Abstract
Purpose :
To determine if RPE utilizes phospholipids from photoreceptor outer segments (OSs) for fatty acid oxidation and ketogenesis.
Methods :
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.
Results :
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.
Conclusions :
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.