A Cell-Based Assay to Investigate Hypolipidemic Effects of Nonalcoholic Fatty Liver Disease Therapeutics

Abstract

In the recent past, there has been a growing interest in developing nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) therapeutics. As a result, a need for in vitro cell models of human hepatic steatosis and high-throughput assays to measure intracellular lipid levels has arisen. To address this growing need, we optimized the conditions based on the current literature to fatten HepG2 hepatocytes by adding a mixture of saturated and unsaturated fatty acids (oleate/palmitate, 2:1 molar ratio) without inducing any overt cytotoxicity. Our results indicate that hepatocytes fatten in a concentration- (0.75–1.5 mM of fatty acids) and time-dependent manner, with a substantial increase in intracellular lipid levels seen within 6 h. Additionally, a method to quantify lipid levels in cells using a fluorescent reagent that is more sensitive than that in conventional assays and adaptable for high-throughput screening is presented. Lastly, the utility of the in vitro cell model and an assay based on AdipoRed to measure hypolipidemic effects of therapeutic drugs is demonstrated using fenofibrate, a molecule that was previously shown to lower lipid levels in the liver.