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Journal of Neuroimmune Pharmacology :... Mar 2015We previously have shown that cerebellar fastigial nucleus (FN) modulates immune function, but pathways or mechanisms underlying this immunomodulation require...
We previously have shown that cerebellar fastigial nucleus (FN) modulates immune function, but pathways or mechanisms underlying this immunomodulation require clarification. Herein, an anterograde and retrograde tracing of nerve tracts between the cerebellar FN and hypothalamus/thalamus was performed in rats. After demonstrating a direct cerebellar FN-hypothalamic/thalamic glutamatergic projection, 6-diazo-5-oxo-L-norleucine (DON), an inhibitor of glutaminase that catalyzes glutamate synthesis, was injected bilaterally in the cerebellar FN and simultaneously, D,L-threo-β-hydroxyaspartic acid (THA), an inhibitor of glutamate transporters on cell membrane, was bilaterally injected in the lateral hypothalamic area (LHA) or the ventrolateral (VL) thalamic nucleus. DON treatment in the FN alone decreased number of glutamatergic neurons that projected axons to the LHA and also diminished glutamate content in both the hypothalamus and the thalamus. These effects of DON were reduced by combined treatment with THA in the LHA or in the VL. Importantly, DON treatment in the FN alone attenuated percentage and cytotoxicity of natural killer (NK) cells and also lowered percentage and cytokine production of T lymphocytes. These DON-caused immune effects were reduced or abolished by combined treatment with THA in the LHA, but not in the VL. Simultaneously, DON treatment elevated level of norepinephrine (NE) in the spleen and mesenteric lymphoid nodes, and THA treatment in the LHA, rather than in the VL, antagonized the DON-caused NE elevation. These findings suggest that glutamatergic neurons in the cerebellar FN regulate innate and adaptive immune functions and the immunomodulation is conveyed by FN-hypothalamic glutamatergic projections and sympathetic nerves that innervate lymphoid tissues.
Topics: Animals; Aspartic Acid; Axons; Cerebellar Nuclei; Diazooxonorleucine; Enzyme Inhibitors; Female; Glutamic Acid; Glutaminase; Hypothalamic Area, Lateral; Hypothalamus; Immunity; Injections; Killer Cells, Natural; Male; Rats; Rats, Sprague-Dawley; Sympathetic Nervous System; T-Lymphocytes; Thalamus
PubMed: 25649846
DOI: 10.1007/s11481-014-9572-y -
Journal of Neurovirology Apr 2015Recovery from encephalomyelitis induced by infection with mosquito-borne alphaviruses is associated with a high risk of lifelong debilitating neurological deficits....
Recovery from encephalomyelitis induced by infection with mosquito-borne alphaviruses is associated with a high risk of lifelong debilitating neurological deficits. Infection of mice with the prototypic alphavirus, Sindbis virus, provides an animal model with which to study disease mechanisms and examine potential therapeutics. Infectious virus is cleared from the brain within a week after infection, but viral RNA is cleared slowly and persists for the life of the animal. However, no studies have examined the effect of infection on neurocognitive function over time. In the present study, we examined neurocognitive function at different phases of infection in 5-week-old C57BL/6 mice intranasally inoculated with Sindbis virus. At the peak of active virus infection, mice demonstrated hyperactivity, decreased anxiety, and marked hippocampal-dependent memory deficits, the latter of which persisted beyond clearance of infectious virus and resolution of clinical signs of disease. Previous studies indicate that neuronal damage during alphavirus encephalomyelitis is primarily due to inflammatory cell infiltration and glutamate excitotoxicity rather than directly by virus infection. Therefore, mice were treated with 6-diazo-5-oxo-l-norleucine (DON), a glutamine antagonist that can suppress both the immune response and excitotoxicity. Treatment with DON decreased inflammatory cell infiltration and cell death in the hippocampus and partially prevented development of clinical signs and neurocognitive impairment despite the presence of infectious virus and high viral RNA levels. This study presents the first report of neurocognitive sequelae in mice with alphavirus encephalomyelitis and provides a model system for further elucidation of the pathogenesis of virus infection and assessment of potential therapies.
