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: 10472510
• 负责人: Daniela Barraza
• 金额: $ 3.55万
• 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/10472510
• 关键词: Acetates; Antibiotics; Bypass; Calcium; Calcium Signaling; Cells; Data; Development; Diabetes Mellitus; Diet; Dietary Supplementation; Drosophila genus; Drosophila melanogaster; Ecdysterone; Ensure; Enteroendocrine Cell; Exhibits; Exocytosis; Exocytosis Induction; Fluorescence; Fluorescence Microscopy; G-Protein-Coupled Receptors; Genes; Genetic Transcription; Homeostasis; ITPR1 gene; Imaging Techniques; Impairment; Insecta; Intestines; Laboratory Finding; Ligands; Link; Lipids; Maintenance; Mediating; Metabolic Diseases; Metabolism; Morbidity - disease rate; Mutation; Nutrient; Obesity; Orphan; Outcome; Pathway interactions; Peptide Synthesis; Peptides; Pharmaceutical Preparations; Phenotype; Physiological Processes; Predisposition; Process; Production; Public Health; Publishing; Quantitative Reverse Transcriptase PCR; Regulation; Regulatory Pathway; Reporting; Repression; Role; Satiation; Signal Pathway; Stains; Stimulant; Stroke; Supplementation; Tachykinin; Testing; Therapeutic; Visualization; Volatile Fatty Acids; Work; calmodulin-dependent protein kinase II; cardiovascular disorder risk; cell type; detection of nutrient; differential expression; experimental study; fly; genetic manipulation; gut microbiota; in vivo; innovation; insight; interest; intestinal homeostasis; knock-down; microbiota; model organism; mortality; mutant; neurotransmitter release; novel; novel therapeutic intervention; novel therapeutics; nutrient absorption; overexpression; preservation; response; steroid hormone; treatment strategy; unpublished works

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.

抽象的 大量证据表明，感知肠腔中的营养物质并做出反应的能力可以 确定肥胖和糖尿病等代谢性疾病的倾向。肠内分泌细胞（EEC） 尽管它是肠道中数量最少的细胞类型，但仍负责此功能。回应 EEC 合成并分泌肠内分泌肽（EEP），控制重要的肠道兴奋剂 生理过程包括饱腹感、肠道收缩和全身代谢。虽然EEP EEP 的功能已被广泛研究，EEP 合成和分泌途径之间的联系是 不太了解。我们的实验室使用果蝇（一种遗传上易于处理的模式生物）来研究 保守的肠道过程。对果蝇的初步研究建立了先前的研究之间的联系 未表征的 EEC 特异性 G 蛋白偶联受体 GPRx 和肠道脂质稳态。 具体来说，GPRx 突变果蝇在肠道中表现出脂滴积累，尽管 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。已发表 研究表明 GPRx 和钙 (Ca2+) 介导的胞吐作用之间可能存在联系，并且进一步 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 众所周知，它可以促进神经递质的产生和释放，并且在相关昆虫物种中，它被证明可以 响应果蝇类固醇激素 20-羟基蜕皮酮 (20E)。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 的共同调节 转录和发布。首先，我将测试 GPRx 通过 Ca2+- 调节 EEC 中 Tk 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 EEC 中钙瞬变的影响。我还将评估胞吐诱导剂绕过 GPRx 的能力 并使用 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 特别感兴趣的是醋酸盐和 20E，因为除了调节 Tk 转录和激活 CaMKII 分别可以挽救在抗生素处理的果蝇中观察到的脂质积累表型 当添加到苍蝇饮食中时。因此，我将首先确定乙酸盐和20E是否调节GPRx表达以及 然后，通过基因操作，评估乙酸盐可用性对 GPRx 维持能力的影响 肠道脂质稳态。最后，我将测试 GPRx 在醋酸盐和 20E 介导的救援中的作用 抗生素处理的果蝇肠道脂质稳态。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) 为调节提供新的见解 EEP表达与分泌之间的联系。因此，研究结果可能会为创新提供信息 治疗代谢性疾病的治疗策略。