
==== Front
Invest Ophthalmol Vis Sci
Invest Ophthalmol Vis Sci
IOVS
Investigative Ophthalmology & Visual Science
0146-0404
1552-5783
The Association for Research in Vision and Ophthalmology

39230994
10.1167/iovs.65.11.5
IOVS-24-39860
Biochemistry and Molecular Biology
Biochemistry and Molecular Biology
Metabolic Phenotyping of Healthy and Diseased Human RPE Cells
Nutrient Use in RPE
Rizwan Saira 1 2
Toothman Beverly 1 2
Li Bo 1 2 3
Engel Abbi J. 4
Lim Rayne R. 4
Niernberger Sheldon 4
Lu Jinyu 1 2
Ratliff Cloe 1 2
Xiang Yinxiao 1 2
Eminhizer Mark 1 2
Chao Jennifer R. 4
Du Jianhai 1 2
1 Department of Ophthalmology and Visual Sciences, West Virginia University, Morgantown, West Virginia, United States
2 Department of Biochemistry and Molecular Medicine, West Virginia University, Morgantown, West Virginia, United States
3 Department of Ophthalmology, Affiliated Hospital of Yangzhou University, Yangzhou University, Yangzhou, Jiangsu Province, China
4 Department of Ophthalmology, University of Washington, Seattle, Washington, United States
# Correspondence: Jianhai Du, One Medical Center Dr, PO Box 9193, WVU Eye Institute, Morgantown, WV 26505, USA; jianhai.du@hsc.wvu.edu.
Jennifer R. Chao, 750 Republican Street, Box 358058, Seattle WA 98109, USA; jrchao@uw.edu.
* SR, BT, and BL contributed equally to the work presented here and should therefore be regarded as equivalent authors.

04 9 2024
9 2024
65 11 514 8 2024
24 2 2024
Copyright 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Purpose

Metabolic defects in the retinal pigment epithelium (RPE) underlie many retinal degenerative diseases. This study aims to identify the nutrient requirements of healthy and diseased human RPE cells.

Methods

We profiled nutrient use of various human RPE cells, including differentiated and dedifferentiated fetal RPE (fRPE), induced pluripotent stem cell–derived RPE (iPSC RPE), Sorsby fundus dystrophy (SFD) patient-derived iPSC RPE, CRISPR-corrected isogenic SFD (cSFD) iPSC RPE, and ARPE-19 cell lines using Biolog Phenotype MicroArray Assays.

Results

Differentiated fRPE cells and healthy iPSC RPE cells can use 51 and 48 nutrients respectively, including sugars, intermediates from glycolysis and tricarboxylic acid (TCA) cycle, fatty acids, ketone bodies, amino acids, and dipeptides. However, when fRPE cells lose their epithelial phenotype through dedifferentiation, nutrient use becomes restricted to 17 nutrients, primarily sugar and glutamine-related amino acids. SFD RPE cells can use 37 nutrients; however, compared to cSFD RPE and healthy iPSC RPE, they are unable to use lactate, some TCA cycle intermediates, and short-chain fatty acids. Nonetheless, they show increased use of branch-chain amino acids (BCAAs) and BCAA-containing dipeptides. Dedifferentiated ARPE-19 cells grown in traditional culture media cannot use lactate and ketone bodies. In contrast, nicotinamide supplementation promotes differentiation toward an epithelial phenotype, restoring the ability to use these nutrients.

Conclusions

Epithelial phenotype confers metabolic flexibility to healthy RPE for using various nutrients. SFD RPE cells have reduced metabolic flexibility, relying on the oxidation of BCAAs. Our findings highlight the potentially important roles of nutrient availability and use in RPE differentiation and diseases.

metabolic phenotyping
nutrient use
retinal pigment epithelium
carbon source
nitrogen source
==== Body
pmcRetinal pigment epithelium (RPE) is crucial in supporting photoreceptor function and survival through various functions, including the visual cycle, nutrient transport, light absorption, phagocytosis of outer segments, and formation of the blood-retina barrier.1 To sustain these critical functions, the RPE relies on a robust mitochondrial metabolism by oxidizing fuels from the photoreceptors and choroidal supply.2 Defects in RPE mitochondrial metabolism can cause epithelial-mesenchymal transition (EMT) and dedifferentiation, with loss of characteristic epithelial traits such as tight junctions, pigmentation and polarity, subsequently leading to photoreceptor death in retinal degenerative diseases, including Sorsby fundus dystrophy (SFD) and age-related macular degeneration (AMD).2–4

