Effects of Ethanol on Insulin Signaling in the Brain

描述（由申请方提供）：妊娠期长期暴露于乙醇具有致畸性，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，乙醇对神经细胞中的胰岛素信号传导具有深刻的抑制作用。使用体外暴露模型，我们检测到乙醇抑制胰岛素信号传导，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（IRS 1）的酪氨酰磷酸化和PI 3激酶的下游激活与受损的存活机制介导的神经元细胞死亡、凋亡增加和线粒体（Mt）功能障碍有关。最近的初步实验表明，小脑发育不全所造成的慢性妊娠暴露于乙醇与类似的类型，但可能更大程度的受损胰岛素信号转导，和神经元的损失介导的细胞凋亡和MT功能障碍。此外，我们获得的证据表明，乙醇损害信号通过IRS 1依赖和IRS 1独立的途径在发育中的大脑。由于妊娠期暴露于乙醇不会抑制胰岛素受体蛋白的表达，我们推测受损的胰岛素信号传导是由与结合亲和力、磷酸化或酪氨酸激酶活化相关的受体功能异常介导的。需要进一步的研究，以了解乙醇发挥其对胰岛素刺激的活力和MT功能在发展过程中的不利影响的机制，并确定在何种程度上发生类似的异常与不同水平的乙醇暴露。在本申请中，我们提出：1）检查低、中、高水平的慢性妊娠期乙醇暴露损害胰岛素刺激的神经元活力和Mt功能的程度; 2）确定乙醇引起Mt功能障碍或凋亡的机制; 3）表征由慢性妊娠期乙醇暴露抑制的胰岛素信号传导途径;以及4）验证特定损伤作为神经元MT功能障碍的介质的作用。这些研究将利用暴露于乙醇和对照大鼠幼仔产生的胰岛素刺激的有丝分裂后原代小脑颗粒神经元培养物，因为小脑是乙醇神经毒性的主要靶点，小脑颗粒神经元对胰岛素有反应，并且功能受到乙醇损害。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.