Lipid composition dictates the rate of lipid peroxidation in artificial lipid droplets.

Cellular and organismal redox imbalance leading to the accumulation of reactive oxygen species significantly enhances lipid peroxidation (LPO). LPO is relatively well studied for phospholipid membranes and to some extent for circulating lipoproteins. However, it is rarely addressed for intracellular lipid droplets (LDs). Here we optimized an model system to investigate oxidizability of different lipid classes within artificial LDs (aLDs). To this end, aLDs were reconstructed using differential centrifugation and characterized by a variety of analytical methods. Influence of different lipid compositions on aLDs size was studied and showed opposing effects of unsaturated phospholipids (PLs), triacyclglycerols (TAGs) and cholesteryl esters (CEs). To address aLDs oxidizability, the LPO sensitive ratiometric probe BODIPY-C11 was infused into aLDs, and lipid peroxidation kinetics, upon LPO activation either by copper/ascorbate or 2,2'-azobis(2-methylpropionamidine), was followed up by fluorescence spectroscopy. Generated lipid peroxidation products were additionally identified and relatively quantified by high-resolution LC-MS/MS. It was demonstrated that lipid composition is detrimental to aLD's oxidation sensitivity. Increasing unsaturation levels in the PL monolayer or the TAG core increases oxidation sensitivity, whereas the presence of CEs in the LD core has a dual effect depending on the acylated fatty acid. Moreover, not only the total level of lipid unsaturation, but also the ratio between different lipid species was shown to play a significant role in LPO propagation. This shows that the lipid composition of aLD's determines their sensitivity to LPO. As LDs lipidome reflects and is dynamically influenced by cellular and organismal metabolic status, our findings provide an important observation linking LD lipid composition and their redox sensitivity.