The RPE utilizes fatty acids for ketogenesis 1 The Retinal Pigment ...

The RPE utilizes fatty acids for ketogenesis

1

The Retinal Pigment Epithelium utilizes Fatty Acids for Ketonegenesis: Implications for Metabolic

Coupling with the Outer Retina

Jeffrey Adijanto1, Jianhai Du

2, Cynthia Moffat

1, Erin L Seifert

1, James B Hurley

2, Nancy J Philp

1.

1From the Dept. of Pathology, Anatomy, & Cell Biology, Thomas Jefferson University, Philadelphia,

Pennsylvania 19107, 2Depts. of Biochemistry and Ophthalmology, University of Washington, Seattle, Washington 98195.

Running title: The RPE utilizes fatty acids for ketogenesis.

To whom correspondences should be addressed: Jeffrey Adijanto and Nancy J Philp, Dept. of

Pathology, Anatomy, & Cell Biology, Thomas Jefferson University. Tel: (215) 503-7854, Fax: (215) 923-

3808, E-mail (Adijanto): [email protected] and E-mail (Philp): [email protected].

Keywords: monocarboxylate transporter 7 (MCT7), fatty acid metabolism, ketone transport and

metabolism, RPE-retina interaction.

Background: RPE cells derive fatty acids from

phagocytized photoreceptor outer segments.

Results: RPE cells metabolize palmitate to

produce beta-hydroxybutyrate (β-HB), a ketone

body that the retina can use as a metabolic

substrate.

Conclusion: RPE cells produce β-HB as a

potential substrate for photoreceptor cells in the

outer retina.

Significance: This is a novel form of RPE-retina

interaction that may be important for retinal cell

health and function.

Abstract

Everyday shortly after light onset, photoreceptor

cells shed approximately a tenth of their outer

segment (OS). The adjacent RPE cells

phagocytize and digest shed photoreceptor OS

(POS), which provides a rich source of fatty acids

that could be utilized as an energy substrate. From

microarray analysis, we found that RPE cells

express particularly high levels of the

mitochondrial 3-hydroxy-3-methylglutaryl-CoA

synthase 2 (Hmgcs2) compared to all other tissues

(except the liver and colon), leading to the

hypothesis that RPE cells, like hepatocytes, can

produce 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 POS, 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 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 metabolites 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 that

are transported to the retina for use as a metabolic

substrate.

Introduction

In the outer retina, the RPE and

photoreceptor cells are physically and

metabolically linked and this symbiotic

relationship supports normal visual function. Each

day, photoreceptor cells shed approximately 10%

of their OS, which are phagocytized by the

underlying RPE (1). These shed photoreceptor OS

(POS) are highly enriched in saturated (14:0, 16:0,

and 18:0) and unsaturated fatty acids (18:1, 18:2,

18:3, 20:4, 22:6) (2). Early radioactive tracer

studies revealed that frog RPE incorporates

docosahexaenoic and arachidonic acids from POS

into triglycerides and phospholipids (3,4). Studies

with cultured porcine RPE also demonstrated that

the RPE can metabolize saturated fatty acids via β-

oxidation (5).

http://www.jbc.org/cgi/doi/10.1074/jbc.M114.565457The latest version is at JBC Papers in Press. Published on June 4, 2014 as Manuscript M114.565457

Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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The RPE utilizes fatty acids for ketogenesis

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While the RPE can metabolize fatty acids

to produce ATP, it was not known whether like the

liver, they can use fatty acids to produce ketones.

Analysis of the publicly available mouse

microarray data (GSE10246; (6)) revealed that

mouse RPE express particularly high levels of

mitochondrial Hmgcs2, the key enzyme required

for ketone production (7). Hmgcs2 mRNA is also

abundant in two other tissues: liver and colon,

both of which are known sites of ketogenesis (8).

Since overexpression of Hmgcs2 alone can induce

fatty acid β-oxidation and ketogenesis in a cell line

that normally lacks these activities (9), the high

expression of Hmgcs2 in the RPE suggested that it

can produce ketones.

In fasted animals, fatty acids taken up by

the liver are catabolized via β-oxidation to

generate acetyl-CoA. While acetyl-CoA readily

enters the TCA cycle, a fraction is shunted into the

ketogenic pathway, which consists of a series of

four reactions that ultimately produce beta-

hydroxybutyrate (β-HB) (8). In the liver, the rate

of β-HB production is dependent on the activity

and expression of Hmgcs2. The Hmgcs2 promoter

contains several regulatory elements–CRE (cAMP

response element), NRRE (Nuclear Receptor

Response Element; interacts with peroxisome

proliferator-activated receptors (PPARs) and

retinoid X receptor (RXR)), and GC box (interacts

with Zinc Finger Protein binding site) (8). During

starvation, influx of fatty acids into the liver

induces activation of PPAR (10), which in turn

promotes ketogenesis through the upregulation of:

(1) Hmgcs2 expression, (2) genes involved in fatty

acid transport including acyl-CoA synthetase

(ACSL) and fatty acid-binding protein (FABP),

(3) genes involved in fatty acid metabolism

peroxisomal acyl-coenzyme A oxidase (ACOX)

and acyl-CoA dehydrogenase (ACAD), and (4)

genes involved in carnitine transport (carnitine

palmitoyltransferase (CPT) and organic

cation/carnitine transporter (OCTN2; encoded by

SLC22A5)). The RPE express all three isoforms

of PPARs (α, δ, and γ), and PPAR-γ expression

was induced in RPE by phagocytosis of POS (11).

These findings suggest that influx of fatty acids

into the RPE from POS phagocytosis can act as a

signal for PPAR-mediated activation of fatty acid

metabolism.

β-HB produced in the liver is transported

out of the hepatocytes into the blood stream. The

transporter for β-HB was recently identified in the

zebrafish model to be monocarboxylate transporter

isoform 7 (MCT7; encoded by Slc16a6) (12).

MCT7 expression in the RPE and retina has not

been reported, but a search in a recently published

microarray data of individual mouse retina cell

types by Siegert et al. (accessible through

http://www.fmi.ch/roska.data/index.php; (13))

revealed that MCT7 is expressed in photoreceptor

cells, suggesting that photoreceptor cells can take

up and metabolize β-HB. Consistent with this

notion, northern blot analysis showed that MCT7

(previously known as MCT6) transcript is

particularly enriched in the brain (14), where the

role of ketones as energy substrates is well-

established. In addition to MCT7, Halestrap and

colleagues demonstrated that MCT1 can transport

β-HB, albeit at a relatively high Km of 10-12 mM

(15). The Km of β-HB transport by MCT7 is

unknown. In the eye, MCT1 is enriched in the

apical processes of RPE and inner segment of

photoreceptor cells (16). However the role of

MCT1 and MCT7 in β-HB transport in the RPE

and retina remains to be determined.

