Role of MitoNEET in regulating osteoblast bioenergetics and catabolism

 描述（由申请人提供）：本提案的目的是确定成骨细胞在体内用于最佳骨形成的关键代谢途径。最近的研究表明，代谢途径的调节是细胞命运和分化的关键决定因素。例如，在活化过程中，免疫T细胞参与有氧糖酵解以产生细胞因子;血管内皮细胞在血管出芽过程中也利用有氧糖酵解。造血干细胞、成骨细胞和破骨细胞依赖于谷氨酰胺代谢以实现最佳分化。在我们的初步研究中，我们观察到成骨细胞在分化的早期阶段上调氧化磷酸化和糖酵解。另一方面，分化的成骨细胞即使在有氧条件下也是高度糖酵解的，这表明它们更喜欢有氧糖酵解（瓦尔堡效应）。目前，成骨细胞利用的底物以及体内成骨细胞分化期间活跃的分解代谢途径在骨骼生物学中尚不清楚。在这个提议中，我们将利用一种新的小鼠模型，在该模型中，我们将在成骨细胞中特异性地过表达线粒体膜蛋白MitoNEET。MitoNEET过表达应导致β-氧化依赖性氧化磷酸化的抑制和糖酵解的增加。我们将通过两个具体目标来实现这一目标。1)在具体的目标1中，我们将利用前成骨细胞MC 3 T3 E1细胞来鉴定 MitoNEET调节成骨细胞分化的机制和代谢途径。我们将产生用rAAVMitoNEET和对照rAAVGFP病毒感染的MC 3 T3 E1细胞，并利用这些细胞来研究OxPhos、Glyc和谷氨酰胺水解。通过操纵葡萄糖、丙酮酸、脂肪酸和氨基酸作为底物，并在体外使用特异性抑制剂，我们可以开始分析哪些代谢途径对最佳分化是必不可少的。我们将利用新的海马细胞外通量技术来研究Escherren途径。2)在具体目标2中，我们将研究MitoNEET在成骨细胞中特异性过表达的体内作用。我们将使用显微计算机断层扫描（μCT）分析和骨组织形态计量学对rtTARunx 2 TREMitoNEET小鼠进行全面的骨骼表型分析。这将确定减少的OxPhos在骨获取中的作用。我们将使用代谢笼研究来测量小鼠的耗氧量和能量消耗，并将这些与骨骼表型相关联。我们将使用四氧化锇染色来表征rtTARunx 2 TREMitoNEET小鼠中的骨髓肥胖。这些实验将提供对β-氧化依赖性氧化磷酸化作用的深入了解，并将开始定义在分化的关键阶段成骨细胞生物能量学的背景特异性性质。

 DESCRIPTION (provided by applicant): The goal of this proposal is to identify the key metabolic pathways utilized by osteoblasts in vivo for optimal bone formation. Recent studies have shown that modulation of metabolic pathways is a key determinant of cell fate and differentiation. For example, during activation immune T-cells engage aerobic glycolysis for cytokine production; vascular endothelial cells also utilize aerobic glycolysis during vessel sprouting. Hematopoietic stem cells, osteoblasts and osteoclasts depend on glutamine metabolism for optimal differentiation. In our preliminary studies we observed that osteoblasts during early stages of differentiation upregulate both oxidative phosphorylation and glycolysis. Differentiated osteoblasts on the other hand are highly glycolytic even under aerobic conditions, suggesting that they prefer aerobic glycolysis (Warburg effect). The substrates that are utilized by osteoblasts and the catabolic pathways that are active during osteoblast differentiation in vivo is currently an unknown in skeletal biology. In this proposal we will utilize a novel mouse model where we will overexpress MitoNEET a mitochondrial membrane protein specifically in the osteoblasts. MitoNEET over- expression should result in suppression of β-Oxidation dependent oxidative phosphorylation and an increase in glycolysis. We will achieve this using two specific aims. 1) In specific aim 1 we will utilize preosteoblast MC3T3E1 cells to identify the mechanisms and metabolic pathways through which MitoNEET regulates osteoblast differentiation. We will generate MC3T3E1 cells infected with rAAVMitoNEET and control rAAVGFP viruses and utilize these cells to study OxPhos, Glyc and glutamynolysis. By manipulating glucose, pyruvate, fatty acids and amino acids as substrates and using specific inhibitors in vitro we can begin to analyze which metabolic pathways are essential for optimal differentiation. We will utilize novel Seahorse extracellular flux technology to study the differen pathways. 2) In specific aim 2 we will study the in vivo effects of MitoNEET over-expression specifically in osteoblasts. We will perform comprehensive skeletal phenotyping of rtTARunx2TREMitoNEET mice using micro-computed tomography (μCT) analysis and bone histomorphometry. This will identify the role of decreased OxPhos in bone acquisition. We will use metabolic cage studies to measure oxygen consumption, and energy expenditure of the mice and correlate these to the skeletal phenotype. We will use osmium tetraoxide staining to characterize marrow adiposity in the rtTARunx2TREMitoNEET mice. These experiments will provide insights into the role of β-Oxidation dependent oxidative phosphorylation, and will begin to define the context specific nature of osteoblast bioenergetics during critical phases of differentiation.