In vitro and ex vivo ¹³C-NMR Spectroscopy Studies of Pyruvate Recycling in Brain

Abstract

Pyruvate recycling is a well established pathway in the liver, but in the brain, the cellular localization of pyruvate recycling remains controversial and its physiological significance is unknown. In cultured cortical astrocytes, pyruvate formed from [U-¹³C]glutamate was shown to re-enter the TCA cycle after conversion to acetyl-CoA, as demonstrated by the labelling patterns in aspartate C-2 and C-3, lactate C-2, and glutamate C-4, which provides evidence for pyruvate recycling in astrocytes. This finding is in agreement with previous studies of astrocytic cultures, in which pyruvate recycling has been described from [U-¹³C]glutamine, in the presence of glutamate, and from [U-¹³C]aspartate. Pyruvate recycling in brain was studied in fasted rats receiving either an intraperitoneal or a subcutaneous injection of [1,2-¹³C]acetate followed by decapitation 30 min later. Extracts of cortical tissue were analysed with ¹³C-NMR spectroscopy and total amounts of amino acids quantified by HPLC. Plasma extracts were analysed with ¹H- and ¹³C-NMR spectroscopy, and showed a significantly larger amount of [1,2-¹³C]acetate in the intraperitoneal group compared to the subcutaneous group. Furthermore, a small amount of label was detected in glucose in both groups. In the subcutaneously injected rats, [4-¹³C]glutamate and [2-¹³C]GABA were less enriched than plasma glucose, which might have been the precursor. In the intraperitoneally injected rats, however, pyruvate formation from [1,2-¹³C]acetate, and re-entry of this pyruvate into the TCA cycle was demonstrated by the presence of greater ¹³C enrichment in [4-¹³C]glutamate and [4-¹³C]glutamine compared to the subcutaneous group, probably resulting from the significantly higher [1,2-¹³C]acetate concentration in brain and plasma.

A comprehensive metabolic profile of cultured astrocytes using isotopic transient metabolic flux analysis and 13C-labeled glucose

“…Pyruvate formed via malic enzyme must re-enter the TCA cycle to be completely oxidized. This pathway is called pyruvate recycling and has been described both in vitro (Sonnewald et al, 1996; Bakken et al, 1997; Waagepetersen et al, 2002) and in vivo (Cerdan et al, 1990; Haberg et al, 1998). As discussed by Waagepetersen et al (2002), this pathway is likely to be more active when cells need to dispose of glutamate and glutamine by oxidative degradation.…”

Metabolic Flux and Compartmentation Analysis in the Brain In vivo

“…This pyruvate formed via malic enzyme must then re-enter the TCA cycle to be oxidized, thus completing the pyruvate recycling pathway. This pathway has been proposed by 13 C MRS of extracts from studies in vitro (34, 75–77) and in vivo (57, 78). This pathway is likely to be more active when cells need to dispose of glutamate and glutamine by oxidative degradation (77), which is an alternative to glutamine efflux from the brain that is generally modeled in 13 C MRS data in vivo ( V efflux , equivalent to V PC ).…”

Mitochondrial Substrate-Level Phosphorylation as Energy Source for Glioblastoma: Review and Hypothesis

“…In addition to the pathways converging toward succinyl-CoA, there are two more ways for producing high-energy phosphates at the substrate level: first through the mitochondrial phosphoenolpyruvate carboxykinase, interconverting PEP and GDP to oxaloacetate and GTP (GTP, GDP, ADP, and ADP can interconvert through nucleoside diphosphate kinase isoforms) and second through the monofunctional C1-tetrahydrofolate (THF) synthase interconverting ADP, phosphate, and 10-formyltetrahydrofolate to ATP, formate, and THF. However, the mitochondrial phosphoenolpyruvate carboxykinase reaction is strongly favored toward PEP formation (thus consuming GTP) in a process called pyruvate recycling pathway (Freidmann et al., 1971; Rognstad and Katz, 1972; Cohen, 1987; Cerdan et al., 1990; Kunnecke et al., 1993; Bakken et al., 1997a, 1997b; Haberg et al., 1998; Chinopoulos, 2013). For this pathway, PEP enters mitochondria through a phosphate/PEP antiporter (McCoy and Doeg, 1975), a protein with isoforms in members C, D, and E of the solute carrier family 35 (Venter et al., 2001; Gerhard et al., 2004; Ota et al., 2004; Skarnes et al., 2011).…”