In the present study we examined whether

the RPE produces β-HB through β-oxidation of

fatty acids and if the ketones produced can be

taken-up and metabolized by photoreceptor cells.

By using a cultured human fetal RPE (hfRPE)

model system, we showed that RPE cells can

metabolize fatty acids to produce β-HB, which

was preferentially released into the apical

compartment. Our data support a model of

metabolic coupling in which RPE cells metabolize

fatty acids derived from shed POS to produce β-

HB that is subsequently transported to the retina to

be used as a substrate for oxidative metabolism.

Experimental Procedures

hfRPE culture model. Fetal human eyes

were obtained from Advanced Bioscience

Resources (Alameda, CA) from random donors

between 18 to 22 weeks of gestation. Eyes were

delivered overnight and tissues were dissected less

than 26 hrs after enucleation. The use of hfRPE

cells in this work conform to the guidelines set by

the NIH institutional review board. hfRPE

monolayers were cultured on T25 flasks (Passage

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0; P0) as previously described (17). T25 flasks of

confluent hfRPE cells were generously provided

for our studies by Drs. Sheldon Miller and

Arvydas Maminishkis. Briefly, hfRPE cells were

trypsinized from a T25 flask and seeded onto 12-

well transwells at ≈ 1.25 x 105 cells/well (passage

1; P1). P1 hfRPE cells were cultured for three to

four weeks to reach maturity (transepithelial

resistance > 500 Ω∙cm2) prior to experimentation.

Transepithelial Resistance (TER) was measured

with Epithelial Volt-Ohm Meter (EVOM) (WPI,

Sarasota, FL) at room temperature.

Western Blot. Mouse tissue samples were

isolated and subsequently homogenized in lysis

buffer : Triton-X (1%), HEPES (25 mM; pH 7.4),

NaCl (150 mM), MgCl2 (5 mM), and n-octyl-β-D-

glucoside (60 mM) and 1X proteinase inhibitor

cocktail (Sigma; P8340). hfRPE cells on transwell

filters were lysed and homogenized using the same

procedure (18). 10 µg total protein lysates were

loaded onto a NuPAGE® 4-12% Tris-Acetate Gel

(Life Technologies) for electrophoresis. Proteins

were subsequently transferred onto PVDF

membranes using XCell II™ Blot Module (Life

Technologies). Nonspecific binding sites were

blocked with TBS (+ 0.1% Tween20) containing

5% w/v BSA. Hmgcs2 antibody (ab137043) was

purchased from Abcam (Cambridge, MA).

Papain dissociation of single retinal cells

from mouse retina. Mouse was sacrificed and its

retina was isolated in ice cold PBS. Next, the

retina was dissociated into single retinal cells

using Papain Dissociation System (Worthington

Biochemical Corporation; Cat#: LK003150)

according to the manufacturer’s protocol. Briefly,

the retina was incubated in a 1.5 mL centrifuge

tube containing 1 mL papain solution (containing

950 µL Papain + 50 µL DNase) for 1 hour at 37oC.

Next, albumin-ovomucoid inhibitor solution (10X)

and DNase was added to the retina to stop the

reaction. A wide-bore flamed tip Pasteur pipette

(without applying any pressure, the pipette leaks

water at a rate of ~1 drop/second) was used to

gently triturate the retina (3 times).

Magnetic bead-mediated purification of

CD73+ rod photoreceptor cells. Dynabeads

protein G (0.75 mg; Cat#10003D; Life

Technologies) was washed with PBS with Tween-

20 (0.02%) and subsequently incubated with 1 μg

of Rat anti-mouse CD73 antibody (Clone: TY/23;

Cat# 550738; BD Pharmigen) in rotation overnight

at 4oC. The next day, the Dynabead Protein G

conjugated with CD73 antibody was washed with

PBS and the Dynabeads-CD73Ab complex was

added to 1 mL of the triturated retinal cell

suspension. The mixture was incubated at room

temperature with rotation for 30 min to allow the

Dynabeads-CD73Ab complex to bind cells.

Unbound cells were removed and the Dynabead-

CD73Ab-Cell complex was washed with PBS

three times before Trizol was added to extract

RNA. RNA was also isolated from unbound cells

using Trizol.

Reverse Transcription and Real-Time

Polymerase chain reaction (qPCR). RNA (0.5

µg/sample) was reverse transcribed using random

hexamers and oligo(dT)20 primers and SuperScript

III (Life Technologies). qPCR reactions were

performed using Power SYBR green Supermix

(Life Technologies) in 20 µL reactions (5 ng

cDNA/RxN). qPCR was performed using ABI

PRISM 7000 Sequence Detection System. Primer

sequences were obtained from a publicly available

database (http://pga.mgh.harvard.edu/primerbank/)

(19). Custom oligos were purchased from

Eurofins MGW Operon (Huntsville, AL). Data is

presented as delta-Ct relative to an endogenous

control, Rplp1. Data is presented as a heat-map

generated in MultiExperiment Viewer, MeV

v4.8.1. Yellow represents high expression (small

delta-Ct) and blue represents low expression (large

delta-Ct).

Microarray Analysis. GSE10246 raw

microarray dataset (GNF Mouse GeneAtlas V3;

Affymetrix Mouse Genome 430 2.0 Array; (6))

was downloaded from NCBI GEO. Each probe ID

(ID_REF) in the dataset was matched with the

most recent annotation file GPL1261-14790,

which is available from Affymetrix’s website.

The median fluorescence value of all probes for

each gene was determined and these values were

converted to the LOG2 scale. Each tissue sample

in the microarray was in duplicates and the

average of the LOG2 (median fluorescence values)

of each tissue duplicates was used for generating

the heatmap. Heatmap is generated in MeV

v4.8.1. Yellow represents high expression (high

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LOG2(intensity)) and blue represents low

expression (low LOG2(intensity)). Single

photoreceptor and Müller cell microarray data by

Roesch and colleagues (20) was downloaded from

the journal website. First, the annotation and gene

names for the probeset were updated using a more

recent annotation file downloaded from

Affymetrix’s website (Mouse430 Annotations,

Release 32, 06/09/11). Next, the gene expression

data was normalized to the average fraction of

Rplp10, Rps12, Rps24, Rpl4, and Rps4x across all

samples. The normalized values were converted

to LOG2 scale and used in the graph. Genes with

LOG2 intensity values below 5 (equivalent to

intensity of 32) are considered absent. For

analysis of retinal gene expression data by Seigert

and colleagues (13), the normalized microarray

data was downloaded from the website

(www.fmi.ch/roska.data/index.php). Of the

photoreceptor samples (b2.1st, b2.2

nd, b2.single,

Chmb4, and d4), b2.1st, b2.2

nd, and b2.single are

rods (expressing Rhodopsin but not Opsins) and

Chmb4 and d4 are cones (expressing Opsins but

not Rhodopsin). Lhx4 of the bipolar cell samples

was excluded because Lhx4 is also expressed in

photoreceptor cells. Similarly, only Rgs5 and

Fbxo32-positive amacrine cell samples and PV,

Drd4, Grik4, and Opn4-positive ganglion cell

samples were used because they were not

expressed in any other retinal cell types. As

indicated by Siegert et al., the threshold for

expression is 20.

