Hepatology
Abstract
Liver damage is typically inferred from serum measurements of cytoplasmic liver enzymes. DNA molecules released from dying hepatocytes are an alternative biomarker, unexplored so far, potentially allowing for quantitative assessment of liver cell death. Here we describe a method for detecting acute hepatocyte death, based on quantification of circulating, cell-free DNA (cfDNA) fragments carrying hepatocyte-specific methylation patterns. We identified 3 genomic loci that are unmethylated specifically in hepatocytes, and used bisulfite conversion, PCR, and massively parallel sequencing to quantify the concentration of hepatocyte-derived DNA in mixed samples. Healthy donors had, on average, 30 hepatocyte genomes/ml plasma, reflective of basal cell turnover in the liver. We identified elevations of hepatocyte cfDNA in patients shortly after liver transplantation, during acute rejection of an established liver transplant, and also in healthy individuals after partial hepatectomy. Furthermore, patients with sepsis had high levels of hepatocyte cfDNA, which correlated with levels of liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Duchenne muscular dystrophy patients, in which elevated AST and ALT derive from damaged muscle rather than liver, did not have elevated hepatocyte cfDNA. We conclude that measurements of hepatocyte-derived cfDNA can provide specific and sensitive information on hepatocyte death, for monitoring human liver dynamics, disease, and toxicity.
Authors
Roni Lehmann-Werman, Judith Magenheim, Joshua Moss, Daniel Neiman, Ofri Abraham, Sheina Piyanzin, Hai Zemmour, Ilana Fox, Talya Dor, Markus Grompe, Giora Landesberg, Bao-Li Loza, Abraham Shaked, Kim Olthoff, Benjamin Glaser, Ruth Shemer, Yuval Dor
Abstract
The gut-liver axis is of clinical importance as a potential therapeutic target in a wide range of liver diseases; however, the mechanisms underlying interactions between microbial products and immune responses in the liver remain unknown. In this study, we demonstrated that IL-10–producing macrophages contribute to immune tolerance in the inflamed liver under intestinal barrier disruption in a murine tandem model of dextran sulfate sodium (DSS) colitis and concanavalin A (Con A) hepatitis. Intestinal barrier disruption protected mice from subsequent liver injury, and the severity of colitis directly affected susceptibility to such injury. The protective effect of DSS–Con A was canceled in gut-sterilized mice, suggesting that gut microbiota play a substantial role in this process. Altered gut microbiota and their metabolites, along with a disrupted intestinal barrier, directly gave rise to immunological permissiveness in the inflamed liver. We identified 1-methylnicotinamide (1-MNA) as a candidate metabolite capable of suppressing liver injury with the potential to induce IL-10–producing macrophages. Consistently, expression of nicotinamide N-methyltransferase, which converts nicotinamide to 1-MNA, was upregulated in the liver of DSS–Con A mice, and this effect was abrogated by gut sterilization. Collectively, our results provide a mechanistic insight into the regulation of immunological balance in the liver via the gut-liver axis.
Authors
Nobuhito Taniki, Nobuhiro Nakamoto, Po-Sung Chu, Yohei Mikami, Takeru Amiya, Toshiaki Teratani, Takahiro Suzuki, Tomoya Tsukimi, Shinji Fukuda, Akihiro Yamaguchi, Shunsuke Shiba, Rei Miyake, Tadashi Katayama, Hirotoshi Ebinuma, Takanori Kanai
Abstract
While several molecular targets are under consideration, mechanistic underpinnings of the transition from uncomplicated nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH) remain unresolved. Here we apply multiscale chemical profiling technologies to mouse models of deranged hepatic ketogenesis to uncover potential NAFLD driver signatures. Use of stable-isotope tracers, quantitatively tracked by nuclear magnetic resonance (NMR) spectroscopy, supported previous observations that livers of wild-type mice maintained long term on a high-fat diet (HFD) exhibit a marked increase in hepatic energy charge. Fed-state ketogenesis rates increased nearly 3-fold in livers of HFD-fed mice, a greater proportionate increase than that observed for tricarboxylic acid (TCA) cycle flux, but both of these contributors to overall hepatic energy homeostasis fueled markedly increased hepatic glucose production (HGP). Thus, to selectively determine the role of the ketogenic conduit on HGP and oxidative hepatic fluxes, we studied a ketogenesis-insufficient mouse model generated by knockdown of the mitochondrial isoform of 3-hydroxymethylglutaryl-CoA synthase (HMGCS2). In response to ketogenic insufficiency, TCA cycle flux in the fed state doubled and HGP increased more than 60%, sourced by a 3-fold increase in glycogenolysis. Finally, high-resolution untargeted metabolomics and shotgun lipidomics performed using ketogenesis-insufficient livers in the fed state revealed accumulation of bis(monoacylglycero)phosphates, which also accumulated in livers of other models commonly used to study NAFLD. In summary, natural and interventional variations in ketogenesis in the fed state strongly influence hepatic energy homeostasis, glucose metabolism, and the lipidome. Importantly, HGP remains tightly linked to overall hepatic energy charge, which includes both terminal fat oxidation through the TCA cycle and partial oxidation via ketogenesis.
Authors
D. André d’Avignon, Patrycja Puchalska, Baris Ercal, YingJu Chang, Shannon E. Martin, Mark J. Graham, Gary J. Patti, Xianlin Han, Peter A. Crawford
Abstract
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.
Authors
Yifeng Wang, Jibiao Li, David Matye, Yuxia Zhang, Katie Dennis, Wen-Xing Ding, Tiangang Li
Abstract
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.
Authors
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
Abstract
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.
Authors
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
Abstract
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.
Authors
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
Abstract
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.
Authors
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
Abstract
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.
Authors
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
Abstract
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.
Authors
Melanie Tran, Sang-Min Lee, Dong-Ju Shin, Li Wang
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