The retinal pigment epithelium utilizes fatty acids for ketogenesis

Abstract

Every day, shortly after light onset, photoreceptor cells shed approximately a tenth of their outer segment. The adjacent retinal pigment epithelial (RPE) cells phagocytize and digest shed photoreceptor outer segment, which provides a rich source of fatty acids that could be utilized as an energy substrate. From a microarray analysis, we found that RPE cells express particularly high levels of the mitochondrial HMG-CoA synthase 2 (Hmgcs2) compared with all other tissues (except the liver and colon), leading to the hypothesis that RPE cells, like hepatocytes, can produce β-hydroxybutyrate (β-HB) from fatty acids. Using primary human fetal RPE (hfRPE) cells cultured on Transwell filters with separate apical and basal chambers, we demonstrate that hfRPE cells can metabolize palmitate, a saturated fatty acid that constitutes .15% of all lipids in the photoreceptor outer segment, to produce β-HB. Importantly, we found that hfRPE cells preferentially release β-HB into the apical chamber and that this process is mediated primarily by monocarboxylate transporter isoform 1 (MCT1). Using a GC-MS analysis of (13)C-labeled metabolites, we showed that retinal cells can take up and metabolize (13)C-labeled β-HB into various TCA cycle intermediates and amino acids. Collectively, our data support a novel mechanism of RPE-retina metabolic coupling in which RPE cells metabolize fatty acids to produce β-HB, which is transported to the retina for use as a metabolic substrate.

FIGURE 1.

hfRPE cells metabolize palmitate as an oxidative substrate. hfRPE cells were incubated in CO₂/HCO₃-free Ringer's solution containing carnitine (2 mm) and glucose (5 mm), and the OCR was measured at 6.5-min intervals to obtain baseline readings. Next, BSA-conjugated palmitate at various concentrations (n = 3 each) was added to each well, and the OCR was measured over the next four time points. OCR values were normalized to the median values of the baseline readings (set to 100%) for each sample.

FIGURE 2.

The RPE expresses Hmgcs2, the key enzyme for ketogenesis. A, acetyl-CoA derived from β-oxidation of fatty acids can be shunted into the HMG-CoA pathway, which is regulated by Hmgcs2 activity. ACAT, acetyl-CoA acetyltransferase; BDH, 3-hydroxybutyrate dehydrogenase; HMGL, HMG-CoA lyase; OXCT, 3-oxoacid-CoA transferase. B, GSE10246 microarray (6) analysis for all enzymes in ketogenesis in mouse RPE versus other tissues. Aacs, acetoacetyl-CoA synthetase. Also shown are qRT-PCR (C) and Western blot (D) analyses for Hmgcs2 expression in mouse RPE versus other tissues.

FIGURE 3.

Human and mouse RPE produce β-HB that is preferentially released apically. A, Western blot analysis of hfRPE total lysates for Hmgcs2 protein expression. B, hfRPE cells were incubated (in both apical (Ap) and basal chambers) with glucose, palmitate, or both, and the apical supernatant was evaluated for β-HB content using a StanBio β-HB kit. NS, not significant. C, hfRPE cells were incubated in Ringer's solution containing glucose, palmitate, or both, and the apical and basal (Ba) supernatant was evaluated for total ketone (β-HB and acetoacetate) content using a Wako total ketone kit. D, hfRPE cells were incubated with [1-¹³C]palmitate with or without unlabeled glucose for 3 h, and ¹³C incorporation into TCA cycle metabolites and β-HB incorporation into the cells was evaluated using GC-MS. E, hfRPE cells were incubated with [1-¹³C]palmitate with or without unlabeled glucose for 3 h, and the apical and basal supernatant was analyzed for ¹³C β-HB levels. F, freshly isolated mouse RPE was incubated in Ringer's solution containing palmitate and glucose for 3 h, and the supernatant was analyzed for β-HB levels using a StanBio β-HB kit. Data were analyzed using Student's t test. * indicates statistical significance with p value < 0.05.

FIGURE 4.

