The role of glycolysis and glucose oxidation in hematopoiesis

项目总结： 营养素的新陈代谢已经用普通组织或体外进行了研究。悬而未决的问题 就是体内干细胞如何代谢营养物质。我们对干细胞新陈代谢的理解 代谢组学通常需要数百万个细胞，而干细胞很少，这一事实限制了研究。我们 开发了描述代谢组并追踪稳定同位素标记的造血细胞营养物质的方法 干细胞(HSCs)和其他从组织中提纯的稀有细胞类型。我们发现T细胞的祖细胞在 与造血干细胞、髓系和B细胞限制的祖细胞相比，胸腺不需要葡萄糖，而不是 普遍的观点认为，造血干细胞比造血祖细胞更能分解糖酵解。稳定同位素示踪 实验表明，在骨髓而不是胸腺，糖酵解和TCA循环是 已断开连接。血液性丙酮酸脱氢酶(PDH)的丢失，这是连接 糖酵解到TCA循环，减少双阳性(DP)T细胞祖细胞的数量，但不影响 造血干细胞或其他造血细胞类型。自相矛盾的是，PDH的丢失并没有损害胸腺中的TCA循环， 但导致丙酮酸的积累和氧化还原平衡的异常。不氧化葡萄糖的细胞 传统上认为TCA循环通过糖酵解通过乳酸脱氢酶将葡萄糖发酵成乳酸 (LDH)。两种LDH亚型之一的LDHA的造血细胞丢失阻碍了红系的发育 造血祖细胞，但不包括造血干细胞、T细胞祖细胞或其他受限的造血祖细胞。单元格类型 造血系统中LDH和PDH需求的特异性提出了为什么不同的问题 干细胞或祖细胞类型选择使用乳酸脱氢酶介导的发酵或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的作用 造血干细胞和受限祖细胞的新陈代谢。我们的假设是T细胞前体细胞需要氧化 葡萄糖通过PDH调节丙酮酸水平和氧化还原动态平衡，与HSCs、髓系细胞和B细胞相反 在新陈代谢方面具有灵活性的祖细胞。在目标1中，我们将测试调节 PDH对DP细胞的影响。在目标2中，我们将确定阻断LDHA/B或 在造血干细胞和限制性祖细胞中，PDH单独或联合使用。在目标3中，我们将调查 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HSCs和祖细胞代谢物池的贡献，系统地测试了这一想法 肝星状细胞是糖酵解细胞，识别中枢碳代谢调节造血的机制。 分化和再生。更广泛地说，我们的实验将解决一个基本的新陈代谢问题 通过测试体内的干细胞或祖细胞是否在葡萄糖发酵或氧化之间切换而提出的问题 教科书上的观点是，如果体内的某些细胞类型耐受两种主要的葡萄糖分解代谢途径的丧失。

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.