Physiological And Metabolic Magnetic Resonance Studies

生理和代谢磁共振研究

• 批准号: 8734089
• 负责人: Robert E London
• 金额: $ 35.67万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8734089
• 关键词: Affinity; Attention; Behavior; Calcium; Cell Extracts; Cells; Chemicals; Consumption; Development; Diet; Dynein ATPase; Elements; Energy-Generating Resources; Enzymes; Evaluation; Exposure to; Fertility; Gene Targeting; Genes; Genetic; Glucose; Glycolysis; Goals; Heavy Metals; Homeostasis; Incubated; Infertility; Inositol; Inositol Phosphate Metabolism Pathway; Inositol Phosphates; Investigation; Ions; Isoenzymes; Isomerism; Knock-out; Knockout Mice; Lactate Dehydrogenase; Link; Magnetic Resonance; Mammalian Cell; Mediating; Metabolic; Metabolism; Metals; Methods; Minerals; Mitochondria; Molecular Analysis; Mus; Organ; Oxidative Phosphorylation; Phosphotransferases; Physiological; Physiological Processes; Phytic Acid; Polyphosphates; Predisposition; Production; Pyruvate; Reactive Oxygen Species; Receptor Activation; Research; Respiration; Role; Sperm Motility; Spermatogenic Cell; Sulfhydryl Compounds; Suspension substance; Suspensions; System; Techniques; Toxin; Wild Type Mouse; Work; absorption; aerobic glycolysis; cell injury; cell motility; environmental agent; environmental chemical; interest; male; novel; oxidation; phytate; sperm cell

This project investigates how chemical toxins or physical factors alter metabolic processes. NMR methods provide a unique approach for the investigation of metabolic and physiological processes in intact systems, perfused organs, cell suspensions, as well as by examination of cell extracts. The main studies performed as part of this research effort during the past year are summarized below: Project 1. Metabolic perturbations of lactate dehydrogenase C knockout. Sperm produce high levels of ATP, 70% of which is utilized by axonemal dynein to drive sperm motility. Glycolysis was shown to be essential for ATP production in sperm by using gene targeting to disrupt some of these genes. The gene for the glycolytic enzyme lactate dehydrogenase C (Ldhc) also is expressed predominantly in spermatogenic cells, and LDHC is abundant in sperm. We demonstrated previously that a knockout (KO) of the lactate dehydrogenase type C (Ldhc) gene disrupted male fertility and caused a considerable reduction in sperm glucose consumption, ATP production, and motility. While that study used mice with a mixed genetic background, the present study used C57BL/6 (B6) and 129S6 (129) Ldhc KO mice. We found that B6 KO males were subfertile and 129 KO males were infertile. Sperm from 129 wild-type (WT) mice have a lower glycolytic rate than sperm from B6 WT mice, resulting in a greater reduction in ATP production in 129 KO sperm than in B6 KO sperm. The lower glycolytic rate in 129 sperm offered a novel opportunity to examine the role of mitochondrial respiration in sperm ATP production and motility. We observed that in media containing a mitochondrial substrate (pyruvate or lactate) as the sole energy source, ATP levels and progressive motility in 129 KO sperm were similar to those in 129 WT sperm. However, when glucose was added, lactate was unable to maintain ATP levels or progressive motility in 129 KO sperm. The rate of respiration (ZO2) was high when 129 KO or WT sperm were incubated with lactate alone, but addition of glucose caused a reduction in ZO2. These results indicate that in the absence of glucose, 129 sperm can produce ATP via oxidative phosphorylation, but in the presence of glucose, oxidative phosphorylation is suppressed and the sperm utilize aerobic glycolysis, a phenomenon known as the Crabtree effect. Project 2. Characterization of inositol phosphate metabolism by NMR. Inositol 1,4,5-trisphosphate (IP3) is the intracellular messenger that links receptor activation to Ca ion mobilization, and has been intensely studied over several decades. The physiological roles of other inositol phosphates have received less attention, but have been of increasing interest in recent years as additional physiological roles have been identified. In general, the inositol phosphates can have multiple positional isomers. These can be distinguished by NMR, but are more difficult to identify using other methods. We have recently worked with the Shears group (LST) in order to identify inositol phosphate metabolites formed under various conditions.

本项目研究化学毒素或物理因素如何改变代谢过程。NMR方法为完整系统、灌注器官、细胞悬液中的代谢和生理过程的研究以及细胞提取物的检查提供了独特的方法。在过去一年中，作为这项研究工作的一部分进行的主要研究概述如下： 项目1。乳酸脱氢酶C基因敲除的代谢紊乱。精子产生高水平的ATP，其中70%被轴丝动力蛋白利用来驱动精子运动。糖酵解被证明是必不可少的ATP生产精子通过使用基因靶向破坏这些基因中的一些。糖酵解酶乳酸脱氢酶C（Ldhc）的基因也主要在生精细胞中表达，LDHC在精子中含量丰富。我们以前证明，敲除（KO）的乳酸脱氢酶C型（Ldhc）基因破坏男性生育能力，并导致精子的葡萄糖消耗，ATP的生产，和活力的显着减少。虽然该研究使用了具有混合遗传背景的小鼠，但本研究使用了C57 BL/6（B6）和129 S6（129）Ldhc KO小鼠。我们发现B6基因敲除的雄性不育，129个基因敲除的雄性不育。来自129野生型（WT）小鼠的精子比来自B6 WT小鼠的精子具有更低的糖酵解速率，导致129 KO精子中的ATP产生比B6 KO精子中的ATP产生减少更大。129个精子的糖酵解速率较低，这为研究线粒体呼吸在精子ATP产生和运动中的作用提供了一个新的机会。我们观察到，在含有线粒体底物（丙酮酸或乳酸）作为唯一能量来源的培养基中，129个KO精子的ATP水平和前向运动力与129个WT精子相似。然而，当加入葡萄糖时，乳酸不能维持129个KO精子的ATP水平或前向运动。当129个KO或WT精子单独与乳酸盐孵育时，呼吸速率（ZO 2）较高，但添加葡萄糖导致ZO 2降低。这些结果表明，在没有葡萄糖的情况下，129精子可以通过氧化磷酸化产生ATP，但在葡萄糖的存在下，氧化磷酸化被抑制，精子利用有氧糖酵解，这种现象被称为克拉布特里效应。 项目2.核磁共振法表征肌醇磷酸代谢。肌醇1，4，5-三磷酸（IP 3）是连接受体活化和钙离子动员的细胞内信使，几十年来一直受到广泛研究。 其他肌醇磷酸盐的生理作用较少受到关注，但近年来随着其他生理作用的确定，人们对肌醇磷酸盐的兴趣越来越大。通常，肌醇磷酸盐可以具有多种位置异构体。这些可以通过NMR来区分，但使用其他方法更难以识别。我们最近与Shears集团（LST）合作，以鉴定在各种条件下形成的肌醇磷酸代谢物。