MicroRNA-375 regulation of enteroendocrine cell biology in diet-induced obesity and bariatric surgery

项目总结/摘要 肠内分泌细胞（AEC）协调多种信号网络以维持代谢稳态。 作为肠上皮的一种罕见的分泌细胞谱系，EECs通过释放一种特异性的细胞因子来感知和响应肠腔刺激。 控制营养感测、食欲、血糖调节和能量平衡的各种激素。饮食- 诱导的肥胖和减肥手术与这些功能的失调和恢复有关， 激素途径，分别。此外，出现了越来越多的药理学策略， 靶向关键EEC信号通路以治疗代谢疾病。然而，尽管有这些进展， 调节EEC生物学的机制仍然没有完全确定。为了弥补这一知识差距，本提案 目的是确定富含EEC的microRNA（miRNA），miR-375在调节饮食效应中的作用。 和手术干预的影响。miRNAs是短的非编码RNA分子， 改变环境背景并在转录后水平调节基因表达。因此，在本发明中， miRNAs是无数生物学途径的关键调节因子，包括肠上皮发育和 功能我们的实验室先前已经证明，miR-375在肠干细胞（ISCs）中高度富集， 和沿着EEC谱系，并且其表达被慢性高脂饮食显著降低。另外我们 初步数据表明减肥手术后ISC中miR-375表达的显著拯救， 与EEC丰度和循环肠道激素水平的增加相一致。因此，我假设 miR-375在饮食诱导的脑缺血的发病和改善过程中对EEC生物学发挥背景特异性作用。 肥胖拟议的研究将通过跨学科的方法使用我们实验室的 建立的miR-375敲除（375-KO）小鼠群体以及尖端的基因组和生物信息学 技术.在目标1中，我将评估miR-375的缺失如何对miR-375的分布产生饮食特异性影响。 通过对小细胞进行高分辨率单细胞RNA测序（scRNA-seq）来区分不同的EEC亚型 来自喂食慢性食物或高脂肪饮食的野生型（WT）和375-KO动物的肠隐窝和绒毛。在 目的2，我将确定miR-375如何促进神经诱导的EEC适应和代谢。 通过scRNA-seq分析来自饮食诱导的肥胖WT和375- 减肥手术或对照程序后的KO小鼠。有了这些单细胞数据集，我将从生物信息学的角度 确定总体EEC丰度、亚型分布（与 循环肠道激素水平）和基因表达（包括候选miR-375靶标的鉴定）。我 还将通过免疫组织化学测定和代谢试验在体内验证这些分子结果。 例如体重和葡萄糖耐量的参数。总而言之，这些发现将进一步加深我们对 的EEC调节，并可能提供新的治疗目标，用于治疗肥胖症及其合并症。

PROJECT SUMMARY / ABSTRACT Enteroendocrine cells (EECs) coordinate a wide variety of signaling networks to maintain metabolic homeostasis. As a rare secretory cell lineage of the gut epithelium, EECs sense and respond to luminal stimuli by releasing a diverse array of hormones that control nutrient sensing, appetite, glycemic regulation, and energy balance. Diet- induced obesity and bariatric surgery have been associated with the dysregulation and restoration of these hormonal pathways, respectively. Moreover, a growing number of pharmacological strategies have emerged that target key EEC signaling pathways to treat metabolic disease. However, despite these advances the molecular mechanisms regulating EEC biology remain incompletely defined. To address this knowledge gap, this proposal aims to determine the role of an EEC-enriched microRNA (miRNA), miR-375, in regulating the effects of dietary and surgical interventions on EEC biology. MiRNAs are short, non-coding RNA molecules that respond to changing environmental contexts and modulate gene expression at the post-transcriptional level. As such, miRNAs are critical regulators of a myriad of biological pathways, including intestinal epithelial development and function. Our lab has previously demonstrated that miR-375 is highly enriched both in intestinal stem cells (ISCs) and along the EEC lineage, and its expression is dramatically reduced by chronic high-fat diet. In addition, our preliminary data demonstrate significant rescue of miR-375 expression in ISCs following bariatric surgery, coinciding with increases in EEC abundance and circulating gut hormone levels. Therefore, I hypothesize that miR-375 exerts context-specific effects on EEC biology during the pathogenesis and amelioration of diet-induced obesity. The proposed studies will test this hypothesis through an interdisciplinary approach using our lab’s established colony of miR-375 knockout (375-KO) mice together with cutting-edge genomic and bioinformatic techniques. In Aim 1, I will assess how the loss of miR-375 exerts diet-specific effects on the distribution of different EEC subtypes by performing high-resolution single-cell RNA-sequencing (scRNA-seq) of small intestinal crypts and villi from wildtype (WT) and 375-KO animals fed either a chronic chow or high-fat diet. In Aim 2, I will determine how miR-375 contributes to surgically-induced EEC adaptations and metabolic improvements through scRNA-seq analyses of crypt and villus samples from diet-induced obese WT and 375- KO mice following bariatric surgery or a control procedure. With these single-cell datasets, I will bioinformatically determine context-dependent changes in overall EEC abundance, subtype distribution (correlated with circulating gut hormone levels), and gene expression (including identification of candidate miR-375 targets). I will also validate these molecular findings in vivo through immunohistochemical assays and metabolic parameters such as body weight and glucose tolerance. Altogether, these findings will further our understanding of EEC regulation and may provide novel therapeutic targets for the treatment of obesity and its comorbidities.