Anne B. Walls

Glutamate receptor-dependent increments in lactate, glucose and oxygen metabolism evoked in rat cerebellum in vivo

Neuronal activity is tightly coupled with brain energy metabolism. Numerous studies have suggested that lactate is equally important as an energy substrate for neurons as glucose. Lactate production is reportedly triggered by glutamate uptake, and independent of glutamate receptor activation. Here we show that climbing fibre stimulation of cerebellar Purkinje cells increased extracellular lactate by 30% within 30 s of stimulation, but not for briefer stimulation periods. To explore whether lactate production was controlled by pre‐ or postsynaptic events we silenced AMPA receptors with CNQX. This blocked all evoked rises in postsynaptic activity, blood flow, and glucose and oxygen consumption. CNQX also abolished rises in lactate concomitantly with marked reduction in postsynaptic currents. Rises in lactate were unaffected by inhibition of glycogen phosphorylase, suggesting that lactate production was independent of glycogen breakdown. Stimulated lactate production in cerebellum is derived directly from glucose uptake, and coupled to neuronal activity via AMPA receptor activation.

Brain glycogen—new perspectives on its metabolic function and regulation at the subcellular level

Glycogen is a complex glucose polymer found in a variety of tissues, including brain, where it is localized primarily in astrocytes. The small quantity found in brain compared to e.g., liver has led to the understanding that brain glycogen is merely used during hypoglycemia or ischemia. In this review evidence is brought forward highlighting what has been an emerging understanding in brain energy metabolism: that glycogen is more than just a convenient way to store energy for use in emergencies—it is a highly dynamic molecule with versatile implications in brain function, i.e., synaptic activity and memory formation. In line with the great spatiotemporal complexity of the brain and thereof derived focus on the basis for ensuring the availability of the right amount of energy at the right time and place, we here encourage a closer look into the molecular and subcellular mechanisms underlying glycogen metabolism. Based on (1) the compartmentation of the interconnected second messenger pathways controlling glycogen metabolism (calcium and cAMP), (2) alterations in the subcellular location of glycogen-associated enzymes and proteins induced by the metabolic status and (3) a sequential component in the intermolecular mechanisms of glycogen metabolism, we suggest that glycogen metabolism in astrocytes is compartmentalized at the subcellular level. As a consequence, the meaning and importance of conventional terms used to describe glycogen metabolism (e.g., turnover) is challenged. Overall, this review represents an overview of contemporary knowledge about brain glycogen and its metabolism and function. However, it also has a sharp focus on what we do not know, which is perhaps even more important for the future quest of uncovering the roles of glycogen in brain physiology and pathology.

Functional significance of brain glycogen in sustaining glutamatergic neurotransmission.

The involvement of brain glycogen in sustaining neuronal activity has previously been demonstrated. However, to what extent energy derived from glycogen is consumed by astrocytes themselves or is transferred to the neurons in the form of lactate for oxidative metabolism to proceed is at present unclear. The significance of glycogen in fueling glutamate uptake into astrocytes was specifically addressed in cultured astrocytes. Moreover, the objective was to elucidate whether glycogen derived energy is important for maintaining glutamatergic neurotransmission, induced by repetitive exposure to NMDA in co‐cultures of cerebellar neurons and astrocytes. In the astrocytes it was shown that uptake of the glutamate analogue d‐[3H]aspartate was impaired when glycogen degradation was inhibited irrespective of the presence of glucose, signifying that energy derived from glycogen degradation is important for the astrocytic compartment. By inhibiting glycogen degradation in co‐cultures it was evident that glycogen provides energy to sustain glutamatergic neurotransmission, i.e. release and uptake of glutamate. The relocation of glycogen derived lactate to the neuronal compartment was investigated by employing d‐lactate, a competitive substrate for the monocarboxylate transporters. Neurotransmitter release was affected by the presence of d‐lactate indicating that glycogen derived energy is important not only in the astrocytic but also in the neuronal compartment.