Cultured human RPE cells are valuable models for investigating RPE function and retinal disease. They exhibit typical RPE morphology, function and signature gene expression, closely resembling native RPE cells.5–8 Human fetal RPE (fRPE) cells, considered the gold standard of RPE culture, have been rigorously characterized in terms of physiology, gene expression and metabolism.5,9,10 Similarly, patient-derived induced pluripotent stem cells (iPSC) RPE have been well characterized and offer the advantage of modeling inherited retinal diseases from patients. SFD, a rare early-onset macular degeneration, results from mutations in the tissue inhibitor of metalloproteinase-3 (TIMP3) gene.11 In our previous study, we showed that iPSC RPE derived from SFD patients carrying the S204C mutation in TIMP3 have irregular extracellular matrices (ECM) and basal laminar deposits, while correction of the S204C variant in SFD iPSC RPE (cSFD) attenuates these findings.12 ARPE-19 cells offer advantages over fRPE and iPSC RPE in terms of easier availability and low cost. ARPE-19 cells show fibroblast-like morphology under traditional culture media. However, supplementation with nicotinamide (NAM), can rapidly induce differentiation of ARPE-19 cells into cells with epithelial characteristics through revitalization of mitochondrial metabolism.13,14

In this study, we conducted extensive metabolic screening of carbon and nitrogen sources across multiple human RPE cultures, including differentiated and dedifferentiated fRPE, healthy and SFD RPE, and ARPE-19 RPE cells. We found that healthy and differentiated RPE cells demonstrate robust metabolic flexibility, allowing them to use diverse nutrient sources. In contrast, dedifferentiated and SFD RPE cells have reduced metabolic flexibility, relying on specific nutrient sources. These cell-specific differences in nutrient use should provide insights into the underlying mechanisms of RPE differentiation and disease.

Methods

Reagent and Key Resources

All the reagents and key resources are detailed in Supplementary Table S1 or methods.

Human RPE Cell Culture

Human fRPE was isolated from human fetal eye cups with no identifiers, obtained from two distinct donors to the Birth Defects Research Laboratory at the University of Washington (UW) and cultured as previously described.9,15 RPE cells at passages 4–6 were used for experiments, and they were seeded at a density of either 5 × 104 cells/well (regular density) or 5 × 103 cells/well (low density) in 96-well plates in MEM RPE media (See details in Supplementary Table S1). Normal control iPSC RPE, SFD patient-derived iPSC RPE and CRISPR-corrected isogenic control (cSFD) RPE were generated in a stepwise procedure as previously reported (see details in Supplementary Methods).12 Human ARPE-19 cells obtained from the American Type Culture Collection (ATCC) were used at passages 5–8 for the experiments. The cells were seeded at 2 × 104 cells/well in 96-well plates and cultured in three different media to mimic dedifferentiated and differentiated states: 1) DMEM/F-12 media with 5% FBS, 2) MEM RPE media, and 3) MEM-NAM (MEM RPE media supplemented with 10 mM NAM as reported.13,14 All RPE cells were cultured for four weeks before metabolic screening.

Metabolic Screening With Biolog Phenotyping MicroArrays

Biolog Phenotyping MicroArrays (Biolog Inc., Hayward, CA, USA) are 96-well plate assays that measure cellular metabolic activity in using various carbon and nitrogen substrates. Each well contains a specific nutrient at 100 µM concentration, except for the negative control. Cells are added to the 96-well in Biolog's proprietary nutrient-limiting media, followed by the addition of a proprietary redox dye. This dye is reduced by cellular NADH generated by substrate use, causing a color change measured by a plate reader. High absorbance indicates greater metabolic activity in using specific substrate through NADH production. We used two Biolog Phenotype MicroArrays (PM-M1 for carbon sources and PM-M2 for nitrogen sources) to assess nutrient use in RPE cells (see details in Supplementary Tables S1–S3). After pre-incubation with 60 µL of nutrient-limiting media (IFM 1 [a proprietary RPMI 1640-based Biolog culture media], glutamine 200 µM, 1% dialyzed FBS, and 1% penicillin and streptomycin) for one hour, RPE cell-containing plates were washed with PBS and incubated with transferred pre-incubation media from PM-M1 or PM-M2 microarrays for 40 hours at 37°C in a 5% CO2 incubator. The redox dye mix MA (10 µL) was then added, and the plates were sealed and read at an absorbance of 595 nm every 15 minutes for four hours.

