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: 9975146
• 负责人: Rachel Eileen Stamateris
• 金额: $ 3.25万
• 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/9975146
• 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. !

项目概要 2 型糖尿病 (T2D) 是美国的一个主要公共卫生问题，约占美国人口的 9.3% 患有该疾病的人口。此外，8600 万人患有糖尿病前期，经济状况不佳 其影响是惊人的，十分之一的医疗保健费用花在了 T2D 及其并发症上。 T2D 结果 来自胰岛素抵抗和β细胞质量减少；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。尽管（来自啮齿动物模型）已经确定β值增加 细胞质量是β细胞增殖增强的结果，新研究表明β细胞去分化 也有助于降低β细胞功能。一些参与细胞周期G1/S转变的蛋白质， 尤其是 Cdk4，对于维持 β 细胞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 细胞质量减少而患上糖尿病，并且 我们之前发现，体外重新表达细胞周期蛋白 D2 可以激活 Cdk4，从而挽救 缺乏 Irs2 的 β 细胞的增殖。因此，我们假设组成型活性的表达 Cdk4（Cdk4 R24C）的形式可能能够挽救 Irs2 KO 小鼠的糖尿病表型。有趣的是， 初步结果表明，Cdk4 R24C 不仅能够完全拯救 β 细胞团，而且还能够完全拯救 β 细胞群。 胰岛素分泌和β细胞分化。有趣的是，最近的研究表明 Cdk4 激酶发挥作用 许多作用与其在细胞周期中的已知活性无关。因此，本提案的目标是 确定这种救援背后的机制并确定 cdk4 在 β 细胞中发挥的非典型作用。在 目标 1，我们将确定 Cdk4 如何拯救 β 细胞增殖，重点关注经典的 Cdk4-Rb- E2F 途径，还将使用 BioID 识别 β 细胞中的新型 Cdk4 相互作用因子。在目标 2 中，我们将 确定 Cdk4 R24C 是否使用两种胰岛灌注来挽救 Irs2 KO 胰岛中的第一或第二阶段胰岛素分泌 和高血糖钳夹研究。我们还将进行分子研究以确定 KATPase 是否 Kir6.2（先前报道为 Cdk4-Rb-E2F1 通路的靶标）有所增加，并且 足以挽救 Irs2 KO 胰岛中的胰岛素分泌。最后，在目标 3 中，我们将探讨 Cdk4 R24C 如何能够 恢复β细胞分化标记。这是令人惊讶和有趣的，因为它违背了数据 表明当β细胞增殖时，它们会失去分化标记，我认为最有可能的是 解释是 Cdk4 具有与其细胞周期作用无关的作用。我会调查Cdk4如何拯救 Pdx1 表达，重点关注 FoxO1 和 PPARγ，这两种调节 Pdx1 表达的转录因子。 使用计算机分析并阅读主要文献，我发现两者都包含 Cdk4 共识 磷酸化位点。因此，我将确定 Cdk4 是否通过其中之一或两者来维持 beta 细胞分化。如果 Cdk4 作为激酶发挥非典型作用，影响 β 细胞生物学的多个方面， 这可能会带来更好的治疗选择，以保留 T2D 中的 β 细胞质量、功能和分化。 ！