Expression of human GLUD2 glutamate dehydrogenase in human tissues: Functional implications

ArticleinNeurochemistry International 61(4):455-62 · June 2012with29 Reads
DOI: 10.1016/j.neuint.2012.06.007 · Source: PubMed
Glutamate dehydrogenase (GDH), a mitochondrial enzyme with a key metabolic role, exists in the human in hGDH1 and hGDH2 isoforms encoded by the GLUD1 and GLUD2 genes, respectively. It seems that GLUD1 was retroposed to the X chromosome where it gave rise to GLUD2 via random mutations and natural selection. Of these, evolutionary Gly456Ala substitution dissociated hGDH2 from GTP control, while replacement of Arg443 by Ser drastically modified basal activity, heat stability, optimal pH, allosteric regulation and migration pattern in SDS-PAGE, thus suggesting an effect on enzyme's conformation. While GLUD2-specific transcripts have been detected in human brain, retina and testis, data on the endogenous hGDH2 protein are lacking. Given the housekeeping nature of hGDH1 and its high homology to hGDH2, the specific detection of hGDH2 in tissues presents a challenge. To develop an antibody specific for hGDH2, we considered that an epitope containing the Arg443Ser change was an attractive target. We accordingly used a peptide that corresponds to residues 436-447, with Ser at position 443, to immunize rabbits and succeeded in raising a polyclonal antibody specific for hGDH2. Western blots showed that human testis contained equal amounts of hGDH2 and hGDH1 and that both isoproteins localized to the mitochondrial fraction. In human brain, however, hGDH2 expression was lower than that of hGDH1. Immuno-histochemical studies on human testis and cerebral cortex, showed punctuate, organelle-like hGDH2 immuno-labeling in sertoli cells and in astrocytes, respectively, consistent with the mitochondrial localization of the enzyme. Similar studies in kidney revealed that hGDH2 is expressed in epithelial cells of the proximal convoluted tubule. As hGDH2 can metabolize glutamate at relatively low pH without the GTP constrain, it may function efficiently under conditions of relative acidification that prevail in astrocytes following glutamate uptake. Similarly, in the kidney, hGDH2 could contribute to enhanced excretion of ammonia under acidosis.
    • "Accordingly, glutamate can be oxidized by astrocytes at higher rate than glucose, 3-hydroxy- butyrate, glutamine, lactate, or malate (McKenna, 2012). Following its uptake by astrocytes, the glutamate fraction escaping the glutamate-glutamine cycle can be converted to a-ketoglutarate, either by transamination reactions or by NADH-producing oxidation catalyzed by GDH, an enzyme abundant in astrocytes (Lovatt et al., 2007; McKenna, 2007; Zaganas et al., 2001 Zaganas et al., , 2012). The oxidative catabolism of glutamate proceeds primarily via GDH in rat astrocytes (McKenna et al., 1996; Westergaard et al., 1996). "
    [Show abstract] [Hide abstract] ABSTRACT: Glucose, the main energy substrate used in the CNS, is continuously supplied by the periphery. Glutamate, the major excitatory neurotransmitter, is foreseen as a complementary energy contributor in the brain. In particular, astrocytes actively take up glutamate and may use it through oxidative glutamate dehydrogenase (GDH) activity. Here, we investigated the significance of glutamate as energy substrate for the brain. Upon glutamate exposure, astrocytes generated ATP in a GDH-dependent way. The observed lack of glutamate oxidation in brain-specific GDH null CnsGlud1(-/-) mice resulted in a central energy-deprivation state with increased ADP/ATP ratios and phospho-AMPK in the hypothalamus. This induced changes in the autonomous nervous system balance, with increased sympathetic activity promoting hepatic glucose production and mobilization of substrates reshaping peripheral energy stores. Our data reveal the importance of glutamate as necessary energy substrate for the brain and the role of central GDH in the regulation of whole-body energy homeostasis.
