367941 Metabolic Switching of Gluconeogenesis in Hepatoma Cells

Wednesday, November 19, 2014: 9:06 AM
214 (Hilton Atlanta)
Woo Suk Ahn, Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA and Maciek R. Antoniewicz, Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE

Diabetes is a metabolic disease characterized by a high level of blood glucose caused by the abnormal regulation of glucose production in the liver. The study of the metabolic regulation of gluconeogenesis in liver can correspondingly provide us valuable information for drug evaluation and diagnosis.  Here, we studied gluconeogenic metabolism using 13C-metabolic flux analysis (13C-MFA), a powerful methodology capable of revealing in vivo metabolic activity. 13C-MFA methodology can quantify reversible, cycling and branched reactions in biochemical networks by the introduction of isotopic tracers, e.g. 13C or 2H atom-based substrates, and mass spectrometry. As a model system for gluconeogenesis, we used a rat hepatoma cell line that can produce glucose in media containing gluconeogenic precursors, e.g. glycerol, lactate and glutamine. We activated gluconeogenesis in hepatoma cells using a cAMP analogue as a phosphoenolpyruvate carboxykinase (PEPCK) transcriptional regulator and by dexamethasone, a glucocorticoid. Based on the culture conditions, we selected isotopic tracers that allowed us to validate the gluconeogenesis model and to estimate central metabolism as well as gluconeogenesis metabolic flux: [U-13C]glycerol to quantify upper gluconeogenesis and the pentose phosphate pathway, [U-13C]glutamine for the TCA cycle and [U-13C]lactate for the pathway validation of gluconeogenesis. Moreover, we constructed a metabolic model based on 13C-labeling information of intracellular metabolites from the multiple tracer experiments. The metabolic model covers gluconeogenesis, glycolysis, the pentose phosphate pathway, pyruvate and TCA cycles, amino acid metabolism, ketone body and albumin production, and fatty acid metabolism. Furthermore, we performed combined 13C-MFA which enables us to observe whole metabolic information by the simultaneous estimation of labeling data from multiple tracer experiments, rather than fragmentized results. Surprisingly, we observed metabolic switching of gluconeogenesis by the cAMP analogue and glucocorticoid. The cAMP analogue showed PEPCK activation by increasing glucose production, but dexamethasone bypassed PEPCK flux to pyruvate kinase as well as glucose production. Dexamethasone shows bifunctional roles in mediating anabolism and catabolism during gluconeogenesis through controlling pyruvate cycling. This study provides a cell-based platform to evaluate metabolic disease and drug efficacy.

Extended Abstract: File Not Uploaded
See more of this Session: Proteomics & Metabolomic Approaches to Systems Biology
See more of this Group/Topical: Topical Conference: Systems Biology