Alpha7 Nicotinic Acetylcholine Receptor Regulation of Glucagon-Like Peptide- 1 Incretin Hormone Action.

本项目的总体目标是建立糖调节的系统生理学，其中α7烟碱型乙酰胆碱受体(α7nAChR)调节胰升糖素样肽-1(GLP-1)的胰岛素激素作用。这个项目可能揭示与我们对2型糖尿病(T2 DM)的理解相关的糖调节的新特征，因为我们发现GTS7nAChR激动剂GTS21刺激小鼠肠道α-1的分泌，提高循环中的GLP-1水平，并改善糖耐量。因此，我们提出了α7nAChR在控制葡萄糖动态平衡中的一个新的和重要的作用，这是因为它能够在器官间控制GLP-1的分泌和作用。为了验证这一假设，我们的目标如下：目标1：我们将使用Cre/Lox技术结合基因敲除或敲入技术来确定α7nAChR激动剂如何降低健康小鼠或糖尿病db/db和ob/ob小鼠模型的血糖水平。第一个目标是评估组织特异性α7nAChR基因敲除小鼠的糖调节基线变化，同时也评估α7nAChR激动剂的作用是如何改变的。第二个目标是确定哪些细胞类型表达α-1受体(GLP-1R)，该受体介导GLP7nAChR激动剂的糖调节作用。存在四种可能性：1)当GTS-21与DPP-4抑制剂联合使用时，小鼠血液中的GLP-1浓度将达到高水平，从而使GLP-1作用于胰腺β细胞GLP-1R，促进胰岛素的分泌；或2)GTS-21由L细胞释放的GLP-1可能在肠壁发挥局部作用，启动刺激胰岛素分泌的神经反射；或3)GTS-21可能作用于后脑孤束核，刺激GLP-1的释放，从而改善葡萄糖稳态。(4)GTS-21可刺激胰腺α细胞释放GLP-1，从而使胰岛内GLP-1在β细胞GLP-1R上发挥旁分泌激素作用。目的2：我们将进行体外研究，以测试GTS-21是否仅作用于L细胞，或者是否也作用于可能表达α7nAChR的其他类型的肠内分泌细胞。例如，GTS-21可能通过刺激K细胞释放GIP来降低血糖水平，从而使GIP作用于β细胞GIP受体来刺激胰岛素的分泌。潜在地，GIP的这种作用将被DPP-4抑制剂增强。由于肽YY(PYY)是从L细胞中与GLP-1共分泌的，我们可能会发现它的释放也受到GTS-21的刺激。这将具有重要意义，因为PYY对于抑制肥胖相关的T2 DM患者的食物摄入量非常重要。最后，我们将测试GTS-21在L细胞和胰腺α细胞中刺激GLP-1生物合成的作用。这种可能性的存在是因为我们发现GTS-21上调了前激素转换酶(PC1/3)的表达，该酶可以从前胰高血糖素中释放GLP-1，同时也上调了GPR119的表达，GPR119是一种刺激胰高血糖素基因转录的GPCR。确定胰岛中的α7nAChR是否调节PC1/3和GPR119的协同表达，从而使α细胞在T2 DM条件下获得分泌GLP-1的能力，将是一个特别有趣的问题。摘要：我们的长期目标是建立α7nAChR控制下的糖调节系统生理学。

The overall goal of this project is to establish the systems physiology of glucoregulation in which alpha 7 nicotinic acetylcholine receptors (α7nAChR) regulate the incretin hormone action of Glucagon-Like Peptide-1 (GLP-1). This project may reveal novel features of glucoregulation that are of relevance to our understanding of type 2 diabetes mellitus (T2DM) since we find that the α7nAChR agonist GTS-21 stimulates intestinal GLP-1 secretion, raises circulating levels of GLP-1, and improves glucose tolerance in mice. Thus, we propose a novel and important role for the α7nAChR in the control of glucose homeostasis by virtue of its ability to exert inter-organ control over GLP-1 secretion and GLP-1 action. To test this Hypothesis, our aims are as follows: Aim 1: We will use Cre/lox technology in combination with gene knockout or knock-in technology to determine how α7nAChR agonists lower levels of blood glucose in healthy mice or db/db and ob/ob mouse models of diabetes. A first goal is to evaluate baseline alterations of glucoregulation in tissue-specific α7nAChR knockout mice, while also assessing how α7nAChR agonist action is modified. A second goal is to identify which cell types express the GLP-1 receptor (GLP-1R) that mediates glucoregulatory actions of α7nAChR agonists. Four possibilities exist: 1) when mice are administered GTS-21 in combination with a DPP-4 inhibitor, the concentration of GLP-1 in the blood will reach high levels so that GLP-1 will act at the pancreatic β-cell GLP-1R to enhance insulin secretion, or 2) GLP-1 released from L-cells in response to GTS-21 might exert a local effect in the intestinal wall to initiate neural reflexes that stimulate insulin secretion, or 3) GTS-21 might act in the hindbrain nucleus tractus solitarius to stimulate GLP-1 release so that glucose homeostasis is improved, or 4) GTS-21 might stimulate GLP-1 release from pancreatic α-cells so that intra-islet GLP-1 will exert a paracrine hormone action at the β-cell GLP-1R. Aim 2: We will perform in vitro studies to test if GTS-21 acts exclusively at L-cells, or if it also acts at other types of enteroendocrine cells that may express the α7nAChR. For example, GTS-21 might lower levels of blood glucose by stimulating the release of GIP from K-cells so that GIP will then act at the β-cell GIP receptor to stimulate insulin secretion. Potentially, this action of GIP will be enhanced by a DPP-4 inhibitor. Since Peptide YY (PYY) is co-secreted with GLP-1 from L-cells, we may find that its release is also stimulated by GTS-21. This would be significant because PYY is important to the suppression of food intake in obesity-related T2DM. Finally, we will test for actions of GTS-21 to stimulate GLP-1 biosynthesis in L-cells and pancreatic α-cells. This possibility exists because we find that GTS-21 upregulates expression of a prohormone convertase (PC1/3) that liberates GLP-1 from proglucagon, while also upregulating expression of GPR119, a GPCR that stimulates glucagon gene transcription. It will be especially interesting to determine if the α7nAChR in pancreatic islets regulates coordinate expression of PC1/3 and GPR119 so that α-cells acquire the ability to secret GLP-1 under conditions of T2DM. Summary: Our long-term goal is to establish the systems physiology of glucoregulation under α7nAChR control.