Metabolic-associated steatotic liver disease (MASLD) is characterised by defective lipid metabolism, inflammation, and can progress to metabolic-associated steatohepatitis (MASH) when accompanied by cell death. Hepatic fibrosis is associated with MASLD, and these effects are partly mediated by the actions of liver-secreted proteins. Hepatic stellate cells (HSC) are the key drivers of hepatic fibrosis, and recent humanised-mouse studies show that hepatic non-parenchymal cells can remodel liver lipid metabolism. Given that liver-secreted proteins can exert pleiotropic autocrine, paracrine, and endocrine actions, we hypothesised that, yet unidentified HSC-derived secreted proteins can regulate MASLD progression and MASLD-associated co-morbidities including hyperglycaemia and adiposity.
Liver was obtained from 118 persons undergoing bariatric surgery. MASLD severity and fibrosis grade were assessed histologically. Precision-cut liver slices (PCLS) were prepared, and proteomics analysis of liver-secreted proteins was performed using liquid chromatography tandem mass spectrometry. We identified 3333 hepatokines and MASH remodelled ~102 liver-secreted proteins compared to MASLD and livers with No MASLD. To identify MASH-inducible HSC-derived secreted protein, we overlayed our liver-secreted proteins with published bulk liver and HSC scRNA-Seq transcriptomics and plasma proteomics datasets. This overlay identified one single target, MFAP4. MFAP4 is a HSC secreted protein that is expressed in HSCs not hepatocytes with no ascribed roles in metabolism or MASLD-associated comorbidities. Expression of MFAP4 in LX2 cells, a HSC line, following excessive palmitic acid, independent of changes in markers of HSC activation. Once-weekly recombinant MFAP4-Fc administration (1 mg/kg) in mice fed a MASLD-inducing high-fat, high-sucrose diet for 4 weeks reduced lean mass and spleen mass, increased adiposity, and liver triglyceride content and hepatic steatosis. MFAP4-Fc treatment in murine PCLS increased fatty acid uptake and this coincided with increased fatty acid incorporation into lipid pools and the expression of fatty acid transporter genes (Fatp1 and Cd36). Further, following six weeks of high-fat diet, mice received once-weekly MFAP4-Fc administration for a further 8 weeks and exhibited hyperglycaemia, increased adiposity and reduced lean mass and liver triglyceride content. To assess whether MFAP4 can promote hepatic fibrosis, we found that LX2 cells, MFAP4-Fc promoted canonical transforming growth factor beta (TGF-β) signalling and augmented TGF-β induced fibrogenesis as determined by SMAD3 phosphorylation and the expression ACTA2, collagen (COL) 1A1 and 1A2 and TIMP1. Administration of MFAP4-Fc on murine PCLS increased expression ACTA2 and COL1A1 expression. Taken together, we have identified MFAP4 a HSC-secreted protein that accelerates the development of MASLD and MASLD-associated co-morbidities and warrants further therapeutic investigation.