Iron Overload Reprograms Glul+ Hepatocyte Fate to Induce Steatosis and Fibrosis via WNT-signaling pathway in Mice

Background & Aims Hepatic iron overload is associated with non-alcoholic fatty liver disease (NAFLD) and cirrhosis in lean individuals. We aimed to explore the underlying mechanism through which excessive iron induces fatty liver and cirrhosis at single-cell resolution. Methods Mouse models were established through feeding a high iron or high fat diet to mimic the condition of iron overload and obesity. Iron overload and obesity induced NAFLD models were confirmed by biochemistry measurements and histological sections. Single-cell RNA-seq (scRNA-seq) and single-nucleus ATAC-seq (snATAC-seq) were conducted to provide insights to the molecular effects of iron overload or obesity on major liver cell types. Pseudo-time analysis and motif activity analysis were performed to gain insight of the differentiation trajectory and the underlying regulatory mechanisms. Cell-cell interaction analysis was utilized to evaluate the hepatic microenvironment in mice with iron overload. Results Iron overload-induced fatty liver is characterized by down-regulation of PPAR signaling pathway. Ghr+ pericentral hepatocytes, expressing key regulator PPARα, are perturbed by iron, in contrast to the increased population in HFD mice. Iron overload and obesity induced distinctive hepatocyte differentiation trajectories. Differentiation of iron-overloaded hepatocytes mitigates oxidative damage but leads to the downregulation of the PPAR signaling pathway. Glul+ pericentral hepatocytes, serves as transit-amplifying cells (TACs) in liver lobule, lose their differentiation ability under high iron diet. The motif analysis of the transcription factors indicated the role of TCF7L2/LEF1 in governing the differentiation of Glul+ hepatocytes through WNT pathway. Reprogrammed Glul+ pericentral hepatocytes under iron overload also established an inflammatory micro-environment involving increased interactions between hepatocytes and non-parenchymal cells (NPCs), contributing to the formation of liver fibrosis. Conclusions This study offers comprehensive evidence elucidating the distinctive molecular and cellular mechanism of iron overload-induced NAFLD and fibrosis. PPARA and TCF7L2 agonists should be considered in developing effective therapeutic strategies for iron overload-induced lean NAFLD.