Characterization of Estrogen Sensitive Ventromedial Hypothalamic Glucose Sensing

描述(申请人提供)：下丘脑腹内侧(VMH)的葡萄糖感觉神经元(GSN)被认为在能量和葡萄糖稳态的调节中发挥作用。然而，GSN履行这一角色的基本机制仍然难以捉摸。目前肥胖症的流行和II型糖尿病(TIIDM)患病率的上升突显了澄清这些机制并潜在地阐明这些疾病的新治疗目标的必要性。这对女性尤其重要，因为肥胖和TIIDM在女性中的发病率更高，但几乎所有关于GSN的研究都是在男性身上进行的。我们之前已经在雄性小鼠身上表明，VMH GSN在TIIDM期间功能障碍。雌激素受体(ER)在两性中的表达与VMH GSNs在VMH腹外侧部的位置之间存在很强的相关性，这表明雌激素对GSNs的调节。该项目假设内质网激活可降低VMH GSN对低糖的敏感性。因此，绝经后雌激素水平的降低可能会增强GSNs对血糖下降的反应性。这可能会导致能源短缺的不适当信号，并在能源充足期间启动不适当的补偿机制。这种反应可能解释了绝经后女性对TIIDM易感性增加的原因。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聚合酶链式反应方法确定葡萄糖兴奋或葡萄糖抑制的神经元是ER阳性的(α或β)。这些神经元的神经递质表型也将被确定，以便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被用来确定雌激素和膜不通透性的BSA偶联雌激素如何调节GSNs的急性活动和葡萄糖敏感性。研究人员还将利用潜在的ER信号分子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。使用相同的技术，该研究员将通过使用类固醇生成因子-1驱动的ER1或ER2基因敲除小鼠(VMH独占基因敲除)和选择性应用ER1或ER2拮抗剂来研究ER1或ER2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，这位研究员将确定雌激素敏感的GSNs是直接投射到垂体前区还是投射到视前区，在那里它们可能会传递影响个体生殖状态的营养状态信息。 公共卫生相关性：女性，尤其是绝经后女性，比男性更容易患上现代世界最严重的两种疾病：肥胖和II型糖尿病(TIIDM)。下丘脑腹内侧的葡萄糖敏感神经元(GSNs)表达雌激素受体，可能在调节血糖和能量平衡方面发挥关键作用。这项提案将调查雌激素是否调节GSNs，从而研究雌激素减少是否会增加绝经后女性对肥胖和TIIDM的易感性。

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. PUBLIC HEALTH RELEVANCE: Females, and especially post-menopausal women, are more prone than males to develop two of the most serious diseases of the modern world: obesity and Type II Diabetes Mellitus (TIIDM). Glucose sensing neurons (GSNs) in the ventromedial hypothalamus, which express the estrogen receptor, may play a key role in regulating glucose and energy homeostasis. This proposal will investigate whether estrogen regulates GSNs and thus, whether diminished estrogen could contribute to the increased susceptibility of post-menopausal women to obesity and TIIDM.