ROBUST GLYCOGEN SHUNT ACTIVITY IN ASTROCYTES: EFFECTS OF GLUTAMATERGIC AND ADRENERGIC AGENTS

The significance and functional roles of glycogen shunt activity in the brain are largely unknown. It represents the fraction of metabolized glucose that passes through glycogen molecules prior to entering the glycolytic pathway. The present study was aimed at elucidating this pathway in cultured astrocytes from mouse exposed to agents such as a high [K+], D-aspartate and norepinephrine (NE) known to affect energy metabolism in response to neurotransmission. Glycogen shunt activity was assessed employing [1,6-13C]glucose, and the glycogen phosphorylase inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) to block glycogen degradation. The label intensity in lactate, reflecting glycolytic activity, was determined by mass spectrometry. In the presence of NE a substantial glycogen shunt activity was observed, accounting for almost 40% of overall glucose metabolism. Moreover, when no metabolic stimulant was applied, a compensatory increase in glycolytic activity was seen when the shunt was inhibited by DAB. Actually the labeling in lactate exceeded that obtained when glycolysis and glycogen shunt both were operational, i.e. supercompensation. A similar phenomenon was seen when astrocytes were exposed to D-aspartate. In addition to glycolysis, tricarboxylic acid (TCA) cycle activity was monitored, analyzing labeling by mass spectrometry in glutamate which equilibrates with alpha-ketoglutarate. Both an elevated [K+] and D-aspartate induced an increased TCA cycle activity, which was altered when glycogen degradation was inhibited. Thus, the present study provides evidence that manipulation of glycogen metabolism affects both glycolysis and TCA cycle metabolism. Altogether, the results reveal a highly complex interaction between glycogenolysis and glycolysis, with the glycogen shunt playing a significant role in astrocytic energy metabolism.

Neuronal glucose but not lactate utilization is positively correlated with NMDA-induced neurotransmission and fluctuations in cytosolic Ca2+ levels

Although the brain utilizes glucose for energy production, individual brain cells may to some extent utilize substrates derived from glucose. Thus, it has been suggested that neurons consume extracellular lactate during synaptic activity. However, the precise role of lactate for fuelling neuronal activity is still poorly understood. Recently, we demonstrated that glucose metabolism is up‐regulated in cultured glutamatergic neurons during neurotransmission whereas that of lactate is not. Here, we show that utilization of glucose but not lactate correlates with NMDA‐induced neurotransmitter glutamate release in cultured cerebellar neurons from mice. Pulses of NMDA at 30, 100, and 300 μM, leading to a progressive increase in both cytosolic [Ca2+] and release of glutamate, increased uptake and metabolism of glucose but not that of lactate as evidenced by mass spectrometric measurement of 13C incorporation into intracellular glutamate. In this manuscript, a cascade of events for the preferential neuronal utilization of glucose during neurotransmission is suggested and discussed in relation to our current understanding of neuronal energy metabolism.

The Glutamine-Glutamate/GABA Cycle: Function, Regional Differences in Glutamate and GABA Production and Effects of Interference with GABA Metabolism

The operation of a glutamine–glutamate/GABA cycle in the brain consisting of the transfer of glutamine from astrocytes to neurons and neurotransmitter glutamate or GABA from neurons to astrocytes is a well-known concept. In neurons, glutamine is not only used for energy production and protein synthesis, as in other cells, but is also an essential precursor for biosynthesis of amino acid neurotransmitters. An excellent tool for the study of glutamine transfer from astrocytes to neurons is [14C]acetate or [13C]acetate and the glial specific enzyme inhibitors, i.e. the glutamine synthetase inhibitor methionine sulfoximine and the tricarboxylic acid cycle (aconitase) inhibitors fluoro-acetate and -citrate. Acetate is metabolized exclusively by glial cells, and [13C]acetate is thus capable when used in combination with magnetic resonance spectroscopy or mass spectrometry, to provide information about glutamine transfer. The present review will give information about glutamine trafficking and the tools used to map it as exemplified by discussions of published work employing brain cell cultures as well as intact animals. It will be documented that considerably more glutamine is transferred from astrocytes to glutamatergic than to GABAergic neurons. However, glutamine does have an important role in GABAergic neurons despite their capability of re-utilizing their neurotransmitter by re-uptake.

Characterization of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) as an inhibitor of brain glycogen shunt activity.

