Characterization of a G Protein-Coupled Receptor Implicated in Intestinal Lipid Homeostasis of Drosophila melanogaster.

摘要 大量证据表明，感知肠腔内营养物质并对其做出反应的能力 确定肥胖和糖尿病等代谢紊乱的易感性。肠内分泌细胞(EEC) 尽管是肠道中最不丰富的细胞类型，但仍负责这一功能。作为对.的回应 肠道刺激剂，EECS合成和分泌控制重要的肠内分泌肽(EEPS) 包括饱腹感、肠道收缩和全身新陈代谢在内的生理过程。虽然EEP 功能已被广泛研究，EEP合成和分泌途径之间的联系是 人们对此知之甚少。我们的实验室使用了果蝇，一种遗传上易驯化的模式生物，来研究 保守的肠道过程。对果蝇的初步研究确定了之前的 EEC特有的G蛋白偶联受体GPRx和肠道中的脂类稳态尚未确定。 具体地说，GPRx突变果蝇在肠道中表现出脂滴堆积，尽管其肠道中的 表达速激肽(TK)的EEC数量，TK是一种抑制果蝇脂肪合成的EEP。已出版 研究表明，GPRx和钙(Ca~(2+))介导的胞吐作用之间可能存在联系，并进一步 我们实验室未发表的工作指出了钙反应因子CaMKII的下游作用。CaMKII是 已知可以促进神经递质的产生和释放，在相关昆虫物种中，它被证明 对果蝇类固醇激素20-羟基蜕皮酮(20E)的反应。这项工作的目的是 阐明GPRx在维持肠道脂质平衡中的作用并评估TK的共同调节 转录和发布。首先，我将检验GPRx通过钙离子调节EECS释放TK的假设。 介导的胞吐作用。为此，我将使用实时成像技术来确定GPRx是否具有 对EECS中钙瞬变的影响。我还将评估胞吐诱导剂绕过GPRx的能力 并挽救在GPRx突变体和击倒果蝇中观察到的脂质积累表型 荧光显微镜。此外，我计划确定触发GPRx刺激的因素(S)。的 特别感兴趣的是乙酸酯和20E，因为除了调节TK转录和激活外， CaMKII和CaMKII分别可以挽救在抗生素处理的果蝇中观察到的脂肪堆积表型 当添加到苍蝇饮食中时。因此，我将首先确定醋酸盐和20E是否调节GPRx的表达和 然后，通过基因操作，评估醋酸盐可获得性对GPRx维持能力的影响 肠道内脂平衡。最后，我将测试GPRx在乙酸盐和20E介导的救援中的作用 抗生素治疗果蝇的肠道脂平衡。总而言之，实验结果将1)阐明 GPRx在维持肠道脂质平衡中的作用和2)为调控提供新的见解 EEP的表达与分泌的关系。因此，这些发现可能会为创新 代谢性疾病的治疗策略。

Abstract Substantial evidence suggests that the ability to sense and respond to nutrients in the intestinal lumen can determine predisposition to metabolic disorders such as obesity and diabetes. The enteroendocrine cell (EEC) is responsible for this function despite being the least abundant cell type in the intestine. In response to intestinal stimulants, EECs synthesize and secrete enteroendocrine peptides (EEPs) that control important physiological processes including satiety, intestinal contractions, and systemic metabolism. Although EEP function has been extensively studied, the connection between EEP synthesis and secretion pathways is poorly understood. Our lab uses Drosophila melanogaster, a genetically tractable model organism, to study conserved intestinal processes. Preliminary studies in Drosophila established a link between the previously uncharacterized, EEC-specific G protein-coupled receptor GPRx and lipid homeostasis in the intestine. Specifically, GPRx mutant flies exhibit lipid droplet accumulation in their intestines despite no changes in the number of EECs expressing tachykinin (Tk), an EEP that represses lipid synthesis in Drosophila. Published studies suggest a possible connection between GPRx and calcium (Ca2+)-mediated exocytosis and further unpublished work in our lab pointed to a downstream role of the calcium response factor CaMKII. CaMKII is known to promote production and release of neurotransmitters and in related insect species, it was shown to act in response to the Drosophila steroid hormone 20-hydroxyecdysone (20E). The aim of this work is to elucidate the function of GPRx in the maintenance intestinal lipid homeostasis and assess co-regulation of Tk transcription and release. First I will test the hypothesis that GPRx modulates Tk release from EECs via Ca2+- mediated exocytosis. To do this, I will employ live imaging techniques to determine whether GPRx has an impact on calcium transients in EECs. I will also assess the ability of an exocytosis inducer to bypass GPRx and rescue the lipid accumulation phenotype observed in GPRx mutant and knockdown flies using fluorescence microscopy. Additionally, I plan to identify the factor(s) that triggers GPRx stimulation. Of particular interest are acetate and 20E because in addition to regulating Tk transcription and activation of CaMKII, respectively, both can rescue the lipid accumulation phenotype observed in antibiotic-treated flies when added to the fly diet. Thus, I will first determine whether acetate and 20E regulate GPRx expression and then, through genetic manipulation, assess the impact of acetate availability on the ability of GPRx to maintain intestinal lipid homeostasis. Lastly, I will test the role of GPRx in acetate- and 20E-mediated rescue of intestinal lipid homeostasis in antibiotic-treated flies. Collectively, experimental outcomes will 1) elucidate the role of GPRx in the maintenance of intestinal lipid homeostasis and 2) provide new insights into the regulatory connection between EEP expression and secretion. Therefore, findings could potentially inform innovative therapeutic strategies for the treatment of metabolic disease.