Modulation of beta-cell PKA activity affects glucose homeostasis

描述（由申请人提供）：提高胰岛细胞中的环AMP（cAMP）在治疗2型糖尿病（T2 DM）中显示出相当大的治疗潜力。升高cAMP增强葡萄糖刺激的胰岛素分泌，增强胰岛素合成以维持胰岛素储存，并通过影响<$-细胞增殖和<$-细胞存活来保护<$-细胞质量。cAMP信号通过两种信号分子，cAMP依赖性蛋白激酶（PKA）和EPAC转导。该项目的目的是使用体内小鼠模型，操纵PKA活性，以了解细胞PKA活性的生理作用。将表达Cre-Lox依赖性组成型活性PKA亚基（caPKA）和显性阴性PKA亚基（dnPKA）的小鼠品系与在胰岛细胞中特异性表达诱导型Cre重组酶（MIP-CreER）的品系杂交，以产生具有增加的<$-细胞PKA活性（<$-caPKA）和抑制的<$-细胞PKA活性（<$-dnPKA）的小鼠。使用同窝小鼠控制的实验将解决三个具体目标：1.确定PKA激活是否能增强胰岛素分泌和葡萄糖清除。2.确定降低的<$-细胞PKA活性是否会降低胰岛素分泌和<$-细胞质量。3.确定改变细胞功能是否影响葡萄糖敏感性。在目标1和2中，PKA活性将通过在10周龄时施用他莫昔芬而改变，并且小鼠在第11周期间表征。目标3是一项长期研究，其中在10周龄时给予他莫昔芬，随后发生胰岛素抵抗，或者在给予他莫昔芬之前建立胰岛素抵抗。目标1和2中的体内和体外研究（使用分离的胰岛）将确定PKA活性如何影响胰岛素分泌、胰岛素合成和<$-细胞质量，以及改变的<$-细胞PKA活性在胰岛素分泌对肠促胰岛素激素的反应中所起的作用。在目标1和2中进行的研究还将确定激活<$-细胞PKA是否存在潜在的不良后果，例如低血糖风险增加、反调节反应受损或未调节<$-细胞质量扩增增加。目的1和2还将探讨肠促胰岛素激素增强胰岛素分泌的作用机制中细胞PKA活性的作用。在目标3中，将给小鼠喂食高脂肪饮食或标准食物对照饮食，以确定β-细胞PKA活性在胰岛素抵抗进展中的作用。这些研究将确定增加β-细胞PKA活性是否可以预防和/或逆转胰岛素抵抗，以及降低PKA活性是否会降低β-细胞活力和/或功能，从而导致T2 DM的发展。这些实验将测试新的假设，即细胞功能的改变可能是外周胰岛素敏感性的主要影响因素。总之，这些实验将确定胰岛细胞中PKA活性在调节胰岛细胞功能和质量中的作用，以及这对全身葡萄糖处置的影响。 公共卫生相关性：环AMP信号传导途径在通过保留胰岛β细胞分泌胰岛素的能力和使β细胞群专利化来治疗糖尿病方面显示出相当大的潜力。本项目探讨主要cAMP信号分子在胰岛<$-细胞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。β细胞PKA活性的操纵将提供对该信号通路的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.