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.
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