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novel therapeutics; nutrient absorption; overexpression; preservation; response; steroid hormone; treatment strategy; unpublished works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，在肠腔中感知和响应营养的能力可以 确定肥胖和糖尿病等代谢疾病的易感性。肠内分泌细胞（EEC） 尽管是肠道中最少的细胞类型，但仍负责此功能。响应 控制重要 生理过程，包括饱腹感，肠收缩和系统性代谢。虽然EEP 功能已经进行了广泛的研究，EEP合成与分泌途径之间的连接为 理解不佳。我们的实验室使用果蝇Melanogaster（一种遗传障碍的模型生物）来研究 保守的肠道过程。果蝇的初步研究建立了以前的 肠中未表征的EEC特异性G蛋白偶联受体GPRX和脂质稳态。 具体而言，尽管没有变化 表达速素（TK）的EEC的数量，一种抑制果蝇中脂质合成的EEP。出版 研究表明GPRX与钙（Ca2+） - 介导的胞吐作用与进一步的联系 在我们的实验室中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生产和释放，在相关昆虫物种中 作用于果蝇类固醇激素20-羟基肌酮（20E）。这项工作的目的是 阐明GPRX在维持肠脂质稳态中的功能并评估TK的共同调节 转录和释放。首先，我将测试GPRX通过Ca2+ - 从EEC释放TK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。我还将评估胞吞作用诱导者绕过GPRX的能力 并挽救在GPRX突变体中观察到的脂质积累表型，并使用 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20E，因为除了调节TK转录和激活外 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 当添加到飞饮食中时。因此，我将首先确定乙酸盐和20E是否调节GPRX表达和 然后，通过遗传操纵，评估乙酸盐可用性对GPRX维持能力的影响 肠脂质稳态。最后，我将测试GPRX在乙酸盐和20E介导的营救中的作用 抗生素处理的果蝇中的肠脂质稳态。总体而言，实验结果将1）阐明 GPRX在维持肠道脂质体内稳态中的作用，2）对调节的新见解 EEP表达与分泌之间的联系。因此，发现可能会为创新提供信息 治疗代谢疾病的治疗策略。