Topics: Alphavirus Infections; Animals; Antimetabolites, Antineoplastic; Behavior, Animal; Diazooxonorleucine; Disease Models, Animal; Encephalitis, Viral; Enzyme-Linked Immunosorbent Assay; Glutamine; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Motor Activity; Sindbis Virus
PubMed: 25645378
DOI: 10.1007/s13365-015-0314-6 -
PloS One 2015Neuroblastomas (NBL) and Ewing's sarcomas (EWS) together cause 18% of all pediatric cancer deaths. Though there is growing interest in targeting the dysregulated...
Neuroblastomas (NBL) and Ewing's sarcomas (EWS) together cause 18% of all pediatric cancer deaths. Though there is growing interest in targeting the dysregulated metabolism of cancer as a therapeutic strategy, this approach has not been fully examined in NBL and EWS. In this study, we first tested a panel of metabolic inhibitors and identified the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON) as the most potent chemotherapeutic across all NBL and EWS cell lines tested. Myc, a master regulator of metabolism, is commonly overexpressed in both of these pediatric malignancies and recent studies have established that Myc causes cancer cells to become "addicted" to glutamine. We found DON strongly inhibited tumor growth of multiple tumor lines in mouse xenograft models. In vitro, inhibition of caspases partially reversed the effects of DON in high Myc expressing cell lines, but not in low Myc expressing lines. We further showed that induction of apoptosis by DON in Myc-overexpressing cancers is via the pro-apoptotic factor Bax. To relieve inhibition of Bax, we tested DON in combination with the Bcl-2 family antagonist navitoclax (ABT-263). In vitro, this combination caused an increase in DON activity across the entire panel of cell lines tested, with synergistic effects in two of the N-Myc amplified neuroblastoma cell lines. Our study supports targeting glutamine metabolism to treat Myc overexpressing cancers, such as NBL and EWS, particularly in combination with Bcl-2 family antagonists.
Topics: Aniline Compounds; Animals; Antimetabolites, Antineoplastic; Apoptosis; Bone Neoplasms; Caspases; Cell Line, Tumor; Cell Proliferation; Diazooxonorleucine; Drug Synergism; Glutamine; Humans; Mice; Neuroblastoma; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Sarcoma, Ewing; Sulfonamides; Xenograft Model Antitumor Assays
PubMed: 25615615
DOI: 10.1371/journal.pone.0116998 -
Analytical Biochemistry Apr 2015Glutamine is an abundant amino acid that plays pivotal roles in cell growth, cell metabolism, and neurotransmission. Dysregulation of glutamine-using pathways has been...
Glutamine is an abundant amino acid that plays pivotal roles in cell growth, cell metabolism, and neurotransmission. Dysregulation of glutamine-using pathways has been associated with pathological conditions such as cancer and neurodegenerative diseases. 6-Diazo-5-oxo-l-norleucine (DON) is a reactive glutamine analog that inhibits enzymes affecting glutamine metabolism such as glutaminase, 2-N-amidotransferase, l-asparaginase, and several enzymes involved in pyrimidine and purine de novo synthesis. As a result, DON is actively used in preclinical models of cancer and neurodegenerative disease. Moreover, there have been several clinical trials using DON to treat a variety of cancers. Considerations of dose and exposure are especially important with DON treatment due to its narrow therapeutic window and significant side effects. Consequently, a robust quantification bioassay is of interest. DON is a polar unstable molecule that has made quantification challenging. Here we report on the characterization of a bioanalytical method to quantify DON in tissue samples involving DON derivatization with 3 N HCl in butanol. The derivatized product is lipophilic and stable. Detection of this analyte by mass spectrometry is fast and specific and can be used to quantify DON in plasma and brain tissue with a limit of detection at the low nanomolar level.
