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.

抽象的 线粒体功能障碍已被提出是胰岛素抵抗，衰老和代谢的主要因素 疾病。 13C NMR体内是评估线粒体通量（如TCA周期和）的主要方法 综合症。 NMR测量13C从标记的底物等标记的底物流动到氨基酸天冬氨酸 和谷氨酸，其理由是，通过13C交换与TCA中间型酮戊二酸，谷氨酸是 与TCA循环中间体混合13C的“陷阱”。由于体内NMR需要主要的技术专长，因此 存在从进入肝素代谢的前体中测量血浆葡萄糖标记的方法 稳态C标记，估计VTCA，尤其是使用C-丙酸。虽然原则上这些方法 13 13 应同意组织测量，在包括 VTCA。差异是最近几个评论，信件和座谈会的主题，但缺乏明确的话题 解决争议是了解线粒体在发病机理和 肝胰岛素抵抗，非酒精性脂肪性肝炎和2型糖尿病的治疗。 争议的解决方案是测量TCA循环中间体的13C位置标记。 nmr in 体内和稳态等离子体葡萄糖方法产生线粒体代谢和 取决于一些关于与胞质谷氨酸和天冬氨酸的关系的未完全测试的假设。 我们最近发布了用于大众同位素多纵谱分析（Mimosa）的平台 全面的，逐步的细胞内代谢综合分析（参见Alves等人，细胞代谢， 2015）。 Mimosa的“质量同位素”方面使用稳定的基于MS/MS的离子碎片分析 同位素标记的代谢产物，以鉴定碳特异性标签位置。 “多边形”方面是主要的 允许直接评估沿着相交途径的标签流的创新，包括线粒体 由于敏感性限制，位置NMR无法访问的中间体。我们在单元中使用了含羞草 模型，发现先前通过稳态谷氨酸标记对旋律的测量也高达3倍 由于线粒体稀释途径，无法测量的线粒体稀释途径高。 我们建议在体内的动物模型中应用含羞草，以建立肝VTCA和 其他关键通量（AIM 1）。我们将使用这些信息来测试体内使用的当前方法的准确性 人类和啮齿动物研究（AIM2）并开发了改进的测量方法。 AIM 3将评估血浆 由组织特异性代谢产生的标记模式，可能会影响组织数据的解释。我们的 初步数据识别脂肪中的糖尿病甘油分流，除了 在体内混淆通量测量。因此，针对此途径可能是一种新颖的治疗方法 糖尿病或其他代谢疾病。一个主要的翻译目标是开发体内交叉验证 分析平台使用MS或NMR人类或啮齿动物。