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Amanda Marie Shaw, Michael L Orr, Zhen-Yang Zhao, Dean P Jones, Young-mi Go, Yan Chen; Phagocytosis of photoreceptor outer segments is an energy source of retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4013. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Phagocytosis of daily shed photoreceptor outer segments (POS) by the retinal pigment epithelium (RPE) is critical for maintaining proper health and function of photoreceptor neurons. On the other hand, phagocytosis elicits cascades of signaling events in the RPE. The goal for the current study is to explore metabolic changes in the RPE in response to phagocytosis of POS, using mass spectrometry-based high resolution metabolomics (HRM).
RPE cells were treated with porcine POS prepared by discontinuous sucrose gradient centrifugation. Metabolites including lactate and glucose, and ATP were measured using commercially available kits based on bioluminescence detection. mRNA levels of key enzymes in the metabolic pathways of interest were measured using quantitative RT-PCR. To examine altered metabolites in their abundance and metabolic pathways in RPE and retina associated with phagocytosis, murine RPE and retina tissues (n=8/group) were harvested during the morning burst of POS shedding. The HRM on these samples were performed using HPLC linked (HILIC column) Q-Exactive mass spectrometer under positive ionization mode.
HRM analyses revealed that during morning burst of POS disc shedding, both RPE and retina tissues underwent metabolic changes. However, the implicated metabolic pathways displayed tissue-specific patterns. In the RPE, the carnitine shuttle and aspartate/asparagine metabolism were the top two pathways being affected. Similar changes were detected in the cultured RPE cells exposed to POS. qRT-PCR assay showed the mRNA levels of key enzymes within the carnitine shuttle and aspartate metabolism, such as glutamic-oxaloacetate transaminase 1 (GOT1) and carnitine palmitoyltransferase 1A (CPT1A), were upregulated in cells challenged with POS. An increase in ATP production was also observed in those cells after phagocytosis.
Upregulation of key enzymes involved in the carnitine shuttle and aspartate metabolism and ATP production during phagocytosis of POS indicated that long-chain fatty acid derived from ingested POS can be used as a source for mitochondrial ATP production. HRM is a reliable high-throughput method that can be used to detect metabolic changes involved in physiological conditions, and can be used in probing early metabolic markers associated with retinal disease conditions.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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