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. 2014 Oct 28;111(43):15579-84.
doi: 10.1073/pnas.1412441111. Epub 2014 Oct 13.

Pyruvate kinase and aspartate-glutamate carrier distributions reveal key metabolic links between neurons and glia in retina

Ken J Lindsay  1 , Jianhai Du  1 , Stephanie R Sloat  1 , Laura Contreras  2 , Jonathan D Linton  1 , Sally J Turner  1 , Martin Sadilek  3 , Jorgina Satrústegui  2 , James B Hurley  4
Affiliations

Pyruvate kinase and aspartate-glutamate carrier distributions reveal key metabolic links between neurons and glia in retina

Ken J Lindsay et al. Proc Natl Acad Sci U S A. .

Abstract

Symbiotic relationships between neurons and glia must adapt to structures, functions, and metabolic roles of the tissues they are in. We show here that Müller glia in retinas have specific enzyme deficiencies that can enhance their ability to synthesize Gln. The metabolic cost of these deficiencies is that they impair the Müller cell's ability to metabolize Glc. We show here that the cells can compensate for this deficiency by using metabolites produced by neurons. Müller glia are deficient for pyruvate kinase (PK) and for aspartate/glutamate carrier 1 (AGC1), a key component of the malate-aspartate shuttle. In contrast, photoreceptor neurons express AGC1 and the M2 isoform of pyruvate kinase, which is commonly associated with aerobic glycolysis in tumors, proliferating cells, and some other cell types. Our findings reveal a previously unidentified type of metabolic relationship between neurons and glia. Müller glia compensate for their unique metabolic adaptations by using lactate and aspartate from neurons as surrogates for their missing PK and AGC1.

Keywords: Müller glia; aerobic glycolysis; glutamine metabolism; photoreceptors; retina.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Glycolytic enzymes in retina. (A) Activities of glycolytic enzymes in homogenates of retina, muscle, liver, and brain. Activities were measured at substrate concentrations above the reported Km for each enzyme. (B) Immunoblot analysis of PK isoforms in homogenates of whole brain, liver, muscle, and retina. DA, dark-adapted; LA, light-adapted. GNAT1−/− retinas were included because they are unresponsive to light. rMC-1 is an SV-40 transformed MC line (18), and HeLa is a cancer cell line. The blot is representative of two experiments. Each lane is loaded with 5 μg of protein. (C) Quantitative immunoblot analysis comparing retina homogenate with known amounts of purified PKM1 and PKM2.
Fig. 2.
Fig. 2.
Distribution of PK in retina. (A, Top) Distribution of PK activity in serial sections of rat retina. (A, Middle) Rhodopsin shows the location of PR outer segments. (A, Bottom) Synaptotagmin shows locations of synapses. Immunoblot shows PKM2 immunoreactivity. (B) Immunohistological localization of PKM2 (Left), CRALBP (Middle), and their overlap (Right) in mouse retina. (C) Immunohistological localization of PKM1 (Left), CRALBP (Middle), and their overlap (Right) in mouse retina.
Fig. 3.
Fig. 3.
Cultured MCs use Asp more effectively than Glc, Lac, and Gln. (A) Accumulation of M3 lactate and pyruvate from 13C Glc in retinas and in cultured MCs. Cultured MCs do not make Lac fast enough for Müller glia to be the primary source of Lac from retinas. Cultured MCs were incubated for 1 h with 5 mM U-13C Lac (B), 5 mM U-13C Glc (C), and 5 mM U-13C Asp (D). The 13C enrichment is expressed as the percentage of total ion intensity of all isotopomers for each metabolite (n = 3). 13C Asp is incorporated more effectively than any other fuel.
Fig. 4.
Fig. 4.
Asp stimulates Glc oxidation. (A) Isolated MCs were incubated with 5 mM U-13C Glc with or without 5 mM unlabeled Asp for 1 h. The medium was collected and analyzed by GC/MS. (B) Isolated MCs were incubated with 5 mM U-13C Glc or with U-13C Lac (5 mM) with vs. without 5 mM unlabeled Asp for 1 h. The percentage of each metabolite in the M2 isotopomer is shown (n = 3). (C) Pyr carboxylase activity is a significant source of citrate and malate, but only in the absence of Asp (n = 3). The M3 isotopomers of citrate and malate are made by carboxylation of Pyr. Total citrate and malate increased only ∼70% and ∼7%, respectively, when Asp was included. (D) Asp (5 mM) increases synthesis and release of Gln into the MC culture medium (n = 3). (E) Asp stimulates synthesis of Gln from U-13C Lac by MCs in intact retinas (note log scale on y axis). (F) Asp and Asn are the most effective anapleurotic substrates for raising the concentration of Gln in retinas. Retinas were incubated with 5 mM lactate and 5 mM additional substrate for 1.5 h (n = 3). The effect on Glu is much smaller. In mouse retinas incubated with Lac alone, the Glu pool size is ∼130-fold larger than the Gln pool size.
Fig. 5.
Fig. 5.
Pulse–chase analysis of U-13C Gln in retina. (A) Schematic model for the role of Asp as a carrier of oxidizing power between retinal neurons and glia. Red circles represent the 13C carbons, and black circles represent the 12C carbons. (B) 13C labeling of Asp, Glu, and Gln from the pulse of U-13C Gln. (Upper) The M5 Gln and Glu are derived directly from the pulse of 5 mM U-13C Gln. After 5 min, the medium was changed to 5 mM unlabeled Lac with no added Gln. Unlabeled Gln was not included in the chase because the intense signal from the added Gln would have obscured the isotopomer signals we intended to quantify. The retinas were subsequently harvested at the indicated times after the pulse. (Lower) The M4 Asp derived from oxidation of Glu via the TCA. The M3 Glu is made by further oxidation via citrate, and M3 Gln is made only in MCs by Gln synthetase. (n = 6).
Fig. 6.
Fig. 6.
Inhibition of Asp transfer to MCs inhibits Gln synthesis in retina. (A) Inhibition of EAAT1 by (2S, 3S)-3-{3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA; 10 μM). (B) Depletion of extracellular Asp by carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD). The medium contained CAD protein (6 μg/mL) and ATP (0.1 mM). (C) Asp, N-acetyl aspartate (NAA) and Gln are diminished in AGC1−/− mouse retinas. (D) Asp boosts Gln levels in AGC1-deficient retinas (n = 4). (E) AOA, an aminotransferase inhibitor, inhibits formation of Asp and promotes formation of citrate, consistent with sequestering oxaloacetate in mitochondria so that it is more likely to be made into citrate. By preventing formation of Asp and by blocking aminotransferase activity in MCs, AOA (1 mM) also causes accumulation of α-ketoglutarate (αKG) and lowers Gln (n = 3).
Fig. 7.
Fig. 7.
Model for relationship between PRs and MCs. MCs are PK-deficient, so glycolytic intermediates can be used for anabolic activities. MCs are AGC1-deficient, so mitochondrial oxaloacetate in MCs is not diverted away from the pathway that leads to Gln synthesis. To compensate for these metabolic deficiencies, PRs produce Lac that MCs can oxidize to Pyr to fuel their mitochondria. PRs also export Asp that MCs can use to oxidize Lac. Nitrogen for the amino group of Gln comes from Asp, and nitrogen for the amide group of Gln comes from NH4+. Mal, malate; OAA, oxaloacetate; OGC, oxoglutarate carrier; Succ, succinate. Double-membrane compartments within the cells represent mitochondria. Green circles represent Glu, and green cylinders represent Glu receptors.
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