探討蛋白磷酸酶參與飲食調控線蟲脂肪儲存及生長速率所扮演的角色

Abstract

食物的含量及營養組成成分是已被證實會影響生物體許多生理功能如生長速率、生殖能力、壽命長短。在我們的實驗中發現，當線蟲餵食 Comamonas sp. DA1877 比餵食 Eschericia coli OP50有較低的脂肪含量。然而，飲食如何改變線蟲的生理現象仍未被完全了解。我們和其他實驗室發現在餵食DA1877的線蟲中，S-腺苷甲硫氨酸合成酶 (S-adenosylmethionine synthetase，sams-1) 會參與在甲基化反應 (Methylation cycle) 途徑中進而影響像是生長加速及脂肪含量下降的生理現象。在酵母菌中，sams-1的產物SAM 透過甲基移轉作用反應酶 (Ppm1p) 造成蛋白磷酸酶2 (PP2Ac)的甲基化進而促進酵母菌的生長以及抑制酵母菌自噬作用的發生。而let-92是PP2Ac在線蟲的同源基因。因此，我們想要探討let-92參與在DA1877調控的脂肪含量下降的角色。我們發現線蟲餵食在兩種不同食物中，體內 PP2Ac 蛋白質表現量是相似的，但餵食DA1877食物的線蟲體內則有較高的甲基化PP2A蛋白表現量。利用RNA干擾實驗我們發現let-92對於線蟲食用DA1877線蟲所造成脂肪降低的情形是必須的。脂肪的消耗及利用主要會經由脂肪自噬作用(lipophagy) 和脂肪分解 (lipolysis)兩種途徑。我們發現自噬作用的起始在DA1877餵食的線蟲中是活化的，而且let-92 對於促進DA1877自噬作用是重要的。接下來，我們發現自噬作用相關基因，unc-51功能缺失時，餵食DA1877食物會造成線蟲體內油滴變大顆，和抑制let-92的情形類似。雖然我們發現let-92經由自噬作用促進脂肪降解， ATGL-1調控的脂肪分解 (lipolysis) 似乎並不參與在其中。另外，我們也探討了PP2A是否參與在DA1877所調控的生長加速。我們發現抑制let-92造成餵食OP50的線蟲生長速率減緩，餵食DA1877的線蟲則沒有改變其生長速度。此外，當線蟲在酵母菌中甲基移轉作用反應酶Ppm1p的同源基因B0285.4及PP2A B regulatory subunit基因，pptr-1和pptr-2並不是DA1877所造成生長加速的主因。總結以上的實驗，我們認為餵食DA1877的線蟲可能透過let-92的後轉譯修飾，促進自噬作用的進行將線蟲脂肪降解，而DA1877所造成的生長加速情形則並非經由let-92的調控。

The amount and nutritional content of diet are essential factors that have been shown to affect many organismal life-history traits, such as development rate, fertility and lifespan. In our previous studies, when fed the soil bacteria Comamonas sp. DA1877, C. elegans showed lower lipid contents compared to worms fed laboratory standard food Escherichia coli OP50. But how diets alter various physiological processes of worms are still unclear and whether they are modulated through similar component remain to be elucidated. We and other lab have shown that S-adenosylmethionine synthetase (sams-1) which participates in the methionine/S-adenosylmethionine (SAM) cycle is required to mediate several physiological processes such as developmental acceleration and lipid reduction on DA1877-fed worms. In budding yeast, SAM, the product of sams-1 promotes growth and inhibits autophagy through the action of methylatransferase Ppm1p, which modifies the catalytic subunit of protein phosphatase 2 (PP2Ac). Thus in this study, we investigated the role of PP2A in DA1877-mediated lipid reduction. We found worms have similar PP2Ac protein on both diets, but more methylated PP2Ac protein than worms fed OP50. We found let-92 is required for DA1877-mediated lipid content decrease in intestinal cells of worms. Consumption and utilization of lipid droplets could through lipophagy or lipolysis. We demonstrated the initiation of autophagy is more active in DA1877-fed worms and let-92 is essential to promote the activation of autophagy. Moreover, deficiency of the autophagy-related gene, unc-51 enlarged lipid droplet sizes in DA1877-fed worms, similar to let-92 (RNAi). While let-92 promotes lipid degradation through autophagy, ATGL-1 dependent lipolysis does not be seem to play a role in this regulation. On the other hand, we investigated the involvement of PP2A in DA1877-mediated developmental acceleration. Knockdown of let-92 resulted in delayed developmental rate in OP50-fed worms than ones fed DA1877. Further, disruption of methyltransferase, B0285.4, which encodes worm homologue of Ppm1p, and PP2A B regulatory subunit genes, pptr-1 and pptr-2 does not suppress DA1877-mediated developmental acceleration. Together, based on our studies, we proposed that diet DA1877 may regulate let-92 activity through post-translational modification to promote autophagy resulting in enhanced worms lipid utilization in the worms, whereas DA1877-mediated developmental acceleration is in a let-92 independent manner.