13C Glutamine Tracing in FRPE Cells

Human fRPE cells seeded at regular and low densities were cultured for four weeks and changed into DMEM with 5 mM glucose and 1 mM 13C glutamine. After incubation for 48 hours, the RPE cells and media were collected for metabolite extraction and analysis of 13C glutamine-derived metabolites with gas chromatography mass spectrometry (GC MS) as previously reported.9,10

Statistics

All data are expressed as the mean ± SD. Fold change of absorbance over negative control without nutrient source >1.5, or P < 0.05 by Student's unpaired two-tailed t-tests using GraphPad Prism 9.0, was considered significant. Heat maps were generated in Microsoft Excel.

Results

Differentiated Human FRPE Cells Demonstrate Remarkable Metabolic Flexibility in Nutrient Use

To study nutrient use in differentiated fRPE cells, fRPE were cultured for four weeks to maturity. RPE demonstrated characteristic cobblestone morphology and pigmentation (Supplementary Figs. S1A–C). The differentiated RPE cultures were switched to nutrient-limiting media with or without a specific nutrient from the PM-M1 plate to screen the use of 91 carbon sources (Fig. 1A, Supplementary Table S2). Remarkably, differentiated RPE could use 23 carbon sources including sugars, glycolysis intermediates (such as sugar phosphates, lactate, and pyruvate), TCA cycle intermediates, nucleosides, fatty acids and ketone bodies (Figs. 1B, 1C). In addition to glucose, differentiated RPE had the capacity to use other sugars such as fructose, mannose, and galactose (Figs. 1B, 1C). Nucleosides, containing ribose moiety, can serve as alternative energy sources. Differentiated RPE could robustly use inosine, adenosine and uridine.

Figure 1. Metabolic phenotyping of differentiated human fRPE cells. (A) A schematic for metabolic phenotyping with carbon sources. Human fRPE cells were grown for four weeks in a 96-well plate and then switched into nutrient-limiting media containing different carbon sources in each well from PM-M1 plate. The use of nutrients leads to NADH production, causing a color change in a redox sensitive dye to purple, which is quantified by a microplate reader at 595 nm. (B) The use of carbon sources by differentiated human fRPE cells and (C) an illustration of the metabolic pathways. (D) A schematic for metabolic phenotyping of nitrogen sources using PM-M2. (E) The use of nitrogen sources by differentiated human fRPE cells and (F) an illustration of the metabolic pathways. N = 3. Fold change over negative control without nutrient source (Ctr) >1.5 or *P < 0.05 over Ctr. Gluc, glucose; Gal, galactose; Mann, Mannose; F1P, fructose 1-phosphophate; R1P, ribose 1-phosphate; G6P, glucose 3-phosphate; G3P, glycerol 3-phosphate; AcAc, acetoacetic acid; 3HB, β-hydroxy butyrate; MelA, melibionic acid.

Similar to PM-M1 carbon source screening, we evaluated the use of 92 nitrogen sources, including 27 amino acids and 60 dipeptides in differentiated RPE using the PM-M2 plate as illustrated in Figure 1D. Differentiated RPE could metabolize 7 amino acids and 17 dipeptides (Fig. 1E). Except for methionine and tryptophan, the remaining five amino acids (proline, ornithine, glutamine, glutamate, and aspartate) follow a similar metabolic pathway in the generation of α-ketoglutarate (αKG) or oxaloacetate, which serve as fuels for the TCA cycle (Figs. 1E, 1F). Notably, glutamine, glutamate, aspartate, and proline are also present in the 17 dipeptides. These findings suggest that differentiated fRPE cells have remarkable metabolic flexibility, enabling them to effectively use diverse nutrient sources to fuel their mitochondrial metabolism (Figs. 1C, 1E).

