Endocrine Abstracts

The incidence of non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, continues to rise. NAFLD is associated with significant liver-specific and cardiovascular morbidity and mortality, including hepatocellular carcinoma (HCC). Currently, there are no licensed therapies, highlighting the importance of understanding the pathogenic mechanisms that drive the condition. Cytochrome p450 oxidoreductase (POR) plays an essential role in activation of all microsomal cytochrome p450s (CYPs) by electron transfer. Rodent models of POR deletion develop hepatic steatosis, but the underpinning mechanisms remain poorly understood. The role of POR in human models to modulate hepatic metabolic phenotype has not been explored in detail. We have tested the hypothesis that POR contributes to NAFLD progression through dysregulation of major metabolic pathways using clinical samples from patients with NAFLD and HCC, in vivo rodent models (American Lifestyle-Induced Obesity Syndrome, ALIOS) and human hepatoma cells. In liver biopsies of NAFLD patients, relative POR mRNA expression was significantly lower compared to non-NAFLD controls (P<0.01). In addition, POR activity as measured by the analysis of urine steroid metabolites, decreased with advancing NAFLD severity (control vs F0-F2, P<0.0001; control vs F3-F4, P<0.0001). In patients with cirrhosis or HCC, POR activity was also decreased when compared to healthy controls (P<0.0001). Mice fed the ALIOS diet (12-months) developed significant hepatic steatosis and fibrosis. In both male and female ALIOS mice, POR mRNA expression was significantly decreased in comparison with normal chow-fed animals (P=0.01, FDR=0.04). In human hepatoma cells (HepG2), lipid loading was associated with decreased POR expression. To determine the cellular impact of decreased POR expression, siRNA knockdown experiments were performed. RNA-sequencing analysis combined with real-time PCR identified multiple metabolic pathways that were dysregulated. Changes in fatty acid metabolism were indicative of futile cycling with increased expression of fatty acid synthesis (AMPK, ACC, FASN) and oxidation (CPT1A) markers. These findings were endorsed with biochemical analysis demonstrating increased triacylglycerol (P<0.01) and 3-hydroxybutyrate (P=0.03) levels in cell culture media, and increased rates of de novo lipogenesis in cellular triacylglycerol (P<0.01). Moreover, there were significant changes in glucose metabolism suggesting increased gluconeogenesis and reduced glucose uptake. Finally, bile acid synthesis was impacted with increased expression of rate limiting synthetic enzymes and altered cellular bile acid production. Altered POR expression and activity has the potential to contribute to the complex metabolic phenotypes associated with NAFLD. Further studies are needed to determine whether this represents a putative cause or consequence of disease.