Macrophage-derived glutamine boosts satellite cells and muscle regeneration.

Muscle regeneration is sustained by infiltrating macrophages and the consequent activation of satellite cells1-4. Macrophages and satellite cells communicate in different ways1-5, but their metabolic interplay has not been investigated. Here we show, in a mouse model, that muscle injuries and ageing are characterized by intra-tissue restrictions of glutamine. Low levels of glutamine endow macrophages with the metabolic ability to secrete glutamine via enhanced glutamine synthetase (GS) activity, at the expense of glutamine oxidation mediated by glutamate dehydrogenase 1 (GLUD1). Glud1-knockout macrophages display constitutively high GS activity, which prevents glutamine shortages. The uptake of macrophage-derived glutamine by satellite cells through the glutamine transporter SLC1A5 activates mTOR and promotes the proliferation and differentiation of satellite cells. Consequently, macrophage-specific deletion or pharmacological inhibition of GLUD1 improves muscle regeneration and functional recovery in response to acute injury, ischaemia or ageing. Conversely, SLC1A5 blockade in satellite cells or GS inactivation in macrophages negatively affects satellite cell functions and muscle regeneration. These results highlight the metabolic crosstalk between satellite cells and macrophages, in which macrophage-derived glutamine sustains the functions of satellite cells. Thus, the targeting of GLUD1 may offer therapeutic opportunities for the regeneration of injured or aged muscles.

Glucose replaces glutamate as energy substrate to fuel glutamate uptake in glutamate dehydrogenase-deficient astrocytes.

Cultured astrocytes treated with siRNA to knock down glutamate dehydrogenase (GDH) were used to investigate whether this enzyme is important for the utilization of glutamate as an energy substrate. By incubation of these cells in media containing different concentrations of glutamate (range 100-500 µM) in the presence or in the absence of glucose, the metabolism of these substrates was studied by using tritiated glutamate or 2-deoxyglucose as tracers. In addition, the cellular contents of glutamate and ATP were determined. The astrocytes were able to maintain physiological levels of ATP regardless of the expression level of GDH and the incubation condition, indicating a high degree of flexibility with regard to regulatory mechanisms involved in maintaining an adequate energy level in the cells. Glutamate uptake was found to be increased in these cells when exposed to increasing levels of extracellular glutamate independently of the GDH expression level. Moreover, increased intracellular glutamate content was observed in the GDH-deficient cells after a 2-hr incubation in the presence of 100 µM glutamate. It is significant that GDH-deficient cells exhibited an increased utilization of glucose in the presence of 250 and 500 µM glutamate, monitored as an increase in the accumulation of tritiated 2-deoxyglucose-6-phosphate. These findings underscore the importance of the expression level of GDH for the ability to utilize glutamate as an energy source fueling its own energy-requiring uptake.

Supplement of TCA cycle intermediates protects against high glucose/palmitate-induced INS-1 beta cell death.

The aim of this study is to investigate the effect of mitochondrial metabolism on high glucose/palmitate (HG/PA)-induced INS-1 beta cell death. Long-term treatment of INS-1 cells with HG/PA impaired energy-producing metabolism accompanying with depletion of TCA cycle intermediates. Whereas an inhibitor of carnitine palmitoyl transferase 1 augmented HG/PA-induced INS-1 cell death, stimulators of fatty acid oxidation protected the cells against the HG/PA-induced death. Furthermore, whereas mitochondrial pyruvate carboxylase inhibitor phenylacetic acid augmented HG/PA-induced INS-1 cell death, supplementation of TCA cycle metabolites including leucine/glutamine, methyl succinate/α-ketoisocaproic acid, dimethyl malate, and valeric acid or treatment with a glutamate dehydrogenase activator, aminobicyclo-heptane-2-carboxylic acid (BCH), significantly protected the cells against the HG/PA-induced death. In particular, the mitochondrial tricarboxylate carrier inhibitor, benzene tricarboxylate (BTA), also showed a strong protective effect on the HG/PA-induced INS-1 cell death. Knockdown of glutamate dehydrogenase or tricarboxylate carrier augmented or reduced the HG/PA-induced INS-1 cell death, respectively. Both BCH and BTA restored HG/PA-induced reduction of energy metabolism as well as depletion of TCA intermediates. These data suggest that depletion of the TCA cycle intermediate pool and impaired energy-producing metabolism may play a role in HG/PA-induced cytotoxicity to beta cells and thus, HG/PA-induced beta cell glucolipotoxicity can be protected by nutritional or pharmacological maneuver enhancing anaplerosis or reducing cataplerosis.

Deletion of glutamate dehydrogenase in beta-cells abolishes part of the insulin secretory response not required for glucose homeostasis.

Insulin exocytosis is regulated in pancreatic ss-cells by a cascade of intracellular signals translating glucose levels into corresponding secretory responses. The mitochondrial enzyme glutamate dehydrogenase (GDH) is regarded as a major player in this process, although its abrogation has not been tested yet in animal models. Here, we generated transgenic mice, named betaGlud1(-/-), with ss-cell-specific GDH deletion. Our results show that GDH plays an essential role in the full development of the insulin secretory response. In situ pancreatic perfusion revealed that glucose-stimulated insulin secretion was reduced by 37% in betaGlud1(-/-). Furthermore, isolated islets with either constitutive or acute adenovirus-mediated knock-out of GDH showed a 49 and 38% reduction in glucose-induced insulin release, respectively. Adenovirus-mediated re-expression of GDH in betaGlud1(-/-) islets fully restored glucose-induced insulin release. Thus, GDH appears to account for about 40% of glucose-stimulated insulin secretion and to lack redundant mechanisms. In betaGlud1(-/-) mice, the reduced secretory capacity resulted in lower plasma insulin levels in response to both feeding and glucose load, while body weight gain was preserved. The results demonstrate that GDH is essential for the full development of the secretory response in beta-cells. However, maximal secretory capacity is not required for maintenance of glucose homeostasis in normo-caloric conditions.

