UCP1 and the regulation of mitochondrial respiration in brown adipose tissue by oxaloacetate

项目总结 通过三氯乙酸循环控制代谢流量的研究可以追溯到许多年前 几十年。然而，仍有一些问题需要澄清。一个重要的，但很重要的 被忽视的问题是草酰乙酸酯(OAA)对琥珀酸脱氢酶的抑制作用 (SDH)在调节线粒体功能中的作用。我实验室的最新发现集中在骨骼上 肌肉线粒体显示，虽然低膜电位(ΔΨ)驱动呼吸，但低ΔΨ 还会启动一系列导致线粒体OAA积累的事件，抑制 SDH，以及随之而来的呼吸作用的抑制或代谢“刹车”。鉴于ΔΨ如此之低 似乎是启动因素，目前的建议集中在高龄津贴在以下方面的作用 调节棕色脂肪组织(BAT)的呼吸，其中ΔΨ天生低，这是由于 解偶联蛋白1(UCP1)的作用 我们的总体假设是线粒体OAA代谢和SDH抑制是 受UCP1控制的ΔΨ和下游事件之间可定义的相互作用调节 这是有生理后果的。我们的目标是解决三个问题。目标1将描述 由UCP1启动的分子事件调节ΔΨ，最终导致OAA抑制O2通量。 AIM 2检查活体小鼠中UCP1的扰动，以确定这是否会转化为OAA效应 线粒体和细胞功能。目的3试图调节OAA对SDH的抑制 这种方式可能被证明对线粒体、细胞和全身新陈代谢有利。 为了实现我们的目标，我们将使用一种新的核磁共振分析来评估线粒体 OAA含量以及其他代谢物。我们了解高龄津贴影响的一个主要原因 缺乏呼吸作用的是，由于代谢物不稳定，OAA很难测定。事实上, 代谢组学服务，无论是商业的还是大学核心的，都不提供量化的 高龄津贴。我们的研究还将使用我们开发的现有技术的新改编 在钳制浓度的ADP下评估线粒体呼吸。这样，我们就可以评估 在通常确定的状态3和状态4之外的有丝分裂呼吸，这两种状态不是 生理状态。最后，我们将使用最新的质谱学方法对目标 代谢物表达和针对代谢流的[13C]同位素示踪研究。 我们的项目适用于肥胖及其并发症的临床问题，因为 了解蝙蝠的生理学和它是如何调节的将增加我们对 全身能量学。

PROJECT SUMMARY Control of metabolic flux through the TCA cycle has been investigated dating back many decades. However, there are issues that still require clarification. One important, but largely neglected, issue regards the role of oxaloacetate (OAA) inhibition of succinate dehydrogenase (SDH) in regulating mitochondrial function. Recent findings in my laboratory focused on skeletal muscle mitochondria show that while low membrane potential (ΔΨ) drives respiration, low ΔΨ also initiates a sequence of events leading to mitochondrial OAA accumulation, inhibition of SDH, and consequent inhibition or metabolic “braking” of respiration. Given that low ΔΨ appears to be the initiating factor, the current proposal is focused on the role of OAA in regulating respiration in brown adipose tissue (BAT) wherein ΔΨ is intrinsically low due to the action of uncoupling protein 1 (UCP1). Our overall hypothesis is that mitochondrial OAA metabolism and inhibition of SDH is regulated by definable interactions between UCP1 controlled ΔΨ and downstream events and that there are physiological consequences. Our aims address three issues. Aim 1 will delineate molecular events initiated by UCP1-regulated ΔΨ and culminating in OAA inhibition of O2 flux. Aim 2 examines UCP1 perturbations in live mice to determine if this translates to OAA effects on mitochondrial and cellular function. Aim 3 attempts to modulate OAA inhibition of SDH in a way that may prove beneficial towards mitochondrial, cellular, and whole-body metabolism. To accomplish our objectives, we will use a novel NMR assay to assess mitochondrial OAA content as well as other metabolites. A major reason why our knowledge of OAA effects on respiration is lacking is that OAA is difficult to assay since the metabolite is not stable. In fact, metabolomics services, whether commercial or in university cores, do not offer quantification of OAA. Our research will also use a novel adaptation of existing technology that we developed to assess mitochondrial respiration at clamped concentrations of ADP. In this way, we can assess mitochodnrial respiration beyond the often-determined state 3 and state 4, which are not physiological states. Finally, we will use up-to-date mass spectroscopy methods for targeted metabolite expression and for [13C]isotopomer tracer studies directed at metabolic flux. Our project is applicable to the clinical issue of obesity and its complications since understanding BAT physiology and how it is regulated will add to our overall knowledge of whole-body energetics.