Physiological And Metabolic Magnetic Resonance Studies

这个项目研究化学毒素或物理因素如何改变新陈代谢过程。核磁共振方法为研究完整系统、灌流器官、细胞悬浮液中的代谢和生理过程以及通过检查细胞提取物提供了一种独特的方法。作为这项研究工作的一部分，过去一年进行的主要研究概述如下： 项目1.乳酸脱氢酶C基因敲除的代谢扰动。精子产生高水平的三磷酸腺苷，其中70%被轴线动力蛋白用来驱动精子的运动。糖酵解被证明对精子中的ATP产生是必不可少的，通过使用基因靶向来破坏其中一些基因。糖酵解酶乳酸脱氢酶C(Ldhc)基因也主要在生精细胞中表达，LDHC在精子中含量丰富。我们以前已经证明，C型乳酸脱氢酶(Ldhc)基因的敲除(KO)扰乱了男性的生育能力，并导致精子葡萄糖消耗、ATP产生和活力显著下降。虽然该研究使用的是具有混合遗传背景的小鼠，但本研究使用的是C57BL/6(B6)和129S6(129)Ldhc KO小鼠。我们发现B6KO雄性不育，129个KO雄性不育。129只野生型(WT)小鼠的精子比B6野生型(WT)小鼠的精子糖酵解率低，导致129 KO小鼠精子比B6 KO小鼠精子产生更多的ATP。129个精子中较低的糖酵解率为研究线粒体呼吸在精子三磷酸腺苷的产生和活力中的作用提供了新的机会。我们观察到，在以线粒体底物(丙酮酸或乳酸)为唯一能量来源的培养液中，129KO精子的ATP水平和前进运动能力与129WT精子相似。然而，当添加葡萄糖时，乳酸不能维持129 KO精子的ATP水平或前进运动能力。当129KO或WT精子单独与乳酸孵育时，呼吸速率(ZO2)较高，但添加葡萄糖可降低ZO2。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，但在葡萄糖存在的情况下，氧化磷酸化被抑制，精子利用有氧糖酵解，这一现象被称为克拉布特里效应。 项目2.核磁共振表征肌醇磷酸代谢。肌醇1，4，5-三磷酸(IP3)是一种连接受体激活和钙离子动员的细胞内信使，几十年来一直被广泛研究。其他肌醇磷酸盐的生理作用受到的关注较少，但近年来随着更多的生理作用被识别出来，人们越来越感兴趣。通常，肌醇磷酸盐可以有多个位置异构体。这些都可以通过核磁共振来区分，但使用其他方法更难识别。我们最近与Shears Group(LST)合作，以确定在不同条件下形成的肌醇磷酸代谢物。

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.