Triglyceride/Cholesterol Ester Ratio Encodes Lipid Droplet Size and Diversity

Abstract Lipid droplet (LD) heterogeneity is a hallmark of their pathophysiological relevance. This is especially evident when cells sequester toxic cholesterol by converting it into cholesterol esters (CEs) within LDs. Since CEs can form liquid crystals (LCs), it remains unclear how such ordered structures are accommodated within the inherently dynamic environment of LDs. Here, we show that the fluidizing properties of triglycerides (TGs) help CE incorporation and influence LD growth and heterogeneity. Seipin, the key regulator of TG-LD formation and size, does not significantly impact CE-rich LD size. Instead, the CE/TG ratio, the sequence of neutral lipid deposition, and the activity of diacylglycerol acyltransferases, especially DGAT2, determine whether LDs enlarge, remain fluid, or lock into LC phases. We found that the LC phase resists LD ripening and acts as a kinetic barrier to lipid entry. Lastly, we observe that perilipins, the most abundant LD surface proteins, differentially target CE-rich LDs: Plin3 and 5 are excluded from them, whereas Plin2 and 4 are favored. These findings highlight the CE/TG ratio as a key organizing principle of lipid storage and LD function, with immediate relevance for diseases linked to sterol accumulation.