Abstract
Purpose :
We have demonstrated that hyperoxia creates a metabolic state that induces glutamine fueled anaplerosis in retinal Müller cells and brain astrocytes. Given our previous findings of the protection against oxygen-induced retinopathy (OIR) by hepatic Hypoxia Inducible Factor (HIF) stabilization during hyperoxia, we hypothesize that HIF stabilization by dimethyloxalylglycine (DMOG) modifies glutamine utilization by hepatocytes.
Methods :
RT-qPCR was used to quantify the expression of malic enzyme 1 and 2 (ME1/2), malate dehydrogenase, and pyruvate carboxylase (PCX) in mRNA from primary hepatocytes isolated from C57BL/6J mice. For steady state labeling experiments, hepatocytes were cultured in media containing [13CU] glutamine with or without DMOG for 24 hours in normoxia, followed by 8 hours in normoxia or hyperoxia. Intracellular metabolites were extracted and processed for GC/MS analysis. IsoCor was used to calculate the mass distribution vectors and 13C atom percent enrichment from peak intensities at selected ions monitored at low resolution for Citrate, α-ketoglutarate, succinate , fumarate, and malate.
Results :
DMOG increased the expression of ME1/2 and PCX. DMOG increased 13C atom percent enrichment in succinate, malate, and fumarate; 13C atom percent enrichment of citrate and α-ketoglutarate were unchanged. DMOG increased M3 malate and M3 fumarate isotopologues in normoxia and hyperoxia.
Conclusions :
DMOG modulates hepatocyte metabolism to increase the relative utilization of glutamine-derived carbon as a substrate for synthesizing TCAC intermediates. While the observed isotopic labeling patterns indicate both branches of TCA metabolism remain active under hypoxiamimesis, the elevation in M3 fumarate and M3 malate, along with upregulated ME1/2, demonstrate a HIF-induced augmentation of reductive TCAC flux even during hyperoxia.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.