(124e) A Hierarchical Approach to Identify Phenotype Relevant Pathways

We applied a hierarchical approach to integrate the phenotypic with metabolitc and gene expression profiles to identify the pathways that regulate free fatty acid (FFA) and tumor necrosis factor–alpha (TNF-alpha)induced cytotoxicity and lipid accumulation in HepG2/C3A cells. FFA and TNF-alpha have been implicated in the pathogenesis of many obesity-related metabolic disorders. Saturated fatty acid was found to be cytotoxic and its toxicity was exacerbated by TNF-alpha. In contrast, unsaturated fatty acids enhanced lipid accumulation in the hepatocytes.

The hierarchical framework consisted of three stages. 1) Different phenotypes induced by TNF-alpha and FFA were characterized, for example, a cytotoxic phenotype was characterized by the level of lactate dehydrogenase (LDH) release. Important metabolites that contribute to separating the phenotype were identified using a discriminant analysis of the twenty seven metabolites. Relevant metabolic pathways were suggested by the identified metabolites. 2) cDNA microarray was used to measure the global gene expression changes induced by FFAs and TNF-alpha. Gene set enrichment analysis (GSEA) of the microarray data identified the relevant pathways in addition to those identified by the metabolite analysis. The pathways identified to be enriched included mitochondria-related pathways, oxidative-stress-related pathways and fatty acid metabolism. 3) Finally, the gene expression and metabolite profiles were integrated with genetic algorithm coupled partial least squares analysis (GA/PLS) to identify the genes most relevant to the metabolites identified in the first step to be highly correlated with the cytotoxicity. GA/PLS was applied to identify the genes relevant to a metabolite, for example intracellular triglyceride accumulation, which suggested potential pathways for modulating the phenotype. The cytotoxic and cytoprotective pathways identified, as well as the experimentally validated pathways will be discussed.