Prenatal Choline Supplement in a Maternal Obesity Model Modulates Offspring Hepatic Lipidomes.

Maternal obesity during pregnancy adversely impacts offspring health, predisposing them to chronic metabolic diseases characterized by insulin resistance, dysregulated macronutrient metabolism, and lipid overload, such as metabolic-associated fatty liver disease (MAFLD). Choline is a semi-essential nutrient involved in lipid and one-carbon metabolism that is compromised during MAFLD progression. Here, we investigated under high-fat (HF) obesogenic feeding how maternal choline supplementation (CS) influenced the hepatic lipidome of mouse offspring. Our results demonstrate that maternal HF+CS increased relative abundance of a subclass of phospholipids called plasmalogens in the offspring liver at both embryonic day 17.5 and after 6 weeks of postnatal HF feeding. Consistent with the role of plasmalogens as sacrificial antioxidants, HF+CS embryos were presumably protected with lower oxidative stress. After postnatal HF feeding, the maternal HF+CS male offspring also had higher relative abundance of both sphingomyelin d42:2 and its side chain, nervonic acid (FA 24:1). Nervonic acid is exclusively metabolized in the peroxisome and is tied to plasmalogen synthesis. Altogether, this study demonstrates that under the influence of obesogenic diet, maternal CS modulates the fetal and postnatal hepatic lipidome of male offspring, favoring plasmalogen synthesis, an antioxidative response that may protect the mouse liver from damages due to HF feeding.

Mouse model of NASH that replicates key features of the human disease and progresses to fibrosis stage 3.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in the United States and the world; with no Food and Drug Administration-approved pharmacological treatment available, it remains an area of unmet medical need. In nonalcoholic steatohepatitis (NASH), the most important predictor of clinical outcome is the fibrosis stage. Moreover, the Food and Drug Administration recommends that clinical trials for drugs to treat this disease include patients with fibrosis stage 2 or greater. Therefore, when using animal models for investigating the pathophysiology of NAFLD and for the preclinical evaluation of new drugs, it is important that the animals develop substantial fibrosis. The aim of this study was to develop a mouse model of NAFLD that replicated the disease in humans, including obesity and progressive liver fibrosis. Agouti yellow mutant mice, which have hyperphagia, were fed a Western diet and water containing high-fructose corn syrup for 16 weeks. Mice became obese and developed glucose intolerance. Their gut microbiota showed dysbiosis with changes that replicate some of the changes described in humans with NASH. They developed NASH with activity scores of 5-6 and fibrosis, which was stage 1 after 16 weeks, and stage 3 after 12 months. Changes in liver gene expression assessed by gene-set enrichment analysis showed 90% similarity with changes in human patients with NASH. Conclusion: Ay mice, when fed a Western diet similar to that consumed by humans, develop obesity and NASH with liver histology, including fibrosis, and gene expression changes that are highly similar to the disease in humans.

Maternal Choline Supplementation and High-Fat Feeding Interact to Influence DNA Methylation in Offspring in a Time-Specific Manner.

Maternal methyl donor supplementation during pregnancy has demonstrated lasting influence on offspring DNA methylation. However, it is unknown whether an adverse postnatal environment, such as high-fat (HF) feeding, overrides the influence of prenatal methyl donor supplementation on offspring epigenome. In this study, we examined whether maternal supplementation of choline (CS), a methyl donor, interacts with prenatal and postnatal HF feeding to alter global and site-specific DNA methylation in offspring. We fed wild-type C57BL/6J mouse dams a HF diet with or without CS throughout gestation. After weaning, the offspring were exposed to HF feeding for 6 weeks resembling a continued obesogenic environment. Our results suggest that maternal CS under the HF condition (HFCS) increased global DNA methylation and DNA methyltransferase 1 (Dnmt1) expression in both fetal liver and brain. However, during the postnatal period, HFCS offspring demonstrated lower global DNA methylation and Dnmt1 expression was unaltered in both the liver and visceral adipose tissue. Site-specific DNA methylation analysis during both fetal and postnatal periods demonstrated that HFCS offspring had higher methylation of CpGs in the promoter of Srebf1, a key mediator of de novo lipogenesis. In conclusion, the influence of maternal CS on offspring DNA methylation is specific to HF feeding status during prenatal and postnatal periods. Without continued CS during the postnatal period, global DNA methylation enhanced by prenatal CS in the offspring was overridden by postnatal HF feeding.

