A new cholesterol diet protocol for zebrafish reveals hypercholesterolemia and fasting-associated hepatic steatosis

Zebrafish are an ideal model organism to study lipid metabolism and to elucidate the molecular underpinnings of human lipid-associated disorders. In this study, we provide a protocol to assay the impact of a high-cholesterol diet (HCD) on lipid deposition and lipoprotein regulation. Fish fed HCD developed hypercholesterolemia as indicated by significantly elevated ApoB-containing lipoproteins (ApoB-LP) and increased plasma levels of cholesterol and cholesterol esters (CE). Feeding of the HCD was also associated with a fatty liver phenotype presented as severe hepatic steatosis in zebrafish larvae after feeding the diet for 8 days, followed by fasting for 24 hours. Additionally, 30 percent of all adult female fish were observed to develop hepatic steatosis after feeding HCD for 2 weeks, followed by 3 days of fasting. The HCD feeding paradigm produced elevated levels of liver triacylglycerol (TG), free cholesterol, and CE compared to fish fed the control diet. The doubling of liver TG was striking because our HCD was only supplemented with cholesterol. The accumulated TG in the liver was not likely due to increase de novo lipogenesis or inhibited beta-oxidation since we identified no differentially expressed genes in these pathways between the livers of fish fed the HCD and the control diets. However, fasted HCD fish had significantly increased expression of the lipogenesis gene fasn in adipose tissue and higher free fatty acids (FFA) in plasma. This suggested that elevated dietary cholesterol resulted in lipid accumulation in adipocytes which supplied more FFA during fasting, promoting hepatic steatosis. In conclusion, our HCD zebrafish protocol represents an effective and reliable approach for studying the temporal characteristics of the physiological and biochemical responses to high levels of dietary cholesterol and provides insights into the mechanisms that may underlie fatty liver disease.