Mary C. McKenna

Exogenous Glutamate Concentration Regulates the Metabolic Fate of Glutamate in Astrocytes

Abstract: The metabolic fate of glutamate in astrocytes has been controversial since several studies reported >80% of glutamate was metabolized to glutamine; however, other studies have shown that half of the glutamate was metabolized via the tricarboxylic acid (TCA) cycle and half converted to glutamine. Studies were initiated to determine the metabolic fate of increasing concentrations of [U‐13C]glutamate in primary cultures of cerebral cortical astrocytes from rat brain. When astrocytes from rat brain were incubated with 0.1 mM [U‐13C]glutamate 85% of the 13C metabolized was converted to glutamine. The formation of [1,2,3‐13C3]glutamate demonstrated metabolism of the labeled glutamate via the TCA cycle. When astrocytes were incubated with 0.2–0.5 mM glutamate, 13C from glutamate was also incorporated into intracellular aspartate and into lactate that was released into the media. The amount of [13C]lactate was essentially unchanged within the range of 0.2–0.5 mM glutamate, whereas the amount of [13C]aspartate continued to increase in parallel with the increase in glutamate concentration. The amount of glutamate metabolized via the TCA cycle progressively increased from 15.3 to 42.7% as the extracellular glutamate concentration increased from 0.1 to 0.5 mM, suggesting that the concentration of glutamate is a major factor determining the metabolic fate of glutamate in astrocytes. Previous studies using glutamate concentrations from 0.01 to 0.5 mM and astrocytes from both rat and mouse brain are consistent with these findings.

The glutamate‐glutamine cycle is not stoichiometric: Fates of glutamate in brain

Although glutamate is usually thought of as the major excitatory neurotransmitter in brain, it is important to note that glutamate has many other fates in brain, including oxidation for energy, incorporation into proteins, and formation of glutamine, γ‐aminobutyric acid (GABA), and glutathione. The compartmentation of glutamate in brain cells is complex and modulated by the presence and concentration of glutamate per se as well as by other metabolites. Both astrocytes and neurons distinguish between exogenous glutamate and glutamate formed endogenously from glutamine via glutaminase. There is evidence of multiple subcellular compartments of glutamate within both neurons and astrocytes, and the carbon skeleton of glutamate can be derived from other amino acids and many energy substrates including glucose, lactate, and 3‐hydroxybutyrate. Both astrocytes and neurons utilize glutamate, albeit for cell‐specific metabolic fates. Glutamate is readily formed in neurons from glutamine synthesized in astrocytes, released into the extracellular space, and taken up by neurons. However, the glutamate‐glutamine cycle is not a stoichiometric cycle but rather an open pathway that interfaces with many other metabolic pathways to varying extents depending on cellular requirements and priorities.

Regulation of energy metabolism in synaptic terminals and cultured rat brain astrocytes: differences revealed using aminooxyacetate.

Several recent studies have demonstrated that the metabolism of energy substrates takes place in multiple compartments in both astrocytes and synaptic terminals from brain. There are a number of differences in the metabolism of astrocytes and synaptic terminals primarily due to the localization of key enzymes such as pyruvate carboxylase and glutamine synthetase in astrocytes. The present study determined the rates of 14CO2 production from several energy substrates by primary cultures of astrocytes and cortical synaptic terminals from rat brain. The rates of 14CO2 production from labelled substrates by astrocytes were 0.96 +/- 0.13, 11.13 +/- 0.67, 10.51 +/- 0.35, 24.92 +/- 1.66 and 4.80 +/- 0.50 for D-[6-14C]glucose, L-[U-14C]lactate, D-3-hydroxy[3-14C]butyrate, L-[U-14C]glutamine and L-[U-14C]ma-late, respectively. The rates of 14CO2 production were also measured in the presence of 5 mM aminooxyacetate (AOAA) to determine the effect of inhibiting the malate-aspartate shuttle and other transaminase reactions on the oxidation of energy substrates. In astrocytes the addition of AOAA decreased the rate of glutamine oxidation 5-fold, consistent with other studies showing that glutamine enters the TCA cycle via transamination. AOAA increased the rate of 14CO2 production from labelled glucose 4-fold, suggesting that inhibition of alanine biosynthesis profoundly alters the utilization of glucose by astrocytes. AOAA also increased the oxidation of lactate and 3-hydroxybutyrate 36 and 58%, respectively. The rates of 14CO2 production from labelled substrates by synaptic terminals were 13.12 +/- 1.05, 35.29 +/- 3.58, 17.66 +/- 1.95, 30.18 +/- 1.10 and 9.95 +/- 1.29, respectively, for glucose, lactate, 3-hydroxybutyrate, glutamine and malate, demonstrating that all substrates were oxidized at a higher rate by synaptic terminals than by astrocytes. The addition of AOAA decreased the rate of 14CO2 production from labelled lactate by 57% suggesting that the use of lactate for energy in synaptic terminals is tightly coupled to the activity of the malate-aspartate shuttle. AOAA had no effect on the rate of 14CO2 production from labelled glutamine, demonstrating that exogenous glutamine enters the TCA cycle in synaptic terminals via glutamate dehydrogenase, not via transamination as is the case with astrocytes. AOAA had no significant effect on the rates of oxidation of glucose, 3-hydroxybutyrate and malate by synaptic terminals. These findings demonstrate that inhibiting transamination with AOAA had very different effects on the oxidation of energy substrates in the two preparations, suggesting that the regulation of metabolism is quite different in astrocytes and synaptic terminals.(ABSTRACT TRUNCATED AT 250 WORDS)