Ringer’s solution and metabolic substrate

preparation. The recipe for hfRPE Ringer’s

solution is based on the hfRPE cell culture

medium: NaCl (116.5 mM), NaHCO3 (26.2 mM),

KCl (5 mM), MgCl2 (0.5 mM), CaCl2 (1.8 mM),

L-carnitine (1 mM), HEPES-NMDG (12 mM

HEPES dissolved in DI H2O in a separate beaker

and titrated to pH 7.4 with a base, N-Methyl-D-

glucamine (NMDG)). The Ringer’s solution for

Seahorse experiments contains 26.2 mM Na-

gluconate and no NaHCO3. The solution was not

equilibrated with CO2. For experiments with

mouse RPE and retina, we used KREBS/HCO3-

Ringer’s solution: NaCl (98.5 mM), NaHCO3

(25.9 mM), KCl (4.9 mM), KH2PO4 (1.2 mM),

MgSO4.7H2O (1.2 mM), CaCl2 (2.6 mM), L-

carnitine (1 mM), HEPES-NMDG (20 mM

HEPES dissolved in DI H2O in a separate beaker

and titrated to pH 7.4 with NMDG). Glucose or

other substrates are excluded from the Ringer and

added prior to the experiment. Before sterile

filtration, Ringer’s solution is equilibrated by

passing the solution with 5% CO2 (balance air).

When fully equilibrated with 5% CO2, the final pH

of the Ringer is 7.4 and the osmolalities of the

hfRPE Ringer and KREBS/HCO3 Ringer’s

solutions (after addition of 5 mM glucose) were

305 mOsm/kg and 300 mOsm/kg, respectively.

To prepare BSA-conjugated palmitate, we first

prepared a 200 mM Na-palmitate stock by

dissolving Na-palmitate powder in 50% methanol

(balance H2O) and incubated at 70oC for 5 min.

Na-palmitate stock was then diluted 100-fold in a

150 mM NaCl solution containing 2% BSA. The

mixture was incubated at 37oC for 1hr to achieve

BSA-palmitate conjugation. The 2 mM palmitate-

conjugate working solution was stored at -20oC

until the day of experiment. The BSA vehicle

control was prepared by diluting 50% methanol

100-fold in 150 mM NaCl + 2% BSA solution.

On the day of the experiment, the 2 mM palmitate-

conjugate stock was added to Ringer solution at a

1:10 fold dilution to achieve palmitate

concentration of 200 μM. For GC-MS analyses of

fatty acid metabolism, 1-13

C1 Na Palmitate (Cat#

CLM-174-1) was purchased from Cambridge

Isotope Laboratories (Tewksbury, MA).

Measurement of RPE oxidative

metabolism using Seahorse XF24 Bioanalyzer.

hfRPE cells were seeded in XF24 cell culture

microplate at 80k cells/well and cultured over 10

days in complete hfRPE medium. On the day of

the experiment, hfRPE cells were switched to

CO2/HCO3-free HEPES-buffered Ringer

(containing 5 mM glucose). The plate was

immediately transferred to the Seahorse XF24

analyzer and baseline oxygen consumption rates

(OCR) were obtained. Next, Ringer’s solution

containing BSA-vehicle or BSA-palmitate was

injected into each well and subsequent changes in

OCR were recorded.

Measurement of β-HB and Total Ketone

Production and Release. hfRPE Ringer’s solution

containing substrates were added to the apical or

basal chambers of hfRPE cells on transwell filters.

Ringer’s solution (70 µL) was collected from

apical and basal chambers at two and three hour

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time-points. For mouse RPE explants, the mouse

was sacrificed and RPE-choroid was micro-

dissected from each eye and transferred to a

KREBS/HCO3 Ringer (at 37oC and 5% CO2)

containing no substrate or glucose + palmitate

(200 μM) in each well in a 96-well plate (80 μL

per well). β-HB was measured using using

StanBio’s β-hydroxybutyrate LiquiColor Reagent

kit (Boerne, TX; Cat#: 2440-058). In this assay,

reagent A and reagent B were mixed in a 6:1 ratio

and 200 μL of the mixture was added to 25 µL of

samples (or β-HB standards) in each well. The

assay plate was incubated at 37oC for 10 min

before absorbance at 505 nm were measured. The

limit of detection for the assay was obtained by

taking the mean of the blank samples + 2X

standard deviation of the blank samples, giving a

confidence level of 95%. For the β-HB assay, the

limit of detection is 43 pmole, which is equivalent

to 1.7 µM in 25 µL. Total ketone (acetoacetate +

β-HB) was measured using Wako Life Sciences

Inc.’s Autokit Total Ketone Body assay

(Richmond, VA; Cat#: 415-73301 and 411-

73401). In this assay, Reagent 1 and Reagent 2

were mixed in a 1:3 ratio and 200 µL of the

mixture was added to 30 µL of the samples (or β-

HB standards) in each well. Immediately,

absorbances at 404 and 650 nm (reference) were

measured at 5 min intervals over four time points.

The rate of increase in (404-650) nm absorbance

was used for measuring total ketone production.

The limit of detection for this assay is 5 pmole

(equivalent to 0.17 µM in a 30 µL volume). Data

is presented as absolute amount (nmole) of β-HB

or ketones released by 1 cm2 of hfRPE monolayer

into the apical or basal chambers after taking into

account the differences in apical vs. basal chamber

volumes: 500 µL and 1500 µL respectively. In

both assays, Ringer’s solution containing no

substrate, glucose (5 mM), palmitate (200 µM), or

glucose + palmitate (200 µM), all gave blank

readouts. We also verified that both assays do not

detect other substrates that are present in the

hfRPE supernatant, including glucose, lactate, and

pyruvate.