Promotion of cholangiocarcinoma growth by diverse cancer-associated fibroblast subpopulations.

Cancer-associated fibroblasts (CAF) are a poorly characterized cell population in the context of liver cancer. Our study investigates CAF functions in intrahepatic cholangiocarcinoma (ICC), a highly desmoplastic liver tumor. Genetic tracing, single-cell RNA sequencing, and ligand-receptor analyses uncovered hepatic stellate cells (HSC) as the main source of CAF and HSC-derived CAF as the dominant population interacting with tumor cells. In mice, CAF promotes ICC progression, as revealed by HSC-selective CAF depletion. In patients, a high panCAF signature is associated with decreased survival and increased recurrence. Single-cell RNA sequencing segregates CAF into inflammatory and growth factor-enriched (iCAF) and myofibroblastic (myCAF) subpopulations, displaying distinct ligand-receptor interactions. myCAF-expressed hyaluronan synthase 2, but not type I collagen, promotes ICC. iCAF-expressed hepatocyte growth factor enhances ICC growth via tumor-expressed MET, thus directly linking CAF to tumor cells. In summary, our data demonstrate promotion of desmoplastic ICC growth by therapeutically targetable CAF subtype-specific mediators, but not by type I collagen.

MicroRNA-21 and Dicer are dispensable for hepatic stellate cell activation and the development of liver fibrosis.

Fibrosis and cancer represent two major complications of chronic liver disease. MicroRNAs have been implicated in the development of fibrosis and cancer, thus constituting potential therapeutic targets. Here, we investigated the role of microRNA-21 (miR-21), a microRNA that has been implicated in the development of fibrosis in multiple organs and has also been suggested to act as an "oncomir." Accordingly, miR-21 was the microRNA that showed the strongest up-regulation in activated hepatic stellate cells (HSCs) in multiple models of fibrogenesis, with an 8-fold to 24-fold induction compared to quiescent HSCs. However, miR-21 antisense inhibition did not suppress the activation of murine or human HSCs in culture or in liver slices. Moreover, genetic deletion of miR-21 in two independently generated knockout mice or miR-21 antisense inhibition did not alter HSC activation or liver fibrosis in models of toxic and biliary liver injury. Despite a strong up-regulation of miR-21 in injury-associated hepatocellular carcinoma and in cholangiocarcinoma, miR-21 deletion or antisense inhibition did not reduce the development of liver tumors. As inhibition of the most up-regulated microRNA did not affect HSC activation, liver fibrosis, or fibrosis-associated liver cancer, we additionally tested the role of microRNAs in HSCs by HSC-specific Dicer deletion. Although Dicer deletion decreased microRNA expression in HSCs and altered the expression of select genes, it only exerted negligible effects on HSC activation and liver fibrosis. CONCLUSION: Genetic and pharmacologic manipulation of miR-21 does not inhibit the development of liver fibrosis and liver cancer. Moreover, suppression of microRNA synthesis does not significantly affect HSC phenotype and activation. (Hepatology 2018;67:2414-2429).

Hepatocyte TAZ/WWTR1 Promotes Inflammation and Fibrosis in Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a leading cause of liver disease worldwide. However, the molecular basis of how benign steatosis progresses to NASH is incompletely understood, which has limited the identification of therapeutic targets. Here we show that the transcription regulator TAZ (WWTR1) is markedly higher in hepatocytes in human and murine NASH liver than in normal or steatotic liver. Most importantly, silencing of hepatocyte TAZ in murine models of NASH prevented or reversed hepatic inflammation, hepatocyte death, and fibrosis, but not steatosis. Moreover, hepatocyte-targeted expression of TAZ in a model of steatosis promoted NASH features, including fibrosis. In vitro and in vivo mechanistic studies revealed that a key mechanism linking hepatocyte TAZ to NASH fibrosis is TAZ/TEA domain (TEAD)-mediated induction of Indian hedgehog (Ihh), a secretory factor that activates fibrogenic genes in hepatic stellate cells. In summary, TAZ represents a previously unrecognized factor that contributes to the critical process of steatosis-to-NASH progression.

