Role of Glycolysis and Fatty Acid Synthesis in the Activation and T Cell-Modulating Potential of Dendritic Cells Stimulated with a TLR5-Ligand Allergen Fusion Protein.

Trained immune responses, based on metabolic and epigenetic changes in innate immune cells, are de facto innate immune memory and, therefore, are of great interest in vaccine development. In previous studies, the recombinant fusion protein rFlaA:Betv1, combining the adjuvant and toll-like receptor (TLR)5-ligand flagellin (FlaA) and the major birch pollen allergen Bet v 1 into a single molecule, significantly suppressed allergic sensitization in vivo while also changing the metabolism of myeloid dendritic cells (mDCs). Within this study, the immune-metabolic effects of rFlaA:Betv1 during mDC activation were elucidated. In line with results for other well-characterized TLR-ligands, rFlaA:Betv1 increased glycolysis while suppressing oxidative phosphorylation to different extents, making rFlaA:Betv1 a suitable model to study the immune-metabolic effects of TLR-adjuvanted vaccines. In vitro pretreatment of mDCs with cerulenin (inhibitor of fatty acid biosynthesis) led to a decrease in both rFlaA:Betv1-induced anti-inflammatory cytokine Interleukin (IL) 10 and T helper cell type (TH) 1-related cytokine IL-12p70, while the pro-inflammatory cytokine IL 1ß was unaffected. Interestingly, pretreatment with the glutaminase inhibitor BPTES resulted in an increase in IL-1ß, but decreased IL-12p70 secretion while leaving IL-10 unchanged. Inhibition of the glycolytic enzyme hexokinase-2 by 2-deoxyglucose led to a decrease in all investigated cytokines (IL-10, IL-12p70, and IL-1ß). Inhibitors of mitochondrial respiration had no effect on rFlaA:Betv1-induced IL-10 level, but either enhanced the secretion of IL-1ß (oligomycin) or decreased IL-12p70 (antimycin A). In extracellular flux measurements, mDCs showed a strongly enhanced glycolysis after rFlaA:Betv1 stimulation, which was slightly increased after respiratory shutdown using antimycin A. rFlaA:Betv1-stimulated mDCs secreted directly antimicrobial substances in a mTOR- and fatty acid metabolism-dependent manner. In co-cultures of rFlaA:Betv1-stimulated mDCs with CD4+ T cells, the suppression of Bet v 1-specific TH2 responses was shown to depend on fatty acid synthesis. The effector function of rFlaA:Betv1-activated mDCs mainly relies on glycolysis, with fatty acid synthesis also significantly contributing to rFlaA:Betv1-mediated cytokine secretion, the production of antimicrobial molecules, and the modulation of T cell responses.

Rutin prevents seizures in kainic acid-treated rats: evidence of glutamate levels, inflammation and neuronal loss modulation.

Rutin, a naturally derived flavonoid molecule with known neuroprotective properties, has been demonstrated to have anticonvulsive potential, but the mechanism of this effect is still unclear. The current study aimed to investigate the probable antiseizure mechanisms of rutin in rats using the kainic acid (KA) seizure model. Rutin (50 and 100 mg kg-1) and carbamazepine (100 mg kg-1) were administered daily by oral gavage for 7 days before KA (15 mg kg-1) intraperitoneal (i.p.) injection. Seizure behavior, neuronal cell death, glutamate concentration, excitatory amino acid transporters (EAATs), glutamine synthetase (GS), glutaminase, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1 and GluA2, N-methyl-D-aspartate (NMDA) receptor subunits GluN2A and GluN2B, activated astrocytes, and inflammatory and anti-inflammatory molecules in the hippocampus were evaluated. Supplementation with rutin attenuated seizure severity in KA-treated rats and reversed KA-induced neuronal loss and glutamate elevation in the hippocampus. Decreased glutaminase and GluN2B, and increased EAATs, GS, GluA1, GluA2 and GluN2A were observed with rutin administration. Rutin pretreatment also suppressed activated astrocytes, downregulated the protein levels of inflammatory molecules [interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), high mobility group Box 1 (HMGB1), interleukin-1 receptor 1 (IL-1R1), and Toll-like receptor-4 (TLR-4)] and upregulated anti-inflammatory molecule interleukin-10 (IL-10) protein expression. Taken together, the results indicate that the preventive treatment of rats with rutin attenuated KA-induced seizures and neuronal loss by decreasing glutamatergic hyperactivity and suppressing the IL-1R1/TLR4-related neuroinflammatory cascade.