Measurement of Transepithelial Lactate

and Ketone Transport. hfRPE on transwells were

transferred to a new 12-well plate and washed with

glucose-free hfRPE Ringer’s solution (apical and

basal chambers) once. Next, the apical chamber

was replaced with Ringer solution containing

either (1) DMSO, (2) AR-C155858 (10 µM; a

specific MCT1 inhibitor (21)), (3) α-cyano-4-

hydroxycinnamate (α-CHC; 5 mM; another MCT1

inhibitor (22)), or (4) both AR-C155858 (10 µM)

& α-CHC (5 mM), and incubated at 37oC and 5%

CO2 for 15 min. In a separate 12-well plate, 1.5

mL of glucose-free Ringer solution was added to

each well and pre-incubated at 37oC and 5% CO2

for 15 min. Ringer solution in the basal chamber

was removed and the apical chambers of each

transwell were replaced with glucose-free Ringer

solution containing 5 mM lactate with or without

AR-C155858 and α-CHC. Next, each transwell

was transferred to the 12-well plate containing 1.5

mL glucose-free media. Ringer solution at the

basal chamber was collected at 30 and 60 min time

points for evaluation of lactate concentration using

a commercially available assay kit (Trinity

Biotech; Cat# 735-10). Glucose was excluded

from the Ringer solution because the RPE rapidly

produces lactate from glucose. After the

experiment, transepithelial resistance of the hfRPE

remained above 300 Ω∙cm2, indicating that the

hfRPE barrier was intact. For β-HB transport

experiments, glucose was included in the Ringer

solution. First, hfRPE on transwells were washed

with Ringer’s solution (apical and basal chambers)

once. Next, the apical chamber was replaced with

Ringer solution containing either (1) DMSO or (2)

AR-C155858 and incubated for 15 min at 37oC

and 5% CO2. Next, each transwell is transferred to

a new 12-well plate with 1.5 mL Ringer (basal

chamber) containing 5 mM β-HB or 0 mM (blank

control), and Ringer in the apical chamber was

collected at 15 and 30 min time points for β-HB

analysis using StanBio’s assay kit. AR-C155858

is colorless and does not interfere with the β-HB

assay.

Gas Chromatography-Mass Spectrometry

(GC-MS) analysis of metabolites. Mouse was

sacrificed and the retina from each eye was

isolated in ice-cold KREBS/HCO3 Ringer

containing unlabeled glucose. Each retina was cut

into three pieces, transferred to fresh Ringer pre-

incubated at 37oC and 5% CO2, and immediately

into KREBS/HCO3 Ringer containing 2,4-13

C2 β-

HB (5 mM) and unlabeled glucose (5 mM). After

incubation (15, 30, or 60 min), the retina pieces

were transferred to ice cold 0.9% NaCl solution

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(using a transfer pipette) for 10 seconds and

picked up with a clean forcep directly into a 1.5

mL centrifuge tube and immediately frozen in dry

ice. Samples were stored at -80oC until

processing. The procedure for processing the

retina or RPE supernatants for GC-MS analysis

was previously published (23,24). An Agilent

7890/5975C GC/MS system (Agilent

Technologies, Santa Clara, CA) with Agilent HP-

5MS column (30 m × 0.25 mm × 0.25 µm film )

column was used for GC separation and analysis

of metabolites and the settings for this system has

been published (23,24). Labeled metabolite data

is presented as relative ion abundances or as % 13

C

labeled metabolites, which was calculated by

dividing the labeled ions with total ion intensity.

Calibration of β-HB was performed using various

concentrations of 2,4-13

C2 β-HB. For mass

isotopomer analysis, single-labeled metabolites are

presented as M1, double-labeled metabolites are

presented as M2, and so forth. The labels are

irrespective of carbon position. The measured

distribution of mass isotopomers was corrected for

natural abundance using defined intensities from

known standards after each experiment. The

natural abundance of the tracers and derivatization

reagents were corrected using IsoCor software. U-13

C Na lactate (Cat#: 485926) and 2,4-13

C2 β-HB

(Cat#: 674117) were purchased from Sigma

Aldrich (St. Louis, MO).

Results

The RPE expresses key enzymes in ketogenesis

The RPE plays an essential role in outer

segment renewal by phagocytosis and digestion of

shed outer segment tips. This process provides the

RPE with a source of lipids that may be

metabolized via β-oxidation to generate acetyl

CoA. To determine if cultured hfRPE cells are

capable of metabolizing fatty acids, we seeded

hfRPE cells on Seahorse XF24 microplates at high

density and cultured them over 10 days to achieve

differentiation, as indicated by formation of fluid-

filled domes. For the Seahorse experiments,

hfRPE cells were first incubated in HEPES-

buffered, CO2/HCO3-free Ringer’s solution

containing carnitine (2 mM) and glucose (5 mM),

and the first 4 measurements of oxygen

consumption rate (OCR) were obtained. Next,

BSA-conjugated palmitate was added to each well

at various concentrations (0, 100, 200, and 400

μM) and the OCR was measured. As shown in

Figure 1, OCR was increased by 30% in hfRPRE

incubated with 200 and 400 μM palmitate whereas

hfRPE cells given BSA vehicle control showed a

decrease in OCR by ≈10%. 2-way ANOVA

analysis using Dunnett’s multiple comparisons test

showed that palmitate-induced increase in OCR

was statistically significant. These palmitate

induced changes in OCR were not observed in the

absence of carnitine, an important co-factor for

fatty acid metabolism (data not shown).

While RPE cells can metabolize lipids as a

source of ATP, it is not known whether it can

oxidize lipids to produce ketones, a well-

established product of lipid metabolism in the

liver. Ketone (acetoacetate + β-HB) production

occurs in the mitochondria, when acetyl-CoA

generated from lipid β-oxidation is shunted to the

3-hydroxy-3-methylglutaryl (HMG)-CoA pathway

(Figure 2A). To explore the ketogenic potential of

RPE cells, we analyzed a publicly available

microarray data-set (GSE10246; (6)) to compare

gene expression of key enzymes in ketogenesis

(i.e., acetyl-CoA acetyltransferase 1 (Acat),

Hmgcs2, HMG-CoA lyase (Hmgcl), β-HB

dehydrogenase (Bdh), and 3-oxoacid-CoA

transferase (Oxct)) in mouse RPE compared to

other tissues (i.e., retina, brain, heart, intestine,

kidney, liver, muscle, and testis). Our analysis

revealed that the rate-limiting enzyme in the

HMG-CoA pathway, the mitochondrial HMG-

CoA synthase (Hmgcs2), was exclusively and

highly expressed in the RPE, liver, and colon with

a high LOG2 intensity of 10.3, 14.1, and 12.3,

respectively (Figure 2B). Hmgcs2 expression of

all other tissues in the array was low, with LOG2

intensities ranging from 3 to 6. To verify this

microarray data, we performed qRT-PCR (Figure

2C) and western blot (Figure 2D) analyses to

compare Hmgcs2 mRNA and protein expression

in the mouse RPE to various other tissues.

Consistent with the microarray data, we found that

only the RPE and liver express Hmgcs2 protein.

The high level of expression of Hmgcs2 in

the RPE suggested that the RPE, like the liver, is

capable of producing ketones. To test if RPE cells

can indeed produce ketones, we turned to a

primary human fetal RPE (hfRPE) culture model

established in Dr. Miller’s laboratory (17). In this

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system, hfRPE cells establish a polarized

monolayer on transwell filters and release

macromolecules into separate apical and

basolateral compartments. Thus ketone

production and their subsequent release into the

apical or basal chambers could be directly

evaluated. First, we verified that Hmgcs2 protein

was indeed expressed in hfRPE cells (Figure 3A).

