antimiRNAs as therapeutic approaches for the prevention and treatment of NAFLD and liver fibrosis.
Recent Insights into the Pathogenesis of Nonalcoholic Fatty Liver Disease.
Arab Juan Pablo,Arrese Marco,Trauner Michael
Annual review of pathology
Nonalcoholic fatty liver disease (NAFLD) is a burgeoning health problem worldwide and an important risk factor for both hepatic and cardiometabolic mortality. The rapidly increasing prevalence of this disease and of its aggressive form nonalcoholic steatohepatitis (NASH) will require novel therapeutic approaches based on a profound understanding of its pathogenesis to halt disease progression to advanced fibrosis or cirrhosis and cancer. The pathogenesis of NAFLD involves a complex interaction among environmental factors (i.e., Western diet), obesity, changes in microbiota, and predisposing genetic variants resulting in a disturbed lipid homeostasis and an excessive accumulation of triglycerides and other lipid species in hepatocytes. Insulin resistance is a central mechanism that leads to lipotoxicity, endoplasmic reticulum stress, disturbed autophagy, and, ultimately, hepatocyte injury and death that triggers hepatic inflammation, hepatic stellate cell activation, and progressive fibrogenesis, thus driving disease progression. In the present review, we summarize the currently available data on the pathogenesis of NAFLD, emphasizing the most recent advances. A better understanding of NAFLD/NASH pathogenesis is crucial for the design of new and efficient therapeutic interventions.
AhR and SHP regulate phosphatidylcholine and S-adenosylmethionine levels in the one-carbon cycle.
Kim Young-Chae,Seok Sunmi,Byun Sangwon,Kong Bo,Zhang Yang,Guo Grace,Xie Wen,Ma Jian,Kemper Byron,Kemper Jongsook Kim
Phosphatidylcholines (PC) and S-adenosylmethionine (SAM) are critical determinants of hepatic lipid levels, but how their levels are regulated is unclear. Here, we show that Pemt and Gnmt, key one-carbon cycle genes regulating PC/SAM levels, are downregulated after feeding, leading to decreased PC and increased SAM levels, but these effects are blunted in small heterodimer partner (SHP)-null or FGF15-null mice. Further, aryl hydrocarbon receptor (AhR) is translocated into the nucleus by insulin/PKB signaling in the early fed state and induces Pemt and Gnmt expression. This induction is blocked by FGF15 signaling-activated SHP in the late fed state. Adenoviral-mediated expression of AhR in obese mice increases PC levels and exacerbates steatosis, effects that are blunted by SHP co-expression or Pemt downregulation. PEMT, AHR, and PC levels are elevated in simple steatosis patients, but PC levels are robustly reduced in steatohepatitis-fibrosis patients. This study identifies AhR and SHP as new physiological regulators of PC/SAM levels.
Ultraconserved element uc.372 drives hepatic lipid accumulation by suppressing miR-195/miR4668 maturation.
Guo Jun,Fang Weiwei,Sun Libo,Lu Yonggang,Dou Lin,Huang Xiuqing,Tang Weiqing,Yu Liqing,Li Jian
Ultraconserved (uc) RNAs, a class of long non-coding RNAs (lncRNAs), are conserved across humans, mice, and rats, but the physiological significance and pathological role of ucRNAs is largely unknown. Here we show that uc.372 is upregulated in the livers of db/db mice, HFD-fed mice, and NAFLD patients. Gain-of-function and loss-of-function studies indicate that uc.372 drives hepatic lipid accumulation in mice by promoting lipogenesis. We further demonstrate that uc.372 binds to pri-miR-195/pri-miR-4668 and suppresses maturation of miR-195/miR-4668 to regulate expression of genes related to lipid synthesis and uptake, including ACC, FAS, SCD1, and CD36. Finally, we identify that uc.372 is located downstream of the insulinoma-associated 2 (INSM2) gene that is transcriptionally activated by upstream transcription factor 1 (USF1). Our findings reveal a novel mechanism by which uc.372 drives hepatic steatosis through inhibition of miR-195/miR-4668 maturation to relieve miR-195/miR-4668-mediated suppression of functional target gene expression.
An Integrated Understanding of the Rapid Metabolic Benefits of a Carbohydrate-Restricted Diet on Hepatic Steatosis in Humans.
Mardinoglu Adil,Wu Hao,Bjornson Elias,Zhang Cheng,Hakkarainen Antti,Räsänen Sari M,Lee Sunjae,Mancina Rosellina M,Bergentall Mattias,Pietiläinen Kirsi H,Söderlund Sanni,Matikainen Niina,Ståhlman Marcus,Bergh Per-Olof,Adiels Martin,Piening Brian D,Granér Marit,Lundbom Nina,Williams Kevin J,Romeo Stefano,Nielsen Jens,Snyder Michael,Uhlén Mathias,Bergström Göran,Perkins Rosie,Marschall Hanns-Ulrich,Bäckhed Fredrik,Taskinen Marja-Riitta,Borén Jan
A carbohydrate-restricted diet is a widely recommended intervention for non-alcoholic fatty liver disease (NAFLD), but a systematic perspective on the multiple benefits of this diet is lacking. Here, we performed a short-term intervention with an isocaloric low-carbohydrate diet with increased protein content in obese subjects with NAFLD and characterized the resulting alterations in metabolism and the gut microbiota using a multi-omics approach. We observed rapid and dramatic reductions of liver fat and other cardiometabolic risk factors paralleled by (1) marked decreases in hepatic de novo lipogenesis; (2) large increases in serum β-hydroxybutyrate concentrations, reflecting increased mitochondrial β-oxidation; and (3) rapid increases in folate-producing Streptococcus and serum folate concentrations. Liver transcriptomic analysis on biopsy samples from a second cohort revealed downregulation of the fatty acid synthesis pathway and upregulation of folate-mediated one-carbon metabolism and fatty acid oxidation pathways. Our results highlight the potential of exploring diet-microbiota interactions for treating NAFLD.
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).
Direct effects of thyroid hormones on hepatic lipid metabolism.
Sinha Rohit A,Singh Brijesh K,Yen Paul M
Nature reviews. Endocrinology
It has been known for a long time that thyroid hormones have prominent effects on hepatic fatty acid and cholesterol synthesis and metabolism. Indeed, hypothyroidism has been associated with increased serum levels of triglycerides and cholesterol as well as non-alcoholic fatty liver disease (NAFLD). Advances in areas such as cell imaging, autophagy and metabolomics have generated a more detailed and comprehensive picture of thyroid-hormone-mediated regulation of hepatic lipid metabolism at the molecular level. In this Review, we describe and summarize the key features of direct thyroid hormone regulation of lipogenesis, fatty acid β-oxidation, cholesterol synthesis and the reverse cholesterol transport pathway in normal and altered thyroid hormone states. Thyroid hormone mediates these effects at the transcriptional and post-translational levels and via autophagy. Given these potentially beneficial effects on lipid metabolism, it is possible that thyroid hormone analogues and/or mimetics might be useful for the treatment of metabolic diseases involving the liver, such as hypercholesterolaemia and NAFLD.
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.
Link between gut-microbiome derived metabolite and shared gene-effects with hepatic steatosis and fibrosis in NAFLD.
Caussy Cyrielle,Hsu Cynthia,Lo Min-Tzu,Liu Amy,Bettencourt Ricki,Ajmera Veeral H,Bassirian Shirin,Hooker Jonathan,Sy Ethan,Richards Lisa,Schork Nicholas,Schnabl Bernd,Brenner David A,Sirlin Claude B,Chen Chi-Hua,Loomba Rohit,
Hepatology (Baltimore, Md.)
Previous studies have shown that gut-microbiome is associated with nonalcoholic fatty liver disease (NAFLD). We aimed to examine if serum metabolites, especially those derived from the gut-microbiome, have a shared gene-effect with hepatic steatosis and fibrosis. This is a cross-sectional analysis of a prospective discovery cohort including 156 well-characterized twins and families with untargeted metabolome profiling assessment. Hepatic steatosis was assessed using magnetic-resonance-imaging proton-density-fat-fraction (MRI-PDFF) and fibrosis using MR-elastography (MRE). A twin additive genetics and unique environment effects (AE) model was used to estimate the shared gene-effect between metabolites and hepatic steatosis and fibrosis. The findings were validated in an independent prospective validation cohort of 156 participants with biopsy-proven NAFLD including shotgun metagenomics sequencing assessment in a subgroup of the cohort. In the discovery cohort, 56 metabolites including 6 microbial metabolites had a significant shared gene-effect with both hepatic steatosis and fibrosis after adjustment for age, sex and ethnicity. In the validation cohort, 6 metabolites were associated with advanced fibrosis. Among them, only one microbial metabolite, 3-(4-hydroxyphenyl)lactate, remained consistent and statistically significantly associated with liver fibrosis in the discovery and validation cohort (fold-change of higher-MRE versus lower-MRE: 1.78, P < 0.001 and of advanced versus no advanced fibrosis: 1.26, P = 0.037, respectively). The share genetic determination of 3-(4-hydroxyphenyl)lactate with hepatic steatosis was R :0.57,95%CI:0.27-0.80, P < 0.001 and with fibrosis was R :0.54,95%CI:0.036-1, P = 0.036. Pathway reconstruction linked 3-(4-hydroxyphenyl)lactate to several human gut-microbiome species. In the validation cohort, 3-(4-hydroxyphenyl)lactate was significantly correlated with the abundance of several gut-microbiome species, belonging only to Firmicutes, Bacteroidetes and Proteobacteria phyla, previously reported as associated with advanced fibrosis. Conclusion: This proof of concept study provides evidence of a link between the gut-microbiome and 3-(4-hydroxyphenyl)lactate that shares gene-effect with hepatic steatosis and fibrosis. (Hepatology 2018).
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).
Specific Hepatic Sphingolipids Relate to Insulin Resistance, Oxidative Stress, and Inflammation in Nonalcoholic Steatohepatitis.
Apostolopoulou Maria,Gordillo Ruth,Koliaki Chrysi,Gancheva Sofia,Jelenik Tomas,De Filippo Elisabetta,Herder Christian,Markgraf Daniel,Jankowiak Frank,Esposito Irene,Schlensak Matthias,Scherer Philipp E,Roden Michael
OBJECTIVE:Insulin resistance and nonalcoholic fatty liver disease have been linked to several lipid metabolites in animals, but their role in humans remains unclear. This study examined the relationship of sphingolipids with hepatic and peripheral metabolism in 21 insulin-resistant obese patients without (NAFL-) or with (NAFL+) nonalcoholic fatty liver and nonalcoholic steatohepatitis (NASH) and 7 healthy lean individuals undergoing tissue biopsies during bariatric or elective abdominal surgery. RESEARCH DESIGN AND METHODS:Hyperinsulinemic-euglycemic clamps with d-[6,6-H]glucose were performed to quantify tissue-specific insulin sensitivity. Hepatic oxidative capacity, lipid peroxidation, and the phosphorylated-to-total c-Jun N-terminal kinase (pJNK-to-tJNK) ratio were measured to assess mitochondrial function, oxidative stress, and inflammatory activity. RESULTS:Hepatic total ceramides were higher by 50% and 33% in NASH compared with NAFL+ and NAFL-, respectively. Only in NASH were hepatic dihydroceramides (16:0, 22:0, and 24:1) and lactosylceramides increased. Serum total ceramides and dihydroceramides (hepatic dihydroceramides 22:0 and 24:1) correlated negatively with whole-body but not with hepatic insulin sensitivity. Hepatic maximal respiration related positively to serum lactosylceramide subspecies, hepatic sphinganine, and lactosylceramide 14:0. Liver lipid peroxides (total ceramides, sphingomyelin 22:0) and the pJNK-to-tJNK ratio (ceramide 24:0; hexosylceramides 22:0, 24:0, and 24:1) all positively correlated with the respective hepatic sphingolipids. CONCLUSIONS:Sphingolipid species are not only increased in insulin-resistant humans with NASH but also correlate with hepatic oxidative stress and inflammation, suggesting that these lipids may play a role during progression of simple steatosis to NASH in humans.
Melatonin improves insulin resistance and hepatic steatosis through attenuation of alpha-2-HS-glycoprotein.
Heo Jee-In,Yoon Dae Wui,Yu Ji Hee,Kim Nam Hoon,Yoo Hye Jin,Seo Ji A,Kim Sin Gon,Choi Kyung Mook,Baik Sei Hyun,Choi Dong Seop,Kim Nan Hee
Journal of pineal research
Melatonin plays an important role in regulating circadian rhythms. It also acts as a potent antioxidant and regulates glucose and lipid metabolism, although the exact action mechanism is not clear. The α2-HS-glycoprotein gene (AHSG) and its protein, fetuin-A (FETUA), are one of the hepatokines and are known to be associated with insulin resistance and type 2 diabetes. The aim of this study was to determine whether melatonin improves hepatic insulin resistance and hepatic steatosis in a FETUA-dependent manner. In HepG2 cells treated with 300 μmol/L of palmitic acid, phosphorylated AKT expression decreased, and FETUA expression increased, but this effect was inhibited by treatment with 10 μmol/L of melatonin. However, melatonin did not improve insulin resistance in FETUA-overexpressing cells, indicating that improvement in insulin resistance by melatonin was dependent on downregulation of FETUA. Moreover, melatonin decreased palmitic acid-induced ER stress markers, CHOP, Bip, ATF-6, XBP-1, ATF-4, and PERK. In addition, in the high-fat diet (HFD) mice, oral treatment with 100 mg/kg/day melatonin for 10 weeks reduced body weight gain to one-third of that of the HFD group and hepatic steatosis. Insulin sensitivity and glucose intolerance improved with the upregulation of muscle p-AKT protein expression. FETUA expression and ER stress markers in the liver and serum of HFD mice were decreased by melatonin treatment. In conclusion, melatonin can improve hepatic insulin resistance and hepatic steatosis through reduction in ER stress and the resultant AHSG expression.
Neutrophil extracellular traps promote inflammation and development of hepatocellular carcinoma in nonalcoholic steatohepatitis.
van der Windt Dirk J,Sud Vikas,Zhang Hongji,Varley Patrick R,Goswami Julie,Yazdani Hamza O,Tohme Samer,Loughran Patricia,O'Doherty Robert M,Minervini Marta I,Huang Hai,Simmons Richard L,Tsung Allan
Hepatology (Baltimore, Md.)
Nonalcoholic steatohepatitis (NASH) is a progressive, inflammatory form of fatty liver disease. It is the most rapidly rising risk factor for the development of hepatocellular carcinoma (HCC), which can arise in NASH with or without cirrhosis. The inflammatory signals promoting the progression of NASH to HCC remain largely unknown. The propensity of neutrophils to expel decondensed chromatin embedded with inflammatory proteins, known as neutrophil extracellular traps (NETs), has been shown to be important in chronic inflammatory conditions and in cancer progression. In this study, we asked whether NET formation occurs in NASH and contributes to the progression of HCC. We found elevated levels of a NET marker in serum of patients with NASH. In livers from STAM mice (NASH induced by neonatal streptozotocin and high-fat diet), early neutrophil infiltration and NET formation were seen, followed by an influx of monocyte-derived macrophages, production of inflammatory cytokines, and progression of HCC. Inhibiting NET formation, through treatment with deoxyribonuclease (DNase) or using mice knocked out for peptidyl arginine deaminase type IV (PAD4 ), did not affect the development of a fatty liver but altered the consequent pattern of liver inflammation, which ultimately resulted in decreased tumor growth. Mechanistically, we found that commonly elevated free fatty acids stimulate NET formation in vitro. CONCLUSION:Our findings implicate NETs in the protumorigenic inflammatory environment in NASH, suggesting that their elimination may reduce the progression of liver cancer in NASH. (Hepatology 2018).
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.
The gut-liver axis and the intersection with the microbiome.
Tripathi Anupriya,Debelius Justine,Brenner David A,Karin Michael,Loomba Rohit,Schnabl Bernd,Knight Rob
Nature reviews. Gastroenterology & hepatology
In the past decade, an exciting realization has been that diverse liver diseases - ranging from nonalcoholic steatohepatitis, alcoholic steatohepatitis and cirrhosis to hepatocellular carcinoma - fall along a spectrum. Work on the biology of the gut-liver axis has assisted in understanding the basic biology of both alcoholic fatty liver disease and nonalcoholic fatty liver disease (NAFLD). Of immense importance is the advancement in understanding the role of the microbiome, driven by high-throughput DNA sequencing and improved computational techniques that enable the complexity of the microbiome to be interrogated, together with improved experimental designs. Here, we review gut-liver communications in liver disease, exploring the molecular, genetic and microbiome relationships and discussing prospects for exploiting the microbiome to determine liver disease stage and to predict the effects of pharmaceutical, dietary and other interventions at a population and individual level. Although much work remains to be done in understanding the relationship between the microbiome and liver disease, rapid progress towards clinical applications is being made, especially in study designs that complement human intervention studies with mechanistic work in mice that have been humanized in multiple respects, including the genetic, immunological and microbiome characteristics of individual patients. These 'avatar mice' could be especially useful for guiding new microbiome-based or microbiome-informed therapies.
High Mobility Group Box-1 Drives Fibrosis Progression Signaling via the Receptor for Advanced Glycation End Products in Mice.
Ge Xiaodong,Arriazu Elena,Magdaleno Fernando,Antoine Daniel J,Dela Cruz Rouchelle,Theise Neil,Nieto Natalia
Hepatology (Baltimore, Md.)
High-mobility group box-1 (HMGB1) is a damage-associated molecular pattern (DAMP) increased in response to liver injury. Because HMGB1 is a ligand for the receptor for advanced glycation endproducts (RAGE), we hypothesized that induction of HMGB1 could participate in the pathogenesis of liver fibrosis though RAGE cell-specific signaling mechanisms. Liver HMGB1 protein expression correlated with fibrosis stage in patients with chronic hepatitis C virus (HCV) infection, primary biliary cirrhosis (PBC), or alcoholic steatohepatitis (ASH). Hepatic HMGB1 protein expression and secretion increased in five mouse models of liver fibrosis attributed to drug-induced liver injury (DILI), cholestasis, ASH, or nonalcoholic steatohepatitis (NASH). HMGB1 was up-regulated and secreted mostly by hepatocytes and Kupffer cells (KCs) following CCl treatment. Neutralization of HMGB1 protected, whereas injection of recombinant HMGB1 promoted liver fibrosis. Hmgb1 ablation in hepatocytes (Hmgb1 ) or in myeloid cells (Hmgb1 ) partially protected, whereas ablation in both (Hmgb1 ) prevented liver fibrosis in vivo. Coculture with hepatocytes or KCs from CCl -injected wild-type (WT) mice up-regulated Collagen type I production by hepatic stellate cells (HSCs); yet, coculture with hepatocytes from CCl -injected Hmgb1 or with KCs from CCl -injected Hmgb1 mice partially blunted this effect. Rage ablation in HSCs (Rage ) and RAGE neutralization prevented liver fibrosis. Last, we identified that HMGB1 stimulated HSC migration and signaled through RAGE to up-regulate Collagen type I expression by activating the phosphorylated mitogen-activated protein kinase kinase (pMEK)1/2, phosphorylated extracellular signal-regulated kinase (pERK)1/2 and pcJun signaling pathway. Conclusion: Hepatocyte and KC-derived HMGB1 participates in the pathogenesis of liver fibrosis by signaling through RAGE in HSCs to activate the pMEK1/2, pERK1/2 and pcJun pathway and increase Collagen type I deposition.
Saturated Fat Is More Metabolically Harmful for the Human Liver Than Unsaturated Fat or Simple Sugars.
Luukkonen Panu K,Sädevirta Sanja,Zhou You,Kayser Brandon,Ali Ashfaq,Ahonen Linda,Lallukka Susanna,Pelloux Véronique,Gaggini Melania,Jian Ching,Hakkarainen Antti,Lundbom Nina,Gylling Helena,Salonen Anne,Orešič Matej,Hyötyläinen Tuulia,Orho-Melander Marju,Rissanen Aila,Gastaldelli Amalia,Clément Karine,Hodson Leanne,Yki-Järvinen Hannele
OBJECTIVE:Nonalcoholic fatty liver disease (i.e., increased intrahepatic triglyceride [IHTG] content), predisposes to type 2 diabetes and cardiovascular disease. Adipose tissue lipolysis and hepatic de novo lipogenesis (DNL) are the main pathways contributing to IHTG. We hypothesized that dietary macronutrient composition influences the pathways, mediators, and magnitude of weight gain-induced changes in IHTG. RESEARCH DESIGN AND METHODS:We overfed 38 overweight subjects (age 48 ± 2 years, BMI 31 ± 1 kg/m, liver fat 4.7 ± 0.9%) 1,000 extra kcal/day of saturated (SAT) or unsaturated (UNSAT) fat or simple sugars (CARB) for 3 weeks. We measured IHTG (H-MRS), pathways contributing to IHTG (lipolysis ([H]glycerol) and DNL (HO) basally and during euglycemic hyperinsulinemia), insulin resistance, endotoxemia, plasma ceramides, and adipose tissue gene expression at 0 and 3 weeks. RESULTS:Overfeeding SAT increased IHTG more (+55%) than UNSAT (+15%, < 0.05). CARB increased IHTG (+33%) by stimulating DNL (+98%). SAT significantly increased while UNSAT decreased lipolysis. SAT induced insulin resistance and endotoxemia and significantly increased multiple plasma ceramides. The diets had distinct effects on adipose tissue gene expression. CONCLUSIONS:Macronutrient composition of excess energy influences pathways of IHTG: CARB increases DNL, while SAT increases and UNSAT decreases lipolysis. SAT induced the greatest increase in IHTG, insulin resistance, and harmful ceramides. Decreased intakes of SAT could be beneficial in reducing IHTG and the associated risk of diabetes.
Mucosal-associated invariant T cells are a profibrogenic immune cell population in the liver.
Hegde Pushpa,Weiss Emmanuel,Paradis Valérie,Wan Jinghong,Mabire Morgane,Sukriti Sukriti,Rautou Pierre-Emmanuel,Albuquerque Miguel,Picq Olivia,Gupta Abhishak Chandra,Ferrere Gladys,Gilgenkrantz Hélène,Kiaf Badr,Toubal Amine,Beaudoin Lucie,Lettéron Philippe,Moreau Richard,Lehuen Agnès,Lotersztajn Sophie
Liver fibrosis is the common response to chronic liver injury, and leads to cirrhosis and its complications. Persistent inflammation is a driving force of liver fibrosis progression. Mucosal-associated invariant T (MAIT) cells are non-conventional T cells that display altered functions during chronic inflammatory diseases. Here, we show that circulating MAIT cells are reduced in patients with alcoholic or non-alcoholic fatty liver disease-related cirrhosis while they accumulate in liver fibrotic septa. Using two models of chronic liver injury, we demonstrate that MAIT cell-enriched mice show increased liver fibrosis and accumulation of hepatic fibrogenic cells, whereas MAIT cell-deficient mice are resistant. Co-culture experiments indicate that MAIT cells enhance the proinflammatory properties of monocyte-derived macrophages, and promote mitogenic and proinflammatory functions of fibrogenic cells, via distinct mechanisms. Our results highlight the profibrogenic functions of MAIT cells and suggest that targeting MAIT cells may constitute an attractive antifibrogenic strategy during chronic liver injury.
Bioactive Lipid Species and Metabolic Pathways in Progression and Resolution of Nonalcoholic Steatohepatitis.
Musso Giovanni,Cassader Maurizio,Paschetta Elena,Gambino Roberto
The prevalence of nonalcoholic steatohepatitis (NASH) is increasing worldwide, yet there are no effective treatments. A decade has passed since the initial lipidomics analyses of liver tissues from patients with nonalcoholic fatty liver disease. We have learned that liver cells from patients with NASH have an abnormal lipid composition and that the accumulation of lipids leads to organelle dysfunction, cell injury and death, and chronic inflammation, called lipotoxicity. We review the lipid species and metabolic pathways that contribute to the pathogenesis of NASH and potential therapeutic targets, including enzymes involved in fatty acid and triglyceride synthesis, bioactive sphingolipids and polyunsaturated-derived eicosanoids, and specialized pro-resolving lipid mediators. We discuss the concept that NASH is a disease that can resolve and the roles of lipid molecules in the resolution of inflammation and regression of fibrosis.
Endoplasmic reticulum stress signalling and the pathogenesis of non-alcoholic fatty liver disease.
Lebeaupin Cynthia,Vallée Deborah,Hazari Younis,Hetz Claudio,Chevet Eric,Bailly-Maitre Béatrice
Journal of hepatology
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.
Urea cycle dysregulation in non-alcoholic fatty liver disease.
De Chiara Francesco,Heebøll Sara,Marrone Giusi,Montoliu Carmina,Hamilton-Dutoit Stephen,Ferrandez Antonio,Andreola Fausto,Rombouts Krista,Grønbæk Henning,Felipo Vicente,Gracia-Sancho Jordi,Mookerjee Rajeshwar P,Vilstrup Hendrik,Jalan Rajiv,Thomsen Karen Louise
Journal of hepatology
BACKGROUND & AIMS:In non-alcoholic steatohepatitis (NASH), the function of urea cycle enzymes (UCEs) may be affected, resulting in hyperammonemia and the risk of disease progression. We aimed to determine whether the expression and function of UCEs are altered in an animal model of NASH and in patients with non-alcoholic fatty liver disease (NAFLD), and whether this process is reversible. METHODS:Rats were first fed a high-fat, high-cholesterol diet for 10 months to induce NASH, before being switched onto a normal chow diet to recover. In humans, we obtained liver biopsies from 20 patients with steatosis and 15 with NASH. Primary rat hepatocytes were isolated and cultured with free fatty acids. We measured the gene and protein expression of ornithine transcarbamylase (OTC) and carbamoylphosphate synthetase (CPS1), as well as OTC activity, and ammonia concentrations. Moreover, we assessed the promoter methylation status of OTC and CPS1 in rats, humans and steatotic hepatocytes. RESULTS:In NASH animals, gene and protein expression of OTC and CPS1, and the activity of OTC, were reversibly reduced. Hypermethylation of Otc promoter genes was also observed. Additionally, in patients with NAFLD, OTC enzyme concentration and activity were reduced and ammonia concentrations were increased, which was further exacerbated in those with NASH. Furthermore, OTC and CPS1 promoter regions were hypermethylated. In primary hepatocytes, induction of steatosis was associated with Otc promoter hypermethylation, a reduction in the gene expression of Otc and Cps1, and an increase in ammonia concentration in the supernatant. CONCLUSION:NASH is associated with a reduction in the gene and protein expression, and activity, of UCEs. This results in hyperammonemia, possibly through hypermethylation of UCE genes and impairment of urea synthesis. Our investigations are the first to describe a link between NASH, the function of UCEs, and hyperammonemia, providing a novel therapeutic target. LAY SUMMARY:In patients with fatty liver disease, the enzymes that convert nitrogen waste into urea may be affected, leading to the accumulation of ammonia, which is toxic. This accumulation of ammonia can lead to scar tissue development, increasing the risk of disease progression. In this study, we show that fat accumulation in the liver produces a reversible reduction in the function of the enzymes that are involved in detoxification of ammonia. These data provide potential new targets for the treatment of fatty liver disease.
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.
Macrophage-Specific Hypoxia-Inducible Factor-1α Contributes to Impaired Autophagic Flux in Nonalcoholic Steatohepatitis.
Wang Xiaojing,de Carvalho Ribeiro Marcelle,Iracheta-Vellve Arvin,Lowe Patrick,Ambade Aditya,Satishchandran Abhishek,Bukong Terence,Catalano Donna,Kodys Karen,Szabo Gyongyi
Hepatology (Baltimore, Md.)
