Succinate Can Shuttle Reducing Power from the Hypoxic Retina to the O2-Rich Pigment Epithelium

Abstract

When O₂ is plentiful, the mitochondrial electron transport chain uses it as a terminal electron acceptor. However, the mammalian retina thrives in a hypoxic niche in the eye. We find that mitochondria in retinas adapt to their hypoxic environment by reversing the succinate dehydrogenase reaction to use fumarate to accept electrons instead of O₂. Reverse succinate dehydrogenase activity produces succinate and is enhanced by hypoxia-induced downregulation of cytochrome oxidase. Retinas can export the succinate they produce to the neighboring O₂-rich retinal pigment epithelium-choroid complex. There, succinate enhances O₂ consumption by severalfold. Malate made from succinate in the pigment epithelium can then be imported into the retina, where it is converted to fumarate to again accept electrons in the reverse succinate dehydrogenase reaction. This malate-succinate shuttle can sustain these two tissues by transferring reducing power from an O₂-poor tissue (retina) to an O₂-rich one (retinal pigment epithelium-choroid).

Keywords: ecosystem; hypoxia; metabolic flux; metabolism; mitochondria; oxygen; retina; retinal pigment epithelium; succinate; succinate dehydrogenase.

Figure 1.. Retinas Release Succinate, which Can Fuel O₂ Consumption in Eyecups

(A) Rate of TCA cycle metabolite release by retinas incubated in 5 mM ¹²C-glucose. Media samples were taken at 30, 60, and 90 min (n = 9 retinas, 3 per time point; error bars indicate SEM). (B) O₂ consumption trace from retinas and eyecups perifused with media containing 5 mM glucose, then 5 mM glucose + 5 mM succinate, and then 5 mM glucose. Vertical gray bars indicate the approximate time when media containing the new metabolite condition reached the tissue (n = 4 retina and 4 eyecup chambers; error bars indicate SEM). (C) O₂ consumption trace of eyecups supplied with 5 mM glucose, then 5 mM glucose + 5 mM of a test metabolite, and then 5 mM glucose + 5 mM test metabolite + 5 mM succinate. Test metabolites are proline (gray), glutamine (teal), lactate (black), and α-ketoglutarate (pink) (n = 3 eyecup chambers per condition; error bars indicate SEM). (D) Change in OCR (relative to 5 mM glucose alone) for eyecups supplied with 5 mM glucose + 5 mM succinate, 5 mM glucose + 20 mM malonate, or 5 mM glucose + 20 mM malonate + 5 mM succinate (n = 4 eyecup chambers for malonate and 2 eyecup chambers for malonate + succinate; error bars indicate SEM). (E) Labeling schematic showing isotopomers of citrate produced by U-¹³C-glucose alone (left) or ¹²C-glucose + U-¹³C-succinate (right). (F) Citrate production in eyecups supplied with 5 mM U-¹³C-glucose alone or 5 mM ¹²C-glucose + 1 mM U-¹³C-succinate (n = 2 eyecups per time point; error bars indicate SEM). (G) Citrate production in retinas supplied with 5 mM U-¹³C-glucose alone or 5 mM ¹²C-glucose + 1 mM U-¹³C-succinate (n = 2 retinas per time point; error bars indicate SEM). (H) Pool size of TCA cycle metabolites in eyecups and retinas supplied with 5 mM ¹²C-glucose + 1 mM U-¹³C-succinate for 10 min relative to tissue supplied with 5 mM ¹²C-glucose alone (n = 2 retinas or eyecups; error bars indicate SEM; ** indicates p < 0.005 using Welch’s t test).

Figure 2.. Succinate Enhances Export of Malate in Eyecups

(A) Rate of TCA cycle metabolite release by eyecups incubated in 5 mM ¹²C-glucose. Media samples were taken at 30, 60, and 90 min (n = 9 eyecups, 3 per time point; error bars indicate SEM). (B) Labeling schematic showing TCA cycle isotopomers produced by eyecups supplied with ¹²C-glucose and U-¹³C-succinate. For simplicity, only isotopomers originating from either metabolite produced in a single turn of the TCA cycle are shown. (C) Rate of TCA cycle metabolite release by eyecups incubated in 5 mM ¹²C-glucose + 2 mM U-¹³C-succinate for 60 and 90 min. The rate at which succinate is consumed by eyecups during incubation is shown on the right (n = 6 eyecups, 3 per time point; error bars indicate SEM). (D) Rate of TCA cycle metabolite release by eyecups incubated in 5 mM ¹²C-glucose + 50 μM U-¹³C-succinate for 30 min. The rate at which succinate is consumed by eyecups during incubation is shown on the right (n = 3 eyecups; error bars indicate SEM).