Dedifferentiated Human FRPE Cells Lose Metabolic Flexibility, Primarily Relying on Sugars and Glutamine as Their Main Nutrient Sources

Seeding fRPE at low density induces their dedifferentiation into a fibroblast-like phenotype.5 To understand nutrient use in dedifferentiated RPE cells, we seeded fRPE at 10% of regular density and cultured them for four weeks. As expected, these cells transitioned from their characteristic cobblestone structure to a fibroblast-like morphology, with upregulated expression of EMT markers including α-smooth muscle actin (αSMA) and vimentin (Fig. 2A, Supplementary Figs. S1D–F, Supplementary Fig. S2). Although different cell densities were noted, total protein content was similar between differentiated and dedifferentiated RPE, indicating similar confluence between culture conditions (Supplementary Fig. S3). Compared to differentiated fRPE, dedifferentiated fRPE used fewer nutrients, with only eight carbon and six nitrogen sources. Intriguingly, almost all carbon sources were sugar and sugar phosphates, whereas all nitrogen sources contained glutamine (Figs. 2A–C). These results suggest that dedifferentiated RPE cells lose the ability to use multiple nutrient sources such as lactate, fatty acids, ketone bodies, and proline. Instead, they become largely restricted to consuming sugars and glutamine (Supplementary Fig. S4). RPE seeded at low density for 1 week displayed a fibroblast-like morphology resembling that of 4-week cultured low-density fRPE cells but expressed less αSMA (Supplementary Figs. S5A, S5B, Supplementary Figs. S1D–F, Supplementary Fig. S2). Nutrient use in one-week cells was largely consistent with those of four-week fRPE, except for the capability to use galactose and adenosine at one week (Supplementary Fig. S5B), suggesting that time in culture has minimal impact on the metabolic phenotype of nutrient use in RPE cells seeded at low density. Consequently, we did not perform PM-M2 screening on one-week fRPE.

Figure 2. Metabolic phenotyping of dedifferentiated human fRPE. (A) Human fRPE were seeded at low density to induce dedifferentiation with a mesenchymal phenotype in a 96-well plate and then switched into nutrient-limiting media containing different carbon (PM-M1) or nitrogen sources (PM-M2) for metabolic phenotyping. (B) Nutrient use of carbon sources by dedifferentiated human fRPE. N = 4. Fold change >1.5 or P < 0.05 over Ctr. (C) Nutrient use of nitrogen sources by dedifferentiated human fRPE. N = 3. Fold change >1.5 or P < 0.05 over Ctr. (D, E) Dedifferentiated RPE consumes more glutamine for their mitochondrial metabolism. 13C-glutamine and its derived metabolites in media (D) and RPE cells (E) were analyzed by GC MS and presented as fold change of 13C labeled abundance over low density RPE. N = 4. *P < 0.05 versus differentiated RPE. (F) Heat maps of altered metabolic genes in dedifferentiated RPE cells induced by TNT (left panel) or multiple passage (right panel) (G) The dedifferentiated RPE cells lose the ability to use multiple nutrient sources but switch to use glutamine, enhancing proline and collagen synthesis. Magenta denotes an increase and blue denotes a decrease in gene expression or metabolic pathways. PFKFB4, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4; LDHA, lactate dehydrogenase A; OGDH, oxoglutarate dehydrogenase; PRODH; proline dehydrogenase; PYCR1, pyrroline-5-carboxylate reductase 1; P4H4, prolyl 4-hydroxylase subunit alpha or beta; GMPPA, GDP-mannose pyrophosphorylase A; PMM2, phosphomannomutase 2.

To investigate how dedifferentiated RPE cells use glutamine, we incubated differentiated and dedifferentiated RPE with 13C glutamine for 48 hours and analyzed 13C glutamine-derived intracellular and extracellular metabolites (Figs. 2D, 2E, Supplementary Fig. S6). Dedifferentiated RPE consumed more 13C glutamine in the media, which was used to produce mitochondrial intermediates and amino acids, especially succinate and proline (Figs. 2D, 2E, Supplementary Fig. S6). In addition to succinate oxidation, succinate can be generated from fumarate by the reverse reaction of succinate dehydrogenase (SDH), which can be quantified by the ratio of labeled M3 fumarate/M3 succinate16 (Supplementary Fig. S7). Our results show that reversal of SDH is significantly increased in dedifferentiated RPE, along with an increase in succinate oxidation. These results suggest that dedifferentiated RPE cells have an impaired electron transport chain and reprogram their metabolism through reverse SDH and proline dehydrogenase towards succinate and proline production.

