Astrocytes take up glutamate in the synaptic area subsequent to glutamatergic transmission by the aid of high affinity glutamate transporters. Glutamate is converted to glutamine or metabolized to support intermediary metabolism and energy production. Glutamate dehydrogenase (GDH) and aspartate aminotransferase (AAT) catalyze the reversible reaction between glutamate and α-ketoglutarate, which is the initial step for glutamate to enter TCA cycle metabolism. In contrast to GDH, AAT requires a concomitant interconversion of oxaloacetate and aspartate. We have investigated the role of GDH in astrocyte glutamate and glucose metabolism employing siRNA mediated knock down (KD) of GDH in cultured astrocytes using stable and radioactive isotopes for metabolic mapping. An increased level of aspartate was observed upon exposure to [U-(13) C]glutamate in astrocytes exhibiting reduced GDH activity. (13) C Labeling of aspartate and TCA cycle intermediates confirmed that the increased amount of aspartate is associated with elevated TCA cycle flux from α-ketoglutarate to oxaloacetate, i.e. truncated TCA cycle. (13) C Glucose metabolism was elevated in GDH deficient astrocytes as observed by increased de novo synthesis of aspartate via pyruvate carboxylation. In the absence of glucose, lactate production from glutamate via malic enzyme was lower in GDH deficient astrocytes. In conclusions, our studies reveal that metabolism via GDH serves an important anaplerotic role by adding net carbon to the TCA cycle. A reduction in GDH activity seems to cause the astrocytes to up-regulate activity in pathways involved in maintaining the amount of TCA cycle intermediates such as pyruvate carboxylation as well as utilization of alternate substrates such as branched chain amino acids.

Glutamate dehydrogenase (GDH), encoded by GLUD1, participates in the breakdown and synthesis of glutamate, the main excitatory neurotransmitter. In the CNS, besides its primary signaling function, glutamate is also at the crossroad of metabolic and neurotransmitter pathways. Importance of brain GDH was questioned here by generation of CNSspecific GDH-null mice (CnsGlud1 À/À ); which were viable, fertile and without apparent behavioral problems. GDH immunoreactivity as well as enzymatic activity were absent in CnsGlud1 À/À brains. Immunohistochemical analyses on brain sections revealed that the pyramidal cells of control animals were positive for GDH, whereas the labeling was absent in hippocampal sections of Cns-Glud1 À/À mice. Electrophysiological recordings showed that deletion of GDH within the CNS did not alter synaptic transmission in standard conditions. Cns-Glud1 À/À mice exhibited deficient oxidative catabolism of glutamate in astrocytes, showing that GDH is required for Krebs cycle pathway. As revealed by NMR studies, brain glutamate levels remained unchanged, whereas glutamine levels were increased. This pattern was favored by upregulation of astrocyte-type glutamate and glutamine transporters and of glutamine synthetase. Present data show that the lack of GDH in the CNS modifies the metabolic handling of glutamate without altering synaptic transmission. Keywords: astrocytes, central nervous system, Glud1, glutamate, glutamate dehydrogenase. J. Neurochem. (2012) 123, 342-348.Glutamate is the major excitatory neurotransmitter in mammalian CNS and is involved in mechanisms of synaptic plasticity, memory, and neuronal or glial cell death. Upon glutamatergic transmission, intersynaptic glutamate clearance is achieved mostly by astrocytes (Bak et al. 2006), preventing toxic accumulation in the extracellular space. In the astrocytes, glutamate is either amidated to glutamine via glutamine synthetase and then recycled back to neurons as part of glutamate-glutamine cycle. Alternatively, glutamate is | 2012 | 123 | 342-348 doi: 10.1111/j.1471-4159.2012.07933.x deaminated to yield a-ketoglutarate, followed by further oxidation in the tricarboxylic acid (TCA) cycle to generate ATP (Yu et al. 1982;McKenna et al. 1996). Such an essential process is commonly attributed to both glutamate dehydrogenase (GDH) and transaminases, although their relative importance has not been established yet. GDH, in rodents encoded solely by GLUD1 (Michaelidis et al. 1993), is a mitochondrial enzyme catalyzing either the anabolic reductive amination of a-ketoglutarate producing glutamate or, conversely, the catabolic oxidative deamination of glutamate (Karaca et al. 2011). In CNS, GDH is predominantly expressed in astrocytes and to lower levels in neurons (Rothe et al. 1994;Mastorodemos et al. 2005). To date, the putative importance of GDH as a player in balanced glutamateglutamine cycle activity and as a supplier of precursors for glutamatergic neurotransmission remains to be established, essentially because we lack loss-...

Müller cells are pivotal in sustaining retinal ganglion cells, and an intact energy metabolism is essential for upholding Müller cell functions. The present study aimed to investigate the impact of lactate on Müller cell survival and function. Primary mice Müller cells and human Müller cell lines (MIO-M1) were treated with or without lactate (10 or 20 mM) for 2 and 24 hours. Simultaneously, Müller cells were incubated with or without 6 mM of glucose. L-lactate exposure increased Müller cell survival independently of the presence of glucose. This effect was abolished by the addition of the monocarboxylate inhibitor 4-cinnamic acid to the treatment media, whereas survival continued to increase in response to addition of D-lactate during glucose restriction. ATP levels decreased over time in MIO-M1 cells and remained stable over time in primary Müller cells. Lactate was preferably metabolized in MIO-M1 cells compared to glucose, and 10 mM of L-Lactate exposure prevented complete glycogen depletion in MIO-M1 cells. Glutamate uptake increased after 2 hours and decreased after 24 hours in glucose-restricted Müller cells compared to cells with glucose supplement. The addition of 10 mM of lactate to the treatment media increased glutamate uptake in glucose supplemented and restricted cells. In conclusion, lactate is a key component in maintaining Müller cell survival and function. Hence, lactate administration may be of great future interest, ultimately leading to novel therapies to rescue retinal ganglion cells.

The amino acid profile and the secretory responses of glutamate dehydrogenase (GDH)-deficient β-cells are characterized. This study shows that GDH is essential for both insulin release and net glutamate synthesis evoked by glucose. Adding cellular glutamate restored the full development of glucose-stimulated insulin secretion, showing the requirement for permissive glutamate levels.

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