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Molecules (Basel, Switzerland) Apr 2020is the aetiologic agent of Chagas disease, which affects people in the Americas and worldwide. The parasite has a complex life cycle that alternates among mammalian...
is the aetiologic agent of Chagas disease, which affects people in the Americas and worldwide. The parasite has a complex life cycle that alternates among mammalian hosts and insect vectors. During its life cycle, passes through different environments and faces nutrient shortages. It has been established that amino acids, such as proline, histidine, alanine, and glutamate, are crucial to survival. Recently, we described that can biosynthesize glutamine from glutamate and/or obtain it from the extracellular environment, and the role of glutamine in energetic metabolism and metacyclogenesis was demonstrated. In this study, we analysed the effect of glutamine analogues on the parasite life cycle. Here, we show that glutamine analogues impair cell proliferation, the developmental cycle during the infection of mammalian host cells and metacyclogenesis. Taken together, these results show that glutamine is an important metabolite for survival and suggest that glutamine analogues can be used as scaffolds for the development of new trypanocidal drugs. These data also reinforce the supposition that glutamine metabolism is an unexplored possible therapeutic target.
Topics: Animals; Azaserine; CHO Cells; Cell Cycle; Cell Proliferation; Cricetulus; Energy Metabolism; Glutamic Acid; Glutamine; Isoxazoles; Life Cycle Stages; Molecular Structure; Trypanocidal Agents; Trypanosoma cruzi
PubMed: 32252252
DOI: 10.3390/molecules25071628 -
BioMed Research International 2020Reprogrammed glucose and glutamine metabolism are essential for tumor initiation and development. As a branch of glucose and metabolism, the hexosamine biosynthesis...
Reprogrammed glucose and glutamine metabolism are essential for tumor initiation and development. As a branch of glucose and metabolism, the hexosamine biosynthesis pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) and contributes to the O-GlcNAcylation process. However, the spectrum of HBP-dependent tumors and the mechanisms by which the HBP promotes tumor aggressiveness remain areas of active investigation. In this study, we analyzed the activity of the HBP and its prognostic value across 33 types of human cancers. Increased HBP activity was observed in pancreatic ductal adenocarcinoma (PDAC), and higher HBP activity predicted a poor prognosis in PDAC patients. Genetic silencing or pharmacological inhibition of the first and rate-limiting enzyme of the HBP, glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), inhibited PDAC cell proliferation, invasive capacity, and triggered cell apoptosis. Notably, these effects can be restored by addition of UDP-GlcNAc. Moreover, similar antitumor effects were noticed by pharmacological inhibition of GFAT1 with 6-diazo-5-oxo-l-norleucine (DON) or Azaserine. PDAC is maintained by oncogenic Wnt/-catenin transcriptional activity. Our data showed that GFAT1 can regulate -catenin expression via modulation of the O-GlcNAcylation process. TOP/FOP-Flash and real-time qPCR analysis showed that GFAT1 knockdown inhibited -catenin activity and the transcription of its downstream target genes and . Ectopic expression of a stabilized form of -catenin restored the suppressive roles of GFAT1 knockdown on PDAC cell proliferation and invasion. Collectively, our findings indicate that higher GFAT1/HBP/O-GlcNAcylation exhibits tumor-promoting roles by maintaining -catenin activity in PDAC.
Topics: Cell Line, Tumor; Cell Proliferation; Databases, Genetic; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); Glycosylation; Hexosamines; Humans; Pancreatic Neoplasms; beta Catenin
PubMed: 32149084
DOI: 10.1155/2020/1921609 -
American Journal of Physiology.... Mar 2020The extent of glucose metabolism during oocyte maturation is closely related to oocyte developmental potential. Thioredoxin-interacting protein (TXNIP) is an α-arrestin...
The extent of glucose metabolism during oocyte maturation is closely related to oocyte developmental potential. Thioredoxin-interacting protein (TXNIP) is an α-arrestin family protein that negatively regulates glucose uptake into cells. However, little information is available regarding the function of TXNIP in bovine oocytes. Accordingly, the present study was performed to investigate the influence of TXNIP on glucose metabolism in bovine oocytes during in vitro maturation. Pharmacological inhibition of TXNIP by azaserine enhanced glucose uptake and imparted a specific metabolic effect on glycolysis and pentose phosphate pathway (PPP). RNA interference (RNAi) was adopted to further determine the biological significance of TXNIP in regulating glucose metabolism. The maturation rate and the developmental competence of TXNIP siRNA-treated oocytes were significantly improved. Knockdown of TXNIP in bovine oocytes significantly increased glycolysis by increasing the activities of phosphofructokinase (PFK), pyruvate kinase, and lactate dehydrogenase; pyruvate and lactate production; and intracellular ATP level, as well as mitochondrial activity. Furthermore, glucose metabolism through PPP was also enhanced by TXNIP depletion, as TXNIP siRNA treatment promoted glucose-6-phosphate dehydrogenase (G6PDH) activity and NADPH content, and helped maintain a high level of glutathione and a low level of reactive oxygen species within the oocytes. Further studies revealed that inhibition of TXNIP resulted increases in glucose transporter 1 (GLUT1) expression, as well as PFK1 platelet isoform () and mRNA levels. These results reveal that TXNIP depletion promotes oocyte maturation by enhancing both glycolysis and the PPP. During in vitro maturation of bovine oocytes, TXNIP serves as a key regulator of glucose uptake by controlling GLUT1 expression.
