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

与果蝇肠道脂质稳态有关的 G 蛋白偶联受体的表征。

• 批准号: 10311129
• 负责人: Daniela Barraza
• 金额: $ 3.98万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10311129
• 关键词: Characterization; Protein; Coupled; Receptor; Implicated

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 Published studies suggest a possible connection between GPRx and calcium (Ca2+)-mediated exocytosis and further unpublished work in our lab pointed to a downstream role of the calcium response factor CaMKII.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.

摘要 大量的证据表明，在肠腔中感知和响应营养物质的能力， 确定易患代谢紊乱如肥胖症和糖尿病。肠内分泌细胞（EEC） 尽管它是肠道中数量最少的细胞类型，但它负责这一功能。响应于 肠内分泌肽（EEPs）是肠内分泌的重要调节因子， 生理过程包括饱腹感、肠道收缩和全身代谢。虽然EEP 功能已经被广泛研究，EEP合成和分泌途径之间的联系， 不太了解。我们的实验室使用果蝇，一种遗传学上易于处理的模式生物，来研究 保存的肠过程。对果蝇的初步研究建立了以前的 未表征的，EEC特异性G蛋白偶联受体GPRx和肠内脂质稳态。 具体而言，GPRx突变果蝇在其肠道中表现出脂滴积累，尽管在肠道中没有改变。 表达速激肽（Tk）的EEC数量，速激肽是一种抑制果蝇脂质合成的EEP。发表 研究表明GPRx和钙（Ca 2+）介导的胞吐作用之间可能存在联系， 我们实验室未发表的工作指出了钙应答因子CaMKII的下游作用。CaMKII是 已知促进神经递质的产生和释放，在相关昆虫物种中， 对果蝇类固醇激素20-羟基蜕皮激素（20 E）起反应。这项工作的目的是 阐明GPRx在维持肠道脂质稳态中的功能并评估Tk的协同调节 转录和释放。首先，我将检验GPRx通过Ca 2 +-ATP调节Tk从EEC释放的假设。 介导的胞吐作用。为此，我将采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突变体和敲低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。的 特别感兴趣的是乙酸和20 E，因为除了调节Tk转录和激活 CaMKII，分别，都可以拯救在杀虫剂处理的苍蝇中观察到的脂质积累表型 加入到苍蝇的食物中。因此，我将首先确定乙酸和20 E是否调节GPRx表达， 然后，通过遗传操作，评估乙酸盐可用性对GPRx维持 肠脂质稳态。最后，我将测试GPRx在乙酸盐和20 E介导的对细胞凋亡的拯救中的作用。 肠道脂质稳态在驱虫处理苍蝇。总的来说，实验结果将1）阐明 GPRx在维持肠道脂质稳态中的作用，2）提供了对调节 EEP表达与分泌之间的关系。因此，研究结果可能会为创新提供信息。 用于治疗代谢疾病的治疗策略。