Molecular mechanisms mediating metabolic benefits of glucagon-like peptide-1 receptor agonists

项目总结 尽管胰升糖素样肽-1受体激动剂刺激胰岛素分泌的能力 减少卡路里摄入量已经被认识了20多年，令人惊讶的是，人们对此知之甚少 这些效应背后的分子机制。我们之前已经证明，激活 下丘脑GLP1R通过参与关键的营养感知机制减少食物摄入量，如 雷帕霉素复合体-1的机制靶点(MTORC1)因为mTORC1也是一个重要的 细胞功能的调节成分，这表明阐明GLP1R激动剂如何调节 MTORC1及其下游目标将解决关于作用机制的关键知识差距 一类重要的糖尿病和减肥药。我们已经发现了一种新的相互作用 通过临床上相关的GLP1R激动剂利拉鲁肽(LIRA)，GLP1R信号的典型靶点， CAMP依赖的蛋白激酶A(PKA)，磷酸化mTORC1调节蛋白Raptor 导致mTORC1信号的增加。我们还鉴定了转录因子缺氧- 诱导因子(HIF)作为GLP1R信号转导的靶标这是相关的，因为HIF刺激糖酵解，一 GLP1R激动剂和增加HIF的厌食和促胰岛素效应所必需的机制 下丘脑和细胞的表达减少了食物的摄入量，刺激了胰岛素的分泌， 分别进行了分析。我们还提供了初步数据显示，Lira不再减轻体重或 新型敲击小鼠的血糖水平用抗PKA的Raptor取代内源性Raptor。我们的 因此，初步数据使我们假设GLP1R-PKA-mTORC1-HIF-糖酵解轴在 下丘脑和细胞介导LIRA减轻体重(目标1)和刺激的能力 胰岛素分泌(目标2)。我们的研究进一步强调了这一点的临床意义 初步数据显示，在人类中发现的GLP1R的两个变体与 心脏代谢结果的改善和对LIRA的反应性的提高也刺激mTORC1 比野生型GLP1R信号转导程度更高。因此，我们将使用小鼠来表达这些人类 GLP1R变种，以检验Lira促进更大程度的减肥和改善血糖的假设 这些小鼠的耐受性通过增强的mTORC1信号(目标3)。我们将通过以下方式实现这些目标 利用我们在评估小鼠代谢表型方面的广泛专业知识，包括实时 胰岛和胰岛能量平衡参数及胰腺功能的测定 活体使用高血糖钳夹。我们将把这些方法应用于一套新的老鼠模型 这使我们能够调节或测量特定细胞类型中目标蛋白的表达和活性。 实现这些目标将描绘出特定的分子机制，可以用来 要么是Lira疗效的提高，要么是设计出更有效的减肥药。

PROJECT SUMMARY Although the ability of glucagon-like peptide-1 receptor (Glp1r) agonists to stimulate insulin secretion and reduce caloric intake has been recognized for over two decades, surprisingly little is known about the molecular mechanisms behind these effects. We have previously shown that activation of the hypothalamic Glp1r reduces food intake by engaging key nutrient sensing mechanisms such as mechanistic Target of Rapamycin Complex-1 (mTORC1). Since mTORC1 is also an important regulatory component of -cell function, this suggests that elucidating how Glp1r agonists regulate mTORC1 and its downstream targets will address a key knowledge gap about the mechanism of action of an important class of diabetes and obesity drugs. We have identified a novel interaction stimulated by the clinically relevant Glp1r agonist liraglutide (Lira) whereby the canonical target of Glp1r signaling, cAMP-dependent protein kinase A (PKA), phosphorylates the mTORC1 regulatory protein Raptor resulting in increased mTORC1 signaling. We have also identified the transcription factor Hypoxia- Inducible Factor (HIF) as a target of Glp1r signaling. This is relevant since HIF stimulates glycolysis, a mechanism necessary for the anorectic and insulinotropic effects of Glp1r agonists, and increased HIF expression in the hypothalamus and -cells reduces food intake and stimulates insulin secretion, respectively. We also provide preliminary data showing that Lira no longer reduces body weight or glucose levels in novel knockin mice replacing endogenous Raptor with a PKA-resistant Raptor. Our preliminary data, therefore, lead us to hypothesize that a Glp1r-PKA-mTORC1-HIF-glycolysis axis in the hypothalamus and -cells mediates the ability of Lira to reduce body weight (Aim 1) and stimulate insulin secretion (Aim 2), respectively. The clinical relevance of this is further emphasized by our preliminary data showing that two variants of the Glp1r found in humans that are associated with improved cardiometabolic outcomes and improved responsiveness to Lira also stimulate mTORC1 signaling to a greater degree than wild-type Glp1r. We will, therefore, use mice expressing these human Glp1r variants to test the hypothesis that Lira promotes greater weight loss and improved glucose tolerance in these mice via enhanced mTORC1 signaling (Aim 3). We will complete these Aims by leveraging our extensive expertise in assessing metabolic phenotypes in mice, including real-time measurements of energy balance parameters as well as pancreatic function in isolated islets and in vivo using hyperglycemic clamps. We will apply these approaches to a suite of novel mouse models that allow us to modulate or measure the expression and activity of target proteins in specific cell types. Accomplishing these Aims will delineate specific molecular mechanisms that can be leveraged towards either the improvement of the effectiveness of Lira or the design of more efficient weight-lowering drugs.