Inflammatory cell activation drives diverse cellular programming during hepatic diseases. Hypoxia-inducible factors (HIFs) have recently been identified as important regulators of immunity and inflammation. In nonalcoholic steatohepatitis (NASH), HIF-1α is upregulated in hepatocytes, where it induces steatosis; however, the role of HIF-1α in macrophages under metabolic stress has not been explored. In this study, we found increased HIF-1α levels in hepatic macrophages in methionine-choline-deficient (MCD) diet-fed mice and in macrophages of patients with NASH compared with controls. The HIF-1α increase was concomitant with elevated levels of autophagy markers BNIP3, Beclin-1, LC3-II, and p62 in both mouse and human macrophages. LysM HIF fl/fl mice, which have HIF-1α levels stabilized in macrophages, showed higher steatosis and liver inflammation compared with HIF fl/fl mice on MCD diet. In vitro and ex vivo experiments reveal that saturated fatty acid, palmitic acid (PA), both induces HIF-1α and impairs autophagic flux in macrophages. Using small interfering RNA-mediated knock-down and overexpression of HIF-1α in macrophages, we demonstrated that PA impairs autophagy via HIF-1α. We found that HIF-1α mediates NF-κB activation and MCP-1 production and that HIF-1α-mediated impairment of macrophage autophagy increases IL-1β production, contributing to MCD diet-induced NASH. Conclusion: Palmitic acid impairs autophagy via HIF-1α activation in macrophages. HIF-1α and impaired autophagy are present in NASH in vivo in mouse macrophages and in human blood monocytes. We identified that HIF-1α activation and decreased autophagic flux stimulate inflammation in macrophages through upregulation of NF-κB activation. These results suggest that macrophage activation in NASH involves a complex interplay between HIF-1α and autophagy as these pathways promote proinflammatory overactivation in MCD diet-induced NASH.
miRNAs in patients with non-alcoholic fatty liver disease: A systematic review and meta-analysis.
Liu Chang-Hai,Ampuero Javier,Gil-Gómez Antonio,Montero-Vallejo Rocío,Rojas Ángela,Muñoz-Hernández Rocío,Gallego-Durán Rocío,Romero-Gómez Manuel
Journal of hepatology
BACKGROUND & AIMS:microRNAs (miRNAs) are deregulated in non-alcoholic fatty liver disease (NAFLD) and have been proposed as useful markers for the diagnosis and stratification of disease severity. We conducted a meta-analysis to identify the potential usefulness of miRNA biomarkers in the diagnosis and stratification of NAFLD severity. METHODS:After a systematic review, circulating miRNA expression consistency and mean fold-changes were analysed using a vote-counting strategy. The sensitivity, specificity, positive and negative likelihood ratios, diagnostic odds ratio and area under the curve (AUC) for the diagnosis of NAFLD or non-alcoholic steatohepatitis (NASH) were pooled using a bivariate meta-analysis. Deeks' funnel plot was used to assess the publication bias. RESULTS:Thirty-seven studies of miRNA expression profiles and six studies of diagnostic accuracy were ultimately included in the quantitative analysis. miRNA-122 and miRNA-192 showed consistent upregulation. miRNA-122 was upregulated in every scenario used to distinguish NAFLD severity. The miRNA expression correlation between the serum and liver tissue was inconsistent across studies. miRNA-122 distinguished NAFLD from healthy controls with an AUC of 0.82 (95% CI 0.75-0.89), and miRNA-34a distinguished non-alcoholic steatohepatitis (NASH) from non-alcoholic fatty liver (NAFL) with an AUC of 0.78 (95% CI 0.67-0.88). CONCLUSION:miRNA-34a, miRNA-122 and miRNA-192 were identified as potential diagnostic markers to segregate NAFL from NASH. Both miRNA-122, in distinguishing NAFLD from healthy controls, and miRNA-34a, in distinguishing NASH from NAFL, showed moderate diagnostic accuracy. miRNA-122 was upregulated in every scenario of NAFL, NASH and fibrosis. LAY SUMMARY: microRNAs are deregulated in non-alcoholic fatty liver disease. The microRNAs, miRNA-34a, miRNA-122 and miRNA-192, were identified as potential biomarkers of non-alcoholic fatty liver and non-alcoholic steatohepatitis, at different stages of disease severity. The correlation between miRNA expression in the serum and in liver tissue was inconsistent, or even inverse.
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.
Small metabolites, possible big changes: a microbiota-centered view of non-alcoholic fatty liver disease.
Chu Huikuan,Duan Yi,Yang Ling,Schnabl Bernd
The spectrum of non-alcoholic fatty liver disease (NAFLD) ranges from simple hepatic steatosis, commonly associated with obesity, to non-alcoholic steatohepatitis, which can progress to fibrosis, cirrhosis and hepatocellular carcinoma. NAFLD pathophysiology involves environmental, genetic and metabolic factors, as well as changes in the intestinal microbiota and their products. Dysfunction of the intestinal barrier can contribute to NAFLD development and progression. Although there are technical limitations in assessing intestinal permeability in humans and the number of patients in these studies is rather small, fewer than half of the patients have increased intestinal permeability and translocation of bacterial products. Microbe-derived metabolites and the signalling pathways they affect might play more important roles in development of NAFLD. We review the microbial metabolites that contribute to the development of NAFLD, such as trimethylamine, bile acids, short-chain fatty acids and ethanol. We discuss the mechanisms by which metabolites produced by microbes might affect disease progression and/or serve as therapeutic targets or biomarkers for NAFLD.
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.
MicroRNA-378 promotes hepatic inflammation and fibrosis via modulation of the NF-κB-TNFα pathway.
Zhang Tianpeng,Hu Junjie,Wang Xiaomei,Zhao Xiaoling,Li Zhuoyu,Niu Junqi,Steer Clifford J,Zheng Guohua,Song Guisheng
Journal of hepatology
BACKGROUND & AIMS:The progression of hepatosteatosis to non-alcoholic steatohepatitis (NASH) is a critical step in the pathogenesis of hepatocellular cancer. However, the underlying mechanism(s) for this progression is essentially unknown. This study was designed to determine the role of miR-378 in regulating NASH progression. METHODS:We used immunohistochemistry, luciferase assays and immunoblotting to study the role of miR-378 in modulating an inflammatory pathway. Wild-type mice kept on a high-fat diet (HFD) were injected with miR-378 inhibitors or a mini-circle expression system containing miR-378, to study loss and gain-of functions of miR-378. RESULTS:MiR-378 expression is increased in livers of dietary obese mice and patients with NASH. Further studies revealed that miR-378 directly targeted Prkag2 that encodes AMP-activated protein kinase γ 2 (AMPKγ2). AMPK signaling negatively regulates the NF-κB-TNFα inflammatory axis by increasing deacetylase activity of sirtuin 1. By targeting Prkag2, miR-378 reduced sirtuin 1 activity and facilitated an inflammatory pathway involving NF-κB-TNFα. In contrast, miR-378 knockdown induced expression of Prkag2, increased sirtuin 1 activity and blocked the NF-κB-TNFα axis. Additionally, knockdown of increased Prkag2 offset the inhibitory effects of miR-378 inhibitor on the NF-κB-TNFα axis, suggesting that AMPK signaling mediates the role of miR-378 in facilitating this inflammatory pathway. Liver-specific expression of miR-378 triggered the development of NASH and fibrosis by activating TNFα signaling. Ablation of TNFα in miR-378-treated mice impaired the ability of miR-378 to facilitate hepatic inflammation and fibrosis, suggesting that TNFα signaling is required for miR-378 to promote NASH. CONCLUSION:MiR-378 plays a key role in the development of hepatic inflammation and fibrosis by positively regulating the NF-κB-TNFα axis. MiR-378 is a potential therapeutic target for the treatment of NASH. LAY SUMMARY:The recent epidemic of obesity has been associated with a sharp rise in the incidence of non-alcoholic fatty liver disease (NAFLD). However, the underlying mechanism(s) remains poorly described and effective therapeutic approaches against NAFLD are lacking. The results establish that microRNA-378 facilitates the development of hepatic inflammation and fibrosis and suggests the therapeutic potential of microRNA-378 inhibitor for the treatment of 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.
Inhibition of Acetyl-CoA Carboxylase by Phosphorylation or the Inhibitor ND-654 Suppresses Lipogenesis and Hepatocellular Carcinoma.
The incidence of hepatocellular carcinoma (HCC) is rapidly increasing due to the prevalence of obesity and non-alcoholic fatty liver disease, but the molecular triggers that initiate disease development are not fully understood. We demonstrate that mice with targeted loss-of-function point mutations within the AMP-activated protein kinase (AMPK) phosphorylation sites on acetyl-CoA carboxylase 1 (ACC1 Ser79Ala) and ACC2 (ACC2 Ser212Ala) have increased liver de novo lipogenesis (DNL) and liver lesions. The same mutation in ACC1 also increases DNL and proliferation in human liver cancer cells. Consistent with these findings, a novel, liver-specific ACC inhibitor (ND-654) that mimics the effects of ACC phosphorylation inhibits hepatic DNL and the development of HCC, improving survival of tumor-bearing rats when used alone and in combination with the multi-kinase inhibitor sorafenib. These studies highlight the importance of DNL and dysregulation of AMPK-mediated ACC phosphorylation in accelerating HCC and the potential of ACC inhibitors for treatment.
LXRα Promotes Hepatosteatosis in Part Through Activation of MicroRNA-378 Transcription and Inhibition of Ppargc1β Expression.
Zhang Tianpeng,Duan Jiangyan,Zhang Lei,Li Zhuoyu,Steer Clifford J,Yan Guiqin,Song Guisheng
Hepatology (Baltimore, Md.)
Nonalcoholic fatty liver disease (NAFLD) is a major risk factor of many end-stage liver diseases. Alterations in microRNA expression have been reported in patients with NAFLD. However, the transcriptional mechanism(s) of dysregulated microRNAs under the state of NAFLD is poorly described, and microRNAs that regulate the pathogenesis of NAFLD synergistically with their regulators remain unknown. Here we report that microRNA-378 expression is significantly increased in fatty livers of mice and patients with NAFLD. Although microRNA-378 locates within the intron of Ppargc1β (peroxisome proliferator-activated receptor γ coactivator 1-beta), there was a significant uncoupling of Ppargc1β mRNA and microRNA-378 levels in both sources of fatty livers. Further studies identified a full-length primary transcript of microRNA-378. LXRα (liver X receptor alpha) functioned as a transcription activator of microRNA-378 and a repressor of Ppargc1β transcription. It is known that miR-378 is an inhibitor of fatty acid oxidation (FAO) and the function of Ppargc1β is opposite to that of miR-378. GW3965 treatment (LXRα agonist) of murine hepatocytes and mice increased microRNA-378 and reduced Ppargc1β, which subsequently impaired FAO and aggravated hepatosteatosis. In contrast, additional treatment of miR-378 inhibitor or Ppargc1β, which knocked down increased miR-378 or recovered expression of Ppargc1β, offset the effects of GW3965. Liver-specific ablation of Lxrα led to decreased miR-378 and increased Ppargc1β, which subsequently improved FAO and reduced hepatosteatosis. Conclusion: Our findings indicated that miR-378 possesses its own transcription machinery, which challenges the well-established dogma that miR-378 transcription is controlled by the promoter of Ppargc1β. LXRα selectively activates transcription of miR-378 and inhibits expression of Ppargc1β, which synergistically impairs FAO. In addition to lipogenesis, impaired FAO by miR-378 in part contributes to LXRα-induced hepatosteatosis.
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.
Dietary cholesterol promotes steatohepatitis related hepatocellular carcinoma through dysregulated metabolism and calcium signaling.
Liang Jessie Qiaoyi,Teoh Narcissus,Xu Lixia,Pok Sharon,Li Xiangchun,Chu Eagle S H,Chiu Jonathan,Dong Ling,Arfianti Evi,Haigh W Geoffrey,Yeh Matthew M,Ioannou George N,Sung Joseph J Y,Farrell Geoffrey,Yu Jun
The underlining mechanisms of dietary cholesterol and nonalcoholic steatohepatitis (NASH) in contributing to hepatocellular carcinoma (HCC) remain undefined. Here we demonstrated that high-fat-non-cholesterol-fed mice developed simple steatosis, whilst high-fat-high-cholesterol-fed mice developed NASH. Moreover, dietary cholesterol induced larger and more numerous NASH-HCCs than non-cholesterol-induced steatosis-HCCs in diethylnitrosamine-treated mice. NASH-HCCs displayed significantly more aberrant gene expression-enriched signaling pathways and more non-synonymous somatic mutations than steatosis-HCCs (335 ± 84/sample vs 43 ± 13/sample). Integrated genetic and expressional alterations in NASH-HCCs affected distinct genes pertinent to five pathways: calcium, insulin, cell adhesion, axon guidance and metabolism. Some of the novel aberrant gene expression, mutations and core oncogenic pathways identified in cholesterol-associated NASH-HCCs in mice were confirmed in human NASH-HCCs, which included metabolism-related genes (ALDH18A1, CAD, CHKA, POLD4, PSPH and SQLE) and recurrently mutated genes (RYR1, MTOR, SDK1, CACNA1H and RYR2). These findings add insights into the link of cholesterol to NASH and NASH-HCC and provide potential therapeutic targets.
A Regulatory Role of Apoptosis Antagonizing Transcription Factor in the Pathogenesis of Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma.
Kumar Divya P,Santhekadur Prasanna K,Seneshaw Mulugeta,Mirshahi Faridoddin,Uram-Tuculescu Cora,Sanyal Arun J
Hepatology (Baltimore, Md.)
Hepatocellular carcinoma (HCC) is increasing as a cause of liver-related mortality largely because of the growing burden of nonalcoholic steatohepatitis (NASH). The mechanisms of HCC development in nonalcoholic fatty liver disease (NAFLD) are incompletely understood. We initially identified apoptosis antagonizing transcription factor (AATF) to be associated with HCC in a mouse model of NASH that develops HCC without the addition of specific carcinogens. AATF, also called che-1, is a transcriptional factor that is highly conserved among eukaryotes. AATF is known to be a central mediator of the cellular responses as it promotes cell proliferation and survival by inducing cell cycle arrest, autophagy, DNA repair, and inhibition of apoptosis. However, the role of AATF in NASH and HCC remains unknown. Here, we provide evidence for AATF as a contributory factor for HCC in NAFLD. AATF overexpression was further verified in human NASH and HCC and multiple human HCC cell lines. Tumor necrosis factor-α (TNFα), known to be increased in NASH, induced AATF expression. Promoter analysis of AATF revealed a sterol regulatory element binding transcription factor 1-c (SREBP-1c) binding site; inhibition of SREBP-1 by using specific inhibitors as well as small interfering RNA decreased TNFα-induced AATF expression. AATF interacted with signal transducer and activator of transcription 3 to increase monocyte chemoattractant protein-1 expression. AATF knockdown decreased cell proliferation, migration, invasion, colony formation, and anchorage-dependent growth in HCC cell lines. Xenograft of QGY-7703 HCC cells with AATF stably knocked down into nonobese diabetic scid gamma mice demonstrated reduced tumorigenesis and metastases. Conclusion: AATF drives NAFLD and hepatocarcinogenesis, offering a potential target for therapeutic intervention.
17-Beta Hydroxysteroid Dehydrogenase 13 Is a Hepatic Retinol Dehydrogenase Associated With Histological Features of Nonalcoholic Fatty Liver Disease.
Ma Yanling,Belyaeva Olga V,Brown Philip M,Fujita Koji,Valles Katherine,Karki Suman,de Boer Ynto S,Koh Christopher,Chen Yanhua,Du Xiaomeng,Handelman Samuel K,Chen Vincent,Speliotes Elizabeth K,Nestlerode Cara,Thomas Emmanuel,Kleiner David E,Zmuda Joseph M,Sanyal Arun J, ,Kedishvili Natalia Y,Liang T Jake,Rotman Yaron
Hepatology (Baltimore, Md.)
Nonalcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease. A single-nucleotide polymorphism (SNP), rs6834314, was associated with serum liver enzymes in the general population, presumably reflecting liver fat or injury. We studied rs6834314 and its nearest gene, 17-beta hydroxysteroid dehydrogenase 13 (HSD17B13), to identify associations with histological features of NAFLD and to characterize the functional role of HSD17B13 in NAFLD pathogenesis. The minor allele of rs6834314 was significantly associated with increased steatosis but decreased inflammation, ballooning, Mallory-Denk bodies, and liver enzyme levels in 768 adult Caucasians with biopsy-proven NAFLD and with cirrhosis in the general population. We found two plausible causative variants in the HSD17B13 gene. rs72613567, a splice-site SNP in high linkage with rs6834314 (r = 0.94) generates splice variants and shows a similar pattern of association with NAFLD histology. Its minor allele generates simultaneous expression of exon 6-skipping and G-nucleotide insertion variants. Another SNP, rs62305723 (encoding a P260S mutation), is significantly associated with decreased ballooning and inflammation. Hepatic expression of HSD17B13 is 5.9-fold higher (P = 0.003) in patients with NAFLD. HSD17B13 is targeted to lipid droplets, requiring the conserved amino acid 22-28 sequence and amino acid 71-106 region. The protein has retinol dehydrogenase (RDH) activity, with enzymatic activity dependent on lipid droplet targeting and cofactor binding site. The exon 6 deletion, G insertion, and naturally occurring P260S mutation all confer loss of enzymatic activity. Conclusion: We demonstrate the association of variants in HSD17B13 with specific features of NAFLD histology and identify the enzyme as a lipid droplet-associated RDH; our data suggest that HSD17B13 plays a role in NAFLD through its enzymatic activity.
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.
Hepatocyte Notch activation induces liver fibrosis in nonalcoholic steatohepatitis.
Science translational medicine
Fibrosis is the major determinant of morbidity and mortality in patients with nonalcoholic steatohepatitis (NASH) but has no approved pharmacotherapy in part because of incomplete understanding of its pathogenic mechanisms. Here, we report that hepatocyte Notch activity tracks with disease severity and treatment response in patients with NASH and is similarly increased in a mouse model of diet-induced NASH and liver fibrosis. Hepatocyte-specific Notch loss-of-function mouse models showed attenuated NASH-associated liver fibrosis, demonstrating causality to obesity-induced liver pathology. Conversely, forced activation of hepatocyte Notch induced fibrosis in both chow- and NASH diet-fed mice by increasing Sox9-dependent Osteopontin (Opn) expression and secretion from hepatocytes, which activate resident hepatic stellate cells. In a cross-sectional study, we found that OPN explains the positive correlation between liver Notch activity and fibrosis stage in patients. Further, we developed a Notch inhibitor [ antisense oligonucleotide ( ASO)] that reduced fibrosis in NASH diet-fed mice. In summary, these studies demonstrate the pathological role and therapeutic accessibility of the maladaptive hepatocyte Notch response in NASH-associated liver fibrosis.
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.
Thioesterase Superfamily Member 2 Promotes Hepatic VLDL Secretion by Channeling Fatty Acids Into Triglyceride Biosynthesis.
Alves-Bezerra Michele,Li Yingxia,Acuña Mariana,Ivanova Anna A,Corey Kathleen E,Ortlund Eric A,Cohen David E
Hepatology (Baltimore, Md.)
In nonalcoholic fatty liver disease (NAFLD), triglycerides accumulate within the liver because the rates of fatty acid accrual by uptake from plasma and de novo synthesis exceed elimination by mitochondrial oxidation and secretion as very low-density lipoprotein (VLDL) triglycerides. Thioesterase superfamily member 2 (Them2) is an acyl-coenzyme A (CoA) thioesterase that catalyzes the hydrolysis of fatty acyl-CoAs into free fatty acids plus CoASH. Them2 is highly expressed in the liver, as well as other oxidative tissues. Mice globally lacking Them2 are resistant to diet-induced obesity and hepatic steatosis, and exhibit improved glucose homeostasis. These phenotypes are attributable, at least in part, to roles of Them2 in the suppression of thermogenesis in brown adipose tissue and insulin signaling in skeletal muscle. To elucidate the hepatic function of Them2, we created mice with liver-specific deletion of Them2 (L-Them2 ). Although L-Them2 mice were not protected against excess weight gain, hepatic steatosis or glucose intolerance, they exhibited marked decreases in plasma triglyceride and apolipoprotein B100 concentrations. These were attributable to reduced rates of VLDL secretion owing to decreased incorporation of plasma-derived fatty acids into triglycerides. The absence of hepatic steatosis in L-Them2 mice fed chow was explained by compensatory increases in rates of fatty acid oxidation and by decreased de novo lipogenesis in high fat-fed mice. Consistent with a role for Them2 in hepatic VLDL secretion, THEM2 levels were increased in livers of obese patients with NAFLD characterized by simple steatosis. Conclusion: Them2 functions in the liver to direct fatty acids toward triglyceride synthesis for incorporation into VLDL particles. When taken together with its functions in brown adipose and muscle, these findings suggest that Them2 is a target for the management of NAFLD and dyslipidemia.
Metabolic aspects in NAFLD, NASH and hepatocellular carcinoma: the role of PGC1 coactivators.
Piccinin Elena,Villani Gaetano,Moschetta Antonio
Nature reviews. Gastroenterology & hepatology
Alterations of hepatic metabolism are critical to the development of liver disease. The peroxisome proliferator-activated receptor-γ coactivators (PGC1s) are able to orchestrate, on a transcriptional level, different aspects of liver metabolism, such as mitochondrial oxidative phosphorylation, gluconeogenesis and fatty acid synthesis. As modifications affecting both mitochondrial and lipid metabolism contribute to the initiation and/or progression of liver steatosis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC), a link between disrupted PGC1 pathways and onset of these pathological conditions has been postulated. However, despite the large quantity of studies, the scenario is still not completely understood, and some issues remain controversial. Here, we discuss the roles of PGC1s in healthy liver and explore their contribution to the pathogenesis and future therapy of NASH and HCC.
Modulation of Insulin Resistance in Nonalcoholic Fatty Liver Disease.
Khan Reenam S,Bril Fernando,Cusi Kenneth,Newsome Philip N
Hepatology (Baltimore, Md.)
Nonalcoholic fatty liver disease (NAFLD) has an estimated prevalence of 25% in the general population, and cirrhosis secondary to nonalcoholic steatohepatitis (NASH) is predicted to become the leading cause of liver transplantation, yet there is a lack of effective licensed treatments for these conditions. There is a close relationship between insulin resistance (IR) and NAFLD, with prevalence of NAFLD being 5-fold higher in patients with diabetes compared to those without. IR is implicated both in pathogenesis of NAFLD and in disease progression from steatosis to NASH. Thus, modulation of IR represents a potential strategy for NAFLD treatment. This review highlights key proposed mechanisms linking IR and NAFLD, such as changes in rates of adipose tissue lipolysis and de novo lipogenesis, impaired mitochondrial fatty acid β-oxidation (FAO), changes in fat distribution, alterations in the gut microbiome, and alterations in levels of adipokines and cytokines. Furthermore, this review will discuss the main pharmacological strategies used to treat IR in patients with NAFLD and their efficacy based on recently published experimental and clinical data. These include biguanides, glucagon-like peptide 1 receptor (GLP-1) agonists, dipeptidyl peptidase 4 (DPP-4) inhibitors, peroxisome proliferator-activated receptor (PPAR-γ/α/δ) agonists, sodium glucose cotransporter 2 (SGLT2) inhibitors, and farnesoid X receptor (FXR) agonists, with further novel treatments on the horizon. Ideally, treatment would improve IR, reduce cardiovascular risk, and produce demonstrable improvements in NASH histology-this is likely to be achieved with a combinatorial approach.
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.
Dual-Specificity Phosphatase 26 Protects Against Nonalcoholic Fatty Liver Disease in Mice Through Transforming Growth Factor Beta-Activated Kinase 1 Suppression.
Ye Ping,Liu Jijun,Xu Wuping,Liu Denghai,Ding Xiangchao,Le Sheng,Zhang Hao,Chen Shanshan,Chen Manhua,Xia Jiahong
Hepatology (Baltimore, Md.)
Nonalcoholic fatty liver disease (NAFLD), which has a wide global distribution, includes different stages ranging from simple steatosis to nonalcoholic steatohepatitis, advanced fibrosis, and liver cirrhosis according to the degree of severity. Chronic low-grade inflammation, insulin resistance, and lipid accumulation are the leading causes of NAFLD. To date, no effective medicine for NAFLD has been approved by governmental agencies. Our study demonstrated that the expression of dual-specificity phosphatase 26 (Dusp26), a member of the Dusp protein family, was decreased in the liver tissue of mice with hepatic steatosis and genetically obese (ob/ob) mice. In our study, hepatic steatosis, inflammatory responses, and insulin resistance were exacerbated in liver-specific Dusp26-knockout (KO) mice but ameliorated in liver-specific Dusp26-transgenic mice induced by a high-fat diet. In addition, the degree of liver fibrosis was aggravated in high-fat high-cholesterol diet-induced Dusp26-KO mice. We further found that the binding of Dusp26 to transforming growth factor beta-activated kinase 1 (TAK1) to block the phosphorylation of TAK1 regulated the TAK1-p38/c-Jun NH2-terminal kinase signaling axis to alleviate hepatic steatosis and metabolic disturbance. Conclusion: These findings suggest that Dusp26 is a good TAK1-dependent therapeutic target for NAFLD.
Deficiency of Mitochondrial Glycerol 3-Phosphate Dehydrogenase Contributes to Hepatic Steatosis.
Zheng Yi,Qu Hua,Xiong Xin,Wang Yuren,Liu Xiufei,Zhang Linlin,Liao Xiaoyu,Liao Qian,Sun Zheng,Ouyang Qin,Yang Gangyi,Zhu Zhiming,Xu Jing,Zheng Hongting
Hepatology (Baltimore, Md.)
Mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) is an integral component of the respiratory chain, and recent studies have suggested that it plays an important role in hepatic glucose homeostasis. However, its function in hepatic lipid metabolism is unclear. Here, we identified a role for mGPDH in nonalcoholic fatty liver disease (NAFLD). Specifically, mGPDH expression and activity were lower in fatty livers from patients and mice with NAFLD (ob/ob, high-fat diet [HFD] and db/db). Liver-specific depletion of mGPDH in mice or mGPDH knockdown in cultured hepatocytes exacerbated diet-induced triglyceride accumulation and steatosis through enhanced lipogenesis. RNA-sequencing revealed that mGPDH regulated endoplasmic reticulum (ER)-related proteins and processes. mGPDH deletion exacerbated tunicamycin (ER stress inducer)-induced hepatic steatosis, whereas tauroursodeoxycholic acid (ER stress inhibitor) rescued mGPDH depletion-induced steatosis on an HFD. Moreover, ER stress induced by mGPDH depletion could be abrogated by the intracellular Ca chelator 1,2-bis (2-aminophenoxy) ethane N,N,N´,N´-tetraacetic acid acetoxymethyl ester, mitochondrial permeability transition pore (mPTP) inhibitor cyclosporine A, or cyclophilin-D (Cyp-D) knockdown. mGPDH promoting Cyp-D ubiquitination was also observed. Finally, liver-specific mGPDH overexpression attenuated hepatic steatosis in ob/ob and HFD mice. Conclusion: mGPDH is a pivotal regulator of hepatic lipid metabolism. Its deficiency induces ER stress by suppressing Cyp-D ubiquitination, a key regulator of the mitochondrial Ca conductance channel mPTP, and results in hepatic steatosis. mGPDH may be a potential therapeutic target for the treatment of NAFLD.
The Role of Innate Immune Cells in Nonalcoholic Steatohepatitis.
Cai Jingjing,Zhang Xiao-Jing,Li Hongliang
Hepatology (Baltimore, Md.)
Inflammation and metabolic dysfunction are hallmarks of nonalcoholic steatohepatitis (NASH), which is one of the fastest-growing liver diseases worldwide. Emerging evidence indicates that innate immune mechanisms are pivotal drivers of inflammation and other pathological manifestations observed in NASH, such as hepatosteatosis, insulin resistance (IR), and fibrosis. This robust innate immune reaction is intrinsic to the liver, which is an important immunological organ that contains a coordinated network of innate immune cells, including Kupffer cells (KCs), dendritic cells (DCs), and lymphocytes. Hepatocytes and liver sinusoidal endothelial cells (LSECs) are not formally innate immune cells, but they take on immune cell function when stressed. These cells can sense excess metabolites and bacterial products and translate those signals into immune responses and pathological hepatic changes during the development of NASH. In this review, we take a historical perspective in describing decades of research that aimed to identify the key molecular and cellular players in the innate immune system in the setting of NASH. Furthermore, we summarize the innate immune cells that are involved in the progression of NASH and illustrate how they sense disturbances in circulating metabolic factors by innate immune receptors and subsequently initiate the intercellular signaling cascades that lead to persistent inflammation and progression of hepatic complications.
