Hepatology
Abstract
Carriers of the hydroxysteroid 17-β dehydrogenase 13 (HSD17B13) gene variant (rs72613567:TA) have a reduced risk of NASH and cirrhosis but not steatosis. We determined its effect on liver histology, lipidome, and transcriptome using ultra performance liquid chromatography-mass spectrometry and RNA-seq. In carriers and noncarriers of the gene variant, we also measured pathways of hepatic fatty acids (de novo lipogenesis [DNL] and adipose tissue lipolysis [ATL] using 2H2O and 2H-glycerol) and insulin sensitivity using 3H-glucose and euglycemic-hyperinsulinemic clamp) and plasma cytokines. Carriers and noncarriers had similar age, sex and BMI. Fibrosis was significantly less frequent while phospholipids, but not other lipids, were enriched in the liver in carriers compared with noncarriers. Expression of 274 genes was altered in carriers compared with noncarriers, consisting predominantly of downregulated inflammation-related gene sets. Plasma IL-6 concentrations were lower, but DNL, ATL and hepatic insulin sensitivity were similar between the groups. In conclusion, carriers of the HSD17B13 variant have decreased fibrosis and expression of inflammation-related genes but increased phospholipids in the liver. These changes are not secondary to steatosis, DNL, ATL, or hepatic insulin sensitivity. The increase in phospholipids and decrease in fibrosis are opposite to features of choline-deficient models of liver disease and suggest HSD17B13 as an attractive therapeutic target.
Authors
Panu K. Luukkonen, Taru Tukiainen, Anne Juuti, Henna Sammalkorpi, P.A. Nidhina Haridas, Onni Niemelä, Johanna Arola, Marju Orho-Melander, Antti Hakkarainen, Petri T. Kovanen, Om Dwivedi, Leif Groop, Leanne Hodson, Amalia Gastaldelli, Tuulia Hyötyläinen, Matej Orešič, Hannele Yki-Järvinen
Abstract
Hepatic inflammasome activation is considered a major contributor to liver fibrosis in NASH. Apoptosis signal–regulating kinase 1 (ASK1) is an apical mitogen-activated protein kinase that activates hepatic JNK and p38 to promote apoptosis, inflammation, and fibrosis. The aim of the current study was to investigate whether pharmacologic inhibition of ASK1 could attenuate hepatic fibrosis driven by inflammasome activation using gain-of-function NOD-like receptor protein 3 (Nlrp3) mutant mice. Tamoxifen-inducible Nlrp3 knock-in (Nlrp3A350V/+CreT-KI) mice and WT mice were administered either control chow diet or diet containing the selective ASK1 inhibitor GS-444217 for 6 weeks. Livers of Nlrp3-KI mice had increased inflammation, cell death, and fibrosis and increased phosphorylation of ASK1, p38, and c-Jun. GS-444217 reduced ASK1 pathway activation, liver cell death, and liver fibrosis. ASK1 inhibition resulted in a significant downregulation of genes involved in collagen production and extracellular matrix deposition, as well as in a reduced hepatic TNF-α expression. ASK1 inhibition also directly reduced LPS-induced gene expression of Collagen 1A1 (Col1a1) in hepatic stellate cells isolated from Nlrp3-KI mice. In conclusion, ASK1 inhibition reduced liver cell death and fibrosis downstream of inflammatory signaling induced by NLRP3. These data provide mechanistic insight into the antifibrotic mechanisms of ASK1 inhibition.