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 mechanisms of cell death and neuroprotection in pediatric ischemic and traumatic brain injury

There are several forms of acute pediatric brain injury, including neonatal asphyxia, pediatric cardiac arrest with global ischemia, and head trauma, that result in devastating, lifelong neurologic impairment. The only clinical intervention that appears neuroprotective is hypothermia initiated soon after the initial injury. Evidence indicates that oxidative stress, mitochondrial dysfunction, and impaired cerebral energy metabolism contribute to the brain cell death that is responsible for much of the poor neurologic outcome from these events. Recent results obtained from both in vitro and animal models of neuronal death in the immature brain point toward several molecular mechanisms that are either induced or promoted by oxidative modification of macromolecules, including consumption of cytosolic and mitochondrial NAD(+) by poly-ADP ribose polymerase, opening of the mitochondrial inner membrane permeability transition pore, and inactivation of key, rate-limiting metabolic enzymes, e.g., the pyruvate dehydrogenase complex. In addition, the relative abundance of pro-apoptotic proteins in immature brains and neurons, and particularly within their mitochondria, predisposes these cells to the intrinsic, mitochondrial pathway of apoptosis, mediated by Bax- or Bak-triggered release of proteins into the cytosol through the mitochondrial outer membrane. Based on these pathways of cell dysfunction and death, several approaches toward neuroprotection are being investigated that show promise toward clinical translation. These strategies include minimizing oxidative stress by avoiding unnecessary hyperoxia, promoting aerobic energy metabolism by repletion of NAD(+) and by providing alternative oxidative fuels, e.g., ketone bodies, directly interfering with apoptotic pathways at the mitochondrial level, and pharmacologic induction of antioxidant and anti-inflammatory gene expression.

Transport ofl-lactate by cultured rat brain astrocytes

Several reports indicate that lactate can serve as an energy substrate for the brain. The rate of oxidation of this substrate by cultured rat brain astrocytes was 3-fold higher than the rate with glucose, suggesting that lactate can serve as an energy source for these cells. Since transport into the astrocytes may play an important role in regulating nutrient use by individuals types of brain cells, we investigated the uptake ofl-[U-14C]lactate by primary cultures of rat brain astrocytes. Measurement of the net uptake suggested two carrier-mediated mechanisms and an Eadie-Hofstee type plot of the data supported this conclusion revealing 2 Km values of 0.49 and 11.38 mM and Vmax values of 16.55 and 173.84 nmol/min/mg protein, respectively. The rate of uptake was temperature dependent and was 3-fold higher at pH 6.2 than at 7.4, but was 50% less at pH 8.2. Although the lactate uptake carrier systems in astrocytes appeared to be labile when incubated in phosphate buffered saline for 20 minutes, the uptake process exhibited an accelerative exchange mechanism. In addition, lactate uptake was altered by several metabolic inhibitors and effectors. Potassium cyanide and α-cyano-4-hydroxycinnamate inhibited lactate uptake, but mersalyl had little or no effect. Phenylpyruvate, α-ketoisocaproate, and 3-hydroxybutyrate at 5 and 10 mM greatly attenuated the rate of lactate uptake. These results suggest that the availability of lactate as an energy source is regulated in part by a biphasic transport system in primary astrocytes.

