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Nature Oct 2013Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is an enzyme with important regulatory functions in the heart and brain, and its chronic activation can be...
Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is an enzyme with important regulatory functions in the heart and brain, and its chronic activation can be pathological. CaMKII activation is seen in heart failure, and can directly induce pathological changes in ion channels, Ca(2+) handling and gene transcription. Here, in human, rat and mouse, we identify a novel mechanism linking CaMKII and hyperglycaemic signalling in diabetes mellitus, which is a key risk factor for heart and neurodegenerative diseases. Acute hyperglycaemia causes covalent modification of CaMKII by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAc modification of CaMKII at Ser 279 activates CaMKII autonomously, creating molecular memory even after Ca(2+) concentration declines. O-GlcNAc-modified CaMKII is increased in the heart and brain of diabetic humans and rats. In cardiomyocytes, increased glucose concentration significantly enhances CaMKII-dependent activation of spontaneous sarcoplasmic reticulum Ca(2+) release events that can contribute to cardiac mechanical dysfunction and arrhythmias. These effects were prevented by pharmacological inhibition of O-GlcNAc signalling or genetic ablation of CaMKIIδ. In intact perfused hearts, arrhythmias were aggravated by increased glucose concentration through O-GlcNAc- and CaMKII-dependent pathways. In diabetic animals, acute blockade of O-GlcNAc inhibited arrhythmogenesis. Thus, O-GlcNAc modification of CaMKII is a novel signalling event in pathways that may contribute critically to cardiac and neuronal pathophysiology in diabetes and other diseases.
Topics: Acetylglucosamine; Animals; Arrhythmias, Cardiac; Benzylamines; Brain; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Diabetes Complications; Diazooxonorleucine; Enzyme Activation; Glucose; Glycosylation; Humans; Hyperglycemia; Mice; Myocardium; Myocytes, Cardiac; Rats; Sarcoplasmic Reticulum; Sulfonamides
PubMed: 24077098
DOI: 10.1038/nature12537 -
Life Sciences Jun 2014Although acute hyperglycemic (AHG) episodes are linked to lower glucose uptake, underlying mechanisms remain unclear. We hypothesized that AHG triggers reactive oxygen...
AIMS
Although acute hyperglycemic (AHG) episodes are linked to lower glucose uptake, underlying mechanisms remain unclear. We hypothesized that AHG triggers reactive oxygen species (ROS) production and increases non-oxidative glucose pathway (NOGP) activation, i.e. stimulation of advanced glycation end products (AGE), polyol pathway (PP), hexosamine biosynthetic pathway (HBP), PKC; thereby decreasing cardiac glucose uptake.
MAIN METHODS
H9c2 cardiomyoblasts were exposed to 25 mM glucose for 24h vs. 5.5mM controls ± modulating agents during the last hour of glucose exposure: a) antioxidant #1 for mitochondrial ROS (250 μM 4-OHCA), b) antioxidant #2 for NADPH oxidase-generated ROS (100 μM DPI), c) NOGP inhibitors - 100 μM aminoguanidine (AGE), 5 μM chelerythrine (PKC); 40 μM DON (HBP); and 10 μM zopolrestat (PP). ROS levels (mitochondrial, intracellular) and glucose uptake were evaluated by flow cytometry.
KEY FINDINGS
AHG elevated ROS, activated NOGPs and blunted glucose uptake. Transketolase activity (pentose phosphate pathway [PPP] marker) did not change. Respective 4-OHCA and DPI treatment blunted ROS production, diminished NOGP activation and normalized glucose uptake. NOGP inhibitory studies identified PKCβII as a key downstream player in lowering insulin-mediated glucose uptake. When we employed an agent (benfotiamine) known to shunt flux away from NOGPs (into PPP), it decreased ROS generation and NOGP activation, and restored glucose uptake under AHG conditions.
SIGNIFICANCE
This study demonstrates that AHG elicits maladaptive events that function in tandem to reduce glucose uptake, and that antioxidant treatment and/or attenuation of NOGP activation (PKC, polyol pathway) may limit the onset of insulin resistance.
Topics: Analysis of Variance; Animals; Benzophenanthridines; Benzothiazoles; Cell Line; Coumaric Acids; Diazooxonorleucine; Flow Cytometry; Glucose; Glycation End Products, Advanced; Guanidines; Hexosamines; Hyperglycemia; Metabolic Networks and Pathways; Models, Biological; Myocardium; Myocytes, Cardiac; Onium Compounds; Phthalazines; Polymers; Rats; Reactive Oxygen Species
PubMed: 24747137
DOI: 10.1016/j.lfs.2014.04.009 -
Antibiotics & Chemotherapy (Northfield,... Aug 1956
Topics: Bacteria; Biological Products; Diazooxonorleucine; Fungi; Humans; Leucine; Neoplasms
PubMed: 24544086
DOI: No ID Found -
Clinical Cancer Research : An Official... Oct 2019Atypical teratoid/rhabdoid tumors (AT/RT) are aggressive infantile brain tumors with poor survival. Recent advancements have highlighted significant molecular...