Targeting Glutaminase by Natural Compounds: Structure-Based Virtual Screening and Molecular Dynamics Simulation Approach to Suppress Cancer Progression.

Cancer cells change their glucose and glutamine (GLU) metabolism to obtain the energy required to continue growing. Glutaminase (GLS) plays a crucial role in promoting cell metabolism for cancer cell growth; targeting GLU metabolism by inhibiting GLS has attracted interest as a potential cancer management strategy. Herein, we employed a sequential screening of traditional Chinese medicine (TCM) database followed by drug-likeness and molecular dynamics simulations against the active site of GLS. We report 12 potent compounds after screening the TCM database against GLS, followed by a drug-likeness filter with Lipinski and Veber rule criteria. Among them, ZINC03978829 and ZINC32296657 were found to have higher binding energy (BE) values than the control compound 6-Diazo-5-Oxo-L-Norleucine, with BEs of -9.3 and -9.7 kcal/mol, respectively, compared to the BE of 6-Diazo-5-Oxo-L-Norleucine (-4.7 kcal/mol) with GLS. Molecular dynamics simulations were used to evaluate the results further, and a 100 ns MD simulation revealed that the hits form stable complexes with GLS and formed 2-5 hydrogen bond interactions. This study indicates that these hits might be employed as GLS inhibitors in the battle against cancer. However, more laboratory tests are a prerequisite to optimize them as GLS inhibitors.

The glutamate/GABA system in the retina of male rats: effects of aging, neurodegeneration, and supplementation with melatonin and antioxidant SkQ1.

Glutamate and -aminobutyric acid (GABA) are the most abundant amino acids in the retina. An imbalance of the glutamate/GABA system is involved in the pathogenesis of various neurodegenerative disorders. Here we for the first time analyzed alterations of expression of glutamate- and GABA-synthesizing enzymes, transporters, and relevant receptors in the retina with age in Wistar rats and in senescence-accelerated OXYS rats who develop AMD-like retinopathy. We noted consistent age-dependent expression changes of GABAergic-system proteins (GAD67, GABA-T, and GAT1) in OXYS and Wistar rats: upregulation by age 3 months and downregulation at age 18 months. At a late stage of AMD-like retinopathy in OXYS rats (18 months), there was significant upregulation of glutaminase and downregulation of glutamine synthetase, possibly indicating an increasing level of glutamate in the retina. AMD-like-retinopathy development in the OXYS strain was accompanied by underexpression of glutamate transporter GLAST. Prolonged supplementation with both melatonin and SkQ1 (separately) suppressed the progression of the AMD-like pathology in OXYS rats without affecting the glutamate/GABA system but worsened the condition of the Wistar rat's retina during normal aging. We observed decreasing protein levels of glutamine synthetase, GLAST, and GABAAR1 and an increasing level of glutaminase in Wistar rats. In summary, both melatonin and mitochondrial antioxidant SkQ1 had different effect on the retinal glutamate / GABA in healthy Wistar and senescence-accelerated OXYS rats.

Filamentous GLS1 promotes ROS-induced apoptosis upon glutamine deprivation via insufficient asparagine synthesis.

GLS1 orchestrates glutaminolysis and promotes cell proliferation when glutamine is abundant by regenerating TCA cycle intermediates and supporting redox homeostasis. CB-839, an inhibitor of GLS1, is currently under clinical investigation for a variety of cancer types. Here, we show that GLS1 facilitates apoptosis when glutamine is deprived. Mechanistically, the absence of exogenous glutamine sufficiently reduces glutamate levels to convert dimeric GLS1 to a self-assembled, extremely low-Km filamentous polymer. GLS1 filaments possess an enhanced catalytic activity, which further depletes intracellular glutamine. Functionally, filamentous GLS1-dependent glutamine scarcity leads to inadequate synthesis of asparagine and mitogenome-encoded proteins, resulting in ROS-induced apoptosis that can be rescued by asparagine supplementation. Physiologically, we observed GLS1 filaments in solid tumors and validated the tumor-suppressive role of constitutively active, filamentous GLS1 mutants K320A and S482C in xenograft models. Our results change our understanding of GLS1 in cancer metabolism and suggest the therapeutic potential of promoting GLS1 filament formation.

Distinct Roles of Glutamine Metabolism in Benign and Malignant Cartilage Tumors With IDH Mutations.

