Mechanism of cdk4 diabetes rescue in IRS2 knockout mice

IRS2敲除小鼠cdk4糖尿病拯救机制

• 批准号: 10197110
• 负责人: Rachel Eileen Stamateris
• 金额: $ 3.3万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10197110
• 关键词: Adipocytes; Alleles; Beta Cell; CCND2 gene; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cell Differentiation process; Cell Proliferation; Cell physiology; Cells; Cellular biology; Closure by clamp; Consensus; Cyclin-Dependent Kinases; Cyclins; Data; Defect; Development; Diabetes Mellitus; Diabetic mouse; Differentiation Antigens; Disease; E2F transcription factors; Epidemic; FOXO1A gene; FRAP1 gene; Failure; Financial compensation; G1/S Transition; Goals; Healthcare; Hyperglycemia; IRS2 gene; Impairment; In Vitro; Insulin; Insulin Resistance; Investigation; Islets of Langerhans; Knock-out; Knockout Mice; Lead; Light; Link; Literature; Maintenance; Mediating; Messenger RNA; Metabolism; Molecular; Mus; Non-Insulin-Dependent Diabetes Mellitus; Nuclear; PPAR gamma; Pancreas; Pathway interactions; Peripheral; Phase; Phenotype; Phosphorylation Site; Phosphotransferases; Play; Population; Prediabetes syndrome; Process; Proliferating; Proteins; Public Health; Reading; Regulation; Reporting; Research; Retinoblastoma; Retinoblastoma Protein; Rodent; Rodent Model; Role; S Phase; Site; Structure of beta Cell of islet; Sum; Testing; Therapeutic; United States; Work; cell dedifferentiation; diabetic; economic impact; improved; in silico; in vivo; insulin secretion; insulin signaling; islet; knock-down; lipid biosynthesis; novel; overexpression; preservation; response; transcription factor; transdifferentiation

PROJECT SUMMARY Type 2 Diabetes (T2D) is a major public health issue in the United States with approximately 9.3% of the population suffering from the disease. Additionally, 86 million people have prediabetes and the economic impact is staggering, with 1 in 10 health care dollars being spent on T2D and its complications. T2D results from insulin resistance and reduced beta cell mass; thus, strategies to increase functional beta cell mass are critical goals for diabetes research. Although it is well established (from rodent models) that increased beta cell mass results from enhanced beta cell proliferation, new research suggests that beta cell dedifferentiation also contributes to reduced beta cell function. Some proteins involved in the G1/S transition of the cell cycle, especially Cdk4, are critical for the maintenance of beta cell proliferation and mass. Insulin receptor substrate 2 knockout (Irs2 KO) mice develop diabetes due to peripheral insulin resistance and reduced beta cell mass, and we previously found that in vitro re-expression of cyclin D2, which activates Cdk4, rescues the loss of proliferation in beta cells lacking Irs2. Therefore, we hypothesized that expression of a constitutively active form of Cdk4 (Cdk4 R24C) might be able to rescue the diabetic phenotype of Irs2 KO mice. Intriguingly, preliminary results suggest that Cdk4 R24C is able to completely rescue not only beta cell mass, but also insulin secretion and beta cell differentiation. Interestingly, recent studies show that the Cdk4 kinase plays many roles independently of its known activity in the cell cycle. Therefore, the goal of this proposal is to determine the mechanisms behind this rescue and determine what atypical roles cdk4 plays in the beta cell. In Aim 1, we will determine how Cdk4 rescues beta cell proliferation, focusing on both the canonical Cdk4-Rb- E2F pathway, and will also identify novel Cdk4 interactors in the beta cell using BioID. In Aim 2, we will determine if Cdk4 R24C rescues 1st or 2nd phase insulin secretion in Irs2 KO islets using both islet perifusion and hyperglycemic clamps studies. We will also perform molecular studies to determine whether the KATPase Kir6.2, which was previously reported to be a target of the Cdk4-Rb-E2F1 pathway, is increased and is sufficient to rescue insulin secretion in Irs2 KO islets. Finally, in Aim 3 we will explore how Cdk4 R24C is able to restore beta cell differentiation markers. This is surprising and interesting, since it goes against the data showing that when beta cells proliferate they lose differentiation markers, and I think the most likely explanation is that Cdk4 is having effects unrelated to its cell cycle actions. I will investigate how Cdk4 rescues Pdx1 expression, with a focus on FoxO1 and PPARγ, two transcription factors that regulate Pdx1 expression. Using in silico analyses and reading the primary literature, I found that both contain Cdk4 consensus phosphorylate sites. Therefore, I will determine whether Cdk4 acts via either or both of these to maintain beta cell differentiation. If Cdk4 plays atypical roles as a kinase to influence multiple aspects of beta cell biology, this may lead to better therapeutic options for preserving beta cell mass, function and differentiation in T2D. !

