Role of MitoNEET in regulating osteoblast bioenergetics and catabolism

MitoNEET 在调节成骨细胞生物能和分解代谢中的作用

• 批准号: 9037135
• 负责人: Anyonya R Guntur
• 金额: $ 7.8万
• 依托单位: MAINEHEALTH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9037135
• 关键词: Adipocytes; Aerobic; Affect; Amino Acids; Bioenergetics; Biology; Blood Vessels; Bone Marrow; Catabolism; Cell Culture Techniques; Cells; Complex; Deoxyglucose; Disease; Electrons; Energy Metabolism; Erythroid; Essential Amino Acids; Fatty Acids; Figs - dietary; Fracture; Genetic Engineering; Genus Hippocampus; Glucose; Glutamine; Glycolysis; Goals; Hematopoietic stem cells; Hepatocyte; Human; Immune; In Vitro; Investigation; Iron; Link; Lipids; Marrow; Measures; Membrane Potentials; Membrane Proteins; Metabolic; Metabolic Pathway; Metabolism; Mitochondria; Mitochondrial Membrane Protein; Modeling; Mus; Nature; Obesity; Osmium; Osteoblasts; Osteoclasts; Osteogenesis; Osteoporosis; Oxidation-Reduction; Oxidative Phosphorylation; Oxygen; Oxygen Consumption; PTH gene; Pathway interactions; Phase; Phenotype; Pioglitazone; Play; Process; Production; Proteins; Pyruvate; Reaction; Role; Staging; Staining method; Stains; Sulfur; Sum; System; T-Lymphocyte; Technology; Testing; Thiazolidinediones; Vascular Endothelial Cell; Virus; Warburg Effect; X-Ray Computed Tomography; Zinc Fingers; aerobic glycolysis; bone; bone mass; cancer cell; cytokine; extracellular; glucose tolerance; immune activation; in vivo; inhibitor/antagonist; insight; mitochondrial membrane; mouse model; novel; novel therapeutic intervention; osteoblast differentiation; overexpression; oxidation; preference; public health relevance; pyruvate carrier; research study; rosiglitazone; skeletal; therapeutic target; uptake

 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.

 描述（由申请人提供）：该提案的目标是确定体内成骨细胞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，在激活过程中，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萌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谷氨酰胺代谢来实现最佳分化。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具有高度糖酵解能力，这表明它们更喜欢有氧糖酵解（瓦尔堡效应）。成骨细胞利用的底物以及在体内成骨细胞分化期间活跃的分解代谢途径目前在骨骼生物学中是未知的。在本提案中，我们将利用一种新型小鼠模型，在该模型中，我们将在成骨细胞中过度表达 MitoNEET（一种线粒体膜蛋白）。 MitoNEET 过度表达应导致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) 在具体目标 1 中，我们将利用前成骨细胞 MC3T3E1 细胞来识别 MitoNEET 调节成骨细胞分化的机制和代谢途径。我们将生成感染 rAAVMitoNEET 和对照 rAAVGFP 病毒的 MC3T3E1 细胞，并利用这些细胞来研究 OxPhos、Glyc 和谷氨酰胺分解。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定抑制剂，我们可以开始分析哪些代谢途径对于最佳分化至关重要。我们将利用新颖的海马细胞外通量技术来研究不同的途径。 2) 在具体目标 2 中，我们将研究 MitoNEET 过度表达的体内效应，特别是在成骨细胞中。我们将使用微计算机断层扫描 (μCT) 分析和骨组织形态计量学对 rtTARunx2TREMitoNEET 小鼠进行全面的骨骼表型分析。这将确定氧化磷酸减少在骨获取中的作用。我们将使用代谢笼研究来测量小鼠的耗氧量和能量消耗，并将其与骨骼表型相关联。我们将使用四氧化锇染色来表征 rtTARunx2TREMitoNEET 小鼠的骨髓肥胖。这些实验将深入了解β-氧化依赖性氧化磷酸化的作用，并将开始定义成骨细胞生物能在分化关键阶段的特定性质。