F-box/WD Repeat-Containing Protein 5 Mediates the Ubiquitination of Apoptosis Signal-Regulating Kinase 1 and Exacerbates Nonalcoholic Steatohepatitis in Mice.
Bai Lan,Chen Ming-Ming,Chen Ze-Dong,Zhang Peng,Tian Song,Zhang Yan,Zhu Xue-Yong,Liu Ye,She Zhi-Gang,Ji Yan-Xiao,Li Hongliang
Hepatology (Baltimore, Md.)
Inhibition of apoptosis signal-regulating kinase 1 (ASK1) activation has emerged as a promising target for the treatment of nonalcoholic steatohepatitis (NASH). Multiple forms of posttranslational modifications determine the activity of ASK1. In addition to phosphorylation, recent studies revealed that ubiquitination is essential for ASK1 activation. However, the endogenous factor that regulates ASK1 ubiquitination and activation remains poorly defined. In this study, we identified the E3 ligase Skp1-Cul1-F-box (SCF) protein F-box/WD repeat-containing protein 5 (FBXW5) as a key endogenous activator of ASK1 ubiquitination. FBXW5 is the central component of the SCF complex (SCF ) that directly interacts with and ubiquitinates ASK1 in hepatocytes during NASH development. An in vivo study showed that hepatocyte-specific overexpression of FBXW5 exacerbated diet-induced systemic and hepatic metabolic disorders, as well as the activation of ASK1-related mitogen-activated protein kinase (MAPK) signaling in the liver. Conversely, hepatocyte-specific deletion of FBXW5 significantly prevented the progression of these abnormalities. Mechanically, FBXW5 facilitated the addition of Lys63-linked ubiquitin to ASK1 and thus exacerbated ASK1-c-Jun N-terminal kinase/p38 MAPK signaling, inflammation, and lipid accumulation. Furthermore, we demonstrated that the N-terminus (S1) and C-terminus (S3) of FBXW5 respectively and competitively ablate the function of FBXW5 on ASK1 activation and served as effective inhibitors of NASH progression. Conclusion: This evidence strongly suggests that SCF is an important activator of ASK1 ubiquitination in the context of NASH. The development of FBXW5(S1) or FBXW5(S3)-mimicking drugs and screening of small-molecular inhibitors specifically abrogating ASK1 ubiquitination-dependent activation are viable approaches for NASH treatment.
Pacer Is a Mediator of mTORC1 and GSK3-TIP60 Signaling in Regulation of Autophagosome Maturation and Lipid Metabolism.
Cheng Xiawei,Ma Xiuling,Zhu Qi,Song Dandan,Ding Xianming,Li Lin,Jiang Xiao,Wang Xinyi,Tian Rui,Su Hua,Shen Zhirong,Chen She,Liu Ting,Gong Weihua,Liu Wei,Sun Qiming
mTORC1 and GSK3 play critical roles in early stages of (macro)autophagy, but how they regulate late steps of autophagy remains poorly understood. Here we show that mTORC1 and GSK3-TIP60 signaling converge to modulate autophagosome maturation through Pacer, an autophagy regulator that was identified in our recent study. Hepatocyte-specific Pacer knockout in mice results in impaired autophagy flux, glycogen and lipid accumulation, and liver fibrosis. Under nutrient-rich conditions, mTORC1 phosphorylates Pacer at serine157 to disrupt the association of Pacer with Stx17 and the HOPS complex and thus abolishes Pacer-mediated autophagosome maturation. Importantly, dephosphorylation of Pacer under nutrient-deprived conditions promotes TIP60-mediated Pacer acetylation, which facilitates HOPS complex recruitment and is required for autophagosome maturation and lipid droplet clearance. This work not only identifies Pacer as a regulator in hepatic autophagy and liver homeostasis in vivo but also reveals a signal integration mechanism involved in late stages of autophagy and lipid metabolism.
Hepatic Interferon Regulatory Factor 6 Alleviates Liver Steatosis and Metabolic Disorder by Transcriptionally Suppressing Peroxisome Proliferator-Activated Receptor γ in Mice.
Tong Jingjing,Han Cui-Juan,Zhang Jia-Zhen,He Wen-Zhi,Zhao Guo-Jun,Cheng Xu,Zhang Lei,Deng Ke-Qiong,Liu Ye,Fan Hui-Fen,Tian Song,Cai Jingjing,Huang Zan,She Zhi-Gang,Zhang Peng,Li Hongliang
Hepatology (Baltimore, Md.)
Nonalcoholic fatty liver disease (NAFLD) has become a worldwide epidemic. A large and growing unmet therapeutic need has inspired numerous studies in the field. Integrating the published genomic data available in the Gene Expression Omnibus (GEO) with NAFLD samples from rodents, we discovered that interferon regulatory factor 6 (IRF6) is significantly downregulated in high-fat diet (HFD)-induced fatty liver. In the current study, we identified IRF6 in hepatocytes as a protective factor in liver steatosis (LS). During HFD challenge, hepatic Irf6 was suppressed by promoter hypermethylation. Severity of HFD-induced LS was exacerbated in hepatocyte-specific Irf6 knockout mice, whereas hepatocyte-specific transgenic mice overexpressing Irf6 (IRF6-HTG) exhibited alleviated steatosis and metabolic disorder in response to HFD feeding. Mechanistic studies in vitro demonstrated that hepatocyte IRF6 directly binds to the promoter of the peroxisome proliferator-activated receptor γ (PPARγ) gene and subsequently halts the transcription of Pparγ and its target genes (e.g., genes that regulate lipogenesis and lipid acid uptake) under physiological conditions. Conclusion: Irf6 is downregulated by promoter hypermethylation upon metabolic stimulus exposure, which fail to inhibit Pparγ and its targets, driving abnormalities of lipid metabolism.
Role of liver sinusoidal endothelial cells in non-alcoholic fatty liver disease.
Hammoutene Adel,Rautou Pierre-Emmanuel
Journal of hepatology
Non-alcoholic fatty liver disease (NAFLD) and its complications are an expanding health problem associated with the metabolic syndrome. Liver sinusoidal endothelial cells (LSECs) are highly specialized endothelial cells localized at the interface between the blood derived from the gut and the adipose tissue on the one side, and other liver cells on the other side. In physiological conditions, LSECs are gatekeepers of liver homeostasis. LSECs display anti-inflammatory and anti-fibrogenic properties by preventing Kupffer cell and hepatic stellate cell activation and regulating intrahepatic vascular resistance and portal pressure. This review focusses on changes occurring in LSECs in NAFLD and on their consequences on NAFLD progression and complications. Capillarization, namely the loss of LSEC fenestrae, and LSEC dysfunction, namely the loss of the ability of LSECs to generate vasodilator agents in response to increased shear stress both occur early in NAFLD. These LSEC changes favour steatosis development and set the stage for NAFLD progression. At the stage of non-alcoholic steatohepatitis, altered LSECs release inflammatory mediators and contribute to the recruitment of inflammatory cells, thus promoting liver injury and inflammation. Altered LSECs also fail to maintain hepatic stellate cell quiescence and release fibrogenic mediators, including Hedgehog signalling molecules, promoting liver fibrosis. Liver angiogenesis is increased in NAFLD and contributes to liver inflammation and fibrosis, but also to hepatocellular carcinoma development. Thus, improving LSEC health appears to be a promising approach to prevent NAFLD progression and complications.
A maresin 1/RORα/12-lipoxygenase autoregulatory circuit prevents inflammation and progression of nonalcoholic steatohepatitis.
Han Yong-Hyun,Shin Kyong-Oh,Kim Ju-Yeon,Khadka Daulat B,Kim Hyeon-Ji,Lee Yong-Moon,Cho Won-Jea,Cha Ji-Young,Lee Bong-Jin,Lee Mi-Ock
The Journal of clinical investigation
Retinoic acid-related orphan receptor α (RORα) is considered a key regulator of polarization in liver macrophages that is closely related to nonalcoholic steatohepatitis (NASH) pathogenesis. However, hepatic microenvironments that support the function of RORα as a polarity regulator were largely unknown. Here, we identified maresin 1 (MaR1), a docosahexaenoic acid (DHA) metabolite with a function of specialized proresolving mediator, as an endogenous ligand of RORα. MaR1 enhanced the expression and transcriptional activity of RORα and thereby increased the M2 polarity of liver macrophages. Administration of MaR1 protected mice from high-fat diet-induced NASH in a RORα-dependent manner. Surprisingly, RORα increased the level of MaR1 through transcriptional induction of 12-lipoxygenase (12-LOX), a key enzyme in MaR1 biosynthesis. Furthermore, we demonstrated that modulation of 12-LOX activity enhanced the protective function of DHA against NASH. Together, these results suggest that the MaR1/RORα/12-LOX autoregulatory circuit could offer potential therapeutic strategies for curing NASH.
Prevalence, incidence, and outcome of non-alcoholic fatty liver disease in Asia, 1999-2019: a systematic review and meta-analysis.
Li Jie,Zou Biyao,Yeo Yee Hui,Feng Yuemin,Xie Xiaoyu,Lee Dong Hyun,Fujii Hideki,Wu Yuankai,Kam Leslie Y,Ji Fanpu,Li Xiaohe,Chien Nicholas,Wei Mike,Ogawa Eiichi,Zhao Changqing,Wu Xia,Stave Christopher D,Henry Linda,Barnett Scott,Takahashi Hirokazu,Furusyo Norihiro,Eguchi Yuichiro,Hsu Yao-Chun,Lee Teng-Yu,Ren Wanhua,Qin Chengyong,Jun Dae Won,Toyoda Hidenori,Wong Vincent Wai-Sun,Cheung Ramsey,Zhu Qiang,Nguyen Mindie H
The lancet. Gastroenterology & hepatology
BACKGROUND:Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease worldwide. Asia is a large, heterogeneous area with substantial variation in socioeconomic status and prevalence of obesity. We estimated the prevalence, incidence, and outcomes of NAFLD in the Asian population to assist stakeholders in understanding NAFLD disease burden. METHODS:We searched PubMed, EMBASE, and the Cochrane Library from database inception to Jan 17, 2019, for studies reporting NAFLD prevalence, incidence, or outcome in Asia. We included only cross-sectional and longitudinal observational studies of patients with NAFLD diagnosed by imaging, serum-based indices, or liver biopsy. Studies that included patients with overlapping liver disease or that did not screen for excess alcohol consumption were excluded. Two investigators independently screened and extracted data. The main outcomes were pooled NAFLD prevalence, incidence, and hepatocellular carcinoma incidence and overall mortality in patients with NAFLD. Summary estimates were calculated using a random-effects model. This study is registered with PROSPERO, number CRD42018088468. FINDINGS:Of 4995 records identified, 237 studies (13 044 518 participants) were included for analysis. The overall prevalence of NAFLD regardless of diagnostic method was 29·62% (95% CI 28·13-31·15). NAFLD prevalence increased significantly over time (25·28% [22·42-28·37] between 1999 and 2005, 28·46% [26·70-30·29] between 2006 and 2011, and 33·90% [31·74-36·12] between 2012 and 2017; p<0·0001). The pooled annual NAFLD incidence rate was 50·9 cases per 1000 person-years (95% CI 44·8-57·4). In patients with NAFLD, the annual incidence of hepatocellular carcinoma was 1·8 cases per 1000 person-years (0·8-3·1) and overall mortality rate was 5·3 deaths per 1000 person-years (1·5-11·4). INTERPRETATION:NAFLD prevalence in Asia is increasing and is associated with poor outcomes including hepatocellular carcinoma and death. Targeted public health strategies must be developed in Asia to target the drivers of this rising epidemic and its associated complications, especially in high-risk groups, such as older obese men. FUNDING:None.
Macrophage p38α promotes nutritional steatohepatitis through M1 polarization.
Zhang Xiang,Fan Lina,Wu Jianfeng,Xu Hongzhi,Leung Wing Yan,Fu Kaili,Wu Jingtong,Liu Ken,Man Kwan,Yang Xiaoyong,Han Jiahuai,Ren Jianlin,Yu Jun
Journal of hepatology
BACKGROUND & AIMS:p38 mitogen-activated protein kinases are important inflammatory factors. p38α alteration has been implicated in both human and mouse inflammatory disease models. Therefore, we aimed to characterize the cell type-specific role of p38α in non-alcoholic steatohepatitis (NASH). METHODS:Human liver tissues were obtained from 27 patients with non-alcoholic fatty liver disease (NAFLD) and 20 control individuals. NASH was established and compared between hepatocyte-specific p38α knockout (p38α), macrophage-specific p38α knockout (p38α) and wild-type (p38α) mice fed with high-fat diet (HFD), high-fat/high-cholesterol diet (HFHC), or methionine-and choline-deficient diet (MCD). p38 inhibitors were administered to HFHC-fed wild-type mice for disease treatment. RESULTS:p38α was significantly upregulated in the liver tissues of patients with NAFLD. Compared to p38α littermates, p38α mice developed significant nutritional steatohepatitis induced by HFD, HFHC or MCD. Meanwhile, p38α mice exhibited less severe steatohepatitis and insulin resistance than p38α mice in response to a HFHC or MCD. The effect of macrophage p38α in promoting steatohepatitis was mediated by the induction of pro-inflammatory factors (CXCL2, IL-1β, CXCL10 and IL-6) secreted by M1 macrophages and associated signaling pathways. p38α mice exhibited M2 anti-inflammatory polarization as demonstrated by increased CD45F4/80CD11bCD206 M2 macrophages and enhanced arginase activity in liver tissues. Primary hepatocytes from p38α mice showed decreased steatosis and inflammatory damage. In a co-culture system, p38α deleted macrophages attenuated steatohepatitic changes in hepatocytes through decreased secretion of pro-inflammatory cytokines (TNF-α, CXCL10 and IL-6), which mediate M1 macrophage polarization in p38α mice. Restoration of TNF-α, CXCL10 or IL-6 induced lipid accumulation and inflammatory responses in p38α hepatocytes co-cultured with p38α macrophages. Moreover, pharmacological p38 inhibitors suppressed HFHC-induced steatohepatitis. CONCLUSIONS:Macrophage p38α promotes the progression of steatohepatitis by inducing pro-inflammatory cytokine secretion and M1 polarization. p38 inhibition protects against steatohepatitis. LAY SUMMARY: p38 mitogen-activated protein kinases are important inflammatory factors. In the present study, we demonstrated that p38α is upregulated in liver tissues of patients with non-alcoholic fatty liver diseases. Genetic deletion of p38α in macrophages led to ameliorated nutritional steatohepatitis in mice through decreased pro-inflammatory cytokine secretion and increased M2 macrophage polarization.
Fructose Promotes Leaky Gut, Endotoxemia, and Liver Fibrosis Through Ethanol-Inducible Cytochrome P450-2E1-Mediated Oxidative and Nitrative Stress.
Hepatology (Baltimore, Md.)
Fructose intake is known to induce obesity, insulin resistance, metabolic syndrome, and nonalcoholic fatty liver disease (NAFLD). We aimed to evaluate the effects of fructose drinking on gut leakiness, endotoxemia, and NAFLD and study the underlying mechanisms in rats, mice, and T84 colon cells. Levels of ileum junctional proteins, oxidative stress markers, and apoptosis-related proteins in rodents, T84 colonic cells, and human ileums were determined by immunoblotting, immunoprecipitation, and immunofluorescence analyses. Fructose drinking caused microbiome change, leaky gut, and hepatic inflammation/fibrosis with increased levels of nitroxidative stress marker proteins cytochrome P450-2E1 (CYP2E1), inducible nitric oxide synthase, and nitrated proteins in small intestine and liver of rodents. Fructose drinking significantly elevated plasma bacterial endotoxin levels, likely resulting from decreased levels of intestinal tight junction (TJ) proteins (zonula occludens 1, occludin, claudin-1, and claudin-4), adherent junction (AJ) proteins (β-catenin and E-cadherin), and desmosome plakoglobin, along with α-tubulin, in wild-type rodents, but not in fructose-exposed Cyp2e1-null mice. Consistently, decreased intestinal TJ/AJ proteins and increased hepatic inflammation with fibrosis were observed in autopsied obese people compared to lean individuals. Furthermore, histological and biochemical analyses showed markedly elevated hepatic fibrosis marker proteins in fructose-exposed rats compared to controls. Immunoprecipitation followed by immunoblot analyses revealed that intestinal TJ proteins were nitrated and ubiquitinated, leading to their decreased levels in fructose-exposed rats. Conclusion: These results showed that fructose intake causes protein nitration of intestinal TJ and AJ proteins, resulting in increased gut leakiness, endotoxemia, and steatohepatitis with liver fibrosis, at least partly, through a CYP2E1-dependent manner.
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.
The Natural History of Advanced Fibrosis Due to Nonalcoholic Steatohepatitis: Data From the Simtuzumab Trials.
Sanyal Arun J,Harrison Stephen A,Ratziu Vlad,Abdelmalek Manal F,Diehl Anna Mae,Caldwell Stephen,Shiffman Mitchell L,Aguilar Schall Raul,Jia Catherine,McColgan Bryan,Djedjos C Stephen,McHutchison John G,Subramanian G Mani,Myers Robert P,Younossi Zobair,Muir Andrew J,Afdhal Nezam H,Bosch Jaime,Goodman Zachary
Hepatology (Baltimore, Md.)
Progression of nonalcoholic steatohepatitis (NASH) is incompletely characterized. We analyzed data on longitudinal changes in liver histology, hepatic venous pressure gradient (HVPG), and serum markers of fibrosis in 475 patients with NASH with bridging fibrosis (F3) or compensated cirrhosis (F4) enrolled in two phase 2b, placebo-controlled trials of simtuzumab. The trials were terminated after 96 weeks because of lack of efficacy, so data from treatment groups were combined. Liver biopsies and HVPG measurements (only for patients with F4 fibrosis) were collected at screening and at weeks 48 and 96. Patients were assessed for Ishak fibrosis stage, hepatic collagen content and alpha-smooth muscle actin (by morphometry), NAFLD Activity Score (NAS), and serum markers of fibrosis. Associations with progression to cirrhosis (in patients with F3 fibrosis) and liver-related clinical events (in patients with F4 fibrosis) were determined. Progression to cirrhosis occurred in 22% (48/217) of F3 patients, and liver-related clinical events occurred in 19% (50/258) of patients with cirrhosis. Factors significantly associated with progression to cirrhosis included higher baseline values of and greater increases in hepatic collagen content, level of alpha-smooth muscle actin, and Enhanced Liver Fibrosis score. Similar factors, plus lack of fibrosis stage improvement (hazard ratio, 9.30; 95% confidence interval, 1.28-67.37), higher HVPG at baseline, and greater increase in HVPG over time, were associated with an increased risk of liver-related clinical events in patients with cirrhosis. Disease progression was not associated with the NAS at baseline or changes in NAS during treatment after adjustment for fibrosis stage. Conclusion: In patients with advanced fibrosis due to NASH, the primary determinant of clinical disease progression is fibrosis and its change over time.
Pharmacologic Inhibition of Epidermal Growth Factor Receptor Suppresses Nonalcoholic Fatty Liver Disease in a Murine Fast-Food Diet Model.
Bhushan Bharat,Banerjee Swati,Paranjpe Shirish,Koral Kelly,Mars Wendy M,Stoops John W,Orr Anne,Bowen William C,Locker Joseph,Michalopoulos George K
Hepatology (Baltimore, Md.)
Epidermal growth factor receptor (EGFR) is a critical regulator of hepatocyte proliferation and liver regeneration. Our recent work indicated that EGFR can also regulate lipid metabolism during liver regeneration after partial hepatectomy. Based on these findings, we investigated the role of EGFR in a mouse model of nonalcoholic fatty liver disease (NAFLD) using a pharmacological inhibition strategy. C57BL6/J mice were fed a chow diet or a fast-food diet (FFD) with or without EGFR inhibitor (canertinib) for 2 months. EGFR inhibition completely prevented development of steatosis and liver injury in this model. In order to study if EGFR inhibition can reverse NAFLD progression, mice were fed the FFD for 5 months, with or without canertinib treatment for the last 5 weeks of the study. EGFR inhibition remarkably decreased steatosis, liver injury, and fibrosis and improved glucose tolerance. Microarray analysis revealed that ~40% of genes altered by the FFD were differentially expressed after EGFR inhibition and, thus, are potentially regulated by EGFR. Several genes and enzymes related to lipid metabolism (particularly fatty acid synthesis and lipolysis), which were disrupted by the FFD, were found to be modulated by EGFR. Several crucial transcription factors that play a central role in regulating these lipid metabolism genes during NAFLD, including peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element-binding transcription factor 1 (SREBF1), carbohydrate-responsive element-binding protein, and hepatocyte nuclear factor 4 alpha, were also found to be modulated by EGFR. In fact, chromatin immunoprecipitation analysis revealed that PPARγ binding to several crucial lipid metabolism genes (fatty acid synthase, stearoyl-coenzyme A desaturase 1, and perilipin 2) was drastically reduced by EGFR inhibition. Further upstream, EGFR inhibition suppressed AKT signaling, which is known to control these transcription factors, including PPARγ and SREBF1, in NAFLD models. Lastly, the effect of EGFR in FFD-induced fatty-liver phenotype was not shared by receptor tyrosine kinase MET, investigated using MET knockout mice. Conclusion: Our study revealed a role of EGFR in NAFLD and the potential of EGFR inhibition as a treatment strategy for NAFLD.
Unexpected Rapid Increase in the Burden of NAFLD in China From 2008 to 2018: A Systematic Review and Meta-Analysis.
Zhou Feng,Zhou Jianghua,Wang Wenxin,Zhang Xiao-Jing,Ji Yan-Xiao,Zhang Peng,She Zhi-Gang,Zhu Lihua,Cai Jingjing,Li Hongliang
Hepatology (Baltimore, Md.)
With rapid lifestyle transitions, the increasing burden of nonalcoholic fatty liver disease (NAFLD) in China has emerged as a major public health issue. To obtain a comprehensive overview of the status of NAFLD over the past decade, we evaluated the epidemiology, risk factors, complications, and management of NAFLD in China through a systematic review and meta-analysis. Five English literature databases and three Chinese databases were searched for relevant topics from 2008 to 2018. A total of 392 studies with a population of 2,054,554 were included. National prevalence of NAFLD was 29.2%, with a heavier disease burden among the middle-aged, males, those in northwest China and Taiwan, regions with a gross domestic product per capita greater than 100,000 yuan, and Uygur and Hui ethnic groups. Currently, original studies on natural history and complications of NAFLD in China are scarce. Several studies revealed that NAFLD is positively correlated with the incidence of extrahepatic tumors, diabetes, cardiovascular disease and metabolic syndrome. The Chinese population may have a higher hereditary risk of NAFLD due to more frequent nonsynonymous mutations in genes regulating lipid metabolism. Ultrasonography is the primary imaging tool in the detection of NAFLD in China. Serum tests and risk stratification algorithms for staging NAFLD remain under investigation. Specific pharmaceutical treatments for NAFLD are still undergoing clinical trials. It is noteworthy that the Chinese are underrepresented compared with their proportion of the NAFLD population in such trials. Conclusion: China experienced an unexpected rapid increase in the burden of NAFLD over a short period. Rising awareness and urgent actions need to be taken in order to control the NAFLD pandemic in China.
Myeloid cells in liver and bone marrow acquire a functionally distinct inflammatory phenotype during obesity-related steatohepatitis.
Krenkel Oliver,Hundertmark Jana,Abdallah Ali T,Kohlhepp Marlene,Puengel Tobias,Roth Tilmann,Branco Diogo Philippini Pontual,Mossanen Jana C,Luedde Tom,Trautwein Christian,Costa Ivan G,Tacke Frank
OBJECTIVE:Bone marrow-derived myeloid cells accumulate in the liver as monocytes and macrophages during the progression of obesity-related non-alcoholic fatty liver disease (NAFLD) to steatohepatitis (NASH). Myeloid cells comprise heterogeneous subsets, and dietary overnutrition may affect macrophages in the liver and bone marrow. We therefore aimed at characterising in depth the functional adaptations of myeloid cells in fatty liver. DESIGN:We employed single-cell RNA sequencing to comprehensively assess the heterogeneity of myeloid cells in the liver and bone marrow during NAFLD, by analysing C57BL/6 mice fed with a high-fat, high-sugar, high-cholesterol 'Western diet' for 16 weeks. We also characterised NAFLD-driven functional adaptations of macrophages in vitro and their functional relevance during steatohepatitis in vivo. RESULTS:Single-cell RNA sequencing identified distinct myeloid cell clusters in the liver and bone marrow. In both compartments, monocyte-derived populations were largely expanded in NASH-affected mice. Importantly, the liver myeloid compartment adapted a unique inflammatory phenotype during NAFLD progression, exemplarily characterised by downregulated inflammatory calprotectin (S100A8/A9) in macrophage and dendritic cell subsets. This distinctive gene signature was also found in their bone marrow precursors. The NASH myeloid phenotype was principally recapitulated by in vitro exposure of bone marrow-derived macrophages with fatty acids, depended on toll-like receptor 4 signalling and defined a characteristic response pattern to lipopolysaccharide stimulation. This imprinted and stable NASH myeloid immune phenotype functionally determined inflammatory responses following acute liver injury (acetaminophen poisoning) in vivo. CONCLUSION:Liver myeloid leucocytes and their bone marrow precursors adapt a common and functionally relevant inflammatory signature during NAFLD progression.
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.
Hepatocyte Deletion of Triglyceride-Synthesis Enzyme Acyl CoA: Diacylglycerol Acyltransferase 2 Reduces Steatosis Without Increasing Inflammation or Fibrosis in Mice.
Gluchowski Nina L,Gabriel Katlyn R,Chitraju Chandramohan,Bronson Roderick T,Mejhert Niklas,Boland Sebastian,Wang Kun,Lai Zon Weng,Farese Robert V,Walther Tobias C
Hepatology (Baltimore, Md.)
Nonalcoholic fatty liver disease (NAFLD) is characterized by excess lipid accumulation in hepatocytes and represents a huge public health problem owing to its propensity to progress to nonalcoholic steatohepatitis, fibrosis, and liver failure. The lipids stored in hepatic steatosis (HS) are primarily triglycerides (TGs) synthesized by two acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. Either DGAT1 or DGAT2 catalyzes this reaction, and these enzymes have been suggested to differentially utilize exogenous or endogenously synthesized fatty acids, respectively. DGAT2 has been linked to storage of fatty acids from de novo lipogenesis, a process increased in NAFLD. However, whether DGAT2 is more responsible for lipid accumulation in NAFLD and progression to fibrosis is currently unknown. Also, it is unresolved whether DGAT2 can be safely inhibited as a therapy for NAFLD. Here, we induced NAFLD-like disease in mice by feeding a diet rich in fructose, saturated fat, and cholesterol and found that hepatocyte-specific Dgat2 deficiency reduced expression of de novo lipogenesis genes and lowered liver TGs by ~70%. Importantly, the reduction in steatosis was not accompanied by increased inflammation or fibrosis, and insulin and glucose metabolism were unchanged. Conclusion: This study suggests that hepatic DGAT2 deficiency successfully reduces diet-induced HS and supports development of DGAT2 inhibitors as a therapeutic strategy for treating NAFLD and preventing downstream consequences.
The Liver as an Endocrine Organ-Linking NAFLD and Insulin Resistance.