Authors
Susanne Schuster-Gaul, Lukas Jonathan Geisler, Matthew D. McGeough, Casey D. Johnson, Anna Zagorska, Li Li, Alexander Wree, Vivian Barry, Igor Mikaelian, Lily J. Jih, Bettina G. Papouchado, Grant Budas, Hal M. Hoffman, Ariel E. Feldstein
Chronic liver disease and impaired hepatic glycogen metabolism in argininosuccinate lyase deficiency
Abstract
Background: Liver disease in urea cycle disorders (UCDs) ranges from hepatomegaly and chronic hepatocellular injury to cirrhosis and end-stage liver disease. However, the prevalence and underlying mechanisms are unclear. Methods: We estimated the prevalence of chronic hepatocellular injury in UCDs using data from a multicenter, longitudinal, natural history study. We also used ultrasound with shear wave elastography and FibroTestTM to evaluate liver stiffness and markers of fibrosis in individuals with argininosuccinate lyase deficiency (ASLD), a disorder with high prevalence of elevated serum alanine aminotransferase (ALT). To understand the human observations, we evaluated the hepatic phenotype of the AslNeo/Neo mouse model of ASLD. Results: We demonstrate a high prevalence of elevated ALT in ASLD (37%). Hyperammonemia and use of nitrogen-scavenging agents, two markers of disease severity, were significantly (p<0.001; p=0.001) associated with elevated ALT in ASLD. In addition, ultrasound with shear wave elastography and FibroTestTM revealed increased echogenicity and liver stiffness even in individuals with ASLD and normal aminotransferases. The AslNeo/Neo mice mimic the human disorder with hepatomegaly, elevated aminotransferases, and excessive hepatic glycogen noted prior to death (3-5 weeks of age). This excessive hepatic glycogen is associated with impaired hepatic glycogenolysis and decreased glycogen phosphorylase and is rescued with helper-dependent adenovirus expressing Asl using a liver specific (ApoE) promoter. Conclusions: Our results link urea cycle dysfunction and impaired hepatic glucose metabolism and identify a mouse model of liver disease in the setting of urea cycle dysfunction. Trial Registration: NCT03721367, NCT00237315 Funding: NIH, Burroughs Wellcome Fund, NUCDF, Genzyme/ACMG Foundation, CPRIT
Authors
Lindsay C. Burrage, Simran Madan, Xiaohui Li, Saima Ali, Mahmoud A. Mohammad, Bridget M. Stroup, Ming-Ming Jiang, Racel Cela, Terry Bertin, Jian Dai, Danielle Guffey, Milton Finegold, Sandesh Nagamani, Charles G. Minard, Juan Marini, Prakash Masand, Deborah Schady, Benjamin L. Shneider, Daniel H. Leung, Deeksha Bali, Brendan Lee
Abstract
BACKGROUND We hypothesized that obesity-associated hepato-steatosis served as a pathophysiologic chemical depot for fat-soluble vitamins and altered normal physiology. Using α-tocopherol (vitamin E) as a model vitamin, pharmacokinetics and kinetics principles were utilized to determine whether excess liver fat sequestered α-tocopherol in women with obesity-associated hepato-steatosis vs healthy controls. METHODS Custom-synthesized deuterated α-tocopherols (d3- and d6-α-tocopherols) were administered to hospitalized healthy women and women with hepato-steatosis under IND guidelines. Serial samples obtained over 72 hours were analyzed by LC/MS. Fluorescent-labelled α-tocopherol was custom-synthesized for cell studies. RESULTS In healthy subjects, 85% of intravenous d6-α-tocopherol disappeared from the circulation within 20 minutes but reappeared within minutes and peaked at 6-8 hours. d3- and d6-α-Tocopherols localized to lipoproteins. Lipoprotein redistribution occurred only in vivo within 1h, indicating a key role of liver in rapid uptake and re-release into the circulation. Compared to healthy subjects, subjects with hepato-steatosis had similar d6-α-tocopherol entry rates into liver, but reduced initial release rates (p<0.001). Similarly, pharmacokinetics parameters of AUC and Maximum Concentration (Cmax) were reduced (AUC0-8 ,p<0.01;Cmax p<0.02) in hepato-steatosis subjects, indicating reduced hepatic d6-α-tocopherol output. Reduced kinetics and pharmacokinetics parameters (AUC and Cmax) in hepato-steatosis subjects who received 2 mg were mirrored by similar reductions in healthy subjects when comparing 5 and 2 mg doses. In vitro, fluorescent-labelled α-tocopherol localized specifically to lipid in fat-loaded hepatocytes, indicating sequestration. CONCLUSIONS The unique role of the liver in vitamin E physiology is dysregulated by excess liver fat. Obesity-associated hepato-steatosis may produce unrecognized hepatic vitamin E sequestration, which might subsequently drive liver disease. Our findings raise the possibility that hepato-steatosis may similarly alter hepatic physiology of other fat-soluble vitamins.