项目总结 在美国，2型糖尿病(T2D)是一个主要的公共卫生问题，约有9.3%的 患有这种疾病的人口。此外，8600万人患有糖尿病前期和经济上的 影响是惊人的，每10个医疗保健美元中就有1个花在T2D及其并发症上。T2D结果 胰岛素抵抗和减少的β细胞质量；因此，增加功能性β细胞质量的策略是 糖尿病研究的关键目标。虽然(从啮齿动物模型)已经得到了很好的证实，但增加Beta 细胞质量是由增强的β细胞增殖引起的，新的研究表明，β细胞去分化 也会降低β细胞的功能。参与细胞周期G1/S转换的一些蛋白质， 尤其是CDK4，对维持β细胞的增殖和质量至关重要。胰岛素受体底物2 基因敲除(Irs2 KO)小鼠由于外周胰岛素抵抗和β细胞质量减少而发展为糖尿病，以及 我们之前发现，在体外重新表达细胞周期蛋白D2，激活CDK4，挽救了细胞周期蛋白D2的丢失 缺乏irs2的β细胞的增殖。因此，我们假设了一种结构性活跃的表达 CDK4(CDK4R24C)有可能挽救Irs2KO小鼠的糖尿病表型。有趣的是， 初步结果表明，CDK4 R24C不仅能够完全挽救β细胞团，而且还能够 胰岛素分泌与β细胞分化。有趣的是，最近的研究表明，CDK4激酶在 许多角色独立于其在细胞周期中的已知活动。因此，这项建议的目标是 确定这种救援背后的机制，并确定CDK4在β细胞中扮演的非典型角色。在……里面 目的1，我们将确定CDK4如何挽救β细胞的增殖，集中在两个典型的CDK4-Rb- E2F途径，并将使用BioID识别β细胞中新的CDK4相互作用因子。在目标2中，我们将 确定CDK4 R24C是否能通过两种胰岛灌流挽救Irs2 KO胰岛的第一或第二时相胰岛素分泌 以及高血糖钳夹研究。我们还将进行分子研究，以确定KATPase 之前被报道为CDK4-Rb-E2F1途径靶点的Kir6.2基因被上调，并被 足以挽救Irs2KO胰岛的胰岛素分泌。最后，在目标3中，我们将探索CDK4 R24C如何能够 以恢复β细胞分化标志物。这是令人惊讶和有趣的，因为它与数据相反 表明当贝塔细胞增殖时，它们会失去分化标志物，我认为最有可能的是 对此的解释是，CDK4的作用与其细胞周期活动无关。我会调查CDK4是如何拯救 Pdx1表达，重点是FoxO1和PPARγ，这两个转录因子调节Pdx1的表达。 在计算机分析和阅读主要文献中，我发现两者都含有CDK4共识 磷酸化部位。因此，我将确定CDK4是否通过这两种方式中的一种或两种来维持beta 细胞分化。如果CDK4扮演着非典型的角色，作为一种激酶来影响β细胞生物学的多个方面， 这可能会导致更好的治疗选择，以保存T2D中的β细胞质量、功能和分化。 好了！