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

项目摘要/摘要 肠内分泌细胞(EECS)协调多种信号网络以维持代谢稳态。 作为肠道上皮的一种罕见的分泌细胞系，EECs通过释放一种细胞因子来感知和响应腔刺激。 控制营养感知、食欲、血糖调节和能量平衡的多种激素。节食- 诱导性肥胖和减肥手术与这些细胞的失调和恢复有关。 分别是荷尔蒙途径。此外，越来越多的药理策略已经出现 针对关键的EEC信号通路治疗代谢性疾病。然而，尽管有这些进展，分子 调控EEC生物学的机制仍未完全确定。为了解决这一知识差距，这项提议 旨在确定EEC富含的microRNA(MiRNA)miR-375在调节饮食影响中的作用 以及对EEC生物学的外科干预。MiRNAs是一种短的、非编码的RNA分子，它对 改变环境背景，并在转录后水平上调节基因表达。因此， MiRNAs是多种生物途径的关键调节者，包括肠上皮发育和 功能。我们的实验室先前已经证明miR-375在肠道干细胞(Isc)中高度丰富。 并且沿着EEC谱系，其表达在慢性高脂饮食中显著减少。此外，我们的 初步数据显示，减肥手术后ISCs中miR-375的表达显著恢复， 与EEC丰度和循环肠道激素水平的增加不谋而合。因此，我假设 MIR-375在饮食诱导的EEC的发病和改善中对EEC生物学的上下文特异性作用 肥胖。拟议的研究将通过使用我们实验室的跨学科方法来验证这一假设 已建立的miR-375基因敲除(375-KO)小鼠群体以及尖端基因组和生物信息学 技巧。在目标1中，我将评估miR-375的丢失如何对 通过对Small进行高分辨率单细胞RNA测序(scRNA-seq)来区分不同的EEC亚型 来自野生型(WT)和375-KO动物的肠腺和绒毛，喂食长期食物或高脂肪食物。在……里面 目标2，我将确定miR-375如何在手术诱导的EEC适应和代谢中发挥作用 通过对饮食诱导的肥胖WT和375-WT的隐窝和绒毛样本的scRNA-seq分析来改进 减肥手术或控制性手术后的KO小鼠。有了这些单细胞数据集，我将在生物信息学上 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(包括识别候选miR-375靶点)。我 还将通过免疫组织化学分析和代谢在体内验证这些分子发现 体重和糖耐量等参数。综上所述，这些发现将加深我们对 并可能为肥胖及其合并症的治疗提供新的治疗靶点。

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.