TY - JOUR

T1 - Direct measurement of backflux between oxaloacetate and fumarate following pyruvate carboxylation

AU - Brekke, Eva

AU - Walls, Anne Byriel

AU - Nørfeldt, Lasse

AU - Schousboe, Arne

AU - Waagepetersen, Helle S

AU - Sonnewald, Ursula

N1 - Copyright © 2011 Wiley Periodicals, Inc.

PY - 2012

Y1 - 2012

N2 - Pyruvate carboxylation (PC) is thought to be the major anaplerotic reaction for the tricarboxylic acid cycle and is necessary for de novo synthesis of amino acid neurotransmitters. In the brain, the main enzyme involved is pyruvate carboxylase, which is predominantly located in astrocytes. Carboxylation leads to the formation of oxaloacetate, which condenses with acetyl coenzyme A to form citrate. However, oxaloacetate may also be converted to malate and fumarate before being regenerated. This pathway is termed the oxaloacetate-fumarate-flux or backflux. Carbon isotope-based methods for quantification of activity of PC lead to underestimation when backflux is not taken into account and critical errors have been made in the interpretation of results from metabolic studies. This study was conducted to establish the degree of backflux after PC in cerebellar and neocortical astrocytes. Astrocyte cultures from cerebellum or neocortex were incubated with either [3-(13) C] or [2-(13) C]glucose, and extracts were analyzed using mass spectrometry or nuclear magnetic resonance spectroscopy. Substantial PC compared with pyruvate dehydrogenase activity was observed, and extensive backflux was demonstrated in both types of astrocytes. The extent of backflux varied between the metabolites, reaffirming that metabolism is highly compartmentalized. By applying our calculations to published data, we demonstrate the existence of backflux in vivo in cat, rat, mouse, and human brain. Thus, backflux should be taken into account when calculating the magnitude of PC to allow for a more precise evaluation of cerebral metabolism.

AB - Pyruvate carboxylation (PC) is thought to be the major anaplerotic reaction for the tricarboxylic acid cycle and is necessary for de novo synthesis of amino acid neurotransmitters. In the brain, the main enzyme involved is pyruvate carboxylase, which is predominantly located in astrocytes. Carboxylation leads to the formation of oxaloacetate, which condenses with acetyl coenzyme A to form citrate. However, oxaloacetate may also be converted to malate and fumarate before being regenerated. This pathway is termed the oxaloacetate-fumarate-flux or backflux. Carbon isotope-based methods for quantification of activity of PC lead to underestimation when backflux is not taken into account and critical errors have been made in the interpretation of results from metabolic studies. This study was conducted to establish the degree of backflux after PC in cerebellar and neocortical astrocytes. Astrocyte cultures from cerebellum or neocortex were incubated with either [3-(13) C] or [2-(13) C]glucose, and extracts were analyzed using mass spectrometry or nuclear magnetic resonance spectroscopy. Substantial PC compared with pyruvate dehydrogenase activity was observed, and extensive backflux was demonstrated in both types of astrocytes. The extent of backflux varied between the metabolites, reaffirming that metabolism is highly compartmentalized. By applying our calculations to published data, we demonstrate the existence of backflux in vivo in cat, rat, mouse, and human brain. Thus, backflux should be taken into account when calculating the magnitude of PC to allow for a more precise evaluation of cerebral metabolism.

KW - Animals

KW - Animals, Newborn

KW - Astrocytes

KW - Carbon

KW - Carbon Isotopes

KW - Cells, Cultured

KW - Cerebellum

KW - Chromatography, High Pressure Liquid

KW - Fumarates

KW - Gas Chromatography-Mass Spectrometry

KW - Glucose

KW - Magnetic Resonance Spectroscopy

KW - Mice

KW - Neocortex

KW - Oxaloacetate

KW - Pyruvic Acid

U2 - 10.1002/glia.21265

DO - 10.1002/glia.21265

M3 - Journal article

C2 - 22052553

VL - 60

SP - 147

EP - 158

JO - GLIA

JF - GLIA

SN - 0894-1491

IS - 1

ER -