Label-free imaging and analysis of the effects of lipolysis products on primary hepatocytes

The increased accumulation of intracellular lipid droplets within hepatocytes is a pathologic hallmark of liver injury of various etiologies, especially non-alcoholic steatohepatitis (NASH). The dynamics, subcellular origin, and chemical composition of lipid droplets under various pathophysiologic conditions, however, remain poorly understood. We used coherent Raman microscopy and spontaneous Raman spectroscopy to monitor and analyze the formation of lipid droplets in living primary rat hepatocytes exposed to triglyceride-rich lipoprotein (TGRL) lipolysis products. After exposure to the complex fatty acid mixture released during the lipolysis process for 30 minutes, new lipid droplets rapidly appeared within hepatocytes and increased in size and number over the total observation period of 205 minutes. Raman spectroscopic analysis of individual intracellular lipid droplets before and after exposure to lipolysis products reveals that the major components of these droplets are esterified unsaturated fatty acids. We find that the fatty acid unsaturation ratio increases with droplet size. Control experiments with defined fatty acid mixtures reveal the complexity of the cellular response to assault by combinations of lipids.