glutaminase and Hypoxia

To meet the demand for energy and biomass, T lymphocytes (T cells) activated to proliferation and clonal expansion, require uptake and metabolism of glucose (Gluc) and the amino acid (AA) glutamine (Gln). Whereas exogenous Gln is converted to glutamate (Glu) by glutaminase (GLS), Gln is also synthesized from the endogenous pool of AA through Glu and activity of glutamine synthase (GS). Most of this knowledge comes from studies on cell cultures under ambient oxygen conditions (normoxia, 21% O2). However, in vivo, antigen induced T-cell activation often occurs under moderately hypoxic (1-4% O2) conditions and at various levels of exogenous nutrients. Here, CD4+ T cells were stimulated for 72 h with antibodies targeting the CD3 and CD28 markers at normoxia and hypoxia (1% O2). This was done in the presence and absence of the GLS and GS inhibitors, Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES) and methionine sulfoximine (MSO) and at various combinations of exogenous Gluc, Gln and pyruvate (Pyr) for the last 12 h of stimulation. We found that T-cell proliferation, viability and levels of endogenous AA were significantly influenced by the availability of exogenous Gln, Gluc and Pyr as well as inhibition of GLS and GS. Moreover, inhibition of GLS and GS and levels of oxygen differentially influenced oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Finally, BPTES-dependent down-regulation of ECAR was associated with reduced hexokinase (HK) activity at both normoxia and hypoxia. Our results demonstrate that Gln availability and metabolism is rate-limiting for CD4+ T-cell activity.

Topics: Amino Acids; CD28 Antigens; CD3 Complex; CD4-Positive T-Lymphocytes; Cell Proliferation; Glucose; Glutamic Acid; Glutaminase; Glutamine; Humans; Hypoxia; Oxygen; Pyruvic Acid

The sex-determining region Y (SRY)-box (SOX) family has a crucial role in carcinogenesis and cancer progression. However, the role of SOX12 and the mechanism by which it is dysregulated in colorectal cancer (CRC) remain unclear. Here we analyzed SOX12 expression patterns in two independent CRC cohorts (cohort I, n = 390; cohort II, n = 363) and found that SOX12 was significantly upregulated in CRC, indicating a poor prognosis in CRC patients. Overexpression of SOX12 promoted CRC cell proliferation and metastasis, whereas downregulation of SOX12 hampered CRC aggressiveness. Mechanistically, SOX12 facilitated asparagine synthesis by transactivating glutaminase (GLS), glutamic oxaloacetic transaminase 2 (GOT2), and asparagine synthetase (ASNS). Downregulation of GLS, GOT2, and ASNS blocked SOX12-mediated CRC cell proliferation and metastasis, whereas ectopic expression of GLS, GOT2, and ASNS attenuated the SOX12 knockdown-induced suppression of CRC progression. In addition, serial deletion, site-directed mutagenesis, luciferase reporter, and chromatin immunoprecipitation (ChIP) assays indicated that hypoxia-inducible factor 1α (HIF-1α) directly binds to the SOX12 promoter and induces SOX12 expression. Administration of L-asparaginase decreased SOX12-mediated tumor growth and metastasis. In human CRC samples, SOX12 expression positively correlated with GLS, GOT2, ASNS, and HIF-1α expression. Based on these results, SOX12 may serve as a prognostic biomarker and L-asparaginase represents a potential novel therapeutic agent for CRC.

Topics: Animals; Asparaginase; Asparagine; Aspartate-Ammonia Ligase; Biomarkers, Tumor; Caco-2 Cells; Cell Movement; Cell Proliferation; Cohort Studies; Colorectal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Glutaminase; HCT116 Cells; HT29 Cells; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Prognosis; SOXC Transcription Factors; Transaminases; Transplantation, Heterologous; Up-Regulation

We studied the effect of hypoxia (days 9-15 of pregnancy) on phosphate-dependent glutaminase activity in the brain of rat offspring aging 18 days and 1, 3, and 6 months. Activity of glutaminase significantly increased in mitochondria from the orbital, visual, and limbic cortex, hypothalamus, and midbrain of 17-day-old offspring. Activity of this enzyme in all brain regions increased at the age of 1 month, but significantly decreased in animals aging 3 and 6 months and prenatally exposed to hypoxia (during organogenesis). Changes in glutaminase activity in various structures of the brain are probably associated with activation of the glutamatergic and GABAergic systems and serve as an adaptive and compensatory reaction of the brain.

Topics: Animals; Brain; Female; Glutaminase; Hypoxia; Male; Organogenesis; Phosphates; Rats; Rats, Wistar

It has been assumed that oxidative phosphorylation (OxPhos) in solid tumors is severely reduced due to cytochrome c oxidase substrate restriction, although the measured extracellular oxygen concentration in hypoxic areas seems not limiting for this activity. To identify alternative hypoxia-induced OxPhos depressing mechanisms, an integral analysis of transcription, translation, enzyme activities and pathway fluxes was performed on glycolysis and OxPhos in HeLa and MCF-7 carcinomas. In both neoplasias exposed to hypoxia, an early transcriptional response was observed after 8h (two times increased glycolysis-related mRNA synthesis promoted by increased HIF-1alpha levels). However, major metabolic remodeling was observed only after 24h hypoxia: increased glycolytic protein content (1-5-times), enzyme activities (2-times) and fluxes (4-6-times). Interestingly, in MCF-7 cells, 24h hypoxia decreased OxPhos flux (4-6-fold), and 2-oxoglutarate dehydrogenase and glutaminase activities (3-fold), with no changes in respiratory complexes I and IV activities. In contrast, 24h hypoxia did not significantly affect HeLa OxPhos flux; neither mitochondria related mRNAs, protein contents or enzyme activities, although the enhanced glycolysis became the main ATP supplier. Thus, prolonged hypoxia (a) targeted some mitochondrial enzymes in MCF-7 but not in HeLa cells, and (b) induced a transition from mitochondrial towards a glycolytic-dependent energy metabolism in both MCF-7 and HeLa carcinomas.