Enchondromas and chondrosarcomas are common cartilage neoplasms that are either benign or malignant, respectively. The majority of these tumors harbor mutations in either IDH1 or IDH2. Glutamine metabolism has been implicated as a critical regulator of tumors with IDH mutations. Using genetic and pharmacological approaches, we demonstrated that glutaminase-mediated glutamine metabolism played distinct roles in enchondromas and chondrosarcomas with IDH1 or IDH2 mutations. Glutamine affected cell differentiation and viability in these tumors differently through different downstream metabolites. During murine enchondroma-like lesion development, glutamine-derived α-ketoglutarate promoted hypertrophic chondrocyte differentiation and regulated chondrocyte proliferation. Deletion of glutaminase in chondrocytes with Idh1 mutation increased the number and size of enchondroma-like lesions. In contrast, pharmacological inhibition of glutaminase in chondrosarcoma xenografts reduced overall tumor burden partially because glutamine-derived non-essential amino acids played an important role in preventing cell apoptosis. This study demonstrates that glutamine metabolism plays different roles in tumor initiation and cancer maintenance. Supplementation of α-ketoglutarate and inhibiting GLS may provide a therapeutic approach to suppress enchondroma and chondrosarcoma tumor growth, respectively. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Therapeutic potentiality of a new flavonoid against ketamine induced glutamatergic dysregulation in schizophrenia: In vivo and in silico approach.

Glutamate and dopamine hypotheses are leading theories of the pathophysiology of schizophrenia. Multiple lines of evidence suggest that dopaminergic and glutamatergic dysfunction is an underlying mechanism in schizophrenia. Since currently available antipsychotic drugs have significant untoward side effects, identification of new neuroprotective compounds from the medicinal plants may prove beneficial in neurodegenerative disorders. In our previous investigation we have isolated, characterized and reported a novel bioactive compound viz. 3-(3, 4-dimethoxy phenyl)-1-(4-methoxy phenyl) prop-2-en-1-one from the Celastrus paniculatus (CP) is used for the current clinical intervention of schizophrenia disease. The present study is mainly aimed to evaluate the neuroprotective potential of the above bioactive compound against ketamine-induced schizophrenia with particular reference to glutamate metabolism using in vivo and in silico methods. The decrease in glutamine content and the activity levels of glutamate dehydrogenase, glutamine synthetase, and glutaminase in different regions of the rat brain suggests lowered oxidative deamination and lowered mobilization of glutamate towards glutamine formation during ketamine-induced schizophrenia. Pre-treatment with the plant compound reversed the alterations in glutamate metabolism and restored the normal glutamatergic neurotransmission akin to the reference drug, clozapine. In addition, the compound has shown strong interaction and exhibited the highest binding energies against selected NMDA receptors with the lowest inhibition constant than the reference drug. Recoveries of these parameters during anti-schizophrenic treatment suggest that administration of plant compound might offer neuroprotection by interrupting the pathological cascade of glutamatergic neurotransmission that occurs during schizophrenia.

Structure-Enabled Discovery of Novel Macrocyclic Inhibitors Targeting Glutaminase 1 Allosteric Binding Site.

The inhibition of glutaminase 1 (GLS1) represents a potential treatment of malignant tumors. Structural analysis led to the design of a novel series of macrocyclic GLS1 allosteric inhibitors. Through extensive structure-activity relationship studies, a promising candidate molecule 13b (LL202) was identified with robust GLS1 inhibitory activity (IC50 = 6 nM) and high GLS1 binding affinity (SPR, Kd = 24 nM; ITC, Kd = 37 nM). The X-ray crystal structure of the 13b-GLS1 complex was resolved, revealing a unique binding mode and providing a novel structural scaffold for GLS1 allosteric inhibitors. Importantly, 13b clearly adjusted the cellular metabolites and induced an increase in the ROS level by blocking glutamine metabolism. Furthermore, 13b exhibited a similar in vivo antitumor activity as CB839. This study adds to the growing body of evidence that macrocyclization provides an alternative and complementary approach for the design of small-molecule inhibitors, with the potential to improve the binding affinity to the targets.

Discovery of IPN60090, a Clinical Stage Selective Glutaminase-1 (GLS-1) Inhibitor with Excellent Pharmacokinetic and Physicochemical Properties.

