NMR studies on energy metabolism of immobilized primary neurons and astrocytes during hypoxia, ischemia and hypoglycemia

Alves, Paula M.; Fonseca, Luis L.; Peixoto, Cristina; Almeida, Angela C. De; Carrondo, Manuel J.T.; Santos, Helena

doi:10.1002/nbm.665

Cited by 32 publications

(20 citation statements)

References 48 publications

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“…Neuronal ATP levels in primary cultures of 'mature' phenotype are unchanged after 1 hr of anoxia (Almeida et al, 2002). Neuronal cultures exposed to glucose/ oxygen free media (ischemia-like conditions) can rapidly restore their ATP and PCr pools upon recovery (Alves et al, 2000).…”

Section: Discussionmentioning

confidence: 99%

Effects of continuous hypoxia on energy metabolism in cultured cerebro-cortical neurons

et al. 2008

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Mechanisms underlying hypoxia-induced neuronal adaptation have not been fully elucidated. In the present study we investigated glucose metabolism and the activities of glycolytic and TCA cycle enzymes in cerebro-cortical neurons exposed to hypoxia (3 days in 1% of O 2 ) or normoxia (room air). Hypoxia led to increased activities of LDH (251%), PK (90%), and HK (24%) and decreased activities of CS (15%) and GDH (34%). Neurons were incubated with [1-13 C]glucose for 45 and 120 min under normoxic or hypoxic (120 min only) conditions and 13 C enrichment determined in the medium and cell extract using 1 H-{ 13 C}-NMR. In hypoxia-treated neurons [3-13 C]lactate release into the medium was 428% greater than in normoxia-treated controls (45-min normoxic incubation) and total flux through lactate was increased by 425%. In contrast glucose oxidation was reduced significantly in hypoxia-treated neurons, even when expressed relative to total cellular protein, which correlated with the reduced activities of the measured mitochondrial enzymes. The results suggest that surviving neurons adapt to prolonged hypoxia by up-regulation of glycolysis and downregulation of oxidative energy metabolism, similar to certain other cell types. The factors leading to adaptation and survival for some neurons but not others remains to be determined.

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Section: Discussionmentioning

confidence: 99%

Effects of continuous hypoxia on energy metabolism in cultured cerebro-cortical neurons

et al. 2008

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“…The ATP content of astrocytes did not change appreciably upon glutaminase addition, but a drastic reduction in the level of PCr was observed (Table 2). This suggests an increased energy demand, but the level of ATP is maintained at the expense of the PCr pool, as observed in response to short times under ischemia (Alves et al, 2000). Decreased PCr /ATP ratios in astrocytes challenged with glutamate in a discontinuous experimental setup have been reported earlier (Sonnewald et al, 1997).…”

Section: Figmentioning

confidence: 92%

Cultures of rat astrocytes challenged with a steady supply of glutamate: New model to study flux distribution in the glutamate–glutamine cycle

Fonseca

Monteiro

Alves

et al. 2005

Glia

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Glutamate metabolism in astrocytes was studied using an experimental setup that simulates the role of neurons (glutamate producers and glutamine consumers) by the addition of glutaminase to the culture medium. Thereby, a steady supply of glutamate was imposed at the expense of glutamine, and the stress intensity was manipulated by changing the glutaminase concentration. Glutamate supply rates in the range 8-23 nmol/min/mg protein were examined for periods of up to 48 h. When the glutamate supply rate exceeded the uptake rate of this amino acid, a transient increase in the extracellular concentration of glutamate was observed. In response to this stress, the fluxes through the glutamate transporter and glutamine synthetase were increased considerably, and the extracellular concentration of glutamate was eventually restored to a low level. The increased levels of glutamine synthetase were demonstrated by immunoblotting analysis. The effect on glutamate metabolism of the transaminase inhibitor, aminooxyacetic acid (AOAA), and of NH4Cl was also investigated. The supply of glutamate caused a concomitant reduction in the levels of phosphocreatine, phosphoethanolamine, and phosphocholine without affecting the ATP pool. Glutamine synthetase was shown to be is a key element in the control of glutamate metabolism in astrocytic cultures. The metabolic fate of glutamate depends greatly on the time of endurance to the challenge: in naive cells, glutamate was primarily metabolized through the transaminase pathway, while in well-adapted cells glutamate was converted almost exclusively through glutamine synthetase.

