Obesity is linked to the development of metabolic dysfunction-associated steatotic liver disease (MASLD). Dysregulated lipid metabolism is a hallmark of MASLD leading to excessive lipid deposition, liver inflammation and fibrosis.
An understudied yet important aspect of hepatic lipid metabolism is understanding how fatty acids (FA) are trafficked from their site of storage in lipid droplets (LD) to their site of oxidation within the mitochondria. However, the identity and functions of proteins residing at LD-mitochondria contact sites remains largely unknown. Accordingly, we conducted a proximity labelling experiment called ‘Split- BioID’ at the LD-mitochondria interface and identified 74 proteins, in HepG2 cells. The second most abundant protein at this interface was zinc finger CCC-H containing antiviral protein-1 (ZC3HAV1). CRISPR-Cas9 mediated deletion of ZC3HAV1 reduced FA oxidation and storage of triglyceride and cholesterol ester content, in HeLa cells. Thus, we hypothesised that ZC3HAV1 deletion would reduce FA oxidation and exacerbate hepatic steatosis development in mice.
We generated hepatocyte-specific deletion of ZC3HAV1 by administering adeno-associated virus encoding distinct guide RNAs targeting ZC3HAV1 into mice expressing Cas9 in hepatocytes (Alb- Cre:lox-STOP-lox-Cas9-GFP mouse). Wildtype (Zc3hav1WT) and ZC3HAV1 knockdown (Zc3hav1KD) mice were fed a high-fat diet for ten weeks to induce mild obesity and hepatic steatosis. Mice exhibited similar body composition, whole-body substrate utilisation and energy expenditure, glucose tolerance and insulin sensitivity. Using radiometric techniques in precision-cut liver slices derived from mice, we report no changes in FA oxidation from triglycerides stored in LDs in Zc3hav1KD compared with Zc3hav1WT mice. There was no difference in steatosis development or MASLD pathology as demonstrated by comparable FA uptake, storage into various lipid types and rates of de novo lipogenesis. This demonstrates that unlike in cultured cells, ZC3HAV1 deletion does not impact FA oxidation or contribute to the development of hepatic steatosis in mice, questioning its role in the pathogenesis of MASLD.