Retinal metabolic remodeling and hepatic nitrogen imbalance in oxygen induced retinopathy (OIR)

Abstract

Purpose : Hyperoxia suppresses blood vessel development in the first phase of OIR. Glutamine, which is only produced by Müller cells in the retina, is necessary for retinal endothelial cell (REC) proliferation and migration. Müller cells produce glutamine via amination of α-ketoglutarate or amidation of glutamate. An alternative pathway for ammonia disposal is the hepatic urea cycle. We here studied effects of hyperoxia on these retinal and hepatic pathways.

Methods : All cell types were cultured in normoxic condition for 24h to establish steady-state and then were moved to normoxic or hyperoxic (75% oxygen) conditions, in media containing stable isotope-labeled substrates. Metabolites were extracted by methanol/chloroform/water and were assayed using gas chromatography mass spectrometry. RT-qPCR was used to evaluate expression of the genes of interest. siRNA targeting HIF1α was used to knockdown HIF1α expression in primary cultured hepatocytes (n=3 or 6, all experiments).

Results : Using [¹³C₆] glucose, we found decreased M2 citrate and M2 glutamate in Müller cells cultured in hyperoxic conditions, implying loss of glycolytic carbon into TCA. This effect was not seen in REC cells. In our [¹³C₅] glutamine labeling experiment, we saw increased amounts of M5 glutamate, M4 aspartate, M4 fumarate, implying increased consumption of glutamine in Müller and REC cells. We confirmed these findings by using [¹³C₅] glutamine and 1-[¹³C] acetate in retinal explants cultures, exposed to hyperoxic conditions. Additionally, expression of GLS1, gene required for glutamine breakdown, was found to be increased in hyperoxic cells. We also saw decreased levels of CPS1 and ARG1 in cultured hepatocytes exposed to hyperoxia and siRNA against HIF1α confirmed it to be HIF1α dependent (p-value < 0.05).

Conclusions : We here demonstrate that the hyperoxia blocks entry of glycolytic carbon into TCA and increases glutaminolysis in Müller glia, which can consequently lead to 1) depleting retinal endothelial cells and neurons of glutamine 2) overproduction of ammonia, which is known to inhibit some enzymes and in addition activates TLR pathway. Decreased hepatocyte specific activity of CPS1 and ARG1 obeserved in hyperoxia can exacerbate the ammonia disposal phenotype.

This is a 2020 ARVO Annual Meeting abstract.