Figure 3.. Retinas Can Use Malate to Produce Succinate via Reverse Electron Transport at SDH

(A) Labeling schematic showing isotopomers of TCA cycle metabolites produced by tissue incubated in U-¹³C-malate + ¹²C-glucose. (B) Isotopomers produced by retinas and eyecups incubated in 5 mM ¹²C-glucose and 5, 50, or 500 μM U-¹³C-malate for 5 min (n = 5 retinas or eyecups per concentration; error bars indicate SEM). (C) Total fumarate levels in retinas and eyecups incubated in 5 mM ¹²C-glucose and 5, 50, or 500 μM U-¹³C-malate for 5 min (n = 5 retinas or eyecups per concentration; error bars indicate SEM; * indicates p < 0.05 using Welch’s t test). (D) m4 fumarate and m4 succinate in retinas supplied with 5 mM ¹²C-glucose + 50 μM U-¹³C-malate in the presence or absence of 20 mM malonate for 5 min (n = 6 retinas; error bars indicate SEM; **** indicates p < 0.0001 using Welch’s t test). (E) m4 fumarate and m4 succinate in WT and AIPL1^−/− retinas supplied with 5 mM ¹²C-glucose + 50 μM U-¹³C-malate for 5 min (n = 6 retinas; error bars indicate SEM; ** indicates p < 0.005 using Welch’s t test). (F) Labeling schematic showing isotopomers produced by 4-²H-glucose. (G) Accumulation of deuterated (m1) malate, fumarate, and succinate α-ketoglutarate in retinas and eyecups incubated in 5 mM 4-²H-glucose for 0.02, 0.5, 1, 2, and 5 min (n = 2 retinas per time point; error bars indicate SEM).

Figure 4.. Reverse SDH Activity Maintains a Significant Portion of the Succinate Pool in Retinas

(A) Fractional enrichment of m2 α-ketoglutarate and m2 succinate in retinas supplied with 5 mM U-¹³C-glucose for 0.02, 0.5, 1, 2, 5, 15, 30, and 60 min (n = 3 to 7 retinas per time point; error bars indicate SEM). (B) Fractional enrichment of m1 fumarate and m1 succinate in retinas supplied with 5 mM 4-²H-glucose for 0.02, 0.5, 1, 2, 5, 15, 30, and 60 min (n = 3 retinas per time point; error bars indicate SEM). (C) Fractional enrichment of m4 fumarate and m4 succinate in retinas supplied with 5 mM ¹²C-glucose + 50 μM U-¹³C-malate for 0.02, 5, 15, 30, and 60 min (n = 3 to 5 retinas per time point; error bars indicate SEM). (D) Graphical description of observed (top) and maximal (bottom) contributions of oxidative TCA cycle activity and reverse SDH activity to maintaining the retinal succinate pool.

Figure 5.. Low COXIV Expression Drives Reversal of SDH in Retinas

(A) Immunoblot of retinas and eyecups using an ETC component cocktail (top blot) and a single antibody against COXIV (bottom blot). ATP5A, ATP synthase subunit 5A; UQRC2, ubiquinol cytochrome c oxidoreductase subunit core 2; CO1, cytochrome c oxidase subunit 1; SDHB, succinate dehydrogenase B; NDUFB8, NADH ubiquinone oxidoreductase subunit B8; COXIV, cytochrome c oxidase subunit 4. (B) Quantification of multiple immunoblots probed with COXIV and ATP5A (each dot represents a biological replicate, n = 3 to 7 retinas per pO₂ condition). (C) m4 fumarate, m4 succinate, and the m4 succinate/m4 fumarate ratio measured in retinas supplied with 5 mM ¹²C-glucose + 50 μM U-¹³C-malate for 5 min after pre-equilibration at specified pO₂ for 2 h (“Fresh” data shown are duplicated from Figure 3B for ease of comparison) (n = 6–9 retinas per pO₂ condition; error bars indicate SEM). (D) m4 fumarate, m4 succinate, and the m4 succinate/m4 fumarate ratio measured in retinas supplied with 5 mM ¹²C-glucose + 50 μM U-¹³C-malate in the presence of 3 mM KCN (n = 6 retinas; error bars indicate SEM; ** indicates p < 0.005 and *** indicates p < 0.001 using Welch’s t test). (E) m1 fumarate, m1 succinate, and the m1 succinate/m1 fumarate ratio in WT and Ndufs4^−/− retinas supplied with 5 mM 4-²H-glucose for 5 min (n = 7 WT and 6 Ndufs4^−/− retinas; error bars indicate SEM; * indicates p < 0.05 using Welch’s t test). (F) m2 fumarate, m2 succinate, and the m2 fumarate/m2 succinate ratio in WT and Ndufs4^−/− retinas supplied with 5 mM U-¹³C-glucose for 5 min (n = 7 WT and 7 Ndufs4^−/− retinas; error bars indicate SEM; ** indicates p < 0.005 and *** indicates p < 0.001 using Welch’s t test). (G) Diagram showing canonical ETC function (left) and proposed ETC function in retinas (right).