Modulation of beta-cell PKA activity affects glucose homeostasis

描述(申请人提供)：在胰岛细胞中提高环磷酸腺苷(CAMP)在治疗2型糖尿病(T2 DM)方面显示出相当大的治疗潜力。升高cAMP可增强葡萄糖刺激的胰岛素分泌，促进胰岛素合成以维持胰岛素的储存，并通过影响细胞增殖和细胞存活来保护细胞质量。CAMP信号通过cAMP依赖的蛋白激酶(PKA)和EPAC两个信号分子进行转导。这个项目的目的是利用体内操纵PKA活性的小鼠模型来了解细胞PKA活性的生理作用。表达依赖Cre-Lox的固有活性PKA亚单位(CaPKA)和显性阴性的PKA亚单位(DnPKA)的小鼠品系将与在胰岛细胞中特异表达可诱导Cre重组酶的品系(MIP-Creer)杂交，以产生细胞PKA活性增强(e-caPKA)和细胞PKA活性被抑制(e-dnPKA)的小鼠。以产仔小鼠为对照的实验将解决三个具体目标：1.确定细胞中激活PKA是否促进胰岛素分泌和葡萄糖清除。2.确定细胞PKA活性降低是否会减少胰岛素分泌和细胞质量。3.确定细胞功能改变是否会影响葡萄糖敏感性。在AIMS 1和2中，10周龄时给予他莫昔芬可改变PKA活性，11周龄小鼠表现为特征。目的3是一项更长期的研究，在这项研究中，他莫昔芬将在10周龄时使用，随后出现胰岛素抵抗，或者在使用他莫昔芬之前建立胰岛素抵抗。AIMS 1和2的体内和体外研究(使用分离的胰岛)将确定PKA活性如何影响胰岛素分泌、胰岛素合成和细胞质量，以及细胞PKA活性改变在胰岛素分泌对胰岛素激素的反应中所起的作用。在AIMS 1和2中进行的研究还将确定激活细胞PKA是否有潜在的不良后果，如低血糖风险增加、逆调节反应受损或不受调节的细胞团块扩张增加。AIMS 1和2还将探索细胞PKA活性在胰岛素激素促进胰岛素分泌的作用机制中的作用。在目标3中，小鼠将被喂以高脂肪饮食或标准饮食对照饮食，以确定细胞PKA活性在胰岛素抵抗进展中的作用。这些研究将确定增加细胞PKA活性是否可以预防和/或逆转胰岛素抵抗，以及降低PKA活性是否会降低细胞活性和/或导致T2 DM发展的功能。这些实验将检验这一新的假设，即细胞功能的变化可能是导致外周胰岛素敏感性的主要因素。总体而言，这些实验将确定胰岛细胞中的PKA活性在调节细胞功能和质量方面所起的作用，以及这对全身葡萄糖处理的影响。 公共卫生相关性：环状AMP信号通路通过保留朗格汉斯胰岛的β细胞分泌胰岛素的能力和对β细胞团的专利保护，在治疗糖尿病方面显示出相当大的潜力。本项目探索了主要的cAMP信号分子在郎格汉斯胰岛细胞内PKA活性中的生理作用。该项目使用新型试剂来研究改变细胞PKA活性的生理后果。对β细胞PKA活性的操纵将提供对这一信号通路治疗潜力背后的基础科学的更好理解。它还将揭示利用这一途径治疗糖尿病的潜在陷阱。

DESCRIPTION (provided by applicant): Raising cyclic AMP (cAMP) in the islet ¿-cells shows considerable therapeutic potential in the treatment of type 2 diabetes mellitus (T2DM). Elevating cAMP potentiates glucose-stimulated insulin secretion, enhances insulin synthesis to maintain insulin stores and protects ¿-cell mass through effects upon ¿-cell proliferation and ¿-cell survival. The cAMP signal is transduced through two signaling molecules, the cAMP-dependent protein kinase (PKA) and EPAC. The aim of this project is to use in vivo mouse models that manipulate PKA activity to understand the physiological role of ¿-cell PKA activity. Mouse strains that express a Cre-Lox-dependent constitutively active PKA subunit (caPKA) and a dominant negative PKA subunit (dnPKA) will be crossed to a strain expressing the inducible Cre-recombinase specifically in the islet ¿-cells (MIP-CreER) to generate mice with increased ¿-cell PKA activity (¿-caPKA) and inhibited ¿-cell PKA activity (¿-dnPKA). Experiments controlled using littermate mice will address three specific aims: 1. Determine whether activation PKA in ¿-cells enhances insulin secretion & glucose clearance. 2. Determine whether reduced ¿-cell PKA activity decreases insulin secretion and ¿-cell mass. 3. Determine whether altering ¿-cell function affects glucose sensitivity. In aims 1 and 2 PKA activity will be altered by administering tamoxifen at 10 weeks of age and the mice characterized during the 11th week. Aim 3 is a longer term study, in which tamoxifen will be administered at 10 weeks of age and the development of insulin resistance followed, or insulin resistance will be established before tamoxifen is administered. In vivo and in vitro studies (using isolated islets) in aims 1 and 2, will determine how PKA activity affects insulin secretion, insulin synthesis and ¿-cell mass and the role that altered ¿-cell PKA activity plays in the response of insulin secretion to incretin hormones. Studies performed in aims 1 and 2 will also determine whether there are potential adverse consequences of activating ¿-cell PKA, such as increased risk of hypoglycemia, an impaired counter-regulatory response or an increased of unregulated ¿-cell mass expansion. Aims 1 and 2 will also explore the role of ¿-cell PKA activity in the mechanisms of action of incretin hormones to potentiate insulin secretion. In aim 3, mice will be fed a high fat diet or a standard chow control diet to determine the role of ¿-cell PKA activity in the progression to insulin resistance. These studies will determine whether increasing ¿-cell PKA activity can prevent and/or reverse insulin resistance and whether decreasing PKA activity reduced ¿-cell viability and/or function that leads to the development of T2DM. These experiments will test the novel hypothesis that alterations in ¿-cell function can be a major contributing factor to peripheral insulin sensitivity. Overall, these experiments will determine the role that PKA activity in islet ¿- cells play in regulating ¿-cell function and mass and the effects that this has upon whole body glucose disposal. PUBLIC HEALTH RELEVANCE: The cyclic AMP signaling pathway shows considerable potential in treating diabetes by preserving the ability of the beta-cells of the islets of Langerhans to secrete insulin and in patenting beta-cell mass. This project explores the physiological role of the main cAMP signaling molecule in ¿-cells PKA activity within the ¿-cells of the pancreatic islets of Langerhans. The project uses of novel reagents to study the physiological consequences of altering the activity of ¿-cell PKA. Manipulation of beta-cell PKA activity will provide greater understanding of the basic science behind the therapeutic potential of this signaling pathway. It will also reveal potential pitfalls of harnessing this pathway for the treatment of diabetes.