PURPOSE
Atypical teratoid/rhabdoid tumors (AT/RT) are aggressive infantile brain tumors with poor survival. Recent advancements have highlighted significant molecular heterogeneity in AT/RT with an aggressive subgroup featuring overexpression of the proto-oncogene. We perform the first comprehensive metabolic profiling of patient-derived AT/RT cell lines to identify therapeutic susceptibilities in high MYC-expressing AT/RT.
EXPERIMENTAL DESIGN
Metabolites were extracted from AT/RT cell lines and separated in ultra-high performance liquid chromatography mass spectrometry. Glutamine metabolic inhibition with 6-diazo-5-oxo-L-norleucine (DON) was tested with growth and cell death assays and survival studies in orthotopic mouse models of AT/RT. Metabolic flux analysis was completed to identify combination therapies to act synergistically to improve survival in high MYC AT/RT.
RESULTS
Unbiased metabolic profiling of AT/RT cell models identified a unique dependence of high MYC AT/RT on glutamine for survival. The glutamine analogue, DON, selectively targeted high MYC cell lines, slowing cell growth, inducing apoptosis, and extending survival in orthotopic mouse models of AT/RT. Metabolic flux experiments with isotopically labeled glutamine revealed DON inhibition of glutathione (GSH) synthesis. DON combined with carboplatin further slowed cell growth, induced apoptosis, and extended survival in orthotopic mouse models of high MYC AT/RT.
CONCLUSIONS
Unbiased metabolic profiling of AT/RT identified susceptibility of high MYC AT/RT to glutamine metabolic inhibition with DON therapy. DON inhibited glutamine-dependent synthesis of GSH and synergized with carboplatin to extend survival in high MYC AT/RT. These findings can rapidly translate into new clinical trials to improve survival in high MYC AT/RT.
Topics: Animals; Antimetabolites, Antineoplastic; Apoptosis; Cell Proliferation; Diazooxonorleucine; Female; Glutamine; Humans; Metabolome; Mice; Mice, Nude; Proto-Oncogene Mas; Proto-Oncogene Proteins c-myc; Rhabdoid Tumor; Teratoma; Tumor Cells, Cultured; Xenograft Model Antitumor Assays
PubMed: 31300448
DOI: 10.1158/1078-0432.CCR-19-0189 -
International Journal of Molecular... May 2021Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to the development of atherosclerosis and restenosis. Glycolysis and...
Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to the development of atherosclerosis and restenosis. Glycolysis and glutaminolysis are increased in rapidly proliferating VSMCs to support their increased energy requirements and biomass production. Thus, it is essential to develop new pharmacological tools that regulate metabolic reprogramming in VSMCs for treatment of atherosclerosis. The effects of 6-diazo-5-oxo-L-norleucine (DON), a glutamine antagonist, have been broadly investigated in highly proliferative cells; however, it is unclear whether DON inhibits proliferation of VSMCs and neointima formation. Here, we investigated the effects of DON on neointima formation in vivo as well as proliferation and migration of VSMCs in vitro. DON simultaneously inhibited FBS- or PDGF-stimulated glycolysis and glutaminolysis as well as mammalian target of rapamycin complex I activity in growth factor-stimulated VSMCs, and thereby suppressed their proliferation and migration. Furthermore, a DON-derived prodrug, named JHU-083, significantly attenuated carotid artery ligation-induced neointima formation in mice. Our results suggest that treatment with a glutamine antagonist is a promising approach to prevent progression of atherosclerosis and restenosis.
Topics: Animals; Antimetabolites, Antineoplastic; Cell Cycle; Cell Movement; Cell Proliferation; Cells, Cultured; Diazooxonorleucine; Glutamine; Glycolysis; Immunohistochemistry; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Mitochondria; Muscle, Smooth, Vascular; Neointima; Oxidative Phosphorylation; Platelet-Derived Growth Factor; Rats; Rats, Sprague-Dawley; Serum Albumin, Bovine
PubMed: 34070527
DOI: 10.3390/ijms22115602 -
Molecular Carcinogenesis Jun 2019Platinum anticancer agents are essential components in chemotherapeutic regimens for non-small-cell lung cancer (NSCLC) patients ineligible for targeted therapy....
