Inhibition of mitochondrial pyruvate transport by zaprinast causes massive accumulation of aspartate at the expense of glutamate in the retina

Abstract

Transport of pyruvate into mitochondria by the mitochondrial pyruvate carrier is crucial for complete oxidation of glucose and for biosynthesis of amino acids and lipids. Zaprinast is a well known phosphodiesterase inhibitor and lead compound for sildenafil. We found Zaprinast alters the metabolomic profile of mitochondrial intermediates and amino acids in retina and brain. This metabolic effect of Zaprinast does not depend on inhibition of phosphodiesterase activity. By providing (13)C-labeled glucose and glutamine as fuels, we found that the metabolic profile of the Zaprinast effect is nearly identical to that of inhibitors of the mitochondrial pyruvate carrier. Both stimulate oxidation of glutamate and massive accumulation of aspartate. Moreover, Zaprinast inhibits pyruvate-driven O2 consumption in brain mitochondria and blocks mitochondrial pyruvate carrier in liver mitochondria. Inactivation of the aspartate glutamate carrier in retina does not attenuate the metabolic effect of Zaprinast. Our results show that Zaprinast is a potent inhibitor of mitochondrial pyruvate carrier activity, and this action causes aspartate to accumulate at the expense of glutamate. Our findings show that Zaprinast is a specific mitochondrial pyruvate carrier (MPC) inhibitor and may help to elucidate the roles of MPC in amino acid metabolism and hypoglycemia.

Keywords: Aspartate; Glutamate; Metabolism; Phosphodiesterases; Pyruvate; Retina.

FIGURE 1.

Zaprinast significantly changes the metabolomic profile in the retina. A, Zaprinast changes metabolites in mouse retina. The retina was cultured for 1 h with 200 μm Zaprinast or with DMSO as a control. Metabolites were extracted, quantified by GC-MS, and normalized to the DMSO alone controls (n = 10). 3-PG, 3-phosphoglyceric acid; PEP, phosphoenolpyruvate; AKG, α-ketoglutarate; GABA, γ-aminobutyric acid; 2-HG, 2-hydroxyglutarate, NAA, N-acetyl aspartate. B, Zaprinast increases pyruvate release from the retina. The medium from A was assayed for metabolites. The data are -fold changes over DMSO control. C, Zaprinast does not change glucose consumption. The medium from A was tested for glucose concentration. NS, no significant difference. * indicates p < 0.05 versus DMSO-treated.

FIGURE 2.

PDE6 is not involved in the effect of Zaprinast on glutamate and aspartate. A, Zaprinast increases cGMP at 50 μm and cAMP at 200 μm (n = 6). B, Zaprinast dose-dependently decreases glutamate and increases aspartate. Glutamate is shown as white bars, and aspartate is shown as black bars in this and in the next two panels. The retina was treated for 1 h. The decrease of glutamate starts at concentrations of Zaprinast as low as 2 μm (n = 5). C, Cngb1 deficiency does not block the effect of 200 μm Zaprinast (Zap) on glutamate and aspartate. The retina was incubated for 1 h (n = 5). D, Zaprinast at 200 μm decreases glutamate and increases aspartate in rd1 mice. Retinas from 3-month-old PDE6b+ and rd1 mice were incubated with Zaprinast for 1 h (n = 4). E, sildenafil increases cGMP in retinas (n = 3). F, glutamate and aspartate do not change in retinas treated with sildenafil (n = 3). * indicates p < 0.05 versus DMSO-treated.

FIGURE 3.

Zaprinast blocks synthesis of citrate, αKG, and glutamate from glucose-derived pyruvate. A, schematic of carbon labeling (black circles for ¹³C) from [¹³C₆]glucose after the first TCA cycle. Pyruvate and lactate are fully labeled on all three carbons. After PDH removes one labeled carbon, the remaining two labeled carbons in acetyl-CoA incorporate into mitochondrial intermediates in the first cycle. OAA, oxaloacetate. B, Zaprinast decreased the enrichment of TCA cycle intermediates from [¹³C₆]glucose. The retina was incubated with 5 mm [¹³C₆]glucose for 15 min. C–J, mass isotopomer distributions of intermediates from B. The fraction of each isotopomer was multiplied by the total metabolite concentration and normalized by protein concentration (pmol or nmol/mg of protein). Zaprinast increases the total incorporation of labeled carbon into pyruvate, but it decreases labeling of TCA intermediates. * indicates p < 0.05 versus DMSO-treated. (n = 3).

FIGURE 4.

Carbons used to increase aspartate come from oxidation of glutamate. A, schematic of carbon labeling (black circles represent ¹³C) from [¹³C₅]glutamine after the first TCA cycle. Most of the ¹³C from [¹³C₅]glutamine labels five carbons in αKG and four carbons for the rest of the intermediates in the first cycle. However, a small fraction (<10%) of the [¹³C₅]glutamine also labels five carbons in citrate by reversal of the isocitrate dehydrogenase reaction. B, Zaprinast increases enrichment of aspartate and malate from [¹³C₅]glutamine. The retina was pulsed with 5 mm [¹³C₅]glutamine for 5 min and then chased with unlabeled glucose for 10 min. C–I, mass isotopomer distribution of intermediates from B. The metabolite concentration was normalized by protein concentration (pmol or nmol/mg protein). Zaprinast increases M4 succinate, malate, and aspartate from [¹³C₅]glutamine. * indicates p < 0.05 versus DMSO treated (n = 3).

FIGURE 5.