We next analyzed the expression of metabolic genes related to nutrient use in two RNA-Seq data sets: (1) differentiated fRPE compared to dedifferentiated RPE induced by multiple passages17 and (2) differentiated primary adult RPE compared to dedifferentiated RPE induced by transforming growth factor-β (TGFβ) and tumor necrosis factor α (TNFα).18 Both dedifferentiated RPE models had significant downregulation of genes involved in fatty acid oxidation, TCA cycle, transporters for lactate and ketone bodies, nucleotide metabolism, and amino acid metabolism, especially proline catabolism and glutamine synthetase (Figs. 2F, 2G, Supplementary Table S4). These data corroborate our findings that dedifferentiated RPE cannot efficiently use fatty acids, lactate, ketone bodies, proline and other nucleosides for mitochondrial metabolism. Importantly, genes involved in proline synthesis, glycolysis, collagen and heparin sulfate were substantially upregulated (Fig. 2F, Supplementary Table S4), suggesting that dedifferentiated RPE cells use sugar and glutamine to activate proline synthesis and the hexosamine biosynthetic pathway to produce collagen and glycan for ECM remodeling (Fig. 2F).

Human IPSC RPE Cells Have Similar Metabolic Flexibility to FRPE Cells but Use More Sugars and TCA Cycle Intermediates

To study nutrient use in human iPSC RPE cells, we cultured iPSC RPE to maturity at four weeks (Supplementary Fig. S8) and then replaced media with carbon or nitrogen sources from PM-M1 or PM-M2 for metabolic phenotyping (Fig. 3A). iPSC RPE could use 31 carbon sources and 17 nitrogen sources (Figs. 3B–D). Similar to fRPE, iPSC RPE demonstrated robust flexibility in using various sugars, fatty acids, lactate, ketone bodies, nucleosides, amino acids, and dipeptides (Figs. 3B–D). However, unlike fRPE, iPSC RPE showed a use preference for more sugars and TCA cycle intermediates and fewer amino acids and dipeptides (Figs. 3E, 3F). These results suggest both healthy fRPE and iPSC RPE possess metabolic flexibility in adapting to different substrates yet also have cell type-specific preferences for certain substrates.

Figure 3. Metabolic phenotyping of healthy human iPSC RPE cells. (A) Human iPSC RPE cells were differentiated in RPE media for four weeks and switched into nutrient-limiting media from PM1 or PM2 for metabolic phenotyping. (B–D) Nutrient use of carbon sources and nitrogen sources by human iPSC RPE cells. N = 3. Fold change >1.5 or P < 0.05 over Ctr. (E) A comparison of number of nutrients that were used between fRPE and iPSC RPE cells. (F) An illustration of nutrient use in iPSC RPE cells. GlcNAc, N-Acetyl-D-glucosamine; Tcba, tricarballylic acid; AlaN, L-alaninamide.

SFD IPSC RPE Cells Use Fewer Intermediates From Glycolytic and TCA Cycle Intermediates but More Branch-Chain Amino Acids (BCAAs)

We reported SFD iPSC RPE cells harboring the TIMP3 S204 mutation have increased extracellular deposits and elevated intracellular 4-hydroxyproline.12 These iPSC RPE and their isogenic controls (cSFD) were grown to maturity at 4 weeks before undergoing metabolic phenotyping with PM-M1 and PM-M2 (Fig. 4A). Both SFD and cSFD had normal cobblestone structure and pigmentation (Supplementary Fig. S9A–B). SFD RPE used 19 carbon sources and 19 nitrogen sources, whereas cSFD RPE used 28 carbon sources and seven nitrogen sources (Figs. 4B–E). SFD RPE used different types of sugar, nucleosides, fatty acids, ketone bodies, amino acids and dipeptides (Figs. 4B, 4D). However, SFD RPE could not use lactate, sugar phosphate, short-chain fatty acids (acetate and butyrate), succinate, αKG and gamma-aminobutyric acid (GABA). Notably, the CRISPR-corrected RPE cells restored the use of these nutrients (Figs. 4C–E). SFD RPE could use proline and dipeptides containing glutamine, alanine, and arginine. Intriguingly, many of the dipeptides used by SFD RPE cells contained BCAAs, leucine, isoleucine and valine (Fig. 4D). These metabolic phenotypes are different from fRPE and normal control iPSC RPE. Although the cSFD RPE cells did not use the BCAAs, they also used significantly fewer nitrogen sources (Fig. 4E, Supplementary Fig. S8C). These results suggest that SFD RPE cells have metabolic defects in recycling nutrients, such as lactate from the neural retina, and rely more on BCAAs, which may contribute to the formation of sub-RPE deposits of lipids and ECM proteins (Fig. 4F).