Next, we incubated hfRPE cells (both apical and

basal chambers) with Ringer containing: (1) no

substrate; (2) glucose (5 mM); (3) BSA-palmitate

(200 μM); or (4) both glucose (5 mM) and BSA-

palmitate (200 μM). Samples of Ringer from

apical and basal chambers were collected at 2 and

3 hr time points and subsequently analyzed for β-

HB using a commercially available assay kit. As

shown in Figure 3B, hfRPE cells fed glucose or

palmitate alone produced low but detectable levels

of β-HB compared to the no-substrate control.

Importantly, hfRPE cells incubated with Ringer

containing both glucose and palmitate produced

and released significantly more β-HB into the

apical chamber (2.80 ± 0.34 nmole/cm2 RPE in

3hr) than those given glucose (1.81 ± 0.35

nmole/cm2 RPE in 3hr; p = 0.025) or palmitate

(1.73 ± 0.17 nmole/cm2 RPE in 3hr; p = 0.015)

alone. While, β-HB was readily detected in the

apical chamber, β-HB in the basal chamber was

below the detection limit of the assay and was

therefore considered not significant and excluded

from the analysis.

In addition to the β-HB kit, we used a total

ketone assay kit that can detect β-HB (+

acetoacetate) at the picomole range (Limit of

detection = 5 pmole). Using this kit, we detected

ketones in the basal compartment at concentrations

well above the detection limit, thus allowing us to

quantify total ketones released into the apical and

basal chambers (Figure 3C). From this

experiment, we verified that hfRPE cells

metabolized palmitate to produce ketones

(consistent with data from the β-HB assay).

Importantly we found that hfRPE cells

preferentially release ketones into the apical

chamber (palmitate + glucose: 2.2 ± 0.2

nmole/cm2 RPE) vs. the basal chamber (palmitate

+ glucose: 1.0 ± 0.2 nmole/cm2 RPE).

To further examine the metabolism of

fatty acids by hfRPE, we incubated hfRPE cells

with 13-carbon labeled Na-palmitate (BSA-

conjugated 1-13

C Na-palmitate; 200 µM) in the

presence or absence of 5 mM unlabeled glucose

for 3 hr and analyzed for 13

C incorporation into

various metabolites in the TCA cycle and β-HB

using gas-chromatography-mass-spectrometry

(GC-MS). As shown in Figure 3D, hfRPE cells

incorporated the first (and only) 13

C-labeled

carbon from palmitate into citrate, glutamate, and

importantly β-HB. There were significant

variations in the data primarily due to the low

levels of 13

C metabolites detected. In addition to

intracellular metabolites, we also analyzed the

Ringer’s solution in the apical and basolateral

compartments of these hfRPE for 13

C metabolites.

We found that hfRPE cells incubated with 1-13

C

palmitate alone released significantly more (7-

fold) labeled β-HB (75.2 ± 49.7 pmole/cm2 RPE in

500 µL volume over 3 hr) into the apical chamber

compared to the basal chamber (10.5 ± 18.3

pmole/cm2 RPE in 1500 µL volume over 3 hr)

(Figure 3E). Similarly, hfRPE cells incubated

with 1-13

C palmitate and unlabeled glucose

released more labeled β-HB into its apical

chamber (157.3 ± 49.0 pmole/cm2 RPE) compared

to its basal chamber (58.8 ± 23.7 pmole/cm2 RPE).

These values were lower (≈ 15 fold) compared to

β-HB detected using assay kits (Figures 3B and

C). Considering that each palmitate can be β-

oxidized into eight acetyl-CoAs and only one was 13

C-labeled (1:7 ratio) and that the ratio of 13

C

labeled vs. unlabeled β-HB was 1:15, it can be

estimated that 50% of palmitate-derived acetyl-

CoA entered the ketogenesis pathway to produce

β-HB while the rest entered the TCA cycle to

produce glutamate and ATP (as demonstrated by

GC-MS data in Figure 3D and Seahorse data in

Figure 1). Collectively, our findings demonstrate

that hfRPE cells metabolize fatty acids to produce

β-HB that was preferentially released across its

apical membrane.

Since hfRPE cells can produce β-HB, we

proceeded to evaluate β-HB production in mouse

RPE. In these experiments, mice (≈ P60) were

euthanized, eyes enucleated and dissected and

freshly isolated RPE was immediately transferred

to KREBS/HCO3 Ringer containing palmitate

(200 μM; in the presence of 5 mM glucose) or no

substrate at 37oC and in 5% CO2 over a three hour

period. As shown in Figure 3F, we found that

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mouse RPE explants incubated with palmitate (and

glucose) consistently produced ≈ 2-fold more β-

HB than mouse RPE without any substrate (n = 4;

p < 0.01; limit of detection = 0.43 nmole).

The retina metabolizes ketones

The preferential release of β-HB across

the RPE apical membrane suggests that RPE cells

produce ketones that can be utilized by the retina

as a metabolic substrate. It is known that cell

types that regularly use β-HB as a source of

energy also express high levels of OXCT1 (3-

oxoacid CoA transferase 1), which catalyzes the

conversion of acetoacetate to acetoacetyl-CoA, a

key step in β-HB catabolism. From qPCR analysis

of CD73+ rods vs. CD73- retinal cells and other

ocular tissues and microarray data from Seigert et

al (13), we showed that mouse photoreceptor cells

are highly enriched in oxct1 mRNA (Figure 4).

To evaluate whether β-HB is metabolized by the

retina at the physiological level, we incubated

freshly isolated mouse retina with 2,4-13

C2 β-HB

(1 hr; in the presence of 5 mM unlabeled glucose)

and found that mouse retina incorporated labeled

carbons from 13

C β-HB into metabolites in the

TCA cycle (citrate, fumarate, and malate), and

amino acids (glutamate and aspartate) (Figure 5A

and B), demonstrating that the retina is capable of

utilizing β-HB as an energy substrate.

Importantly, a large fraction of the β-HB-derived

acetyl-CoA that enters the TCA cycle was

converted into glutamate (as indicated by low 13

C

incorporation into TCA intermediates: succinate,

fumarate, malate, and aspartate) (Figure 5B).

Mass isotopomer analysis showed that 13

C-labeled

β-HB was metabolized into citrate in the TCA

cycle within 15 min (Figure 5C). Furthermore, it

took less than 15 min for each molecule of β-HB

to be metabolized into glutamate (single-labeled

glutamate (M1) was detected in 15 min, but not

double-labeled glutamate (M2); Fig 5D) and

between 30-60 min for β-HB to be metabolized

into aspartate (double-labeled aspartate (M2) was

detected in 60 min, but not in 15 and 30 min;

Figure 5E).

