Estradiol Regulation of Hypothalamic Astrocyte Glycogen

摘要： 糖原是大脑神经元在高度活动或过度兴奋状态下使用的可氧化燃料的关键储备。 葡萄糖缺乏医源性低血糖是一种反复出现的并发症， 胰岛素依赖型糖尿病，并有严重的神经损伤风险。因此， 以最大限度地保护脑糖原免受低血糖的有害影响。我们的研究表明 雌二醇和后脑儿茶酚胺神经递质信号会聚调节腹内侧核 下丘脑核（VMH）糖原代谢酶蛋白表达。该项目将解决 星形胶质细胞腺苷5 '-磷酸活化蛋白激酶（AMPK）调节 VMH糖原代谢，雌二醇通过经典的受体介导的信号传导来控制 基础和低血糖增强的后脑去甲肾上腺素能输入对传感器活性的影响。雌激素 据推测，糖原燃料储备的增加与局部神经细胞 低血糖时能量稳定。拟议的研究由三个具体目标指导：1） 雌二醇对正常人下丘脑代谢感觉结构糖原含量的影响 评估VMH星形胶质细胞雌激素受体的作用 AMPK介导的糖原代谢对葡萄糖缺乏反应的变体;和3）确定底物 脑内儿茶酚胺与雌激素相互作用对VMH星形胶质细胞糖原的调节 新陈代谢.本项目将使用原位免疫细胞化学/激光捕获 显微切割/高灵敏度qPCR和Western blot方法用于定量细胞类型特异性 分子谱;操纵糖原代谢酶活性的药理学工具;纳米 选择性敲低星形胶质细胞蛋白表达的技术装置;成对糖原 光密度组织化学和神经细胞AMPK/磷酸化AMPK蛋白质印迹分析， 具有VMH代谢感觉神经细胞AMPK活性的高分辨率定量糖原图;以及 扫描粒子诱导X射线发射光谱法进行电解质的单电池微量分析 相关VMH神经元电活动，并量化雌二醇对低血糖模式的影响 锰的跨突触顺行运输，神经元激活的可靠指标， A2去甲肾上腺素能神经元和VMH。这项工作将利用一个创新的调查组合， 雌激素调节星形胶质细胞糖原的细胞和分子机制 质量，知识，可以利用，以促进发展的治疗策略，为神经， 脑糖原水平的保护性放大。

Abstract: Glycogen is a critical reserve of oxidizable fuel for brain neuron use during states of heightened activity or glucoprivation. Iatrogenic hypoglycemia is a recurring complication of obligatory meticulous control of insulin-dependent diabetes mellitus and poses a serious risk of neural injury. There is thus keen interest to maximize protection against harmful effects of hypoglycemia by cerebral glycogen. Our studies show that estradiol and hindbrain catecholamine neurotransmitter signals converge to regulate ventromedial hypothalamus nucleus (VMH) glycogen metabolic enzyme protein expression. This project will address the hypothesis that astrocyte adenosine 5’-mono-phosphate-activated protein kinase (AMPK) regulates VMH glycogen metabolism, and that estradiol acts via classical receptor-mediated signaling to control effects of basal and hypoglycemia-enhanced hindbrain noradrenergic input on sensor activity. Estrogen augmentation of the glycogen fuel reserve is presumed to correlate with prolonged local nerve cell energetic stability during hypoglycemia. Proposed research is guided by three Specific Aims: 1) Determine effects of estradiol on hypothalamic metabolic sensory structure glycogen content in normo- and hypoglycemic ovariectomized female rats; 2) Assess the role of VMH astrocyte estrogen receptor variants in AMPK- mediated glycogen metabolic responses to glucoprivation; and 3) Determine substrates for interaction of hindbrain catecholamine and estrogen regulation of VMH astrocyte glycogen metabolism. This project will use a combinatory in situ immunocytochemistry/laser-capture microdissection/high-sensitivity qPCR and Western blot approach for quantitative cell type-specific molecular profiling; pharmacologic tools for manipulation of glycogen metabolic enzyme activity; nano- technological devices for selective knockdown of astrocyte protein expression; paired glycogen densitometric histochemistry and nerve cell AMPK/phosphoAMPK Western blot analyses to correlate high-resolution quantitative glycogen maps with VMH metabolic sensory nerve cell AMPK activity; and scanning particle-induced X-ray emission spectrometry to perform single-cell microanalysis of electrolyte correlates of VMH neuron electrical activity, and to quantify effects of estradiol on hypoglycemic patterns of transynaptic anterograde transport of Mn, a reliable indicator of neuron activation, between hindbrain A2 noradrenergic neurons and the VMH. This work will utilize an innovative assembly of investigative strategies to identify cellular and molecular mechanisms of estrogen regulation of astrocyte glycogen mass, knowledge that can be leveraged to advance development of therapeutic strategies for neuro- protective amplification of brain glycogen levels.