Biology of IGFs in Bone

描述(由申请人提供)： 胰岛素样生长因子是在低等动物体内进化而来的一种生长因子，它可以在嗅觉、食物消耗、新陈代谢、生长、繁殖和休眠等多种生理过程中发挥作用。这些功能是通过激活一个共同的跨膜受体蛋白的多个相关配体的作用来完成的。在包括哺乳动物在内的高等生物中，胰岛素和IGF配体及其受体发生了分歧，承担了更多受限的功能。当代的观点认为，胰岛素样生长因子在细胞增殖、存活和生物生长中起主导作用，而胰岛素的主要功能包括调节燃料积累、储存和能量消耗。然而，这种简单化的模型往往掩盖了这样一个事实，即胰岛素和IGF-1在几个生理过程中继续发挥重叠的作用。在上一次资助期间，我们使用在成骨细胞中缺乏IGF-1或胰岛素受体的小鼠来开始区分IGF-1和胰岛素在骨骼中的作用。这些研究使我们发现了胰岛素信号在骨骼中的两个以前不为人知的作用。首先，胰岛素抑制Runx2抑制剂Twist2，后者促进正常骨形成所需的成骨细胞分化。第二，胰岛素诱导骨钙素(OC)的产生，影响葡萄糖的利用和能量消耗。这些结果将在提案的下一节中描述，这表明成骨细胞中存在一个新的内分泌调节环路，通过该环路，成骨细胞中的胰岛素信号控制出生后骨发育并刺激OC的产生，后者反过来调节胰腺胰岛素的分泌。该项目的总体目标将在两个假定的效应细胞中测试该模型的有效性：成骨细胞和胰腺b细胞。这些研究分为两个具体目标。具体目标1：鉴定成骨细胞中的胰岛素作用。上一次资助期间的研究表明，成骨细胞中的胰岛素信号对正常的成骨细胞分化和出生后骨积累是必不可少的，但也直接刺激OC的产生和g-羧化。在这个目标中，我们将定义胰岛素在成骨细胞中的全面作用。我们将确定胰岛素转录激活OC产生和翻译后G-羧化的机制。我们将描述胰岛素调节成骨细胞葡萄糖转运的机制，并确定成骨细胞葡萄糖转运蛋白4在全球葡萄糖利用中的作用。最后，为了测试体内假定的骨-胰腺内分泌环路的有效性，我们将确定高胰岛素-正常血糖钳夹对野生型和成骨细胞中缺乏GLUT4的小鼠循环中低羧化OC水平的影响。本质上，这些研究将检验新的假设，即成骨细胞在维持葡萄糖稳态中发挥关键作用。具体目标2：明确OC影响胰腺SS细胞功能的机制初步研究表明OC通过增加细胞内钙离子([Ca~(2+)]_i)来增加胰腺胰岛素分泌，我们推测这种作用是通过与B细胞上的G蛋白受体相互作用而发生的。为此，我们将在体外和体内进行研究，以确定可能的OC的G蛋白受体。我们将描述低羧化OC对胰岛素分泌的影响，并测试在胰腺SS细胞中表达的候选GPC(GRPC6a)的意义。同时，我们将筛选对OC有反应的新型GPCR。这些研究将与Bryan Roth和国家精神卫生研究所的精神活性药物筛选计划(NIMH-PDSP)合作进行，以确定OC是否可以激活不同的肽能和非肽能GPCR。在这些筛选中发现的候选者将通过评估OC诱导的信号在OC非反应细胞类型中的异位表达而被询问。对这一途径的理解最终应该能够更好地管理糖尿病退伍军人，并可以想象到设计一种单一的治疗方法，可以同时针对骨质疏松症和2型糖尿病。

DESCRIPTION (provided by applicant): The insulin-like growth factors evolved in lower animalsto enable a wide range of physiological processes including smell, food consumption, metabolism, growth, reproduction and dormancy. These functions were accomplished by the actions of multiple related ligands that activated a common transmembrane receptor protein. In higher organisms including mammals, the insulin and IGF ligands and their receptors diverged and assumed more circumscribed functions. The contemporary view is that IGFs serve dominant roles in cell proliferation, survival and organismal growth, whereas insulin's primaryfunctionsinclude the regulation of fuel accumulation, storage and energy expenditure. However, such a simplistic modeltends to obscure the fact that insulin and IGF-1 continue to perform overlapping roles in several physiological processes. During the last funding period, we used mice lacking either IGF-1 or insulin receptor in osteoblasts to begin to distinguish actions of IGF-1 and insulin in the skeleton. These studies led to our discovery of two previously unappreciated roles for insulin signaling in bone. First, insulin suppresses the Runx2 inhibitor Twist2, which promotes osteoblast differentiation necessary for normal bone formation. Second, insulin induces production of osteocalcin (OC), which influences glucose utilization and energy expenditure. These results, which are described in the next section of the proposal, suggest the existence of a novel endocrine regulatory loop through which insulin signaling in the osteoblast controls postnatal bone development and stimulates OC production, which in turn, regulates pancreatic insulin secretion. The overall goal of this project will test the validity of this model in two putative effector cells: the osteoblast and pancreatic b-cell. The studiesare divided into two Specific Aims. Specific Aim 1: Characterize insulin actionsin the osteoblast. Studies during the last funding period indicate that insulin signaling in the osteoblasts is essential for normal osteoblast differentiation and postnatal bone accumulation but also directly stimulates OC production and g-carboxylation. In this aim, we will define the full range of insulin actions in osteoblasts. We will determine the mechanisms responsible for insulin transcriptional activation of OC production and posttranslational g-carboxylation. We will characterize the mechanisms through which insulin regulates glucose transport in the osteoblast and determine the role of the osteoblast glucose transporter-4 in global glucose utilization. Finally, to test the validity of putative bone-pancreas endocrine loop in vivo, we will determine the effect of a hyperinsulinemic-euglycemic clamp on circulating undercarboxylated OC levels in both wild-type and mice lacking Glut4 in osteoblasts. In essence, these studies will test the novel hypothesis that the osteoblast plays a critical role in the maintenance of glucose homeostasis. Specific Aim 2: Define the mechanisms through which OC influences pancreatic ss-cell function Preliminary studies suggest that OC increases pancreatic insulin secretion by increasing intracellular calcium ([Ca2+]i ), which we postulate occurs secondarily to interaction with a G-protein receptor on the b-cell. In this aim, we will perform in vitro and in vivo studies to identify the putative G-protein receptor for OC. We will characterize the effects of undercarboxylated OC on insulin secretion and test the significance of a candidate GPCR (GRPC6a) that is expressed in the pancreatic ss-cell. In parallel we will screen for novel GPCRs that respond to OC. These studies will be performed in collaboration with Bryan Roth and the National Institute of Mental Health's Psychoactive Drug Screening Program (NIMH-PDSP), to determine if OC can activate distinct peptidergic and non-peptidergic GPCRs. Candidates found in these screens will be interrogated by assessment of OC induced signals following ectopic expression in OC non- responsive cell types. An understanding of this pathway should ultimately enable better management of diabetic veterans and conceivably lead to the design a single therapy, which can simultaneously target both osteoporosis and type 2 diabetes.