The RPE and retina transports ketones via

MCTs

The fact that β-HB (with pKa of 4.39) is

ionized at physiological pH and the observation

that β-HB is released by the RPE and can be taken

up and metabolized in the retina suggest the

presence of facilitated transporters that mediate the

transfer of these metabolites across the RPE and

retinal cell membranes. Two promising

candidates for facilitated ketone transporter are

monocarboxylate transporters (MCT) isoforms 1

(MCT1; encoded by Slc16a1) and 7 (MCT7;

encoded by Slc16a6), both of which are known to

transport β-HB (12,15). While MCT1 is expressed

exclusively at the RPE apical membrane and

enriched at the photoreceptor inner segment (16),

MCT7 expression in the RPE and retina has not

been studied. Microarray analysis (GSE10246;

(6)) revealed that MCT7 was highly expressed in

the RPE and retina (Figure 6A) compared to all

other tissues (e.g., brain, heart, intestine, kidney,

liver, muscle). Analysis of single cell microarray

data (20) comparing rod photoreceptor cells

(adult) vs. Müller cells (Adult) and other retinal

cells revealed that MCT7 is particularly enriched

in rod photoreceptor cells (Figure 6B).

Corroborating this finding is a publicly available

microarray data set by Siegert and colleagues (13),

who achieved mRNA profiling of almost all

retinal cell types (Rod and cone photoreceptor

cells, and horizontal, bipolar, amacrine, ganglion,

and microglia cells) in the murine model. Part of

their data is represented in Figure 6C, which

shows that Slc16a6 was highly expressed in rod

(b2.1st, b2.2

nd, and b2.single) and cone (Chrnb4

and d4) photoreceptor cells, as well as microglia

(Csf2rb2), but not in any other retinal cell types

tested (horizontal, bipolar, amacrine, and ganglion

cells). To confirm the microarray data, we used a

dynabead®-conjugated CD73 antibody to pull

down CD73+ rod photoreceptor cells (from

papain-dissociated mouse retina) for qRT-PCR

analysis. CD73 is a cell surface antigen that is

specifically expressed on rod photoreceptor cells

and can be used to select for rods from a mixed

pool of retinal cells (25). We verified that the

CD73+ rods express Crx, rhodopsin (Rho) and

Pde6a, but not cone opsin (short wavelength; Opn-

sw), RPE65 (RPE marker), or slc6a1 (bipolar cell

marker) (Figure 6D). Importantly, we found that

these CD73+ retinal cells are enriched in MCT1

and MCT7 (Figure 6D), consistent with the

microarray data analyses. Collectively, our data

support a model in which β-HB released from the

RPE into the interphotoreceptor space is

transported into the retina via MCT1 and MCT7.

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To further test this model, we directly

evaluated β-HB transport across the RPE and

retina. While there is currently no known inhibitor

for Mct7, we were able to test the role of Mct1 in

β-HB transport across the RPE using an MCT1/2

inhibitor (AR-C155858, (21)). Fully differentiated

and polarized hfRPE cells on transwell filters (RT>

500 Ω∙cm2) were used in these transport

experiments. Glucose-free Ringer solution

containing lactate (5 mM) was added to the apical

bath in the presence or absence of AR-C155858

(10 μM; apical chamber) (or another well-

established MCT1 inhibitor, α-CHC; 5 mM).

Ringer’s solution in the basal chamber (which

initially contained no lactate) was then analyzed

for lactate concentration at 30 and 60 min time

points. Over this 1 hr timespan, RT remained well

above 500 Ω∙cm2, indicating that the barrier

properties of the hfRPE cells were intact

throughout the experiment. As shown in Figure

7A, both AR-C155858 and α-CHC (a competitive

inhibitor of MCT1) significantly decreased

transepithelial lactate transport by ~40-50%. AR-

C155858 together with α-CHC decreased lactate

transport by 65-70%. Next, we evaluated the role

of MCT1 in β-HB transport across the RPE. In

this experiment, Ringer containing β-HB (5 mM)

was added to the hfRPE basal chamber and Ringer

in the apical chamber was analyzed for β-HB at 15

and 30 min time points. As shown in Figure 7B,

AR-C155858 significantly inhibited β-HB

transport by ~75-80%, indicating that MCT1 plays

a major role in β-HB transport across the RPE

apical membrane. Since MCT1 is also known to

transport acetoacetate (15), we evaluated

acetoacetate transport across the RPE using the

same strategy: Ringer’s solution containing

lithium acetoacetate (5 mM) was added to the

hfRPE basal chamber and Ringer’s solution in the

apical chamber was analyzed at 15 and 30 min for

acetoacetate using the total ketone kit. Figure 7C

shows that hfRPE can mediate transepithelial

acetoacetate transport, and like β-HB transport,

this process was significantly inhibited by AR-

C155858, suggesting that the RPE can potentially

release acetoacetate as a metabolic substrate for

the retina.

To evaluate the role of MCT1 in β-HB

uptake by the retina, we incubated freshly isolated

mouse retina with either U-13

C lactate as a positive

control or 2,4-13

C2 β-HB for 30 min in the

presence or absence of AR-C155858 (10 μM)

(Figure 8). Samples were subsequently analyzed

by GC-MS to determine 13

C incorporation into

various downstream metabolites. 2-way ANOVA

with Sidak’s multiple comparisons test was used

for data analysis. As expected, mouse retinas

given 13

C lactate and treated with AR-C155858

contained significantly lower levels of 13

C

metabolites (lactate, citrate, and glutamate) than

control (Figure 8A), indicating that lactate

transport into the retina is predominantly mediated

by MCT1/2. In contrast, the effect of AR-

C155858 on 13

C metabolite incorporation from

2,4-13

C2 β-HB into citrate and glutamate were not

significant (Figure 8B), suggesting that MCT7

may be the dominant transporter for β-HB in the

retina, more specifically, photoreceptor cells.

Discussion

Previous studies have shown that the RPE

is capable of metabolizing POS, enriched in

saturated fatty acids (2,26)), via mitochondrial β-

oxidation (5,27). Direct measurement of oxygen

consumption by hfRPE cells using the Seahorse

XF24 Bioanalyzer confirmed that hfRPE cells

could use palmitate as a substrate for oxidative

respiration (Figure 1). This is consistent with our

finding that hfRPE cells incorporate 13

C palmitate

into key TCA cycle metabolites, citrate and

glutamate (Figure 3D). However, in addition to

using palmitate as an energy substrate, we found

that RPE cells used ≈ 50% of acetyl-CoA derived

from palmitate to produce β-HB. Furthermore, β-

HB was preferentially released across its apical

membrane towards the retina, suggesting that β-

HB plays an important role in retinal metabolism

and health. These findings led us to hypothesize a

model of metabolic coupling between the RPE and

the retina in which the RPE metabolizes fatty acids

derived from phagocytized photoreceptor outer

segment (POS) discs and produces β-HB as an

end-product of fatty acid β-oxidation (as illustrated

in Figure 9). β-HB is transported across the RPE

apical membrane via MCT1, and into

photoreceptor cells via MCT1 and MCT7.