Watt Matthew J,Miotto Paula M,De Nardo William,Montgomery Magdalene K
The liver is a dynamic organ that plays critical roles in many physiological processes, including the regulation of systemic glucose and lipid metabolism. Dysfunctional hepatic lipid metabolism is a cause of nonalcoholic fatty liver disease (NAFLD), the most common chronic liver disorder worldwide, and is closely associated with insulin resistance and type 2 diabetes. Through the use of advanced mass spectrometry "omics" approaches and detailed experimentation in cells, mice, and humans, we now understand that the liver secretes a wide array of proteins, metabolites, and noncoding RNAs (miRNAs) and that many of these secreted factors exert powerful effects on metabolic processes both in the liver and in peripheral tissues. In this review, we summarize the rapidly evolving field of "hepatokine" biology with a particular focus on delineating previously unappreciated communication between the liver and other tissues in the body. We describe the NAFLD-induced changes in secretion of liver proteins, lipids, other metabolites, and miRNAs, and how these molecules alter metabolism in liver, muscle, adipose tissue, and pancreas to induce insulin resistance. We also synthesize the limited information that indicates that extracellular vesicles, and in particular exosomes, may be an important mechanism for intertissue communication in normal physiology and in promoting metabolic dysregulation in NAFLD.
Sestrin 3 Protects Against Diet-Induced Nonalcoholic Steatohepatitis in Mice Through Suppression of Transforming Growth Factor β Signal Transduction.
Hepatology (Baltimore, Md.)
Sestrin 3 (Sesn3) belongs to the three-member sestrin protein family. Sestrins have been implicated in antioxidative stress, adenosine monophosphate-activated protein kinase and mammalian target of rapamycin signal transduction, and metabolic homeostasis. However, the role of Sesn3 in the development of nonalcoholic steatohepatitis (NASH) has not been previously studied. In this work, we generated Sesn3 whole-body knockout and liver-specific transgenic mice to investigate the hepatic function of Sesn3 in diet-induced NASH. With only 4 weeks of dietary treatment, Sesn3 knockout mice developed severe NASH phenotype as characterized by hepatic steatosis, inflammation, and fibrosis. Strikingly, after 8-week feeding with a NASH-inducing diet, Sesn3 transgenic mice were largely protected against NASH development. Transcriptomic analysis revealed that multiple extracellular matrix-related processes were up-regulated, including transforming growth factor β (TGF-β) signaling and collagen production. Further biochemical and cell biological analyses have illustrated a critical control of the TGF-β-mothers against decapentaplegic homolog (Smad) pathway by Sesn3 at the TGF-β receptor and Smad3 levels. First, Sesn3 inhibits the TGF-β receptor through an interaction with Smad7; second, Sesn3 directly inhibits the Smad3 function through protein-protein interaction and cytosolic retention. Conclusion: Sesn3 is a critical regulator of the extracellular matrix and hepatic fibrosis by suppression of TGF-β-Smad3 signaling.
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.
miRNAs and NAFLD: from pathophysiology to therapy.
Gjorgjieva Monika,Sobolewski Cyril,Dolicka Dobrochna,Correia de Sousa Marta,Foti Michelangelo
Non-alcoholic fatty liver disease (NAFLD) is associated with a thorough reprogramming of hepatic metabolism. Epigenetic mechanisms, in particular those associated with deregulation of the expressions and activities of microRNAs (miRNAs), play a major role in metabolic disorders associated with NAFLD and their progression towards more severe stages of the disease. In this review, we discuss the recent progress addressing the role of the many facets of complex miRNA regulatory networks in the development and progression of NAFLD. The basic concepts and mechanisms of miRNA-mediated gene regulation as well as the various setbacks encountered in basic and translational research in this field are debated. miRNAs identified so far, whose expressions/activities are deregulated in NAFLD, and which contribute to the outcomes of this pathology are further reviewed. Finally, the potential therapeutic usages in a short to medium term of miRNA-based strategies in NAFLD, in particular to identify non-invasive biomarkers, or to design pharmacological analogues/inhibitors having a broad range of actions on hepatic metabolism, are highlighted.
Microbiota-driven gut vascular barrier disruption is a prerequisite for non-alcoholic steatohepatitis development.
Mouries Juliette,Brescia Paola,Silvestri Alessandra,Spadoni Ilaria,Sorribas Marcel,Wiest Reiner,Mileti Erika,Galbiati Marianna,Invernizzi Pietro,Adorini Luciano,Penna Giuseppe,Rescigno Maria
Journal of hepatology
BACKGROUND & AIMS:Fatty liver disease, including non-alcoholic fatty liver (NAFLD) and steatohepatitis (NASH), has been associated with increased intestinal barrier permeability and translocation of bacteria or bacterial products into the blood circulation. In this study, we aimed to unravel the role of both intestinal barrier integrity and microbiota in NAFLD/NASH development. METHODS:C57BL/6J mice were fed with high-fat diet (HFD) or methionine-choline-deficient diet for 1 week or longer to recapitulate aspects of NASH (steatosis, inflammation, insulin resistance). Genetic and pharmacological strategies were then used to modulate intestinal barrier integrity. RESULTS:We show that disruption of the intestinal epithelial barrier and gut vascular barrier (GVB) are early events in NASH pathogenesis. Mice fed HFD for only 1 week undergo a diet-induced dysbiosis that drives GVB damage and bacterial translocation into the liver. Fecal microbiota transplantation from HFD-fed mice into specific pathogen-free recipients induces GVB damage and epididymal adipose tissue enlargement. GVB disruption depends on interference with the WNT/β-catenin signaling pathway, as shown by genetic intervention driving β-catenin activation only in endothelial cells, preventing GVB disruption and NASH development. The bile acid analogue and farnesoid X receptor agonist obeticholic acid (OCA) drives β-catenin activation in endothelial cells. Accordingly, pharmacologic intervention with OCA protects against GVB disruption, both as a preventive and therapeutic agent. Importantly, we found upregulation of the GVB leakage marker in the colon of patients with NASH. CONCLUSIONS:We have identified a new player in NASH development, the GVB, whose damage leads to bacteria or bacterial product translocation into the blood circulation. Treatment aimed at restoring β-catenin activation in endothelial cells, such as administration of OCA, protects against GVB damage and NASH development. LAY SUMMARY:The incidence of fatty liver disease is reaching epidemic levels in the USA, with more than 30% of adults having NAFLD (non-alcoholic fatty liver disease), which can progress to more severe non-alcoholic steatohepatitis (NASH). Herein, we show that disruption of the intestinal epithelial barrier and gut vascular barrier are early events in the development of NASH. We show that the drug obeticholic acid protects against barrier disruption and thereby prevents the development of NASH, providing further evidence for its use in the prevention or treatment of NASH.
Hepatocyte Stress Increases Expression of Yes-Associated Protein and Transcriptional Coactivator With PDZ-Binding Motif in Hepatocytes to Promote Parenchymal Inflammation and Fibrosis.
Mooring Meghan,Fowl Brendan H,Lum Shelly Z C,Liu Ye,Yao Kangning,Softic Samir,Kirchner Rory,Bernstein Aaron,Singhi Aatur D,Jay Daniel G,Kahn C Ronald,Camargo Fernando D,Yimlamai Dean
Hepatology (Baltimore, Md.)
BACKGROUND AND AIMS:Activated hepatocytes are hypothesized to be a major source of signals that drive cirrhosis, but the biochemical pathways that convert hepatocytes into such a state are unclear. We examined the role of the Hippo pathway transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in hepatocytes to facilitate cell-cell interactions that stimulate liver inflammation and fibrosis. APPROACH AND RESULTS:Using a variety of genetic, metabolic, and liver injury models in mice, we manipulated Hippo signaling in hepatocytes and examined its effects in nonparenchymal cells to promote liver inflammation and fibrosis. YAP-expressing hepatocytes rapidly and potently activate the expression of proteins that promote fibrosis (collagen type I alpha 1 chain, tissue inhibitor of metalloproteinase 1, platelet-derived growth factor c, transforming growth factor β2) and inflammation (tumor necrosis factor, interleukin 1β). They stimulate expansion of myofibroblasts and immune cells, followed by aggressive liver fibrosis. In contrast, hepatocyte-specific YAP and YAP/TAZ knockouts exhibit limited myofibroblast expansion, less inflammation, and decreased fibrosis after CCl injury despite a similar degree of necrosis as controls. We identified cellular communication network factor 1 (CYR61) as a chemokine that is up-regulated by hepatocytes during liver injury but is expressed at significantly lower levels in mice with hepatocyte-specific deletion of YAP or TAZ. Gain-of-function and loss-of-function experiments with CYR61 in vivo point to it being a key chemokine controlling liver fibrosis and inflammation in the context of YAP/TAZ. There is a direct correlation between levels of YAP/TAZ and CYR61 in liver tissues of patients with high-grade nonalcoholic steatohepatitis. CONCLUSIONS:Liver injury in mice and humans increases levels of YAP/TAZ/CYR61 in hepatocytes, thus attracting macrophages to the liver to promote inflammation and fibrosis.
Hepatic IRF2BP2 Mitigates Nonalcoholic Fatty Liver Disease by Directly Repressing the Transcription of ATF3.
Fang Jing,Ji Yan-Xiao,Zhang Peng,Cheng Lin,Chen Yue,Chen Jun,Su Yanfang,Cheng Xu,Zhang Yan,Li Tianyu,Zhu Xuehai,Zhang Xiao-Jing,Wei Xiang
Hepatology (Baltimore, Md.)
BACKGROUND AND AIMS:Although knowledge regarding the pathogenesis of nonalcoholic fatty liver disease (NAFLD) has profoundly grown in recent decades, the internal restrictive mechanisms remain largely unknown. We have recently reported that the transcription repressor interferon regulatory factor-2 binding protein 2 (IRF2BP2) is enriched in cardiomyocytes and inhibits pathological cardiac hypertrophy in mice. Notably, IRF2BP2 is abundantly expressed in hepatocytes and dramatically down-regulated in steatotic livers, whereas the role of IRF2BP2 in NAFLD is unknown. APPROACH AND RESULTS:Herein, using gain-of-function and loss-of-function approaches in mice, we demonstrated that while hepatocyte-specific Irf2bp2 knockout exacerbated high-fat diet-induced hepatic steatosis, insulin resistance and inflammation, hepatic Irf2bp2 overexpression protected mice from these metabolic disorders. Moreover, the inhibitory role of IRF2BP2 on hepatosteatosis is conserved in a human hepatic cell line in vitro. Combinational analysis of digital gene expression and chromatin immunoprecipitation sequencing identified activating transcription factor 3 (ATF3) to be negatively regulated by IRF2BP2 in NAFLD. Chromatin immunoprecipitation and luciferase assay substantiated the fact that IRF2BP2 is a bona fide transcription repressor of ATF3 gene expression via binding to its promoter region. Functional studies revealed that ATF3 knockdown significantly relieved IRF2BP2 knockout-exaggerated hepatosteatosis in vitro. CONCLUSION:IRF2BP2 is an integrative restrainer in controlling hepatic steatosis, insulin resistance, and inflammation in NAFLD through transcriptionally repressing ATF3 gene expression.
Rilpivirine attenuates liver fibrosis through selective STAT1-mediated apoptosis in hepatic stellate cells.
Martí-Rodrigo Alberto,Alegre Fernando,Moragrega Ángela B,García-García Francisco,Martí-Rodrigo Pablo,Fernández-Iglesias Anabel,Gracia-Sancho Jordi,Apostolova Nadezda,Esplugues Juan V,Blas-García Ana
OBJECTIVE:Liver fibrosis constitutes a major health problem worldwide due to its rapidly increasing prevalence and the lack of specific and effective treatments. Growing evidence suggests that signalling through cytokine-activated Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways regulates liver fibrosis and regeneration. Rilpivirine (RPV) is a widely used anti-HIV drug not reported to produce hepatotoxicity. We aimed to describe the potential hepatoprotective effects of RPV in different models of chronic liver injury, focusing on JAK-STAT signalling regulation. DESIGN:The effects of RPV on hepatic steatosis, inflammation and fibrogenesis were studied in a nutritional mouse model of non-alcoholic fatty liver disease, carbon tetrachloride-induced fibrosis and bile duct ligation-induced fibrosis. Primary human hepatic stellate cells (hHSC) and human cell lines LX-2 and Hep3B were used to investigate the underlying molecular mechanisms. RESULTS:RPV exerted a clear anti-inflammatory and antifibrotic effect in all the in vivo models of liver injury employed, and enhanced STAT3-dependent proliferation in hepatocytes and apoptosis in HSC through selective STAT1 activation. These results were reproduced in vitro; RPV undermined STAT3 activation and triggered STAT1-mediated pathways and apoptosis in HSC. Interestingly, this selective pro-apoptotic effect completely disappeared when STAT1 was silenced. Conditioned medium experiments showed that HSC apoptosis activated STAT3 in hepatocytes in an interleukin-6-dependent mechanism. CONCLUSION:RPV ameliorates liver fibrosis through selective STAT1-dependent induction of apoptosis in HSC, which exert paracrinal effects in hepatocytes, thus promoting liver regeneration. RPV's actions may represent an effective strategy to treat chronic liver diseases of different aetiologies and help identify novel therapeutic targets.
Hepatocyte-derived extracellular vesicles promote endothelial inflammation and atherogenesis via microRNA-1.
Jiang Fangjie,Chen Qi,Wang Wei,Ling Yan,Yan Yan,Xia Pu
Journal of hepatology
BACKGROUND & AIMS:Clinical evidence has indicated a close link between non-alcoholic fatty liver disease (NAFLD) and cardiovascular disease (CVD). However, the underlying mechanism remains to be elucidated. This study aimed to explore a potential role of hepatocyte-derived extracellular vesicles (EVs) in endothelial inflammation and atherogenesis in the context of NAFLD. METHODS:EVs were isolated, quantified and characterized from steatotic hepatocytes. An endothelial cell-specific PCR array was used to screen the functional properties of EVs. Profiling of global microRNA expression was conducted in EVs. The expression level and biological function of microRNA-1 (miR-1) was determined by quantitative PCR, immunoblot and reporter gene assays, respectively. The in vivo effect of miR-1 on atherogenesis was investigated in apolipoprotein E (ApoE)-deficient mice administered with a miR-1-specific inhibitor, antagomiR-1. RESULTS:Steatotic hepatocytes released more EVs, which had significantly altered miRNA expression profiles compared to the EVs released by control hepatocytes. Endothelial cells co-cultured with steatotic hepatocytes, or treated with their EVs or miR-1, expressed significantly more proinflammatory molecules, as well as exhibiting increased NF-κB activity and reduced Kruppel-like factor 4 (KLF4) expression. EV-induced endothelial inflammation was prevented by either downregulation or inhibition of miR-1. While miR-1 treatment suppressed KLF4 expression and reporter gene activity, overexpression of KLF4 dramatically abolished the miR-1-induced endothelial inflammation. Moreover, not only did the miR-1 inhibitor reduce endothelial inflammation in vitro, but it also attenuated atherogenesis in ApoE-deficient mice. CONCLUSION:Steatotic hepatocyte-derived EVs promote endothelial inflammation and facilitate atherogenesis by miR-1 delivery, KLF4 suppression and NF-κB activation. The findings illustrate an important role of hepatocyte-derived EVs in distant communications between the liver and vasculature, suggesting a new mechanism underlying the link between NAFLD and CVD. LAY SUMMARY:Non-alcoholic fatty liver disease (NAFLD), a condition highly prevalent in obese and/or diabetic patients, is emerging as an independent risk factor of cardiovascular disease. Herein, we demonstrated that extracellular vesicles, released by hepatocytes under NAFLD conditions, cause vascular endothelial inflammation and promote atherosclerosis. Within these toxic vesicles, we identified a small molecular cargo that acted as a potent inducer of endothelial inflammation. By inhibiting this cargo's function, a specific gene-based inhibitor profoundly attenuated atherogenesis in mice, uncovering a novel mechanism which may be used to prevent or treat cardiovascular disease in patients with NAFLD.
The gut microbiome and metabolic syndrome.
Dabke Kruttika,Hendrick Gustaf,Devkota Suzanne
The Journal of clinical investigation
The metabolic syndrome (MetS) is a constellation of risk factors that, if left untreated, will often progress to greater metabolic defects such as type 2 diabetes and nonalcoholic fatty liver disease. While these risk factors have been established for over 40 years, the definition of MetS warrants reconsideration in light of the substantial data that have emerged from studies of the gut microbiome. In this Review we present the existing recent literature that supports the gut microbiome's potential influence on the various risk factors of MetS. The interplay of the intestinal microbiota with host metabolism has been shown to be mediated by a myriad of factors, including a defective gut barrier, bile acid metabolism, antibiotic use, and the pleiotropic effects of microbially produced metabolites. These data show that events that start in the gut, often in response to external cues such as diet and circadian disruption, have far-reaching effects beyond the gut.
Controlled-release mitochondrial protonophore (CRMP) reverses dyslipidemia and hepatic steatosis in dysmetabolic nonhuman primates.
Goedeke Leigh,Peng Liang,Montalvo-Romeral Valle,Butrico Gina M,Dufour Sylvie,Zhang Xian-Man,Perry Rachel J,Cline Gary W,Kievit Paul,Chng Keefe,Petersen Kitt Falk,Shulman Gerald I
Science translational medicine
Nonalcoholic fatty liver disease (NAFLD) is estimated to affect up to one-third of the general population, and new therapies are urgently required. Our laboratory previously developed a controlled-release mitochondrial protonophore (CRMP) that is functionally liver-targeted and promotes oxidation of hepatic triglycerides. Although we previously demonstrated that CRMP safely reverses hypertriglyceridemia, fatty liver, hepatic inflammation, and fibrosis in diet-induced rodent models of obesity, there remains a critical need to assess its safety and efficacy in a model highly relevant to humans. Here, we evaluated the impact of longer-term CRMP treatment on hepatic mitochondrial oxidation and on the reversal of hypertriglyceridemia, NAFLD, and insulin resistance in high-fat, fructose-fed cynomolgus macaques ( 6) and spontaneously obese dysmetabolic rhesus macaques ( 12). Using positional isotopomer nuclear magnetic resonance tracer analysis (PINTA), we demonstrated that acute CRMP treatment (single dose, 5 mg/kg) increased rates of hepatic mitochondrial fat oxidation by 40%. Six weeks of CRMP treatment reduced hepatic triglycerides in both nonhuman primate models independently of changes in body weight, food intake, body temperature, or adverse reactions. CRMP treatment was also associated with a 20 to 30% reduction in fasting plasma triglycerides and low-density lipoprotein (LDL)-cholesterol in dysmetabolic nonhuman primates. Oral administration of CRMP reduced endogenous glucose production by 18%, attributable to a 20% reduction in hepatic acetyl-coenzyme A (CoA) content [as assessed by whole-body β-hydroxybutyrate (β-OHB) turnover] and pyruvate carboxylase flux. Collectively, these studies provide proof-of-concept data to support the development of liver-targeted mitochondrial uncouplers for the treatment of metabolic syndrome in humans.
Adaptive immunity: an emerging player in the progression of NAFLD.
Nature reviews. Gastroenterology & hepatology
In the past decade, nonalcoholic fatty liver disease (NAFLD) has become a leading cause of chronic liver disease and cirrhosis, as well as an important risk factor for hepatocellular carcinoma (HCC). NAFLD encompasses a spectrum of liver lesions, including simple steatosis, steatohepatitis and fibrosis. Although steatosis is often harmless, the lobular inflammation that characterizes nonalcoholic steatohepatitis (NASH) is considered a driving force in the progression of NAFLD. The current view is that innate immune mechanisms represent a key element in supporting hepatic inflammation in NASH. However, increasing evidence points to the role of adaptive immunity as an additional factor promoting liver inflammation. This Review discusses data regarding the role of B cells and T cells in sustaining the progression of NASH to fibrosis and HCC, along with the findings that antigens originating from oxidative stress act as a trigger for immune responses. We also highlight the mechanisms affecting liver immune tolerance in the setting of steatohepatitis that favour lymphocyte activation. Finally, we analyse emerging evidence concerning the possible application of immune modulating treatments in NASH therapy.
Yes-Associated Protein in Kupffer Cells Enhances the Production of Proinflammatory Cytokines and Promotes the Development of Nonalcoholic Steatohepatitis.
Song Kyoungsub,Kwon Hyunjoo,Han Chang,Chen Weina,Zhang Jinqiang,Ma Wenbo,Dash Srikanta,Gandhi Chandrashekhar R,Wu Tong
Hepatology (Baltimore, Md.)
BACKGROUND AND AIMS:Yes-associated protein (YAP) plays an important role in hepatocarcinogenesis, although the potential role of YAP in non-neoplastic liver diseases remains largely unknown. We report herein that YAP in Kupffer cells (KCs) enhances the production of proinflammatory cytokines and promotes the development of nonalcoholic steatohepatitis (NASH). Our data show that the expression of YAP is significantly increased in KCs of wild-type mice fed a high-fat diet (HFD). APPROACH AND RESULTS:We generated mice with macrophage/monocyte-specific deletion of YAP (YAP ) or Toll-like receptor 4 (TLR4; TLR4 ), and animals were fed an HFD or treated with lipopolysaccharide (LPS). Our data showed that YAP mice fed an HFD exhibited lower serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) levels and less hepatic inflammation when compared to their littermate controls. LPS treatment induced accumulation of YAP in KCs in vitro and in mice, which was prevented by macrophage/monocyte-specific deletion of TLR4 (TLR4 ). LPS transcriptionally activates YAP through activator protein 1 in macrophages/KCs. LPS-induced YAP further enhances expression of proinflammatory cytokines (including monocyte chemoattractant protein 1, tumor necrosis factor alpha, and interleukin 6) through YAP association with the TEA domain-binding motif in the promoter region of inflammatory cytokines. Forced overexpression of active YAP (YAP5SA) in KCs enhanced the production of proinflammatory cytokines. Treatment of HFD-fed mice with verteporfin inhibited KC activation, reduced liver inflammation, and decreased serum ALT/AST levels. Analyses of liver tissues from NASH patients reveal that YAP is increased in KCs and that level of YAP in human liver tissues is positively correlated with expression of proinflammatory cytokines. CONCLUSIONS:This study describes an important role of YAP in KCs for regulation of liver inflammation in NASH. Our findings suggest that inhibition of YAP may represent an effective therapeutic strategy for NASH treatment.
The gut-liver axis in liver disease: Pathophysiological basis for therapy.
Albillos Agustín,de Gottardi Andrea,Rescigno María
Journal of hepatology
The gut-liver axis refers to the bidirectional relationship between the gut and its microbiota, and the liver, resulting from the integration of signals generated by dietary, genetic and environmental factors. This reciprocal interaction is established by the portal vein which enables transport of gut-derived products directly to the liver, and the liver feedback route of bile and antibody secretion to the intestine. The intestinal mucosal and vascular barrier is the functional and anatomical structure that serves as a playground for the interactions between the gut and the liver, limiting the systemic dissemination of microbes and toxins while allowing nutrients to access the circulation and to reach the liver. The control of microbial communities is critical to maintaining homeostasis of the gut-liver axis, and as part of this bidirectional communication the liver shapes intestinal microbial communities. Alcohol disrupts the gut-liver axis at multiple interconnected levels, including the gut microbiome, mucus barrier, epithelial barrier and at the level of antimicrobial peptide production, which increases microbial exposure and the proinflammatory environment of the liver. Growing evidence indicates the pathogenetic role of microbe-derived metabolites, such as trimethylamine, secondary bile acids, short-chain fatty acids and ethanol, in the pathogenesis of non-alcoholic fatty liver disease. Cirrhosis by itself is associated with profound alterations in gut microbiota and damage at the different levels of defence of the intestinal barrier, including the epithelial, vascular and immune barriers. The relevance of the severe disturbance of the intestinal barrier in cirrhosis has been linked to translocation of live bacteria, bacterial infections and disease progression. The identification of the elements of the gut-liver axis primarily damaged in each chronic liver disease offers possibilities for intervention. Beyond antibiotics, upcoming therapies centred on the gut include new generations of probiotics, bacterial metabolites (postbiotics), faecal microbial transplantation, and carbon nanoparticles. FXR-agonists target both the gut and the liver and are currently being tested in different liver diseases. Finally, synthetic biotic medicines, phages that target specific bacteria or therapies that create physical barriers between the gut and the liver offer new therapeutic approaches.
Protective and Detrimental Roles of p38α Mitogen-Activated Protein Kinase in Different Stages of Nonalcoholic Fatty Liver Disease.
Hepatology (Baltimore, Md.)
BACKGROUND AND AIMS:Neutrophil infiltration is a hallmark of nonalcoholic steatohepatitis (NASH), but how this occurs during the progression from steatosis to NASH remains obscure. Human NASH features hepatic neutrophil infiltration and up-regulation of major neutrophil-recruiting chemokines (e.g., chemokine [C-X-C motif] ligand 1 [CXCL1] and interleukin [IL]-8). However, mice fed a high-fat diet (HFD) only develop fatty liver without significant neutrophil infiltration or elevation of chemokines. The aim of this study was to determine why mice are resistant to NASH development and the involvement of p38 mitogen-activated protein kinase (p38) activated by neutrophil-derived oxidative stress in the pathogenesis of NASH. APPROACH AND RESULTS:Inflamed human hepatocytes attracted neutrophils more effectively than inflamed mouse hepatocytes because of the greater induction of CXCL1 and IL-8 in human hepatocytes. Hepatic overexpression of Cxcl1 and/or IL-8 promoted steatosis-to-NASH progression in HFD-fed mice by inducing liver inflammation, injury, and p38 activation. Pharmacological inhibition of p38α/β or hepatocyte-specific deletion of p38a (a predominant form in the liver) attenuated liver injury and fibrosis in the HFD -induced NASH model that is associated with strong hepatic p38α activation. In contrast, hepatocyte-specific deletion of p38a in HFD-induced fatty liver where p38α activation is relatively weak exacerbated steatosis and liver injury. Mechanistically, weak p38α activation in fatty liver up-regulated the genes involved in fatty acid β-oxidation through peroxisome proliferator-activated receptor alpha phosphorylation, thereby reducing steatosis. Conversely, strong p38α activation in NASH promoted caspase-3 cleavage, CCAAT-enhancer-binding proteins homologous protein expression, and B cell lymphoma 2 phosphorylation, thereby exacerbating hepatocyte death. CONCLUSIONS:Genetic ablation of hepatic p38a increases simple steatosis but ameliorates oxidative stress-driven NASH, indicating that p38α plays distinct roles depending on the disease stages, which may set the stage for investigating p38α as a therapeutic target for the treatment of NASH.
Acetyl-CoA Derived from Hepatic Peroxisomal β-Oxidation Inhibits Autophagy and Promotes Steatosis via mTORC1 Activation.
He Anyuan,Chen Xiaowen,Tan Min,Chen Yali,Lu Dongliang,Zhang Xiangyu,Dean John M,Razani Babak,Lodhi Irfan J
Autophagy is activated by prolonged fasting but cannot overcome the ensuing hepatic lipid overload, resulting in fatty liver. Here, we describe a peroxisome-lysosome metabolic link that restricts autophagic degradation of lipids. Acyl-CoA oxidase 1 (Acox1), the enzyme that catalyzes the first step in peroxisomal β-oxidation, is enriched in liver and further increases with fasting or high-fat diet (HFD). Liver-specific Acox1 knockout (Acox1-LKO) protected mice against hepatic steatosis caused by starvation or HFD due to induction of autophagic degradation of lipid droplets. Hepatic Acox1 deficiency markedly lowered total cytosolic acetyl-CoA levels, which led to decreased Raptor acetylation and reduced lysosomal localization of mTOR, resulting in impaired activation of mTORC1, a central regulator of autophagy. Dichloroacetic acid treatment elevated acetyl-CoA levels, restored mTORC1 activation, inhibited autophagy, and increased hepatic triglycerides in Acox1-LKO mice. These results identify peroxisome-derived acetyl-CoA as a key metabolic regulator of autophagy that controls hepatic lipid homeostasis.
Acyl-Coenzyme A Thioesterase 9 Traffics Mitochondrial Short-Chain Fatty Acids Toward De Novo Lipogenesis and Glucose Production in the Liver.
