Background Non-alcoholic fatty liver disease (NAFLD) begins as simple hepatic steatosis, but further progress to chronic liver diseases results in severe liver damage and hepatic failure. However, therapeutic options are scarce due to the lack of reliable human in vitro liver models for understanding disease progression mechanisms and developing therapies. Results We describe here a novel method for generating 3D hepatic spheroids using HepaRG cells, vascular endothelial cells, and mesenchymal stem cells cultured on a thick layer of soft matrix in a narrow conical tube; this method improved self-organization efficiency and functional competence. We further developed a 3D hepatic steatosis model with excess glucose and palmitate, accurately recapitulating steatosis phenotypes such as neutral lipid accumulation, enhanced expression of lipogenesis and gluconeogenesis markers, increased intracellular triglyceride content, and reduced glucose uptake. The expression and activity of cytochrome P450 4A (CYP4A), a hepatic glucose and lipid homeostasis enzyme, that is highly expressed in liver tissues from NAFLD patients, was induced in our in vitro steatosis model, and inhibiting CYP4A with the selective inhibitor HET0016 or a specific siRNA ameliorated steatosis-related pathology through reduced ER stress and improved insulin signaling. Conclusions We provide here a novel 3D human cell-based hepatic model that can be easily generated and reliably simulate hepatic steatosis pathology. We have experimentally validated its potential for target validation and drug evaluation by focusing on CYP4A, which may serve as a translational platform for drug development. Electronic supplementary material The online version of this article (10.1186/s13036-019-0198-8) contains supplementary material, which is available to authorized users.

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Skatole (3-methylindole, 3MI) is a natural-origin compound derived from plants, insects, and microbial metabolites in human intestines. Skatole has an anti-lipid peroxidation effect and is a biomarker for several diseases. However, its effect on hepatocyte lipid metabolism and lipotoxicity has not been elucidated. Hepatic lipotoxicity is induced by excess saturated free fatty acids in hyperlipidemia, which directly damages the hepatocytes. Lipotoxicity is involved in several metabolic diseases and hepatocytes, particularly affecting nonalcoholic fatty liver disease (NAFLD) progression. NAFLD is caused by the accumulation of fat by excessive free fatty acids (FFAs) in the blood and is accompanied by hepatic damage, such as endoplasmic reticulum (ER) stress, abnormal glucose and insulin metabolism, oxidative stress, and lipoapoptosis with lipid accumulation. Hepatic lipotoxicity causes multiple hepatic damages in NAFLD and has a directly effect on the progression from NAFLD to nonalcoholic steatohepatitis (NASH). This study confirmed that the natural compound skatole improves various damages to hepatocytes caused by lipotoxicity in hyperlipidemic conditions. To induce lipotoxicity, we exposed HepG2, SNU-449, and Huh7 cells to palmitic acid, a saturated fatty acid, and confirmed the protective effect of skatole. Skatole inhibited fat accumulation in the hepatocytes, reduced ER and oxidative stress, and recovered insulin resistance and glucose uptake. Importantly, skatole reduced lipoapoptosis by regulating caspase activity. In conclusion, skatole ameliorated multiple types of hepatocyte damage induced by lipotoxicity in the presence of excess free fatty acids.

The transcriptional regulator YAP, which plays important roles in the development, regeneration, and tumorigenesis, is activated when released from inhibition by the Hippo kinase cascade. The regulatory mechanism of YAP in Hippo-low contexts is poorly understood. Here, we performed a genome-wide RNA interference screen to identify genes whose loss of function in a Hippo-null background affects YAP activity. We discovered that the coatomer protein complex I (COPI) is required for YAP nuclear enrichment and that COPI dependency of YAP confers an intrinsic vulnerability to COPI disruption in YAP-driven cancer cells. We identified MAP2K3 as a YAP regulator involved in inhibitory YAP phosphorylation induced by COPI subunit depletion. The endoplasmic reticulum stress response pathway activated by COPI malfunction appears to connect COPI and MAP2K3. In addition, we provide evidence that YAP inhibition by COPI disruption may contribute to transcriptional up-regulation of PTGS2 and proinflammatory cytokines. Our study offers a resource for investigating Hippo-independent YAP regulation as a therapeutic target for cancers and suggests a link between YAP and COPI-associated inflammatory diseases.