Osteoblastic Respiration and IRS Signaling

描述(由申请人提供)：本提案的长期目标是描述骨细胞中的能量利用是如何被调节的，以及基质的可用性如何影响成骨细胞的分化和骨的形成。在整个生命周期中，最佳的骨形成是骨获取峰值和骨骼重塑所必需的。我们先前已经证明IGF-I是通过mTORC1途径实现OB终末分化所必需的。此外，甲状旁腺激素诱导骨骼IGF-I，这种多肽是甲状旁腺素对骨的合成代谢作用所必需的。以前我们注意到，在C57BL/6J(B6)和C3H/HeJ(C3H)小鼠中，后者表现出更高的骨密度、更多的骨形成和更多的骨骼Igf1表达。重要的是，C3HCOB比B6具有更高的氧化磷酸化(OxPhos)和糖酵解(Glyc)速率。然后，我们确定在OB分化末期，糖酵解(GYC)在两个菌株中比OxPhos更受欢迎，PTH刺激IGF-I，在体外增加COB和头盖骨中的GIGC。为了了解IGF通路在OB分化的生物能量学中的作用，我们一直在研究SML/SML小鼠，它的mTOR信号缺陷是由于IRS1基因的‘功能丧失’突变造成的。这些小鼠的骨量非常低，骨形成减少，但OB数量和类骨体积正常。值得注意的是，在体外和体内，SML/SML Cobs显著减少了OB分化过程中的OxPhos和Glyc。综上所述，这些数据表明了能量代谢在骨形成过程中的重要性，以及IGF-I在细胞呼吸中的核心作用。因此，在这个方案中，我们的总体假设是，甲状旁腺素通过诱导IGF-I增强血糖来刺激骨形成，这反过来又激活IRS1/mTORC/AKT信号。因此，我们在一种高风险、高影响的策略中提出了两个具体目标，以利用新颖的单细胞成像和对OBS和颅骨的生物能量学研究来描述甲状旁腺素诱导骨形成过程中底物可获得性的重要性：1-确定OB呼吸和甘氨酸的上下文特异性性质及其与体外和体外甲状旁腺激素反应的骨形成的关系：我们认为，当甘氨酸被激活时，甲状旁腺素影响下的COB分化过程中存在一个时间依赖性的开关，导致基质的合成和矿化增强。新的原位成像和细胞呼吸研究将被用来确定PTH对单细胞生物能量学的作用，并将与体外研究进行比较。2-确定IRS/mTORC1、mTORC2/AKT信号通路在甲状旁腺素刺激的分化OB能量平衡改变中的重要性。我们推测，在PTH后OB分化过程中，通过IRS/AKT途径激活mTORC2是上调血糖的关键。我们将描述mTOR途径的单个信号成分与OB生物能量学的关系。这些研究的发现可能导致对OBS的基础生物学的新见解，并增加治疗骨骼疾病的新的合成代谢方法的可能性。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to delineate how energy utilization in bone cells is regulated and in turn, how substrate availability affects osteoblast differentiation and bone formation. Optimal bone formation is required for peak bone acquisition and skeletal remodeling across the lifespan. We previously showed IGF-I is necessary for terminal OB differentiation through the mTORC1 pathway. Moreover, PTH induces skeletal IGF-I and this peptide is required for the anabolic actions of PTH on bone. Previously we noted that in C57BL/6J (B6) and C3H/HeJ (C3H) mice, the latter exhibits higher bone density, greater bone formation, and more skeletal Igf1 expression. Importantly, C3H COBs have higher rates of oxidative phosphorylation (OxPhos) and glycolysis (Glyc) than B6. We then established that during terminal OB differentiation glycolysis (Glyc) is favored over OxPhos in both strains and that PTH, which stimulates IGF-I, increases Glyc in both COBs and calvariae ex vivo. To understand the role of the IGF pathway in the bioenergetics of OB differentiation, we have been studying the sml/sml mouse that has a defect in mTOR signaling due to a 'loss of function' mutation in the Irs1 gene. These mice have very low bone mass, reduced bone formation but normal OB number and osteoid volume. Remarkably, sml/sml COBs have significantly reduced OxPhos and Glyc during OB differentiation in vitro and in vivo. Taken together these data point to the importance of energy metabolism during bone formation, and the central role of IGF-I in cellular respiration. As such, in this proposal our over- arching hypothesis is that PTH stimulates bone formation by enhancing Glyc through the induction of IGF-I, which in turn activates IRS1/ mTorc/AKT signaling. Thus we propose 2 specific aims in a high risk, high impact strategy to delineate the importance of substrate availability during PTH induced bone formation using novel single cell imaging and bioenergetic studies of OBs and calvariae: 1-Determine the context-specific nature of OB respiration and Glyc as well as its relationship to bone formation in response to PTH in +/+ and sml/sml mice in vitro and ex vivo: We propose there is a time dependent switch during COB differentiation under the influence of PTH when Glyc is activated, leading to enhanced synthesis and mineralization of matrix. Novel in situ imaging and cellular respiration studies will be employed to determine PTH actions on single cell bioenergetics and will be compared with in vitro studies. 2-Define the importance of the IRS/ mTORC1 mTORC2/AKT signaling pathway for PTH-stimulated changes in energy balance in differentiated OBs. We postulate that mTORC2 activation via the IRS/AKT pathway is critical for up regulating Glyc during the OB differentiation following PTH. We will delineate te relationship of individual signaling components of the mTOR pathway to OB bioenergetics. Findings from these studies could lead to novel insights into the fundamental biology of OBs and enhance the possibility of newer anabolic approaches for skeletal disorders.