中国科学院昆明动物研究所机构知识库

大量研究发现，长寿突变体往往会积累更多的脂肪，有选择性的延长生物的寿命也会伴随着脂肪积累的增加，因此寿命与能量代谢之间有非常密切的联系，但是二者之间具体的调节机制仍然不清楚。胰岛素信号通路在调控寿命与能量代谢中发挥非常重要的作用，而且在秀丽线虫（Caenorhabditis elegans）到哺乳动物中非常保守。在秀丽线虫中，胰岛素受体突变体daf-2脂肪含量高同时寿命延长。DAF-2调控下游能量代谢及寿命相关靶基因依赖于叉头蛋白转录因子DAF-16。在细胞中，脂肪储存在脂滴（lipid droplets，LDs）中。脂滴是一种从酵母到哺乳动物都非常保守的球形细胞器，由单层磷脂膜包含甘油三酯和胆固醇酯构成，脂滴表面有很多脂滴蛋白。本研究将脂滴做为切入点，探讨寿命与能量代谢之间的作用机制。本研究提取纯化野生型和daf-2突变体的脂滴，分析二者脂滴蛋白的差异，共找到167个脂滴及脂滴相关蛋白表达量发生显著性变化。通过RNAi筛选，找到类固醇脱氢酶LET-767，抑制let-767能够降低daf-2突变体脂肪含量，缩短daf-2突变体寿命。脂滴蛋白组结果显示，在daf-2突变体脂滴中，LET-767表达量是野生型的三倍。实验发现，LET-767蛋白仅仅募集于daf-2突变体脂滴上。同时发现，在daf-2突变体中，LET-767总蛋白表达量增加。let-767干扰后，三酰甘油水解酶1（adipose triglyceride lipase 1，ATGL-1）的表达量增加，脂肪分解加速，从而降低daf-2和age-1的脂肪含量。ATGL-1过表达能够恢复daf-2的突变体寿命至野生型水平。let-767调节ATGL-1表达依赖于转录因子DAF-16/FoxO；同时let-767 干扰促进DAF-16转录因子的入核。综上所述，LET-767通过DAF-16转录因子调节ATGL-1的表达，调控秀丽线虫脂肪积累和寿命。目前公认脂滴起源于内质网，本论文另外一部分研究工作讨论内质网在脂滴形成中发挥的重要作用。通过EMS筛选找到kun55突变体，其脂肪含量非常低。通过经典遗传学定位结合DNA测序，确定kun55突变体的突变基因为anc-1。anc-1编码秀丽线虫KASH蛋白家族中的ANC-1，与哺乳动物中的SYNE1和SYNE2蛋白同源。ANC-1蛋白参与调节细胞核和线粒体的定位。由于ANC-1蛋白分子量高达956.5kDa，因此我们设计了原位整合的新技术，将GFP蛋白插入线虫基因组的特定位置，稳定遗传并表达，从而构建了ANC-1::GFP转基因线虫。ANC-1蛋白表达在细胞质中，在肠道细胞的细胞核周围表达较丰富。通过研究发现，在anc-1(kun55) 和 anc-1(e1873)突变体中，内质网形态严重被破坏，由正常的网状结构，聚缩成球状分布于细胞质中。大量研究显示，内质网在脂滴的形成及扩大过程中发挥非常重要的作用。研究发现，内质网形态结构被破坏后，导致脂滴标记蛋白DHS-3和DGAT-2表达及分布紊乱。因此，ANC-1通过维持内质网形态调节脂滴的形成和扩大。

 A lot of studies find that there is a very close connection between engery metabolism and longevity. From C. elegans to mammals, insulin/IGF signaling pathway plays an important role in energy metabolism and longevity conservatively. In Caenorhabditis elegans, insulin/IGF-1 receptor mutant daf-2 showed high lipid storage and extended lifespan. DAF-2 controls the target genes which regulate lifespan and lipid metabolism depended on DAF-16, a FOXO transcription factor. But the detailed mechanism between fat accumulation and longevity is still unclear. Lipid stored in cellular organelles named lipid droplets (LDs) which are conserved from yeast to human. LDs are made up of phospholipids monolayer and triglycerides (TAGs)-cholesterol ester (CE) core, and there are many proteins decorating the surface of LDs.We purified the lipid droplets of daf-2 mutant and WT worms, and then compared the surface proteins of LDs of daf-2 to WT, and identified 167 proteins have different expression level. Then we confirmed these genes by RNAi and identified LET-767(a steroid dehydrogenase) plays an important role in the regulation of fat storage. Fat content of daf-2 mutant showed significantly decreased and the lifespan of daf-2 shorted notably in let-767 RNAi background. To identify whether LET-767 localized on LDs, we purified LDs of LET-767::GFP. In daf-2 mutant, LET-767::GFP recruited on LDs. In the daf-2 LDs proteomics, expression level of LET-767 is 3 times higher than WT. The total expression of LET-767 also increased in daf-2 mutant. We find that let-767 RNAi decreased the fat accumulation of daf-2 and age-1 dependent on the high expression level of ATGL-1(adipose triglyceride lipase 1). Moreover, the lifespan of daf-2;atgl-1::GFP is shortened to WT levels. And then we found that daf-16 can rescue the let-767 RNAi induced ATGl-1 expression. let-767 RNAi can also increased the DAF-16 nuclear localization. Taken together, our results identify ATGL-1 as a critical regulator of LET-767 specifically dependent on DAF-16/FoxO in the fat accumulation and lifespan. LDs originated from the ER acknowledgedly. And another work was about the critical function of ER in the formation of LDs. From EMS screening, we identified a new mutant-kun55 that showed decreased fat content. kun55 is a new mutant of anc-1. anc-1 encodes ANC-1 that is the homology of mammalian SYNE1 and SYNE2 proteins. ANC-1 is a member of the KASH proteins family in C. elegans. ANC-1 affects the positioning of nuclei and mitochondria within the cytoskeleton. ANC-1 is huge protein that is 956.5 kDa. We designed a new technology named in-suit integration that can integrate the GFP into the specific location of the genome, and we constructed the transgenic line-ANC-1::GFP. ANC-1 localized cytoplasmically and was enriched at the nuclear periphery in the intestine.The structure of endoplasmic reticulum(ER) in anc-1(kun55) and anc-1(e1873) mutant was destroyed, from normal reticulate structure to ball structure. ER plays a very important role in the formation and expansion of LDs. The destroyed structure of ER confused the expression and location of the LDs marker protein DHS-3 and DGAT-2. So, ANC-1 affects the formation and positioning of LDs within the ER structure.