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The RPE consumes mitochondrial fuels, and RPE cells are in direct contact with the retina and metabolites exchange between these tissues within an eye (Adijanto et al. For these reasons, we tested the capacity of RPE cells to consume succinate. We measured O2 consumed by intact mouse retinas and eyecups fueled with succinate. For these experiments we used a custom-built perifusion oximeter (Sweet et al.

Succinate causes a 0. Succinate injections OCR in retinas by only 0. The Km for eyecup O2 consumption bayer code fueled with succinate is 2. No other glycolytic or TCA cycle metabolite we tested stimulates O2 consumption as much as succinate, and cold sensitive tooth metabolites we tested did not Isordil (Isosorbide Dinitrate)- Multum enhance the succinate-induced OCR (Figure 1C).

Addition of the downstream TCA cycle metabolites fumarate and malate partially suppress O2 consumption in eyecups stimulated by succinate (consistent with mass action) (Supplemental Figure 1B). We confirmed the specificity of this effect to succinate dehydrogenase (SDH), as succinate-stimulated increases in OCR are blocked by Isordil (Isosorbide Dinitrate)- Multum SDH inhibitor malonate (Figure 1D).

We used isotopic Isordil (Isosorbide Dinitrate)- Multum to determine if the presence of succinate enhances the rate of TCA cycle reactions. We supplied retinas and eyecups Isordil (Isosorbide Dinitrate)- Multum either 5 mM U-13C-glucose, or with 5 mM 12C-glucose and 1 mM U-13C-succinate for times ranging from 0 to 5 minutes.

As a readout of TCA cycle flux, we compared the rate of m2 citrate formed from glucose alone (left panel of Fig. In striking contrast, retinas make citrate using carbons from glucose (m2) faster than with carbons from Isordil (Isosorbide Dinitrate)- Multum (m4) (Fig. Rates for other TCA cycle bendroflumethiazide are similarly enhanced in eyecups but not retinas (Supplemental Fig.

Eyecup preparations were used for these experiments because removing the RPE (mechanically or enzymatically) from the eyecups can damage it and alter its metabolic features.

Since eyecups contain multiple cell types (RPE, choroidal endothelial, and sclera) we repeated the flux analysis with cultured human fetal RPE (hfRPE) at 2 minutes to assess if RPE uses succinate. These fully differentiated RPE cells make twice as much citrate from succinate carbons (m4) as citrate from glucose carbons (m2) (Supplemental Fig. The findings in Fig. To determine if eyecups release a metabolite that retinas can use to enhance succinate production, we supplied eyecups with 5 mM 12C-glucose and quantified TCA cycle metabolites released into the incubation medium Isordil (Isosorbide Dinitrate)- Multum 30, 60 and 90 minutes.

We then determined if succinate influences the export of any of these metabolites by Isordil (Isosorbide Dinitrate)- Multum eyecups with U-13C-succinate in addition to 12C-glucose (Figure 2B). When we Isordil (Isosorbide Dinitrate)- Multum eyecups with 5 mM 12C-glucose and 2 mM U-13C-succinate for 30 minutes, they release 15-fold more malate and 271-fold more fumarate compared to when they were supplied with 5 mM glucose alone (Figure 2C).

The majority of malate and fumarate released is m4, indicating it was made from U-13C-succinate. TCA cycle metabolites released by eyecups bal in 5 mM 12C-glucose. For simplicity, only isotopomers produced in a single turn of the TCA cycle are shown.

Succinate consumed by eyecups during incubation is shown on the right panel. Remarkably, eyecups export equivalent amounts of m4 malate (made from succinate) and m0 malate (made from glucose). This is despite being incubated in a 100-fold lower concentration of succinate compared to glucose.

Even at low concentrations of succinate, eyecups export significant quantities of malate. We next tested whether malate exported by eyecups can influence retinal metabolism. Retinas in an eye are in a hypoxic environment, and hypoxic tissues Isordil (Isosorbide Dinitrate)- Multum produce succinate by both canonical oxidative TCA cycle activity as well as through reverse electron transport Isordil (Isosorbide Dinitrate)- Multum SDH (Chouchani et al. Since retinas export succinate (Fig.

Based on this premise, Isordil (Isosorbide Dinitrate)- Multum tested if retinas might use malate for anaplerosis to sustain succinate synthesis by reverse electron transport at SDH.

We used U-13C-malate to quantify reverse electron transport at SDH in both retinas and eyecups (Figure 3A). At Isordil (Isosorbide Dinitrate)- Multum concentrations, retinas form much more m4 succinate than eyecups (Figure 3B, 3C). None of these concentrations of malate increase the size of the fumarate pool in retinas (Figure 3C).

This shows Isordil (Isosorbide Dinitrate)- Multum the formation of m4 succinate we observe in retinas is not driven only by an increase in concentration grief the reactant of the reverse SDH reaction (fumarate) and is instead a property that is inherent to retinas and not eyecups.

Total metabolite levels for other metabolites are Isordil (Isosorbide Dinitrate)- Multum in Supplemental 3A. Retinas are composed of several types of neurons, so we next investigated if a specific cell type in the retina favors reverse SDH activity.

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