Steensels Sandra,Qiao Jixuan,Zhang Yanzhen,Maner-Smith Kristal M,Kika Nourhan,Holman Corey D,Corey Kathleen E,Bracken W Clay,Ortlund Eric A,Ersoy Baran A
Hepatology (Baltimore, Md.)
BACKGROUND AND AIMS:Obesity-induced pathogenesis of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) is associated with increased de novo lipogenesis (DNL) and hepatic glucose production (HGP) that is due to excess fatty acids. Acyl-coenzyme A (CoA) thioesterase (Acot) family members control the cellular utilization of fatty acids by hydrolyzing (deactivating) acyl-CoA into nonesterified fatty acids and CoASH. APPROACH AND RESULTS:Using Caenorhabditis elegans, we identified Acot9 as the strongest regulator of lipid accumulation within the Acot family. Indicative of a maladaptive function, hepatic Acot9 expression was higher in patients with obesity who had NAFLD and NASH compared with healthy controls with obesity. In the setting of excessive nutrition, global ablation of Acot9 protected mice against increases in weight gain, HGP, steatosis, and steatohepatitis. Supportive of a hepatic function, the liver-specific deletion of Acot9 inhibited HGP and steatosis in mice without affecting diet-induced weight gain. By contrast, the rescue of Acot9 expression only in the livers of Acot9 knockout mice was sufficient to promote HGP and steatosis. Mechanistically, hepatic Acot9 localized to the inner mitochondrial membrane, where it deactivated short-chain but not long-chain fatty acyl-CoA. This unique localization and activity of Acot9 directed acetyl-CoA away from protein lysine acetylation and toward the citric acid (TCA) cycle. Acot9-mediated exacerbation of triglyceride and glucose biosynthesis was attributable at least in part to increased TCA cycle activity, which provided substrates for HGP and DNL. β-oxidation and ketone body production, which depend on long-chain fatty acyl-CoA, were not regulated by Acot9. CONCLUSIONS:Taken together, our findings indicate that Acot9 channels hepatic acyl-CoAs toward increased HGP and DNL under the pathophysiology of obesity. Therefore, Acot9 represents a target for the management of NAFLD.
Blocking integrin αβ-mediated CD4 T cell recruitment to the intestine and liver protects mice from western diet-induced non-alcoholic steatohepatitis.
Rai Ravi P,Liu Yunshan,Iyer Smita S,Liu Silvia,Gupta Biki,Desai Chirayu,Kumar Pradeep,Smith Tekla,Singhi Aatur D,Nusrat Asma,Parkos Charles A,Monga Satdarshan P,Czaja Mark J,Anania Frank A,Raeman Reben
Journal of hepatology
BACKGROUND & AIMS:The heterodimeric integrin receptor αβ regulates CD4 T cell recruitment to inflamed tissues, but its role in the pathogenesis of non-alcoholic steatohepatitis (NASH) is unknown. Herein, we examined the role of αβ-mediated recruitment of CD4 T cells to the intestine and liver in NASH. METHODS:Male littermate F11r (control) and junctional adhesion molecule A knockout F11r mice were fed a normal diet or a western diet (WD) for 8 weeks. Liver and intestinal tissues were analyzed by histology, quantitative reverse transcription PCR (qRT-PCR), 16s rRNA sequencing and flow cytometry. Colonic mucosa-associated microbiota were analyzed using 16s rRNA sequencing. Liver biopsies from patients with NASH were analyzed by confocal imaging and qRT-PCR. RESULTS:WD-fed knockout mice developed NASH and had increased hepatic and intestinal αβ CD4 T cells relative to control mice who developed mild hepatic steatosis. The increase in αβ CD4 T cells was associated with markedly higher expression of the αβ ligand mucosal addressin cell adhesion molecule 1 (MAdCAM-1) in the colonic mucosa and livers of WD-fed knockout mice. Elevated MAdCAM-1 expression correlated with increased mucosa-associated Proteobacteria in the WD-fed knockout mice. Antibiotics reduced MAdCAM-1 expression indicating that the diet-altered microbiota promoted colonic and hepatic MAdCAM-1 expression. αβ blockade in WD-fed knockout mice significantly decreased αβ CD4 T cell recruitment to the intestine and liver, attenuated hepatic inflammation and fibrosis, and improved metabolic indices. MAdCAM-1 blockade also reduced hepatic inflammation and fibrosis in WD-fed knockout mice. Hepatic MAdCAM-1 expression was elevated in patients with NASH and correlated with higher expression of α and β integrins. CONCLUSIONS:These findings establish αβ/MAdCAM-1 as a critical axis regulating NASH development through colonic and hepatic CD4 T cell recruitment. LAY SUMMARY:Non-alcoholic steatohepatitis (NASH) is an advanced and progressive form of non-alcoholic fatty liver disease (NAFLD), and despite its growing incidence no therapies currently exist to halt NAFLD progression. Herein, we show that blocking integrin receptor αβ-mediated recruitment of CD4 T cells to the intestine and liver not only attenuates hepatic inflammation and fibrosis, but also improves metabolic derangements associated with NASH. These findings provide evidence for the potential therapeutic application of αβ antibody in the treatment of human NASH.
Hepatic HuR modulates lipid homeostasis in response to high-fat diet.
Zhang Zhuojun,Zong Chen,Jiang Mingyang,Hu Han,Cheng Xiaolei,Ni Juhua,Yi Xia,Jiang Bin,Tian Feng,Chang Ming-Wen,Su Wen,Zhu Lijun,Li Jinfan,Xiang Xueping,Miao Congxiu,Gorospe Myriam,de Cabo Rafael,Dou Yali,Ju Zhenyu,Yang Jichun,Jiang Changtao,Yang Zhongzhou,Wang Wengong
Lipid transport and ATP synthesis are critical for the progression of non-alcoholic fatty liver disease (NAFLD), but the underlying mechanisms are largely unknown. Here, we report that the RNA-binding protein HuR (ELAVL1) forms complexes with NAFLD-relevant transcripts. It associates with intron 24 of Apob pre-mRNA, with the 3'UTR of Uqcrb, and with the 5'UTR of Ndufb6 mRNA, thereby regulating the splicing of Apob mRNA and the translation of UQCRB and NDUFB6. Hepatocyte-specific HuR knockout reduces the expression of APOB, UQCRB, and NDUFB6 in mice, reducing liver lipid transport and ATP synthesis, and aggravating high-fat diet (HFD)-induced NAFLD. Adenovirus-mediated re-expression of HuR in hepatocytes rescues the effect of HuR knockout in HFD-induced NAFLD. Our findings highlight a critical role of HuR in regulating lipid transport and ATP synthesis.
Novel antisense inhibition of diacylglycerol O-acyltransferase 2 for treatment of non-alcoholic fatty liver disease: a multicentre, double-blind, randomised, placebo-controlled phase 2 trial.
Loomba Rohit,Morgan Erin,Watts Lynnetta,Xia Shuting,Hannan Lisa A,Geary Richard S,Baker Brenda F,Bhanot Sanjay
The lancet. Gastroenterology & hepatology
BACKGROUND:Diacylglycerol-O-acyltransferase 2 (DGAT2) is one of two enzyme isoforms that catalyse the final step in the synthesis of triglycerides. IONIS-DGAT2 is an antisense oligonucleotide inhibitor of DGAT2 that is under clinical investigation for the treatment of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). The aim of this trial was to examine the safety, tolerability, and efficacy of IONIS-DGAT2 versus placebo in reducing liver fat in patients with type 2 diabetes and NAFLD. METHODS:This double-blind, randomised, placebo-controlled, phase 2 study consisted of a 2-week screening period, a run-in period of up to 4 weeks, a 13-week treatment period of once-weekly dosing, and a 13-week post-treatment follow-up period. The study was done at 16 clinical research sites in Canada, Poland, and Hungary. Eligible participants were aged 18-75 years, had a body-mass index at screening between 27 kg/m and 39 kg/m, haemoglobin A (HbA) levels from 7·3% to 9·5%, and liver fat content 10% or greater before randomisation, and agreed to maintain a stable diet and exercise routine throughout the study. Enrolled participants were stratified on the basis of liver fat content during the run-in period (<20% or ≥20%) and then centrally randomised (2:1) to receive once weekly subcutaneous injection of 250 mg IONIS-DGAT2 or placebo for 13 weeks. Participants, investigators, funder personnel, and the clinical research organisation staff, including central readers of MRI scans, were all masked to treatment identity. The primary endpoints were the safety, tolerability, and pharmacodynamic effect of IONIS-DGAT2 on hepatic steatosis, according to absolute reduction from baseline in liver fat percentage as quantified by MRI-estimated proton density fat fraction and assessed in the per-protocol population. Pharmacodynamic performance was determined in the per-protocol population by the change in liver fat content from baseline to 2 weeks after the last dose. The per-protocol population included all randomised participants who received at least ten doses of study drug, with the first four doses administered in the first 5 weeks, did not miss more than three consecutive weekly doses, and who had no protocol deviations that might affect efficacy. All randomised participants who received at least one dose of study drug were included in the safety analysis. This study is registered with ClinicalTrials.gov, NCT03334214. FINDINGS:Between Nov 3, 2017, and Nov 28, 2018, we screened 173 people for eligibility. 44 were enrolled and randomly assigned to receive either IONIS-DGAT2 (29 participants) or placebo (15 participants). After 13 weeks of treatment, the mean absolute reduction from baseline was -5·2% (SD 5·4) in the IONIS-DGAT2 group compared with -0·6% (6·1) in the placebo group (treatment difference -4·2%, 95% CI -7·8 to -0·5, p=0·026). Reductions in liver fat were not accompanied by hyperlipidaemia, elevations in serum aminotransferases or plasma glucose, changes in bodyweight, or gastrointestinal side-effects compared with placebo. Six serious adverse events occurred in four patients treated with IONIS-DGAT2. No serious adverse events were reported in the placebo group. One of four patients reported three serious adverse events: acute exacerbation of chronic obstructive pulmonary disease, cardiac arrest, and ischaemic cerebral infarction, each considered severe and not related to study drug. Three of four patients reported one serious adverse event of increased blood triglycerides (severe, unrelated to study drug), deep-vein thrombosis (severe, unlikely to be related to study drug), and acute pancreatitis (mild, unrelated to study drug). INTERPRETATION:Our results suggest that DGAT2 antisense inhibition could be a safe and efficacious strategy for treatment of NAFLD and support further investigation in patients with biopsy-proven NASH. Based on the pharmacological target, the response to treatment observed in this study population could extend to the broader population of patients with NAFLD. FUNDING:Ionis Pharmaceuticals.
Liver X Receptor Alpha Activation Inhibits Autophagy and Lipophagy in Hepatocytes by Dysregulating Autophagy-Related 4B Cysteine Peptidase and Rab-8B, Reducing Mitochondrial Fuel Oxidation.
Kim Yun Seok,Nam Hyeon Joo,Han Chang Yeob,Joo Min Sung,Jang Kiseok,Jun Dae Won,Kim Sang Geon
Hepatology (Baltimore, Md.)
BACKGROUND AND AIMS:Fat accumulation results from increased fat absorption and/or defective fat metabolism. Currently, the lipid-sensing nuclear receptor that controls fat utilization in hepatocytes is elusive. Liver X receptor alpha (LXRα) promotes accumulation of lipids through the induction of several lipogenic genes. However, its effect on lipid degradation is open for study. Here, we investigated the inhibitory role of LXRα in autophagy/lipophagy in hepatocytes and the underlying basis. APPROACH AND RESULTS:In LXRα knockout mice fed a high-fat diet, or cell models, LXRα activation suppressed the function of mitochondria by inhibiting autophagy/lipophagy and induced hepatic steatosis. Gene sets associated with "autophagy" were enriched in hepatic transcriptome data. Autophagy flux was markedly augmented in the LXRα knockout mouse liver and primary hepatocytes. Mechanistically, LXRα suppressed autophagy-related 4B cysteine peptidase (ATG4B) and Rab-8B, responsible for autophagosome and -lysosome formation, by inducing let-7a and microRNA (miR)-34a. Chromatin immunoprecipitation assay enabled us to find LXRα as a transcription factor of let-7a and miR-34a. Moreover, 3' untranslated region luciferase assay substantiated the direct inhibitory effects of let-7a and miR-34a on ATG4B and Rab-8B. Consistently, either LXRα activation or the let-7a/miR-34a transfection lowered mitochondrial oxygen consumption rate and mitochondrial transmembrane potential and increased fat levels. In obese animals or nonalcoholic fatty liver disease (NAFLD) patients, let-7a and miR-34a levels were elevated with simultaneous decreases in ATG4B and Rab-8B levels. CONCLUSIONS:LXRα inhibits autophagy in hepatocytes through down-regulating ATG4B and Rab-8B by transcriptionally activating microRNA let-7a-2 and microRNA 34a genes and suppresses mitochondrial biogenesis and fuel consumption. This highlights a function of LXRα that culminates in the progression of liver steatosis and steatohepatitis, and the identified targets may be applied for a therapeutic strategy in the treatment of NAFLD.
Impaired Kupffer Cell Self-Renewal Alters the Liver Response to Lipid Overload during Non-alcoholic Steatohepatitis.
Tran Sophie,Baba Ines,Poupel Lucie,Dussaud Sébastien,Moreau Martine,Gélineau Adélaïde,Marcelin Geneviève,Magréau-Davy Elissa,Ouhachi Melissa,Lesnik Philippe,Boissonnas Alexandre,Le Goff Wilfried,Clausen Björn E,Yvan-Charvet Laurent,Sennlaub Florian,Huby Thierry,Gautier Emmanuel L
Kupffer cells (KCs) are liver-resident macrophages that self-renew by proliferation in the adult independently from monocytes. However, how they are maintained during non-alcoholic steatohepatitis (NASH) remains ill defined. We found that a fraction of KCs derived from Ly-6C monocytes during NASH, underlying impaired KC self-renewal. Monocyte-derived KCs (MoKCs) gradually seeded the KC pool as disease progressed in a response to embryo-derived KC (EmKC) death. Those MoKCs were partly immature and exhibited a pro-inflammatory status compared to EmKCs. Yet, they engrafted the KC pool for the long term as they remained following disease regression while acquiring mature EmKC markers. While KCs as a whole favored hepatic triglyceride storage during NASH, EmKCs promoted it more efficiently than MoKCs, and the latter exacerbated liver damage, highlighting functional differences among KCs with different origins. Overall, our data reveal that KC homeostasis is impaired during NASH, altering the liver response to lipids, as well as KC ontogeny.
An Endoplasmic Reticulum Stress-MicroRNA-26a Feedback Circuit in NAFLD.
Xu Haixia,Tian Yan,Tang Dongmei,Zou Sailan,Liu Geng,Song Jiulin,Zhang Guixiang,Du Xiao,Huang Wei,He Bin,Lin Weiqiang,Jin Liang,Huang Wendong,Yang Jiayin,Fu Xianghui
Hepatology (Baltimore, Md.)
BACKGROUND AND AIMS:Endoplasmic reticulum (ER) stress is an adaptive response to excessive ER demand and contributes to the development of numerous diseases, including nonalcoholic fatty liver disease (NAFLD), which is hallmarked by the accumulation of lipid within hepatocytes. However, the underlying mechanisms remain elusive. MicroRNAs (miRNAs) play an indispensable role in various stress responses, but their implications in ER stress have not yet been systemically investigated. In this study, we identify a negative feedback loop consisting of hepatic ER stress and miR-26a in NAFLD pathogenesis. APPROACH AND RESULTS:Combining miRNA dot blot array and quantitative PCR, we find that miR-26a is specifically induced by ER stress in liver cells. This induction of miR-26a is critical for cells to cope with ER stress. In human hepatoma cells and murine primary hepatocytes, overexpression of miR-26a markedly alleviates chemical-induced ER stress, as well as palmitate-triggered ER stress and lipid accumulation. Conversely, deficiency of miR-26a exhibits opposite effects. Mechanistically, miR-26a directly targets the eukaryotic initiation factor 2α, a core ER stress effector controlling cellular translation. Intriguingly, miR-26a is reduced in the livers of patients with NAFLD. Hepatocyte-specific restoration of miR-26a in mice significantly mitigates high-fat diet-induced ER stress and hepatic steatosis. In contrast, deficiency of miR-26a in mice exacerbates high-fat diet-induced ER stress, lipid accumulation, inflammation and hepatic steatosis. CONCLUSIONS:Our findings suggest ER stress-induced miR-26a up-regulation as a regulator for hepatic ER stress resolution, and highlight the ER stress/miR-26a/eukaryotic initiation factor 2α cascade as a promising therapeutic strategy for NAFLD.
IRE1A Stimulates Hepatocyte-Derived Extracellular Vesicles That Promote Inflammation in Mice With Steatohepatitis.
Dasgupta Debanjali,Nakao Yasuhiko,Mauer Amy S,Thompson Jill M,Sehrawat Tejasav S,Liao Chieh-Yu,Krishnan Anuradha,Lucien Fabrice,Guo Qianqian,Liu Mengfei,Xue Fei,Fukushima Masanori,Katsumi Tomohiro,Bansal Aditya,Pandey Mukesh K,Maiers Jessica L,DeGrado Timothy,Ibrahim Samar H,Revzin Alexander,Pavelko Kevin D,Barry Michael A,Kaufman Randal J,Malhi Harmeet
BACKGROUND & AIMS:Endoplasmic reticulum to nucleus signaling 1 (ERN1, also called IRE1A) is a sensor of the unfolded protein response that is activated in the livers of patients with nonalcoholic steatohepatitis (NASH). Hepatocytes release ceramide-enriched inflammatory extracellular vesicles (EVs) after activation of IRE1A. We studied the effects of inhibiting IRE1A on release of inflammatory EVs in mice with diet-induced steatohepatitis. METHODS:C57BL/6J mice and mice with hepatocyte-specific disruption of Ire1a (IRE1α) were fed a diet high in fat, fructose, and cholesterol to induce development of steatohepatitis or a standard chow diet (controls). Some mice were given intraperitoneal injections of the IRE1A inhibitor 4μ8C. Mouse liver and primary hepatocytes were transduced with adenovirus or adeno-associated virus that expressed IRE1A. Livers were collected from mice and analyzed by quantitative polymerase chain reaction and chromatin immunoprecipitation assays; plasma samples were analyzed by enzyme-linked immunosorbent assay. EVs were derived from hepatocytes and injected intravenously into mice. Plasma EVs were characterized by nanoparticle-tracking analysis, electron microscopy, immunoblots, and nanoscale flow cytometry; we used a membrane-tagged reporter mouse to detect hepatocyte-derived EVs. Plasma and liver tissues from patients with NASH and without NASH (controls) were analyzed for EV concentration and by RNAscope and gene expression analyses. RESULTS:Disruption of Ire1a in hepatocytes or inhibition of IRE1A reduced the release of EVs and liver injury, inflammation, and accumulation of macrophages in mice on the diet high in fat, fructose, and cholesterol. Activation of IRE1A, in the livers of mice, stimulated release of hepatocyte-derived EVs, and also from cultured primary hepatocytes. Mice given intravenous injections of IRE1A-stimulated, hepatocyte-derived EVs accumulated monocyte-derived macrophages in the liver. IRE1A-stimulated EVs were enriched in ceramides. Chromatin immunoprecipitation showed that IRE1A activated X-box binding protein 1 (XBP1) to increase transcription of serine palmitoyltransferase genes, which encode the rate-limiting enzyme for ceramide biosynthesis. Administration of a pharmacologic inhibitor of serine palmitoyltransferase to mice reduced the release of EVs. Levels of XBP1 and serine palmitoyltransferase were increased in liver tissues, and numbers of EVs were increased in plasma, from patients with NASH compared with control samples and correlated with the histologic features of inflammation. CONCLUSIONS:In mouse hepatocytes, activated IRE1A promotes transcription of serine palmitoyltransferase genes via XBP1, resulting in ceramide biosynthesis and release of EVs. The EVs recruit monocyte-derived macrophages to the liver, resulting in inflammation and injury in mice with diet-induced steatohepatitis. Levels of XBP1, serine palmitoyltransferase, and EVs are all increased in liver tissues from patients with NASH. Strategies to block this pathway might be developed to reduce liver inflammation in patients with NASH.
Loss of hepatic Mboat7 leads to liver fibrosis.
Thangapandi Veera Raghavan,Knittelfelder Oskar,Brosch Mario,Patsenker Eleonora,Vvedenskaya Olga,Buch Stephan,Hinz Sebastian,Hendricks Alexander,Nati Marina,Herrmann Alexander,Rekhade Devavrat Ravindra,Berg Thomas,Matz-Soja Madlen,Huse Klaus,Klipp Edda,Pauling Josch K,Wodke Judith Ah,Miranda Ackerman Jacobo,Bonin Malte von,Aigner Elmar,Datz Christian,von Schönfels Witigo,Nehring Sophie,Zeissig Sebastian,Röcken Christoph,Dahl Andreas,Chavakis Triantafyllos,Stickel Felix,Shevchenko Andrej,Schafmayer Clemens,Hampe Jochen,Subramanian Pallavi
OBJECTIVE:The rs641738C>T variant located near the membrane-bound O-acyltransferase domain containing 7 (MBOAT7) locus is associated with fibrosis in liver diseases, including non-alcoholic fatty liver disease (NAFLD), alcohol-related liver disease, hepatitis B and C. We aim to understand the mechanism by which the rs641738C>T variant contributes to pathogenesis of NAFLD. DESIGN:Mice with hepatocyte-specific deletion of MBOAT7 (Mboat7) were generated and livers were characterised by histology, flow cytometry, qPCR, RNA sequencing and lipidomics. We analysed the association of rs641738C>T genotype with liver inflammation and fibrosis in 846 NAFLD patients and obtained genotype-specific liver lipidomes from 280 human biopsies. RESULTS:Allelic imbalance analysis of heterozygous human liver samples pointed to lower expression of the MBOAT7 transcript on the rs641738C>T haplotype. Mboat7 mice showed spontaneous steatosis characterised by increased hepatic cholesterol ester content after 10 weeks. After 6 weeks on a high fat, methionine-low, choline-deficient diet, mice developed increased hepatic fibrosis as measured by picrosirius staining (p0.05), hydroxyproline content (p0.05) and transcriptomics, while the inflammatory cell populations and inflammatory mediators were minimally affected. In a human biopsied NAFLD cohort, MBOAT7 rs641738C>T was associated with fibrosis (p0.004) independent of the presence of histological inflammation. Liver lipidomes of Mboat7 mice and human rs641738TT carriers with fibrosis showed increased total lysophosphatidylinositol levels. The altered lysophosphatidylinositol and phosphatidylinositol subspecies in MBOAT7 livers and human rs641738TT carriers were similar. CONCLUSION:Mboat7 deficiency in mice and human points to an inflammation-independent pathway of liver fibrosis that may be mediated by lipid signalling and a potentially targetable treatment option in NAFLD.
iRhom2 Promotes Hepatic Steatosis by Activating MAP3K7-Dependent Pathway.
Xu Minxuan,Ge Chenxu,Zhu Liancai,Qin Yuting,Du Chengjiang,Lou Deshuai,Li Qiang,Hu Linfeng,Sun Yan,Dai Xianling,Xiong Mingxin,Long Tingting,Zhan Jianxia,Kuang Qin,Li Huanhuan,Yang Qiufeng,Huang Ping,Teng Xuepeng,Feng Jing,Wu Yekuan,Dong Wei,Wang Bochu,Tan Jun
Hepatology (Baltimore, Md.)
BACKGROUND AND AIMS:Nonalcoholic fatty liver disease (NAFLD) has been widely recognized as a precursor to metabolic complications. Elevated inflammation levels are predictive of NAFLD-associated metabolic disorder. Inactive rhomboid-like protein 2 (iRhom2) is regarded as a key regulator in inflammation. However, the precise mechanisms by which iRhom2-regulated inflammation promotes NAFLD progression remain to be elucidated. APPROACH AND RESULTS:Here, we report that insulin resistance, hepatic steatosis, and specific macrophage inflammatory activation are significantly alleviated in iRhom2-deficient (knockout [KO]) mice, but aggravated in iRhom2 overexpressing mice. We further show that, mechanistically, in response to a high-fat diet (HFD), iRhom2 KO mice and mice with iRhom2 deficiency in myeloid cells only showed less severe hepatic steatosis and insulin resistance than controls. Inversely, transplantation of bone marrow cells from healthy mice to iRhom2 KO mice expedited the severity of insulin resistance and hepatic dyslipidemia. Of note, in response to HFD, hepatic iRhom2 binds to mitogen-activated protein kinase kinase kinase 7 (MAP3K7) to facilitate MAP3K7 phosphorylation and nuclear factor kappa B cascade activation, thereby promoting the activation of c-Jun N-terminal kinase/insulin receptor substrate 1 signaling, but disturbing AKT/glycogen synthase kinase 3β-associated insulin signaling. The iRhom2/MAP3K7 axis is essential for iRhom2-regulated liver steatosis. CONCLUSIONS:iRhom2 may represent a therapeutic target for the treatment of HFD-induced hepatic steatosis and insulin resistance.
A Universal Gut-Microbiome-Derived Signature Predicts Cirrhosis.
Oh Tae Gyu,Kim Susy M,Caussy Cyrielle,Fu Ting,Guo Jian,Bassirian Shirin,Singh Seema,Madamba Egbert V,Bettencourt Ricki,Richards Lisa,Yu Ruth T,Atkins Annette R,Huan Tao,Brenner David A,Sirlin Claude B,Downes Michael,Evans Ronald M,Loomba Rohit
Dysregulation of the gut microbiome has been implicated in the progression of non-alcoholic fatty liver disease (NAFLD) to advanced fibrosis and cirrhosis. To determine the diagnostic capacity of this association, we compared stool microbiomes across 163 well-characterized participants encompassing non-NAFLD controls, NAFLD-cirrhosis patients, and their first-degree relatives. Interrogation of shotgun metagenomic and untargeted metabolomic profiles by using the random forest machine learning algorithm and differential abundance analysis identified discrete metagenomic and metabolomic signatures that were similarly effective in detecting cirrhosis (diagnostic accuracy 0.91, area under curve [AUC]). Combining the metagenomic signature with age and serum albumin levels accurately distinguished cirrhosis in etiologically and genetically distinct cohorts from geographically separated regions. Additional inclusion of serum aspartate aminotransferase levels, which are increased in cirrhosis patients, enabled discrimination of cirrhosis from earlier stages of fibrosis. These findings demonstrate that a core set of gut microbiome species might offer universal utility as a non-invasive diagnostic test for cirrhosis.
Silencing hepatic MCJ attenuates non-alcoholic fatty liver disease (NAFLD) by increasing mitochondrial fatty acid oxidation.
Barbier-Torres Lucía,Fortner Karen A,Iruzubieta Paula,Delgado Teresa C,Giddings Emily,Chen Youdinghuan,Champagne Devin,Fernández-Ramos David,Mestre Daniela,Gomez-Santos Beatriz,Varela-Rey Marta,de Juan Virginia Gutiérrez,Fernández-Tussy Pablo,Zubiete-Franco Imanol,García-Monzón Carmelo,González-Rodríguez Águeda,Oza Dhaval,Valença-Pereira Felipe,Fang Qian,Crespo Javier,Aspichueta Patricia,Tremblay Frederic,Christensen Brock C,Anguita Juan,Martínez-Chantar María Luz,Rincón Mercedes
Nonalcoholic fatty liver disease (NAFLD) is considered the next major health epidemic with an estimated 25% worldwide prevalence. No drugs have yet been approved and NAFLD remains a major unmet need. Here, we identify MCJ (Methylation-Controlled J protein) as a target for non-alcoholic steatohepatitis (NASH), an advanced phase of NAFLD. MCJ is an endogenous negative regulator of the respiratory chain Complex I that acts to restrain mitochondrial respiration. We show that therapeutic targeting of MCJ in the liver with nanoparticle- and GalNAc-formulated siRNA efficiently reduces liver lipid accumulation and fibrosis in multiple NASH mouse models. Decreasing MCJ expression enhances the capacity of hepatocytes to mediate β-oxidation of fatty acids and minimizes lipid accumulation, which results in reduced hepatocyte damage and fibrosis. Moreover, MCJ levels in the liver of NAFLD patients are elevated relative to healthy subjects. Thus, inhibition of MCJ emerges as an alternative approach to treat NAFLD.
