The role of glycolysis and glucose oxidation in hematopoiesis

糖酵解和葡萄糖氧化在造血中的作用

• 批准号: 10340134
• 负责人: Michalis Agathocleous
• 金额: $ 40.36万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10340134
• 关键词: Ablation; Acetyl Coenzyme A; Address; B-Lymphocytes; Biochemical; Bone Marrow; Bone Marrow Cells; Carbon; Cell Respiration; Cells; Cellular Metabolic Process; Cellular biology; Citric Acid Cycle; Dependence; Embryo; Enzymes; Equilibrium; Erythropoiesis; Fermentation; Frequencies; Gatekeeping; Genetic; Glucose; Glutathione; Glycolysis; Hematopoiesis; Hematopoietic; Hematopoietic System; Hematopoietic stem cells; Homeostasis; Impairment; In Vitro; Individual; Isotopes; Label; Lactate Dehydrogenase; Measures; Mediating; Metabolic; Metabolic Pathway; Metabolism; Methods; Myelogenous; Myeloid Cells; NADH; Natural regeneration; Nature; Neurons; Nutrient; Oxidation-Reduction; Pathway interactions; Positioning Attribute; Production; Protein Isoforms; Pyruvate; Role; Specificity; Stable Isotope Labeling; T-Lymphocyte; Testing; Textbooks; Thymus Gland; Tissues; Work; cell type; experimental study; flexibility; glucose metabolism; hematopoietic differentiation; in vivo; innovation; metabolome; metabolomics; mouse model; nerve stem cell; novel; nutrient metabolism; oxidation; progenitor; pyruvate dehydrogenase; stable isotope; stem cell function; stem cells; thymic regeneration

PROJECT SUMMARY: The metabolism of nutrients has been studied using unfractionated tissues, or in vitro. An unresolved question is how nutrients are metabolized by stem cells in vivo. Our understanding of stem cell metabolism has been limited by the fact that metabolomics typically requires millions of cells, while stem cells are rare. We developed methods to profile the metabolome and to trace stable isotope labeled nutrients in hematopoietic stem cells (HSCs) and other rare cell types purified from tissues. We found that T cell progenitors in the thymus are glucose avid as compared to HSCs, myeloid and B cell restricted progenitors, in contrast to the prevailing view that HSCs are more glycolytic than hematopoietic progenitors. Stable isotope tracing experiments showed that in the bone marrow but not the thymus, glycolysis and the TCA cycle are disconnected. Hematopoietic loss of pyruvate dehydrogenase (PDH), the gatekeeper enzyme that connects glycolysis to the TCA cycle, reduced the number of double positive (DP) T cell progenitors but did not affect HSCs or other hematopoietic cell types. Loss of PDH paradoxically did not impair the TCA cycle in the thymus, but caused accumulation of pyruvate and aberrant redox balance. Cells which do not oxidize glucose in the TCA cycle are classically thought to ferment glucose through glycolysis to lactate via lactate dehydrogenase (LDH). Hematopoietic loss of LDHA, one of the two LDH isoforms, impaired development of erythroid progenitors but not HSCs, T cell progenitors or other restricted hematopoietic progenitors. The cell type specificity in the requirement of LDH and PDH in the hematopoietic system raises the question of why different stem or progenitor cell types choose to use LDH-mediated fermentation or PDH-mediated oxidation in vivo. This application’s objective is to systematically dissect the role of glycolytic as compared to oxidative metabolism in HSCs and restricted progenitors. Our hypothesis is that T cell progenitors require oxidation of glucose via PDH to regulate pyruvate levels and redox homeostasis, in contrast to HSCs, myeloid and B cell progenitors which are metabolically flexible. In Aim 1 we will test the metabolic mechanisms which mediate the effects of PDH on DP cells. In Aim 2 we will determine the cellular and metabolic effects of blocking LDHA/B or PDH alone or in combination in HSCs and restricted progenitors. In Aim 3 we will investigate the role of LDHA/B and PDH in hematopoietic and thymopoietic regeneration. These experiments will identify the contribution of glucose to metabolite pools in HSCs and progenitors in vivo, systematically test the idea that HSCs are glycolytic, and identify mechanisms by which central carbon metabolism regulates hematopoietic differentiation and regeneration. More generally our experiments will address a fundamental metabolic question by testing if stem or progenitor cells in vivo switch between glucose fermentation or oxidation, as is the textbook view, or if some cell types in vivo tolerate the loss of both major glucose catabolic pathways.

项目概要： 营养物质的代谢已使用未分级的组织或体外进行了研究。一个未解决的问题 是体内干细胞代谢营养物质的方式。我们对干细胞代谢的理解是 受限于代谢组学通常需要数百万个细胞，而干细胞很少的事实。我们 开发了分析代谢组和追踪造血系统中稳定同位素标记营养素的方法 干细胞 (HSC) 和从组织中纯化的其他稀有细胞类型。我们发现 T 细胞祖细胞 与 HSC、骨髓和 B 细胞限制性祖细胞相比，胸腺对葡萄糖有强烈的渴求。 普遍观点认为，HSC 比造血祖细胞更具糖酵解能力。稳定同位素示踪 实验表明，在骨髓中，而不是在胸腺中，糖酵解和 TCA 循环是 已断开连接。丙酮酸脱氢酶（PDH）的造血损失，丙酮酸脱氢酶是连接细胞的看门酶 糖酵解对 TCA 循环的影响，减少了双阳性 (DP) T 细胞祖细胞的数量，但不影响 HSC 或其他造血细胞类型。矛盾的是，PDH 的丧失并没有损害胸腺中的 TCA 循环， 但引起丙酮酸积累和氧化还原平衡异常。不氧化葡萄糖的细胞 TCA 循环传统上被认为通过乳酸脱氢酶通过糖酵解将葡萄糖发酵为乳酸 （LDH）。 LDHA（两种 LDH 亚型之一）的造血损失，红细胞发育受损 祖细胞，但不包括 HSC、T 细胞祖细胞或其他受限造血祖细胞。细胞类型 造血系统中 LDH 和 PDH 需求的特异性提出了一个问题：为什么不同 干细胞或祖细胞类型选择使用LDH介导的发酵或PDH介导的体内氧化。 本申请的目的是系统地剖析糖酵解与氧化作用相比的作用 HSC 和受限祖细胞的代谢。我们的假设是 T 细胞祖细胞需要氧化 与 HSC、骨髓细胞和 B 细胞相比，葡萄糖通过 PDH 调节丙酮酸水平和氧化还原稳态 代谢灵活的祖细胞。在目标 1 中，我们将测试介导 PDH对DP细胞的影响。在目标 2 中，我们将确定阻断 LDHA/B 或 PDH 单独或组合存在于 HSC 和限制性祖细胞中。在目标 3 中，我们将研究以下角色的作用： LDHA/B 和 PDH 在造血和胸腺再生中的作用。这些实验将确定 葡萄糖对体内 HSC 和祖细胞代谢池的贡献，系统地测试了以下想法： HSC 具有糖酵解功能，可识别中央碳代谢调节造血的机制 分化和再生。更一般地说，我们的实验将解决基本的代谢问题 通过测试体内干细胞或祖细胞是否在葡萄糖发酵或氧化之间切换来提出问题 教科书的观点，或者体内某些细胞类型是否能够耐受两种主要葡萄糖分解代谢途径的丧失。