Mechanism of cdk4 diabetes rescue in IRS2 knockout mice

项目摘要 2型糖尿病（T2D）是美国的一个主要公共卫生问题，约9.3%的美国人患有2型糖尿病。 受疾病困扰的人群。此外，8600万人患有前驱糖尿病， 影响是惊人的，十分之一的医疗保健费用用于T2D及其并发症。T2D结果 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。尽管已经很好地建立了（从啮齿动物模型）， 细胞质量的结果从增强β细胞增殖，新的研究表明，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转换的蛋白质， 尤其是Cdk 4，对于维持β细胞增殖和质量至关重要。胰岛素受体底物蛋白2 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糖尿病， 我们以前发现，在体外重新表达细胞周期蛋白D2，它激活Cdk4，挽救了细胞周期蛋白D2的丢失。 在缺乏Irs2的β细胞中增殖。因此，我们假设，表达的组成性活性， Cdk4 R24C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，Cdk 4 R24 C不仅能够完全拯救β细胞质量，而且能够完全拯救β细胞质量。 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， 许多作用独立于其在细胞周期中的已知活性。因此，本提案的目标是 确定这种拯救背后的机制，并确定cdk 4在β细胞中发挥的非典型作用。在 目的1，我们将确定Cdk4如何拯救β细胞增殖，重点是经典的Cdk4-Rb- E2F途径，并将使用BioID识别β细胞中的新型Cdk4相互作用物。在目标2中，我们将 确定Cdk4 R24C是否使用两种胰岛灌流挽救Irs2 KO胰岛中的第1或第2时相胰岛素分泌 和高血糖钳夹研究。我们还将进行分子研究，以确定KATP酶是否 Kir6.2，先前报道为Cdk4-Rb-E2F1通路的靶标，增加并且是 足以挽救Irs2 KO胰岛中的胰岛素分泌。最后，在目标3中，我们将探索Cdk4 R24C如何能够 来恢复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，它们失去了分化标记，我认为最有可能的是， Cdk4的作用与其细胞周期作用无关。我将研究Cdk4如何拯救 Pdx 1表达，重点是FoxO1和PPAR γ，这两种转录因子调节Pdx 1表达。 使用计算机分析和阅读主要文献，我发现两者都包含Cdk 4共识 磷酸化位点。因此，我将确定Cdk4是否通过其中一个或两个来维持β 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， 这可能导致在T2D中保留β细胞质量、功能和分化的更好的治疗选择。 !

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. !