Topics: 1-Butanol; Animals; Brain; Chlorine; Chromatography, High Pressure Liquid; Diazooxonorleucine; Esters; Male; Mice, Inbred C57BL; Reference Standards; Tandem Mass Spectrometry; Time Factors
PubMed: 25584882
DOI: 10.1016/j.ab.2015.01.001 -
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 -
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 -
Reproduction (Cambridge, England) Feb 2014Most of the previous studies on ovarian hyaluronan (HA) have focused on mature antral follicles or corpora lutea, but scarcely on small preantral follicles. Moreover,...
Most of the previous studies on ovarian hyaluronan (HA) have focused on mature antral follicles or corpora lutea, but scarcely on small preantral follicles. Moreover, the origin of follicular HA is unknown. To clarify the localization of HA and its synthases in small growing follicles, involvement of HA in follicle growth, and gonadotropin regulation of HA synthase (Has) gene expression, in this study, perinatal, immature, and adult ovaries of Wistar-Imamichi rats were examined histologically and biochemically and by in vitro follicle culture. HA was detected in the extracellular matrix of granulosa and theca cell layers of primary follicles and more advanced follicles. Ovarian HA accumulation ontogenetically started in the sex cords of perinatal rats, and its primary site shifted to the intrafollicular region of primary follicles within 5 days of birth. The Has1-3 mRNAs were expressed in the ovaries of perinatal, prepubertal, and adult rats, and the expression levels of Has1 and Has2 genes were modulated during the estrous cycle in adult rats and following administration of exogenous gonadotropins in immature acyclic rats. The Has1 and Has2 mRNAs were predominantly localized in the theca and granulosa cell layers of growing follicles respectively. Treatments with chemicals known to reduce ovarian HA synthesis induced follicular atresia. More directly, the addition of Streptomyces hyaluronidase, which specifically degrades HA, induced the arrest of follicle growth in an in vitro culture system. These results indicate that gonadotropin-regulated HA synthesis is involved in normal follicle growth.
Topics: Animals; Diazooxonorleucine; Estrous Cycle; Female; Follicular Atresia; Gene Expression; Glucuronosyltransferase; Gonadotropins, Equine; Granulosa Cells; Hyaluronan Synthases; Hyaluronic Acid; Hyaluronoglucosaminidase; Hymecromone; Ovarian Follicle; Ovary; RNA, Messenger; Rats; Rats, Wistar; Theca Cells; Tissue Culture Techniques
PubMed: 24218629
DOI: 10.1530/REP-13-0464 -
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 -
Journal of Molecular Medicine (Berlin,... Oct 2013The rapid growth of cancer cells is permitted by metabolic changes, notably increased aerobic glycolysis and increased glutaminolysis. Aerobic glycolysis is also evident...
UNLABELLED
The rapid growth of cancer cells is permitted by metabolic changes, notably increased aerobic glycolysis and increased glutaminolysis. Aerobic glycolysis is also evident in the hypertrophying myocytes in right ventricular hypertrophy (RVH), particularly in association with pulmonary arterial hypertension (PAH). It is unknown whether glutaminolysis occurs in the heart. We hypothesized that glutaminolysis occurs in RVH and assessed the precipitating factors, transcriptional mechanisms, and physiological consequences of this metabolic pathway. RVH was induced in two models, one with PAH (Monocrotaline-RVH) and the other without PAH (pulmonary artery banding, PAB-RVH). Despite similar RVH, ischemia as determined by reductions in RV VEGFα, coronary blood flow, and microvascular density was greater in Monocrotaline-RVH versus PAB-RVH. A sixfold increase in (14)C-glutamine metabolism occurred in Monocrotaline-RVH but not in PAB-RVH. In the RV working heart model, the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON) decreased glutaminolysis, caused a reciprocal increase in glucose oxidation, and elevated cardiac output. Consistent with the increased glutaminolysis in RVH, RV expressions of glutamine transporters (SLC1A5 and SLC7A5) and mitochondrial malic enzyme were elevated (Monocrotaline-RVH > PAB-RVH > control). Capillary rarefaction and glutamine transporter upregulation also occurred in RVH in patients with PAH. cMyc and Max, known to mediate transcriptional upregulation of glutaminolysis, were increased in Monocrotaline-RVH. In vivo, DON (0.5 mg/kg/day × 3 weeks) restored pyruvate dehydrogenase activity, reduced RVH, and increased cardiac output (89 ± 8, vs. 55 ± 13 ml/min, p < 0.05) and treadmill distance (194 ± 71, vs. 36 ±7 m, p < 0.05) in Monocrotaline-RVH. Glutaminolysis is induced in the RV in PAH by cMyc-Max, likely as a consequence of RV ischemia. Inhibition of glutaminolysis restores glucose oxidation and has a therapeutic benefit in vivo.