Authors
Pierre-Christian Violet, Ifechukwude C. Ebenuwa, Yu Wang, Mahtab Niyyati, Sebastian J. Padayatty, Brian Head, Kenneth Wilkins, Stacey Chung, Varsha Thakur, Lynn Ulatowski, Jeffrey Atkinson, Mikel Ghelfi, Sheila Smith, Hongbin Tu, Gerd Bobe, Chia-Ying Liu, David W. Herion, Robert D. Shamburek, Danny Manor, Maret G. Traber, Mark Levine
Abstract
Background: Hepatic encephalopathy (HE) is associated with poor outcomes. A prior randomized, pilot trial demonstrated safety after oral capsular FMT in HE with favorable changes in microbial composition and cognition. However, microbial functional changes are unclear. Aim: Determine impact of FMT on gut-brain axis compared to placebo using microbial function based on bile acids (BA), inflammation (serum IL-6, lipopolysaccharide-binding protein,LBP), and EncephalApp. Methods: 20 cirrhotic patients were randomized 1:1 into receiving one-time FMT capsules from a donor enriched in Lachnospiraceae and Ruminococcaceae, or placebo capsules with 5-month follow-up for safety outcomes. Stool microbiota and BA, serum IL-6, BA and LBP, and EncephalApp were analyzed at baseline and 4-weeks post-FMT/placebo. Correlation networks between microbiota, BAs, EncephalApp, IL-6 and LBP were performed pre/post-FMT. Results: FMT-assigned participants had one HE recurrence and 2 unrelated infections. Six placebo-assigned participants developed negative outcomes. FMT, but not placebo, was associated with reduced serum IL-6 and LBP and improved EncephalApp. FMT-assigned participants demonstrated higher deconjugation and secondary BA formation in feces and serum compared to baseline. No change was seen in placebo. Correlation networks showed greater complexity post-FMT compared to baseline. Beneficial taxa such as Ruminococcaceae, Verrucomicrobiaceae and Lachnospiraceae were correlated with cognitive improvement and decrease in inflammation post-FMT. Fecal/serum secondary/primary ratios and PiCRUST secondary BA pathways did not increase in participants who developed poor outcomes. Conclusions: Gut microbial function in cirrhosis is beneficially affected by capsular FMT with improved inflammation and cognition. Lower secondary BAs in FMT recipients could select for participants who develop negative outcomes.
Authors
Jasmohan S. Bajaj, Nita Salzman, Chathur Acharya, Hajime Takei, Genta Kakiyama, Andrew Fagan, Melanie B. White, Edith A. Gavis, Mary L. Holtz, Michael Hayward, Hiroshi Nittono, Philip B. Hylemon, I. Jane Cox, Roger Williams, Simon D. Taylor-Robinson, Richard K. Sterling, Scott C. Matherly, Michael Fuchs, Hannah Lee, Puneet Puri, R. Todd Stravitz, Arun J. Sanyal, Lola Ajayi, Adrien Le Guennec, R. Andrew Atkinson, Mohammad S. Siddiqui, Velimir A. Luketic, William M. Pandak, Masoumeh Sikaroodi, Patrick M. Gillevet
Abstract
Thymic stromal lymphopoietin (TSLP) is a cytokine mainly released by epithelial cells that plays important roles in inflammation, autoimmune disease, and cancer. While TSLP is expressed in the liver at high levels, the role of TSLP in liver ischemia/reperfusion (I/R) injury remains unknown. Experiments were carried out to determine the role of TSLP in liver I/R injury. Wild-type (WT) and TSLP receptor–knockout (TSLPR–/–) mice were subjected to liver partial warm I/R injury. Liver injury was assessed by measuring serum alanine aminotransferase (ALT) level, necrotic areas by liver histology, hepatocyte death, and local hepatic inflammatory responses. Signal pathways were explored in vivo and in vitro to identify possible mechanisms for TSLP in I/R injury. TSLP and TSLPR protein expression increased during liver I/R in vivo and following hepatocyte hypoxia/reoxygenation in vitro. Deletion of TSLPR or neutralization of TSLP with anti-TSLP antibody exacerbated liver injury in terms of serum ALT levels as well as necrotic areas in liver histology. Administration of exogenous recombinant mouse TSLP to WT mice significantly