Characterization of Estrogen Sensitive Hypothalamic Glucose Sensing Neurons

雌激素敏感下丘脑葡萄糖传感神经元的表征

• 批准号: 8715788
• 负责人: Ammy Marie Santiago
• 金额: $ 2.93万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8715788
• 关键词: 5' -AMP-activated protein kinase; Ablation; Action Potentials; Acute; Anterior Pituitary Gland; Attenuated; Bovine Serum Albumin; Cell Nucleus; Cells; Conjugated Estrogens; Data; Disease; Eating; Electrophysiology (science); Epidemic; Estrogen Receptors; Estrogen receptor positive; Estrogens; Exhibits; Female; Fertility; Frequencies; Gluconeogenesis; Glucose; Gonadotropin Hormone Releasing Hormone; Homeostasis; Hormones; Hypoglycemia; Hypothalamic structure; Incidence; Individual; Insulin; Insulin Resistance; Knock-out; Knockout Mice; Lead; Leptin; Location; Medial; Membrane; Menopause; Methodology; Middle Hypothalamus; Mus; Neurons; Neurotransmitters; Nitric Oxide Synthase Type I; Non-Insulin-Dependent Diabetes Mellitus; Nuclear; Nutrient; Nutritional status; Obesity; Patch-Clamp Techniques; Phenotype; Phosphotransferases; Physiological; Play; Postabsorptive Hypoglycemia; Postmenopause; Potassium Channel; Predisposition; Preoptic Areas; Prevalence; Protein Isoforms; Receptor Activation; Receptor Signaling; Regulation; Risk; Role; SF1; Satiation; Signal Pathway; Signal Transduction; Signaling Molecule; Techniques; Testing; Tracer; Western Blotting; Woman; analog; blood glucose regulation; combat; inhibitor/antagonist; male; new therapeutic target; pre-doctoral; receptor expression; reproductive; response; sex; sexual dimorphism

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.

描述（由申请人提供）：下丘脑腹内侧（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发挥作用。然而，GSN 发挥这一作用的基本机制仍然难以捉摸。当前的肥胖流行和 II 型糖尿病 (TIIDM) 患病率的上升强调了阐明这些机制并可能阐明这些疾病的新治疗靶点的必要性。这对女性尤其重要，因为女性肥胖和 TIIDM 的发病率更高，但几乎所有 GSN 的研究都是在男性中进行的。我们之前已经在雄性小鼠中证明，VMH GSN 在 TIIDM 期间功能失调。两性雌激素受体 (ER) 表达与腹外侧 VMH 内 VMH GSN 的位置之间存在很强的相关性，表明雌激素调节 GSN。该项目假设 ER 激活会减弱 VMH GSN 对葡萄糖降低的敏感性。因此，绝经后雌激素水平的降低可能会增强 GSN 对葡萄糖降低的反应。这可能会导致能源不足的不适当信号，并在能源充足期间启动不适当的补偿机制。这种反应可以解释绝经后女性对 TIIDM 的易感性增加。为了检验这一假设，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用于确定雌激素和膜不可渗透的 BSA 结合雌激素如何调节 GSN 的急性活动以及葡萄糖敏感性。该研究员还将利用潜在 ER 信号分子的抑制剂和蛋白质印迹分析来了解雌激素调节 VMH GSN 敏感性的机制。使用这些相同的技术，该研究员将通过使用类固醇生成因子 1 驱动的 ER1 或 ER2 敲除小鼠（VMH 独家敲除）和选择性 ER1 或 ER2 拮抗剂应用，研究 ER1 或 ER2 消融对 VMH GSN 葡萄糖敏感性的影响。最后，研究人员将使用荧光逆行示踪剂确定雌激素敏感的 GSN 是否直接投射到垂体前叶或视前区，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。