CRISPR Cas9‐mediated deletion of Biliverdin reductase A (BVRA) in mouse liver cells models a broad spectrum of NAFLD Phenotype

Abstract

Obesity is the leading cause of Non Alcoholic Fatty Liver Disease (NAFLD) which is also associated with insulin resistance and diabetes. NAFLD include a spectrum of pathologies which starts with fat accumulation (steatosis). The presence of a ‘second hit’ drives the continuum from steatosis to steatohepatitis, fibrosis, cirrhosis and liver failure/cancer. We previously showed that bilirubin treatment protects mice from hepatic steatosis. Biliverdin reductase (BVRA) ‐the key enzyme for bilirubin synthesis, also signals through MAPK/AKT which is an important insulin‐regulated pathway.The aim of this study was to test the hypothesis that BVRA protects against steatosis via regulation of intracellular fat processing through a combination of direct signaling and bilirubin generation.We used the CRISPR‐Cas9 system to delete/disrupt a 16.5kbp fragment between Exon1 and Exon 5 of the BVRA gene in a mouse liver cell line –Hepa 1c1c7 and studied NAFLD‐related phenotypes. Using a novel protein UnaG which fluoresces only when bound to bilirubin, we show that both intracellular bilirubin and conversion of biliverdin to bilirubin (BVRA activity) by live cells and by cell lysates were markedly reduced in these BVRA‐KO cells. With no treatment, these cells recapitulated the transcriptomic signature of drug‐induced steatosis as FOXA1, HHEX and SREB1F –genes associated with fatty acid processing, were changed in a manner that favors fat accumulation. Consequently, when exposed to free‐fatty acids, Nile‐Red staining coupled with flow cytometry showed that BVRA‐KO cells accumulate 42% more fat (p=0.007) and 100% more triglycerides (p=0.002) compared to wildtype cells. They also showed a blunting of insulin‐stimulated AKT phosphorylation (up to 41% less) compared to wildtype cells. Lastly, untreated BVRA‐KO cells express significantly higher levels (1.7–3.2 folds) of markers of fibrosis versus wildtype cells.In conclusion, our data shows that BVRA plays a critical role in protecting against lipid accumulation and in the pathophysiology of NAFLD. Studies are on‐going to determine the independent contributions of the signaling and reductase activities of BVRA and thus to identify the best BVRA‐directed therapeutic target. Also, given that the deletion of this important gene required no [additional] second‐hit to precipitate phenotypes like fibrosis which represents a later stage in the NAFLD continuum, these cells may be a novel in‐vitro model for studying a broader spectrum of NAFLD phenotypes.Support or Funding InformationNIH grant PO1HL 51971 and P20GM‐104357