Irene B. Hopkins

Hyperoxic Reperfusion After Global Ischemia Decreases Hippocampal Energy Metabolism

Background and Purpose— Previous reports indicate that compared with normoxia, 100% ventilatory O2 during early reperfusion after global cerebral ischemia decreases hippocampal pyruvate dehydrogenase activity and increases neuronal death. However, current standards of care after cardiac arrest encourage the use of 100% O2 during resuscitation and for an undefined period thereafter. Using a clinically relevant canine cardiac arrest model, in this study we tested the hypothesis that hyperoxic reperfusion decreases hippocampal glucose metabolism and glutamate synthesis. Methods— After 10 minutes of cardiac arrest, animals were resuscitated and ventilated for 1 hour with 100% O2 (hyperoxic) or 21% to 30% O2 (normoxic). At 30 minutes reperfusion, [1-13C]glucose was infused, and at 2 hours, brains were rapidly removed and frozen. Extracted metabolites were analyzed by 13C nuclear magnetic resonance spectroscopy. Results— Compared with nonischemic controls, the hippocampi from hyperoxic animals had elevated levels of unmetabolized 13C-glucose and decreased incorporation of 13C into all isotope isomers of glutamate. These findings indicate impaired neuronal metabolism via the pyruvate dehydrogenase pathway for carbon entry into the tricarboxylic acid cycle and impaired glucose metabolism via the astrocytic pyruvate carboxylase pathway. No differences were observed in the cortex, indicating that the hippocampus is more vulnerable to metabolic changes induced by hyperoxic reperfusion. Conclusions— These results represent the first direct evidence that hyperoxia after cardiac arrest impairs hippocampal oxidative energy metabolism in the brain and challenge the rationale for using excessively high resuscitative ventilatory O2.

Differential distribution of the enzymes glutamate dehydrogenase and aspartate aminotransferase in cortical synaptic mitochondria contributes to metabolic compartmentation in cortical synaptic terminals.

There have been numerous studies on the activity and localization of aspartate aminotransferase (AAT) and glutamate dehydrogenase (GDH) in brain tissue. However, there is still a controversy as to the specific roles and relative importance of these enzymes in glutamate and glutamine metabolism in astrocytes and neurons or synaptic terminals. There are many reports documenting GDH activity in synaptic terminals, yet the misconception that it is a glial enzyme persists. Furthermore, there is evidence that this tightly regulated enzyme may have an increased role in synaptic metabolism in adverse conditions such as low glucose and hyperammonemia that could compromise synaptic function. In the present study, we report high activity of both AAT and GDH in mitochondrial subfractions from cortical synaptic terminals. The relative amount of GDH/AAT activity was higher in SM2 mitochondria, compared to SM1 mitochondria. Such a differential distribution of enzymes can contribute significantly to the compartmentation of metabolism. There is evidence that the metabolic capabilities of the SM1 and SM2 subfractions of synaptic mitochondria are compatible with the compartments A and B of neuronal metabolism proposed by Waagepetersen et al. (1998b. Dev. Neurosci. 20, 310-320).

Mitochondrial malic enzyme activity is much higher in mitochondria from cortical synaptic terminals compared with mitochondria from primary cultures of cortical neurons or cerebellar granule cells.

Most of the malic enzyme activity in the brain is found in the mitochondria. This isozyme may have a key role in the pyruvate recycling pathway which utilizes dicarboxylic acids and substrates such as glutamine to provide pyruvate to maintain TCA cycle activity when glucose and lactate are low. In the present study we determined the activity and kinetics of malic enzyme in two subfractions of mitochondria isolated from cortical synaptic terminals, as well as the activity and kinetics in mitochondria isolated from primary cultures of cortical neurons and cerebellar granule cells. The synaptic mitochondrial fractions had very high mitochondrial malic enzyme (mME) activity with a Km and a Vmax of 0.37 mM and 32.6 nmol/min/mg protein and 0.29 mM and 22.4 nmol/min mg protein, for the SM2 and SM1 fractions, respectively. The Km and Vmax for malic enzyme activity in mitochondria isolated from cortical neurons was 0.10 mM and 1.4 nmol/min/mg protein and from cerebellar granule cells was 0.16 mM and 5.2 nmol/min/mg protein. These data show that mME activity is highly enriched in cortical synaptic mitochondria compared to mitochondria from cultured cortical neurons. The activity of mME in cerebellar granule cells is of the same magnitude as astrocyte mitochondria. The extremely high activity of mME in synaptic mitochondria is consistent with a role for mME in the pyruvate recycling pathway, and a function in maintaining the intramitochondrial reduced glutathione in synaptic terminals.

Energy metabolism in cortical synaptic terminals from weanling and mature rat brain: evidence for multiple compartments of tricarboxylic acid cycle activity.

