The role of the peripheral liver circadian clock and diurnal gut microbial mediators in the regulation of hepatic gluconeogenesis

项目总结： 代谢综合征在美国影响超过25%的成年人，由于其多因素和多因素的影响而难以治疗 复杂的自然界。葡萄糖调节失调，无论是葡萄糖水平过高还是不足，都是新陈代谢的标志。 具有短期和长期有害生理影响的综合症。肝糖异生，即 肝脏内源性葡萄糖的产生，是维持体内葡萄糖平衡的关键。 长时间的禁食。尽管糖异生作用受到多种系统的影响，但强有力的证据表明 宿主的昼夜节律和肠道微生物群提供的线索对这一过程有很大贡献。 任何一个系统的破坏都会导致肝脏糖异生的异常，尽管几乎没有机制的见解提供 解释这些后果是如何产生的，以及这两个系统是如何联系在一起的。使用小说 实验方法，我将梳理这些关系，以定义具体的作用机制 肝脏核心生物钟和肠道微生物区系在调节血糖稳态中的作用。因此，我将谈到 对代谢综合征进行有针对性的有效治疗干预的需求尚未得到满足。 我的初步数据显示，肝脏核心生物钟基因BMal1的定向缺失会导致 无特定病原体(SPF)但不是无菌患者的葡萄糖清除增加和糖异生减少 (Gf)小鼠。这种表型差异可以通过将完整的微生物群落移植到GF中来恢复 肝脏BMal1缺陷小鼠，暗示肠道微生物是正常肝脏BMal1的必要条件和充分条件 糖异生的调节。连续48小时收集的粪便微生物16S rRNA测序揭示 缺乏肝脏BMal1的小鼠表现出几乎两倍于振荡的梭状芽胞杆菌分类群，这表明 在微生物群落中，动态和功能可以反馈改变宿主的新陈代谢。这些数据让我了解到 假设肝脏糖异生是由肝脏之间的双向相互作用驱动的 特定种类的肠道微生物和代谢物的生物钟和昼夜模式。我两个都要用 SPF和GF Bm al1-Flow白蛋白-Cre转基因小鼠，其中Bm ar1仅在肝细胞中缺失。二 中心目标被提出：1)检查肝钟在转导肠道微生物线索中的中心作用 调节宿主肝脏GNG和葡萄糖代谢，以及2)确定已知的肠道微生物产品如何具有 已被证明调节肝脏时钟功能，调节时钟控制的肝脏GNG和系统血糖 监管。我将应用活体实验模型，灵知生物技术，功能元基因组分析， 和小鼠常规化实验。这一战略将填补与以下方面相关的知识空白 肠道微生物和外周生物钟之间的相互作用，以及控制每个生物钟如何 系统对肝脏的糖异生有独特的影响。这些学习是一种杰出的训练 这是我培养实验和批判性思维技能的工具，这是成为一名富有成效和 独立研究人员专注于宿主新陈代谢的微生物基础。

PROJECT SUMMARY: Metabolic syndrome affects over 25% of adults in the US and is difficult to treat due to its multifactorial and complex nature. Glucose dysregulation, either excess or deficiency of glucose levels, is a hallmark of metabolic syndrome with both short- and long-term detrimental physiological effects. Hepatic gluconeogenesis, the endogenous production of glucose in the liver, is critical for maintenance of glucose homeostasis during periods of prolonged fasting. Although gluconeogenesis is influenced by a myriad of systems, strong evidence suggests that host circadian rhythms and cues provided by the gut microbiome significantly contribute to this process. Disruption of either system leads to aberrant hepatic gluconeogenesis, although few mechanistic insights provide an explanation for how these consequences arise and how the two systems are connected. Using novel experimental approaches, I will tease apart these relationships to define the specific mechanisms of action of the hepatic core circadian clock and gut microbiota in regulating glucose homeostasis. Thus, I will address the unmet need for targeted and effective therapeutic interventions for metabolic syndrome. My preliminary data show that targeted deletion of the hepatic core circadian clock gene Bmal1 leads to increased glucose clearance and reduced gluconeogenesis in Specific Pathogen Free (SPF) but not germ-free (GF) mice. This phenotypic difference can be restored by transplantation of a full microbial community into GF hepatic Bmal1 deficient mice, implying that gut microbes are necessary and sufficient for proper hepatic Bmal1 regulation of gluconeogenesis. Microbial 16S rRNA sequencing of stool serially collected over 48 hours reveals that mice lacking hepatic Bmal1 exhibit nearly twice the number of oscillating Clostridia taxa, suggesting changes in microbial community dynamics and function could feed back to alter host metabolism. These data led me to hypothesize that hepatic gluconeogenesis is driven by bidirectional interactions between the hepatic circadian clock and diurnal patterns of specific classes of gut microbes and metabolites. I will utilize both SPF and GF Bmal1-floxed Albumin-Cre transgenic mice, where Bmal1 is deleted only in hepatocytes. Two central aims are proposed: 1) Examine the central role of the liver clock in transducing gut microbial cues that regulate host hepatic GNG and glucose metabolism, and 2) Identify how known gut microbial products that have been shown to modulate liver clock function mediate clock-controlled hepatic GNG and systemic glucose regulation. I will apply in vivo experimental models, gnotobiotic technology, functional metagenomic analyses, and murine conventionalization experiments. This strategy will fill crucial gaps in knowledge relevant to the interactions between gut microbes and peripheral circadian clocks, as well as mechanisms governing how each system imposes unique influence on hepatic gluconeogenesis. These studies serve as an outstanding training vehicle for me to develop the experimental and critical thinking skills necessary to become a productive and independent researcher focused on the microbial basis of host metabolism.