Bile acids are signaling molecules that critically control hepatocellular function. Disrupted bile acid homeostasis may be implicated in the pathogenesis of chronic liver diseases. Glutathione is an important antioxidant that protects the liver against oxidative injury. Various forms of liver disease share the common characteristics of reduced cellular glutathione and elevated oxidative stress. This study reports a potentially novel physiological function of bile acids in regulating hepatic sulfur amino acid and glutathione metabolism. We found that bile acids strongly inhibited the cysteine dioxygenase type-1–mediated (CDO1-mediated) cysteine catabolic pathway via a farnesoid X receptor–dependent mechanism. Attenuating this bile acid repressive effect depleted the free cysteine pool and reduced the glutathione concentration in mouse liver. Upon acetaminophen challenge, cholestyramine-fed mice showed impaired hepatic glutathione regeneration capacity and markedly worsened liver injury, which was fully prevented by N-acetylcysteine administration. These effects were recapitulated in CDO1-overexpressing hepatocytes. Findings from this study support the importance of maintaining bile acid homeostasis under physiological and pathophysiological conditions, as altered hepatic bile acid signaling may negatively impact the antioxidant defense mechanism and sensitivity to oxidative injury. Furthermore, this finding provides a possible explanation for the reported mild hepatotoxicity associated with the clinical use of bile acid sequestrants in human patients.
Yifeng Wang, Jibiao Li, David Matye, Yuxia Zhang, Katie Dennis, Wen-Xing Ding, Tiangang Li
Excess lipid accumulation is an early signature of nonalcoholic fatty liver disease (NAFLD). Although liver receptor homolog 1 (LRH-1) (encoded by NR5A2) is suppressed in human NAFLD, evidence linking this phospholipid-bound nuclear receptor to hepatic lipid metabolism is lacking. Here, we report an essential role for LRH-1 in hepatic lipid storage and phospholipid composition based on an acute hepatic KO of LRH-1 in adult mice (LRH-1AAV8-Cre mice). Indeed, LRH-1–deficient hepatocytes exhibited large cytosolic lipid droplets and increased triglycerides (TGs). LRH-1–deficient mice fed high-fat diet displayed macrovesicular steatosis, liver injury, and glucose intolerance, all of which were reversed or improved by expressing wild-type human LRH-1. While hepatic lipid synthesis decreased and lipid export remained unchanged in mutants, elevated circulating free fatty acid helped explain the lipid imbalance in LRH-1AAV8-Cre mice. Lipidomic and genomic analyses revealed that loss of LRH-1 disrupts hepatic phospholipid composition, leading to lowered arachidonoyl (AA) phospholipids due to repression of Elovl5 and Fads2, two critical genes in AA biosynthesis. Our findings reveal a role for the phospholipid sensor LRH-1 in maintaining adequate pools of hepatic AA phospholipids, further supporting the idea that phospholipid diversity is an important contributor to healthy hepatic lipid storage.
