Hormonal regulation of a Ca2+/AMPK signaling pathway

描述(申请人提供)：总体目标是确定下丘脑神经元中调节食欲和体重的钙依赖信号通路，以提供对人类肥胖的新见解。AMPK最初被发现用于保护细胞免受耗尽ATP的压力，是Ghrelin和Leptin作用于下丘脑弓状核(ARM)神经元以控制NPY的产生从而控制食欲、能量平衡和体重的关键酶。钙离子是一种普遍存在的第二信使，它介导激素和生长因子启动的信号级联反应，其中钙调素(CaM)是钙离子受体，钙/钙调素依赖的蛋白激酶(CaMK)转导钙/钙调素信号，引起多种细胞反应。已确定的“CaMK级联”由Ca~(2+)/CaM、CaMK(a或P)和CaMK(I或IV)组成。最近，CaMKK也被证明作为AMPKK发挥作用，并需要在一些人类细胞中激活AMPK。CaMKKp是一种大脑特有的酶，在手臂上表达，CaMKKp“‘”小鼠表现出下丘脑AMPK磷酸化/活性以及NPY/AgRP mRNAs的降低。事实上，CaMKKp“‘”小鼠与NPY或NPY受体缺失的小鼠有许多共同的表型。将唯一的选择性CaMKK拮抗剂STO-609注入成年野生型小鼠的第三脑室，导致进食量和体重以及下丘脑NPY/AgRP mRNAs含量的急剧下降。我们假设CaMKKp在NPY/AgRP神经元中是一种主要的AMPKK，该酶的急性抑制会导致NPY的产生减少，从而导致食物摄入量减少，能量平衡改变和体重减轻。为了评估这一假说，我们将：1)利用转基因小鼠来研究CaMKK、p和a作为AMPKK在小鼠下丘脑和培养细胞中的生理相关作用(S)，以阐明调节NPY基因表达和丙二酰辅酶A水平的途径；以及2)通过使用分子和生化技术来确定CaMKK和AMPK之间的物理相互作用，以了解CaMKK/AMPK信号复合体的性质以及如何抑制该复合体。我们的结果将验证CaMKKp作为药物发现的目标，这最终可能导致发现治疗肥胖症的新疗法。

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.