Characterization of Estrogen Sensitive Hypothalamic Glucose Sensing Neurons

描述（由申请人提供）：腹内侧下丘脑（VMH）的葡萄糖感应神经元（GSN）被认为在能量和葡萄糖稳态的调节中起作用。然而，GSN履行这一角色的潜在机制仍然难以捉摸。目前肥胖症的流行和II型糖尿病（TIIDM）患病率的上升强调了澄清这些机制的必要性，并可能阐明这些疾病的新治疗靶点。这对女性尤其重要，因为女性肥胖和TIIDM的发生率更高，但几乎所有GSN研究都是在男性中进行的。我们以前已经表明，在雄性小鼠中，VMH GSN在TIIDM期间功能障碍。雌激素受体（ER）的表达，在两种性别，和VMH GSNs的腹外侧VMH内的位置之间的强相关性表明，雌激素调节GSNs。该项目假设ER激活减弱VMH GSN对葡萄糖降低的敏感性。因此，绝经后雌激素水平降低可能会增强GSN对葡萄糖降低的反应性。这可能导致能量不足的不适当信号，并在能量充足期间启动不适当的补偿机制。这种反应可以解释绝经后女性对TIIDM的易感性增加。为了验证这一假设，博士前研究员将首先使用免疫组织化学和单细胞PCR方法确定葡萄糖兴奋或葡萄糖抑制神经元是否为ER阳性（α或β）。这些神经元的神经递质表型也将被确定，以更好地了解它们在能量稳态中的作用。其次，电生理膜片钳技术将用于确定雌激素和膜不可渗透的BSA结合雌激素如何调节GSNs的急性活性以及葡萄糖敏感性。该研究员还将利用潜在的ER信号分子的抑制剂和蛋白质印迹分析来了解VMH GSNs敏感性的雌激素调节机制。使用这些相同的技术，研究员将通过使用类固醇生成因子-1驱动的ER 1或ER 2敲除小鼠（VMH排他性敲除）和选择性ER 1或ER 2拮抗剂应用，研究ER 1或ER 2消融对VMH GSN葡萄糖敏感性的影响。最后，使用荧光逆行示踪剂，该研究员将确定雌激素敏感的GSNs是否直接投射到垂体前叶或视前区，在那里它们可能中继影响个体生殖状态的营养状态信息。

DESCRIPTION (provided by applicant): Glucose sensing neurons (GSN) of the ventromedial hypothalamus (VMH) are believed to play a role in the regulation of energy and glucose homeostasis. However, the underlying mechanisms by which GSNs fulfill this role remain elusive. The current obesity epidemic and the rise in the prevalence of Type II Diabetes Mellitus (TIIDM) underscore the necessity to clarify these mechanisms and potentially illuminate novel therapeutic targets for these diseases. This is especially important for females since there is a greater incidence of obesity and TIIDM in females yet virtually all studies of GSNs have been performed in males. We have previously shown, in male mice, that VMH GSNs are dysfunctional during TIIDM. The strong correlation between estrogen receptor (ER) expression, in both sexes, and the location of VMH GSNs within the ventrolateral VMH suggests that estrogen regulates GSNs. This project hypothesizes that ER activation attenuates VMH GSN sensitivity to decreased glucose. Thus diminished estrogen levels following menopause may enhance responsiveness of GSNs to glucose decreases. This could lead to inappropriate signals of energy deficit and initiation of inappropriate compensatory mechanisms during energy sufficiency. Such a response may explain the increased susceptibility to TIIDM in post-menopausal females. To test this hypothesis, the pre-doctoral fellow will first determine whether glucose-excited or glucose-inhibited neurons are ER positive (either alpha or beta) using immunohistochemical and single-cell PCR methodologies. The neurotransmitter phenotype of these neurons will also be determined in order to better understand their role in energy homeostasis. Secondly, electrophysiological patch-clamp techniques will be used to determine how estrogen and the membrane impermeable BSA-conjugated estrogen regulate the acute activity as well as the glucose sensitivity of GSNs. The fellow will also utilize inhibitors of potential ER signaling molecules and western blot analysis to understand the mechanism of estrogenic regulation of VMH GSNs sensitivity. Using these same techniques, the fellow will investigate the effects of ER1 or ER2 ablation on the glucose sensitivity of VMH GSNs via the use of steroidogenic factor-1 driven ER1- or ER2-knockout mice (VMH exclusive knockout) and selective ER1 or ER2 antagonist application. Finally, using a fluorescent retrograde tracer, the fellow will determine whether estrogen-sensitive GSNs project directly to the anterior pituitary or to the preoptic area where they could potentially relay nutrient status information influencing the reproductive state of the individual.