FACTORS CONTROLLING METABOLIC FLUX IN THE LIVER

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 老鼠是生物学研究的基石。研究人类生理学和疾病的第一个显而易见的选择是在人类身上进行实验，但如果出于伦理或安全方面的考虑，这是不可能的，第二个最好的模型系统是老鼠。与其他动物模型相比，小鼠更便宜，需要更少的空间，但小鼠的主要优势是，它相对直接地操纵它的基因组。这一优势为基因在疾病期间如何调控和失调提供了丰富的信息。它导致了以前未知的激素和受体的发现，这些激素和受体催生了全新的临床药物类别。它使我们能够识别新陈代谢控制中的脆弱点，这些点可能最终被证明是几种疾病的根本原因。利用小鼠作为研究人类疾病的工具的关键是找到更好的方法来衡量基因干预对生理的功能结果。我的DBP需要研究资源开发尖端技术来监测代谢流量，主要目标是了解疾病的分子和代谢基础。我的工作分为三个方面。 项目1利用资源中开发的新陈代谢流量测量技术来支持生物学家，这些生物学家创造了重要的遗传小鼠模型，但没有能力对小鼠进行表型鉴定。这个子项目推动了研究资源对老鼠新陈代谢的研究方式进行了革命性的改变。在这项资助的开始，并不确定磁共振是否可以用来测量小鼠的代谢流量。通过对大鼠和人类实验方案的小型化，我们能够通过汇集3只小鼠的样本来完成类似的测量。这是一个实质性的进步，因为第一次，代谢流量可以通过核磁共振在基因改变的途径中测量到。在过去的5年里，在资源中心设备和人员的支持下，我们已经能够改进这些实验，现在我们可以在一只鼠标上进行同样的测量。这一进展是巨大的，因为我们需要研究的许多小鼠模型发生在罕见的基因类型或脆弱的表型中。换句话说，每一只鼠标都是一种宝贵的资源。 在项目2中，我们利用研究资源提供的技术，在经典的糖异生酶PEPCK靶向和分级丢失的小鼠模型中，测量通过肝脏葡萄糖产生途径的代谢通量。我们证明，与课本上的生物化学相反，这种酶不是糖异生的速率决定因素。这一发现颠覆了40年前的研究范式，即糖尿病高血糖的发生是因为糖尿病肝脏过度表达PEPCK。为了进一步研究控制肝脏代谢流量的因素，我们需要在研究资源中进行额外的技术开发，特别是通过混合质谱学和核磁共振方法来实现技术的微型化。将MS与当前的RR方法相结合来测量通量是一个重要的目标，因为MS比核磁共振敏感得多，而且还可以检测代谢物中的同位素示踪剂的掺入。 在项目3中，我们将需要RR来提供x核低温探测器。据我们所知，冷冻探针技术还没有被用来在完整的功能正常的小鼠心脏中实现实时光谱分析。我们之前利用该资源开发了一种间接检测技术来测量跳动的小鼠心脏中的底物氧化。该方法提供了良好的灵敏度，但以损失~(13)C化学位移分散为代价。因此，在代谢产物中存在显著的13C富集物时，不能测量13C谷氨酸标记动力学，这些代谢物在1H维度与谷氨酸共振，最显著的是13C富集脂。在600 MHz光谱仪上获取10 mm x核冷探头将使我们能够监测浓缩动力学，其灵敏度与间接检测相当，但具有13C化学位移分散的明显好处。利用这一优势，我们将能够监测多种代谢物的浓缩动力学。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The mouse is a cornerstone of biological research. The first obvious choice to study human physiology and disease is to perform experiments in Man, but when that is not possible, because of ethical or safety concerns, the next best model system is the mouse. The mouse is less expensive and requires less room to house than other animal models, but the primary advantage of the mouse is that it is relatively straight forward to manipulate its genome. This advantage has provided a bonanza of information about how genes are regulated and dysregulated during disease. It has lead to the discovery of previously unknown hormones and receptors that have birthed entire new classes of clinical drugs. It has allowed us to identify vulnerable points in the control of metabolism that may turn out to be the root cause of several diseases. The key to capitalizing on the mouse as a tool to study human disease is finding better ways to measure the functional outcome of genetic interventions on physiology. My DBP requires the Research Resource to develop cutting edge techniques to monitor metabolic flux with the primary goal of understanding the molecular and metabolic basis of disease. My work is divided into three efforts. Project 1 utilizes technologies for measuring metabolic flux developed in the Resource to support biologists who have created important genetic mouse models, but do not have the capacity to phenotype the mice. This subproject has driven the Research Resource to revolutionize the way mouse metabolism is studied. At the outset of this grant it was not certain that magnetic resonance could be used to measure metabolic flux in mice. Through miniaturization of rat and human protocols we were able accomplish similar measurements by pooling samples from 3 mice. This was a substantial advance, because for the first time, metabolic flux could be measured in a genetically altered pathway by NMR. In the past 5 years, with support of Resource equipment and personnel, we've been able to improve these experiments to the point that we can now make the same measurements in a single mouse. This advancement is enormous because many of the mouse models we need to study occur in rare genotypes or fragile phenotypes. In other words, every mouse is a precious resource. In Project 2, we utilize the technology provided by the Research Resource to measure metabolic flux through liver pathways of glucose production in mouse models with targeted and graded loss of the classic gluconeogenic enzyme PEPCK. We demonstrated that, contrary to textbook biochemistry, this enzyme is not rate determining for gluconeogenesis. This finding reversed the 4 decade old paradigm, that diabetic hyperglycemia occurs because the diabetic liver overexpresses PEPCK. To further investigate the factors controlling metabolic flux in liver we require additional technology development in the Research Resource, in particular with regard to miniaturization of the technique by hybridizing mass spectrometry and NMR approaches. Integration of MS with current RR methods to measure flux is an important goal because MS is far more sensitive than NMR and also detects incorporation of isotope tracers into metabolites. In Project 3, we will need the RR to provide an x nucleus cryoprobe. To our knowledge cryoprobe technology has not been utilized to achieve real-time spectroscopy in the intact functioning mouse heart. We previously utilized the Resource to develop an indirect detection technique to measure substrate oxidation in the beating mouse heart. The method provided excellent sensitivity but at the expense of lost 13C chemical shift dispersion. Consequently 13C glutamate labeling kinetics could not be measured in the presence of significant 13C enrichment in metabolites that co-resonated with glutamate in the 1 H dimension, most notably, 13C enriched lipid. Acquisition of the 10 mm x nucleus cold probe on the 600 MHz spectrometer will allow us to monitor enrichment kinetics, at a sensitivity comparable to indirect detection, but with the obvious benefit of 13C chemical shift dispersion. Using this advantage, we will be able to monitor the enrichment kinetics of multiple classes of metabolites.