It is well documented that the brain preferentially utilizes alternative substrates for energy during brain development; however, less is known about the use of these substrates by synaptic terminals. The present study compared the rates of 14CO2 production from 1 mM D-[6-14C]glucose, L-[U-14C]glutamine, D-3-hydroxy[3-14C]butyrate, L-[U-14C]lactate and L-[U-14C]malate by synaptic terminals isolated from 17- to 18-day-old and 7- to 8-week-old rat brain. The rates of 14CO2 production from glucose, glutamine, 3-hydroxybutyrate, lactate and malate were 8.55 +/- 0.78, 25.90 +/- 4.58, 42.28 +/- 3.54, 48.42 +/- 2.09, and 9.31 +/- 1.61 nmol/h/mg protein (mean +/- SEM), respectively, in synaptic terminals isolated from 17- to 18-day-old rat brain and 12.95 +/- 1.64, 30.62 +/- 4.19, 16.09 +/- 2.62, 40.33 +/- 6.77, and 8.25 +/- 1.69 nmol/h/mg protein (mean +/- SEM), respectively, in synaptic terminals isolated from 7- to 8-week-old rat brain. In competition studies using unlabelled added substrates, the addition of 3-hydroxybutyrate, lactate or glutamine greatly decreased the rate of 14CO2 production from labelled glucose. Added unlabelled glucose increased the rate of 14CO2 production from 3-hydroxybutyrate in synaptic terminals from 7- to 8-week-old rat brain, but had no effect on 14CO2 production from any other substrates. Lactate also increased 14CO2 production from 3-hydroxybutyrate at 7-8 weeks, whereas the addition of 3-hydroxybutyrate decreased 14CO2 production from lactate only in synaptic terminals from 17- to 18-day-old rat brain. None of the added substrates altered the rate of 14CO2 production from labelled glutamine or malate suggesting that these substrates are metabolized in relatively distinct compartments within synaptic terminals. Overall the data demonstrate that synaptic terminals from both weanling and adult rat brain can utilize a variety of substrates for energy. In addition, the competition studies demonstrate that the interactions of substrates change with age and suggest that there are multiple compartments of energy metabolism (or tricarboxylic acid cycle activity) in isolated synaptic terminals.

Metabolism of acetyl-L-carnitine for energy and neurotransmitter synthesis in the immature rat brain.

J. Neurochem. (2010) 114, 820–831.

α-cyano-4-hydroxycinnamate decreases both glucose and lactate metabolism in neurons and astrocytes: Implications for lactate as an energy substrate for neurons

The rates of uptake and oxidation of [U‐14C]lactate and [U‐14C]glucose were determined in primary cultures of astrocytes and neurons from rat brain, in the presence and absence of the monocarboxylic acid transport inhibitor α‐cyano‐4‐hydroxycinnamate (4‐CIN). The rates of uptake for 1 mM lactate and glucose were 7.45 ± 1.35 and 8.80 ± 1.0 nmol/30 sec/mg protein in astrocytes and 2.36 ± 0.19 and 1.93 ± 0.16 nmol/30 sec/mg protein in neuron cultures, respectively. Lactate transport into both astrocytes and neurons was significantly decreased by 0.25–1.0 mM 4‐CIN; however, glucose uptake was not affected. The rates of 14CO2 formation from 1 mM lactate and glucose were 12.49 ± 0.77 and 3.42 ± 0.67 nmol/hr/mg protein in astrocytes and 29.32 ± 2.81 and 10.04 ± 1.79 nmol/hr/mg protein in neurons, respectively. Incubation with 0.25 mM 4‐CIN decreased the oxidation of lactate and glucose to 57.1% and 54.1% of control values in astrocytes and to 13.2% and 41.6% of the control rates in neurons, respectively. Preincubation with 4‐CIN further decreased the oxidation of both glucose and lactate. Studies with glucose specifically labeled in the one and six positions demonstrated that 4‐CIN decreased mitochondrial glucose oxidation but did not impair the metabolism of glucose via the pentose phosphate pathway in the cytosol. The lack of effect of 4‐CIN on glutamate oxidation demonstrated that overall mitochondrial metabolism was not impaired. These findings suggest that the impaired neuronal function and tissue damage in the presence of 4‐CIN observed in other studies may be due in part to decreased uptake of lactate; however, the effects of 4‐CIN on mitochondrial transport would significantly decrease the oxidative metabolism of pyruvate derived from both glucose and lactate.