Zaprinast inhibits pyruvate-driven oxygen consumption. Zaprinast inhibits O₂ consumption in brain mitochondria when fueled by pyruvate but not when fueled by glutamate or succinate (Suc) in conditions starting with either glutamate/malate (Glu/Mal, A and B) or pyruvate/malate (Pyr/Mal, C and D). The large dip upon the addition of both Zaprinast (200 μm) and DMSO is an artifact caused by differences in O₂ solubility between water and DMSO. The substrate was added at the time point indicated by arrows. B and D are increments (ΔOCR) over the previous rate by subtracting OCR initiated by the substrate added before. OCR, oxygen consumption rate (n = 3). E and F, MPC inhibitor UK5099 inhibits pyruvate-driven oxygen consumption. Like Zaprinast, UK5099 at 10 nm inhibits oxygen consumption fueled by pyruvate but not glutamate and succinate. E is a representative trace from F, the normalized data (n = 3). * indicates p < 0.05 versus DMSO treated. Drug indicates the adding of DMSO, Zaprinast, or UK5099. * indicates p < 0.05 versus DMSO treated (n = 3).

FIGURE 6.

Zaprinast inhibits transport of pyruvate into mitochondria but does not inhibit PDH. A and B, Zaprinast (Zap) inhibits pyruvate influx into liver mitochondria. A, isolated liver mitochondria was preincubated with DMSO or Zaprinast at 100 μm for 5 min before adding 15 μm [2-¹⁴C]pyruvate for 1 min. As a positive control, 10 mm unlabeled pyruvate (Cold Pyr) was added into mitochondria instantly after [2-¹⁴C]pyruvate addition (n = 5). B, Zaprinast dose-dependently inhibits mitochondrial pyruvate transport (n = 5). C, Zaprinast does not affect PDH activity. PDH protein was incubated with either Zaprinast at different concentrations or PDH inhibitor 3-fluropyruvate (3-FP) at 5 mm for 10 min. PDH activity was measured by cycling assay (n = 4). D–E, both Zaprinast and UK5099 inhibit citrate and glutamate synthesis from [¹³C₃]pyruvate in brain mitochondria with intact membranes but not when mitochondrial membranes are disrupted. Membrane intact and disrupted mitochondria were incubated with 1 mm [¹³C₃]pyruvate and Zaprinast (100 μm) or UK5099 (UK, 100 μm) for 10 min at 30 °C. The ¹³C citrate and glutamate were measured by GC-MS. Mem, membrane (n = 3). F–G, MPC inhibitors increase pyruvate and aspartate but decrease glutamate in the retina. Retinas were treated with α-cyano-4-hydroxycinnamic acid (4CIN, 100 μm), UK5099 (100 μm), and Zaprinast (100 μm) for 1 h. (n = 3). * indicates p < 0.05 versus DMSO treated.

FIGURE 7.

Disruption of AGC1 activity does not prevent Zaprinast from stimulating oxidation of glutamate into aspartate. A, schematic of malate-aspartate shuttle. OAA, oxaloacetate; OGC, 2-oxoglutarate carrier. B, the retina from AGC1^−/− mice has lower aspartate and pyruvate, but Zaprinast (Zap) induces similar metabolic changes. The retina was incubated with DMSO or Zaprinast for 1 h in the presence of glucose. C–F, pyruvate, citrate, glutamate, and aspartate labeled from [¹³C₆]glucose. Retinas from WT, AGC1^+/−, and AGC1^−/− mice were incubated with 5 mm [¹³C₆]glucose for 15 min. Data were expressed as -fold increase of ¹³C label over the DMSO control. * indicates p < 0.05 versus DMSO treated within groups, and # indicates versus DMSO or Zaprinast treated in the WT group. (n = 5).

FIGURE 8.

Zaprinast causes accumulation of aspartate inside mitochondria and decreases aspartate utilization. A–C, most of the aspartate increase induced by Zaprinast (Zap) does not come from [¹³C₃]pyruvate. Brain mitochondria were incubated with 1 mm [¹³C₃]pyruvate and 100 μm Zaprinast for 10 min supplemented with EGTA 100 μm, 1 mm glutamate, 0.5 mm malate, 2.5 mm ADP, and 50 μm Ca²⁺. Metabolites from both mitochondria and medium were analyzed (n = 3). OAA, oxaloacetate. D, schematic of carbon labeling (black circles represent ¹³C) from [¹³C₄]aspartate. E, [¹³C₄]aspartate increased glutamate enrichment and TCA cycle intermediates. The retina was incubated with 250 μm [¹³C₄]aspartate and unlabeled glucose for 1 h (n = 3). F–H, M4 aspartate increased, but M4 citrate and M3 glutamate decreased in response to Zaprinast (100 μm). I, Zaprinast decreased the lactate/pyruvate ratio (n = 3). J, Zaprinast decreased the level of NADH in the retina. Retinas were treated with Zaprinast for 1 h. * indicates p < 0.05 versus DMSO treated or without Zaprinast (n = 3).

FIGURE 9.

MPC inhibition causes accumulation of aspartate at the expense of glutamate. Inhibition of MPC by Zaprinast or UK5099 blocks entry of pyruvate into mitochondria, which decreases production of acetyl-CoA, citrate, αKG, glutamate, and NADH and increases oxaloacetate (OAA) and aspartate. Aspartate exits mitochondria in exchange for glutamate via AGC1 to oxidize more glutamate into aspartate. In the cytosol the glutamate may be transaminated into αKG, which exchanges with malate or enters the mitochondria through either AGC1 or glutamate carrier (GC). Metabolites with red represent increases, blue represents decrease, and black represents unchanged or untested. Thick lines represent more flux in that direction. GOT1, cytosolic glutamate oxoglutarate transaminase; GOT2, mitochondrial glutamate oxoglutarate transaminase.