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

全面、跨平台验证的组织内和组织间代谢的 13C 通量测量

• 批准号: 9196135
• 负责人: Richard G Kibbey
• 金额: $ 52.04万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9196135
• 关键词: Acetates; Adipose tissue; Aging; Agreement; Alanine; Amino Acids; Animal Model; Appearance; Aspartate; Bypass; Carbon; Cell model; Cellular Metabolic Process; Citric Acid Cycle; Continuous Infusion; Data; Data Analyses; Diabetes Mellitus; Disease; Etiology; Evolution; Fatty acid glycerol esters; Freezing; Glucose; Glutamates; Glutamine; Glycerol; Goals; Hepatic; High Fat Diet; Human; In Situ; Insulin Resistance; Ions; Isotope Labeling; Kinetics; Label; Letters; Liver; Mass Spectrum Analysis; Measurement; Measures; Metabolic; Metabolic Diseases; Metabolic Pathway; Metabolism; Methods; Mitochondria; Muscle; Non-Insulin-Dependent Diabetes Mellitus; Organ; Pathogenesis; Pathologic; Pathway interactions; Pattern; Plasma; Play; Positioning Attribute; Propionates; Published Comment; Publishing; Pyruvate; Rattus; Resolution; Rodent; Role; Shunt Device; Side; Stable Isotope Labeling; Technical Expertise; Testing; Time; Tissues; Tracer; abstracting; awake; basal insulin; base; feeding; glucose metabolism; glucose production; hepatic gluconeogenesis; improved; in vivo; innovation; insulin sensitivity; liver metabolism; mitochondrial dysfunction; mitochondrial metabolism; nonalcoholic steatohepatitis; novel; symposium

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.

摘要 线粒体功能障碍已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 NMR是评估线粒体通量如TCA循环的主要方法， 回补NMR测量从标记底物如[3- 13 C]乳酸盐到氨基酸天冬氨酸的13 C流 和谷氨酸，其原理是通过与TCA中间体β-酮戊二酸的13 C交换，谷氨酸被 用于13 C与TCA循环中间体混合的“阱”。由于体内核磁共振需要主要的技术专长， 存在从进入肝代谢的前体和从 稳态C标记，估计VTCA，特别是使用C-丙酸。虽然原则上这些方法 13 13 应该与组织测量一致，但在几个比率中观察到了很大的差异，包括 VTCA。这种分歧是最近几篇评论、信件和研讨会的主题，但缺乏一个明确的 分辨率解决这一争议是理解线粒体在发病机制中的作用的关键， 治疗肝脏胰岛素抵抗、非酒精性脂肪性肝炎和2型糖尿病。 解决争议的方法是测量TCA循环中间体的13 C位置标记。NMR在 体内和稳态血浆葡萄糖方法产生线粒体代谢的间接测量， 依赖于一些关于细胞溶质谷氨酸和天冬氨酸的关系的不完全测试的假设。 我们最近发布了质量同位素多纵标光谱分析（MIMOSA）平台，用于 细胞内代谢的全面、逐步、综合分析（参见Alves等人，细胞代谢， 2015年）的报告。MIMOSA的“质量同位素异构体”方面使用基于MS/MS的稳定同位素的离子碎裂分析。 同位素标记的代谢物以鉴定碳特异性标记位置。“多纵”方面是一大 创新，允许直接评估标记流沿着交叉途径，包括线粒体 由于灵敏度限制，位置NMR无法获得中间体。我们在一个细胞中使用了MIMOSA 模型，并发现先前通过稳态谷氨酸标记的回补措施也高达3倍 高是由于线粒体稀释途径，否则无法测量。 我们建议在体内动物模型中应用MIMOSA，以建立肝脏VTCA的基础事实， 其他关键通量（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（目标2），并开发改进的测量方法。目标3将评估血浆 由组织特异性代谢产生的标记模式可能影响组织数据的解释。我们 初步数据确定脂肪中的乳酸-甘油分流可能具有病理作用， 干扰体内通量测量。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。一个主要的转化目标是开发一个交叉验证的体内 使用MS或NMR分析平台，用于人类或啮齿动物。