reduced liver damage compared with controls, but failed to prevent I/R injury in TSLPR–/– mice. TSLP induced autophagy in hepatocytes during liver I/R injury. Mechanistically, Akt was activated in WT mice during liver I/R injury. The opposite results were observed in TSLPR–/– mice. In addition, TSLP could directly induce Akt activation in hepatocytes independent of nonparenchymal cells in vitro. Furthermore, the Akt agonist, insulin-like growth factor-1 (IGF-1), prevented I/R injury in TSLPR–/– mice and an Akt inhibitor, LY294002, blocked the protective effects of TSLP in WT mice subjected to I/R. Our data indicate that TSLP protects against liver I/R injury via activation of the PI3K/Akt pathway. Through this pathway, TSLP induces autophagy in hepatocytes. Thus, TSLP is a potent inhibitor of stress-induced hepatocyte necrosis.
Authors
Shilai Li, Zhongjie Yi, Meihong Deng, Melanie J. Scott, Chenxuan Yang, Wenbo Li, Zhao Lei, Nicole M. Santerre, Patricia Loughran, Timothy R. Billiar
Abstract
Worldwide, over a billion people suffer from chronic liver diseases, which often lead to fibrosis and then cirrhosis. Treatments for fibrosis remain experimental, in part because no unifying mechanism has been identified that initiates liver fibrosis. Necroptosis has been implicated in multiple liver diseases. Here, we report that O-linked β-N-acetylglucosamine (O-GlcNAc) modification protects against hepatocyte necroptosis and initiation of liver fibrosis. Decreased O-GlcNAc levels were seen in patients with alcoholic liver cirrhosis and in mice with ethanol-induced liver injury. Liver-specific O-GlcNAc transferase–KO (OGT-LKO) mice exhibited hepatomegaly and ballooning degeneration at an early age and progressed to liver fibrosis and portal inflammation by 10 weeks of age. OGT-deficient hepatocytes underwent excessive necroptosis and exhibited elevated protein expression levels of receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL), which are key mediators of necroptosis. Furthermore, glycosylation of RIPK3 by OGT is associated with reduced RIPK3 protein stability. Taken together, these findings identify OGT as a key suppressor of hepatocyte necroptosis, and OGT-LKO mice may serve as an effective spontaneous genetic model of liver fibrosis.
Authors
Bichen Zhang, Min-Dian Li, Ruonan Yin, Yuyang Liu, Yunfan Yang, Kisha A. Mitchell-Richards, Jin Hyun Nam, Rui Li, Li Wang, Yasuko Iwakiri, Dongjun Chung, Marie E. Robert, Barbara E. Ehrlich, Anton M. Bennett, Jun Yu, Michael H. Nathanson, Xiaoyong Yang
Abstract
Neutrophils play critical roles during the initial phase of hepatic ischemia/reperfusion injury (HIRI). However, the regulation of neutrophil activation, infiltration, and proinflammatory cytokine secretion has not been fully elucidated. In this study, we revealed that OX40 was expressed by neutrophils, its expression in neutrophils was time-dependently upregulated following HIRI, and Ox40 knockout markedly alleviated liver injury. Compared with wild-type neutrophils, the adoptive transfer of Ox40–/– neutrophils decreased HIRI in neutrophil-depleted Rag2/Il2rg–/– or Ox40–/– mice. Moreover, consistently, the in vitro experiments showed that Ox40 not only prolonged neutrophil survival but also promoted proinflammatory cytokines, ROS production, and even neutrophil chemotaxis. Further investigation demonstrated that the knockout of Ox40 in neutrophils inhibited NF-κB signaling via the TRAF1/2/4 and IKKα/IKKβ/IκBα pathways. OX40L and OX86 stimulation could enhance neutrophil activation and survival in vitro and in vivo. In conclusion, our study provides a new understanding of OX40, which is expressed not only in adaptive immune cells but also in innate immune cells, i.e., neutrophils, contributing to the activation and survival of neutrophils. These findings provide a novel potential therapeutic target for the prevention of HIRI during liver transplantation or hepatic surgery.
