Comprehensive, Cross Platform-Validated 13C Flux Measures of Intra-and Inter-tissue Metabolism

摘要 线粒体功能障碍被认为是胰岛素抵抗、衰老和代谢的主要因素。 疾病。体内~(13)C核磁共振一直是评估线粒体通量的主要方法，如TCA循环和 复原力。核磁共振测量13C从标记底物(如[3-13C]乳酸)流入氨基酸天冬氨酸 和谷氨酸，其原理是通过与三氯乙酸中间体-酮戊二酸进行13C交换，谷氨酸是 ~(13)C与三氯乙烷循环中间体混合的“陷阱”。由于体内核磁共振需要主要的技术专长， 有方法可以测量进入肝脏代谢的前体的血糖标记，并从 稳态C标记，估计VTCA，特别是使用C-丙酸。尽管原则上这些方法 13 13 如果与组织测量结果一致，在几个比率上观察到了很大的差异，包括 VTCA。这种分歧是最近几篇评论、信件和座谈会的主题，但缺乏明确的 决议。解决这一争议是了解线粒体在发病机制中作用的关键。 治疗肝脏胰岛素抵抗、非酒精性脂肪性肝炎和2型糖尿病。 争议的一个解决方案是测量TCA循环中间体的13C位置标记。核磁共振In 体内和稳态血糖方法产生线粒体代谢的间接测量和 依赖于一些关于胞浆中谷氨酸和天冬氨酸关系的不完全测试的假设。 我们最近发布了质量同分异构体多坐标光谱分析(MIMOSA)平台 全面的、逐步的、综合的细胞内代谢分析(见Alves等人的《细胞代谢》， 2015年)。含羞草的“质量同分异构体”方面使用了基于MS/MS的离子碎裂分析稳定的- 同位素标记的代谢物，以识别碳特定的标记位置。“多纵坐标”方面是一个主要方面。 允许直接评估包括线粒体在内的交叉路径上的标记流的创新 由于灵敏度限制，位置核磁共振无法接触到的中间体。我们在一间牢房里用含羞草 模型，并发现以前通过稳态谷氨酸标记的抗逆反应措施也高达3倍 高是由于线粒体稀释途径，否则无法测量。 我们建议在活体动物模型中应用Mimosa来建立肝脏VTCA和 其他关键流量(目标1)。我们将使用这些信息来测试目前在体内使用的方法的准确性 人类和啮齿动物研究(AIM2)，并开发改进的测量方法。AIM 3将评估血浆 由组织特定代谢产生的标记模式可能会影响组织数据的解释。我们的 初步数据表明，脂肪中的乳酸-甘油分流可能具有病理效应 体内的混杂通量测量。因此，靶向这一途径可能是一种新的治疗方法 糖尿病或其他代谢疾病。一个主要的翻译目标是开发一种在体内交叉验证的 使用MS或核磁共振的分析平台，无论是人类还是啮齿动物。

Abstract Mitochondrial dysfunction has been proposed as a major factor in insulin resistance, aging, and metabolic diseases. 13C NMR in vivo has been the main method to assess mitochondrial fluxes like the TCA cycle and anaplerosis. NMR measures 13C flow from labeled substrates like [3-13C]lactate into the amino acids aspartate and glutamate, with the rationale that via 13C exchange with the TCA intermediate -ketoglutarate, glutamate is a “trap” for 13C mixing with TCA cycle intermediates. Because NMR in vivo requires major technical expertise, methods exist to measure plasma glucose labeling from precursors that enter hepatic metabolism and, from steady-state C labeling, estimate VTCA, particularly using C-propionate. Although in principle these methods 13 13 should agree with tissue measurements, large discrepancies have been observed in several rates, including VTCA. The divergence is the subject of several recent commentaries, letters, and symposia but lacks a clear resolution. Resolving the controversy is key to understand the role of mitochondria in the pathogenesis and treatment of hepatic insulin resistance, nonalcoholic steatohepatitis, and type 2 diabetes. A solution to the controversy is to measure 13C positional labeling of TCA cycle intermediates. NMR in vivo and steady-state plasma glucose methods yield indirect measures of mitochondrial metabolism and depend on some incompletely tested assumptions about relationships with cytosolic glutamate and aspartate. We recently published the Mass Isotopomeric Multi Ordinate Spectral Analysis (MIMOSA) platform for comprehensive, stepwise, integrated analysis of intracellular metabolism (see Alves et al., Cell Metabolism, 2015). The “mass isotopomer” aspect of MIMOSA uses MS/MS-based ion fragmentation analysis of stable- isotope-labeled metabolites to identify carbon-specific label positions. The “multi-ordinate” aspect is a major innovation that allows direct assessment of label flow along intersecting pathways, including mitochondrial intermediates that are inaccessible by positional NMR due to sensitivity limitations. We used MIMOSA in a cell model and found that previous measures of anaplerosis by steady-state glutamate labeling were up to 3x too high due to mitochondrial dilution pathways that could not otherwise be measured. We propose to apply MIMOSA in an animal model in vivo to establish the ground truth for hepatic VTCA and other key fluxes (Aim 1). We will use the information to test the accuracy of present methods used in vivo for human and rodent studies (Aim2) and develop improved measurement methods. Aim 3 will assess plasma labeling patterns resulting from tissue-specific metabolism that can impact the interpretation of tissue data. Our preliminary data identify a lactate-glycerol shunt in adipose that may have pathologic effects in addition to confounding flux measurements in vivo. Consequently, targeting this pathway may be a novel treatment for diabetes or other metabolic diseases. A major translational goal is to develop a cross-validated in vivo analytic platform using either MS or NMR either humans or rodents.