    Full-text · Article · Oct 2015
    • "Müller cells possess enzymes that are involved in the de novo synthesis of glutamate from pyruvate, e.g., pyruvate carboxylase, that catalyzes the carboxylation of pyruvate to oxaloacetate as substrate of the Krebs cycle, and glutamate dehydrogenase, that converts α-ketoglutarate to glutamate (Gebhard, 1992; Ola et al., 2011a). Glutamate dehydrogenase is able to metabolize glutamate at relatively low pH (Zaganas et al., 2012) that prevails in glial cells following glutamate uptake (Bouvier et al., 1992 ). The activity of the malate-aspartate shuttle in Müller cells is low (LaNoue et al., 2001 ) due to the low expression of the aspartate aminotransferase (Gebhard, 1991) and of glutamate-aspartate exchangers (Xu et al., 2007 ). "
    [Show abstract] [Hide abstract] ABSTRACT: Müller cells, the principal glial cells of the retina, support the synaptic activity by the uptake and metabolization of extracellular neurotransmitters. Müller cells express uptake and exchange systems for various neurotransmitters including glutamate and γ-aminobutyric acid (GABA). Müller cells remove the bulk of extracellular glutamate in the inner retina and contribute to the glutamate clearance around photoreceptor terminals. By the uptake of glutamate, Müller cells are involved in the shaping and termination of the synaptic activity, particularly in the inner retina. Reactive Müller cells are neuroprotective, e.g., by the clearance of excess extracellular glutamate, but may also contribute to neuronal degeneration by a malfunctioning or even reversal of glial glutamate transporters, or by a downregulation of the key enzyme, glutamine synthetase. This review summarizes the present knowledge about the role of Müller cells in the clearance and metabolization of extracellular glutamate and GABA. Some major pathways of GABA and glutamate metabolism in Müller cells are described; these pathways are involved in the glutamate-glutamine cycle of the retina, in the defense against oxidative stress via the production of glutathione, and in the production of substrates for the neuronal energy metabolism.
    Full-text · Article · Apr 2013
    • "Whereas hGDH1 shows high levels in liver, brain and kidney, hGDH2 is found mainly in human testis, brain and kidney. In human brain, hGDH2 is expressed in astrocytes, whereas in human kidney it is expressed in the epithelial cells lining the proximal convoluted tubules (Zaganas et al. 2012). At the functional level, hGDH1 and hGDH2 differ markedly in their regulatory properties. "
    [Show abstract] [Hide abstract] ABSTRACT: Glutamate dehydrogenase (GDH) uses ammonia to reversibly convert α-ketoglutarate to glutamate using NADP(H) and NAD(H) as cofactors. While GDH in most mammals is encoded by a single GLUD1 gene, humans and other primates have acquired a GLUD2 gene with distinct tissue expression profile. The two human isoenzymes (hGDH1 and hGDH2), though highly homologous, differ markedly in their regulatory properties. Here we obtained hGDH1 and hGDH2 in recombinant form and studied their K(m) for ammonia in the presence of 1.0 mM ADP. The analyses showed that lowering the pH of the buffer (from 8.0 to 7.0) increased the K(m) for ammonia substantially (hGDH1: from 12.8 ± 1.4 mM to 57.5 ± 1.6 mM; hGDH2: from 14.7 ± 1.6 mM to 62.2 ± 1.7 mM), thus essentially precluding reductive amination. Moreover, lowering the ADP concentration to 0.1 mM not only increased the K(0.5) [NH(4) (+)] of hGDH2, but also introduced a positive cooperative binding phenomenon in this isoenzyme. Hence, intra-mitochondrial acidification, as occurring in astrocytes during glutamatergic transmission should favor the oxidative deamination of glutamate. Similar considerations apply to the handling of glutamate by the proximal convoluted tubules of the kidney during systemic acidosis. The reverse could apply for conditions of local or systemic hyperammonemia or alkalosis.
    Full-text · Article · Feb 2013
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