Figure 4. Metabolic phenotyping of iPSC SFD and cSFD RPE cells. (A) Human iPSC SFD RPE, carrying TIMP3 S204C mutation and mutation corrected cSFD RPE cells, were differentiated in RPE media for four weeks and then switched to nutrient-limiting media with carbon or nitrogen sources from PM-M1 or PM-M2 for metabolic phenotyping. (B–E) Nutrient use of (B, C) carbon sources and (D, E) nitrogen sources in iPSC SFD RPE cells. N = 3. Fold change >1.5 or P < 0.05 over Ctr. (F) An illustration of altered nutrient use in SFD iPSC RPE cells. L-Mal, L-Malic Acid; mmSuc, mono methyl succinate; 3HB, β-hydroxy butyrate; BCAAs, branch-chained amino acids.

Culture Media Composition Controls the Differentiation and Nutrient Use in ARPE-19 Cells

ARPE-19 cells, typically cultured in DMEM/F-12 media per manufacturer's instructions, undergo rapid differentiation into a differentiated RPE-like phenotype grown MEM-α-based RPE culture media supplemented with NAM.13,14 To investigate the influence of media composition on nutrient use, we cultured ARPE-19 cells under three media (DMEM/F12, MEM RPE media and MEM RPE media plus NAM) for four weeks and focused only on the screening of carbon sources using the PM-M1 plate (Fig. 5A). DMEM/F-12 cultured cells (fibroblast-like morphology, Supplementary Fig. S10A) used 16 carbon sources (Fig. 5B) but, similar to dedifferentiated fRPE, could not metabolize lactate, ketone bodies, or xylitol (Fig. 5B, Fig. 6A). MEM RPE media partially differentiated ARPE-19 cells (cobblestone structure, Supplementary Fig. S10B) and increased their carbon source use to 24 (covering 94% of those used by DMEM/F12 cells, Fig. 5C). Although they gained the ability to use ketone bodies and some sugars, lactate and xylitol remained unused (Fig. 5C). Supplementation with NAM in MEM RPE media induced differentiated RPE-like morphology (Supplementary Fig. S10C) and allowed ARPE-19 cells to use lactate, ketone bodies, sugar phosphates, xylose, and tagatose (Figs. 5D, 5E). This metabolic phenotype resembles that of differentiated fRPE and iPSC RPE, although ARPE-19 cells reduced use of short-chain fatty acids and maltose (Fig. 6A). These findings highlight the critical role of nutrient availability and use in RPE differentiation.

Figure 5. Metabolic phenotyping of ARPE-19 cells cultured in different media. (A) ARPE-19 cells were cultured in three different media: DMEM/F-12, MEM RPE media, and MEM RPE media with 10 mM NAM for four weeks. All the cells were then changed into nutrient-limiting media with carbon sources from PM-M1 for metabolic phenotyping. (B–D) Nutrient use of carbon sources of ARPE-19 cells cultured in (B) DMEM/F12, (C) MEM RPE media, and (D) MEM RPE media with NAM. Fold change >1.5 or P < 0.05 over Ctr. (E) A comparison of number of nutrients that were used in ARPE-19 cells grown in different culture media. (F, G) An illustration of altered nutrient use in APRE-19 cells cultured between DMEM/F12 and MEM RPE media with NAM. Meso-Tar, meso tartatic acid; mmSuc, mono methyl succinate; AcAc, acetoacetic acid; 3HB, β-hydroxy butyrate.