Inhibition of glutaminase-1 (GLS-1) hampers the proliferation of tumor cells reliant on glutamine. Known glutaminase inhibitors have potential limitations, and in vivo exposures are potentially limited due to poor physicochemical properties. We initiated a GLS-1 inhibitor discovery program focused on optimizing physicochemical and pharmacokinetic properties, and have developed a new selective inhibitor, compound 27 (IPN60090), which is currently in phase 1 clinical trials. Compound 27 attains high oral exposures in preclinical species, with strong in vivo target engagement, and should robustly inhibit glutaminase in humans.

Solution Structures and Dynamic Assembly of the 24-Meric Plasmodial Pdx1-Pdx2 Complex.

Plasmodium species are protozoan parasites causing the deadly malaria disease. They have developed effective resistance mechanisms against most antimalarial medication, causing an urgent need to identify new antimalarial drug targets. Ideally, new drugs would be generated to specifically target the parasite with minimal or no toxicity to humans, requiring these drug targets to be distinctly different from the host's metabolic processes or even absent in the host. In this context, the essential presence of vitamin B6 biosynthesis enzymes in Plasmodium, the pyridoxal phosphate (PLP) biosynthesis enzyme complex, and its absence in humans is recognized as a potential drug target. To characterize the PLP enzyme complex in terms of initial drug discovery investigations, we performed structural analysis of the Plasmodium vivax PLP synthase domain (Pdx1), glutaminase domain (Pdx2), and Pdx1-Pdx2 (Pdx) complex (PLP synthase complex) by utilizing complementary bioanalytical techniques, such as dynamic light scattering (DLS), X-ray solution scattering (SAXS), and electron microscopy (EM). Our investigations revealed a dodecameric Pdx1 and a monodispersed Pdx complex. Pdx2 was identified in monomeric and in different oligomeric states in solution. Interestingly, mixing oligomeric and polydisperse Pdx2 with dodecameric monodisperse Pdx1 resulted in a monodispersed Pdx complex. SAXS measurements revealed the low-resolution dodecameric structure of Pdx1, different oligomeric structures for Pdx2, and a ring-shaped dodecameric Pdx1 decorated with Pdx2, forming a heteromeric 24-meric Pdx complex.

Differentiation of NSC-34 cells is characterized by expression of NGF receptor p75, glutaminase and NCAM L1, activation of mitochondria, and sensitivity to fatty acid intervention.

Motor neuronal damage due to diseases, traumatic insults or de-afferentation of the spinal cord is often incurable because of poor intrinsic regenerative capacity. Hence, medical basic research has to provide a better understanding of development-/regeneration-related cellular processes as only way to develop new and successful therapeutic strategies. Here, we investigated the neuronal differentiation of the NSC-34 hybrid cell line, which is an accepted model for spinal cord motor neurons. Their differentiation was stimulated by switching from normal to differentiation medium and by supplementation with palmitic and oleic acid. To characterize neuro-differentiation of NSC-34 cells, expression of nicotinic acetylcholine receptor alpha 4, NGF p75 receptor, IGF I alpha receptor, glutaminase, NCAM L1, ADAM10 and myelin basic protein as well as activation of mitochondria were analyzed. Both switch from normal to differentiation medium and fatty acid application stimulated NSC-34 differentiation. Differentiation was characterized by diminishing expression of the nicotinic acetylcholine receptor alpha 4 and enhancing expression of the NGF receptor p75, of glutaminase, of NCAM L1 and it's partially transformation from the cell surface into the cell. Fatty acid intervention stabilized the expression of the nicotinic acetylcholine receptor alpha 4, diminished the expression of the NGF receptor p75, consolidated the expression profile of NCAM L1, and intensified the expression of the relevant for NCAM L1 cleavage ADAM10. However, NCAM L1 cleavage itself was unaffected by fatty acid intervention, as was the differentiation-relevant activation of mitochondria and their transformation into neuronal filopodia.

Hyperoxia induces glutamine-fuelled anaplerosis in retinal Müller cells.

Although supplemental oxygen is required to promote survival of severely premature infants, hyperoxia is simultaneously harmful to premature developing tissues such as in the retina. Here we report the effect of hyperoxia on central carbon metabolism in primary mouse Müller glial cells and a human Müller glia cell line (M10-M1 cells). We found decreased flux from glycolysis entering the tricarboxylic acid cycle in Müller cells accompanied by increased glutamine consumption in response to hyperoxia. In hyperoxia, anaplerotic catabolism of glutamine by Müller cells increased ammonium release two-fold. Hyperoxia induces glutamine-fueled anaplerosis that reverses basal Müller cell metabolism from production to consumption of glutamine.

