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Jianhai Du, Aya Yanagida, Anh Huan Vo, James B Hurley, Jennifer R Chao; Reductive carboxylation of 2-oxoglutarate to citrate in retinal pigment epithelium . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4637.
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© ARVO (1962-2015); The Authors (2016-present)
Mitochondrial dysfunction in retinal pigment epithelium (RPE) can contribute to genetic and age-related ocular diseases. The regulation of mitochondrial metabolism in healthy and diseased RPE is not yet understood. We have used 13C tracer analysis to study the mitochondrial metabolic flux in human fetal RPE (hfRPE) and induced pluripotent stem cell (iPSC)-derived RPE. We also analyzed iPSC-RPE derived from patients with Kearns Sayre Syndrome (KSS) phenotype, a mitochondrial disease with pigmentary retinopathy.
hfRPE, iPSC-RPE and ARPE 19 cells were grown for 4-6 weeks and then switched into culture medium with U-13C labeled glutamine. Metabolites were extracted and analyzed by GC-MS and LC-MS. Isotopomers of glutamine-derived mitochondrial intermediates were calculated by MSD ChemStation and corrected for natural abundance.
U-13C glutamine rapidly labeled mitochondrial intermediates in hfRPE cells within 1 hour. Strikingly, M5 citrate from reverse carboxylation of 2-oxoglutarate (2-OG) was 2 fold higher in enrichment than M4 citrate which derives from classical forward TCA cycle activity. This reverse flux to citrate from 2-OG was as high as 36% (M5 citrate/M5 2-OG) in hfRPE and was between 35%-45% in iPSC RPE, ARPE 19 and mouse RPE cells. However, the reverse flux to citrate was much lower in mouse retina and other neuronal tissue (<10%). NADP-dependent isocitrate dehydrogenase (IDH1/IDH2) catalyzes this reductive carboxylation. We found that both isoforms are highly expressed in fRPE. KSS iPSC RPE had diminished reductive carboxylation and elevated oxidation of glutathione.
Compared to retina and other neuronal tissue, RPE has a high-rate of reductive carboxylation through NADP-dependent IDH activity. It is likely that this reductive pathway regulates cellular redox homeostasis. Dys-regulation of this pathway might contribute to oxidative damage and the pathogenesis of pigmentary retinopathy in KSS patients.
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