Topics: Breast Neoplasms; Carcinoma; Electron Transport Complex I; Energy Metabolism; Female; Glutaminase; Glycolysis; HeLa Cells; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Ketoglutarate Dehydrogenase Complex; Mitochondria; Oxidative Phosphorylation; Uterine Cervical Neoplasms

Exposure to high altitude causes loss of body mass and alterations in metabolic processes, especially carbohydrate and protein metabolism. The present study was conducted to elucidate the role of glutamine synthetase, glutaminase and glycogen synthetase under conditions of chronic intermittent hypoxia. Four groups, each consisting of 12 male albino rats (Wistar strain), were exposed to a simulated altitude of 7620 m in a hypobaric chamber for 6 h per day for 1, 7, 14 and 21 days, respectively. Blood haemoglobin, blood glucose, protein levels in the liver, muscle and plasma, glycogen content, and glutaminase, glutamine synthetase and glycogen synthetase activities in liver and muscle were determined in all groups of exposed and in a group of unexposed animals. Food intake and changes in body mass were also monitored. There was a significant reduction in body mass (28-30%) in hypoxia-exposed groups as compared to controls, with a corresponding decrease in food intake. There was rise in blood haemoglobin and plasma protein in response to acclimatization. Over a three-fold increase in liver glycogen content was observed following 1 day of hypoxic exposure (4.76 +/- 0.78 mg.g-1 wet tissue in normal unexposed rats; 15.82 +/- 2.30 mg.g-1 wet tissue in rats exposed to hypoxia for 1 day). This returned to normal in later stages of exposure. However, there was no change in glycogen synthetase activity except for a decrease in the 21-days hypoxia-exposed group. There was a slight increase in muscle glycogen content in the 1-day exposed group which declined significantly by 56.5, 50.6 and 42% following 7, 14, and 21 days of exposure, respectively. Muscle glycogen synthetase activity was also decreased following 21 days of exposure. There was an increase in glutaminase activity in the liver and muscle in the 7-, 14- and 21-day exposed groups. Glutamine synthetase activity was higher in the liver in 7- and 14-day exposed groups; this returned to normal following 21 days of exposure. Glutamine synthetase activity in muscle was significantly higher in the 14-day exposed group (4.32 mumol gamma-glutamyl hydroxamate formed.g protein-1.min-1) in comparison to normal (1.53 mumol gamma-glutamyl hydroxamate formed.g protein-1.min-1); this parameter had decreased by 40% following 21 days of exposure. These results suggest that since no dramatic changes in the levels of protein were observed in the muscle and liver, there is an alteration in glutaminase and glutamine synthetase

Topics: Albinism; Altitude; Altitude Sickness; Animals; Glutamate-Ammonia Ligase; Glutaminase; Glycogen Synthase; Hypoxia; Liver; Male; Rats; Rats, Wistar

Topics: Acyltransferases; Amidohydrolases; Ammonia; Animals; Asparaginase; Brain; Cats; Central Nervous System; Dogs; Energy Metabolism; Enzymes; Epilepsy; Glutamate Dehydrogenase; Glutaminase; Hepatic Encephalopathy; Humans; Hypercapnia; Hypoxia; Mice; Rats; Synapses; Transglutaminases; Urea

Topics: Acidosis; Ammonia; Animals; Gluconeogenesis; Glutamate Dehydrogenase; Glutaminase; Hypoxia; Kidney; Male; Phosphoenolpyruvate Carboxylase; Phosphoric Monoester Hydrolases; Rats

The effect of hypobaric hypoxia on the activities of glutamine synthetase, glutaminase and cyclic 3'5' AMP phosphodiesterase in rat brain was studied after exposure to 25,000' for 6 h. Glutamine synthetase activity was increased in all the regions of brain studied, and addition of gamma amino butyric acid, serotonin and cortisol in vitro produced a differential response. Glutaminase activity decreased in the whole brain. Cyclic 3'5' AMP phosphodiesterase activity decreased in cerebellum, medulla, hypothalamus and pituitary showing an accumulation of cyclic 3'5' AMP in these regions. The results suggest that glutamine synthesis and degradation are regulated in the central nervous system by cyclic AMP and cortisol: Gamma aminoburyric acid and other compounds can modulate the activity of glutamine synthetase and glutaminase.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Aminobutyrates; Animals; Brain; Cerebellum; Cerebral Cortex; gamma-Aminobutyric Acid; Glutamate-Ammonia Ligase; Glutaminase; Hydrocortisone; Hypoxia; Male; Medulla Oblongata; Phosphoric Diester Hydrolases; Rats; Serotonin