Glutamate Pays Its Own Way in Astrocytes

In vitro and in vivo studies have shown that glutamate can be oxidized for energy by brain astrocytes. The ability to harvest the energy from glutamate provides astrocytes with a mechanism to offset the high ATP cost of the uptake of glutamate from the synaptic cleft. This brief review focuses on oxidative metabolism of glutamate by astrocytes, the specific pathways involved in the complete oxidation of glutamate and the energy provided by each reaction.

New Insights into the Compartmentation of Glutamate and Glutamine in Cultured Rat Brain Astrocytes

Studies from several groups have provided evidence that glutamate and glutamine are metabolized in different compartments in astrocytes. In the present study we measured the rates of 14CO2 production from U-[14C]glutamate and U-[14C]glutamine, and utilized both substrate competition experiments and the transaminase inhibitor aminooxyacetic acid (AOAA) to obtain more information about the compartmentation of these substrates in cultured rat brain astrocytes. The rates of oxidation of 1 mM glutamine and glutamate were 26.4 +/- 1.4 and 63.0 +/- 7.4 nmol/h/mg protein, respectively. The addition of 1 mM glutamate decreased the rate of oxidation of glutamine to 26.3% of the control rate, demonstrating that glutamate can effectively compete with the oxidation of glutamine by astrocytes. In contrast, the addition of 1 mM glutamine had little or no effect on the rate of oxidation of glutamate by astrocytes, demonstrating that the glutamate produced intracellularly from exogenous glutamine does not dilute the glutamate taken up from the media. The addition of 5 mM AOAA decreased the rate of 14CO2 production from glutamine to 29.2% of the control rate, consistent with earlier studies by our group. The addition of 5 mM AOAA decreased the rate of oxidation of concentrations of glutamate < or = 0.1 mM by approximately 50%, but decreased the oxidation of 0.5-1 mM glutamate by only approximately 20%, demonstrating that a substantial portion of glutamate enters the tricarboxylic acid (TCA) cycle via glutamate dehydrogenase (GDH) rather than transamination, and that as the concentration of glutamate increases the relative proportion entering the TCA cycle via GDH also increases. To determine if the presence of an amino group acceptor (i.e. a ketoacid) would increase the rate of metabolism of glutamate, pyruvate was added in some experiments. Addition of 1 mM pyruvate increased the rate of oxidation of glutamate, and the increase was inhibited by AOAA, consistent with enhanced entry of glutamate into the TCA cycle via transamination in the presence of pyruvate. Enzymatic studies showed that pyruvate increased the activity of mitochondrial aspartate aminotransferase (AAT). Overall, the data demonstrate that glutamate formed intracellularly from glutamine enters the TCA cycle primarily via transamination, but does not enter the same TCA cycle compartment as glutamate taken up from the extracellular milieu. In contrast, extracellular glutamate enters the TCA cycle in astrocytes via both transamination and GDH, and can compete with, or dilute, the oxidation of glutamate produced intracellularly from glutamine.

Glutamate Metabolism in the Brain Focusing on Astrocytes

Metabolism of glutamate, the main excitatory neurotransmitter and precursor of GABA, is exceedingly complex and highly compartmentalized in brain. Maintenance of these neurotransmitter pools is strictly dependent on the de novo synthesis of glutamine in astrocytes which requires both the anaplerotic enzyme pyruvate carboxylase and glutamine synthetase. Glutamate is formed directly from glutamine by deamidation via phosphate activated glutaminase a reaction that also yields ammonia. Glutamate plays key roles linking carbohydrate and amino acid metabolism via the tricarboxylic acid (TCA) cycle, as well as in nitrogen trafficking and ammonia homeostasis in brain. The anatomical specialization of astrocytic endfeet enables these cells to rapidly and efficiently remove neurotransmitters from the synaptic cleft to maintain homeostasis, and to provide glutamine to replenish neurotransmitter pools in both glutamatergic and GABAergic neurons. Since the glutamate-glutamine cycle is an open cycle that actively interfaces with other pathways, the de novo synthesis of glutamine in astrocytes helps to maintain the operation of this cycle. The fine-tuned biochemical specialization of astrocytes allows these cells to respond to subtle changes in neurotransmission by dynamically adjusting their anaplerotic and glycolytic activities, and adjusting the amount of glutamate oxidized for energy relative to direct formation of glutamine, to meet the demands for maintaining neurotransmission. This chapter summarizes the evidence that astrocytes are essential and dynamic partners in both glutamatergic and GABAergic neurotransmission in brain.