Intestinal Virome Signature Associated With Severity of Nonalcoholic Fatty Liver Disease.
Lang Sonja,Demir Münevver,Martin Anna,Jiang Lu,Zhang Xinlian,Duan Yi,Gao Bei,Wisplinghoff Hilmar,Kasper Philipp,Roderburg Christoph,Tacke Frank,Steffen Hans-Michael,Goeser Tobias,Abraldes Juan G,Tu Xin M,Loomba Rohit,Stärkel Peter,Pride David,Fouts Derrick E,Schnabl Bernd
BACKGROUND & AIMS:Alterations in the gut microbiome have been associated with the severity of nonalcoholic fatty liver disease (NAFLD). Previous studies focused exclusively on the bacteria in the microbiome; we investigated changes in the viral microbiome (virome) in patients with NAFLD. METHODS:In a prospective, cross-sectional, observational study, we extracted RNA and DNA virus-like particles from fecal samples from 73 patients with NAFLD: 29 patients had an NAFLD Activity Score (NAS) of 0-4, 44 patients had an NAS of 5-8 or liver cirrhosis (LCI), 37 patients had F0-F1 fibrosis, and 36 patients had F2-F4 fibrosis. As controls, 9 individuals without liver disease and 13 patients with mild primary biliary cholangitis were included in the analysis. We performed shotgun metagenomic sequencing of virus-like particles. RESULTS:Patients with NAFLD and NAS 5-8/LCI had a significant decrease in intestinal viral diversity compared with patients with NAFLD and NAS 0-4 or control individuals. The presence of more advanced NAFLD was associated with a significant reduction in the proportion of bacteriophages compared with other intestinal viruses. Using multivariate logistic regression analysis with leave-1-out cross validation, we developed a model, including a viral diversity index and simple clinical variables, that identified patients with NAS 5-8/LCI with an area under the curve of 0.95 (95% confidence interval, 0.91-0.99) and F2-F4 fibrosis with an area under the curve of 0.88 (95% confidence interval, 0.80-0.95). Addition of data on viral diversity significantly improved multivariate models, including those based on only clinical parameters or bacterial diversity. CONCLUSIONS:In a study of fecal viromes from patients with NAFLD and control individuals, we associated histologic markers of NAFLD severity with significant decreases in viral diversity and proportion of bacteriophages. We developed a model based on fecal viral diversity and clinical data that identifies patients with severe NAFLD and fibrosis more accurately than models based only on clinical or bacterial data.
AGER1 downregulation associates with fibrosis in nonalcoholic steatohepatitis and type 2 diabetes.
Dehnad Ali,Fan Weiguo,Jiang Joy X,Fish Sarah R,Li Yuan,Das Suvarthi,Mozes Gergely,Wong Kimberly A,Olson Kristin A,Charville Gregory W,Ali Mohammed,Török Natalie J
The Journal of clinical investigation
Type 2 diabetes is clinically associated with progressive necroinflammation and fibrosis in nonalcoholic steatohepatitis (NASH). Advanced glycation end-products (AGEs) accumulate during prolonged hyperglycemia, but the mechanistic pathways that lead to accelerated liver fibrosis have not been well defined. In this study, we show that the AGEs clearance receptor AGER1 was downregulated in patients with NASH and diabetes and in our NASH models, whereas the proinflammatory receptor RAGE was induced. These findings were associated with necroinflammatory, fibrogenic, and pro-oxidant activity via the NADPH oxidase 4. Inhibition of AGEs or RAGE deletion in hepatocytes in vivo reversed these effects. We demonstrate that dysregulation of NRF2 by neddylation of cullin 3 was linked to AGER1 downregulation and that induction of NRF2 using an adeno-associated virus-mediated approach in hepatocytes in vivo reversed AGER1 downregulation, lowered the level of AGEs, and improved proinflammatory and fibrogenic responses in mice on a high AGEs diet. In patients with NASH and diabetes or insulin resistance, low AGER1 levels were associated with hepatocyte ballooning degeneration and ductular reaction. Collectively, prolonged exposure to AGEs in the liver promotes an AGER1/RAGE imbalance and consequent redox, inflammatory, and fibrogenic activity in NASH.
Dietary cholesterol drives fatty liver-associated liver cancer by modulating gut microbiota and metabolites.
Zhang Xiang,Coker Olabisi Oluwabukola,Chu Eagle Sh,Fu Kaili,Lau Harry C H,Wang Yi-Xiang,Chan Anthony W H,Wei Hong,Yang Xiaoyong,Sung Joseph J Y,Yu Jun
OBJECTIVE:Non-alcoholic fatty liver disease (NAFLD)-associated hepatocellular carcinoma (HCC) is an increasing healthcare burden worldwide. We examined the role of dietary cholesterol in driving NAFLD-HCC through modulating gut microbiota and its metabolites. DESIGN:High-fat/high-cholesterol (HFHC), high-fat/low-cholesterol or normal chow diet was fed to C57BL/6 male littermates for 14 months. Cholesterol-lowering drug atorvastatin was administered to HFHC-fed mice. Germ-free mice were transplanted with stools from mice fed different diets to determine the direct role of cholesterol modulated-microbiota in NAFLD-HCC. Gut microbiota was analysed by 16S rRNA sequencing and serum metabolites by liquid chromatography-mass spectrometry (LC-MS) metabolomic analysis. Faecal microbial compositions were examined in 59 hypercholesterolemia patients and 39 healthy controls. RESULTS:High dietary cholesterol led to the sequential progression of steatosis, steatohepatitis, fibrosis and eventually HCC in mice, concomitant with insulin resistance. Cholesterol-induced NAFLD-HCC formation was associated with gut microbiota dysbiosis. The microbiota composition clustered distinctly along stages of steatosis, steatohepatitis and HCC. and increased sequentially; while and were depleted in HFHC-fed mice, which was corroborated in human hypercholesteremia patients. Dietary cholesterol induced gut bacterial metabolites alteration including increased taurocholic acid and decreased 3-indolepropionic acid. Germ-free mice gavaged with stools from mice fed HFHC manifested hepatic lipid accumulation, inflammation and cell proliferation. Moreover, atorvastatin restored cholesterol-induced gut microbiota dysbiosis and completely prevented NAFLD-HCC development. CONCLUSIONS:Dietary cholesterol drives NAFLD-HCC formation by inducing alteration of gut microbiota and metabolites in mice. Cholesterol inhibitory therapy and gut microbiota manipulation may be effective strategies for NAFLD-HCC prevention.
FXR Isoforms Control Different Metabolic Functions in Liver Cells via Binding to Specific DNA Motifs.
Ramos Pittol Jose Miguel,Milona Alexandra,Morris Imogen,Willemsen Ellen C L,van der Veen Suzanne W,Kalkhoven Eric,van Mil Saskia W C
BACKGROUND & AIMS:The nuclear receptor subfamily 1 group H member 4 (NR1H4, also called FXR) is a ligand-activated transcription factor that, upon binding of bile acids, regulates the expression of genes involved in bile acid, fat, sugar, and amino acid metabolism. Transcript variants encode the FXR isoforms alpha 1, alpha 2, alpha 3, and alpha 4, which activate different genes that regulate metabolism. Little is known about the mechanisms by which the different isoforms regulate specific genes or how the expression of these genes affects the outcomes of patients given drugs that target FXR. METHODS:We determined genome-wide binding of FXR isoforms in mouse liver organoids that express individual FXR isoforms using chromatin immunoprecipitation, followed by sequencing analysis and DNA motif discovery. We validated regulatory DNA sequences by mobility shift assays and with luciferase reporters using mouse and human FXR isoforms. We analyzed mouse liver organoids and HepG2 cells that expressed the FXR isoforms using chromatin immunoprecipitation, quantitative polymerase chain reaction, and immunoblot assays. Organoids were analyzed for mitochondrial respiration, lipid droplet content, and triglyceride excretion. We used the FXR ligand obeticholic acid to induce FXR activity in organoids, cell lines, and mice. We collected data on the binding of FXR in mouse liver and the expression levels of FXR isoforms and gene targets in human liver tissue and primary human hepatocytes from the Gene Expression Omnibus. RESULTS:In mouse liver cells, 89% of sites that bound FXR were bound by only FXRα2 or FXRα4, via direct interactions with the DNA sequence motif ER-2. Via DNA binding, these isoforms regulated metabolic functions in liver cells, including carbon metabolism and lipogenesis. Incubation with obeticholic acid increased mitochondrial pyruvate transport and reduced insulin-induced lipogenesis in organoids that expressed FXRα2 but not FXRα1. In human liver tissues, levels of FXRα2 varied significantly and correlated with expression of genes predicted to be regulated via an ER-2 motif. CONCLUSIONS:Most metabolic effects regulated by FXR in mouse and human liver cells are regulated by the FXRα2 isoform via specific binding to ER-2 motifs. The expression level of FXRα2 in liver might be used to predict responses of patients to treatment with FXR agonists.
Targeting NFATc4 attenuates non-alcoholic steatohepatitis in mice.
Du Meng,Wang Xiaojing,Yuan Lin,Liu Bing,Mao Xiaoxiang,Huang Dandan,Yang Liu,Huang Kun,Zhang Fengxiao,Wang Yan,Luo Xi,Wang Cheng,Peng Jiangtong,Liang Minglu,Huang Dan,Huang Kai
Journal of hepatology
BACKGROUND & AIMS:The nuclear factor of activated T-cells (NFAT) family was first recognised to play an important role in the differentiation of T cells, but has since been shown to regulate multiple pathophysiological processes. However, whether it is involved in the pathogenesis of non-alcoholic steatohepatitis (NASH) remains unknown. METHODS:Hepatic NFATc expression and localisation were analysed in C57BL/6 mice on a methionine-choline-deficient diet, as well as in samples from non-alcoholic fatty liver disease patients. Gain- or loss-of-function approaches were used to investigate the role of NFATc4 in NASH. RESULTS:NFATc4 translocates from the cytoplasm to the nucleus in hepatocytes of both humans and rodents with NASH. NFATc4 knockdown resulted in decreased hepatic steatosis, inflammation, and fibrosis during NASH progression. Mechanistically, we found that activated NFATc4 directly bound to peroxisome proliferator-activated receptor α (PPARα) in the nucleus and negatively regulated its transcriptional activity, thereby impairing the hepatic fatty acid oxidation pathway and increasing lipid deposition in the liver. Moreover, NFATc4 activation increased the production and secretion of osteopontin (OPN) from hepatocytes, which subsequently enhanced the macrophage-mediated inflammatory response and hepatic stellate cell-mediated fibrosis progression via paracrine signalling. CONCLUSIONS:Hepatic NFATc4 activation accelerates the progression of NASH by suppressing PPARα signalling and increasing OPN expression. Genetic or pharmacological inhibition of NFATc4 may have potential for future therapy of NASH. LAY SUMMARY:NFATc4 is activated in the non-alcoholic steatohepatitis of mice and patients. Inhibition of NFATc4 activation alleviates lipid deposition, inflammatory response, and fibrosis progression in the liver.
Reduced Nogo expression inhibits diet-induced metabolic disorders by regulating ChREBP and insulin activity.
Zhang Shuang,Guo Fangling,Yu Miao,Yang Xiaoxiao,Yao Zhi,Li Qi,Wei Zhuo,Feng Ke,Zeng Peng,Zhao Dan,Li Xiaoju,Zhu Yan,Miao Qing Robert,Iwakiri Yasuko,Chen Yuanli,Han Jihong,Duan Yajun
Journal of hepatology
BACKGROUND & AIMS:Chronic overconsumption of a high-carbohydrate diet leads to steatosis and its associated metabolic disorder and, eventually, to non-alcoholic fatty liver disease. Carbohydrate-responsive element binding protein (ChREBP) and insulin regulate de novo lipogenesis from glucose. Herein, we studied the effect of reticulon-4 (Nogo) expression on diet-induced metabolic disorders in mice. METHODS:Nogo-deficient (Nogo) and littermate control [wild-type (WT)] mice were fed a high-glucose or high-fructose diet (HGD/HFrD) to induce metabolic disorders. The effects of Nogo small interfering (si) RNA (siRNA) on HFrD-induced metabolic disorders were investigated in C57BL/6J mice. RESULTS:HGD/HFrD induced steatosis and its associated metabolic disorders in WT mice by activating ChREBP and impairing insulin sensitivity. They also activated Nogo-B expression, which in turn inhibited insulin activity. In response to HGD/HFrD feeding, Nogo deficiency enhanced insulin sensitivity and energy metabolism to reduce the expression of ChREBP and lipogenic molecules, activated AMP-activated catalytic subunit α, peroxisome proliferator activated receptor α and fibroblast growth factor 21, and reduced endoplasmic reticulum (ER) stress and inflammation, thereby blocking HGD/HFrD-induced hepatic lipid accumulation, insulin resistance and other metabolic disorders. Injection of Nogo siRNA protected C57BL/6J mice against HFrD-induced metabolic disorders by ameliorating insulin sensitivity, ChREBP activity, ER stress and inflammation. CONCLUSIONS:Our study identified Nogo as an important mediator of insulin sensitivity and ChREBP activity. Reduction of Nogo expression is a potential strategy for the treatment of high-carbohydrate diet-induced metabolic complications. LAY SUMMARY:Nogo deficiency blocks high-carbohydrate diet-induced glucose intolerance and insulin resistance, while increasing glucose/lipid utilisation and energy expenditure. Thus, reduction of Nogo expression protects against high-carbohydrate diet-induced body-weight gain, hepatic lipid accumulation and the associated metabolic disorders, indicating that approaches inhibiting Nogo expression can be applied for the treatment of diseases associated with metabolic disorders.
Hepatocyte pyroptosis and release of inflammasome particles induce stellate cell activation and liver fibrosis.
Gaul Susanne,Leszczynska Aleksandra,Alegre Fernando,Kaufmann Benedikt,Johnson Casey D,Adams Leon A,Wree Alexander,Damm Georg,Seehofer Daniel,Calvente Carolina J,Povero Davide,Kisseleva Tatiana,Eguchi Akiko,McGeough Matthew D,Hoffman Hal M,Pelegrin Pablo,Laufs Ulrich,Feldstein Ariel E
Journal of hepatology
BACKGROUND & AIMS:Increased hepatocyte death contributes to the pathology of acute and chronic liver diseases. However, the role of hepatocyte pyroptosis and extracellular inflammasome release in liver disease is unknown. METHODS:We used primary mouse and human hepatocytes, hepatocyte-specific leucine 351 to proline Nlrp3CreA mice, and Gsdmd mice to investigate pyroptotic cell death in hepatocytes and its impact on liver inflammation and damage. Extracellular NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes were isolated from mutant NLRP3-YFP HEK cells and internalisation was studied in LX2 and primary human hepatic stellate cells. We also examined a cohort of 154 adult patients with biopsy-proven non-alcoholic fatty liver disease (Sir Charles Gairdner Hospital, Nedlands, Western Australia). RESULTS:We demonstrated that primary mouse and human hepatocytes can undergo pyroptosis upon NLRP3 inflammasome activation with subsequent release of NLRP3 inflammasome proteins that amplify and perpetuate inflammasome-driven fibrogenesis. Pyroptosis was inhibited by blocking caspase-1 and gasdermin D activation. The activated form of caspase-1 was detected in the livers and in serum from patients with non-alcoholic steatohepatitis and correlated with disease severity. Nlrp3CreA mice showed spontaneous liver fibrosis under normal chow diet, and increased sensitivity to liver damage and inflammation after treatment with low dose lipopolysaccharide. Mechanistically, hepatic stellate cells engulfed extracellular NLRP3 inflammasome particles leading to increased IL-1β secretion and α-smooth muscle actin expression. This effect was abrogated when cells were pre-treated with the endocytosis inhibitor cytochalasin B. CONCLUSIONS:These results identify hepatocyte pyroptosis and release of inflammasome components as a novel mechanism to propagate liver injury and liver fibrosis development. LAY SUMMARY:Our findings identify a novel mechanism of inflammation in the liver. Experiments in cell cultures, mice, and human samples show that a specific form of cell death, called pyroptosis, leads to the release of complex inflammatory particles, the NLRP3 inflammasome, from inside hepatocytes into the extracellular space. From there they are taken up by other cells and thereby mediate inflammatory and pro-fibrogenic stress signals. The discovery of this mechanism may lead to novel treatments for chronic liver diseases in the future.
17-Beta Hydroxysteroid Dehydrogenase 13 Deficiency Does Not Protect Mice From Obesogenic Diet Injury.
Hepatology (Baltimore, Md.)
BACKGROUND AND AIMS:17-Beta hydroxysteroid dehydrogenase 13 (HSD17B13) is genetically associated with human nonalcoholic fatty liver disease (NAFLD). Inactivating mutations in HSD17B13 protect humans from NAFLD-associated and alcohol-associated liver injury, fibrosis, cirrhosis, and hepatocellular carcinoma, leading to clinical trials of anti-HSD17B13 therapeutic agents in humans. We aimed to study the in vivo function of HSD17B13 using a mouse model. APPROACH AND RESULTS:Single-cell RNA-sequencing and quantitative RT-PCR data revealed that hepatocytes are the main HSD17B13-expressing cells in mice and humans. We compared Hsd17b13 whole-body knockout (KO) mice and wild-type (WT) littermate controls fed regular chow (RC), a high-fat diet (HFD), a Western diet (WD), or the National Institute on Alcohol Abuse and Alcoholism model of alcohol exposure. HFD and WD induced significant weight gain, hepatic steatosis, and inflammation. However, there was no difference between genotypes with regard to body weight, liver weight, hepatic triglycerides (TG), histological inflammatory scores, expression of inflammation-related and fibrosis-related genes, and hepatic retinoid levels. Compared to WT, KO mice on the HFD had hepatic enrichment of most cholesterol esters, monoglycerides, and certain sphingolipid species. Extended feeding with the WD for 10 months led to extensive liver injury, fibrosis, and hepatocellular carcinoma, with no difference between genotypes. Under alcohol exposure, KO and WT mice showed similar hepatic TG and liver enzyme levels. Interestingly, chow-fed KO mice showed significantly higher body and liver weights compared to WT mice, while KO mice on obesogenic diets had a shift toward larger lipid droplets. CONCLUSIONS:Extensive evaluation of Hsd17b13 deficiency in mice under several fatty liver-inducing dietary conditions did not reproduce the protective role of HSD17B13 loss-of-function mutants in human NAFLD. Moreover, mouse Hsd17b13 deficiency induces weight gain under RC. It is crucial to understand interspecies differences prior to leveraging HSD17B13 therapies.
Efficacy and Safety of Aldafermin, an Engineered FGF19 Analog, in a Randomized, Double-Blind, Placebo-Controlled Trial of Patients With Nonalcoholic Steatohepatitis.
Harrison Stephen A,Neff Guy,Guy Cynthia D,Bashir Mustafa R,Paredes Angelo H,Frias Juan P,Younes Ziad,Trotter James F,Gunn Nadege T,Moussa Sam E,Kohli Anita,Nelson Kristin,Gottwald Mildred,Chang William C G,Yan Andrew Z,DePaoli Alex M,Ling Lei,Lieu Hsiao D
BACKGROUND & AIMS:Aldafermin, an engineered analog of fibroblast growth factor 19, inhibits bile acid synthesis and regulates metabolic homeostasis. We report results from a 24-week, phase 2 study, with serial liver biopsies, of patients with nonalcoholic steatohepatitis (NASH). METHODS:We performed a double-blind study of 78 patients with NASH at 9 centers in the United States. Key inclusion criteria were biopsy-proven NASH with Nonalcoholic Fatty Liver Disease Activity Score ≥4, stage 2 or 3 fibrosis by NASH Clinical Research Network classification, and absolute liver fat content ≥8%, measured by magnetic resonance imaging-proton density fat fraction. Patients were randomly assigned (1:2) to groups given subcutaneous placebo (n = 25) or aldafermin 1 mg (n = 53) daily for 24 weeks. The primary outcome was change in absolute liver fat content from baseline at week 24. Secondary outcomes included serum markers and histologic measures of fibrosis improvement and NASH resolution. RESULTS:At week 24, the aldafermin group had a significant reduction in absolute liver fat content (reduction of 7.7%) compared with placebo (reduction of 2.7%; difference, reduction of 5.0%; 95% confidence interval, reduction of 8.0%-1.9%; P = .002). Aldafermin produced significantly greater decreases in levels of 7α-hydroxy-4-cholesten-3-one, bile acids, alanine and aspartate aminotransferases, and neoepitope-specific N-terminal pro-peptide of type III collagen (Pro-C3) than placebo. Fibrosis improvement (≥1 stage) with no worsening of NASH was achieved in 38% of patients receiving aldafermin vs 18% of patients receiving placebo (P = .10). NASH resolution with no worsening of fibrosis was observed in 24% of patients given aldafermin vs 9% of patients given placebo (P = .20). Discontinuations due to adverse events occurred in no patients in the aldafermin group and 4% of patients in the placebo group. CONCLUSIONS:In a phase 2 trial of patients with NASH, aldafermin reduced liver fat and produced a trend toward fibrosis improvement. ClinicalTrials.gov, Number: NCT02443116.
Microbiota and Fatty Liver Disease-the Known, the Unknown, and the Future.
Lang Sonja,Schnabl Bernd
Cell host & microbe
The liver communicates with the intestine via the portal vein, biliary system, and mediators in the circulation. Microbes in the intestine maintain liver homeostasis but can also serve as a source of pathogens and molecules that contribute to fatty liver diseases. We review changes in the gut microbiota that can promote development or progression of alcohol-associated and non-alcoholic fatty liver disease-the most common chronic liver diseases in Western countries. We discuss how microbes and their products contribute to liver disease pathogenesis, putative microbial biomarkers of disease, and potential treatment approaches based on manipulation of the gut microbiota. Increasing our understanding of interactions between the intestinal microbiome and liver might help us identify patients with specific disease subtypes and select specific microbiota-based therapies.
The Commensal Microbe Veillonella as a Marker for Response to an FGF19 Analog in NASH.
Loomba Rohit,Ling Lei,Dinh Duy M,DePaoli Alex M,Lieu Hsiao D,Harrison Stephen A,Sanyal Arun J
Hepatology (Baltimore, Md.)
BACKGROUND AND AIMS:The composition of the human gut microbiota is linked to health and disease, and knowledge of the impact of therapeutics on the microbiota is essential to decipher their biological roles and to gain new mechanistic insights. Here we report the effect of aldafermin, an analog of the gut hormone FGF19, versus placebo on the gut microbiota in a prospective, phase 2 study in patients with NASH. APPROACH AND RESULTS:A total of 176 patients with biopsy-confirmed nonalcoholic steatohepatitis (NASH) (nonalcoholic fatty liver disease activity score ≥ 4), fibrosis (F1-F3 by NASH Clinical Research Network criteria), and elevated liver fat content (≥ 8% by magnetic resonance imaging-proton density fat fraction) received 0.3 mg (n = 23), 1 mg (n = 49), 3 mg (n = 49), and 6 mg (n = 28) aldafermin or placebo (n = 27) for 12 weeks. Stool samples were collected on day 1 and week 12 and profiled using 16S ribosomal RNA gene sequencing; 122 patients had paired stool microbiome profiles at both day 1 and week 12. Overall, the state of the gut microbial community was distinctly stable in patients treated with aldafermin, with all major phyla and genera unaltered during therapy. Patients treated with aldafermin showed a significant, dose-dependent enrichment in the rare genus Veillonella, a commensal microbe known to have lactate-degrading and performance-enhancing properties, which correlated with changes in serum bile acid profile. CONCLUSIONS:Veillonella may be a bile acid-sensitive bacteria whose enrichment is enabled by aldafermin-mediated suppression of bile acid synthesis and, in particular, decreases in toxic bile acids. This study provides an integrated analysis of gut microbiome, serum bile acid metabolome, imaging, and histological measurements in clinical trials testing aldafermin for NASH. Our results provide a better understanding of the intricacies of microbiome-host interactions (clinicaltrials.gov trial No. NCT02443116).
miR-552-3p modulates transcriptional activities of FXR and LXR to ameliorate hepatic glycolipid metabolism disorder.
Fan Lei,Lai Rongtao,Ma Ningning,Dong Yunxia,Li Yu,Wu Qian,Qiao Junwen,Lu Henglei,Gong Likun,Tao Zhouteng,Chen Jing,Xie Qing,Ren Jin
Journal of hepatology
BACKGROUND & AIMS:The nuclear location of miRNAs has been known for more than a decade, but the exact function of miRNAs in the nucleus has not been fully elucidated. We previously discovered that intranuclear miR-552-3p has an inhibitory role on gene transcription and contains a particular AGGTCA-like sequence, the cis-elements of the NR1 subfamily of nuclear receptors. Here, we aim to explore the potential effect of miR-552-3p and its AGGTCA-like sequence on NR1s and its possible application in improving hepatic glycolipid metabolism. METHODS:RNA-seq, mass spectrometry, and bioinformatics analysis were used to reveal the possible pathways influenced by miR-552-3p. High fat-high fructose diet-fed mice and db/db mice transfected with AAV2/8-miR-552-3p were established to investigate the in vivo effects of miR-552-3p on hepatic glycolipid metabolism. Fluorescence resonance energy transfer, pull-down, electrophoretic mobility shift, and chromatin immunoprecipitation assays were performed to explore the mechanism by which miR-552-3p regulates NR1s. RT-PCR was conducted to analyse miR-552-3p levels in liver biopsies from patients with NAFLD and normal controls. RESULTS:MiR-552-3p could inhibit metabolic gene expression in vitro and displayed beneficial effects on glycolipid metabolism in vivo. Intranuclear miR-552-3p primarily regulated the LXRα and FXR pathways; this was achieved by its binding to the complementary sequence of AGGTCA to modulate the transcriptional activities of LXRα and FXR. Moreover, LXRα and FXR ligands could restore the effects of miR-552-3p on gene expression and glycolipid metabolism. Additionally, the hepatic miR-552-3p level was significantly decreased in liver samples from patients with NAFLD compared to normal controls. CONCLUSIONS:The mechanism by which miR-552-3p modulates LXRα and FXR has revealed a new method of miRNA-mediated gene regulation. In addition, the beneficial effects in vivo and clinical relevance of miR-552-3p suggest that it might be a potential therapeutic target for the treatment of glycolipid metabolic disease. LAY SUMMARY:Glycolipid metabolic diseases, which have become a major public health concern worldwide, are triggered by abnormalities in lipid and glucose metabolism. Herein, we show that miR-552-3p has the ability to ameliorate hepatic glycolipid metabolic diseases by modulating the transcriptional activities of LXRα and FXR in the nucleus. These findings provide evidence that miR-552-3p may serve as a potential therapeutic target.
Not Chopped Liver-A Careful, Fate-Mapping Study of Macrophages in NASH.
Moreno-Fernandez Maria E,Miraldi Emily R,Divanovic Senad
Macrophages are central to the pathogenesis of non-alcoholic steatohepatitis (NASH). However, the identities and functional relationships between tissue-resident and tissue-recruited macrophages in NASH remain poorly understood. A recent study from Seidman et al. (2020) elucidates, at single-cell resolution, the fates, niches, and regulatory landscapes of liver tissue-resident and tissue-recruited macrophage populations in NASH.
A Membrane-Bound Diacylglycerol Species Induces PKCϵ-Mediated Hepatic Insulin Resistance.