Platinum anticancer agents are essential components in chemotherapeutic regimens for non-small-cell lung cancer (NSCLC) patients ineligible for targeted therapy. However, platinum-based regimens have reached a plateau of therapeutic efficacy; therefore, it is critical to implement novel approaches for improvement. The hexosamine biosynthesis pathway (HBP), which produces amino-sugar N-acetyl-glucosamine for protein glycosylation, is important for protein function and cell survival. Here we show a beneficial effect by the combination of cisplatin with HBP inhibition. Expression of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme of HBP, was increased in NSCLC cell lines and tissues. Pharmacological inhibition of GFAT activity or knockdown of GFATimpaired cell proliferation and exerted synergistic or additive cytotoxicity to the cells treated with cisplatin. Mechanistically, GFAT positively regulated the expression of binding immunoglobulin protein (BiP; also known as glucose-regulated protein 78, GRP78), an endoplasmic reticulum chaperone involved in unfolded protein response (UPR). Suppressing GFAT activity resulted in downregulation of BiP that activated inositol-requiring enzyme 1α, a sensor protein of UPR, and exacerbated cisplatin-induced cell apoptosis. These data identify GFAT-mediated HBP as a target for improving platinum-based chemotherapy for NSCLC.
Topics: A549 Cells; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Diazooxonorleucine; Down-Regulation; Drug Synergism; Endoplasmic Reticulum Chaperone BiP; Gene Expression Regulation, Neoplastic; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); Heat-Shock Proteins; Hexosamines; Humans; Lung Neoplasms
PubMed: 30790354
DOI: 10.1002/mc.22992 -
The Journal of Biological Chemistry Sep 1983Reaction of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase with 6-diazo-5-oxo-L-norleucine resulted in complete loss of its ability to catalyze...
Reaction of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase with 6-diazo-5-oxo-L-norleucine resulted in complete loss of its ability to catalyze glutamine-dependent phosphoribosylamine formation and its glutaminase activity, whereas its ability to catalyze ammonia-dependent phosphoribosylamine formation and to hydrolyze phosphoribosylpyrophosphate was increased. The site of reaction with 6-diazo-5-oxo-L-norleucine was the NH2-terminal cysteine residue. The NH2-terminal sequence of the B. subtilis enzyme was homologous with that of the corresponding amidotransferase from Escherichia coli, for which the NH2-terminal cysteine is also essential for glutamine utilization (Tso, J. Y., Hermodson, M. A., and Zalkin, H. (1982) J. Biol. Chem. 257, 3532-3536). The fact that the metal-free E. coli amidotransferase contains a glutamine-utilizing structure that is very similar to that found in B. subtilis amidotransferase, which contains an essential [4Fe-4S] center, indicates that the iron-sulfur center probably plays no role in glutamine utilization.
Topics: Amidophosphoribosyltransferase; Amino Acid Sequence; Bacillus subtilis; Binding Sites; Diazooxonorleucine; Glutamine; Pentosyltransferases
PubMed: 6411716
DOI: No ID Found -
Scientific Reports Jan 2014Glutaminase is a metabolic enzyme responsible for glutaminolysis, a process harnessed by cancer cells to feed their accelerated growth and proliferation. Among the...
Glutaminase is a metabolic enzyme responsible for glutaminolysis, a process harnessed by cancer cells to feed their accelerated growth and proliferation. Among the glutaminase isoforms, human kidney-type glutaminase (KGA) is often upregulated in cancer and is thus touted as an attractive drug target. Here we report the active site inhibition mechanism of KGA through the crystal structure of the catalytic domain of KGA (cKGA) in complex with 6-diazo-5-oxo-L-norleucine (DON), a substrate analogue of glutamine. DON covalently binds with the active site Ser286 and interacts with residues such as Tyr249, Asn335, Glu381, Asn388, Tyr414, Tyr466 and Val484. The nucleophilic attack of Ser286 sidechain on DON releases the diazo group (N2) from the inhibitor and results in the formation of an enzyme-inhibitor complex. Mutational studies confirmed the key role of these residues in the activity of KGA. This study will be important in the development of KGA active site inhibitors for therapeutic interventions.
Topics: Binding Sites; Catalytic Domain; Crystallography, X-Ray; Diazooxonorleucine; Glutaminase; Glutamine; Humans; Kidney; Kinetics; Models, Molecular; Mutation; Protein Binding; Protein Conformation; Substrate Specificity
PubMed: 24451979
DOI: 10.1038/srep03827 -
Differentiation; Research in Biological... 1980Adult rat adrenal cortical cells maintained in medium supplemented with horse serum (HS) from cohesive epithelial islands secrete large amounts of corticosterone. Such...
