Abstract
Purpose :
We have previously demonstrated near complete protection against oxygen induced retinopathy (OIR) through pharmacological stabilization of Hypoxia Inducible Factor (HIF) during hyperoxic phase of the disease model. The purpose of this study was to investigate changes in retinal cell metabolism responsible for the therapeutic protection of retinal vasculature by HIF stabilizer Roxadustat (RXD).
Methods :
Cultured at 80% confluence, human retinal endothelial cells (RECs) and an immortalized muller glial cell line (MIO-M1) were exposed to RXD or PBS control for 24 h. Initial and final Glutamine (Gln) concentrations in the conditioned media were quantified by GC/MS using U-13C5 Gln as an internal standard. The rate of Gln utilization was calculated as the change in extracellular glutamine concentration. For the Glucose labeling experiment in MIO-M1 cells, unlabeled Glucose was replaced with an equal concentration of U-13C6 Glucose in the treatment media +/-RXD. After 24 hours, intracellular metabolites were extracted and processed for GC/MS analysis. Mass Isotopomer Distributions (MIDs) and Mean Enrichment (ME) were calculated using isocor. Unlabeled controls and cell-free blanks were included in each plate.
Results :
RXD lowered the overall rate of Gln utilization in RECs (Δ[Gln] = -0.857 mM for PBS vs -0.596 mM in RXD-treated cells t(10)=3.88, p=0.003. Likewise, Gln depletion from the media was reduced by RXD in MIO-M1cells (Δ[Gln] = -1.0mM for PBS vs -0.86mM in RXD-treated cells t(8.5)=3.4, p=0.0091. In MIO-M1 cultures, RXD increased ME of Alanine (83% vs 78.8%; p=1.55e-10) and Serine (13.47% vs 10.74%; p=2.70e-8). RXD decreased ME of Aspartate (3.92% vs 14%; p=9.0e-19), Fumarate (4.54% vs 14.29%; p=8.03e-18), Succinate (3.15% vs 11.3%; p=1.213e-11), and Proline (3.68% vs 9.76%; p=1.31e-17). There were also significant differences in the MIDs of these metabolites.
Conclusions :
While the decreased enrichment in TCA intermediates corroborates a well-documented HIF induced inhibition of glycolytic carbon entry into the TCA cycle, our findings of the increased serine and alanine enrichment support involvement of one-carbon and transamination pathways in the therapeutic effect of RXD. The reduction in Gln uptake under hypoxiamimesis also demonstrates that HIF stabilization modulates glutamine metabolism in RECs and MIO-M1 cells.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.