Metabolic Reprogramming of Host Cells in Response to Enteroviral Infection.

Enterovirus 71 (EV71) infection is an endemic disease in Southeast Asia and China. We have previously shown that EV71 virus causes functional changes in mitochondria. It is speculative whether EV71 virus alters the host cell metabolism to its own benefit. Using a metabolomics approach, we demonstrate that EV71-infected Vero cells had significant changes in metabolism. Glutathione and its related metabolites, and several amino acids, such as glutamate and aspartate, changed significantly with the infectious dose of virus. Other pathways, including glycolysis and tricarboxylic acid cycle, were also altered. A change in glutamine/glutamate metabolism is critical to the viral infection. The presence of glutamine in culture medium was associated with an increase in viral replication. Dimethyl α-ketoglutarate treatment partially mimicked the effect of glutamine supplementation. In addition, the immunoblot analysis revealed that the expression of glutamate dehydrogenase (GDH) and trifunctional carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) increased during infection. Knockdown of expression of glutaminase (GLS), GDH and CAD drastically reduced the cytopathic effect (CPE) and viral replication. Furthermore, we found that CAD bound VP1 to promote the de novo pyrimidine synthesis. Our findings suggest that virus may induce metabolic reprogramming of host cells to promote its replication through interactions between viral and host cell proteins.

Studies on the potential link between antidepressant effect of Xiaoyao San and its pharmacological activity of hepatoprotection based on multi-platform metabolomics.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine (TCM) theory, depression is considered to be "liver qi stagnation", and relieving "liver qi stagnation" is regarded as an effective method for treating depression. Xiaoyao San (XYS) is a well-known TCM formula for the treatment of depression by relieving "liver qi stagnation". This formula consists of Radix Paeoniae Alba (Paeonia lactiflora Pall.), Radix Bupleuri (Bupleurum chinense DC.), Poria (Poria cocos (Schw.) Wolf), Rhizoma Atractylodis Macrocephalae (Atractylodes macrocephala Koidz.), Radix Angelicae Sinensis (Angelica sinensis (Oliv.) Diels), Radix Glycyrrhizae (Glycyrrhiza uralensis Fisch.), Rhizoma Zingiberis Recens (Zingiber officinale Roscoe) and Herba Menthae Haplocalycis (Mentha haplocalyx Briq.). AIM OF THE STUDY: Several studies have suggested that depression is associated with liver injury. XYS was a well-known TCM formula for the treatment of depression and liver stagnancy. However, it was still unknown whether the antidepressant effect of XYS is related to the pharmacological activity of hepatoprotection. The aim of this study was to elucidate the potential link between the antidepressant and hepatoprotective effect of XYS. MATERIALS AND METHODS: A depression rat model was established by the CUMS (chronic unpredictable mild stress) procedure. The antidepressant effect of XYS was assessed by the behavioral indicators, and the hepatoprotective effect of XYS was evaluated through biochemical assays. 1H-NMR and LC/MS-based liver metabolomics were performed to discover key metabolic pathways involved in the antidepressant and hepatoprotective effects of XYS. Further, the key pathway was validated using commercial kits. RESULTS: The results demonstrated that XYS pretreatment could significantly improve the depressive symptom induced by CUMS. More importantly, the results demonstrated that liver injury was observed in the CUMS model rats, and XYS had a hepatoprotective effect by reducing the activities of AST and ALT in serum, increasing the levels of SOD and GSH-Px and reducing the contents of MDA, IL-6, and IL-1ß in the liver. In addition, the NMR and LC/MS-based metabolomics results indicated that XYS improved 23 of the 35 perturbed potential liver biomarkers that were induced by CUMS. Among them, 9 biomarkers were significantly correlated with both depression and liver pathology, according to Pearson correlation analysis. Metabolic pathway analyses of these 9 biomarkers showed that glutamine and glutamate metabolism were the most important metabolic pathways. Furthermore, to verify glutamine and glutamate metabolism, the levels of glutamine and glutamate, and the activity of glutamine synthetase (GS) and glutaminase (GLS) were quantitatively determined in the liver by commercial kits, and these results were consistent with the metabolomics results. CONCLUSIONS: XYS could significantly improve the depressive and liver injury symptoms induced by CUMS. The metabolomics results indicate that the regulation of glutamine and glutamate metabolism to maintain the balance of ammonia and promote energy metabolism is a potential junction between the antidepressant and hepatoprotective effects of XYS.