Adult rat adrenal cortical cells maintained in medium supplemented with horse serum (HS) from cohesive epithelial islands secrete large amounts of corticosterone. Such cells do not produce detectable extracellular material (ECM) and are not motile. Cultures exposed to fetal calf serum supplements (FCS) produce metachromatic ECM, modulate to a fibroblastic morphology, and become motile. Within 24 h, steroid production by these cells drop 100-fold. Cells now resemble myofibroblastic "stem" cells of the adrenal cortical capsule, and express structural and functional bimorphism by exhibiting a myofibroblastic phenotype while retaining responsiveness to adrenocorticotropic hormone (ACTH) and limited corticosteroid secreting capacity. Exposure of the myofibroblastic cells to ACTH in FCS overrides the effect of FSC: ECM disappears, steroid production increases several fold, and cells develop an epithelial morphology. The possibility that ECM produced in response to FCS may be responsible for the alteration from a highly differentiated, non-motile adrenocortical cell to a less differentiated, motile adrenocortical stem cell was investigated by inhibition studies using 6-diazo-5-oxo-L-nor-leucine (DON) and by exogenously added components of ECM. DON, a glutamine analogue, inhibited the synthesis of metachromatic ECM in FCS, and prevented the modulation to a fibroblastic morphology, onset of motility, and decrease in steroid production. Addition of hyaluronic acid, but not of chondroitin sulfate, to the epithelioid secretory cells promoted a drop in steroid production and slight alteration in morphology and movement. Both results are consistent with the possibility that metachromatic ECM production is responsible for the reversion of the steroid secretory to the myofibroblastic phenotype. This effect was mimicked by maintaining cells on polystyrene surfaces that were sulfonated to a negative charge density similar to that of ECM. This result implies that the negative charge of ECM may contribute to the expression of the adrenocortical stem cell phenotype, and that its effect is extracellular. A possible physiologic role for ECM-mediated control of adrenal cortical differentiation is proposed.
Topics: Adrenal Cortex; Adrenocorticotropic Hormone; Animals; Cell Differentiation; Cells, Cultured; Chondroitin Sulfates; Corticosterone; Culture Media; Diazooxonorleucine; Fetal Blood; Hyaluronic Acid; Rats; Rats, Inbred F344
PubMed: 6256247
DOI: 10.1111/j.1432-0436.1980.tb01085.x -
Neurochemistry International 2006Brain edema and the subsequent increase in intracranial pressure are the major neurological complications in fulminant hepatic failure (FHF). Brain edema in FHF is...
Brain edema and the subsequent increase in intracranial pressure are the major neurological complications in fulminant hepatic failure (FHF). Brain edema in FHF is predominantly "cytotoxic" due principally to astrocyte swelling. It is generally believed that ammonia plays a key role in this process, although the mechanism by which ammonia brings about such swelling is yet to be defined. It has been postulated that glutamine accumulation in astrocytes subsequent to ammonia detoxification results in increased osmotic forces leading to cell swelling. While the hypothesis is plausible and has gained support, it has never been critically tested. In this study, we examined whether a correlation exists between cellular glutamine levels and the degree of cell swelling in cultured astrocytes exposed to ammonia. Cultured astrocytes derived from rat brain cortices were exposed to ammonia (5 mM) for different time periods and cell swelling was measured. Cultures treated with ammonia for 1-3 days showed a progressive increase in astrocyte cell volume (59-127%). Parallel treatment of astrocyte cultures with ammonia showed a significant increase in cellular glutamine content (60-80%) only at 1-4 h, a time when swelling was absent, while glutamine levels were normal at 1-3 days, a time when peak cell swelling was observed. Thus no direct correlation between cell swelling and glutamine levels was detected. Additionally, acute increase in intracellular levels of glutamine by treatment with the glutaminase inhibitor 6-diazo-5-oxo-L-norleucine (DON) after ammonia exposure also did not result in swelling. On the contrary, DON treatment significantly blocked (66%) ammonia-induced astrocyte swelling at a later time point (24 h), suggesting that some process resulting from glutamine metabolism is responsible for astrocyte swelling. Additionally, ammonia-induced free radical production and induction of the mitochondrial permeability transition (MPT) were significantly blocked by treatment with DON, suggesting a key role of glutamine in the ammonia-induced free radical generation and the MPT. In summary, our findings indicate a lack of direct correlation between the extent of cell swelling and cellular levels of glutamine. While glutamine may not be acting as an osmolyte, we propose that glutamine-mediated oxidative stress and/or the MPT may be responsible for the astrocyte swelling by ammonia.
Topics: Ammonia; Ammonium Chloride; Animals; Animals, Newborn; Astrocytes; Cell Size; Cells, Cultured; Cerebral Cortex; Diazooxonorleucine; Free Radicals; Glutaminase; Glutamine; Hydrolysis; Membrane Potentials; Mitochondria; Permeability; Rats
PubMed: 16517020
DOI: 10.1016/j.neuint.2005.11.017