Authors
Hua Jin, Chunpan Zhang, Chengyang Sun, Xinyan Zhao, Dan Tian, Wen Shi, Yue Tian, Kai Liu, Guangyong Sun, Hufeng Xu, Dong Zhang
Abstract
The common patatin-like phospholipase domain–containing protein 3 (PNPLA3) variant I148M predisposes to nonalcoholic liver disease but not its metabolic sequelae. We compared the handling of labeled polyunsaturated fatty acids (PUFAs) and saturated fatty acids (SFA) in vivo in humans and in cells harboring different PNPLA3 genotypes. In 148M homozygous individuals, triglycerides (TGs) in very low–density lipoproteins (VLDL) were depleted of PUFAs both under fasting and postprandial conditions compared with 148I homozygotes, and the PUFA/SFA ratio in VLDL-TGs was lower relative to the chylomicron precursor pool. In human PNPLA3-148M and PNPLA3-KO cells, PUFA but not SFA incorporation into TGs was increased at the expense of phosphatidylcholines, and under lipolytic conditions, PUFA-containing diacylglycerols (DAGs) accumulated compared with PNPLA3-148I cells. Polyunsaturated TGs were increased, while phosphatidylcholines (PCs) were decreased in the human liver in 148M homozygous individuals as compared with 148I homozygotes. We conclude that human PNPLA3-I148M is a loss-of-function allele that remodels liver TGs in a polyunsaturated direction by impairing hydrolysis/transacylation of PUFAs from DAGs to feed phosphatidylcholine synthesis.
Authors
Panu K. Luukkonen, Auli Nick, Maarit Hölttä-Vuori, Christoph Thiele, Elina Isokuortti, Susanna Lallukka-Brück, You Zhou, Antti Hakkarainen, Nina Lundbom, Markku Peltonen, Marju Orho-Melander, Matej Orešič, Tuulia Hyötyläinen, Leanne Hodson, Elina Ikonen, Hannele Yki-Järvinen
Abstract
Non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) are liver manifestations of the metabolic syndrome and can progress to hepatocellular carcinoma (HCC). Loss of Growth Hormone (GH) signaling is reported to predispose to NAFLD and NASH through direct actions on the liver. Here, we report that aged mice lacking hepatocyte Jak2 (JAK2L), an obligate transducer of Growth Hormone (GH) signaling, spontaneously develop the full spectrum of phenotypes found in patients with metabolic liver disease, beginning with insulin resistance and lipodystrophy and manifesting as NAFLD, NASH and even HCC, independent of dietary intervention. Remarkably, insulin resistance, metabolic liver disease, and carcinogenesis are prevented in JAK2L mice via concomitant deletion of adipocyte Jak2 (JAK2LA). Further, we demonstrate that GH increases hepatic lipid burden but does so indirectly via signaling through adipocyte JAK2. Collectively, these data establish adipocytes as the mediator of GH-induced metabolic liver disease and carcinogenesis. In addition, we report a new spontaneous model of NAFLD, NASH, and HCC that recapitulates the natural sequelae of human insulin resistance-associated disease progression. The work presented here suggests a attention be paid towards inhibition of adipocyte GH signaling as a therapeutic target of metabolic liver disease.
Authors
Kevin C. Corbit, Camella G. Wilson, Dylan Lowe, Jennifer L. Tran, Nicholas B. Vera, Michelle Clasquin, Aras N. Mattis, Ethan J. Weiss
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