Diego A. Miranda, William C. Krause, Amaury Cazenave-Gassiot, Miyuki Suzawa, Hazel Escusa, Juat Chin Foo, Diyala S. Shihadih, Andreas Stahl, Mark Fitch, Edna Nyangau, Marc Hellerstein, Markus R. Wenk, David L. Silver, Holly A. Ingraham
BACKGROUND. Cirrhosis is associated with gut microbial changes, but current 16S rDNA techniques sequence both dead and live bacteria. We aimed to determine the rRNA content compared with DNA from the same stool sample to evaluate cirrhosis progression and predict hospitalizations. METHODS. Cirrhotics and controls provided stool for RNA and DNA analysis. Comparisons were made between cirrhotics/controls and within cirrhosis (compensated/decompensated, infected/uninfected, renal dysfunction/not, rifaximin use/not) with respect to DNA and RNA bacterial content using linear discriminant analysis. A separate group was treated with omeprazole for 14 days with longitudinal microbiota evaluation. Patients were followed for 90 days for hospitalizations. Multivariable models for hospitalizations with clinical data with and without DNA and RNA microbial data were created. RESULTS. Twenty-six controls and 154 cirrhotics (54 infected, 62 decompensated, 20 renal dysfunction, 18 rifaximin) were included. RNA and DNA analysis showed differing potentially pathogenic taxa but similar autochthonous taxa composition. Thirty subjects underwent the omeprazole study, which demonstrated differences between RNA and DNA changes. Thirty-six patients were hospitalized within 90 days. In the RNA model, MELD score and Enterococcus were independently predictive of hospitalizations, while in the DNA model MELD was predictive and Roseburia protective. In both models, adding microbiota significantly added to the MELD score in predicting hospitalizations. CONCLUSION. DNA and RNA analysis of the same stool sample demonstrated differing microbiota composition, which independently predicts the hospitalization risk in cirrhosis. RNA and DNA content of gut microbiota in cirrhosis are modulated differentially with disease severity, infections, and omeprazole use. TRIAL REGISTRATION. NCT01458990. FUNDING. VA Merit I0CX001076.
Jasmohan S. Bajaj, Leroy R. Thacker, Andrew Fagan, Melanie B. White, Edith A. Gavis, Phillip B. Hylemon, Robert Brown, Chathur Acharya, Douglas M. Heuman, Michael Fuchs, Swati Dalmet, Masoumeh Sikaroodi, Patrick M. Gillevet
A DNA methylation (DNAm) signature (the “Horvath clock”) has been proposed as a measure of human chronological and biological age. We determined peripheral blood DNAm in patients with nonalcoholic steatohepatitis (NASH) and assessed whether accelerated aging occurs in these patients. DNAm signatures were obtained in patients with biopsy-proven NASH and stage 2–3 fibrosis. The DNAm profile from one test and two validation cohorts served as controls. Age acceleration was calculated as the difference between DNAm age and the predicted age based on the linear model derived from controls. Hepatic collagen content was assessed by quantitative morphometry. The Horvath clock accurately predicts the chronological age of the entire cohort. Age acceleration was observed among NASH subjects compared with control data sets and our test controls. Age acceleration in NASH subjects did not differ by fibrosis stage but correlated with hepatic collagen content. A set of 152 differentially methylated CpG islands between NASH subjects and controls identified gene set enrichment for transcription factors and developmental pathways. Patients with NASH exhibit epigenetic age acceleration that correlates with hepatic collagen content.
Rohit Loomba, Yevgeniy Gindin, Zhaoshi Jiang, Eric Lawitz, Stephen Caldwell, C. Stephen Djedjos, Ren Xu, Chuhan Chung, Robert P. Myers, G. Mani Subramanian, Zachary Goodman, Michael Charlton, Nezam H. Afdhal, Anna Mae Diehl
The scaffold protein synectin plays a critical role in the trafficking and regulation of membrane receptor pathways. As platelet-derived growth factor receptor (PDGFR) is essential for hepatic stellate cell (HSC) activation and liver fibrosis, we sought to determine the role of synectin on the PDGFR pathway and development of liver fibrosis. Mice with deletion of synectin from HSC were found to be protected from liver fibrosis. mRNA sequencing revealed that knockdown of synectin in HSC demonstrated reductions in the fibrosis pathway of genes, including PDGFR-β. Chromatin IP assay of the PDGFR-β promoter upon synectin knockdown revealed a pattern of histone marks associated with decreased transcription, dependent on p300 histone acetyltransferase. Synectin knockdown was found to downregulate PDGFR-α protein levels, as well, but through an alternative mechanism: protection from autophagic degradation. Site-directed mutagenesis revealed that ubiquitination of specific PDGFR-α lysine residues was responsible for its autophagic degradation. Furthermore, functional studies showed decreased PDGF-dependent migration and proliferation of HSC after synectin knockdown. Finally, human cirrhotic livers demonstrated increased synectin protein levels. This work provides insight into differential transcriptional and posttranslational mechanisms of synectin regulation of PDGFRs, which are critical to fibrogenesis.
