Metabolic Cellular Stress and its Regulatory Mechanism in Diabetic Embryopathy

摘要 糖尿病妇女所生的婴儿中，有高达10%的婴儿会发生先天性畸形。最佳血糖 控制是很难实现和维持的，即使是短暂的高血糖暴露也可能导致 畸形。这个项目是在我们强大的初步数据的基础上制定的。我们已找到1) 高血糖损害自噬，增加有缺陷的线粒体的积累， 发育中的神经上皮中的功能障碍蛋白和肿胀的内质网(ER)； 无毒的自噬激活剂海藻糖逆转高血糖诱导的自噬损害和 神经管缺陷(NTDS)；3)PKCA基因缺失，p70S6K1抑制剂，ER伴侣(4-PBA)和 在神经管中过表达sirtuin2(SIRT2)组蛋白脱乙酰酶，均可降低高血糖- 诱导NTDS；4)p70S6K1抑制剂和Sir2过表达均可恢复自噬水平 标记，LC 3-II。我们测试了一种新的假设，即母体糖尿病引起的自噬损害 通过破坏细胞内稳态，导致内质网应激和细胞凋亡，从而导致NTD的形成 母体高血糖会激活p70S6K1，导致自噬功能受损。恢复 海藻糖的自噬作用可预防高血糖诱导的NTDS。此外，降低了SIRT2和 SIRT6的表达介导了p70S6K1的作用。目标1将确定海藻糖是否可以防止 通过纠正导致内质网应激和内质网损伤的自噬损伤，高血糖诱导NTDS 细胞凋亡。我们假设母体糖尿病引起ATg1和Sqsmt1的异常变化 表达，调节自噬，导致自噬损伤，从而诱导细胞凋亡和 NTDS和海藻糖逆转自噬损伤，将恢复细胞内环境平衡，从而 预防糖尿病引起的NTDS。目标2将研究激活机制和作用 P70S6K1在糖尿病胚胎病变自噬损伤和神经管畸形形成中的作用我们的工作 假说是PKCA激活p70S6K1，导致自噬损伤，DNA- 甲基转移酶(DNMT)和SIRT2、6基因表达降低导致NTD的形成。 目标3将确定降低的SIRT2和SIRT6基因的潜在机制和作用 在导致糖尿病胚胎病变的自噬损伤中的表达。我们将检验这一假设 启动子超甲基化和随后降低的转录因子结合活性导致SIRT2 和SIRT6基因减少，通过调节Atg1和Atg1的表达而导致自噬功能受损 Sqsmt1通过FOXO转录因子的脱乙酰基。我们的研究将为 自噬、内质网应激、p70S6K1和SIRT2/6作为治疗干预的靶点。

ABSTRACT Congenital malformations occur in up to 10% of babies born to diabetic women. Optimal glycemic control is difficult to achieve and maintain, and even transient exposure to hyperglycemia can cause malformations. This project is formulated on the basis of our strong preliminary data. We have found 1) hyperglycemia impairs autophagy and increases the accumulation of defective mitochondria, dysfunctional proteins and swollen endoplasmic reticulum (ER) in the developing neuroepithelium; 2) the non-toxic autophagy activator, trehalose, reverses hyperglycemia-induced autophagy impairment and neural tube defects (NTDs); 3) PKCa gene deletion, a p70S6K1 inhibitor, an ER chaperone (4-PBA) and overexpression of sirtuin 2 (SIRT2) histone deacetylase in the neural tube, all reduce hyperglycemia- induced NTDs; 4) both p70S6K1 inhibitor and SIR2 overexpression restore levels of the autophagy marker, LC3-II. We test a novel hypothesis that maternal diabetes-induced autophagy impairment causes NTD formation by disrupting cellular homeostasis leading to ER stress and apoptosis, and that maternal hyperglycemia activates p70S6K1 resulting in autophagy impairment. Restoration of autophagy by trehalose prevents hyperglycemia-induced NTDs. In addition, reduced SIRT2 and SIRT6 expression mediates the effect of p70S6K1. Aim 1 will determine whether trehalose prevents hyperglycemia-induced NTDs by correcting autophagy impairment that causes ER stress and apoptosis. We hypothesize that maternal diabetes induces aberrant changes of Atg1 and Sqsmt1 expression, which regulate autophagy, leading to autophagy impairment which induces apoptosis and NTDs, and reversal of autophagy impairment by trehalose, will restore cellular homeostasis and thus prevent diabetes-induced NTDs. Aim 2 will investigate the activation mechanism and the role of p70S6K1 in autophagy impairment and NTD formation in diabetic embryopathy. Our working hypothesis is that PKCa activates p70S6K1 which causes autophagy impairment, an increase in DNA- methyltransferases (DNMTs) and a decrease in SIRT 2 and 6 gene expression leading to NTD formation. Aim 3 will determine the underlying mechanism and the role of reduced sirt2 and sirt6 gene expression in autophagy impairment that leads to diabetic embryopathy. We will test the hypothesis that promoter hypermethylation and subsequent reduced transcription factor binding activities cause sirt2 and sirt6 gene reduction which lead to autophagy impairment by modulating the expression of Atg1 and Sqsmt1 via deacetylation of Foxo transcription factors. Our studies will provide mechanistic evidence for autophagy, ER stress, p70S6K1 and SIRT2/6 as targets for therapeutic interventions.