Monounsaturated fat prevent, whereas saturated fat promote, cholesterol crystals in cytoplasmic lipid droplets

Objective: Metabolic dysfunction associated steatohepatitis (MASH) may, in part, result from excessive storage of cholesterol in hepatic lipid droplets that transition into a crystalline state. The mechanism by which crystals form in lipid droplets of hepatocytes and other cells such as macrophage is incompletely understood. Recently, we showed cholesterol must be esterified for crystals to form in lipid droplets. As the physicochemical properties of saturated cholesteryl ester render it more susceptible to crystallize than unsaturated cholesteryl ester, we investigated whether lipid droplet crystals are composed of saturated cholesteryl ester. Method: Lipid droplet crystals were measured in cholesterol-loaded Hep3B hepatocyte-like and RAW 264.7 macrophage-like cells co-treated with vehicle, monounsaturated fat, or agents that increase cellular saturated fat level. Lipidomics was used to quantify cholesteryl ester species. We also used Fourier Transform Infrared (FTIR) spectromicroscopy on livers of mice with MASH to determine the molecular identity of crystals in hepatic lipid droplets. Results: Cholesterol loading increased cholesteryl ester content and lipid droplet crystals. Monounsaturated fat treatment (e.g., oleate) increased the ratio of unsaturated to saturated cholesteryl ester and this corresponded with a substantial reduction in lipid droplet crystals. Treatments increasing saturated fat level did not exhibit this effect. (FTIR) spectromicroscopy identified cholesteryl palmitate as the prominent molecular constituent of hepatic lipid droplet crystals in mice with diet-induced MASH. Conclusion: Hepatic lipid droplet crystals in mice with diet-induced MASH are composed of saturated cholesteryl ester and unsaturated fat intake supports the cellular capacity to sustain non-crystalline liquid disordered cholesteryl ester in lipid droplets.