Aspects of glucose metabolism in vitro, in the healthy developing brain and after neonatal hypoxia-ischemia : with particular focus on the pentose phosphate pathway and pyruvate carboxylation
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Glucose is used for more than energy production, such as the pentose phosphate pathway (PPP) and neurotransmitter synthesis dependent on anaplerosis. Glucose metabolism has been thoroughly investigated in the adult brain. However, little is known about glucose metabolism in the neonatal brain and after neonatal hypoxiaischemia (HI), an important cause of cerebral palsy. In the PPP, glucose is metabolized to provide the cells with pentoses for nucleotide synthesis and NADPH for lipid biosynthesis as well as regeneration of reduced glutathione. Consequently, this pathway is important during growth and it plays an important role in the defense against oxidative stress. Pyruvate carboxylation is required for de novo synthesis of neurotransmitters. This is necessary to increase the amount of glutamate to reach adult levels, but also produces glutamate which can be released during energy failure, causing excitotoxicity. In the adult brain, hypoxia-ischemia leads to oxidative stress and reduced activity of pyruvate carboxylase (PC). Oxidative stress and glutamate excitotoxicity are two of the mechanisms for tissue damage during neonatal HI. Thus, it is relevant to study both the PPP and pyruvate carboxylation in the neonatal brain during physiological conditions and after neonatal HI. However, there is also a need for more detailed knowledge of the PPP and pyruvate carboxylation at a cellular level. Therefore, we have conducted cell culture experiments using [2-13C]- and [3-13C]glucose in combination with 13C-Nuclear Magnetic Resonance (NMR) Spectroscopy, Gas Chromatography – Mass Spectrometry and High- Performance Liquid Chromatography (HPLC). In Paper 1, we show that pyruvate carboxylation is coupled with extensive backflux, a possible confounder when interpreting results from metabolic studies conducted using [1-13/14C]labeled glucose. We could also demonstrate that this backflux was present in vivo based on published material. In Paper 2, we investigated the PPP activity in cultured neurons from neocortex and cerebellum. We show that neurons do not label extracellular lactate while they do label TCA cycle intermediates and related amino acids via the PPP. Nonetheless, when comparing labeled isotopomers of glutamate, the major proportion of glucose was metabolized via glycolysis. Based on these results, we chose to study metabolism in a neonatal animal model using [1,2-13C]glucose in combination with 1H- and 13C-NMR spectroscopy and HPLC. Labeling from [1,2-13C]glucose in TCA cycle intermediates is not affected by backflux, and allows for investigation of pyruvate carboxylation as well as the PPP in relation to both lactate and glutamate production in vivo. In Paper 3 we also used a combined injection of [1-13C]glucose and [1,2-13C]acetate to study the metabolic interactions between neurons and astrocytes. In Paper 3, we found relatively high PC and PPP activities relative to other pathways of glucose metabolism compared to values from experiments on adult animals. We also found a generally lower rate of oxidative glucose metabolism in the neonatal compared to adult animals. The transfer of glutamine from astrocytes to neurons was relatively higher in the neonatal animals while the transfer of glutamate from neurons to astrocytes was much lower than in the adult animals. This could indicate that the neurons are less active, that the uptake of glutamate by astrocytes is lower, or both. After neonatal HI (Paper 4), we demonstrated that PPP activity was reduced, which could render the neonatal brain more susceptible to oxidative injury. Moreover, the amount of pyruvate metabolized via PC was reduced, but it was relatively preserved compared to the amount of pyruvate metabolized via pyruvate dehydrogenase. Moreover, as argued in the discussion of this thesis, the transfer of glutamine from astrocytes to neurons appeared to be maintained. This could indicate that the astrocytes were providing metabolic support for the neurons. However, the glutamine the neurons received from the astrocytes can also be used to make new glutamate that can be released if the neurons experience a second energy failure. Furthermore, we found increased amounts of GABA, probably due to decreased breakdown. This increase could also contribute to excitotoxicity because GABA can have a depolarizing effect at this developmental stage. In conclusion, we found that there is substantial backflux following pyruvate carboxylation, and when studying the PPP in neurons, it may be better to study incorporation of label into TCA cycle intermediates rather than extracellular lactate. Moreover, we found that both PC and PPP used a relatively larger proportion of the available glucose in the neonatal brain compared to the adult brain. After neonatal HI, PPP was reduced while PC activity was relatively preserved, which could render the neonatal brain more susceptible to oxidative injury as well as excitotoxicity in the case of energy failure in the recovery phase.