Abstract

Introduction: Non-Alcoholic Fatty Liver Disease (NAFLD) is the main cause of chronic hepatic disease. This pathology is characterized by the accumulation of lipids in the liver in the form of lipid droplets (LDs). The number, size and composition of LDs vary greatly within cells reflecting the diversification of lipid droplet function and destiny. A LD-selective type of autophagy, called Lipophagy, has been recognized as the key mechanism for hepatic lipid degradation, which integrates the participation of macroautophagy (MA), chaperone mediated autophagy (CMA) as well as the action of neutral and acid lipases. However, the specific effects of MA and CMA activation on LD diversity has not been clarified. Materials and Methods: We developed an automated microscopy image analysis method for the evaluation of LD content, size and intensity in single cells, using the fluorescent dye Bodipy. We tested a battery of 15 small molecule activators of MA and CMA primary rat hepatocytes to analyze changes in LD populations in conditions of lipid overload. Results: Our method was able to discriminate changes in number, size and intensity of LDs in response to lipid overload and nutrient deprivation in hepatic cells. We observed significant differences in LD size and number in cells treated with different autophagy activators indicating important differences between the mechanisms of autophagy activation in lipid droplets dynamics in primary hepatocytes. Discussion: Our results indicate that pharmacologic activation of specific autophagy pathways are more relevant in LD breakdown than others in the context of lipid overload, opening an opportunity for deciphering specific molecular targets for pharmacological treatment of hepatic steatosis. Acknowledgements: Fondecyt 11161056, Proyecto FCPV Apoyo Basal AFB170004.