Topics: Adenosine Triphosphate; Animals; Azaserine; Carrier Proteins; Cattle; Female; Gene Knockdown Techniques; Glucose; Glycolysis; In Vitro Oocyte Maturation Techniques; Infertility, Female; Intracellular Space; Mitochondria; Oocytes; Oxidation-Reduction; RNA Interference; RNA, Small Interfering
PubMed: 31935112
DOI: 10.1152/ajpendo.00057.2019 -
Journal of Cancer Research and... 2019The effect of acetylsalicylic acid (ASA) on thiol levels was studied in a rat model of azaserine carcinogenesis.
AIM
The effect of acetylsalicylic acid (ASA) on thiol levels was studied in a rat model of azaserine carcinogenesis.
MATERIALS AND METHODS
ASA and azaserine were applied to the animals to research changes in cellular sulfhydryl (-SH) content and variations in free and protein-bound molecules containing the -SH group. Such effects in rats injected with azaserine were investigated at low (200 ppm) and high (400 ppm) concentrations of ASA over a relatively short (6 months) and a relatively long (12 months) period.
RESULTS
Changes in the hepatic, pancreatic, and renal -SH contents were also determined.
CONCLUSION
Compared to the other tissues studied, the liver contained the highest levels of both free and protein-bound -SH.
Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Aspirin; Azaserine; Carcinogens; Dose-Response Relationship, Drug; Humans; Kidney; Liver; Male; Neoplasms; Neoplasms, Experimental; Pancreas; Rats; Rats, Wistar; Sulfhydryl Compounds; Treatment Outcome
PubMed: 30880783
DOI: 10.4103/jcrt.JCRT_1319_16 -
Oxidative Medicine and Cellular... 2018Remote ischemic preconditioning (RIPC) is an adaptive response, manifesting when local short-term ischemic preconditioning reduces damage to adjacent or distant tissues...
Remote ischemic preconditioning (RIPC) is an adaptive response, manifesting when local short-term ischemic preconditioning reduces damage to adjacent or distant tissues or organs. O-linked -N-acetylglucosamine (O-GlcNAc) glycosylation of intracellular proteins denotes a type of posttranslational modification that influences multiple cytoplasmic and nuclear protein functions. Growing evidence indicates that stress can induce an acute increase in O-GlcNAc levels, which can be cytoprotective. The current study aimed to determine whether RIPC can provide renoprotection against contrast-induced acute kidney injury (CI-AKI) by augmenting O-GlcNAc signaling. We established a stable model of CI-AKI using 5/6 nephrectomized rats exposed to dehydration followed by iohexol injection via the tail vein. We found that RIPC increased UDP-GlcNAc levels through the hexosamine biosynthetic pathway as well as global renal O-GlcNAcylation. RIPC-induced elevation of O-GlcNAc signaling ameliorated CI-AKI based on the presence of less tubular damage and apoptosis and the amount of reactive oxygen species. In addition, the use of alloxan, an O-GlcNAc transferase inhibitor, and azaserine, a glutamine fructose-6-phosphate amidotransferase inhibitor, neutralized the protective effect of RIPC against oxidative stress and tubular apoptosis. In conclusion, RIPC attenuates local oxidative stress and tubular apoptosis induced by contrast exposure by enhancing O-GlcNAc glycosylation levels; this can be a potentially useful approach for lowering the risk of CI-AKI.
Topics: Acute Kidney Injury; Animals; Contrast Media; Humans; Male; N-Acetylglucosaminyltransferases; Rats; Rats, Sprague-Dawley
PubMed: 30186544
DOI: 10.1155/2018/4895913 -
Cell Reports Feb 2018Sirtuin 3 (SIRT3) is a NAD-dependent deacetylase downregulated in aging and age-associated diseases such as cancer and neurodegeneration and in high-fat diet...