Figure 6. Comparison of metabolic phenotyping profiles of human fRPE cells, dedifferentiated fRPE, iPSC RPE cells, SFD RPE, cSFD RPE, and ARPE-19 cells grown under different culture media. Heat map visualization of nutrients that were differentially used by different RPE cells from (A) PM-M1 nutrients and (B) PM-M2 nutrient microarray plates. De-fRPE, dedifferentiated fRPE with low-density seedings; ARPE, ARPE-19 cells; MelA, melibionic acid; GlcNAc, N-Acetyl-D-glucosamine; Tcba, tricarballylic acid; mmSuc, mono methyl succinate; AlaN, alaninamide.

Discussion

In this study, we identified distinctive metabolic phenotypes of nutrient use in healthy and diseased human RPE cells (Fig. 6). Differentiated fRPE and iPSC RPE are metabolically flexible, capable of adapting to various nutrient sources. Conversely, dedifferentiated RPE cells and SFD patient-derived iPSC RPE cells have reduced flexibility and are biased toward fewer, specific substrates. NAM supplementation promoted an RPE-like metabolic adaptation in ARPE-19 cells, although their nutrient use remains distinct from differentiated RPE.

The RPE must manage a substantial nutrient influx originating daily from phagocytosed lipid- and protein-rich photoreceptor outer segments, “waste products” such as lactate produced by the neural retina, and nutrient-laden blood through the choroidal circulation.1,2 In addition to the direct transport of nutrients to the neural retina, RPE can recycle and generate nutrients via lysosomes and mitochondria to support retinal metabolism, thereby establishing a metabolic ecosystem between the outer retina and RPE.1,2,19 The ability to use a variety of nutrients is crucial for maintaining this metabolic ecosystem, and disruption of this balance because of mitochondrial or lysosomal dysfunction in RPE can lead to retinal degeneration.3,4,20 Healthy human RPE cells are versatile in using different nutrients, including glucose, galactose, lactate, fatty acids, succinate, proline, glutamine, and other amino acids.9,19,21–24 Our results confirm this versatility, as demonstrated by their full use in healthy fRPE and iPSC RPE cells, compared to the partial use by NAM-treated ARPE-19 cells.

Furthermore, we found that human RPE cells use a wide range of other nutrients including various sugars, ketone bodies, nucleosides and dipeptides. Mannose and galactose, for example, are components of glycoproteins found in abundance in outer segment proteins such as rhodopsin and peripherin 2.25–27 The ability to use these sugars from the daily degradation of outer segments may allow RPE to preserve more glucose to supply for photoreceptors. Additionally, like the liver, human RPE cells can synthesize ketone bodies, which can be used by the neural retina.22,28 Interestingly, we found that differentiated human RPE, but not dedifferentiated RPE cells, can readily use ketone bodies, suggesting that the ability to use ketone bodies might be a characteristic of RPE metabolism. Consistent with this metabolic capability, differentiated RPE expresses key genes involved in ketone body degradation,22 a feature distinct from the liver, which cannot use ketone bodies. Our findings underscore the metabolic flexibility of healthy differentiated RPE in nutrient use, which is an essential function for RPE to support retinal metabolism and health.

RPE EMT and dedifferentiation have been implicated in the pathogenesis of proliferative vitreoretinopathy and AMD.29 We found dedifferentiated RPE cells were unable to use alternative fuels like lactate, fatty acids, ketone bodies, and proline, but, instead, prefer to use sugars and glutamine. RNA-seq data from EMT models show reduced expression of genes involved in the TCA cycle, lactate transport, ketone body use and proline catabolism and glutamine synthesis. In contrast, gene expression associated with glycolysis, proline synthesis, collagen synthesis, and glycoproteins are upregulated (Fig. 2E, Supplementary Table S4). Consistently, dedifferentiated RPE cells have been reported to switch from oxidative phosphorylation to glycolysis and lose the ability to use proline as a fuel.5,15 Inhibition of mitochondrial metabolism in human RPE cells or mouse RPE is sufficient to cause the activation of glycolysis, RPE dedifferentiation and retinal degeneration.4,30 Furthermore, mitochondrial respiration is diminished in cultured primary RPE cells from AMD donors.31 Inhibiting mitochondrial respiration impedes proline consumption while increasing glucose consumption and lactate production.32 The inability of affected RPE to use exogenous lactate and other alternative nutrient sources may be an important mechanism in AMD pathogenesis.