In this study, we focused on β-HB as the

major substrate that is shuttled from the RPE and

retina. However, acetoacetate, the other ketone

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body, can also potentially fulfill this role. It is

important to mention that a similar mechanism of

ketone shuttle and metabolism has been proposed

as a form of neuron-astrocyte interaction (28).

This mechanism is based on the ability of

astrocytes to metabolize fatty acids, producing

ketone bodies β-HB and acetoacetate (29,30) that

can be utilized by adjacent neurons as oxidative

substrates. Drawing parallels between the neuron-

astrocyte and RPE-retina systems, one could posit

that the RPE could also produce and release

acetoacetate and β-HB as a substrate for

photoreceptor metabolism. Consistent with this

notion, both β-HB and acetoacetate are well-

established transport substrates of MCT1, and we

showed that transepithelial acetoacetate transport

across the RPE was significantly inhibited by the

specific MCT1 inhibitor, AR-C155858. Since the

retina expresses MCT1 (and MCT7) and that

acetoacetate is the direct downstream product of β-

HB catabolism, photoreceptor cells are likely to

metabolize acetoacetate as an oxidative substrate

as they do with β-HB.

Since POS phagocytosis is regulated by

the circadian rhythm (1), the utilization of shed

POS as a source of lipid substrate for RPE

metabolism suggests that ketone production by the

RPE follows the same diurnal schedule of POS

shedding, an event that is characterized by a

transient burst of activity in the morning upon

light-onset. Thus this may be a mechanism

through which photoreceptor cells signal to the

RPE, which responds by ramping up production

and transport of β-HB to photoreceptor cells to

help fulfill the metabolic demands of POS

renewal. At the molecular level, POS

phagocytosis can activate two potential pathways

in the RPE that enhance ketone synthesis: (1)

InsP3/Ca2+

/cAMP signaling (31) and (2)

peroxisome proliferator-activated receptors

(PPAR) (11). The former is supported by a recent

study that used high-throughput RNA-Seq analysis

to show that POS phagocytosis activates genetic

pathways associated with InsP3/Ca2+

signaling

(31), which can activate Ca2+

-sensitive adenylyl

cyclase type 1 (ADCY 1; which is enriched in the

RPE) to produce cAMP and stimulate Hmgcs2

expression via a CRE element at its promoter.

Free fatty acids (released from digested POS) can

act as ligands of PPAR (32), which forms a

heterodimer with the retinoid X receptor, to

stimulate Hmgcs2 expression by binding to the

Nuclear Receptor Response Element (NRRE) at its

promoter (10). Consistent with this hypothesis,

analysis of RNA-Seq data by Mustafi et al. (31)

also revealed that mouse RPE isolated at 1.5 hr

after light onset express 30% higher Hmgcs2

transcript than RPE isolated at 9 hrs. Furthermore,

PPAR-gamma coactivator 1 alpha (PGC-1α;

encoded by PPARGC1A), the master regulator of

mitochondrial biogenesis and oxidative

metabolism (33), is expressed at 50% higher levels

in mouse RPE isolated at 1.5 vs. 9 hr after light

onset. This data supports our hypothesis that

photoreceptor phagocytosis is followed by

activation of fatty acid β-oxidation and

ketogenesis pathways.

Our data showed that the retina rapidly

takes up β-HB from the extracellular space and

metabolized it via oxidative metabolism. While

the retina is composed of many different cell

types, RPE-generated β-HB is most likely taken up

and metabolized in the photoreceptor or Müller

glia cells due to their close proximity with the

RPE. Here, we demonstrated that the recently

identified β-HB transporter, MCT7, is expressed

highly and specifically in mouse photoreceptor

cells (Figure 6), suggesting that a large fraction of

β-HB produced by the RPE are transported into

photoreceptor cells. In addition to transport,

photoreceptor cells possess a high mitochondrial

capacity that allows them to effectively metabolize

β-HB (33). Müller cells on the other hand, have

low mitochondria density and are primarily

glycolytic (34,35). The oxidative nature of

photoreceptor cells provides two key factors that

are needed for efficient β-HB metabolism: (1) a

high NAD+/NADH ratio, which promotes BDH-

mediated conversion of β-HB to acetoacetate, and

(2) a source of succinyl-CoA (a TCA cycle

metabolite), which is required as a co-factor for

OXCT1-mediated conversion of acetoacetate to

acetoacetyl-CoA, a key step in β-HB catabolism in

the mitochondria. Importantly, OXCT1 is highly

expressed in photoreceptor cells, as determined by

qPCR analysis of CD73+ rod cells and microarray

data of retina cells from Seigert et al. (13) (Figure

4). While ketone metabolism generally occurs in

the mitochondria, there is an enzyme, acetoacetyl-

CoA synthetase (Aacs) that catalyzes the same

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reaction as Oxct1, but in the cytosol. However,

Aacs is not expressed in mouse retina according to

two microarray datasets (GSE10246 and Siegert’s

data), suggesting that mitochondrial Oxct1 is the

dominant pathway through which β-HB is

metabolized in photoreceptor cells. Taken

together, our analysis support the notion that β-HB

produced by the RPE is primarily metabolized in

photoreceptor cells.

In this study, we found that glutamate is a

the major product of β-HB metabolism. This is

consistent with a recent study that reported that β-

HB is a preferred substrate for glutamate synthesis

in neurons (36) where glutamate acts as an

important neurotransmitter and as a co-factor for

glutathione and amino acid synthesis. In our

experiments, we found that 13

C β-HB metabolism

was unaffected by the presence or absence of

unlabeled glucose (data not shown), suggesting

that glucose metabolism pathways does not

compete with β-HB metabolism. Beyond the role

of β-HB as a metabolic substrate, β-HB also

suppresses oxidative stress by acting as an

inhibitor of HDAC1 in various cell types (37).

This notion is backed by numerous studies

showing that increasing β-HB levels, by

administering a ketogenic diet or direct β-HB

infusion, can ameliorate symptoms of

neurodegenerative diseases such as Alzheimer’s

and Huntington’s disease (reviewed in (38)). In

the context of RPE and retina health, the

neuroprotective effects of β-HB present a useful

mechanism through which the RPE could shield

photoreceptor cells (and potentially Müller cells)

from the high stress environment of the subretinal

space. The RPE itself is a likely beneficiary of its

own β-HB production, which helps it overcome

the toxicity of oxidative stressors that it inherits

from phagocytized POS. An important

consideration for this mechanism is whether the

RPE is producing enough β-HB to exert a

physiological significant impact on RPE and retina

health. Considering that the adult human eye has a

subretinal space volume of 10 µL (39) across the

retina surface area of 12 cm2 (40). β-HB

production at a rate of ~ 1 nmole/hr.cm2 RPE (as

estimated from our data) will generate ~ 1 mM β-

HB concentration in one hour. Since β-HB has

been shown to induce histone acetylation in the

kidney at concentrations as low as 0.2 mM, we

expect the role of β-HB in RPE and retinal cell

health to be non-trivial.

