Estradiol Regulation of Hypothalamic Astrocyte Glycogen

摘要： 糖原是脑神经元在高活动状态下使用的关键的可氧化燃料储备 葡萄糖酸化。医源性低血糖是一种反复出现的并发症，必须严格控制 胰岛素依赖型糖尿病，并构成神经损伤的严重风险。因此，人们对此有浓厚的兴趣 最大限度地保护大脑糖原对低血糖的有害影响。我们的研究表明 雌激素和后脑儿茶酚胺神经递质信号汇聚，调节腹内侧 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‘-单磷酸激活蛋白激酶(AMPK)调控假说 VMH糖原代谢，而雌二醇通过经典的受体介导的信号来控制 基础和低血糖增强的后脑去甲肾上腺素能输入对感觉器活动的影响。雌激素 推测糖原燃料储备的增加与局部神经细胞的延长有关 低血糖时的能量稳定性。建议的研究以三个具体目标为指导：1) 雌二醇对正常大鼠下丘脑代谢性感觉结构糖原含量的影响 2)观察VMH星形胶质细胞雌激素受体的作用 AMPK介导的糖原代谢反应的变异体；3)决定底物 后脑儿茶酚胺与雌激素对VMH星形胶质细胞糖原调节的相互作用 新陈代谢。该项目将使用原位免疫细胞化学/激光捕获相结合的方法 显微切割/高灵敏度qPCR和Western印迹定量检测细胞类型特异性 分子图谱；操纵糖原代谢酶活性的药理学工具；纳米 选择性抑制星形胶质细胞蛋白表达的技术装置；配对糖原 密度组织化学与神经细胞AMPK/磷酸AMPK蛋白印迹分析的相关性 高分辨率定量糖原图谱显示VMH代谢感觉神经细胞AMPK活性； 扫描粒子诱导X射线发射光谱法用于电解液的单细胞微量分析 VMH神经元电活动的相关性以及雌二醇对降糖模式的影响 后脑之间跨突触顺行转运的锰，一个可靠的神经元激活指标 A2去甲肾上腺素能神经元和VMH。这项工作将利用调查人员的创新集合 雌激素调节星形胶质细胞糖原的细胞和分子机制研究进展 海量，知识，可以被用来推动神经治疗策略的发展- 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.