Dopamine action in pancreatic islet function

本研究的长期目标是确定多巴胺信号对朗格汉斯岛激素分泌的机制和影响。血糖稳态依赖于胰岛2细胞的葡萄糖刺激胰岛素分泌（GSIS），但我们也知道非葡萄糖信号对调节胰岛素分泌很重要，并且已知许多这些信号通过g蛋白偶联受体进行交流。多巴胺通过许多g蛋白偶联受体起作用，但其在2细胞中的信号传导尚未被很好地表征。目前所有的抗精神病药物都阻断D2多巴胺受体和其他靶点，最有效的第二代抗精神病药物会引起显著的代谢副作用，导致肥胖和糖尿病的风险增加。不幸的是，关于副作用的分子机制还没有达成共识。由于所有这些抗精神病药物都能阻断D2受体，但并不是所有的药物都有显著的代谢副作用，因此人们认为这些副作用是通过与D2受体以外的分子相互作用介导的。作为这些药物的潜在靶点，2细胞几乎没有引起人们的注意，大概是因为没有已知的多巴胺能神经元终止于胰岛，也没有足够水平的多巴胺在血液中循环。然而，左旋多巴确实以适当的水平在血液中循环，2细胞含有将左旋多巴转化为多巴胺所需的分子机制。2-细胞也表达D2受体和VMAT-2，将多巴胺装载到分泌颗粒中。因此，D2受体抑制和2细胞功能障碍之间可能存在联系，但对这种联系的研究将取决于对多巴胺在2细胞中的作用的更全面的了解。基于这一认识，我们假设2细胞摄取左旋多巴，合成多巴胺并将其包装在胰岛素分泌颗粒中，刺激的多巴胺分泌通过自分泌回路下调随后的胰岛素分泌。为了验证这一假设，我们将利用最先进的活细胞成像方法，其中许多是我们实验室首创的。这些新技术使我们能够以前所未有的特异性和分辨率检查细胞内和多细胞事件。我们还开发了几种用于胰岛及其分泌产物研究的新型微流体装置，这些装置都可以与我们独特的定量成像方法相结合，为许多拟议的实验提供理想的平台。在本提案中，我们将完善和扩展我们已建立的活细胞成像方法，以解决以下四个具体目标：1)确定左旋多巴对2细胞多巴胺产生的影响，并由此调节GSIS中涉及的细胞内信号通路；2)确定2细胞内多巴胺受体激活的胞内信号通路和功能靶点；3)确定D2受体和多巴胺转运体在这些信号通路中的表达和转运的作用；4)确定各种第二代抗精神病药物对这些通路的影响。

DESCRIPTION (provided by applicant): Project Summary The long term goal of this research is to determine the mechanism and effect of dopamine signaling on hormone secretion from the islet of Langerhans. Blood glucose homeostasis depends on glucose-stimulated insulin secretion (GSIS) from islet 2 cells, but we also know that non-glucose signals are important for modulating insulin secretion, and many of these signals are known to communicate through G-protein coupled receptors. Dopamine acts through a number of G-protein coupled receptors, but its signaling in the 2-cell has not been well-characterized. All current antipsychotic drugs block D2 dopamine receptors as well as other targets, and the most effective second generation antipsychotics cause significant metabolic side-effects leading to increased risks of obesity and diabetes. Unfortunately, there is no consensus about the molecular mechanisms underlying the side-effects. Since all of these anti-psychotics block D2 receptors, but not all have notable metabolic side-effects, it has been assumed that the side-effects are mediated by interactions with molecules other than D2 receptors. The 2-cell has drawn little attention as a potential target of these drugs, presumably because there are no known dopaminergic neurons terminating in the islets, nor do sufficient levels of dopamine circulate through the blood stream. However, L-DOPA does circulate in the blood stream at appropriate levels, and 2-cells contain the molecular machinery needed to convert L-DOPA to dopamine. 2- cells also express the D2 receptor and VMAT-2, which loads dopamine into secretory granules. Thus, a link may exist between D2 receptor inhibition and 2-cell dysfunction, but investigations of this link will depend on a more complete understanding of dopamine action in the 2-cell. Towards this understanding, we hypothesize that 2-cells take up L-DOPA, synthesize dopamine and package it in the insulin secretory granules, and that stimulated secretion of dopamine acts through an autocrine loop to down-regulate subsequent insulin secretion. To test this hypothesis, we will utilize state-of-the-art live cell imaging methods, many of which have been pioneered by our laboratory. These novel techniques allow us to examine intracellular and multicellular events with unprecedented specificity and resolution. We have also developed several novel microfluidic devices for the study of pancreatic islets and their secreted products, and these can all be coupled with our unique quantitative imaging approaches to provide an ideal platform for many of the proposed experiments. In this proposal, we will refine and extend our established live cell imaging approaches to address the following four specific aims: 1) to determine the effects of L-DOPA on 2-cells in terms of the generation of dopamine and resulting modulation of the intracellular signaling pathways involved in GSIS; 2) to determine the intracellular signaling pathways and functional targets of dopamine receptor activation in the 2-cell; 3) to determine the role of expression and trafficking of the D2 receptor and the dopamine transporter in these signaling pathways; 4) to determine the effects of various second generation antipsychotic drugs on these pathways. PUBLIC HEALTH RELEVANCE: Narrative Dopamine is known to affect brain function and is a target for antipsychotic drugs, but it can also cause changes throughout the body. We are studying the relationship between dopamine and insulin. The knowledge gained from our work about how dopamine affects insulin production in the pancreas will be important for designing new diabetes treatments and for understanding the observed links between many antipsychotic drugs, weight gain, and an increased risk of diabetes.