Effects of Ethanol on Insulin Signaling in the Brain

描述（由申请人提供）：妊娠期慢性暴露于乙醇是致畸的，并导致中枢神经系统（CNS）的主要结构异常，包括小头畸形和小脑发育不全。胰岛素介导中枢神经系统的生长、发育和功能，乙醇对神经元细胞中的胰岛素信号传导具有深远的抑制作用。使用体外暴露模型，我们检测到乙醇对胰岛素信号的抑制，从其受体水平开始，并通过调节神经元存活的途径向下游延伸。重要的是，乙醇抑制胰岛素刺激的胰岛素受体底物-1 （IRS1）酪氨酸磷酸化和PI3激酶的下游活化与神经元细胞死亡有关，神经元细胞死亡是由存活机制受损、细胞凋亡增加和线粒体（Mt）功能障碍介导的。最近的初步实验表明，慢性妊娠乙醇暴露引起的小脑发育不全与相似类型但可能更大程度的胰岛素信号受损有关，并且神经元损失是由细胞凋亡和Mt功能障碍介导的。此外，我们获得的证据表明，在发育中的大脑中，乙醇通过irs1依赖性和irs1非依赖性途径损害信号传导。由于妊娠期暴露于乙醇中不会抑制胰岛素受体蛋白的表达，我们假设胰岛素信号的受损是由与结合亲和力、磷酸化或酪氨酸激酶激活相关的受体功能异常介导的。需要进一步的研究来了解乙醇在发育过程中对胰岛素刺激的生存能力和Mt功能产生不利影响的机制，并确定不同水平乙醇暴露下类似异常发生的程度。在这个应用中，我们建议：1)检查低、中等或高水平的慢性妊娠乙醇暴露对胰岛素刺激的神经元活力和Mt功能的损害程度；2)确定乙醇引起Mt功能障碍或细胞凋亡的机制；3)研究妊娠期慢性乙醇暴露对胰岛素信号通路的抑制作用；4)验证特异性损伤作为神经元Mt功能障碍介质的作用。由于小脑是乙醇神经毒性的主要靶点，而且小脑颗粒神经元既对胰岛素有反应，又受到乙醇的功能损害，因此研究将利用乙醇刺激的有丝分裂后初级小脑颗粒神经元培养物和对照大鼠幼崽。这些研究可能会导致新的方法来拯救神经细胞从慢性妊娠暴露于乙醇的不良影响。

DESCRIPTION (provided by applicant): Chronic gestational exposure to ethanol is teratogenic and causes major structural abnormalities in the central nervous system (CNS) including microcephaly and cerebellar hypoplasia. Insulin mediates CNS growth, development, and function, and ethanol has profound inhibitory effects on insulin signaling in neuronal cells. Using in vitro exposure models, we detected ethanol inhibition of insulin signaling, beginning at the level of its receptor and extending downstream through pathways that regulate neuronal survival. Importantly, ethanol inhibition of insulin-stimulated tyrosyl phosphorylation of the insulin receptor substrate-1 (IRS1) and downstream activation of PI3 kinase have been linked neuronal cell death mediated by impaired survival mechanisms, increased apoptosis, and mitochondrial (Mt) dysfunction. Recent preliminary experiments showed that cerebellar hypoplasia caused by chronic gestational exposure to ethanol is associated with similar types but probably greater degrees of impaired insulin signaling, and that neuronal loss is mediated by both apoptosis and Mt dysfunction. In addition, we obtained evidence that ethanol impairs signaling through both IRS1-dependent and IRS1-independent pathways in the developing brain. Since gestational exposure to ethanol does not inhibit insulin receptor protein expression, we hypothesize that the impaired insulin signaling is mediated by abnormalities in receptor function related to binding affinity, phosphorylation, or tyrosine kinase activation. Further studies are required to understand the mechanisms by which ethanol exerts its adverse effects on insulin-stimulated viability and Mt function during development, and determine the degree to which similar abnormalities occur with different levels of ethanol exposure. In this application, we propose to: 1) examine the degrees to which low, moderate, or high levels of chronic gestational ethanol exposure impair insulin stimulated neuronal viability and Mt function; 2) determine the mechanisms by which ethanol causes Mt dysfunction or apoptosis; 3) characterize the insulin signaling pathways that are inhibited by chronic gestational exposure to ethanol; and 4) validate the roles of specific impairments as mediators of neuronal Mt dysfunction. The studies will utilize insulin-stimulated post-mitotic primary cerebellar granule neuron cultures generated from ethanol-exposed and control rat pups since the cerebellum is a major target of ethanol neurotoxicity and cerebellar granule neurons are both responsive to insulin and functionally impaired by ethanol. These investigations could lead to new approaches for rescuing neuronal cells from the adverse effects of chronic gestational exposure to ethanol.