Enhanced de novo lipogenesis mediated by sterol regulatory element–binding proteins (SREBPs) is thought to be involved in nonalcoholic steatohepatitis (NASH) pathogenesis. In this study, we assessed the impact of SREBP inhibition on NASH and liver cancer development in murine models. Unexpectedly, SREBP inhibition via deletion of the SREBP cleavage–activating protein (SCAP) in the liver exacerbated liver injury, fibrosis, and carcinogenesis despite markedly reduced hepatic steatosis. These phenotypes were ameliorated by restoring SREBP function. Transcriptome and lipidome analyses revealed that SCAP/SREBP pathway inhibition altered the fatty acid (FA) composition of phosphatidylcholines due to both impaired FA synthesis and disorganized FA incorporation into phosphatidylcholine via lysophosphatidylcholine acyltransferase 3 (LPCAT3) downregulation, which led to endoplasmic reticulum (ER) stress and hepatocyte injury. Supplementation with phosphatidylcholines significantly improved liver injury and ER stress induced by SCAP deletion. The activity of the SCAP/SREBP/LPCAT3 axis was found to be inversely associated with liver fibrosis severity in human NASH. SREBP inhibition also cooperated with impaired autophagy to trigger liver injury. Thus, excessively strong and broad lipogenesis inhibition was counterproductive for NASH therapy; this will have important clinical implications in NASH treatment.

Progression of fibrosis and the development of cirrhosis are responsible for the liver-related morbidity and mortality associated with chronic liver diseases. There is currently a great unmet need for effective antifibrotic strategies. Stem cells play a central role in wound-healing responses to restore liver homeostasis following injury. Here we tested the hypothesis that extracellular vesicles (EVs) isolated from induced pluripotent stem cells (iPSCs) modulate hepatic stellate cell (HSC) activation and may have antifibrotic effects. Human iPSCs were generated by reprogramming primary skin fibroblasts. EVs were isolated by differential centrifugation, quantified by flow cytometry (FACS), and characterized by dynamic light scattering and transmission electron microscopy (TEM). Primary human HSCs were activated with TGF-β (10 ng/mL) and exposed to iPSC-EVs. Efficacy of iPSC-EVs was tested on HSCs in vitro and in 2 murine models of liver injury (CCl ₄ and bile duct ligation). Characterization of iPSC-derived EVs by flow cytometry identified a large population of EVs released by iPSCs, primarily with a diameter of 300 nm, that could be visualized by TEM as round, cup-shaped objects. Fluorescent tracing assays detected iPSC-EVs in HSC cytosol after a short incubation, and EV uptake by HSCs resulted in both decrease of profibrogenic markers α–smooth muscle actin, CollagenIα1, fibronectin, and tissue inhibitor of metalloproteinases–1 and HSC profibrogenic responses, such as chemotaxis and proliferation. Genomics analyses of iPSC-EV miRNA cargo revealed 22 highly expressed miRNAs, among which miR-92a-3p was the most abundant. Transcriptome analysis identified 60 genes downmodulated and 235 upregulated in TGF-β–primed HSCs in the presence or absence of iPSC-EVs. Intravenous injection of iPSC-EVs in CCl ₄ -induced and bile duct ligation–induced liver fibrosis resulted in antifibrotic effects at protein and gene levels. Results of this study identify iPSC-EVs as a potentially novel antifibrotic approach that may reduce or reverse liver fibrosis in patients with chronic liver disease.

Background: Hepatic sarcopenia is one of many complications associated with chronic liver disease (CLD) and has a high mortality rate; however, the liver-muscle axis is not fully understood. Therefore, few effective treatments exist for hepatic sarcopenia, the best of which being branched-chain amino acid (BCAA) supplementation to help increase muscle mass. Our aim was to investigate the molecular mechanism(s) of hepatic sarcopenia focused on bile acid (BA) composition.Methods: The correlation between serum BA levels and psoas muscle mass index (PMI) was examined in 73 CLD patients. Gastrocnemius muscle phenotype and serum BA levels were assessed in CLD rats treated with BCAA. Mouse skeletal muscle cells (C2C12) were incubated with lithocholic acid (LCA), G-protein-coupled receptor 5 (TGR5) agonist or TGR5 antagonist to assess skeletal muscle hypertrophy.Results: In human CLD, serum LCA levels were the sole factor positively correlated with PMI and were significantly decreased in both the low muscle mass group and the deceased group. Serum LCA levels were also shown to predict patient survival. Gastrocnemius muscle weight significantly increased in CLD rats treated with BCAA via suppression of protein degradation pathways, coupled with a significant increase in serum LCA levels. LCA treated C2C12 hypertrophy occurred in a concentration-dependent manner linked with TGR5-Akt pathways based upon inhibition results via a TGR5 antagonist.Conclusions: Our results indicate LCA-mediated skeletal muscle hypertrophy via activation of TGR5-IGF1-Akt signaling pathways. In addition, serum LCA levels were associated with skeletal muscle mass in cirrhotic rats, as well as CLD patients, and predicted overall patient survival.Funding: This research was supported by JSPS KAKENHI Grant Number 22K08011 and 21H02892, and AMED under Grant Number JP21fk0210090 and JP22fk0210115. Maintaining cirrhotic rats were partially supported by Otsuka Pharmaceutical Company.