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  • CRISPR Cas9-mediated deletion of biliverdin reductase A (BVRA) in mouse liver cells induces oxidative stress and lipid accumulation.

CRISPR Cas9-mediated deletion of biliverdin reductase A (BVRA) in mouse liver cells induces oxidative stress and lipid accumulation.

Archives of biochemistry and biophysics (2019-08-20)
Darren M Gordon, Samuel O Adeosun, Somtochukwu I Ngwudike, Christopher D Anderson, John E Hall, Terry D Hinds, David E Stec
ABSTRACT

Obesity is the predominant cause of non-alcoholic fatty liver disease (NAFLD), which is associated with insulin resistance and diabetes. NAFLD includes a spectrum of pathologies that starts with simple steatosis, which can progress to non-alcoholic steatohepatitis (NASH) with the commission of other factors such as the enhancement of reactive oxygen species (ROS). Biliverdin reductase A (BVRA) reduces biliverdin to the antioxidant bilirubin, which may serve to prevent NAFLD, and possibly the progression to NASH. To further understand the role of BVRA in hepatic function, we used CRISPR-Cas9 technology to target the Blvra gene in the murine hepa1c1c7 hepatocyte cell line (BVRA KO). BVRA activity and protein levels were significantly lower in BVRA KO vs. wild-type (WT) hepatocytes. Lipid accumulation under basal and serum-starved conditions was significantly (p < 0.05) higher in BVRA KO vs. WT cells. The loss of BVRA resulted in the reduction of mitochondria number, decreased expression of markers of mitochondrial biogenesis, uncoupling, oxidation, and fusion, which paralleled reduced mitochondrial oxygen consumption. BVRA KO cells exhibited increased levels of ROS generation and decreased levels of superoxide dismutase mRNA expression. In conclusion, our data demonstrate a critical role for BVRA in protecting against lipid accumulation and oxidative stress in hepatocytes, which may serve as a future therapeutic target for NAFLD and its progression to NASH.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Protease Inhibitor Cocktail, for use with mammalian cell and tissue extracts, DMSO solution
Sigma-Aldrich
Phosphatase Inhibitor Cocktail 2, aqueous solution (dark coloration may develop upon storage, which does not affect the activity)
Sigma-Aldrich
β-Nicotinamide adenine dinucleotide phosphate sodium salt hydrate
Sigma-Aldrich
L-(−)-Glucose, ≥99%
Sigma-Aldrich
2′,7′-Dichlorofluorescein diacetate, BioReagent, suitable for fluorescence, ≥95% (HPLC)
Sigma-Aldrich
Tetramethylrhodamine ethyl ester perchlorate, suitable for fluorescence, ≥90% (HPCE)