Vitamin B3, nicotinamide, enhances mitochondrial metabolism to promote differentiation of the retinal pigment epithelium

Abstract

In the mammalian retina, a metabolic ecosystem exists in which photoreceptors acquire glucose from the choriocapillaris with the help of the retinal pigment epithelium (RPE). While the photoreceptor cells are primarily glycolytic, exhibiting Warburg-like metabolism, the RPE is reliant on mitochondrial respiration. However, the ways in which mitochondrial metabolism affect RPE cellular functions are not clear. We first used the human RPE cell line, ARPE-19, to examine mitochondrial metabolism in the context of cellular differentiation. We show that nicotinamide induced rapid differentiation of ARPE-19 cells, which was reversed by removal of supplemental nicotinamide. During the nicotinamide-induced differentiation, we observed using quantitative PCR, Western blotting, electron microscopy, and metabolic respiration and tracing assays that (1) mitochondrial gene and protein expression increased, (2) mitochondria became larger with more tightly folded cristae, and (3) mitochondrial metabolism was enhanced. In addition, we show that primary cultures of human fetal RPE cells responded similarly in the presence of nicotinamide. Furthermore, disruption of mitochondrial oxidation of pyruvate attenuated the nicotinamide-induced differentiation of the RPE cells. Together, our results demonstrate a remarkable effect of nicotinamide on RPE metabolism. We also identify mitochondrial respiration as a key contributor to the differentiated state of the RPE and thus to many of the RPE functions that are essential for retinal health and photoreception. In the mammalian retina, a metabolic ecosystem exists in which photoreceptors acquire glucose from the choriocapillaris with the help of the retinal pigment epithelium (RPE). While the photoreceptor cells are primarily glycolytic, exhibiting Warburg-like metabolism, the RPE is reliant on mitochondrial respiration. However, the ways in which mitochondrial metabolism affect RPE cellular functions are not clear. We first used the human RPE cell line, ARPE-19, to examine mitochondrial metabolism in the context of cellular differentiation. We show that nicotinamide induced rapid differentiation of ARPE-19 cells, which was reversed by removal of supplemental nicotinamide. During the nicotinamide-induced differentiation, we observed using quantitative PCR, Western blotting, electron microscopy, and metabolic respiration and tracing assays that (1) mitochondrial gene and protein expression increased, (2) mitochondria became larger with more tightly folded cristae, and (3) mitochondrial metabolism was enhanced. In addition, we show that primary cultures of human fetal RPE cells responded similarly in the presence of nicotinamide. Furthermore, disruption of mitochondrial oxidation of pyruvate attenuated the nicotinamide-induced differentiation of the RPE cells. Together, our results demonstrate a remarkable effect of nicotinamide on RPE metabolism. We also identify mitochondrial respiration as a key contributor to the differentiated state of the RPE and thus to many of the RPE functions that are essential for retinal health and photoreception. Correction: Vitamin B3, nicotinamide, enhances mitochondrial metabolism to promote differentiation of the retinal pigment epitheliumJournal of Biological ChemistryVol. 298Issue 10PreviewIn the Experimental procedures of the original article (page 12, “Conditions for cell culture” section), the concentrations of stock solutions for taurine, hydrocortisone, and triiodothyronine were mistakenly reported rather than the final concentrations of those components in the medium. Additionally, nonessential amino acids (NEAA) were mistakenly omitted from the medium recipe. Full-Text PDF Open Access In the early 20th century, the German physiologist Otto Warburg investigated the metabolism of rapidly dividing cancer cells, which he showed utilized aerobic glycolysis to fuel their anabolic activities, including DNA replication and lipid synthesis (1Warburg O.P. Negelein E. On the metabolism of carcinoma cells.Biochem. Z. 1924; 152: 309-344Google Scholar, 2Rajala R.V.S. Aerobic glycolysis in the retina: functional roles of pyruvate kinase isoforms.Front. Cell Dev. Biol. 2020; 8: 266Crossref PubMed Scopus (27) Google Scholar). Interestingly, Warburg (3Warburg O. On the origin of cancer cells.Science. 1956; 123: 309-314Crossref PubMed Scopus (10185) Google Scholar, 4Warburg O. On respiratory impairment in cancer cells.Science. 1956; 124: 269-270Crossref PubMed Google Scholar) also observed this type of metabolism in the postmitotic cells of the retina. Since then, there have been several studies demonstrating that the light-sensitive photoreceptor cells of the retina are among the most energy-demanding cells in the body (5Niven J.E. Laughlin S.B. Energy limitation as a selective pressure on the evolution of sensory systems.J. Exp. Biol. 2008; 211: 1792-1804Crossref PubMed Scopus (719) Google Scholar, 6Wong-Riley M. Energy metabolism of the visual system.Eye Brain. 2010; 2: 99-116Crossref PubMed Google Scholar). 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To fuel this type of metabolism, the photoreceptor cells are heavily dependent on an adjacent monolayer of cells called the retinal pigment epithelium (RPE). The apical surface of the RPE faces the outer segment of the photoreceptors, whereas its basal surface is juxtaposed to the nutrient-rich blood supply of the choriocapillaris. The polarity of this epithelium is essential to the many functions that it performs to maintain the health of the photoreceptors, including (1) vectorial transport of water, ions, nutrients, and waste products, (2) regeneration of the visual chromophore, (3) growth factor secretion, and (4) phagocytosis of distal photoreceptor outer segment disk membranes (10Strauss O. The retinal pigment epithelium in visual function.Physiol. Rev. 2005; 85: 845-881Crossref PubMed Scopus (2072) Google Scholar, 11Lakkaraju A. Umapathy A. Tan L.X. Daniele L. Philp N.J. Boesze-Battaglia K. cell of the retinal pigment 2020; Scopus Google Scholar). In the retinal metabolic glucose by the choriocapillaris the which this fuel and it to the photoreceptor cells K. of the retina and retinal pigment epithelium a metabolic ecosystem in the 2017; PubMed Scopus Google Scholar). In the photoreceptor cells transport the of glucose to the it as a fuel in the RPE to which is to the mitochondrial and to ATP oxidative Interestingly, this metabolic photoreceptor and RPE cells is to that and cells, which on and glucose as fuel E. a from to PubMed Scopus Google Scholar, G.L. of mitochondrial Full Text Full Text PDF PubMed Scopus Google Scholar). In to photoreceptor cells, the RPE been to heavily dependent on oxidative and mitochondrial metabolism. is because RPE cells glucose consumption to a of it to to the the RPE K. of the retina and retinal pigment epithelium a metabolic ecosystem in the 2017; PubMed Scopus Google Scholar), M. from the retina to the pigment 2020; Full Text Full Text PDF PubMed Scopus Google Scholar), A. K. 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