新型RNA结合蛋白丙酮酸羧化酶调控胰岛β细胞增殖的作用及机制研究

Type 2 diabetes mellitus (T2DM) and its complications are becoming a major global health burden. β-cell proliferation and compensatory hyperplasia is an important mechanism under the pathological conditions, including insulin resistance, and contributes to maintaining the glucose homeostasis. It is an important potential therapeutic target for diabetes to promote the proliferation of existing β cell, increase the levels of endogenous insulin level, and protect against hyperglycemia. Although β cell proliferation dysfunction 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. Pyruvate carboxylase (PC) is one of the key enzymes from Krebs cycle in mitochondrial, thereby regulates insulin secretion in β cell. Apart from these established roles, recently, we showed that PC is activated in β-cell of high fat diet induced obese mice and female pregnant mice under the insulin resistant condition. Overexpression of PC may induce β cell proliferation, and glucose induced β cell proliferation may blocked by PC knockdown in MIN6 β cell. Such a defect is perhaps due to aberrant effect of PC to MDM2-p53 signaling pathway, especially on the mature MDM2 mRNA level. Based on these findings, we proposed that that a tight control of the PC and its downstream MDM2-p53 axis is important for maintaining β-cell proliferation function. In the early stage of insulin resistance, short term of metabolic stress (eg. obesity or nutrient overload) may induce PC, suppress p53 expression via MDM2, thereby promote β cell proliferation. However, defection of PC may cause β-cell proliferation dysfunction, and result in diabetes in the condition of insulin resistance. In this project, we will test whether genetic deletion of PC in β-cells results in an impaired adaptive β cell proliferation, leads to decreased β cell mass, and causes the onset of severe diabetes phenotype in insulin resistance animal model. In addition, we will investigate the role of PC on β cell proliferation by modulating a novel PC downstream MDM2-p53 signaling pathway. We will also investigate whether inactivation of p53 in β-cells can alleviate PC deletion induced deleterious effects on β-cell proliferation function. The results will provide new insight into the fundamental mechanisms of β-cell function and regulation of β cell mass, 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细胞增殖、代偿性增生是胰岛素抵抗状态等病理状态下维持机体糖调节稳态的重要机制。促进现有β细胞增殖，增加内源性胰岛素分泌水平是糖尿病重要的潜在治疗靶点和研究热点。我们前期研究发现，丙酮酸羧化酶（PC）在高脂饮食，孕期等胰岛素抵抗状态小鼠β细胞表达上调；PC过表达促进体外β细胞增殖；PC通过上调成熟MDM2mRNA表达抑制p53蛋白表达水平；我们还建立了PC胰岛β细胞选择性敲除小鼠模型和p53胰岛β细胞选择性敲除小鼠模型，并初步观察了β细胞功能和糖代谢的特征。因此，PC很可能参与β细胞增殖调控的重要机制。我们将分别从动物、细胞、分子水平，进一步明确PC对β细胞增殖功能的影响作用，确定MDM2-p53通路在其中的作用环节，以及PC作为新型RNA结合蛋白对MDM2进行转录后调控的具体机制。研究结果将阐明PC-MDM2-p53自反馈通路调控胰岛β细胞增殖功能的机制，为防治2型糖尿病提供新靶点。

丙酮酸羧化酶（pyruvate carboxylase, PC）在非小细胞肺癌等部分肿瘤细胞中具有调控细胞增殖的作用。在慢性糖脂毒性和炎症状态下，胰岛β细胞内p53蛋白水平上调，p53可通过阻滞细胞周期和诱导细胞凋亡等途径抑制胰岛β细胞增殖。p53表达水平受其上游负性调节因子鼠双微体基因（mouse double minute 2, mdm2）的严格调控。MDM2-p53信号轴在细胞增殖调控中具有重要作用，既往研究发现苹果酸酶可通过此通路参与细胞的增殖调控。我们的研究旨在探究PC对胰岛β细胞增殖的作用及其可能的分子机制。我们通过Cre-loxp系统构建β细胞选择性敲除PC小鼠（PCβKO），腹腔葡萄糖耐量试验结果提示β细胞选择性敲除PC导致高脂饮食喂养小鼠出现糖耐量异常的表型，其空腹血糖升高，空腹胰岛素水平降低。CCK-8增殖试验提示，PC可以促进INS-1E细胞/小鼠原代胰岛β细胞增殖；Ki-67免疫荧光染色等增殖水平评估提示，β细胞选择性敲除PC导致小鼠胰岛β细胞增殖水平降低；在急性β细胞增殖模型中，β-PCKO小鼠胰岛β细胞代偿性增殖能力显著降低。体外实验通过western blot，QPCR，RTPCR，RNA稳定性分析等研究，证实过表达PC后，MDM2蛋白水平增高、p53蛋白水平降低； PC调控β细胞增殖的作用可能是由MDM2介导，提示PC可能通过转录后调控机制增加MDM2 mRNA的稳定性，从而上调其蛋白表达水平，抑制p53表达。PC调控β细胞增殖的作用可能是由MDM2介导，PC可能通过转录后调控机制增加MDM2 mRNA的稳定性，从而上调其蛋白表达水平，进一步通过抑制p53水平促进β细胞增殖。通过上述研究结果提示，早期胰岛素抵抗、短期营养过剩等轻度代谢异常状态可诱导β细胞PC表达，上调MDM2进而抑制p53表达，促进β细胞代偿性增殖并维持正常胰岛素分泌水平，维持正常糖代谢。随着胰岛素抵抗状态的持续加重，长期慢性代谢应激状态导致PC表达水平降低，p53水平增高，导致β细胞增殖缺陷及胰岛素分泌水平降低的双重异常，最终造成血糖显著增高。