Mouse models of liver cancer.

Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide, and the third leading cause of cancer mortality. The great majority of patients are not eligible for curative therapies, and therapeutic approaches for advanced disease show only limited efficacy. Difficulties to treat HCC are due to the heterogenous genetic alterations of HCC, profound alterations in the hepatic microenvironment, and incomplete understanding of HCC biology. Mouse models of HCC will be helpful to improve our understanding of HCC biology, the contributions of the specific pathways and genetic alterations to carcinogenesis. In addition, mouse models of HCC may contribute to elucidate the role of the tumor microenvironment, and serve as models for preclinical studies. As no single mouse model is appropriate to study all of the above, we discuss key features and limitations of commonly used models. Furthermore, we provide detailed protocols for select models, in which HCC is induced genetically, chemically or by transplantation of tumor cells.

The HMGB1/RAGE axis triggers neutrophil-mediated injury amplification following necrosis.

In contrast to microbially triggered inflammation, mechanisms promoting sterile inflammation remain poorly understood. Damage-associated molecular patterns (DAMPs) are considered key inducers of sterile inflammation following cell death, but the relative contribution of specific DAMPs, including high-mobility group box 1 (HMGB1), is ill defined. Due to the postnatal lethality of Hmgb1-knockout mice, the role of HMGB1 in sterile inflammation and disease processes in vivo remains controversial. Here, using conditional ablation strategies, we have demonstrated that epithelial, but not bone marrow-derived, HMGB1 is required for sterile inflammation following injury. Epithelial HMGB1, through its receptor RAGE, triggered recruitment of neutrophils, but not macrophages, toward necrosis. In clinically relevant models of necrosis, HMGB1/RAGE-induced neutrophil recruitment mediated subsequent amplification of injury, depending on the presence of neutrophil elastase. Notably, hepatocyte-specific HMGB1 ablation resulted in 100% survival following lethal acetaminophen intoxication. In contrast to necrosis, HMGB1 ablation did not alter inflammation or mortality in response to TNF- or FAS-mediated apoptosis. In LPS-induced shock, in which HMGB1 was considered a key mediator, HMGB1 ablation did not ameliorate inflammation or lethality, despite efficient reduction of HMGB1 serum levels. Our study establishes HMGB1 as a bona fide and targetable DAMP that selectively triggers a neutrophil-mediated injury amplification loop in the setting of necrosis.

Different fatty acids inhibit apoB100 secretion by different pathways: unique roles for ER stress, ceramide, and autophagy.

Although short-term incubation of hepatocytes with oleic acid (OA) stimulates secretion of apolipoprotein B100 (apoB100), exposure to higher doses of OA for longer periods inhibits secretion in association with induction of endoplasmic reticulum (ER) stress. Palmitic acid (PA) induces ER stress, but its effects on apoB100 secretion are unclear. Docosahexaenoic acid (DHA) inhibits apoB100 secretion, but its effects on ER stress have not been studied. We compared the effects of each of these fatty acids on ER stress and apoB100 secretion in McArdle RH7777 (McA) cells: OA and PA induced ER stress and inhibited apoB100 secretion at higher doses; PA was more potent because it also increased the synthesis of ceramide. DHA did not induce ER stress but was the most potent inhibitor of apoB100 secretion, acting via stimulation of autophagy. These unique effects of each fatty acid were confirmed when they were infused into C57BL6J mice. Our results suggest that when both increased hepatic secretion of VLDL apoB100 and hepatic steatosis coexist, reducing ER stress might alleviate hepatic steatosis but at the expense of increased VLDL secretion. In contrast, increasing autophagy might reduce VLDL secretion without causing steatosis.