Delayed cerebral oxidative glucose metabolism after traumatic brain injury in young rats

Traumatic brain injury (TBI) results in a cerebral metabolic crisis that contributes to poor neurologic outcome. The aim of this study was to characterize changes in oxidative glucose metabolism in early periods after injury in the brains of immature animals. At 5 h after controlled cortical impact TBI or sham surgery to the left cortex, 21–22 day old rats were injected intraperitoneally with [1,6‐13C]glucose and brains removed 15, 30 and 60 min later and studied by ex vivo 13C‐NMR spectroscopy. Oxidative metabolism, determined by incorporation of 13C into glutamate, glutamine and GABA over 15–60 min, was significantly delayed in both hemispheres of brain from TBI rats. The most striking delay was in labeling of the C4 position of glutamate from neuronal metabolism of glucose in the injured, ipsilateral hemisphere which peaked at 60 min, compared with the contralateral and sham‐operated brains, where metabolism peaked at 30 and 15 min, respectively. Our findings indicate that (i) neuronal‐specific oxidative metabolism of glucose at 5–6 h after TBI is delayed in both injured and contralateral sides compared with sham brain; (ii) labeling from metabolism of glucose via the pyruvate carboxylase pathway in astrocytes was also initially delayed in both sides of TBI brain compared with sham brain; (iii) despite this delayed incorporation, at 6 h after TBI, both sides of the brain showed apparent increased neuronal and glial metabolism, reflecting accumulation of labeled metabolites, suggesting impaired malate aspartate shuttle activity. The presence of delayed metabolism, followed by accumulation of labeled compounds is evidence of severe alterations in homeostasis that could impair mitochondrial metabolism in both ipsilateral and contralateral sides of brain after TBI. However, ongoing oxidative metabolism in mitochondria in injured brain suggests that there is a window of opportunity for therapeutic intervention up to at least 6 h after injury.

Postischemic Oxidative Stress Promotes Mitochondrial Metabolic Failure in Neurons and Astrocytes

Oxidative stress and mitochondrial dysfunction have been closely associated in many subcellular, cellular, animal, and human studies of both acute brain injury and neurodegenerative diseases. Our animal models of brain injury caused by cardiac arrest illustrate this relationship and demonstrate that both oxidative molecular modifications and mitochondrial metabolic impairment are exacerbated by reoxygenation of the brain using 100% ventilatory O2 compared to lower levels that maintain normoxemia. Numerous molecular mechanisms may be responsible for mitochondrial dysfunction caused by oxidative stress, including oxidation and inactivation of mitochondrial proteins, promotion of the mitochondrial membrane permeability transition, and consumption of metabolic cofactors and intermediates, for example, NAD(H). Moreover, the relative contribution of these mechanisms to cell injury and death is likely different among different types of brain cells, for example, neurons and astrocytes. In order to better understand these oxidative stress mechanisms and their relevance to neurologic disorders, we have undertaken studies with primary cultures of astrocytes and neurons exposed to O2 and glucose deprivation and reoxygenation and compared the results of these studies to those using a rat model of neonatal asphyxic brain injury. These results support the hypothesis that release and or consumption of mitochondrial NAD(H) is at least partially responsible for respiratory inhibition, particularly in neurons.

Thyroid hormone action on glucose transporter activity in astrocytes

In astrocytes from rat brain cultured in thyroid hormone-deficient media cytochalasin B-binding was decreased 80%; addition of L-T3 increased binding to 75% of control levels. Saponin-treatment of controls increased accessibility of binding sites to 60% above untreated cells. Saponin also increased binding in deficient cells; however, the level was less than in treated controls, suggesting L-T3 deficiency decreases total glucose transporters. Addition of L-T3 appeared to convert most (90%) of the binding sites from unavailable to accessible status. Changes in binding to plasma membranes in response to L-T3 level were similar to those in intact cells. No binding to Golgi was detectable, thus no evidence for translocation of carriers was obtained. L-T3 may activate the glucose transporter by increasing its accessibility in brain cells.

Synthesis of glutamate and glutamine in dibutyryl cyclic AMP-treated astrocytes

The relative contributions of radioactively labeled fatty acids and glucose to synthesis of glutamate and glutamine were compared in native and dibutyryl cyclic AMP (diBcAMP)-treated primary rat astrocytes. The intracellular specific activities of glutamate and glutamine were 10-fold greater than the specific activities of aspartate or alanine. Butyrate, octanoate and palmitate were equally as effective as precursors for glutamate and glutamine while glucose was 50% as effective as the fatty acids. The specific activity of glutamate and glutamine were identical in the absence of diBcAMP. In diBcAMP treated cells the specific activity of glutamine was greater than that of glutamate when octanoate and palmitate were the labeled precursors. This suggests that cultured astrocytes preferentially utilize free fatty acids for glutamate/glutamine synthesis and that diBcAMP-treated astrocytes contain more than one glutamate compartment.