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如何被调节，反过来，底物可用性如何影响成骨细胞分化和骨形成。在整个生命周期中，最佳骨形成是骨骼获取和骨骼重塑的高峰。我们之前发现IGF-I是通过mTORC1途径向OB终末期分化所必需的。此外，甲状旁腺激素诱导骨骼igf - 1，这种肽是甲状旁腺激素对骨骼的合成代谢作用所必需的。先前我们注意到，在C57BL/6J （B6）和C3H/HeJ （C3H）小鼠中，后者表现出更高的骨密度、更大的骨形成和更多的骨骼Igf1表达。重要的是，C3H cob具有比B6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，两种菌株的糖酵解（Glyc）比OxPhos更有利，而PTH刺激IGF-I，增加cob和颅骨体内的Glyc。为了了解IGF通路在OB分化的生物能量学中的作用，我们一直在研究由于Irs1基因“功能丧失”突变而导致mTOR信号缺陷的sml/sml小鼠。这些小鼠骨量非常低，骨形成减少，但OB数和类骨体积正常。值得注意的是，在体外和体内OB分化过程中，sml/sml cob显著降低了OxPhos和Glyc。综上所述，这些数据表明了骨形成过程中能量代谢的重要性，以及igf - 1在细胞呼吸中的核心作用。因此，在本研究中，我们的总体假设是PTH通过诱导igf - 1来增强Glyc，进而激活IRS1/ mTorc/AKT信号，从而刺激骨形成。因此，我们提出了高风险、高影响策略中的两个具体目标，利用新的单细胞成像和OBs和颅骨的生物能量研究来描述PTH诱导骨形成过程中底物可用性的重要性：1 .在体外和离体实验中，确定+/+和sml/sml小鼠OB呼吸和糖的环境特异性及其与PTH反应中骨形成的关系；我们认为在PTH的影响下，COB分化过程中存在一个时间依赖的开关，当Glyc被激活时，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用于确定甲状旁腺素对单细胞生物能量学的作用，并将与体外研究进行比较。2 .明确IRS/ mTORC1 mTORC2/AKT信号通路在pth刺激的分化OBs能量平衡变化中的重要性。我们假设mTORC2通过IRS/AKT通路激活对于PTH后OB分化过程中Glyc的上调至关重要。我们将描述mTOR通路中各个信号组分与OB生物能量学的关系。这些研究的发现可能会导致对OBs基础生物学的新见解，并增加骨骼疾病的新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.