Identifying genes required for digestive physiology and lipid metabolism

肠道对膳食营养素的吸收是导致人类多种代谢疾病的重要过程。动物对高脂肪食物的反应需要消化组织和肠道微生物群之间的协调。肠上皮细胞（EC）是肠道的吸收细胞，其制备脂质，用于在含载脂蛋白B（Apo B）的脂蛋白中循环至远端组织。感觉肠内分泌细胞（EEC）通过钙依赖性激素释放将营养信息传递给其他细胞和组织。然而，介导EC和EEC餐后对高脂肪餐的反应的转录和信号通路尚不清楚，微生物群如何影响这些相互作用也是未知的。为了解决这些知识差距，我们的研究团队开创了斑马鱼系统，用于研究脂质代谢和宿主-微生物群相互作用。这包括（1）对消化器官脂质摄取、运输和储存成像的新方法，（2）从消失的少量材料中定量含ApoB的脂蛋白的大小和数量的第一个报告物线，（3）允许分析对膳食营养素的体内实时响应的EEC活性的荧光指示剂，和（4）用于成本有效的gnotobiotic操纵的方法。利用这些工具的高分辨率在体内成像只可能在斑马鱼幼虫，我们已经发现了一个动态的整合途径EC，EEC，和微生物餐后饮食脂质的反应。这包括EC中宿主转录因子和脂蛋白合成酶途径之间的早期餐后相互作用，以及由微生物群介导的EEC的晚期餐后适应性反应。该提案的目的是确定这些EC和EEC餐后对膳食脂质反应的分子机制，以及由微生物群控制的具体步骤。我们将测试中心假设，即微生物群促进膳食脂肪分解为脂肪酸，脂肪酸被EC吸收，导致转录程序的激活和含ApoB的脂蛋白颗粒的合成，这些脂蛋白颗粒被EEC共同感知以改变其活性。这种竞争性的更新利用了三个领域领先的实验室之间的长期合作伙伴关系，这些实验室在前一个资助期内开发了一套强大的突变体和新型转基因报告细胞系。拟议研究的预期结果预计将产生重大影响，因为它们可能导致合理操纵EC，EEC和微生物群相互作用以及对膳食脂肪的反应的新策略，可用于降低人类代谢疾病的发病率和严重程度。

Intestinal absorption of dietary nutrients is an important process contributing to the etiology of multiple metabolic diseases in humans. An animal’s response to a high-fat meal requires coordination between digestive tissues and intestinal microbiota. Enterocytes (EC) are the absorptive cells of the intestine that prepare lipids for circulation to distal tissues in Apolipoprotein B (ApoB)-containing lipoproteins. Sensory enteroendocrine cells (EEC) communicate nutrient information to other cells and tissues via calcium- dependent hormone release. However, the transcriptional and signaling pathways mediating EC and EEC postprandial responses to a high-fat meal are unclear, and how microbiota influence these interactions is unknown. To address these knowledge gaps, our research team has pioneered the zebrafish system for studies of lipid metabolism and host-microbiota interaction. This includes (1) novel methods to image digestive organ lipid uptake, transport and storage, (2) the first reporter line to quantify the size and numbers of ApoB- containing lipoproteins from vanishing small amounts of material, (3) a fluorescent indicator of EEC activity that permits analysis of in vivo real-time responses to dietary nutrients, and (4) methods for cost-efficient gnotobiotic manipulation. Leveraging these tools for high-resolution in vivo imaging only possible in the larval zebrafish, we have uncovered a dynamic integrative pathway underlying EC, EEC, and microbial postprandial responses to dietary lipids. This includes early postprandial interactions in EC between a host transcription factor and the lipoprotein synthesis enzyme pathway, and late postprandial adaptive responses by EEC that are mediated by microbiota. The objective of this proposal is to define the molecular mechanisms underlying these EC and EEC postprandial responses to dietary lipid, and the specific steps controlled by microbiota. We will test the central hypothesis that microbiota promote lipolysis of dietary fat into fatty acids that are absorbed by EC leading to activation of a transcriptional program and synthesis of ApoB-containing lipoprotein particles, which are collectively perceived by EEC to alter their activity. This competitive renewal leverages long-standing partnerships between three field-leading labs with a powerful set of mutant and novel transgenic reporter lines developed during the prior funding period. The expected outcomes of the proposed research are expected to have a significant impact because they are likely to lead to new strategies for rationally manipulating EC, EEC, and microbiota interactions and responses to dietary fat which could be used to reduce incidence and severity of metabolic diseases in humans.