KEY MESSAGE
Patients with pulmonary artery hypertension (PAH) have evidence of cardiac glutaminolysis. Cardiac glutaminolysis is associated with microvascular rarefaction/ischemia. As in cancer, cardiac glutaminolysis results from activation of cMyc-Max. The specific glutaminolysis inhibitor DON regresses right ventricular hypertrophy. DON improves cardiac function and exercise capacity in an animal model of PAH.
Topics: Animals; Basic-Leucine Zipper Transcription Factors; Carrier Proteins; Diazooxonorleucine; Disease Models, Animal; Familial Primary Pulmonary Hypertension; Glucose; Glutamine; Heart Ventricles; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Male; Models, Biological; Oxidation-Reduction; Proto-Oncogene Proteins c-myc; Rats
PubMed: 23794090
DOI: 10.1007/s00109-013-1064-7 -
Molecular Endocrinology (Baltimore, Md.) Mar 2013Plasma membrane cholesterol accumulation has been implicated in cellular insulin resistance. Given the role of the hexosamine biosynthesis pathway (HBP) as a sensor of...
Plasma membrane cholesterol accumulation has been implicated in cellular insulin resistance. Given the role of the hexosamine biosynthesis pathway (HBP) as a sensor of nutrient excess, coupled to its involvement in the development of insulin resistance, we delineated whether excess glucose flux through this pathway provokes a cholesterolgenic response induced by hyperinsulinemia. Exposing 3T3-L1 adipocytes to physiologically relevant doses of hyperinsulinemia (250pM-5000pM) induced a dose-dependent gain in the mRNA/protein levels of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR). These elevations were associated with elevated plasma membrane cholesterol. Mechanistically, hyperinsulinemia increased glucose flux through the HBP and O-linked β-N-acetylglucosamine (O-GlcNAc) modification of specificity protein 1 (Sp1), known to activate cholesterolgenic gene products such as the sterol response element-binding protein (SREBP1) and HMGR. Chromatin immunoprecipitation demonstrated that increased O-GlcNAc modification of Sp1 resulted in a higher binding affinity of Sp1 to the promoter regions of SREBP1 and HMGR. Luciferase assays confirmed that HMGR promoter activity was elevated under these conditions and that inhibition of the HBP with 6-diazo-5-oxo-l-norleucine (DON) prevented hyperinsulinemia-induced activation of the HMGR promoter. In addition, both DON and the Sp1 DNA-binding inhibitor mithramycin prevented the hyperinsulinemia-induced increases in HMGR mRNA/protein and plasma membrane cholesterol. In these mithramycin-treated cells, both cortical filamentous actin structure and insulin-stimulated glucose transport were restored. Together, these data suggest a novel mechanism whereby increased HBP activity increases Sp1 transcriptional activation of a cholesterolgenic program, thereby elevating plasma membrane cholesterol and compromising cytoskeletal structure essential for insulin action.
Topics: 3T3-L1 Cells; Animals; Biosynthetic Pathways; Cell Nucleus; Cholesterol; DNA; Diazooxonorleucine; Glucose; Glycosylation; Hexosamines; Humans; Hydroxymethylglutaryl CoA Reductases; Hyperinsulinism; Insulin; Insulin Resistance; Mice; Promoter Regions, Genetic; Protein Binding; Protein Transport; Sp1 Transcription Factor; Sterol Regulatory Element Binding Protein 1; Transcriptional Activation
PubMed: 23315940
DOI: 10.1210/me.2012-1213