Diabetes Genome Anatomy Project |
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Projects > Project 7
Project 7:
Interfacing Small Molecules with the Diabetes Genome Anatomy Project
Primary Investigator: Stuart L. Schreiber, Ph.D. (Harvard University)
Specific Aims
- Develop high-throughput phenotypic cell-based assays toward identifying small molecules with mechanisms of action important to the insulin pathway
- Develop quantitative means of measuring multiple sets of metabolites from different insulin-related cell types and different cellular conditions
Summary
Chemical genomics aims to use small molecules to probe genetic variation. Specifically, experimental design in chemical genomic screening takes into account genetic and genomic differences in identifying cellular reactions to small molecules. Further, this approach provides a temporal control over cell states not normally achieved by molecular biology methods. We are using chemical genomic methods in order to probe the effects of small molecules within the cellular context of type 2 diabetes.
In an effort to develop cell-based assays with relevance to type 2 diabetes, we are probing the effects of small molecules on adipocyte differentiation and on insulin signaling in liver cells. First, using an immortalized brown preadipocyte cell line developed by the Kahn lab, we will screen small-molecule collections for the ability to induce differentiation in the absence of a conventional differentiation cocktail. Other preadipocyte cell lines derived from IRS-1-/- and IRS-2-/- will be used in screening as well, establishing a matrix of experiments in which the genetic variation of these cell lines determines the cellular responses to small molecules. Differences in the ability of a small molecule to induce adipogenesis in these cell lines will inform us of the mechanism of action of these compounds. Second, using an insulin receptor tyrosine kinase inhibitor, we will screen small molecules for their ability to suppress the cellular effects of this inhibitor on a hepatoma cell line. Such small molecules will be likely to have stimulatory effects on the insulin signaling pathway in these cells.
A comprehensive identification and quantification of metabolites will be invaluable in the characterization of the cell lines derived from the various IRS knockout mice, as well as in the responses of these cell lines to small molecules. Cellular extracts will be derivatized and analyzed using gas chromatography-mass spectrometry (GC-MS) and high-pressure liquid chromatography (HPLC). We will then quantify in the aqueous fractions organic acids, glycolytic or gluconeogenic intermediates, and molecules and cofactors involved in nucleotide biosynthesis (such as ATP, ADP, NAD, and NADH). An approach resulting in the identification and quantification of lipid-metabolism intermediates such as fatty acids, triacylglycerol, and glycerol-3-phosphate will also be developed. The fluxes of metabolites obtained can be used either to develop a metabolite profile of cells either deleted in key insulin pathway genes or treated with small molecules arising from the planned screens, or to aid in hypothesis generation, by using these metabolites as inputs in computational models for established biochemical pathways. In the same way that chemical genomics aims to develop a quantitative basis for chemical and, eventually, biological space, metabolic profiling of cell lines will further our understanding and definition of metabolic space.