LINKING GENOMICS TO FUNCTION VIA METABOLIC PHENOTYPING

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Recombinant strains with well defined genetic backgrounds are often found to exhibit small functional differences despite specific changes at the genetic level while in other cases, single gene alterations result in profound phenotypic variations. Although a first step in explaining such macroscopic differences is to probe the full detail of the expression phenotype by genome-wide expression measurements, transcription data alone are insufficient to elucidate the actual metabolic state of a cell and its functions. The latter require information about intracellular metabolic fluxes, which constitute fundamental determinants of cell physiology and excellent metrics of cell function. "Metabolic phenotyping" is the process and methods of determining intracellular fluxes as determinants of the cellular metabolic state. Combined with transcription data, the investigators provide a complete framework for analyzing the effect of drugs and studying disease. This application integrates the expertise of three participating laboratories for the purpose of combining metabolic and expression phenotyping to elucidate central carbon and lipid metabolism in model mouse hepatoma and hepatocyte cultures. Determination of intracellular fluxes will follow a systems approach termed metabolic reconstruction whereby the entire metabolic network is configured such as to best represent macroscopic rate and isotopic label distribution measurements made by GC-MS. Of particular attention are issues of observability, redundancy, and solution stability to ensure method feasibility and accuracy of the results. Differential transcription data will be obtained by DNA microarrays for mouse genes involved in central carbon metabolic, gluconeogenic and lipid biosynthetic pathways, as well as for other genes with particular expression variability that will be identified in the course of the research. Bioinformatics methods and programs, developed over the past 12 years will be deployed for this purpose. The general goal of the research is to identify relationships between the metabolic phenotype as defined above and the transcriptional state as defined by expression data of consequence in pathways important to diabetes. Specific aims will focus on flux quantification in mouse hepatoma and hepatocyte cultures to elucidate glutamine metabolism and lipogenesis, other central metabolic pathways and cholesterol synthesis, the effect of nutrients, hormones and drugs like Metformin and finally, pleiotrophic effects generated by altering the normal expression of a single gene, such as over-expressing the truncated verion of sterol-regulatory element binding protein-1a in transgenic mice. The broader contribution of this research is to extend the paradigm of holistic transcriptional investigation introduced by DNA microarray technologies to the study of metabolic level processes by metabolic phenotyping. As such, it holds the promise of identifying most, if not all points in metabolism affected by the action of drugs or genetic modifications thus guiding future programs of drug development and gene therapy.