Optical photothermal infrared imaging of fatty acid esterification in the ER of living cells

Lipotoxicity is an accumulation of lipids that leads to cell death and metabolic disease. Saturated fatty acids are more likely to cause lipotoxicity, however, the mechanism remains unclear due to challenges visualizing reactions in live cells. Here, we use optical photothermal infrared (OPTIR) microspectroscopy to investigate palmitic acid (PA) metabolism in hepatocytes with sub-micron spatial resolution. Upon PA feeding, we discover a time-dependent ester carbonyl stretch localized to the ER and lipid droplet-ER contact sites of lipid droplets with abnormal morphology. This stretch is assigned to diacylglycerol intermediates in the glycerol-3-phosphate pathway. C-D stretches of deuterated PA provide complementary molecular details, supporting a model whereby PA acyl chain packing in the ER reduces enzyme diffusion slowing PA metabolism. Our results provide a deeper understanding of how phase changes induced by high melting temperature fatty acids and their metabolites change ER chemistry as well as provides a tool for detecting chemical and environmental changes associated with lipotoxicity in live cells. TeaserSub-micron IR imaging of palmitic acid metabolism in live cells reveals diacylglycerol buildup and gel phase changes in the ER.