Testing direct effects of reproduction on lifespan with controlled feeding in grasshoppers

 描述(由申请人提供)：通过延缓衰老和延缓与年龄有关的疾病的发病，可以增加健康跨度。最近对线虫的研究开始揭示减少繁殖如何延长寿命。同时，减少喂食(即限制饮食)也可以延长寿命。摄食率 没有报告减少繁殖时蠕虫的数量，而且可能无法可靠地测量。繁殖减少会间接减少摄食量。因此，关于减少繁殖如何延长蠕虫寿命的研究可能会因为无意中减少摄食而混乱。本项目将使用一种大型、低成本的无脊椎动物来测试减少繁殖如何在不依赖日常喂养的情况下延长寿命。具有不同繁殖水平的蝗虫(卵巢切除或假手术)，但在匹配的每日摄食率下，将被用来确定由于繁殖减少而直接延长寿命的机制。由于卵巢切除后喂食量减少，每天喂食等量的假手术动物将受到饮食限制。这种蝗虫的脂肪身体和大脑的转录本已经获得，这使得许多基因的mRNA定量和RNAi敲除成为可能。通过这种方式，我们将研究两条已知的影响寿命的途径。对于每一种，我们首先比较了卵巢切除和全饮食动物以及假手术和饮食限制动物和匹配喂养的动物中途径成分的mRNA水平。其次，RNAi将被用来敲除该通路，以确定它是否是卵巢切除后延长生命所必需的。目的1以雷帕霉素(TOR)途径为靶点，检测细胞生长。TOR基因敲除可延长蠕虫、苍蝇和小鼠的寿命。我预测TOR途径的组成部分将在延长生命的饮食限制下减少，但在延长卵巢切除后不会减少。接下来，我预测，TOR基因敲除后的寿命延长与卵巢切除的寿命延长是相加的，而不是饮食限制。目的2将研究神经肽F/Y刺激摄食的作用。神经肽F/Y参与小鼠的寿命延长，但可能不是通过改变生长途径。我预测神经肽F的mRNA水平在饮食限制后会增加，但在卵巢切除后不会增加。神经肽F的基因敲除预计会延长寿命，并与卵巢切除相加，但不会限制饮食。此外，我还将测试神经肽F是否能减少生物分子损伤(蛋白质羰基、脂质过氧化)。一项新的目标3将通过限制饮食来测试半胱氨酸产生气态硫化氢在延长寿命中的作用。最近证明，硫化氢对于限制饮食对小鼠的健康影响是必不可少的。我将测试摄入半胱氨酸的氧化和分配的变化是否与硫化氢产生的变化有关。我预测减少饮食会使硫化氢含量升高，但生殖减少时不会。了解生殖减少是如何延长寿命的，可能会揭示药物开发的另一个目标，即改善人类健康寿命。与R15计划一致，学生将参与所有数据收集和展示。

 DESCRIPTION (provided by applicant): Increased health span could be accomplished by slowing aging and delaying the onset of age-related diseases. Recent work in C. elegans worms is beginning to reveal how reduced reproduction may extend lifespan. At the same time, reduced feeding (i.e., dietary restriction) also is well known to extend lifespan. The feeding rate of worms upon reduced reproduction was not reported, and it may not be able to be measured reliably. Reduced reproduction can indirectly reduce feeding. Hence, studies on how reduced reproduction extends lifespan in worms may be confounded by inadvertent reductions in feeding. The present project will use a large, low-cost invertebrate to test how reduced reproduction extends lifespan, independent of daily feeding. Grasshoppers with different levels of reproduction (ovariectomized or sham-operated) but on matched daily feeding rates will be used to identify mechanisms of life-extension directly due to reduced reproduction. Because feeding is reduced upon ovariectomy, the sham-operated animals fed the same amount daily will be on dietary restriction. Transcriptomes for fat body and brain for this grasshopper have been acquired, making possible mRNA quantification and RNAi knockdown of many genes. In this way we will examine two pathways known to effect lifespan. For each, we first compare mRNA levels of pathway components in ovariectomized & full diet animals and sham-operated & dietary restricted animals with matched feeding. Second, RNAi will be used to knockdown the pathway to determine if it is required for life-extension upon ovariectomy. Aim 1 will test cellula growth by the Target of Rapamycin (TOR) pathway. Knockdown of TOR extends lifespan in worms, flies, and mice. I predict that components of the TOR pathway will be reduced upon life-extending dietary restriction, but not upon life-extending ovariectomy. Next, I predict that life-extension upon TOR knockdown be additive with life-extension by ovariectomy, but not dietary restriction. Aim 2 will examine the role of the stimulation of feeding by neuropeptide F/Y. This peptide is involved in life-extension in mice, but perhaps not via altering growth pathways. I predict that mRNA levels for neuropeptide F will increase upon dietary restriction, but not upon ovariectomy. Knockdown of neuropeptide F is predicted to increase lifespan and be additive with ovariectomy, but not dietary restriction. In addition, I will test whether neuropeptide F reduces bio-molecular damage (protein carbonyls, lipid peroxidation). A new Aim 3 will test the role of production of gaseous hydrogen sulfide from cysteine in life-extension by dietary restriction. H2S has recently been shown to be essential for the salubrious effects of dietary restriction in mice. I will test whether changes in oxidation and allocation of ingested cysteine are associated with altered H2S production. I predict H2S will be higher upon reduced diet, but not upon reduced reproduction. Understanding how reduced reproduction increases lifespan may reveal an additional target for drug development toward improving human health span. Consistent with the R15 program, students will be involved in all data collection and presentation.