Photoreceptor cells express OXCT1. A, qRT-PCR analysis for Oxct1 expression in Dynabead®-purified CD73-positive versus CD73-negative rod photoreceptor cells and other mouse ocular tissues. Expression levels in various tissues are relative to the RPE (set to 100%). B, microarray data (13) analysis for Oxct1 expression in rod and cone photoreceptor cells versus other retinal cell types. PV, parvalbumin.

FIGURE 5.

The mouse retina metabolizes ¹³C β-HB to produce TCA cycle metabolites and amino acids. A, schematic of 2,4-¹³C₂ β-HB metabolism through one TCA cycle (ending at oxaloacetate). For each metabolite, the ¹³C carbon is highlighted in gray at the expected carbon position. Each 2-¹³C acetyl-CoA that enters the TCA cycle will produce single labeled metabolites. In the second turn, new 2-¹³C acetyl-CoA will add to a 2-¹³C₁ oxaloacetate to produce 2,4-¹³C₂ citrate, and so on. OXCT, 3-oxoacid-CoA transferase; ACAT, acetyl-CoA acetyltransferase; BDH, 3-hydroxybutyrate dehydrogenase; CoA-SH, coenzyme A; CS, citrate synthase. B, a freshly isolated mouse retina was incubated in Krebs/HCO₃ Ringer's solution containing 2,4-¹³C₂ β-HB over 15, 30, or 60 min, and the whole tissue was analyzed for ¹³C-labeled metabolites using GC-MS. Shown is the percent ¹³C-labeled versus unlabeled citrate (C), glutamate (D), and aspartate (E) isotopomers in a mouse retina incubated with 2,4-¹³C₂ β-HB for 15, 30, or 60 min. M1 represents single ¹³C-labeled metabolites (irrespective of carbon position), M2 represents double ¹³C-labeled metabolites, and so forth. Data are presented as percent of ¹³C-labeled metabolite relative to the total (both labeled and unlabeled) within the sample.

FIGURE 6.

MCT1 and MCT7 are expressed specifically in photoreceptor cells. A, GSE10246 microarray (6) analysis of all MCT isoforms in the RPE and retina versus other tissues. B, microarray data (20) analysis of single mouse rod photoreceptor and Müller cells for Mct1 and Mct7 expression. C, microarray data (13) analysis for Mct7 expression in rod and cone photoreceptor cells versus other retinal cell types. D, qRT-PCR analysis of photoreceptor specific genes (Crx, Rho, Pde6a, and Opn1-sw) and Mct1 and Mct7 expression in Dynabead®-purified CD73-positive versus CD73-negative rod photoreceptor cells and other mouse ocular tissues.

FIGURE 7.

hfRPE cells transport β-HB across their apical membranes via MCT1. A, apical-to-basal transport of lactate across hfRPE cells on Transwells in the presence of AR-C155858 (10 μm), α-CHC (5 mm), or both in the apical bath. DMSO, dimethyl sulfoxide. B, basal-to-apical transport of β-HB across hfRPE cells on Transwells in the presence of AR-C155858 (10 μm) in the apical (Ap) bath. C, basal-to-apical transport of acetoacetate across hfRPE cells on Transwells in the presence of AR-C155858 (10 μm) in the apical bath. Data were analyzed using Student's t test. * indicates statistical significance with p value < 0.05.

FIGURE 8.

β-HB transport into the retina was independent of MCT1 activity. A freshly isolated retina was incubated with [U-¹³C]lactate (5 mm) (A) or 2,4-¹³C₂ β-HB (5 mm) (B) for 30 min in the presence or absence of AR-C155858 (10 μm). Retina samples were analyzed for ¹³C metabolites using GC-MS. Data are presented as percent of ¹³C-labeled metabolite relative to the total (both labeled and unlabeled) within the sample. Data were analyzed using two-way ANOVA. * indicates statistical significance with p value < 0.05.

FIGURE 9.

Model of metabolic coupling between photoreceptor cells and the RPE. Shed ROS is phagocytized by the RPE and degraded to release fatty acids that are converted into β-HB. MCT1 at the RPE apical microvilli mediates transport of β-HB into the interphotoreceptor space. Photoreceptor cells take up β-HB via MCT1 and MCT7 and metabolize it in the mitochondria to generate amino acid intermediates and ATP.