Elucidating how transcription factors MAFA and MAFB and mitochondrial activity control human β cell identity and function

项目摘要 2型糖尿病(T2D)可归因于β细胞特性的丧失或去分化，其特征是获得 不成熟的细胞标志物和胰岛素表达和分泌的丧失。而T2D中β细胞不成熟的病因 核编码的线粒体基因表达和转录因子(TF)的损伤尚不清楚 表达式发生。此外，线粒体结构和功能的缺陷会导致血糖受损- 刺激胰岛素分泌(GSI)，并在人类T2D患者的β细胞中被报道。有趣的是， 在β细胞未成熟期间发生的转录变化包括核编码的线粒体的丢失 基因表达程序。我的研究将验证这样的假设，即T2D中β细胞的不成熟是由 线粒体功能基因由转录因子A(MAFA)或B(MAFB)调控。 此外，我预测MAFA/MAFB本身就是有核串扰的逆行目标 线粒体功能缺陷引起的信号级联反应。 我将通过以下途径阐明MAFA和MAFB在人β细胞代谢控制中的作用 线粒体功能的调节(目标1)。我将确定MAFA和/或MAFB的损失如何影响 线粒体功能与β细胞特性的相关性研究 MAFb基因敲除人伪胰岛和EnDoc-βH3β细胞株。代谢组学将在恩多克- βH3β细胞株，以确定MAFA/B如何影响燃料利用率。我的初步数据显示基因缺失 丝裂原吞噬(即线粒体生物生成和周转的平衡)会降低β细胞的成熟度。这包括 生理、代谢和转录特征与代谢过载、氧化损伤和 综合应激反应(ISR)。众所周知，Mafa(可能是MAFB)对氧化应激更敏感 与T2Dβ-细胞功能障碍相关的患者比其他功能障碍患者更多。而β细胞线粒体基因的核表达 众所周知，线粒体反馈驱动β-细胞核基因表达(逆行信号)尚未 被分析过了。I将描述在线粒体功能障碍时，TF水平和β细胞成熟度是否发生改变 (目标2)。此外，我观察到，对ISR的药理抑制可以缓解胰岛中未成熟的标记。 有丝分裂缺陷的小鼠。利用药理学工具分析人类伪胰岛的基因表达 关于移植和EnDoc-βH3细胞，我将询问这些条件对人类β细胞的影响。我希望如此 由于对ISR的敏感性，MAFA(可能还有MAFB)水平将会降低。此外，我将确定是否 抑制ISR可恢复MAFa/MAFb的表达并逆转β-细胞的不成熟 线粒体受损。

Project Summary Type 2 diabetes (T2D) can be attributed to loss of β-cell identity or de-differentiation, marked by acquisition of immature cell markers and loss of insulin expression and secretion. While the etiology of β-cell immaturity in T2D is unclear, impairments in nuclear-encoded mitochondrial gene expression and transcription factor (TF) expression occur. Additionally, defects in mitochondrial structure and function leads to impaired glucose- stimulated insulin secretion (GSIS) and have been reported in β-cells of human T2D patients. Interestingly, the transcriptional changes that occur during β-cell immaturity involve loss of the nuclear-encoded mitochondrial gene expression program. My studies will test the hypothesis that β-cell immaturity in T2D is driven by loss of mitochondrial functional gene regulation by the TFs MAF bZIP transcription factor A (MAFA) or B (MAFB). Furthermore, I predict that MAFA/MAFB are themselves targets of mito-nuclear crosstalk through a retrograde signaling cascade induced by defects in mitochondrial function. I will elucidate the contribution of MAFA and MAFB on metabolic control in human β cells through regulation of mitochondrial function (Aim 1). I will determine how loss of MAFA and/or MAFB affects mitochondrial function and β cell identity by assaying oxygen consumption and gene expression in MAFA and/or MAFB knockdown human pseudoislets and EndoC-βH3 β cell lines. Metabolomics will be performed on EndoC- βH3 β cell lines to determine how MAFA/B influences fuel utilization. My preliminary data shows that genetic loss of mitophagy (i.e., the balance of mitochondrial biogenesis and turnover) reduces β-cell maturity. This includes physiologic, metabolic, and transcriptional signatures consistent with metabolic overload, oxidative damage, and the integrated stress response (ISR). MAFA (and likely MAFB) is known to be more sensitive to oxidative stress associated with T2D β-cell dysfunction than other TFs. While nuclear expression of β-cell mitochondrial genes are well known, mitochondrial feedback to drive β-cell nuclear gene expression (retrograde signaling) has not been analyzed. I will delineate if TF levels and β-cell maturity are altered in response to mitochondrial dysfunction (Aim 2). Further, I observed that pharmacological inhibition of the ISR relieves markers of immaturity in islets of mitophagy-deficient mice. Utilizing pharmacologic tools and analysis of gene expression in human pseudoislet transplants and EndoC-βH3 cells, I will interrogate how such conditions impact human β cells. I expect that MAFA (and possibly MAFB) levels will be reduced because of their ISR sensitivity. Moreover, I will determine if inhibition of the ISR restores MAFA/MAFB expression and reverses β-cell immaturity in the background of mitochondrial damage.