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“…It has been successfully used to assess the metabolic state of a variety of tissues under various conditions. [19][20][21] The ATP/DNA ratio suffers similar limitations, but DNA does not degrade as quickly in dead cells as either ATP or ADP. Because of this profound difference in the kinetics of degradation of DNA under conditions of stress, the ATP/DNA ratio permits the inclusion of dead cells into a viability estimate once the apoptotic process is fully executed.…”

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confidence: 99%

The ATP/DNA Ratio Is a Better Indicator of Islet Cell Viability Than the ADP/ATP Ratio

Suszynski¹,

Wildey²,

Falde³

et al. 2008

Transplantation Proceedings

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Real-time, accurate assessment of islet viability is critical for avoiding transplantation of nontherapeutic preparations. Measurements of the intracellular ADP/ATP ratio have been recently proposed as useful prospective estimates of islet cell viability and potency. However, dead cells may be rapidly depleted of both ATP and ADP, which would render the ratio incapable of accounting for dead cells. Since the DNA of dead cells is expected to remain stable over prolonged periods of time (days), we hypothesized that use of the ATP/DNA ratio would take into account dead cells and may be a better indicator of islet cell viability than the ADP/ATP ratio. We tested this hypothesis using mixtures of healthy and lethally heat-treated (HT) rat insulinoma cells and human islets. Measurements of ATP/DNA and ADP/ATP from the known mixtures of healthy and HT cells and islets were used to evaluate how well these parameters correlated with viability. The results indicated that ATP and ADP were rapidly (within 1 hour) depleted in HT cells. The fraction of HT cells in a mixture correlated linearly with the ATP/DNA ratio, whereas the ADP/ADP ratio was highly scattered, remaining effectively unchanged. Despite similar limitations in both ADP/ADP and ATP/ DNA ratios, in that ATP levels may fluctuate significantly and reversibly with metabolic stress, the results indicated that ATP/DNA was a better measure of islet viability than the ADP/ATP ratio.Islet cell transplantation is emerging as a promising therapy for the treatment of type 1 diabetes. [1][2][3][4][5] Despite recent advances, the transplantation of islets poses a unique challenge with respect to achieving a consistent clinical outcome. Part of this challenge is being able to reliably and rapidly assess clinical islet quality through the quantification of viability and function before transplantation. Current viability assays are limited in their ability to accurately predict transplantation outcomes in vivo. 6 Consequently, to improve the clinical islet transplantation outcomes, it is imperative to develop more accurate viability assays.A proposed method to assess islet viability and potency before transplantation is quantification of the ADP/ATP ratio. 6 -10 This ratio has been specifically applied in discriminating islet preparations that are suitable for clinical transplantation from those that are not. 6 However, this application may be problematic under certain conditions. Intracellular ADP and ATP levels fluctuate rapidly because these high-energy phosphate molecules are rapidly produced and consumed in many intracellular biochemical reactions. It is widely known that viable cells turn over entire ATP stores on the order of minutes, and that dead cells are incapable of replenishing To better account for dead cells and to more accurately assess the viability of an islet preparation, we suggest the use of an ATP/DNA ratio. DNA does not degrade as rapidly as ADP or ATP in a dead cell. Therefore, using direct measurements of DNA, dead cells in an islet preparation...

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NMR studies on energy metabolism of immobilized primary neurons and astrocytes during hypoxia, ischemia and hypoglycemia

Cited by 32 publications

References 48 publications

Effects of continuous hypoxia on energy metabolism in cultured cerebro-cortical neurons

Effects of continuous hypoxia on energy metabolism in cultured cerebro-cortical neurons

Cultures of rat astrocytes challenged with a steady supply of glutamate: New model to study flux distribution in the glutamate–glutamine cycle

The ATP/DNA Ratio Is a Better Indicator of Islet Cell Viability Than the ADP/ATP Ratio

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