Background and Aims In nonalcoholic fatty liver disease (NAFLD), fibrosis is the most important factor contributing to NAFLD‐associated morbidity and mortality. Prevention of progression and reduction in fibrosis are the main aims of treatment. Even in early stages of NAFLD, hepatic and systemic hyperammonemia is evident. This is due to reduced urea synthesis; and as ammonia is known to activate hepatic stellate cells, we hypothesized that ammonia may be involved in the progression of fibrosis in NAFLD. Approach and Results In a high‐fat, high‐cholesterol diet–induced rodent model of NAFLD, we observed a progressive stepwise reduction in the expression and activity of urea cycle enzymes resulting in hyperammonemia, evidence of hepatic stellate cell activation, and progressive fibrosis. In primary, cultured hepatocytes and precision‐cut liver slices we demonstrated increased gene expression of profibrogenic markers after lipid and/or ammonia exposure. Lowering of ammonia with the ammonia scavenger ornithine phenylacetate prevented hepatocyte cell death and significantly reduced the development of fibrosis both in vitro in the liver slices and in vivo in a rodent model. The prevention of fibrosis in the rodent model was associated with restoration of urea cycle enzyme activity and function, reduced hepatic ammonia, and markers of inflammation. Conclusions The results of this study suggest that hepatic steatosis results in hyperammonemia, which is associated with progression of hepatic fibrosis. Reduction of ammonia levels prevented progression of fibrosis, providing a potential treatment for NAFLD.

Precision cut liver slices (PCLSs) retain the structure and cellular composition of the native liver and represent an improved system to study liver fibrosis compared to two‐dimensional mono‐ or co‐cultures. The aim of this study was to develop a bioreactor system to increase the healthy life span of PCLSs and model fibrogenesis. PCLSs were generated from normal rat or human liver, or fibrotic rat liver, and cultured in our bioreactor. PCLS function was quantified by albumin enzyme‐linked immunosorbent assay (ELISA). Fibrosis was induced in PCLSs by transforming growth factor beta 1 (TGFβ1) and platelet‐derived growth factor (PDGFββ) stimulation ± therapy. Fibrosis was assessed by gene expression, picrosirius red, and α‐smooth muscle actin staining, hydroxyproline assay, and soluble ELISAs. Bioreactor‐cultured PCLSs are viable, maintaining tissue structure, metabolic activity, and stable albumin secretion for up to 6 days under normoxic culture conditions. Conversely, standard static transwell‐cultured PCLSs rapidly deteriorate, and albumin secretion is significantly impaired by 48 hours. TGFβ1/PDGFββ stimulation of rat or human PCLSs induced fibrogenic gene expression, release of extracellular matrix proteins, activation of hepatic myofibroblasts, and histological fibrosis. Fibrogenesis slowly progresses over 6 days in cultured fibrotic rat PCLSs without exogenous challenge. Activin receptor‐like kinase 5 (Alk5) inhibitor (Alk5i), nintedanib, and obeticholic acid therapy limited fibrogenesis in TGFβ1/PDGFββ‐stimulated PCLSs, and Alk5i blunted progression of fibrosis in fibrotic PCLS. Conclusion: We describe a bioreactor technology that maintains functional PCLS cultures for 6 days. Bioreactor‐cultured PCLSs can be successfully used to model fibrogenesis and demonstrate efficacy of antifibrotic therapies.

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Reporting of In Vivo Experiments AST Aspartate aminotransferase αSMA α-smooth muscle actin ATG16L Autophagy Related 16L ATG7 Autophagy Related 7 AAV Adeno-associated virus BODIPY 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene BNIP BCL2 interacting protein 3 BNIP3L/NIX BCL2 interacting protein 3 like BBVA Banco Bilbao Vizcaya Argentaria°C Celsius Degrees CCPG1 Cell-Cycle Progression Gene 1 CMA Chaperon Mediated Autophagy Co-IP Co-Immuno Precipitation Col1a1 Type IA1 collagen CPT1 Carnitine PalmitoylTransferase I Ctrl Control Financial support: This work was supported by C0120R3166, C0245R4032 and BH182173 from Newcastle University. MG-M is a Sara Borrell Postdoctoral fellow (CD18/00203

During postprandial state, the liver is exposed to high levels of dietary fatty acids (FAs) and carbohydrates. FAs are re-esterified into triglycerides, which can be stored in lipid droplets (LDs) in the liver. Aberrant accumulation of LDs can lead to diseases such as alcoholic liver disease and non-alcoholic fatty liver disease, the latter being the most common liver pathology in western countries. Improved understanding of LD biology has potential to unlock new treatments for these liver diseases. The Perilipin (Plin) family is the group of proteins that coat LDs, controlling their biogenesis, stabilization, and preventing their degradation. Recent studies have revealed that autophagy is involved in LD degradation and, therefore, may be crucial to avoid lipid accumulation. Here, we show that a phosphorylated form of Plin3 is required for selective degradation of LDs in fibroblasts, primary hepatocytes and human liver slices. We demonstrate that oleic acid treatment induces the recruitment of the autophagy machinery to the surface of LDs. When Plin3 is silenced, this recruitment is suppressed resulting in accumulation of lipid. Plin3 pulldowns revealed interactions with the autophagy initiator proteins Fip200 and Atg16L indicating that Plin3 may function as a docking protein involved in lipophagy activation. Of particular importance, we define Plin3 as a substrate for mTORC1-dependent phosphorylation and show that this event is decisive for lipophagy. Our study therefore reveals that Plin3, and its phosphorylation by mTORC1, is crucial for degradation of LDs by autophagy. We propose that stimulating this pathway to enhance LD autophagy in hepatocytes will help protect the liver from lipid-mediated toxicity thus offering new therapeutic opportunities in human steatotic liver diseases.