Complement Component 4 Suppression Accompanies Abatement Of Liver Steatosis In Hepatocyte-specific Angiotensinogen Deficient Mice

Background: Steatosis is a frequently associated with chronic liver disease. Western diet (WD)-induced steatosis has been shown consistently to be abated by hepatocyte-specific angiotensinogen (AGT) deficiency, independently of the synthesis of angiotensin peptides. However, the molecular mechanism by which AGT facilitates WD-induced steatosis is unknown. This study aims to determine the contribution of AGT to WD-induced transcriptomic changes in the liver. Methods and Results: To determine the molecular basis of liver steatosis, we performed temporal transcriptomic analyses by bulk RNA sequencing in mice fed a WD to induce steatosis. Livers from low-density lipoprotein receptor deficient (LDLR-/-) mice fed WD (42% of calories from fat) for 5, 14, or 42 days were evaluated against mice fed normal diet. Gene ontology analysis revealed that upregulated genes were related to inflammation at each interval. Liver transcriptomes underwent a transition at 14 days WD that persisted at 42 days, evidenced by principle component analysis of unfiltered genes. To explore the contribution of AGT in steatosis, we next performed additional liver transcriptomic analyses comparing hepatocyte-specific AGT deficient (hepAGT -/- ) mice and wild type (hepAGT +/+ ) littermates at 14 and 42 days of WD feeding. Compared to hepAGT +/+ littermates, there were 128 and 23 differentially expressed genes (DEGs) in hepAGT -/- mice at 14 and 42 days of WD, respectively. Interestingly, four DEGs were overlapped between intervals. The four DEGs were Agt , Mup21 - a urinary protein with no human homolog, Moxd1 - a predicted enzyme with no known substrate, and C4a - a pro-inflammatory zymogen critical in all three complement pathways. Since the gene ontology analysis identified the contribution of multiple genes related to inflammation, we focused on C4a . RT-qPCR detected a 2.7-fold decrease in C4a mRNA expression in hepAGT -/- mice at 42 days of WD (p=0.008 by Rank Sum Test). Conclusions: Inflammatory genes were upregulated early in the development of steatosis. Hepatocyte-specific AGT deficiency suppressed C4a mRNA. Future studies will determine which complement pathways are activated by WD and characterize the spatial distribution of C4a and its proteolytic derivatives.