A key feature in dedifferentiated cells is their overproduction of ECM proteins, primarily collagens and glycoproteins, through activation of cytokine signaling such as TGFβ and TNFα.33,34 In fibroblasts, TGFβ stimulates the oxidation of glucose and glutamine to synthesize proline and other amino acids crucial for ECM protein production.35 Interestingly, proline synthesis acts as a vent for growth when cells are under mitochondrial redox stress or hypoxia by recycling NADH into NAD+, thus enhancing the oxidation of glutamine and glucose.35,36 Proline synthesis may be particularly crucial in dedifferentiated RPE cells, given their inhibited mitochondrial metabolism and heavy reliance on lactate production for NAD+ regeneration. Similarly, to compensate for the deficiency in the electron transport chain or hypoxia, fumarate can serve as an electron acceptor to produce succinate,16 which is accumulated in dedifferentiated RPE. Strategies aimed at relieving mitochondrial redox stress and reverting proline synthesis to proline catabolism or succinate production could hold promise for treating RPE dedifferentiation.

TIMP3, a risk gene for AMD, is secreted by the RPE and plays a critical role in ECM remodeling by inhibiting matrix metalloproteinases.37 Specific mutations in TIMP3 result in SFD, which is characterized by a thickened layer of basal laminar drusen-like sub-RPE deposits containing lipids and ECM proteins.12,38 Unlike healthy or mutation-corrected RPE cells, SFD RPE cells use more free BCAAs or dipeptides containing BCAAs (Figs. 4F, 6). Leucine and isoleucine are ketogenic amino acids, and their oxidation promotes the synthesis and transport of fatty acids and cholesterol.39,40 BCAA oxidation is closely associated with dysregulated glucose and lipid metabolism in diabetes and non-alcoholic fatty liver diseases.39,41,42 Interestingly, intermediates of the BCAA degradation pathway are significantly elevated in the plasma of AMD patients,43 and BCAT1, a key gene in BCAA degradation, is upregulated in the RPE/choroid from AMD donors.44 Moreover, in addition to proline, ECM proteins are highly enriched in BCAAs.45 Our findings suggest that SFD RPE may upregulate the BCAA degradation pathway by using BCAAs from free amino acids or protein degradation, contributing to the formation of sub-RPE deposits.

There are a few limitations in this study. Although total cellular protein content was similar between dedifferentiated and differentiated RPE, differences in cell number and size may influence metabolic phenotype. Nutrient use in additional dedifferentiation models need to be conducted in future studies. Separately, high absorbance reflects greater reduction of NADH generated from a specific nutrient. The Biolog assays cannot detect the reduction of FADH2 or other metabolic pathways such as the synthesis of amino acids, nucleotides and fatty acids. In addition, the metabolic phenotyping assay can only measure the capability and capacity of using a specific nutrient but not preferences of nutrient use because only one nutrient is available at a time. Therefore the use might be different in vivo, and additional functional studies with nutrient supplements or genetic manipulations of metabolic pathways are needed to interpret these findings. Finally, testing additional iPSC RPE lines from a larger cohort of normal and affected donors will help control for variability in genetic and environmental backgrounds.

In conclusion, metabolic phenotyping offers a sensitive and powerful platform for measuring nutrient use in healthy and diseased human RPE cells. The distinct patterns of nutrient use of various RPE cells provide valuable insights into the mechanisms of RPE dedifferentiation and disease.

Supplementary Material

Supplement 1

Supplement 2

Acknowledgments

Supported by National Institutes of Health Grant EY034364 (J. R. C. and J. D), EY03459 (J. R. C. and J. D), EY031324 (JD), EY032462(JD), the Retina Research Foundation (JD), and funds for Core facilities P20 GM103434 and P20 GM144230 (WV INBRE grant). Fetal RPE tissue samples were made available through Dr. Ian Glass and the Birth Defects Research Laboratory at the University of Washington (UWR24HD000836).

Disclosure: S. Rizwan, None; B. Toothman, None; B. Li, None; A.J. Engel, None; R.R. Lim, None; S. Niernberger, None; J. Lu, None; C. Ratliff, None; Y. Xiang, None; M. Eminhizer, None; J.R. Chao, None; J. Du, None
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