Small-interfering-RNA-mediated silencing of human glutamate dehydrogenase induces apoptosis in neuroblastoma cells.

In the nervous system, GDH (glutamate dehydrogenase) is enriched in astrocytes and is important for recycling glutamate, a major excitatory neurotransmitter. The function of hGDH (human GDH) may be important in neurodegenerative diseases such as Parkinson's disease. To test the effect of decreased hGDH expression, several vector-based plasmidlinked hGDH siRNAs (small interfering RNAs) were expressed intracellularly in BE(2)C human neuroblastoma cells. Immunoblotting and reverse-transcription-PCR confirmed that expression of hGDH protein and mRNA was knocked down by co-transfection with phGDH-siRNA vectors in BE(2)C human neuroblastoma cells. TUNEL (terminal uridine deoxynucleotidyl transferase dUTP nick-end labelling) and DNA fragmentation assays 48 h after transfection of phGDH-siRNAs revealed that inhibition of hGDH expression induced cellular apoptosis and activated phospho-ERK1/2 (phospho-extracellular-signal-regulated kinase 1/2). These findings show that inhibition of hGDH expression by siRNA is related to apoptosis in neuronal cells.

Control of the synthesis and subcellular targeting of the two GDH genes products in leaves and stems of Nicotiana plumbaginifolia and Arabidopsis thaliana.

Although the physiological role of the enzyme glutamate dehydrogenase which catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate remains to be elucidated, it is now well established that in higher plants the enzyme preferentially occurs in the mitochondria of phloem companion cells. The Nicotiana plumbaginifolia and Arabidopis thaliana enzyme is encoded by two distinct genes encoding either an alpha- or a beta-subunit. Using antisense plants and mutants impaired in the expression of either of the two genes, we showed that in leaves and stems both the alpha- and beta-subunits are targeted to the mitochondria of the companion cells. In addition, we found in both species that there is a compensatory mechanism up-regulating the expression of the alpha-subunit in the stems when the expression of the beta-subunit is impaired in the leaves, and of the beta-subunit in the leaves when the expression of the alpha-subunit is impaired in the stems. When one of the two genes encoding glutamate dehydrogenase is ectopically expressed, the corresponding protein is targeted to the mitochondria of both leaf and stem parenchyma cells and its production is increased in the companion cells. These results are discussed in relation to the possible signalling and/or physiological function of the enzyme which appears to be coordinated in leaves and stems.

Prominent glutamine oxidation activity in mitochondria of avian transplantable hepatoma induced by MC-29 virus.

Well coupled mitochondria were isolated from transplantable chicken hepatoma induced by MC-29 virus. The mitochondrial phosphate-dependent and phosphate-independent glutaminase activities were increased compared with those from normal chicken liver. Glutamate dehydrogenase was undetectable in the tumor mitochondria. Oxypolarographic tests showed the following: glutamine oxidation was prominent in the tumor mitochondria and was mediated through an NAD-linked reaction, while mitochondria from the liver showed a feeble glutamine oxidation; glutamine oxidation by tumor mitochondria was inhibited either by aminooxyacetate, inhibitor of transaminases, or prior incubation of mitochondria with DON (6-diazo-5-oxonorleucine), which inhibited mitochondrial glutaminases. Bromofuroate, inhibitor of glutamate dehydrogenase, had little or no effect; and glutamate oxidation was also inhibited by aminooxyacetate, while it was not affected by DON. These findings clearly show a high glutamate oxidation activity in the hepatoma and indicate that the product of glutamine hydrolysis, glutamate, is catabolized via transamination in the mitochondria to supply ATP.

Glutamate dehydrogenase deficiency in three patients with spinocerebellar syndrome.

Four nicotinamide-adenine dinucleotide phosphate-requiring enzymes were measured in disrupted cultured skin fibroblasts from a 19-year-old patient with juvenile onset of a spinocerebellar and extrapyramidal syndrome. There was marked reduction in the activity of glutamate dehydrogenase (GDH) (22% of mean control activity); GDH activity was also decreased in homogenates of leukocytes from this patient (38% of mean control activity). GDH activity was measured in the leukocytes of two siblings afflicted with adult-onset spinocerebellar syndrome and found to be decreased in both (29% and 31% of mean control activity); an unaffected sibling had normal GDH activity. Mixing experiments with control fibroblast and leukocyte homogenates did not show the presence of a GDH inhibitor in cells from these patients. This allosterically regulated enzyme was stimulated by adenosine 5'-diphosphate (10(-3) M) and inhibited by guanosine 5'-triphosphate (10(-3) M) in both fibroblast and leukocyte homogenates; these changes occurred in equal proportions in the patients and controls. The decreased fibroblast and leukocyte GDH activity persisted at different concentrations of the enzyme's substrates and with successive passages of cultured fibroblasts. GDH may have an important role in the metabolism of glutamate, a putative neurotransmitter in cerebellum, brainstem, and spinal cord. A genetic deficiency of GDH may underlie some forms of spinocerebellar ataxias.