秀丽隐杆线虫持久幼虫的脂质分子基础：在进入、维持和退出持久幼虫期间，膜动态调节脂质代谢的生理意义

The proposed study aims to employ Caenorhabditis elegans (C elegans) dauer entry, survival and exit as a paradigm to investigate global regulation of lipid metabolism in response to varying nutritional and environmental inputs, with particular emphasis on the possible roles of changing membrane dynamics in influencing the storage and mobilization of fat depots. Due to the unique membrane features of C elegans, the study is expected to reveal novel insights pertaining to membrane regulation of fat depot accumulation and utilization. We observed PUFA-enriched membrane compositions in dauers that may reduce the solubility of sphingolipids and cholesterols. Accordingly, reductions in sphingomyelins (SM), ceramides (Cer), and sphingosyl PE (PECer), as well as free cholesterols (Cho) were observed in dauers compared to other larval stages. Our preliminary findings therefore suggest that dauer larva may have vastly different membrane lipid dynamics, based on their phospholipidome and sphingolipidome compositions, compared to other larval stages. The physiological significance of these PUFA-enriched membrane regions awaits further verification. We hypothesize that dauers utilize these PUFA-enriched regions to exclude sphingolipids/cholesterol-enriched domains operative in other larval stages in order to set forth an alternative array of metabolic pathways unique to dauer entry and endurance. Distinct cluster of triacylglycerides (TAGs) features comprising at least one saturated or monounsaturated fatty acyl moieties were specifically increased in dauers. The selective accumulation of these TAGs would also translate to the inhibition of saturated and monounsaturated fatty acids release in dauers. The selective hydrolysis of TAGs may have specific physiological relevance for dauer endurance, for instance, in preventing the worms from exiting the dauer state. How the worms coordinate various lipid-related enzymes in concert to orchestrate such defined patterns of TAG hydrolysis, however, warrants further investigation. The proposed study aims to obtain a global, temporal map of changing membrane dynamics during dauer entry, dauer recovery as well as prolonged dauer survival and to unravel the possible associations between specific membrane signatures with fat mobilization and/or fat storage under varying nutritional inputs. The physiological impact of these membrane signatures would be verified using RNAi of specific lipid-related genes or in relevant mutants. Research on these alternative forms of membrane regulation on fat metabolism may confer novel insights on the pathogenesis of human obesity and the metabolic syndrome; as such mechanisms may operate in a temporal- or regional-dependent manner in higher mammalian systems.

项目旨在利用秀丽线虫持久幼虫作为范例来研究应对不同营养环境的脂代谢整体调节，并强调膜动态变化可能对脂肪存储/动员的影响。我们发现与其他幼虫阶段相比，持久幼虫的膜组分富含多不饱和脂肪酸(PUFA)。相应地，持久幼虫鞘磷脂和游离胆固醇含量较少。因此， 持久幼虫有着截然不同的脂膜动态。持久幼虫很可能利用PUFA-富集区域来排除在其他幼虫阶段操作的鞘脂/胆固醇富集域，从而提供一种进入和维持持久幼虫阶段特有的代谢途径。并且，我们发现持久幼虫中含有饱和或单不饱和脂肪酸的三酰基甘油(TAG)特异增加。此项目期望通过组学途径取得在进入、恢复及延长寿命阶段持久幼虫膜动态的全面了解，并阐明在不同营养环境中特异膜特征与脂肪动员或存储之间的关联。由于线虫脂膜成分独特，此研究期望揭示关于膜调节脂肪存储和利用的新机制。这些特异的膜机制或在哺乳动物中在特定时间或区域操作，并对人类肥胖和代谢综合征的发病机理赋予新的见解。

膜调节脂肪代谢的研究可为人类肥胖和代谢综合征的发病机制提供新的见解，并揭示人类肥胖和2型糖尿病的新治疗目标。Dauers是线虫发育的休眠期，具有独特的膜脂组成，与其它线虫发育期相比，中性脂储备极为丰富。Dauers能够对其脂质储备进行定量分配，以确保甘油三酯的逐步水解，从而达到缓慢释放能量以确保持久生存，还能够在食物再供应后迅速调动其三酰甘油储存，恢复正常发育。本研究以Dauer为研究范式，研究了膜脂对脂肪动员的调节作用。采用基于液相色谱连接质谱的脂组学方伐 （包括对不同脂肪酰部分的多个反应监测转变），我们证明，与其他发育阶段相比，秀丽线虫Dauer幼虫的膜磷脂中含有独特的多不饱和脂肪酸富集。磷脂中的酯化多不饱和脂肪酸在dauer维持整个过程中呈现暂时性积累，随后在滞育终止前急剧下降。酯化的多不饱和脂肪酸的减少伴随着未结合多不饱和脂肪酸及其相应的下游氧化衍生物（即类花生酸）的增加。广泛磷脂谱图揭示，磷脂化的多不饱和脂肪酸对于维持dauer休眠非常重要，主要通过抑制类花生酸的产生；并且维持膜脂稳态的失败与dauer阶段的终止有关。目前，对从脂组学结果鉴定的脂肪代谢候选基因后续的RNAi和突变体研究工作对Dauer存活、Dauer进入和Dauer退出的影响仍然在进行中。