Inhibition of Mitochondrial Pyruvate Transport by Zaprinast Causes Massive Accumulation of Aspartate at the Expense of Glutamate in the Retina

Du, Jianhai; Cleghorn, Whitney M.; Contreras, Laura; Lindsay, Ken J.; Rountree, Austin M.; Chertov, Andrei O.; Turner, Sean; Sahaboglu, Ayse; Linton, Jonathan D.; Sadı́lek, Martin; Satrústegui, Jorgina; Sweet, Ian R.; Paquet-Durand, François; Hurley, James B.

doi:10.1074/jbc.m113.507285

Cited by 75 publications

(83 citation statements)

References 48 publications

(60 reference statements)

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“…The decrease in glucose concentration in the media during the course of those experiments was negligible. For fatty acid analysis, RPE cells were labeled with 2 mM U- 13 C glutamine in DMEM with 1% FBS for 48 h. RPE cells were homogenized, and the metabolites were extracted, dried, derived with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (TBDMS), and analyzed by GC-MS (Agilent 7890/5975C) as described in detail (60)(61)(62).…”

Section: Methodsmentioning

confidence: 99%

Reductive carboxylation is a major metabolic pathway in the retinal pigment epithelium

Yanagida

Knight

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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102

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The retinal pigment epithelium (RPE) is a monolayer of pigmented cells that requires an active metabolism to maintain outer retinal homeostasis and compensate for oxidative stress. Using 13 C metabolic flux analysis in human RPE cells, we found that RPE has an exceptionally high capacity for reductive carboxylation, a metabolic pathway that has recently garnered significant interest because of its role in cancer cell survival. The capacity for reductive carboxylation in RPE exceeds that of all other cells tested, including retina, neural tissue, glial cells, and a cancer cell line. Loss of reductive carboxylation disrupts redox balance and increases RPE sensitivity to oxidative damage, suggesting that deficiencies of reductive carboxylation may contribute to RPE cell death. Supporting reductive carboxylation by supplementation with an NAD + precursor or its substrate α-ketoglutarate or treatment with a poly(ADP ribose) polymerase inhibitor protects reductive carboxylation and RPE viability from excessive oxidative stress. The ability of these treatments to rescue RPE could be the basis for an effective strategy to treat blinding diseases caused by RPE dysfunction.reductive carboxylation | RPE | metabolism | age-related macular degeneration | oxidative stress T he retinal pigment epithelium (RPE) is a monolayer of postmitotic cells situated between the photoreceptors of the retina and the choroidal blood supply. The interaction of the RPE and photoreceptors is critical to maintaining vision. Functions of the RPE include phagocytosis of shed photoreceptor outer segments, recycling of retinoids, production and secretion of cytokines and chemokines, and mediating the exchange of nutrients and metabolites between the choroid and photoreceptors (1, 2). RPE cells provide crucial metabolic support for the retina.RPE dysfunction can lead to photoreceptor death and retinal degenerative disease, such as age-related macular degeneration (AMD), which is the leading cause of irreversible vision loss in the elderly human population (3-5). RPE cells are exposed to ongoing oxidative stress from the combined effects of light, choroidal O 2 , polyunsaturated fatty acids, and retinoids (6). The resulting impairment in RPE energy metabolism and function by oxidative stress is one likely mechanism for the pathogenesis of AMD (3, 7-11).Mitochondria support the active energy metabolism of the RPE (10-12). A recent report showed that RPE is less stable and less able to support the retina when it is forced to rely on glycolytic rather than mitochondrial metabolism (13). Another recent report supports the importance of mitochondria in RPE by showing that bolstering mitochondrial activity makes these cells more resilient to oxidative damage (14).In mitochondria, citrate can be generated from acetyl CoA and oxaloacetate as part of the TCA cycle. However, under hypoxic conditions, some cells also produce citrate via reductive carboxylation of α-ketoglutarate (αKG) through the action of NADPH-dependent isocitrate dehydrogenases (IDH) (15-17)....

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Section: Methodsmentioning

confidence: 99%

Reductive carboxylation is a major metabolic pathway in the retinal pigment epithelium

Yanagida

Knight

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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102

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“…Retinas were cultured in Krebs-Ringer/HEPES/bicarbonate (KRB) buffer at 37°C in a 5% CO 2 incubator as we previously reported (6,19).…”

Section: Production Of Energy In a Cell Must Keep Pace With Demandmentioning

confidence: 99%

“…Retinal O 2 Consumption ex Vivo-A flow culture system (20) was used to measure O 2 consumption rate as in our previous report with minor modification (19,21). Retinas were isolated into a nutrient mix containing 5 mM glucose, 5 mM lactate, 500 M pyruvate, 1 mM glutamine, and 1 mM leucine in KRB buffer cut into four pieces and loaded into each chamber sandwiched with Cytodex beads.…”

Section: Production Of Energy In a Cell Must Keep Pace With Demandmentioning

confidence: 99%

Phototransduction Influences Metabolic Flux and Nucleotide Metabolism in Mouse Retina