Sirtuin 3 (SIRT3) is a NAD-dependent deacetylase downregulated in aging and age-associated diseases such as cancer and neurodegeneration and in high-fat diet (HFD)-induced metabolic disorders. Here, we performed a small-molecule screen and identified an unexpected metabolic vulnerability associated with SIRT3 loss. Azaserine, a glutamine analog, was the top compound that inhibited growth and proliferation of cells lacking SIRT3. Using stable isotope tracing of glutamine, we observed its increased incorporation into de novo nucleotide synthesis in SIRT3 knockout (KO) cells. Furthermore, we found that SIRT3 KO cells upregulated the diversion of glutamine into de novo nucleotide synthesis through hyperactive mTORC1 signaling. Overexpression of SIRT3 suppressed mTORC1 and growth in vivo in a xenograft tumor model of breast cancer. Thus, we have uncovered a metabolic vulnerability of cells with SIRT3 loss by using an unbiased small-molecule screen.
Topics: Amino Acid Sequence; Animals; Azaserine; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Fibroblasts; Glutamine; Mechanistic Target of Rapamycin Complex 1; Mice, Knockout; Mice, Nude; Nucleotides; Promoter Regions, Genetic; Signal Transduction; Sirtuin 3; Small Molecule Libraries; Up-Regulation
PubMed: 29466723
DOI: 10.1016/j.celrep.2018.01.076 -
Frontiers in Microbiology 2017Previous studies showed differences in the regulatory response to C/N balance in with respect to other cyanobacteria, but no information was available about its causes,...
Previous studies showed differences in the regulatory response to C/N balance in with respect to other cyanobacteria, but no information was available about its causes, or the ecological advantages conferred to thrive in oligotrophic environments. We addressed the changes in key enzymes (glutamine synthetase, isocitrate dehydrogenase) and the gene (the global nitrogen regulator) involved in C/N metabolism and its regulation, in three model strains: MED4, SS120, and MIT9313. We observed a remarkable level of diversity in their response to azaserine, a glutamate synthase inhibitor which increases the concentration of the key metabolite 2-oxoglutarate, used to sense the C/N balance by cyanobacteria. Besides, we studied the binding between the global nitrogen regulator (NtcA) and the promoter of the gene in the same strains, and its dependence on the 2-oxoglutarate concentration, by using isothermal titration calorimetry, surface plasmon resonance, and electrophoretic mobility shift. Our results show a reduction in the responsiveness of NtcA to 2-oxoglutarate in , especially in the MED4 and SS120 strains. This suggests a trend to streamline the regulation of C/N metabolism in late-branching strains (MED4 and SS120), in adaptation to the rather stable conditions found in the oligotrophic ocean gyres where this microorganism is most abundant.
PubMed: 29375510
DOI: 10.3389/fmicb.2017.02641 -
Nature Communications Jan 2018AMP-activated protein kinase (AMPK) has been shown to inhibit cardiac hypertrophy. Here, we show that submaximal AMPK activation blocks cardiomyocyte hypertrophy without...
AMP-activated protein kinase (AMPK) has been shown to inhibit cardiac hypertrophy. Here, we show that submaximal AMPK activation blocks cardiomyocyte hypertrophy without affecting downstream targets previously suggested to be involved, such as p70 ribosomal S6 protein kinase, calcineurin/nuclear factor of activated T cells (NFAT) and extracellular signal-regulated kinases. Instead, cardiomyocyte hypertrophy is accompanied by increased protein O-GlcNAcylation, which is reversed by AMPK activation. Decreasing O-GlcNAcylation by inhibitors of the glutamine:fructose-6-phosphate aminotransferase (GFAT), blocks cardiomyocyte hypertrophy, mimicking AMPK activation. Conversely, O-GlcNAcylation-inducing agents counteract the anti-hypertrophic effect of AMPK. In vivo, AMPK activation prevents myocardial hypertrophy and the concomitant rise of O-GlcNAcylation in wild-type but not in AMPKα2-deficient mice. Treatment of wild-type mice with O-GlcNAcylation-inducing agents reverses AMPK action. Finally, we demonstrate that AMPK inhibits O-GlcNAcylation by mainly controlling GFAT phosphorylation, thereby reducing O-GlcNAcylation of proteins such as troponin T. We conclude that AMPK activation prevents cardiac hypertrophy predominantly by inhibiting O-GlcNAcylation.
