(535c) Quantifying Metabolism of Differentiated Adipocytes Using 13C-Metabolic Flux Analysis

Obesity and type II diabetes are increasing health epidemics in the United States and around the world and are characterized by dysregulation of lipid metabolism.  A more complete understanding of de novo lipid synthesis at the metabolic level is required to develop novel therapeutics for the treatment of these disorders.  Towards this end, we are implementing a systems biology approach using metabolic flux analysis and stable isotope ¹³C-tracers to determine the active reaction pathways in an adipocyte cell line 3T3-L1.  Differentiated white adipocytes were incubated with [U-¹³C]glucose, [1,2-¹³C]glucose, [U-¹³C]glutamine, and [5-¹³C]glutamine and the incorporation of ¹³C-atoms into metabolites from each tracer was analyzed with gas chromatography mass spectrometry (GC-MS). Metabolic fluxes were then determined using Metran software based on the EMU framework.

[U-¹³C]glucose and [1,2-¹³C]glucose did not reach isotopic steady-state within 24 hours.  We speculate that an initially unlabeled metabolite pool is exchanging with central metabolism and slowly becomes enriched.  [U-¹³C]glutamine and [5-¹³C]glutamine achieved isotopic steady-state in intracellular metabolites within 6 hours.  For glutamine tracers, the mass isotopomer distribution of citrate indicated activity of reductive carboxylation in which reverse citric acid cycle flux occurs from a-ketoglutarate to citrate.  This result provided evidence for isocitrate dehydrogenase as a potential modulator of adipocyte metabolism. Fatty acid profiling and mass isotopomer analysis of the differentiated adipocytes was conducted.  Fatty acids varied from 14 to 18 carbons in chain length, included even and odd chain length, and were saturated and monounsaturated.  For glutamine tracers, isotopomer spectral analysis (ISA) was conducted on the mass isotopomer distributions for fatty acids.  ISA suggested that odd-chain fatty acid synthesis proceeded from elongation of a three-carbon precursor rather than by alpha-oxidation as was previously assumed. Together, these results provide valuable new information regarding de novo lipogenesis in adipocytes that can be used to identify new drug targets to combat excessive fat accumulation.