The pharmacological properties of 1,4‐dideoxy‐1,4‐imino‐d‐arabinitol (DAB), a potent inhibitor of glycogen phosphorylase and synthase activity in liver preparations, were characterized in different brain tissue preparations as a prerequisite for using it as a tool to investigate brain glycogen metabolism. Its inhibitory effect on glycogen phosphorylase was studied in homogenates of brain tissue and astrocytes and IC50‐values close to 400 nM were found. However, the concentration of DAB needed for inhibition of glycogen shunt activity, i.e. glucose metabolism via glycogen, in intact astrocytes was almost three orders of magnitude higher. Additionally, such complete inhibition required a pre‐incubation period, a finding possibly reflecting a limited permeability of the astrocytic membrane. DAB did not affect the accumulation of 2‐deoxyglucose‐6‐phosphate indicating that the transport of DAB is not mediated by the glucose transporter. DAB had no effect on enzymes involving glucose‐6‐phosphate, i.e. glucose‐6‐phosphate dehydrogenase, phosphoglucoisomerase and hexokinase. Furthermore, DAB was evaluated in a functional preparation of the isolated mouse optic nerve, in which its presence severely reduced the ability to sustain evoked compound action potentials in the absence of glucose, a condition in which glycogen serves as an important energy substrate. Based on the experimental findings, DAB can be used to evaluate glycogen shunt activity and its functional importance in intact brain tissue and cells at a concentration of 300–1000 μM and a pre‐incubation period of 1 h.

GAD65 is essential for synthesis of GABA destined for tonic inhibition regulating epileptiform activity

J. Neurochem. (2010) 115, 1398–1408.

Quantitative importance of the pentose phosphate pathway determined by incorporation of 13C from [2-13C]- and [3-13C]glucose into TCA cycle intermediates and neurotransmitter amino acids in functionally intact neurons

The brain is highly susceptible to oxidative injury, and the pentose phosphate pathway (PPP) has been shown to be affected by pathological conditions, such as Alzheimers disease and traumatic brain injury. While this pathway has been investigated in the intact brain and in astrocytes, little is known about the PPP in neurons. The activity of the PPP was quantified in cultured cerebral cortical and cerebellar neurons after incubation in the presence of [2-13C]glucose or [3-13C]glucose. The activity of the PPP was several fold lower than glycolysis in both types of neurons. While metabolism of 13C-labeled glucose via the PPP does not appear to contribute to the production of releasable lactate, it contributes to labeling of tricarboxylic acid (TCA) cycle intermediates and related amino acids. Based on glutamate isotopomers, it was calculated that PPP activity accounts for ∼6% of glucose metabolism in cortical neurons and ∼4% in cerebellar neurons. This is the first demonstration that pyruvate generated from glucose via the PPP contributes to the synthesis of acetyl CoA for oxidation in the TCA cycle. Moreover, the fact that 13C labeling from glucose is incorporated into glutamate proves that both the oxidative and the nonoxidative stages of the PPP are active in neurons.

Knockout of GAD65 has major impact on synaptic GABA synthesized from astrocyte-derived glutamine

γ-Aminobutyric acid (GABA) synthesis from glutamate is catalyzed by glutamate decarboxylase (GAD) of which two isoforms, GAD65 and GAD67, have been identified. The GAD65 has repeatedly been shown to be important during intensified synaptic activity. To specifically elucidate the significance of GAD65 for maintenance of the highly compartmentalized intracellular and intercellular GABA homeostasis, GAD65 knockout and corresponding wild-type mice were injected with [1-13C]glucose and the astrocyte-specific substrate [1,2-13C]acetate. Synthesis of GABA from glutamine in the GABAergic synapses was further investigated in GAD65 knockout and wild-type mice using [1,2-13C]acetate and in some cases c-vinylGABA (GVG, Vigabatrin), an inhibitor of GABA degradation. A detailed metabolic mapping was obtained by nuclear magnetic resonance (NMR) spectroscopic analysis of tissue extracts of cerebral cortex and hippocampus. The GABA content in both brain regions was reduced by ~20%. Moreover, it was revealed that GAD65 is crucial for maintenance of biosynthesis of synaptic GABA particularly by direct synthesis from astrocytic glutamine via glutamate. The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism. Furthermore, a severe neuronal hypometabolism, involving glycolysis and tricarboxylic acid (TCA) cycle activity, was observed in cerebral cortex of GAD65 knockout mice.