MDM2-p53信号通路调控胰岛β细胞胰岛素分泌功能的机制研究

Type 2 diabetes mellitus (T2DM) and its complications are becoming a major global health burden. Defective glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells is a key feature of T2DM, and contributes to the development of hyperglycemia. Although chronic metabolic stress (eg. obesity or nutrient overload) has been shown to impair β-cell function, the underlying mechanism remains unclear. A thorough understanding of the pathogenesis of β-cell failure will help to develop more therapeutic strategies against diabetes by relieving metabolic stress in β-cells. The tumor suppressor p53 is stabilized and becomes transcriptionally active in response to genotoxic stress, thereby controlling cell cycle. Apart from these established roles, p53 has recently been shown to contribute to the development of metabolic disorders such as insulin resistance and premature aging. Recently, we showed that p53 is stabilized and activated in β-cells of obese mice with β-cell dysfunction, which is accompanied with an elevation of mouse double minute 2 (MDM2), a principle negative regulator of p53. Treatment with the MDM2 inhibitor Nutlin-3a, which stabilizes and activates p53 by disrupting the interaction between MDM2 and p53, causes defective GSIS in β-cell. Such a defect is perhaps due to aberrant tricarboxylic acid (TCA) cycle and diminished ATP production in mitochondria. Based on these results, we proposed that a tight control of the MDM2-p53 axis is important for maintaining β-cell function. Chronic metabolic stress activates p53, leading to aberrant mitochondrial functions, thereby resulting in β-cell dysfunction and T2DM. In this project, we will test whether genetic deletion of MDM2 in β-cells results in an uncontrolled activation of p53, leading to defective GSIS and glucose intolerance, prove the concept that chronic activation of p53 by deleting its negative regulator MDM2 leads to the onset of T2DM, and inactivation of p53 in β-cells can alleviate MDM2 deletion induced deleterious effects on β-cell function. In addition, we will investigate how the interplay between p53 and MDM2 fine-tune GSIS by modulating TCA cycle and ATP production via a novel p53 target pyruvate carboxylase (PC) gene. The results will provide new insight into the fundamental mechanisms of β-cell function and regulation of insulin secretion, and to shed new light on the pathogenesis of glucose dysregulation in diabetes. The unique mouse model established in this study will serve as a useful tool to identify and validate novel drug targets for the development of therapeutics to combat diabetes, one of the major chronic disease in China.

胰岛β细胞功能异常与2型糖尿病密切相关,但其调控机制尚不明确。MDM2-p53信号通路参与细胞应激和葡萄糖代谢的调控机制，迄今对其与β细胞功能的关系知之甚少。我们的预实验结果发现肥胖小鼠胰岛β细胞MDM2，p53水平显著上调；MDM2抑制剂导致β细胞p53上调，胰岛素分泌缺陷和线粒体功能异常。因此我们提出假说：MDM2-p53可能通过抑制线粒体代谢影响β细胞胰岛素分泌功能。为验证这一假说，我们将利用已建立的MDM2胰岛β细胞选择性敲除小鼠、离体胰岛、及MIN6 β细胞为研究模型，通过胰岛素分泌功能测定、线粒体功能测定、慢病毒感染系统、生物信息学等手段，从分子、细胞、组织以及动物水平等层次探讨MDM2-p53对β细胞分泌功能的调控作用， 明确MDM2-p53通过对PC转录调控影响线粒体功能的机制。本研究将从MDM2-p53这个新视点揭示调控β细胞功能的基本机制，为防治2型糖尿病提供新靶点。

在胰腺β细胞中，线粒体代谢对葡萄糖刺激的胰岛素分泌（GSIS）至关重要，然而我们对调控线粒体中间代谢产物稳态的分子机制却知之甚少。本研究显示，在小鼠中通过基因敲除或药物抑制p53的负性调控子MDM2，可激活β细胞中的p53，并损害GSIS，导致糖耐受不良。其机制是，p53激活可抑制线粒体中丙酮酸脱羧酶（PC）的表达，导致三羧酸循环的中间产物草酰乙酸和NADPH产量减少，并损害耗氧量。在缺乏MDM2的胰岛中受损的GSIS和线粒体代谢可通过恢复PC的表达逆转。在致糖尿病的情况下，小鼠β细胞中MDM2和p53表达上调，而PC减少。药物抑制p53可通过恢复PC表达来缓解糖尿病胰岛中的GSIS受损。因此，MDM2-p53-丙酮酸羧化酶信号轴可将线粒体代谢和胰岛素分泌、葡萄糖稳态联系在一起，代表了一个新的糖尿病治疗潜在靶点。

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