PHLPP1/2 phosphatases as new target for beta-cell-directed therapy in diabetes

1型糖尿病(T1D)和2型糖尿病(T2D)都是由于胰岛β细胞功能和/或质量下降所致。迫切需要开发治疗方法来恢复糖尿病患者的β细胞质量。细胞凋亡和产生胰岛素的β细胞功能受损是β细胞衰竭的标志，也是糖尿病的根本原因。调节β细胞凋亡和/或增强再生能力是治疗糖尿病的一种有吸引力的治疗方法。丝氨酸-苏氨酸磷酸酶(STPs)，如PHLPP1/2(PH域富含亮氨酸重复蛋白磷酸酶-1/2)，调节细胞死亡，是药物开发的潜在靶点。了解PHLPPs在β细胞死亡调控中的作用具有重要的生物学意义和临床意义。我们发现PHLPP1和PHLPP2在体外糖尿病诱生条件下的β细胞中高度上调，在人类T1D和T2D中以及在糖尿病小鼠模型中。PHLPP1/2过表达会损害β细胞的存活和功能。PHLPP1/2诱导的β细胞凋亡是通过失活AKT促生存信号和激活促凋亡的MST1激酶这两种PHLPP底物来实现的。在DFG资助的最初阶段，我们发现：(I)PHLPP、AKT和MST1形成了一个控制β细胞凋亡的自我抑制三角。(Ii)在糖尿病条件下，mTORC1超激活调节PHLPPs的上调、MST1的激活和β细胞的死亡。(Iii)在体外和体内糖尿病小鼠模型中，对PHLPP的遗传和药物抑制恢复了β细胞的存活和胰岛素的分泌。在本提案中，我们继续通过评估PHLPP在细胞中如何上调以及mTORC1是否在翻译水平上控制PHLPP来全面揭示糖尿病条件下PHLPP上调的机制。我们还将进行胰岛移植研究；将来自PHLPP1-KO小鼠和野生型对照的胰岛移植到糖尿病小鼠中，以揭示和区分胰岛的特定作用和体内PHLPP1缺失的系统代谢益处。最终，我们将研究实验室中可用的特定化学PHLPP抑制剂的实用性、药代动力学和基于MALDI的药物定位，并随后在临床前研究中测试它们在体外恢复β细胞存活和功能以及在体内恢复糖尿病小鼠模型中血糖、β细胞存活和功能的有效性。本研究的目的是进一步阐明PHLPP在胰岛中的调控和作用的细胞和分子机制，以便将我们最初非常有希望的结果推进到翻译研究中；建立以前未知的PHLPP1/2信号通路，作为β细胞治疗性药物干预的新靶点，以恢复糖尿病患者的胰岛β细胞团。

Both type 1 diabetes (T1D) and type 2 diabetes (T2D) result from a decline in pancreatic beta-cell function and/or mass. There is a critical need to develop therapeutic approaches to restore beta-cell mass in patients with diabetes. Apoptosis as well as impaired function of insulin producing beta-cell are hallmarks of beta-cell failure and the fundamental cause of diabetes. Modulation of beta-cell apoptosis and/or enhancement of regenerative capacity represent an attractive therapeutic approach to the treatment of diabetes. Serine-threonine phosphatases (STPs) such as PHLPP1/2 (PH domain leucine-rich repeat protein phosphatase-1/2) regulate cell death and serve as potential targets for drug development. Our understanding of the function of PHLPPs in beta-cell death regulation has important biological and clinical significance. We show that PHLPP1 and PHLPP2 are highly up-regulated in beta-cells under diabetogenic conditions in vitro, in human T1D and T2D and in mouse models of diabetes. PHLPP1/2 overexpression impairs beta-cell survival and function. PHLPP1/2 induced beta-apoptosis is mediated through the inactivation of AKT pro-survival signaling and the activation of pro-apoptotic MST1 kinase, two well-established PHLPP substrates. During the initial DFG-funded period, we have found: (i) PHLPP, AKT and MST1 form an auto-inhibitory triangle that controls beta-cell apoptosis. (ii) mTORC1 hyper-activation regulates PHLPPs up-regulation, MST1 activation and beta-cell death under diabetic conditions. (iii) Genetic and pharmacological inhibition of PHLPP restores beta-cell survival and insulin secretion in vitro and in in vivo mouse models of diabetes.In the present proposal, we continue to fully discover the mechanism of PHLPP upregulation under diabetic conditions by assessing how PHLPP is upregulated in the -cell and whether mTORC1 controls PHLPPs at the translation level. We will also perform islet transplantation studies; islets from PHLPP1-KO mice and wild-type controls are transplanted into diabetic mice in order to uncover and distinguish an islet specific action and a systemic metabolic benefit of PHLPP1 deletion in vivo. Ultimately, we will investigate the utility, pharmacokinetics and MALDI-based drug localization of specific chemical PHLPP inhibitors available in the lab, and subsequently test their efficacy to restore beta-cell survival and function in vitro and to normalize glycemia, beta-cell survival and function in vivo in a mouse model of diabetes within a preclinical study. The purpose of this application is to further elucidate the cellular and molecular mechanisms of PHLPP regulation and action in pancreatic islets in order to move our initial highly promising results forward into translational research; to establish the previously uncharacterized PHLPP1/2 signaling pathway as a novel target for beta-cell curative pharmacological intervention to restore a functional pancreatic beta-cell mass in diabetes.