Mary C. Drinane, Usman Yaqoob, Haibin Yu, Fanghong Luo, Thomas Greuter, Juan P. Arab, Enis Kostallari, Vikas K. Verma, Jessica Maiers, Thiago Milech De Assuncao, Michael Simons, Debabrata Mukhopadhyay, Tatiana Kisseleva, David A. Brenner, Raul Urrutia, Gwen Lomberk, Yandong Gao, Giovanni Ligresti, Daniel J. Tschumperlin, Alexander Revzin, Sheng Cao, Vijay H. Shah
Although recent evidence has pointed to the role of organ- and pathogenesis-specific macrophage subsets, it is still unclear which subsets are critically involved in the pathogenesis of nonalcoholic steatohepatitis (NASH). Using melanocortin-4 receptor–deficient (MC4R-KO) mice fed Western diet (WD), which exhibit liver phenotypes similar to those of human NASH, we found a histological structure, termed hepatic crown-like structure (hCLS), in which CD11c+ macrophages surround dead/dying hepatocytes, a prominent feature of NASH. Here, we demonstrate that hCLS-constituting macrophages could be a novel macrophage subset that drives hepatocyte death-triggered liver fibrosis. In an “inducible NASH model,” hepatocyte death induces hCLS formation and liver fibrosis sequentially in the short term. In combination with the long-term WD feeding model, we also showed that resident macrophages are a major cellular source of CD11c+ macrophages constituting hCLS, which exhibited gene expression profiles distinct from CD11c– macrophages scattered in the liver. Moreover, depletion of CD11c+ macrophages abolished hCLS formation and fibrogenesis in NASH. Our clinical data suggest the role of CD11c+ macrophages in the disease progression from simple steatosis to NASH. This study sheds light on the role of resident macrophages, in addition to recruited macrophages, in the pathogenesis of NASH.
Michiko Itoh, Takayoshi Suganami, Hideaki Kato, Sayaka Kanai, Ibuki Shirakawa, Takeru Sakai, Toshihiro Goto, Masahiro Asakawa, Isao Hidaka, Hiroshi Sakugawa, Koji Ohnishi, Yoshihiro Komohara, Kenichi Asano, Isao Sakaida, Masato Tanaka, Yoshihiro Ogawa
Accumulation of lipid droplets and inflammatory cell infiltration is the hallmark of nonalcoholic steatohepatitis (NASH). The roles of noncoding RNAs in NASH are less known. We aim to elucidate the function of miR-141/200c in diet-induced NASH. WT and miR-141/200c–/– mice were fed a methionine and choline deficient (MCD) diet for 2 weeks to assess markers of steatosis, liver injury, and inflammation. Hepatic miR-141 and miR-200c RNA levels were highly induced in human patients with NASH fatty liver and in WT MCD mice. miR-141/200c–/– MCD mice had reduced liver weights and triglyceride (TG) levels, which was associated with increased microsomal TG transfer protein (MTTP) and PPARα but reduced SREBP1c and FAS expression. Inflammation was attenuated and F4/80 macrophage activation was suppressed in miR-141/200c–/– mice, as evidenced by decreased serum aminotransferases and IL-6 and reduced hepatic proinflammatory, neutrophil, and profibrotic genes. Treatment with LPS in BM-derived macrophages isolated from miR-200c/141–/– mice polarized macrophages toward the M2 antiinflammatory state by increasing Arg1 and IL-10 levels while decreasing the M1 marker iNOS. In addition, elevated phosphorylated AMPK (p-AMPK), p-AKT, and p-GSK3β and diminished TLR4 and p-mTOR/p-4EBP1 proteins were observed. Lipidomics and metabolomics revealed alterations of TG and phosphatidylcholine (PC) lipid species by miR-141/200c deficiency. In summary, miR-141/200c deficiency diminished NASH-associated hepatic steatosis and inflammation by reprogramming lipid and inflammation signaling pathways.
