Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Leipzig University, Leipzig, Germany.
Center for Biotechnology and Biomedicine, Leipzig University, Leipzig, Germany.
Free Radic Res. 2021 Apr;55(4):469-480. doi: 10.1080/10715762.2021.1898603. Epub 2021 Apr 19.
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.
细胞和机体氧化还原失衡导致活性氧（ROS）积累，显著增强脂质过氧化（LPO）。LPO 在磷脂膜方面得到了相对较好的研究，在一定程度上也在循环脂蛋白方面有所涉及。然而，对于细胞内的脂滴（LDs）却很少涉及。在这里，我们优化了一个模型系统，以研究不同脂质类别的可氧化性，这些脂质存在于人工脂滴（aLDs）中。为此，我们使用差速离心法重建了 aLDs，并通过多种分析方法对其进行了表征。研究了不同脂质组成对 aLDs 大小的影响，结果表明不饱和磷脂（PLs）、三酰基甘油（TAGs）和胆固醇酯（CEs）具有相反的作用。为了研究 aLDs 的可氧化性，我们将 LPO 敏感的比率探针 BODIPY-C11 注入 aLDs，通过荧光光谱法跟踪 LPO 激活后（由铜/抗坏血酸或 2,2'-偶氮双（2-甲基丙脒））的脂质过氧化动力学。此外，还通过高分辨率 LC-MS/MS 对生成的脂质过氧化产物进行了鉴定和相对定量。结果表明，脂质组成对 aLD 的氧化敏感性有害。PL 单层或 TAG 核中不饱和程度的增加会增加氧化敏感性，而 CE 存在于 LD 核中会根据酰化脂肪酸而产生双重影响。此外，不仅总脂质不饱和水平，而且不同脂质种类之间的比例也被证明在 LPO 传播中起着重要作用。这表明 aLD 的脂质组成决定了它们对 LPO 的敏感性。由于 LDs 的脂质组反映并受到细胞和机体代谢状态的动态影响，我们的研究结果提供了一个重要的观察结果，将 LD 脂质组成与其氧化还原敏感性联系起来。
