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

本项目的总体目标是建立葡萄糖调节的系统生理学，其中α 7烟碱乙酰胆碱受体（α 7 nAChR）调节胰高血糖素样肽-1（GLP-1）的肠促胰岛素激素作用。该项目可能揭示与我们对2型糖尿病（T2 DM）的理解相关的葡萄糖调节的新特征，因为我们发现α 7 nAChR激动剂GTS-21刺激肠道GLP-1分泌，提高GLP-1的循环水平，并改善小鼠的葡萄糖耐量。因此，我们提出了一种新的和重要的作用，α 7 nAChR在控制葡萄糖稳态凭借其能够发挥器官间控制GLP-1分泌和GLP-1的行动。为了验证这一假设，我们的目标如下：目的1：我们将使用Cre/lox技术结合基因敲除或敲入技术来确定α 7 nAChR激动剂如何降低健康小鼠或db/db和ob/ob糖尿病小鼠模型的血糖水平。第一个目标是评估组织特异性α 7 nAChR敲除小鼠中葡萄糖调节的基线变化，同时评估α 7 nAChR激动剂作用如何改变。第二个目标是确定哪些细胞类型表达GLP-1受体（GLP-1 R），该受体介导α 7 nAChR激动剂的葡萄糖调节作用。存在四种可能性：1）当给予小鼠GTS-21与DPP-4抑制剂组合时，血液中GLP-1的浓度将达到高水平，使得GLP-1将作用于胰腺β细胞GLP-1 R以增强胰岛素分泌，或2）响应于GTS-21从L细胞释放的GLP-1可能在肠壁中发挥局部作用以启动刺激胰岛素分泌的神经反射，或3）GTS-21可能在后脑孤束核中起作用，刺激GLP-1释放，从而改善葡萄糖稳态，或4）GTS-21可能刺激胰腺α细胞释放GLP-1，从而胰岛内GLP-1将对β细胞GLP-1 R发挥旁分泌激素作用。目标二：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α 7 nAChR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随后作用于β细胞GIP受体以刺激胰岛素分泌。DPP-4抑制剂可能会增强GIP的这种作用。由于肽YY（PYY）与GLP-1从L细胞共分泌，我们可以发现其释放也被GTS-21刺激。这将是重要的，因为PYY对抑制肥胖相关T2 DM的食物摄入很重要。最后，我们将测试GTS-21刺激L细胞和胰腺α细胞中GLP-1生物合成的作用。存在这种可能性是因为我们发现GTS-21上调了从胰高血糖素原释放GLP-1的激素原转化酶（PC 1/3）的表达，同时也上调了GPR 119（一种刺激胰高血糖素基因转录的GPCR）的表达。确定胰岛中的α 7 nAChR是否调节PC 1/3和GPR 119的协调表达，从而使α细胞获得在T2 DM条件下分泌GLP-1的能力，将是特别有趣的。我们的长期目标是建立α 7 nAChR控制下的葡萄糖调节系统生理学。

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.