Effects of Ethanol on Insulin Signaling in the Brain

描述(申请人提供)：妊娠长期接触乙醇是致畸的，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良。胰岛素介导中枢神经系统的生长、发育和功能，而乙醇对神经细胞中的胰岛素信号转导有深刻的抑制作用。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，向下游延伸到调节神经元存活的途径。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激酶的下游激活与存活机制受损、凋亡增加和线粒体(MT)功能障碍所介导的神经细胞死亡有关。最近的初步实验表明，慢性妊娠酒精暴露引起的小脑发育不良与胰岛素信号通路受损的类型相似，但可能程度更大，神经元的丢失是由细胞凋亡和线粒体功能障碍共同介导的。此外，我们还获得了证据表明，在发育中的大脑中，乙醇通过IRS1依赖和IRS1非依赖的途径来损害信号传递。由于孕期接触乙醇并不抑制胰岛素受体蛋白的表达，我们推测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所介导的。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活性和mt功能产生不利影响的机制，并确定不同水平的乙醇暴露时发生类似异常的程度。在这一应用中，我们建议：1)检测低、中、高水平的慢性妊娠酒精暴露对胰岛素刺激的神经元活性和线粒体功能的损害程度；2)确定乙醇导致线粒体功能障碍或细胞凋亡的机制；3)表征长期妊娠酒精暴露抑制的胰岛素信号通路；以及4)验证特定损伤作为神经元MT功能障碍的介导者的作用。这些研究将利用胰岛素刺激的有丝分裂后原代小脑颗粒神经元培养，这些神经元是乙醇暴露和对照大鼠幼鼠产生的，因为小脑是乙醇神经毒性的主要靶点，而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。这些研究可能会带来新的方法，将神经细胞从长期妊娠酒精暴露的不利影响中拯救出来。

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.