Melanie Tran, Sang-Min Lee, Dong-Ju Shin, Li Wang
We developed an in vitro model system where induced pluripotent stem cells (iPSCs) differentiate into 3-dimensional human hepatic organoids (HOs) through stages that resemble human liver during its embryonic development. The HOs consist of hepatocytes, and cholangiocytes, which are organized into epithelia that surround the lumina of bile duct–like structures. The organoids provide a potentially new model for liver regenerative processes, and were used to characterize the effect of different JAG1 mutations that cause: (a) Alagille syndrome (ALGS), a genetic disorder where NOTCH signaling pathway mutations impair bile duct formation, which has substantial variability in its associated clinical features; and (b) Tetralogy of Fallot (TOF), which is the most common form of a complex congenital heart disease, and is associated with several different heritable disorders. Our results demonstrate how an iPSC-based organoid system can be used with genome editing technologies to characterize the pathogenetic effect of human genetic disease-causing mutations.
Yuan Guan, Dan Xu, Phillip M. Garfin, Ursula Ehmer, Melissa Hurwitz, Greg Enns, Sara Michie, Manhong Wu, Ming Zheng, Toshihiko Nishimura, Julien Sage, Gary Peltz
Alcoholic steatohepatitis (ASH) and nonalcoholic steatohepatitis (NASH) are among the most frequent causes of chronic liver disease in the United States. Although the two entities are triggered by different etiologies — chronic alcohol consumption (ASH) and obesity-associated lipotoxicity (NASH) — they share overlapping histological and clinical features owing to common pathogenic mechanisms. These pathogenic processes include altered hepatocyte lipid metabolism, organelle dysfunction (i.e., ER stress), hepatocyte apoptosis, innate immune system activation, and hepatic stellate cell activation. Nonetheless, there are several disease-specific molecular signaling pathways, such as differential pathway activation downstream of TLR4 (MyD88-dependence in NASH versus MyD88-independence in ASH), inflammasome activation and IL-1β signaling in ASH, insulin resistance and lipotoxicity in NASH, and dysregulation of different microRNAs, which clearly highlight that ASH and NASH are two distinct biological entities. Both pathogenic similarities and differences have therapeutic implications. In this Review, we discuss these pathogenic mechanisms and their therapeutic implications for each disease, focusing on both shared and distinct targets.
Thomas Greuter, Harmeet Malhi, Gregory J. Gores, Vijay H. Shah
With the increase in obesity worldwide, its associated comorbidities, including nonalcoholic steatohepatitis (NASH), have become a public health problem that still lacks effective therapy. We have previously reported that mixed-lineage kinase 3–deficient (MLK3-deficient) mice are protected against diet-induced NASH. Given the critical need to identify new therapeutic agents, we sought to examine whether the small-molecule MLK3 inhibitor URMC099 would be effective in reversing diet-induced murine NASH. C57BL/6J mice were fed either a diet high in saturated fat, fructose, and cholesterol (FFC), or a chow diet for 24 weeks. Mice were treated with either URMC099 (10 mg/kg) twice daily by intraperitoneal injection or its vehicle during the last 2 weeks of the feeding study. FFC-fed mice receiving URMC099 had similar body weight, caloric intake, homeostatic model assessment of insulin resistance, metabolic phenotype, and hepatic steatosis compared with vehicle-treated mice. Furthermore, FFC-fed mice treated with URMC099 had less hepatic macrophage infiltration, activation, and proinflammatory polarization, as well as less liver injury and fibrosis when compared with vehicle-treated mice. In conclusion, URMC099 is well tolerated in mice without obvious toxicities and appears to be efficacious in reversing diet-induced NASH. Hence, URMC099 may serve as a therapeutic agent in human NASH.
Kyoko Tomita, Rohit Kohli, Brittany L. MacLaurin, Petra Hirsova, Qianqian Guo, Luz H. Gutierrez Sanchez, Harris A. Gelbard, Burns C. Blaxall, Samar H. Ibrahim
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