Loss of hepatic Lgr4 and Lgr5 promotes nonalcoholic fatty liver disease

Background & Aims The Rspo-Lgr4/5-Znrf3/Rnf43 module is a master regulator of hepatic Wnt/β-catenin signaling and metabolic zonation, but its impact on nonalcoholic fatty liver disease (NAFLD) remains unclear. We studied whether liver-specific loss of the Wnt/β-catenin modulators Leucine-Rich Repeat-Containing G Protein-Coupled Receptor 4/5 (Lgr4/5) promotes nonalcoholic fatty liver disease (NAFLD). Methods Mice with liver-specific deletion of both receptors Lgr4/5 (Lgr4/5dLKO) were fed with normal diet (ND) or high fat diet (HFD). Livers of these mice were analyzed for lipid and fibrotic content by tissue staining and immunohistochemistry (IHC), and lipoproteins, inflammation and liver enzyme markers were measured in blood. Mechanistic insights into hepatic lipid accumulation were obtained by using ex vivo primary hepatocyte cultures derived from the Lgr4/5dLKO mice. Lipid analysis of mouse livers was performed by mass spectrometry (MS)-based untargeted lipidomic analysis. Results We demonstrated that liver-specific ablation of Lgr4/5-mediated Wnt signaling resulted in hepatic steatosis, impaired bile acid (BA) secretion and predisposition to liver fibrosis. Under HFD conditions, we observed progressive intrahepatic fat accumulation, developing into macro-vesicular steatosis. Serum lipoprotein levels in HFD-fed Lgr4/5dLKO mice were decreased, rather than increased, suggesting that accumulation of fat in the liver was due to impaired lipid secretion by hepatocytes. Our lipidome analysis revealed a severe alteration of several lipid species in livers of Lgr4/5dLKO mice, including triacylglycerol estolides (TG-EST), a storage form of bioactive free fatty acid (FA) esters of hydroxy FAs (FAHFAs). Conclusions Loss of hepatic Wnt/β-catenin activity by Lgr4/5 deletion led to deregulation of lipoprotein pathways, loss of BA secretion, intrinsic alterations of lipid homeostasis and the onset of NAFLD. Lay summary The Wnt/β-catenin pathway plays an important role during development and tissue homeostasis. Loss of Wnt/β-catenin activity in mouse liver leads to loss of liver zonation, but the impact on nonalcoholic fatty liver disease (NAFLD) remains unclear. We show that livers of mice developed steatosis upon deletion of the positive pathway regulators Lgr4/5. Livers of knock-out (KO) mice exhibited altered lipid composition due to impaired lipid secretion. Furthermore, livers of these mice developed a nonalcoholic steatohepatitis (NASH)-like phenotype and fibrotic features derived from activated hepatic stellate cells. Our data demonstrate a protective role of Wnt/β-catenin pathway activity towards the development of NAFLD. Highlights ### Competing Interest Statement All authors except M.L.M.G, A.G.M, C.G. and L.T. are or have been employed by and/or shareholders of Novartis Pharma AG. * Alb-Cre : Cre recombinase under the hepatocyte-specific albumin promoter αSMA : Alpha Smooth Muscle Actin ALT : alanine aminotransferase AST : aspartate aminotransferase AXIN2 : Axis inhibition protein 2 BA : bile acid CDH1 : Cadherin 1 c-HDL : cholesterol-high density lipoprotein CLDN2 : Claudin-2 CLF : cholyl-L-lysyl-fluorescein CTNNB1 : Catenin beta 1 DG : diacylglycerol DNL : de novo lipogenesis FA : fatty acid FAHFA : fatty acid esters of hydroxy fatty acids FPLC : fast protein liquid chromatography-based sizeexclusion GTT : glucose tolerance test HFD : high fat diet HSC : hepatic stellate cells IBA1 : Ionized Calcium-Binding Adapter Molecule 1 IHC : immunohistochemistry IL6 : Interleukin 6 ISH : in situ hybridization ITT : insulin tolerance test KO : knock-out KRT19 : Keratin 19 LGR : Leucine-Rich RepeatContaining G Protein-Coupled Receptor Lgr4/5dLKO : Leucine-Rich Repeat-Containing G Protein-Coupled Receptor 4 and 5 double liver knock-out LDL : low density lipoprotein LRP6 : LDL-Receptor Related Protein 6 MCD : methionine- and choline-deficient diet NAFLD : nonalcoholic fatty liver disease NASH : nonalcoholic steatohepatitis ND : normal diet OxTG : oxidized triglycerol RNF43 : Ring Finger Protein 43 RSPO : Roof Plate-Specific Spondin SD, SD : standard deviation SEM : standard error of the mean SLC : solute carrier SWE : shear wave elastography t-BA : total bile acids t-Chol : total cholesterol TEM : Transmission electron microscopy TG : triglycerol TG-EST : triacylglycerol estolides TJ : tight junctions TNFα : tumor necrosis factor alpha VLDL : very low density lipoprotein VLDLR : very low density lipoprotein receptor Wnt : Wingless-related integration site WT : wild-type ZNRF3 : Zinc And Ring Finger 3 ZO-1 : Zona Occludens Protein-1