Lyu Kun,Zhang Ye,Zhang Dongyan,Kahn Mario,Ter Horst Kasper W,Rodrigues Marcos R S,Gaspar Rafael C,Hirabara Sandro M,Luukkonen Panu K,Lee Seohyuk,Bhanot Sanjay,Rinehart Jesse,Blume Niels,Rasch Morten Grønbech,Serlie Mireille J,Bogan Jonathan S,Cline Gary W,Samuel Varman T,Shulman Gerald I
Nonalcoholic fatty liver disease is strongly associated with hepatic insulin resistance (HIR); however, the key lipid species and molecular mechanisms linking these conditions are widely debated. We developed a subcellular fractionation method to quantify diacylglycerol (DAG) stereoisomers and ceramides in the endoplasmic reticulum (ER), mitochondria, plasma membrane (PM), lipid droplets, and cytosol. Acute knockdown (KD) of diacylglycerol acyltransferase-2 in liver induced HIR in rats. This was due to PM sn-1,2-DAG accumulation, which promoted PKCϵ activation and insulin receptor kinase (IRK)-T1160 phosphorylation, resulting in decreased IRK-Y1162 phosphorylation. Liver PM sn-1,2-DAG content and IRK-T1160 phosphorylation were also higher in humans with HIR. In rats, liver-specific PKCϵ KD ameliorated high-fat diet-induced HIR by lowering IRK-T1160 phosphorylation, while liver-specific overexpression of constitutively active PKCϵ-induced HIR by promoting IRK-T1160 phosphorylation. These data identify PM sn-1,2-DAGs as the key pool of lipids that activate PKCϵ and that hepatic PKCϵ is both necessary and sufficient in mediating HIR.
rs641738C>T near MBOAT7 is associated with liver fat, ALT and fibrosis in NAFLD: A meta-analysis.
Teo Kevin,Abeysekera Kushala W M,Adams Leon,Aigner Elmar,Anstee Quentin M,Banales Jesus M,Banerjee Rajarshi,Basu Priyadarshi,Berg Thomas,Bhatnagar Pallav,Buch Stephan,Canbay Ali,Caprio Sonia,Chatterjee Ankita,Ida Chen Yii-Der,Chowdhury Abhijit,Daly Ann K,Datz Christian,de Gracia Hahn Dana,DiStefano Johanna K,Dong Jiawen,Duret Amedine, ,Emdin Connor,Fairey Madison,Gerhard Glenn S, ,Guo Xiuqing,Hampe Jochen,Hickman Matthew,Heintz Lena,Hudert Christian,Hunter Harriet,Kelly Matt,Kozlitina Julia,Krawczyk Marcin,Lammert Frank,Langenberg Claudia,Lavine Joel,Li Lin,Lim Hong Kai,Loomba Rohit,Luukkonen Panu K,Melton Phillip E,Mori Trevor A,Palmer Nicholette D,Parisinos Constantinos A,Pillai Sreekumar G,Qayyum Faiza,Reichert Matthias C,Romeo Stefano,Rotter Jerome I,Im Yu Ri,Santoro Nicola,Schafmayer Clemens,Speliotes Elizabeth K,Stender Stefan,Stickel Felix,Still Christopher D,Strnad Pavel,Taylor Kent D,Tybjærg-Hansen Anne,Umano Giuseppina Rosaria,Utukuri Mrudula,Valenti Luca,Wagenknecht Lynne E,Wareham Nicholas J,Watanabe Richard M,Wattacheril Julia,Yaghootkar Hanieh,Yki-Järvinen Hannele,Young Kendra A,Mann Jake P
Journal of hepatology
BACKGROUND & AIMS:A common genetic variant near MBOAT7 (rs641738C>T) has been previously associated with hepatic fat and advanced histology in NAFLD; however, these findings have not been consistently replicated in the literature. We aimed to establish whether rs641738C>T is a risk factor across the spectrum of NAFLD and to characterise its role in the regulation of related metabolic phenotypes through a meta-analysis. METHODS:We performed a meta-analysis of studies with data on the association between rs641738C>T genotype and liver fat, NAFLD histology, and serum alanine aminotransferase (ALT), lipids or insulin. These included directly genotyped studies and population-level data from genome-wide association studies (GWAS). We performed a random effects meta-analysis using recessive, additive and dominant genetic models. RESULTS:Data from 1,066,175 participants (9,688 with liver biopsies) across 42 studies were included in the meta-analysis. rs641738C>T was associated with higher liver fat on CT/MRI (+0.03 standard deviations [95% CI 0.02-0.05], p = 4.8×10) and diagnosis of NAFLD (odds ratio [OR] 1.17 [95% CI 1.05-1.3], p = 0.003) in Caucasian adults. The variant was also positively associated with presence of advanced fibrosis (OR 1.22 [95% CI 1.03-1.45], p = 0.021) in Caucasian adults using a recessive model of inheritance (CC + CT vs. TT). Meta-analysis of data from previous GWAS found the variant to be associated with higher ALT (p = 0.002) and lower serum triglycerides (p = 1.5×10). rs641738C>T was not associated with fasting insulin and no effect was observed in children with NAFLD. CONCLUSIONS:Our study validates rs641738C>T near MBOAT7 as a risk factor for the presence and severity of NAFLD in individuals of European descent. LAY SUMMARY:Fatty liver disease is a common condition where fat builds up in the liver, which can cause liver inflammation and scarring (including 'cirrhosis'). It is closely linked to obesity and diabetes, but some genes are also thought to be important. We did this study to see whether one specific change ('variant') in one gene ('MBOAT7') was linked to fatty liver disease. We took data from over 40 published studies and found that this variant near MBOAT7 is linked to more severe fatty liver disease. This means that drugs designed to work on MBOAT7 could be useful for treating fatty liver disease.
Osteopontin Expression Identifies a Subset of Recruited Macrophages Distinct from Kupffer Cells in the Fatty Liver.
Remmerie Anneleen,Martens Liesbet,Thoné Tinne,Castoldi Angela,Seurinck Ruth,Pavie Benjamin,Roels Joris,Vanneste Bavo,De Prijck Sofie,Vanhockerhout Mathias,Binte Abdul Latib Mushida,Devisscher Lindsey,Hoorens Anne,Bonnardel Johnny,Vandamme Niels,Kremer Anna,Borghgraef Peter,Van Vlierberghe Hans,Lippens Saskia,Pearce Edward,Saeys Yvan,Scott Charlotte L
Metabolic-associated fatty liver disease (MAFLD) represents a spectrum of disease states ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). Hepatic macrophages, specifically Kupffer cells (KCs), are suggested to play important roles in the pathogenesis of MAFLD through their activation, although the exact roles played by these cells remain unclear. Here, we demonstrated that KCs were reduced in MAFLD being replaced by macrophages originating from the bone marrow. Recruited macrophages existed in two subsets with distinct activation states, either closely resembling homeostatic KCs or lipid-associated macrophages (LAMs) from obese adipose tissue. Hepatic LAMs expressed Osteopontin, a biomarker for patients with NASH, linked with the development of fibrosis. Fitting with this, LAMs were found in regions of the liver with reduced numbers of KCs, characterized by increased Desmin expression. Together, our data highlight considerable heterogeneity within the macrophage pool and suggest a need for more specific macrophage targeting strategies in MAFLD.
Role of Patatin-Like Phospholipase Domain-Containing 3 Gene for Hepatic Lipid Content and Insulin Resistance in Diabetes.
Zaharia Oana P,Strassburger Klaus,Knebel Birgit,Kupriyanova Yuliya,Karusheva Yanislava,Wolkersdorfer Martin,Bódis Kálmán,Markgraf Daniel F,Burkart Volker,Hwang Jong-Hee,Kotzka Jörg,Al-Hasani Hadi,Szendroedi Julia,Roden Michael,
OBJECTIVE:The rs738409(G) single nucleotide polymorphism (SNP) in the patatin-like phospholipase domain-containing 3 () gene associates with increased risk and progression of nonalcoholic fatty liver disease (NAFLD). As the recently described severe insulin-resistant diabetes (SIRD) cluster specifically relates to NAFLD, this study examined whether this SNP differently associates with hepatic lipid content (hepatocellular lipids [HCL]) and insulin sensitivity in recent-onset diabetes. RESEARCH DESIGN AND METHODS:A total of 917 participants in the German Diabetes Study (GDS) underwent genotyping, hyperinsulinemic-euglycemic clamps with stable isotopic tracer dilution, and MRS. RESULTS:The G allele associated positively with HCL (β = 0.36, < 0.01), independent of age, sex, and BMI across the whole cohort, but not in the individual clusters. Those with SIRD exhibited lowest whole-body insulin sensitivity compared with those with severe insulin-deficient (SIDD), moderate obesity-related (MOD), moderate age-related (MARD), and severe autoimmune diabetes (SAID) clusters (all < 0.001). Interestingly, the SIRD group presented with higher prevalence of the rs738409(G) SNP compared with other clusters and the glucose-tolerant control group ( < 0.05). HCL was higher in the SIRD group (median 13.6% [1st quartile 5.8; 3rd quartile 19.1] compared with the MOD (6.4 % [2.1; 12.4], < 0.05), MARD (3.0% [1.0; 7.9], < 0.001), SAID (0.4% [0.0; 1.5], < 0.001), and glucose-tolerant (0.9% [0.4; 4.9), < 0.001) group. Although the polymorphism did not directly associate with whole-body insulin sensitivity in SIRD, the G-allele carriers had higher circulating free fatty acid concentrations and greater adipose tissue insulin resistance compared with noncarriers (both < 0.001). CONCLUSIONS:Members of the SIRD cluster are more frequently carriers of the rs738409(G) variant. The SNP-associated adipose tissue insulin resistance and excessive lipolysis may contribute to their NAFLD.
Targeting Mitochondria-Located circRNA SCAR Alleviates NASH via Reducing mROS Output.
Zhao Qiyi,Liu Jiayu,Deng Hong,Ma Ruiying,Liao Jian-You,Liang Huixin,Hu Jingxiong,Li Jiaqian,Guo Zhiyong,Cai Junchao,Xu Xiaoding,Gao Zhiliang,Su Shicheng
Mitochondria, which play central roles in immunometabolic diseases, have their own genome. However, the functions of mitochondria-located noncoding RNAs are largely unknown due to the absence of a specific delivery system. By circular RNA (circRNA) expression profile analysis of liver fibroblasts from patients with nonalcoholic steatohepatitis (NASH), we observe that mitochondrial circRNAs account for a considerable fraction of downregulated circRNAs in NASH fibroblasts. By constructing mitochondria-targeting nanoparticles, we observe that Steatohepatitis-associated circRNA ATP5B Regulator (SCAR), which is located in mitochondria, inhibits mitochondrial ROS (mROS) output and fibroblast activation. circRNA SCAR, mediated by PGC-1α, binds to ATP5B and shuts down mPTP by blocking CypD-mPTP interaction. Lipid overload inhibits PGC-1α by endoplasmic reticulum (ER) stress-induced CHOP. In vivo, targeting circRNA SCAR alleviates high fat diet-induced cirrhosis and insulin resistance. Clinically, circRNA SCAR is associated with steatosis-to-NASH progression. Collectively, we identify a mitochondrial circRNA that drives metaflammation and serves as a therapeutic target for NASH.
Two Roads Diverge in the Sick Liver, Monocytes Travel Both.
Zigmond Ehud,Varol Chen
Liver macrophages play critical roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). In this issue of Immunity, Tran et al. and Remmerie et al. reveal that Ly6C monocytes in NASH replace dying resident Kupffer cells, while concomitantly are differentiating into distinct lipid associated macrophages.
Non-alcoholic fatty liver disease and risk of incident diabetes mellitus: an updated meta-analysis of 501 022 adult individuals.
Mantovani Alessandro,Petracca Graziana,Beatrice Giorgia,Tilg Herbert,Byrne Christopher D,Targher Giovanni
OBJECTIVE:Follow-up studies have shown that non-alcoholic fatty liver disease (NAFLD) is associated with an increased risk of incident diabetes, but currently, it is uncertain whether this risk changes with increasing severity of NAFLD. We performed a meta-analysis of relevant studies to quantify the magnitude of the association between NAFLD and risk of incident diabetes. DESIGN:We systematically searched PubMed, Scopus and Web of Science databases from January 2000 to June 2020 using predefined keywords to identify observational studies with a follow-up duration of at least 1 year, in which NAFLD was diagnosed by imaging techniques or biopsy. Meta-analysis was performed using random-effects modelling. RESULTS:33 studies with 501 022 individuals (30.8% with NAFLD) and 27 953 cases of incident diabetes over a median of 5 years (IQR: 4.0-19 years) were included. Patients with NAFLD had a higher risk of incident diabetes than those without NAFLD (n=26 studies; random-effects HR 2.19, 95% CI 1.93 to 2.48; =91.2%). Patients with more 'severe' NAFLD were also more likely to develop incident diabetes (n=9 studies; random-effects HR 2.69, 95% CI 2.08 to 3.49; =69%). This risk markedly increased across the severity of liver fibrosis (n=5 studies; random-effects HR 3.42, 95% CI 2.29 to 5.11; =44.6%). All risks were independent of age, sex, adiposity measures and other common metabolic risk factors. Sensitivity analyses did not alter these findings. Funnel plots did not reveal any significant publication bias. CONCLUSION:This updated meta-analysis shows that NAFLD is associated with a ~2.2-fold increased risk of incident diabetes. This risk parallels the underlying severity of NAFLD.
Activating Adenosine Monophosphate-Activated Protein Kinase Mediates Fibroblast Growth Factor 1 Protection From Nonalcoholic Fatty Liver Disease in Mice.
Lin Qian,Huang Zhifeng,Cai Genxiang,Fan Xia,Yan Xiaoqing,Liu Zhengshuai,Zhao Zehua,Li Jingya,Li Jia,Shi Hongxue,Kong Maiying,Zheng Ming-Hua,Conklin Daniel J,Epstein Paul N,Wintergerst Kupper A,Mohammadi Moosa,Cai Lu,Li Xiaokun,Li Yu,Tan Yi
Hepatology (Baltimore, Md.)
BACKGROUND AND AIMS:Fibroblast growth factor (FGF) 1 demonstrated protection against nonalcoholic fatty liver disease (NAFLD) in type 2 diabetic and obese mice by an uncertain mechanism. This study investigated the therapeutic activity and mechanism of a nonmitogenic FGF1 variant carrying 3 substitutions of heparin-binding sites (FGF1 ) against NAFLD. APPROACH AND RESULTS:FGF1 administration was effective in 9-month-old diabetic mice carrying a homozygous mutation in the leptin receptor gene (db/db) with NAFLD; liver weight, lipid deposition, and inflammation declined and liver injury decreased. FGF1 reduced oxidative stress by stimulating nuclear translocation of nuclear erythroid 2 p45-related factor 2 (Nrf2) and elevation of antioxidant protein expression. FGF1 also inhibited activity and/or expression of lipogenic genes, coincident with phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and its substrates. Mechanistic studies on palmitate exposed hepatic cells demonstrated that NAFLD-like oxidative damage and lipid accumulation could be reversed by FGF1 . In palmitate-treated hepatic cells, small interfering RNA (siRNA) knockdown of Nrf2 abolished only FGF1 antioxidative actions but not improvement of lipid metabolism. In contrast, AMPK inhibition by pharmacological agent or siRNA abolished FGF1 benefits on both oxidative stress and lipid metabolism that were FGF receptor (FGFR) 4 dependent. Further support of these in vitro findings is that liver-specific AMPK knockout abolished therapeutic effects of FGF1 against high-fat/high-sucrose diet-induced hepatic steatosis. Moreover, FGF1 improved high-fat/high-cholesterol diet-induced steatohepatitis and fibrosis in apolipoprotein E knockout mice. CONCLUSIONS:These findings indicate that FGF1 is effective for preventing and reversing liver steatosis and steatohepatitis and acts by activation of AMPK through hepatocyte FGFR4.
Osteopontin Takes Center Stage in Chronic Liver Disease.
Song Zhuolun,Chen Wei,Athavale Dipti,Ge Xiaodong,Desert Romain,Das Sukanta,Han Hui,Nieto Natalia
Hepatology (Baltimore, Md.)
Osteopontin (OPN) was first identified in 1986. The prefix osteo- means bone; however, OPN is expressed in other tissues, including liver. The suffix -pontin means bridge and denotes the role of OPN as a link protein within the extracellular matrix. While OPN has well-established physiological roles, multiple "omics" analyses suggest that it is also involved in chronic liver disease. In this review, we provide a summary of the OPN gene and protein structure and regulation. We outline the current knowledge on how OPN is involved in hepatic steatosis in the context of alcoholic liver disease and non-alcoholic fatty liver disease. We describe the mechanisms whereby OPN participates in inflammation and liver fibrosis and discuss current research on its role in hepatocellular carcinoma and cholangiopathies. To conclude, we highlight important points to consider when doing research on OPN and provide direction for making progress on how OPN contributes to chronic liver disease.
The multi-faces of Angptl8 in health and disease: Novel functions beyond lipoprotein lipase modulation.
Abu-Farha Mohamed,Ghosh Anindya,Al-Khairi Irina,Madiraju S R Murthy,Abubaker Jehad,Prentki Marc
Progress in lipid research
Angiopoietin-like protein (ANGPTL) family members, mainly ANGPTL3, ANGPTL4 and ANGPTL8, are physiological inhibitors of lipoprotein lipase (LPL), and play a critical role in lipoprotein and triglyceride metabolism in response to nutritional cues. ANGPTL8 has been described by different names in various studies and has been ascribed various functions at the systemic and cellular levels. Circulating ANGPTL8 originates mainly from the liver and to a smaller extent from adipose tissues. In the blood, ANGPTL8 forms a complex with ANGPTL3 or ANGPTL4 to inhibit LPL in fed or fasted conditions, respectively. Evidence is emerging for additional intracellular and receptor-mediated functions of ANGPTL8, with implications in NFκB mediated inflammation, autophagy, adipogenesis, intra-cellular lipolysis and regulation of circadian clock. Elevated levels of plasma ANGPTL8 are associated with metabolic syndrome, type 2 diabetes, atherosclerosis, hypertension and NAFLD/NASH, even though the precise relationship is not known. Whether ANGPTL8 has direct pathogenic role in these diseases, remains to be explored. In this review, we develop a balanced view on the proposed association of this protein in the regulation of several pathophysiological processes. We also discuss the well-established functions of ANGPTL8 in lipoprotein metabolism in conjunction with the emerging novel extracellular and intracellular roles of ANGPTL8 and the implicated metabolic and signalling pathways. Understanding the diverse functions of ANGPTL8 in various tissues and metabolic states should unveil new opportunities of therapeutic intervention for cardiometabolic disorders.
Distinct signatures of gut microbiome and metabolites associated with significant fibrosis in non-obese NAFLD.
Lee Giljae,You Hyun Ju,Bajaj Jasmohan S,Joo Sae Kyung,Yu Junsun,Park Seoyeon,Kang Hyena,Park Jeong Hwan,Kim Jung Ho,Lee Dong Hyeon,Lee Seonhwa,Kim Won,Ko GwangPyo
Nonalcoholic fatty liver disease (NAFLD) is associated with obesity but also found in non-obese individuals. Gut microbiome profiles of 171 Asians with biopsy-proven NAFLD and 31 non-NAFLD controls are analyzed using 16S rRNA sequencing; an independent Western cohort is used for external validation. Subjects are classified into three subgroups according to histological spectra of NAFLD or fibrosis severity. Significant alterations in microbiome diversity are observed according to fibrosis severity in non-obese, but not obese, subjects. Ruminococcaceae and Veillonellaceae are the main microbiota associated with fibrosis severity in non-obese subjects. Furthermore, stool bile acids and propionate are elevated, especially in non-obese subjects with significant fibrosis. Fibrosis-related Ruminococcaceae and Veillonellaceae species undergo metagenome sequencing, and four representative species are administered in three mouse NAFLD models to evaluate their effects on liver damage. This study provides the evidence for the role of the microbiome in the liver fibrosis pathogenesis, especially in non-obese subjects.
Role of CC chemokine receptor 9 in the progression of murine and human non-alcoholic steatohepatitis.
Morikawa Rei,Nakamoto Nobuhiro,Amiya Takeru,Chu Po-Sung,Koda Yuzo,Teratani Toshiaki,Suzuki Takahiro,Kurebayashi Yutaka,Ueno Akihisa,Taniki Nobuhito,Miyamoto Kentaro,Yamaguchi Akihiro,Shiba Shunsuke,Katayama Tadashi,Yoshida Kosuke,Takada Yoshiaki,Ishihara Rino,Ebinuma Hirotoshi,Sakamoto Michiie,Kanai Takanori
Journal of hepatology
BACKGROUND & AIMS:The number of patients with non-alcoholic steatohepatitis (NASH) is increasing globally. Recently, specific chemokine receptors have garnered interest as therapeutic targets in NASH. This is the first report to examine the role of the C-C chemokine receptor 9 (CCR9)/C-C chemokine receptor ligand 25 (CCL25) axis, and to reveal its therapeutic potential in NASH. METHODS:Patients with biopsy-proven non-alcoholic liver disease (NAFLD) were recruited and their serum and hepatic chemokine expression was examined. Furthermore, wild-type (WT) and Ccr9 mice were fed a high-fat high-cholesterol (HFHC) diet for 24 weeks to establish NASH. RESULTS:Serum CCL25, and hepatic CCR9 and CCL25 expression levels were increased in patients with NASH compared to healthy volunteers. Furthermore, Ccr9 mice were protected from HFHC diet-induced NASH progression both serologically and histologically. Flow cytometry and immunohistochemistry analysis showed that CCR9CD11b inflammatory macrophages accumulated in the inflamed livers of HFHC diet-fed mice, while the number was reduced in Ccr9 mice. Consistent with human NASH livers, CCR9 was also expressed on hepatic stellate cells (HSCs) in mice with NASH, while CCR9-deficient HSCs showed less fibrogenic potential in vitro. Administration of a CCR9 antagonist hampered further fibrosis progression in mice with NASH, supporting its potential clinical application. Finally, we showed that CCR9 blockade attenuated the development of NAFLD-related hepatocellular carcinoma in HF diet-fed mice injected with diethylnitrosamine. CONCLUSIONS:These results highlight the role of the CCR9/CCL25 axis on macrophage recruitment and fibrosis formation in a murine NASH model, providing new insights into therapeutic strategies for NASH. LAY SUMMARY:Herein, we show that a specific chemokine axis involving a receptor (CCR9) and its ligand (CCL25) contributes to the progression of non-alcoholic steatohepatitis and carcinogenesis in humans and mice. Furthermore, treatment with a CCR9 antagonist ameliorates the development of steatohepatitis and holds promise for the treatment of patients with non-alcoholic steatohepatitis.
Maladaptive regeneration - the reawakening of developmental pathways in NASH and fibrosis.
Zhu Changyu,Tabas Ira,Schwabe Robert F,Pajvani Utpal B
Nature reviews. Gastroenterology & hepatology
With the rapid expansion of the obesity epidemic, nonalcoholic fatty liver disease is now the most common chronic liver disease, with almost 25% global prevalence. Nonalcoholic fatty liver disease ranges in severity from simple steatosis, a benign 'pre-disease' state, to the liver injury and inflammation that characterize nonalcoholic steatohepatitis (NASH), which in turn predisposes individuals to liver fibrosis. Fibrosis is the major determinant of clinical outcomes in patients with NASH and is associated with increased risks of cirrhosis and hepatocellular carcinoma. NASH has no approved therapies, and liver fibrosis shows poor response to existing pharmacotherapy, in part due to an incomplete understanding of the underlying pathophysiology. Patient and mouse data have shown that NASH is associated with the activation of developmental pathways: Notch, Hedgehog and Hippo-YAP-TAZ. Although these evolutionarily conserved fundamental signals are known to determine liver morphogenesis during development, new data have shown a coordinated and causal role for these pathways in the liver injury response, which becomes maladaptive during obesity-associated chronic liver disease. In this Review, we discuss the aetiology of this reactivation of developmental pathways and review the cell-autonomous and cell-non-autonomous mechanisms by which developmental pathways influence disease progression. Finally, we discuss the potential prognostic and therapeutic implications of these data for NASH and liver fibrosis.
Nonalcoholic steatohepatitis: the role of peroxisome proliferator-activated receptors.
Francque Sven,Szabo Gyongyi,Abdelmalek Manal F,Byrne Christopher D,Cusi Kenneth,Dufour Jean-François,Roden Michael,Sacks Frank,Tacke Frank
Nature reviews. Gastroenterology & hepatology
The increasing epidemic of obesity worldwide is linked to serious health effects, including increased prevalence of type 2 diabetes mellitus, cardiovascular disease and nonalcoholic fatty liver disease (NAFLD). NAFLD is the liver manifestation of the metabolic syndrome and includes the spectrum of liver steatosis (known as nonalcoholic fatty liver) and steatohepatitis (known as nonalcoholic steatohepatitis), which can evolve into progressive liver fibrosis and eventually cause cirrhosis. Although NAFLD is becoming the number one cause of chronic liver diseases, it is part of a systemic disease that affects many other parts of the body, including adipose tissue, pancreatic β-cells and the cardiovascular system. The pathomechanism of NAFLD is multifactorial across a spectrum of metabolic derangements and changes in the host microbiome that trigger low-grade inflammation in the liver and other organs. Peroxisome proliferator-activated receptors (PPARs) are a group of nuclear regulatory factors that provide fine tuning for key elements of glucose and fat metabolism and regulate inflammatory cell activation and fibrotic processes. This Review summarizes and discusses the current literature on NAFLD as the liver manifestation of the systemic metabolic syndrome and focuses on the role of PPARs in the pathomechanisms as well as in the potential targeting of disease.
Hepatocyte Nuclear Factor 4α Prevents the Steatosis-to-NASH Progression by Regulating p53 and Bile Acid Signaling (in mice).
Hepatology (Baltimore, Md.)
BACKGROUND AND AIMS:Hepatocyte nuclear factor 4α (HNF4α) is highly enriched in the liver, but its role in the progression of nonalcoholic liver steatosis (NAFL) to NASH has not been elucidated. In this study, we investigated the effect of gain or loss of HNF4α function on the development and progression of NAFLD in mice. APPROACH AND RESULTS:Overexpression of human HNF4α protected against high-fat/cholesterol/fructose (HFCF) diet-induced steatohepatitis, whereas loss of Hnf4α had opposite effects. HNF4α prevented hepatic triglyceride accumulation by promoting hepatic triglyceride lipolysis, fatty acid oxidation, and VLDL secretion. Furthermore, HNF4α suppressed the progression of NAFL to NASH. Overexpression of human HNF4α inhibited HFCF diet-induced steatohepatitis in control mice but not in hepatocyte-specific p53 mice. In HFCF diet-fed mice lacking hepatic Hnf4α, recapitulation of hepatic expression of HNF4α targets cholesterol 7α-hydroxylase and sterol 12α-hydroxylase and normalized hepatic triglyceride levels and attenuated steatohepatitis. CONCLUSIONS:The current study indicates that HNF4α protects against diet-induced development and progression of NAFLD by coordinating the regulation of lipolytic, p53, and bile acid signaling pathways. Targeting hepatic HNF4α may be useful for treatment of NASH.
MicroRNAs as regulators, biomarkers and therapeutic targets in liver diseases.
Wang Xiaolin,He Yong,Mackowiak Bryan,Gao Bin
MicroRNAs (miRNAs) are small, non-coding RNAs that post-transcriptionally regulate gene expression by binding to specific mRNA targets and promoting their degradation and/or translational inhibition. miRNAs regulate both physiological and pathological liver functions. Altered expression of miRNAs is associated with liver metabolism dysregulation, liver injury, liver fibrosis and tumour development, making miRNAs attractive therapeutic strategies for the diagnosis and treatment of liver diseases. Here, we review recent advances regarding the regulation and function of miRNAs in liver diseases with a major focus on miRNAs that are specifically expressed or enriched in hepatocytes (miR-122, miR-194/192), neutrophils (miR-223), hepatic stellate cells (miR-29), immune cells (miR-155) and in circulation (miR-21). The functions and target genes of these miRNAs are emphasised in alcohol-associated liver disease, non-alcoholic fatty liver disease, drug-induced liver injury, viral hepatitis and hepatocellular carcinoma, as well liver fibrosis and liver failure. We touch on the roles of miRNAs in intercellular communication between hepatocytes and other types of cells via extracellular vesicles in the pathogenesis of liver diseases. We provide perspective on the application of miRNAs as biomarkers for early diagnosis, prognosis and assessment of liver diseases and discuss the challenges in miRNA-based therapy for liver diseases. Further investigation of miRNAs in the liver will help us better understand the pathogeneses of liver diseases and may identify biomarkers and therapeutic targets for liver diseases in the future.