Topics: AMP-Activated Protein Kinases; Acetylglucosamine; Acylation; Animals; Animals, Newborn; Azaserine; Azo Compounds; Biphenyl Compounds; Cardiomegaly; Enzyme Activation; Enzyme Activators; Gene Expression Regulation; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); Glycosylation; Heart Ventricles; Male; Mice; Mice, Knockout; Myocardium; Myocytes, Cardiac; Nitrogenous Group Transferases; Norleucine; Phosphorylation; Primary Cell Culture; Pyrones; Rats; Rats, Wistar; Signal Transduction; Thiophenes; Troponin T
PubMed: 29371602
DOI: 10.1038/s41467-017-02795-4 -
MSystems 2017requires the capability to accommodate to environmental changes in order to proliferate in oligotrophic oceans, in particular regarding nitrogen availability. A precise...
requires the capability to accommodate to environmental changes in order to proliferate in oligotrophic oceans, in particular regarding nitrogen availability. A precise knowledge of the composition and changes in the proteome can yield fundamental insights into such a response. Here we report a detailed proteome analysis of the important model cyanobacterium SS120 after treatment with azaserine, an inhibitor of ferredoxin-dependent glutamate synthase (GOGAT), to simulate extreme nitrogen starvation. In total, 1,072 proteins, corresponding to 57% of the theoretical proteome, were identified-the maximum proteome coverage obtained for any strain thus far. Spectral intensity, calibrated quantification by the Hi3 method, was obtained for 1,007 proteins. Statistically significant changes ( value of <0.05) were observed for 408 proteins, with the majority of proteins (92.4%) downregulated after 8 h of treatment. There was a strong decrease in ribosomal proteins upon azaserine addition, while many transporters were increased. The regulatory proteins P and PipX were decreased, and the global nitrogen regulator NtcA was upregulated. Furthermore, our data for indicate that NtcA also participates in the regulation of photosynthesis. responds to the lack of nitrogen by slowing down translation, while inducing photosynthetic cyclic electron flow and biosynthesis of proteins involved in nitrogen uptake and assimilation. is the most abundant photosynthetic organism on Earth, contributing significantly to global primary production and playing a prominent role in biogeochemical cycles. Here we study the effects of extreme nitrogen limitation, a feature of the oligotrophic oceans inhabited by this organism. Quantitative proteomics allowed an accurate quantification of the proteome, finding three main responses to nitrogen limitation: upregulation of nitrogen assimilation-related proteins, including transporters; downregulation of ribosome proteins; and induction of the photosystem II cyclic electron flow. This suggests that nitrogen limitation affects a range of metabolic processes far wider than initially believed, with the ultimate goal of saving nitrogen and maximizing the nitrogen uptake and assimilation capabilities of the cell.
PubMed: 28593196
DOI: 10.1128/mSystems.00008-17 -
Oncotarget Dec 2016The hexosamine biosynthetic pathway (HBP) requires two key nutrients glucose and glutamine for O-linked N-acetylglucosamine (O-GlcNAc) cycling, a post-translational...
The hexosamine biosynthetic pathway (HBP) requires two key nutrients glucose and glutamine for O-linked N-acetylglucosamine (O-GlcNAc) cycling, a post-translational protein modification that adds GlcNAc to nuclear and cytoplasmic proteins. Increased GlcNAc has been linked to regulatory factors involved in cancer cell growth and survival. However, the biological significance of GlcNAc in diffuse large B-cell lymphoma (DLBCL) is not well defined. This study is the first to show that both the substrate and the endpoint O-GlcNAc transferase (OGT) enzyme of the HBP were highly expressed in DLBCL cell lines and in patient tumors compared with normal B-lymphocytes. Notably, high OGT mRNA levels were associated with poor survival of DLBCL patients. Targeting OGT via small interference RNA in DLBCL cells inhibited activation of GlcNAc, nuclear factor kappa B (NF-κB), and nuclear factor of activated T-cells 1 (NFATc1), as well as cell growth. Depleting both glucose and glutamine in DLBCL cells or treating them with an HBP inhibitor (azaserine) diminished O-GlcNAc protein substrate, inhibited constitutive NF-κB and NFATc1 activation, and induced G0/G1 cell-cycle arrest and apoptosis. Replenishing glucose-and glutamine-deprived DLBCL cells with a synthetic glucose analog (ethylenedicysteine-N-acetylglucosamine [ECG]) reversed these phenotypes. Finally, we showed in both in vitro and in vivo murine models that DLBCL cells easily take up radiolabeled technetium-99m-ECG conjugate. These findings suggest that targeting the HBP has therapeutic relevance for DLBCL and underscores the imaging potential of the glucosamine analog ECG in DLBCL.
Topics: Acetylglucosamine; Animals; Antineoplastic Agents; Apoptosis; Azaserine; Cell Line, Tumor; Cell Proliferation; Contrast Media; Cysteine; Enzyme Inhibitors; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glycosylation; Hexosamines; Humans; Lymphoma, Large B-Cell, Diffuse; Mice, Inbred NOD; Mice, SCID; N-Acetylglucosaminyltransferases; NF-kappa B; NFATC Transcription Factors; Organotechnetium Compounds; RNA Interference; RNA, Messenger; RNAi Therapeutics; Signal Transduction; Transfection
PubMed: 27716624
DOI: 10.18632/oncotarget.12413