Rountree

Cleghorn³

et al. 2016

Journal of Biological Chemistry

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Production of energy in a cell must keep pace with demand.Photoreceptors use ATP to maintain ion gradients in darkness, whereas in light they use it to support phototransduction. Matching production with consumption can be accomplished by coupling production directly to consumption. Alternatively, production can be set by a signal that anticipates demand. In this report we investigate the hypothesis that signaling through phototransduction controls production of energy in mouse retinas. We found that respiration in mouse retinas is not coupled tightly to ATP consumption. By analyzing metabolic flux in mouse retinas, we also found that phototransduction slows metabolic flux through glycolysis and through intermediates of the citric acid cycle. We also evaluated the relative contributions of regulation of the activities of ␣-ketoglutarate dehydrogenase and the aspartate-glutamate carrier 1. In addition, a comprehensive analysis of the retinal metabolome showed that phototransduction also influences steady-state concentrations of 5-GMP, ribose-5-phosphate, ketone bodies, and purines. Warburg et al.(1) and Krebs (2) reported in the 1920s that tumors and retinas rely on aerobic glycolysis. Some of the biochemical mechanisms by which cancer cells adapt to aerobic glycolysis have been gleaned from investigations of specific metabolic adaptations of cancer cells either in culture or in a tumor (3, 4). Retinas offer distinct advantages for investigating aerobic glycolysis. They have high metabolic rates, a uniquely laminated structure, and the primary signaling pathway by which retinas respond to light is defined clearly.Retinas convert 80 -96% of glucose they consume into lactic acid (5-9), similar to the extent of aerobic glycolysis that fuels cancer cells (3). Aerobic glycolysis occurs primarily in photoreceptors (10) where the energy demands are very different in darkness than in light (5,7,(11)(12)(13). In darkness energy is consumed within the inner segments to support ion pumping (5, 13). In light energy is consumed by the outer segments (OS) 2 to support phototransduction and regeneration of visual pigments. A photoreceptor neuron also performs anabolic metabolism to replace the ϳ10% of its OS material that is lost each day to phagocytosis by the retinal pigmented epithelium (14, 15).Some of the carbons in glucose consumed by a retina reach the mitochondrial matrix where they are oxidized in biochemical reactions that reduce NAD ϩ to NADH. Transfer of electrons from NADH to O 2 then generates a proton gradient across the mitochondrial inner membrane. In some tissues dissipation of the proton gradient is coupled tightly to ATP demand. In others, proton leakage can dissipate the gradient even without ATP synthesis (16). In this report we show that mitochondria in retinas are more uncoupled than mitochondria in other tissues.The ability of a photoreceptor to respond to light, to release neurotransmitter, to regenerate visual pigment, to renew itself, and to remain viable requires that production of energy keeps pace...

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“…Twenty-one, male, Sprague Dawley rats were included in the post-mortem study (42 eyes in total) and six rats were included in the tissue comparison study; all rats were in the age-range of [7][8][9][10][11][12] weeks. The rats were kept in the same room under standard laboratory conditions and fed with standard chow.…”

Section: Animalsmentioning

confidence: 99%

“…[6][7][8][9][10] It has also been used to study ageing, a variety of ocular pathologies, metabolite transport in the retina, effects of drugs and metabolic changes during corneal organ culture for transplantation. [11][12][13][14][15] Aqueous and vitreous are the most accessible intraocular samples that can be obtained in clinical studies, and these have been used to interrogate the metabolic activities of surrounding ocular structures. 11,15 However, it is unclear whether sampling ocular biofluids provides robust and reproducible information on the metabolic activity of surrounding tissues and ideally, the metabolome of the ocular tissue of interest should be studied directly.…”

Section: Introductionmentioning

confidence: 99%

Characterisation of the metabolome of ocular tissues and post-mortem changes in the rat retina

Tan

Mullard

Hollywood

et al. 2016

Experimental Eye Research

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and 48 hours post-mortem. To study the metabolite composition of rat ocular tissues, eyes were dissected immediately after culling to isolate the cornea, lens, vitreous and retina, prior to storing at -80 o C. Tissue extracts were subjected to Gas Chromatograph Mass Spectrometry (GC-MS) and Ultra High Performance Liquid Chromatography Mass Spectrometry (UHPLC-MS). Generally, the metabolic composition of the retina was stable for 8 hours post-mortem when eyes were stored at 4 o C, but showed increasing changes thereafter. However, some more rapid changes were observed such as increases in TCA cycle metabolites after 2 hours post-mortem, whereas some metabolites such as fatty acids only showed decreases in concentration from 24 hours. A total of 42 metabolites were identified across the ocular tissues by GC-MS (MSI level 1) and 2782 metabolites were annotated by UHPLC-MS (MSI level 2) according to MSI reporting standards. Many of the metabolites detected were common to all of the tissues but some metabolites showed partitioning between different ocular structures with 655, 297, 93 and 13 metabolites being uniquely detected in the retina, lens, cornea and vitreous respectively. Only a small percentage (1.6%) of metabolites found in the vitreous were exclusively found in the retina and not other tissues. In conclusion, mass

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Inhibition of Mitochondrial Pyruvate Transport by Zaprinast Causes Massive Accumulation of Aspartate at the Expense of Glutamate in the Retina

Cited by 75 publications

References 48 publications

Reductive carboxylation is a major metabolic pathway in the retinal pigment epithelium

Reductive carboxylation is a major metabolic pathway in the retinal pigment epithelium

Phototransduction Influences Metabolic Flux and Nucleotide Metabolism in Mouse Retina

Characterisation of the metabolome of ocular tissues and post-mortem changes in the rat retina

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