Identification of a Metabolic, Transcriptomic, and Molecular Signature of Patatin-Like Phospholipase Domain Containing 3-Mediated Acceleration of Steatohepatitis.
Banini Bubu A,Kumar Divya P,Cazanave Sophie,Seneshaw Mulugeta,Mirshahi Faridoddin,Santhekadur Prasanna K,Wang Liangsu,Guan Hong Ping,Oseini Abdul M,Alonso Cristina,Bedossa Pierre,Koduru Srinivas V,Min Hae-Ki,Sanyal Arun J
Hepatology (Baltimore, Md.)
BACKGROUND AND AIMS:The mechanisms by which the I148M mutant variant of the patatin-like phospholipase domain-containing 3 (PNPLA3 ) drives development of nonalcoholic steatohepatitis (NASH) are not known. The aim of this study was to obtain insights on mechanisms underlying PNPLA3 -induced acceleration of NASH. APPROACH AND RESULTS:Hepatocyte-specific overexpression of empty vector (luciferase), human wild-type PNPLA3, or PNPLA3 was achieved using adeno-associated virus 8 in a diet-induced mouse model of nonalcoholic fatty liver disease followed by chow diet or high-fat Western diet with ad libitum administration of sugar in drinking water (WDSW) for 8 weeks. Under WDSW, PNPLA3 overexpression accelerated steatohepatitis with increased steatosis, inflammation ballooning, and fibrosis (P < 0.001 versus other groups for all). Silencing PNPLA3 after its initial overexpression abrogated these findings. PNPLA3 caused 22:6n3 docosahexanoic acid depletion and increased ceramides under WDSW in addition to increasing triglycerides and diglycerides, especially enriched with unsaturated fatty acids. It also increased oxidative stress and endoplasmic reticulum stress. Increased total ceramides was associated with signature of transducer and activator of transcription 3 (STAT3) activation with downstream activation of multiple immune-inflammatory pathways at a transcriptomic level by network analyses. Silencing PNPLA3 reversed STAT3 activation. Conditioned media from HepG2 cells overexpressing PNPLA3 increased procollagen mRNA expression in LX2 cells; this was abrogated by hepatocyte STAT3 inhibition. CONCLUSIONS:Under WDSW, PNPLA3 overexpression promotes steatosis and NASH by metabolic reprogramming characterized by increased triglycerides and diglycerides, n3 polyunsaturated fatty acid depletion, and increased ceramides with resultant STAT3 phosphorylation and downstream inflammatory pathway activation driving increased stellate cell fibrogenic activity.
TANK-Binding Kinase 1 Regulates the Localization of Acyl-CoA Synthetase ACSL1 to Control Hepatic Fatty Acid Oxidation.
Huh Jin Young,Reilly Shannon M,Abu-Odeh Mohammad,Murphy Anne N,Mahata Sushil K,Zhang Jinyu,Cho Yoori,Seo Jong Bae,Hung Chao-Wei,Green Courtney R,Metallo Christian M,Saltiel Alan R
Hepatic TANK (TRAF family member associated NFκB activator)-binding kinase 1 (TBK1) activity is increased during obesity, and administration of a TBK1 inhibitor reduces fatty liver. Surprisingly, liver-specific TBK1 knockout in mice produces fatty liver by reducing fatty acid oxidation. TBK1 functions as a scaffolding protein to localize acyl-CoA synthetase long-chain family member 1 (ACSL1) to mitochondria, which generates acyl-CoAs that are channeled for β-oxidation. TBK1 is induced during fasting and maintained in the unphosphorylated, inactive state, enabling its high affinity binding to ACSL1 in mitochondria. In TBK1-deficient liver, ACSL1 is shifted to the endoplasmic reticulum to promote fatty acid re-esterification in lieu of oxidation in response to fasting, which accelerates hepatic lipid accumulation. The impaired fatty acid oxidation in TBK1-deficient hepatocytes is rescued by the expression of kinase-dead TBK1. Thus, TBK1 operates as a rheostat to direct the fate of fatty acids in hepatocytes, supporting oxidation when inactive during fasting and promoting re-esterification when activated during obesity.
Hepatokines and adipokines in NASH-related hepatocellular carcinoma.
Kucukoglu Ozlem,Sowa Jan-Peter,Mazzolini Guillermo Daniel,Syn Wing-Kin,Canbay Ali
Journal of hepatology
The incidence of hepatocellular carcinoma (HCC) is increasing in industrialised societies; this is likely secondary to the increasing burden of non-alcoholic fatty liver disease (NAFLD), its progressive form non-alcoholic steatohepatitis (NASH), and the metabolic syndrome. Cumulative studies suggest that NAFLD-related HCC may also develop in non-cirrhotic livers. However, prognosis and survival do not differ between NAFLD- or virus-associated HCC. Thus, research has increasingly focused on NAFLD-related risk factors to better understand the biology of hepatocarcinogenesis and to develop new diagnostic, preventive, and therapeutic strategies. One important aspect thereof is the role of hepatokines and adipokines in NAFLD/NASH-related HCC. In this review, we compile current data supporting the use of hepatokines and adipokines as potential markers of disease progression in NAFLD or as early markers of NAFLD-related HCC. While much work must be done to elucidate the mechanisms and interactions underlying alterations to hepatokines and adipokines, current data support the possible utility of these factors - in particular, angiopoietin-like proteins, fibroblast growth factors, and apelin - for detection or even as therapeutic targets in NAFLD-related HCC.
The Nuclear Orphan Receptor NR2F6 Promotes Hepatic Steatosis through Upregulation of Fatty Acid Transporter CD36.
Zhou Bing,Jia Lijing,Zhang Zhijian,Xiang Liping,Yuan Youwen,Zheng Peilin,Liu Bin,Ren Xingxing,Bian Hua,Xie Liwei,Li Yao,Lu Jieli,Zhang Huijie,Lu Yan
Advanced science (Weinheim, Baden-Wurttemberg, Germany)
Nuclear receptors (NRs) are a superfamily of transcription factors which sense hormonal signals or nutrients to regulate various biological events, including development, reproduction, and metabolism. Here, this study identifies nuclear receptor subfamily 2, group F, member 6 (NR2F6), as an important regulator of hepatic triglyceride (TG) homeostasis and causal factor in the development of non-alcoholic fatty liver disease (NAFLD). Adeno-associated virus (AAV)-mediated overexpression of NR2F6 in the liver promotes TG accumulation in lean mice, while hepatic-specific suppression of NR2F6 improves obesity-associated hepatosteatosis, insulin resistance, and methionine and choline-deficient (MCD) diet-induced non-alcoholic steatohepatitis (NASH). Mechanistically, the fatty acid translocase CD36 is identified as a transcriptional target of NR2F6 to mediate its steatotic role. NR2F6 is able to bind directly onto the CD36 promoter region in hepatocytes and increases the enrichment of nuclear receptor coactivator 1 (SRC-1) and histone acetylation at its promoter. Of pathophysiological significance, NR2F6 is significantly upregulated in the livers of obese mice and NAFLD patients. Moreover, treatment with metformin decreases NR2F6 expression in obese mice, resulting in suppression of CD36 and reduced hepatic TG contents. Therefore, these results provide evidence for an unpredicted role of NR2F6 that contributes to liver steatosis and suggest that NR2F6 antagonists may present a therapeutic strategy for reversing or treating NAFLD/NASH pathogenesis.
Nomenclature and definition of metabolic-associated fatty liver disease: a consensus from the Middle East and north Africa.
Shiha Gamal,Alswat Khalid,Al Khatry Maryam,Sharara Ala I,Örmeci Necati,Waked Imam,Benazzouz Mustapha,Al-Ali Fuad,Hamed Abd Elkhalek,Hamoudi Waseem,Attia Dina,Derbala Moutaz,Sharaf-Eldin Mohamed,Al-Busafi Said A,Zaky Samy,Bamakhrama Khaled,Ibrahim Nazir,Ajlouni Yousef,Sabbah Meriam,Salama Mohsen,Anushiravani Amir,Afredj Nawel,Barakat Salma,Hashim Almoutaz,Fouad Yasser,Soliman Reham
The lancet. Gastroenterology & hepatology
With the increasing prevalence of obesity and type 2 diabetes, fatty liver disease associated with metabolic dysfunction is a global health problem, especially because it is one of the earliest consequences of obesity and it precedes diabetes development. Fatty liver disease associated with metabolic dysfunction is of particular concern in the Middle East and north Africa, where its prevalence is greater than that in the rest of the world. Despite the magnitude of the problem, no regional guidelines have been developed to address this disease. This Review describes suggestions of redefining fatty liver disease associated with metabolic dysfunction, including its terminology and criteria for diagnosis. Experts have raised serious concerns on the current nomenclature, which labels the disease as non-alcoholic fatty liver disease (NAFLD), and its diagnostic criteria. The panel reached a consensus that the disease should be renamed as metabolic-associated fatty liver disease (MAFLD) and that the disease should be diagnosed by positive criteria. The aim is now to work with authorities across the region to implement these proposed changes and reflect them in health-care policy and to improve health care for patients in this region.
Neutral Ceramidase Mediates Nonalcoholic Steatohepatitis by Regulating Monounsaturated Fatty Acids and Gut IgA B Cells.
Gu Xuemei,Sun Rui,Chen Liang,Chu Shenghui,Doll Mark A,Li Xiaohong,Feng Wenke,Siskind Leah,McClain Craig J,Deng Zhongbin
Hepatology (Baltimore, Md.)
BACKGROUND AND AIMS:Nonalcoholic steatohepatitis (NASH) is associated with obesity and an increased risk for liver cirrhosis and cancer. Neutral ceramidase (NcDase), which is highly expressed in the intestinal brush border of the small intestine, plays a critical role in digesting dietary sphingolipids (ceramide) to regulate the balance of sphingosine and free fatty acids. It remains unresolved whether obesity-associated alteration of NcDase contributes to the manifestation of NASH. Here, we revealed that NcDase deficiency in murine models of NASH prevents hepatic inflammation and fibrosis but not steatosis. APPROACH AND RESULTS:NcDase mice display reduced stearoyl-CoA desaturase (SCD) 1 expression with a compositional decrease of monounsaturated fatty acids (MUFAs) under the different dietary conditions. We further found that NcDase is a functional regulator of intestinal B cells and influences the abundance and quality of the secretory IgA response toward commensal bacteria. Analysis of composition of the gut microbiota found that Clostridiales colonization was increased in NcDase mice. The colonization of germ-free mice with gut microbiota from NcDase mice resulted in a greater decrease in the expression of SCD1 and the level of MUFAs in the liver relative to gut microbiota from wild-type littermates, which are associated with the alternation of IgA-bound bacteria, including increase of Ruminococcaceae and reduction of Desulfovibrio. Mechanistically, NcDase is a crucial link that controls the expression of SCD1 and MUFA-mediated activation of the Wnt/β-catenin. Very importantly, our experiments further demonstrated that Wnt3a stimulation can enhance the activity of NcDase in hepatocytes. CONCLUSIONS:Thus, the NcDase-SCD1-Wnt feedback loop promotes the diet-induced steatohepatitis and fibrosis through the regulation of intestinal IgA immune cells.
Hepatic Krüppel-like factor 16 (KLF16) targets PPARα to improve steatohepatitis and insulin resistance.
Sun Nannan,Shen Chuangpeng,Zhang Lei,Wu Xiaojie,Yu Yuanyuan,Yang Xiaoying,Yang Chen,Zhong Chong,Gao Zhao,Miao Wei,Yang Zehong,Gao Weihang,Hu Ling,Williams Kevin,Liu Changhui,Chang Yongsheng,Gao Yong
OBJECTIVE:Impaired hepatic fatty acids oxidation results in lipid accumulation and redox imbalance, promoting the development of fatty liver diseases and insulin resistance. However, the underlying pathogenic mechanism is poorly understood. Krüppel-like factor 16 (KLF16) is a transcription factor that abounds in liver. We explored whether and by what mechanisms KLF16 affects hepatic lipid catabolism to improve hepatosteatosis and insulin resistance. DESIGN:KLF16 expression was determined in patients with non-alcoholic fatty liver disease (NAFLD) and mice models. The role of KLF16 in the regulation of lipid metabolism was investigated using hepatocyte-specific KLF16-deficient mice fed a high-fat diet (HFD) or using an adenovirus/adeno-associated virus to alter KLF16 expression in mouse primary hepatocytes (MPHs) and in vivo livers. RNA-seq, luciferase reporter gene assay and ChIP analysis served to explore the molecular mechanisms involved. RESULTS:KLF16 expression was decreased in patients with NAFLD, mice models and oleic acid and palmitic acid (OA and PA) cochallenged hepatocytes. Hepatic KLF16 knockout impaired fatty acid oxidation, aggravated mitochondrial stress, ROS burden, advancing hepatic steatosis and insulin resistance. Conversely, KLF16 overexpression reduced lipid deposition and improved insulin resistance via directly binding the promoter of peroxisome proliferator-activated receptor α (PPARα) to accelerate fatty acids oxidation and attenuate mitochondrial stress, oxidative stress in and HFD mice. PPARα deficiency diminished the KLF16-evoked protective effects against lipid deposition in MPHs. Hepatic-specific PPARα overexpression effectively rescued KLF16 deficiency-induced hepatic steatosis, altered redox balance and insulin resistance. CONCLUSIONS:These findings prove that a direct KLF16-PPARα pathway closely links hepatic lipid homeostasis and redox balance, whose dysfunction promotes insulin resistance and hepatic steatosis.
XBP1 links the 12-hour clock to NAFLD and regulation of membrane fluidity and lipid homeostasis.
Meng Huan,Gonzales Naomi M,Lonard David M,Putluri Nagireddy,Zhu Bokai,Dacso Clifford C,York Brian,O'Malley Bert W
A distinct 12-hour clock exists in addition to the 24-hour circadian clock to coordinate metabolic and stress rhythms. Here, we show that liver-specific ablation of X-box binding protein 1 (XBP1) disrupts the hepatic 12-hour clock and promotes spontaneous non-alcoholic fatty liver disease (NAFLD). We show that hepatic XBP1 predominantly regulates the 12-hour rhythmicity of gene transcription in the mouse liver and demonstrate that perturbation of the 12-hour clock, but not the core circadian clock, is associated with the onset and progression of this NAFLD phenotype. Mechanistically, we provide evidence that the spliced form of XBP1 (XBP1s) binds to the hepatic 12-hour cistrome to directly regulate the 12-hour clock, with a periodicity paralleling the harmonic activation of the 12-hour oscillatory transcription of many rate-limiting metabolic genes known to have perturbations in human metabolic disease. Functionally, we show that Xbp1 ablation significantly reduces cellular membrane fluidity and impairs lipid homeostasis via rate-limiting metabolic processes in fatty acid monounsaturated and phospholipid remodeling pathways. These findings reveal that genetic disruption of the hepatic 12-hour clock links to the onset and progression of NAFLD development via transcriptional regulator XBP1, and demonstrate a role for XBP1 and the 12-hour clock in the modulation of phospholipid composition and the maintenance of lipid homeostasis.
Current Concepts, Opportunities, and Challenges of Gut Microbiome-Based Personalized Medicine in Nonalcoholic Fatty Liver Disease.
Nonalcoholic fatty liver disease (NALFD) is now a leading cause of chronic liver disease worldwide, in part, as a consequence of rapidly rising levels of obesity and metabolic syndrome and is a major risk factor for cirrhosis, hepatocellular carcinoma, and liver-related mortality. From NAFLD stems a myriad of clinical challenges related to both diagnosis and management. A growing body of evidence suggests an intricate linkage between the gut microbiome and the pathogenesis of NAFLD. We highlight how our current knowledge of the gut-liver axis in NAFLD may be leveraged to develop gut microbiome-based personalized approaches for disease management, including its use as a non-invasive biomarker for diagnosis and staging, as a target for therapeutic modulation, and as a marker of drug response. We will also discuss current limitations of these microbiome-based approaches. Ultimately, a better understanding of microbiota-host interactions in NAFLD will inform the development of novel preventative strategies and precise therapeutic targets.
Hepatocyte-specific NRF2 activation controls fibrogenesis and carcinogenesis in steatohepatitis.
Mohs Antje,Otto Tobias,Schneider Kai Markus,Peltzer Mona,Boekschoten Mark,Holland Christian H,Hudert Christian A,Kalveram Laura,Wiegand Susanna,Saez-Rodriguez Julio,Longerich Thomas,Hengstler Jan G,Trautwein Christian
Journal of hepatology
BACKGROUND & AIMS:In chronic liver diseases, inflammation induces oxidative stress and thus may contribute to the progression of liver injury, fibrosis, and carcinogenesis. The KEAP1/NRF2 axis is a major regulator of cellular redox balance. In the present study, we investigated whether the KEAP1/NRF2 system is involved in liver disease progression in humans and mice. METHODS:The clinical relevance of oxidative stress was investigated by liver RNA sequencing in a well-characterized cohort of patients with non-alcoholic fatty liver disease (n = 63) and correlated with histological and clinical parameters. For functional analysis, hepatocyte-specific Nemo knockout (NEMO) mice were crossed with hepatocyte-specific Keap1 knockout (KEAP1) mice. RESULTS:Immunohistochemical analysis of human liver sections showed increased oxidative stress and high NRF2 expression in patients with chronic liver disease. RNA sequencing of liver samples in a human pediatric NAFLD cohort revealed a significant increase of NRF2 activation correlating with the grade of inflammation, but not with the grade of steatosis, which could be confirmed in a second adult NASH cohort. In mice, microarray analysis revealed that Keap1 deletion induces NRF2 target genes involved in glutathione metabolism and xenobiotic stress (e.g., Nqo1). Furthermore, deficiency of one of the most important antioxidants, glutathione (GSH), in NEMO livers was rescued after deleting Keap1. As a consequence, NEMO/KEAP1 livers showed reduced apoptosis compared to NEMO livers as well as a dramatic downregulation of genes involved in cell cycle regulation and DNA replication. Consequently, NEMO/KEAP1 compared to NEMO livers displayed decreased fibrogenesis, lower tumor incidence, reduced tumor number, and decreased tumor size. CONCLUSIONS:NRF2 activation in patients with non-alcoholic steatohepatitis correlates with the grade of inflammation, but not steatosis. Functional analysis in mice demonstrated that NRF2 activation in chronic liver disease is protective by ameliorating fibrogenesis, initiation and progression of hepatocellular carcinogenesis. LAY SUMMARY:The KEAP1 (Kelch-like ECH-associated protein-1)/NRF2 (erythroid 2-related factor 2) axis has a major role in regulating cellular redox balance. Herein, we show that NRF2 activity correlates with the grade of inflammation in patients with non-alcoholic steatohepatitis. Functional studies in mice actually show that NRF2 activation, resulting from KEAP1 deletion, protects against fibrosis and cancer.
Global epidemiology of NAFLD-related HCC: trends, predictions, risk factors and prevention.
Huang Daniel Q,El-Serag Hashem B,Loomba Rohit
Nature reviews. Gastroenterology & hepatology
One quarter of the global population is estimated to have nonalcoholic fatty liver disease (NAFLD). The incidence of nonalcoholic steatohepatitis (NASH) is projected to increase by up to 56% in the next 10 years. NAFLD is already the fastest growing cause of hepatocellular carcinoma (HCC) in the USA, France and the UK. Globally, the prevalence of NAFLD-related HCC is likely to increase concomitantly with the growing obesity epidemic. The estimated annual incidence of HCC ranges from 0.5% to 2.6% among patients with NASH cirrhosis. The incidence of HCC among patients with non-cirrhotic NAFLD is lower, approximately 0.1 to 1.3 per 1,000 patient-years. Although the incidence of NAFLD-related HCC is lower than that of HCC of other aetiologies such as hepatitis C, more people have NAFLD than other liver diseases. Urgent measures that increase global awareness and tackle the metabolic risk factors are necessary to reduce the impending burden of NAFLD-related HCC. Emerging evidence indicates that reduced immune surveillance, increased gut inflammation and gut dysbiosis are potential key steps in tumorigenesis. In this Review, we discuss the global epidemiology, projections and risk factors for NAFLD-related HCC, and propose preventive strategies to tackle this growing problem.
RIPK3 acts as a lipid metabolism regulator contributing to inflammation and carcinogenesis in non-alcoholic fatty liver disease.
Afonso Marta B,Rodrigues Pedro M,Mateus-Pinheiro Miguel,Simão André L,Gaspar Maria M,Majdi Amine,Arretxe Enara,Alonso Cristina,Santos-Laso Alvaro,Jimenez-Agüero Raul,Eizaguirre Emma,Bujanda Luis,Pareja Maria Jesus,Banales Jesus M,Ratziu Vlad,Gautheron Jeremie,Castro Rui E,Rodrigues Cecília M P
OBJECTIVE:Receptor-interacting protein kinase 3 (RIPK3) is a key player in necroptosis execution and an emerging metabolic regulator, whose contribution to non-alcoholic fatty liver disease (NAFLD) is controversial. We aimed to clarify the impact of RIPK3 signalling in the pathogenesis of human and experimental NAFLD. DESIGN:RIPK3 levels were evaluated in two large independent cohorts of patients with biopsy proven NAFLD diagnosis and correlated with clinical and biochemical parameters. Wild-type (WT) or -deficient ( ) mice were fed a choline-deficient L-amino acid-defined diet (CDAA) or an isocaloric control diet for 32 and 66 weeks. RESULTS:RIPK3 increased in patients with non-alcoholic steatohepatitis (NASH) in both cohorts, correlating with hepatic inflammation and fibrosis. Accordingly, deficiency ameliorated CDAA-induced inflammation and fibrosis in mice at both 32 and 66 weeks. WT mice on the CDAA diet for 66 weeks developed preneoplastic nodules and displayed increased hepatocellular proliferation, which were reduced in mice. Furthermore, 3 deficiency hampered tumourigenesis. Intriguingly, mice displayed increased body weight gain, while lipidomics showed that deletion of shifted hepatic lipid profiles. Peroxisome proliferator-activated receptor γ (PPARγ) was increased in mice and negatively correlated with hepatic RIPK3 in patients with NAFLD. Mechanistic studies established a functional link between RIPK3 and PPARγ in controlling fat deposition and fibrosis. CONCLUSION:Hepatic RIPK3 correlates with NAFLD severity in humans and mice, playing a key role in managing liver metabolism, damage, inflammation, fibrosis and carcinogenesis. Targeting RIPK3 and its intricate signalling arises as a novel promising approach to treat NASH and arrest disease progression.
Hepatocyte-specific IL11 cis-signaling drives lipotoxicity and underlies the transition from NAFLD to NASH.
Dong Jinrui,Viswanathan Sivakumar,Adami Eleonora,Singh Brijesh K,Chothani Sonia P,Ng Benjamin,Lim Wei Wen,Zhou Jin,Tripathi Madhulika,Ko Nicole S J,Shekeran Shamini G,Tan Jessie,Lim Sze Yun,Wang Mao,Lio Pei Min,Yen Paul M,Schafer Sebastian,Cook Stuart A,Widjaja Anissa A
IL11 is important for fibrosis in non-alcoholic steatohepatitis (NASH) but its role beyond the stroma in liver disease is unclear. Here, we investigate the role of IL11 in hepatocyte lipotoxicity. Hepatocytes highly express IL11RA and secrete IL11 in response to lipid loading. Autocrine IL11 activity causes hepatocyte death through NOX4-derived ROS, activation of ERK, JNK and caspase-3, impaired mitochondrial function and reduced fatty acid oxidation. Paracrine IL11 activity stimulates hepatic stellate cells and causes fibrosis. In mouse models of NASH, hepatocyte-specific deletion of Il11ra1 protects against liver steatosis, fibrosis and inflammation while reducing serum glucose, cholesterol and triglyceride levels and limiting obesity. In mice deleted for Il11ra1, restoration of IL11 cis-signaling in hepatocytes reconstitutes steatosis and inflammation but not fibrosis. We found no evidence for the existence of IL6 or IL11 trans-signaling in hepatocytes or NASH. These data show that IL11 modulates hepatocyte metabolism and suggests a mechanism for NAFLD to NASH transition.
Gut microbiota impact on the peripheral immune response in non-alcoholic fatty liver disease related hepatocellular carcinoma.
Behary Jason,Amorim Nadia,Jiang Xiao-Tao,Raposo Anita,Gong Lan,McGovern Emily,Ibrahim Ragy,Chu Francis,Stephens Carlie,Jebeili Hazem,Fragomeli Vincenzo,Koay Yen Chin,Jackson Miriam,O'Sullivan John,Weltman Martin,McCaughan Geoffrey,El-Omar Emad,Zekry Amany
The gut microbiota is reported to modulate the immune response in hepatocellular carcinoma (HCC). Here, we employ metagenomic and metabolomic studies to characterise gut microbiota in patients with non-alcoholic fatty liver disease (NAFLD) related cirrhosis, with or without HCC, and evaluate its effect on the peripheral immune response in an ex vivo model. We find that dysbiosis characterises the microbiota of patients with NAFLD-cirrhosis, with compositional and functional shifts occurring with HCC development. Gene function of the microbiota in NAFLD-HCC supports short chain fatty acid production, and this is confirmed by metabolomic studies. Ex vivo studies show that bacterial extracts from the NAFLD-HCC microbiota, but not from the control groups, elicit a T cell immunosuppressive phenotype, characterised by expansion of regulatory T cells and attenuation of CD8 + T cells. Our study suggest that the gut microbiota in NAFLD-HCC is characterised by a distinctive microbiome/metabolomic profile, and can modulate the peripheral immune response.
Mast Cells Promote Nonalcoholic Fatty Liver Disease Phenotypes and Microvesicular Steatosis in Mice Fed a Western Diet.
Hepatology (Baltimore, Md.)
BACKGROUND AND AIMS:Nonalcoholic fatty liver disease (NAFLD) is simple steatosis but can develop into nonalcoholic steatohepatitis (NASH), characterized by liver inflammation, fibrosis, and microvesicular steatosis. Mast cells (MCs) infiltrate the liver during cholestasis and promote ductular reaction (DR), biliary senescence, and liver fibrosis. We aimed to determine the effects of MC depletion during NAFLD/NASH. APPROACH AND RESULTS:Wild-type (WT) and Kit (MC-deficient) mice were fed a control diet (CD) or a Western diet (WD) for 16 weeks; select WT and Kit WD mice received tail vein injections of MCs 2 times per week for 2 weeks prior to sacrifice. Human samples were collected from normal, NAFLD, or NASH mice. Cholangiocytes from WT WD mice and human NASH have increased insulin-like growth factor 1 expression that promotes MC migration/activation. Enhanced MC presence was noted in WT WD mice and human NASH, along with increased DR. WT WD mice had significantly increased steatosis, DR/biliary senescence, inflammation, liver fibrosis, and angiogenesis compared to WT CD mice, which was significantly reduced in Kit WD mice. Loss of MCs prominently reduced microvesicular steatosis in zone 1 hepatocytes. MC injection promoted WD-induced biliary and liver damage and specifically up-regulated microvesicular steatosis in zone 1 hepatocytes. Aldehyde dehydrogenase 1 family, member A3 (ALDH1A3) expression is reduced in WT WD mice and human NASH but increased in Kit WD mice. MicroRNA 144-3 prime (miR-144-3p) expression was increased in WT WD mice and human NASH but reduced in Kit WD mice and was found to target ALDH1A3. CONCLUSIONS:MCs promote WD-induced biliary and liver damage and may promote microvesicular steatosis development during NAFLD progression to NASH through miR-144-3p/ALDH1A3 signaling. Inhibition of MC activation may be a therapeutic option for NAFLD/NASH treatment.