Abstract
Purpose :
The mouse retina and proximal RPE-choroid tissue are essential for vision. To maintain function the retina rapidly uses metabolic fuel in the form of glucose. The RPE-choroid is more versatile and may metabolize a variety of fuels including glucose, lactate, pyruvate, succinate, and proline. This versatility allows for use of fuels supplied by circulation, but RPE cells also contain internal stores of glycogen and lipids from phagocytosed photoreceptor outer segments. Whether these internal fuels can support metabolism and to what extent are unknown. Here we investigate whether glycogen or lipids make substantial contributions to mitochondrial activity.
Methods :
RPE-choroid tissue was dissected from 2-5 month old C57BL/6J mice immediately before the experiment. To measure mitochondrial activity of RPE-choroid, we used a custom-built flow culture system that determines O2 concentration using the phosphorescent lifetime of the O2-sensitive dye, polymerized Pt(II) Meso-tetra(pentafluorophenyl)porphine. Tissue is bathed in Krebs-ringer-bicarbonate buffer equilibrated with O2 in an artificial lung, so O2 consumption rate (OCR) is calculated from the flow rate and amount of O2 depleted from the buffer. All experiments are ended by adding KCN, a cytochrome c oxidase inhibitor, to isolate mitochondrial respiration.
Results :
RPE-choroid tissue OCR is nearly constant over a broad range of glucose concentrations. In the complete absence of added glucose or any other external fuel, OCR decays only ~35% over 9 hours. In the absence of external fuel, the CPTI inhibitor etomoxir and the nonselective lipase inhibitor CAY10499 each inhibited OCR only mildly. The alkylating agent iodoacetate (IAA) completely inhibits OCR in the absence of external fuel. IAA is known to inhibit the GAPDH reaction by reacting with cysteine, suggesting that glycolysis is part of the pathway that contributes a substrate for O2 consumption in the absence of external fuel.
Conclusions :
Together these data show that RPE-choroid are utilizing an internal store of fuel to reduce O2. This store is likely to consist both of outer segment lipid-derived fatty acids and glycogen stores. The relative proportion to which each of them fuels RPE-choroid metabolism, and whether these internal substrates are normally oxidized in the presence of external metabolic fuels, are topics for future study.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.