Hormonal regulation of a Ca2+/AMPK signaling pathway

描述（由申请人提供）：总体目标是定义下丘脑神经元中调节食欲和体重的Ca2+依赖信号通路，为人类肥胖提供新的见解。AMPK最初被发现可以保护细胞免受消耗ATP的应激，是胃饥饿素和瘦素作用于弓状核（ARM）下丘脑神经元控制NPY产生的关键酶，从而控制食欲、能量稳态和体重。Ca2+是一种普遍存在的第二信使，介导由激素和生长因子引发的信号级联反应，其中钙调蛋白（CaM）是Ca2+受体，Ca2+/CaM依赖性蛋白激酶（CaMK）转导Ca2+/CaM信号，导致许多细胞反应。CaMK级联由Ca2+/CaM、CaMKK （A或P）和CaMK （I或IV）组成。最近，CaMKKs也被证明具有AMPKKs的功能，并且需要在一些人类细胞中激活AMPK。CaMKKp是一种脑特异性酶，在ARM中表达，CaMKKp“'”小鼠显示下丘脑AMPK磷酸化/活性以及NPY/AgRP mrna降低。事实上，CaMKKp“'”小鼠与缺乏NPY或NPY受体的小鼠共享许多表型。将唯一的选择性CaMKK拮抗剂sto609输注到成年野生型小鼠的第三脑室，可导致食物摄入量和体重的急性减少，以及下丘脑NPY/AgRP mrna的含量。我们假设CaMKKp在NPY/AgRP神经元中作为主要的AMPKK，并且该酶的急性抑制导致NPY的产生减少，从而导致食物摄入量减少，能量稳态改变和体重减轻。为了验证这一假设，我们将：1)利用转基因小鼠研究CaMKKs、p和a作为AMPKKs在小鼠下丘脑和培养细胞中的生理相关作用，以阐明调节NPY基因表达和丙二酰辅酶a水平的途径；2)通过使用分子和生化技术来了解CaMKK/AMPK信号复合物的性质以及如何抑制该复合物，定义CaMKKs和AMPK之间的物理相互作用。我们的研究结果应该验证CaMKKp作为药物发现的靶点，这可能最终导致确定治疗肥胖的新疗法。

DESCRIPTION (provided by applicant): The overall objective is to define the Ca2+-dependent signaling pathway in hypothalamic neurons that regulates appetite and body weight to provide novel insights to human obesity. AMPK, originally discovered to protect cells against stresses that deplete ATP, is a key enzyme in the pathways by which ghrelin and leptin act on arcuate nucleus (ARM) hypothalamic neurons to control production of NPY and, thus, appetite, energy homeostasis and body weight. Ca2+ is a ubiquitous 2nd messenger that mediates signaling cascades initiated by hormones and growth factors, in which calmodulin (CaM) is the Ca2+ receptor and Ca2+/CaM- dependent protein kinases (CaMK) transduce the Ca2+/CaM signal resulting in many cell responses. A "CaMK cascade" has been identified composed of Ca2+/CaM, a CaMKK (a or P) and a CaMK (I or IV). Recently the CaMKKs have also been shown to function as AMPKKs and are required to activate AMPK in some human cells. CaMKKp, a brain-specific enzyme, is expressed in the ARM and CaMKKp"'" mice show decreased hypothalamic AMPK phosphorylation/activity as well as NPY/AgRP mRNAs. Indeed, CaMKKp"'" mice share many phenotypes with mice null for NPY or an NPY receptor. Infusion of the only selective CaMKK antagonist, STO-609, into the 3rd ventricle of adult wild-type mice results in acute decreases in food intake and body weight as well as hypothalamic content of NPY/AgRP mRNAs. We hypothesize that CaMKKp serves as a primary AMPKK in NPY/AgRP neurons and that acute inhibition of this enzyme leads to decreased production of NPY which causes decreased food intake, altered energy homeostasis and weight loss. To evaluate this hypothesis we will: 1) utilize genetically altered mice to investigate the physiologically relevant role(s) of the CaMKKs, p and a as AMPKKs in mouse hypothalamus and cultured cells to clarify pathways that regulate NPY gene expression and malonyl CoA level; and 2) define the physical interaction between the CaMKKs and AMPK by employing molecular and biochemical technology to understand the nature of the CaMKK/AMPK signaling complex and how this complex can be inhibited. Our results should validate CaMKKp as a target for drug discovery, which could eventually lead to identification of new therapeutics to treat obesity.