Summary

In this study, we show for the first time

that mouse and human RPE cells can metabolize

fatty acids to produce ketones that are

preferentially released into the retinal

microenvironment. Furthermore, we demonstrate

that the retina can take up and metabolize β-HB as

a metabolic substrate. However, our study also

brought about many new questions involving the

regulation of β-HB production in the RPE and the

role of β-HB in the retina: (1) Does phagocytosis

of POS induce ketogenesis? Does β-HB

production in the RPE in vivo follow the diurnal

cycle of POS phagocytosis? (2) Does

InsP3/Ca2+

/cAMP signaling and PPAR activate

RPE ketogenesis? (3) Can β-HB inhibit oxidative

stress in the RPE and retina? Can it slow retinal

degeneration in disease models? These questions

explore the mechanism of β-HB production in the

RPE and the role of β-HB in retinal health and

function.

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Footnotes

The authors thank Drs. Sheldon Miller and Arvydas Maminishkis for kindly providing us the hfRPE cell

culture model developed in their laboratory. This work was supported by NIH grant EY012042 to NJP

and NIH grants to EY017863 and EY006641 to JBH.

Figure Legends

Figure 1: hfRPE cells metabolize palmitate as an oxidative substrate. hfRPE cells were incubated in

CO2/HCO3-free Ringer 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 reads (set to 100%) for each

sample. “*” indicates p < 0.05 and “**” indicates p < 0.01.

Figure 2: The RPE expresses Hmgcs2, the key enzyme for ketogenesis. (A) Acetyl-CoA derived from

beta-oxidation of fatty acids can be shunted into the HMG-CoA pathway, which is regulated by Hmgcs2

activity. (B) GSE10246 microarray (Lattin et al., 2008) analysis for all enzymes in ketogenesis in mouse

RPE vs. other tissues. (C) qRT-PCR and (D) western blot analysis for Hmgcs2 expression in mouse RPE

vs. 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 and basal chambers) with glucose, palmitate, or both, and the apical supernatant

was evaluated for β-HB content using StanBio’s β-HB kit. (C) hfRPE cells were incubated in Ringer

containing glucose, palmitate, or both, and the apical and basal supernatant was evaluated for total ketone

(β-HB + acetoacetate) content using Wako’s total ketone kit. (D) hfRPE cells were incubated with 1-13

C

palmitate with or without unlabeled glucose for three hours and 13

C incorporation into TCA cycle

metabolites and β-HB into the cells was evaluated using GC-MS. (E) hfRPE cells were incubated with 1-13

C palmitate with or without unlabeled glucose for three hours and the apical and basal supernatant was

analyzed for 13

C β-HB levels. (F) Freshly isolated mouse RPE was incubated in Ringer containing

palmitate and glucose for three hours and the supernatant was analyzed for β-HB level using StanBio’s β-

HB kit.

Figure 4: Photoreceptor cells express OXCT1. (A) qRT-PCR analysis for Oxct1 expression in

dynabead®-purified CD73-positive vs. 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 (Seigert et

al., 2012) analysis for Oxct1 expression in rod and cone photoreceptor cells vs. other retinal cell types.

Figure 5: Mouse retina metabolizes 13

C β-HB to produce TCA cycle metabolites and amino acids. (A) Schematic of 2,4-

13C2 β-HB metabolism through one cycle of the TCA cycle (ending at oxaloacetate).

For each metabolite, the 13

C carbon/s is highlighted in gray at the expected carbon position. Each 2-13

C

acetyl-CoA that enters the TCA cycle will produce single labeled metabolites. In the second turn, new 2-13

C acetyl-CoA will add to a 2-13

C1 oxaloacetate to produce 2,4-13

C2 citrate and so on. (B) Freshly

isolated mouse retina was incubated in KREBS/HCO3 Ringer containing 2,4-13

C2 β-HB over 15, 30, or 60

min and the whole tissue was analyzed for 13

C labeled metabolites using GC-MS. % 13

C-labeled vs.

unlabeled (C) citrate, (D) glutamate, and (E) aspartate isotopomers in mouse retina incubated with 2,4-13

C2 β-HB for 15, 30, or 60 min. M1 represents single 13

C-labeled metabolites (irrespective of carbon

position) and M2 represents double 13

C-labeled metabolites and so forth. Data is presented as % of 13

C-

labeled metabolite relative to the total (both labeled and unlabeled) within the sample.

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Figure 6: MCT1 and MCT7 are specifically expressed in photoreceptor cells. (A) GSE10246

microarray (Lattin et al., 2008) analysis of all MCT isoforms in the RPE and retina vs. other tissues. (B)

Microarray data (Roesch et al., 2008) analysis of single mouse rod photoreceptor and Müller cells for

Mct1 and Mct7 expression. (C) Microarray data (Siegert et al., 2012) analysis for Mct7 expression in rod

and cone photoreceptor cells vs. 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 vs. negative rod photoreceptor cells and other mouse ocular tissues.

Figure 7: hfRPE cells transports β-HB across its apical membrane 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. (B) Basal to apical transport of β-HB across the hfRPE cells on

transwells in the presence of AR-C155858 (10 µM) in the apical bath. (C) Basal to apical transport of

acetoacetate across the hfRPE cells on transwells in the presence of AR-C155858 (10 µM) in the apical

bath.

Figure 8: β-HB transport into the retina was independent of MCT1 activity. (A) Freshly isolated

retina was incubated with U-13

C lactate (5 mM) or (B) 2,4-13

C2 β-HB (5 mM) for 30 min in the presence

or absence of AR-C155858 (10 µM). Retina samples were analyzed for 13

C metabolites using GC-MS.

Data is presented as % of 13

C-labeled metabolite relative to the total (both labeled and unlabeled) within

the sample.

Figure 9: Model of metabolic coupling between photoreceptor cells and the RPE. Shed ROS are

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.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Figure 8

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Figure 9

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PhilpJeffrey Adijanto, Jianhai Du, Cynthia Moffat, Erin Seifert, James B. Hurley and Nancy J.

for Metabolic Coupling with the Outer RetinaThe Retinal Pigment Epithelium utilizes Fatty Acids for Ketonegenesis: Implications

published online June 4, 2014J. Biol. Chem.

10.1074/jbc.M114.565457Access the most updated version of this article at doi:

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