pH Regulation of Fat Storage in the Nematode Intestine

描述(由申请人提供)： 缺乏Na+/H+或CI-/HCO3-交换影响了哺乳动物肠道的液体和电解质运输，但酸碱运输在营养吸收过程和脂肪代谢中的最终作用相对较少。此外，关于肠道PHI如何影响细胞信号事件对营养物质可获得性的反应，目前还知之甚少。我们假设Phi作为一个协同信使，并提供了一个代谢环境，通过它可以解释不同的细胞和跨细胞信号通路的作用。在这一应用中，我们提供了将线虫中的酸碱运输与脂肪储存和相关信号事件联系起来的初步证据，并提出如下建议：目标1涉及通过首先评估线虫中SLC4和SLC26CI-/HCO3-同源物的分布，然后使用重组蛋白表达和体内功能，使用遗传编码的pH指示剂和RNAi来表征线虫中的SLC4和SLC26CI-/HCO3-同源物。AIM 2旨在测试肠道PHI的变化如何影响调节脂肪储存的经典细胞信号过程，以及哪些细胞信号过程参与传递细胞酸中毒的表型效应。AIM 3旨在利用遗传、反向遗传和功能基因组分析来确定减弱细胞内pH的调节蛋白，以及对细胞内酸化的全球转录反应。这项提议的优势在于，它将使用一个强大的模型系统和新的试剂来监测活体的pH值，从一个独特的角度研究脂肪生物学：酸碱动态平衡。

DESCRIPTION (provided by applicant): Deficiencies in Na+/H+ or CI-/HCO3- exchange compromise fluid and electrolyte transport in the mammalian intestine, but the ultimate role of acid-base transport in nutrient uptake processes and lipid metabolism is relatively poorly understood. In addition, little is known regarding how intestinal pHi influences cell signaling events that response to nutrient availability. We hypothesis that pHi acts as a synergistic messenger, and provides a metabolic context through which the actions of diverse cellular and trans-cellular signaling pathways are interpreted. In this application, we offer preliminary evidence linking acid-base transport to fat storage and related signaling events in the nematode C. elegans, and propose the following: Aim 1 involves characterizing the SLC4 and SLC26 CI-/HCO3- homologs in C. elegans by first assessing their distribution, then examining function both in situ, using recombinant protein expression and in vivo, using a genetically encoded pH indicator and RNAi. Aim 2 is designed to test how changes in intestinal pHi affect classic cell signaling processes that regulate fat storage, and conversely what cell signaling processes are involved in conveying the phenotypic effects of cellular acidosis. Aim 3 is designed to utilize genetic, reverse-genetic, and functional genomic assays to identify regulatory proteins that attenuate intracellular pH, as well as the global transcriptional responses to intracellular acidification. The strength of this proposal is that it will use a powerful model system and novel reagents for monitoring pH in living organisms to study fat biology from a unique perspective: that of acid-base homeostasis.