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Juan Reyes-Reveles, Kathleen Boesze-Battaglia, Desiree Alexander, Anuradha Dhingra, Alvina Bragin, Nancy J Philp; Retinal pigment epithelial cells oxidize fatty acid from ingested photoreceptor outer segments to produce ketone bodies.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1755.
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© 2017 Association for Research in Vision and Ophthalmology.
To determine if RPE utilizes phospholipids from photoreceptor outer segments (OSs) for fatty acid oxidation and ketogenesis.
Polarized Human fetal RPE (hfRPE) and ARPE19 cells were fed, palmitate, docosahexaenoic acid (DHA) or outer segments for various periods of time and β-Hydroxybutyrate (β-HB) levels were measured using a commercially available kit (LiquiColor, Stanbio). The ketogenic enzymes, 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) and β-hydroxybutyrate dehydrogenase (BDH1) were accessed by immunoblotting and subcellular localization determined by immunostaining and confocal microscopy.
HMGCS2, the enzyme catalyzing the committed step in ketogenesis was detected in lysates prepared from ARPE-19 and hfRPE. Consistent with previous studies showing that hfRPE cells utilize the 16 carbon fatty acid, when cells were incubated with the 22:6 substrate, DHA, β-HB was released. Twice as much β-HB was secreted by the apical RPE when the cells were fed DHA as compared to the 16 carbon palmitate. To determine if the endogenous substrate for RPE-ketogenesis is derived from ingested OS, polarized hfRPE or ARPE19 were fed OS in the apical chamber and β-HB levels measured. Ingestion of OSs in the presence of glucose stimulated ketogenesis resulting in the preferential release of β-HB into the apical medium in both hfRPE and to a lesser extent in ARPE19, with no detectable β-HB in the basal media. The extent of β-HB released was does dependent, with 0.3+/- 0.028 nmoles of β-HB released with 25uM OS and 1.65 +/-0.148 nmoles released when cells were fed 250uM OS. The addition of OS also resulted in an increase in the free fatty acid content of the RPE cells, to 20+/-1.87 uM with OS addition. No β-HB release was observed when the RPE cells were challenged with beads and decreased levels of β-HB were released upon challenge with oxidized OS, from 3.16+/- 0.29 nmoles with OS to 0.42 +/- 0.54 nmoles (oxOS). In both human and mouse RPE, HMGCS2 co-localized with the mitochondrial marker COX-4.
Human RPE cells can utilize both 16:0 and 22:6 fatty acid substrates and OS to generate β-HB, which is preferentially exported across the apical membrane and serves as a metabolic substrate or neuroprotectant for photoreceptor cells.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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