Pyruvate recycling in cultured neurons from cerebellum

Abstract

Pyruvate recycling is a pathway for complete oxidation of glutamate. The cellular location and the physiological significance of such recycling has been debated during the last decade. The present study was aimed at elucidating whether recycling takes place in neuron-enriched cultures of dissociated cerebella, consisting mainly of glutamatergic granule cells, some GABAergic neurons, and few astrocytes. These cultures and cultures of astrocytes from cerebellum were incubated in medium containing [U-(13)C]glutamate, and cell extracts were analyzed by gas chromatography and mass spectrometry. Additionally, in the case of the neuron-enriched cultures, a magnetic resonance (MR) spectrum was obtained. It could be shown that the atom percentage excess of the isotopomer representing pyruvate recycling in glutamate (M + 4) was similar for astrocytes and neuron-enriched cultures. However, the latter showed more recycling in glutamine (synthesized in the small fraction of astrocytes) than the pure astrocyte cultures, whereas the reverse was the case for aspartate. In fact, the atom percentage excess of the isotopomer representing pyruvate recycling in glutamine was slightly but significantly higher than that in glutamate in the neuron-enriched cultures. It can be concluded that pyruvate recycling is clearly present in neurons, and this was verified by MR spectroscopy.

Glutamate-Induced ATP Synthesis: Relationship between Plasma Membrane Na+/Ca2+Exchanger and Excitatory Amino Acid Transporters in Brain and Heart Cell Models

“…Both plasma membrane NCX and EAAT activities are required to elicit this metabolic response. This experimental evidence expanded our previous study in which we demonstrated a key role of the mitochondrial NCX and EAAT in Glu-induced ATP synthesis (Magi et al, 2012) and strengthened the earlier postulated role of Glu in brain energy metabolism (McKenna et al, 1996;Olstad et al, 2007). It is well known that as a neurotransmitter, the released Glu exerts its signaling function by interacting with specific receptors until it is removed from the extracellular fluid by the rapid uptake operated by EAATs (Kanai et al, 1993;Danbolt, 2001).…”

“…It has already been established that Glu per se is able to activate neuronal and glial energy metabolism (Hertz and Hertz, 2003;Panov et al, 2009;Magi et al, 2012). After being picked up by astrocytes or neurons, Glu can be converted to a-keto-glutarate, which, as an intermediate of the Krebs cycle, can increase energy metabolism (Olstad et al, 2007;Amaral et al, 2011). The metabolic fate of Glu in the cells is influenced by its extracellular concentration as observed in astrocytes, in which Glu is preferentially metabolized via the Krebs cycle when its extracellular levels rise up to the low millimolar range (McKenna et al, 1996).…”

Complex Glutamate Labeling from [U-13C]glucose or [U-13C]lactate in Co-cultures of Cerebellar Neurons and Astrocytes

“…Another study using [3-13 C]glutamate confirmed that recycling takes place in astrocytes but not in neurons or cerebral cortical co-cultures [7]. Using MS analysis, which is more sensitive than MRS, recent experiments have shown that pyruvate recycling from [U-13 C]glutamate is taking place in cerebellar neurons [35]. Interestingly, when cerebellar co-cultures were superfused with [U-13 C]glucose or [U-13 C]lactate considerable recycling was detected.…”

13C metabolic flux analysis in neurons utilizing a model that accounts for hexose phosphate recycling within the pentose phosphate pathway

“…Thus, at least one other reaction must contribute to labeling of cytoplasmic pyruvate. Previous studies with neurons have shown that malic enzyme can, at least under some conditions, be a significant source of pyruvate (Amaral et al, 2011b; Bak et al, 2007; Cruz et al, 1998; Olstad et al, 2007) that could affect labeling of extracellular lactate. The contribution of malic enzyme to the pyruvate pool, J 7 /(2 J 2 a + J 3 b + J 7 ), was introduced into the model as a second solver-optimized flux ratio (Fig.…”