Molecular cloning, structural modeling and characterization of a novel glutaminase-free L-asparaginase from Cobetia amphilecti AMI6.

The exploration of new sources of L-asparaginase with low glutaminase activity is of great interest in both medical and food applications. In the current study, a novel L-asparaginase gene (CobAsnase) from halotolerant Cobetia amphilecti AMI6 was cloned and over-expressed in Escherichia coli. The enzyme had a molecular mass of 37 kDa on SDS-PAGE and dynamic light scattering (DLS) analysis revealed that CobAsnase is a homotetramer in solution. The purified enzyme showed optimum activity at pH and temperature of 7 and 60 °C, respectively, with obvious thermal stability. It exhibited strict substrate specificity towards L-asparagine with no detectable activity on L-glutamine. Pre-treatment of potato slices by CobAsnase prior to frying reduced the acrylamide contents in the processed chips up to 81% compared with untreated control. These results suggest that CobAsnase is a potential candidate for applications in the food industry for mitigation of acrylamide formation in fried potato and baked foods.

An l-glutaminase enzyme reactor based on porous bamboo sticks and its application in enzyme inhibitors screening.

Inspired by the porous and fibrous structure of commercially available bamboo, herein we created an l-glutaminase enzyme reactor based on bamboo sticks. The enzyme was immobilized onto the bamboo sticks through a glutaraldehyde modification to achieve covalent bonding. The enzymatic hydrolysis efficiency of the prepared l-glutaminase@bamboo sticks based porous enzyme reactor was evaluated by chiral ligand exchange capillary electrochromatography using l-glutamine as the substrate. l-glutaminase@bamboo exhibited improved enzymatic hydrolysis performances, including high hydrolysis efficiency (maximum rate Vmax: two fold higher than the free enzyme), prolonged stability (14 days) and good reusability. l-Glutaminase@bamboo sticks also expanded application capability in pharmaceutical industry in enzyme inhibitor screening. These excellent properties could be attributed to the micropores of bamboo sticks, which led to the fast enzymatic kinetics. The results suggest that the pores of bamboo sticks played an important role in the proposed enzyme reactor during the hydrolysis of l-glutamine and l-glutaminase inhibitor screening.

Effect of glutenin and gliadin modified by protein-glutaminase on retrogradation properties and digestibility of potato starch.

The glutenin (Glu) and gliadin (Gli) were modified by protein-glutaminase (PG) to obtain soluble glutenin (PG-Glu) and gliadin (PG-Gli), and PG-Glu or PG-Gli was added to potato starch (PS) according to different amounts (0.5%, 1.0%, and 1.5%, based on dry starch weight, w/w) to explore the effect of modified proteins on the retrogradation behavior and digestibility of PS. The results showed that the long-term retrogradation of PS was accelerated by the addition of PG-Glu or PG-Gli. The addition of PG-Glu or PG-Gli led to an increase in hydrogen bonds within starch molecules and induced a significant increase in resistant starch content. The hydrolysis kinetic parameters, C∞ and K, both decreased with the increasing level of modified protein, indicating the deceleration of hydrolysis rate by the addition of PG-Glu or PG-Gli. In summary, the addition of PG-Glu or PG-Gli could promote the retrogradation of PS and mitigate the digestion of starch.

In Vivo Preclinical Molecular Imaging of Repeated Exposure to an N-methyl-d-aspartate Antagonist and a Glutaminase Inhibitor as Potential Glutamatergic Modulators.

Glutamate is the principal excitatory neurotransmitter in the brain and is at the base of a wide variety of neuropathologies, including epilepsy, autism, Fragile X, and obsessive compulsive disorder. Glutamate has also become the target for novel drugs in treatment and in fundamental research settings. However, much remains unknown on the working mechanisms of these drugs and the effects of chronic administration on the glutamatergic system. This study investigated the chronic effects of two glutamate-modulating drugs with imaging techniques to further clarify their working mechanisms for future research opportunities. Animals were exposed to saline (1 ml/kg), (5S,10R)-(+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) (0.3 mg/kg), or ebselen (10 mg/kg) for 7 consecutive days. At the sixth injection, animals underwent a positron emission tomography (PET)/computed tomography (CT) with (3-(6-methyl-pyridin-2-ylethynyl)-cyclohex-2-enone-O-11C-methyl-oxime) (ABP-688) to visualize the metabotropic G protein-coupled glutamate receptor 5 (mGluR5). After the seventh injection, animals underwent a magnetic resonance spectroscopy (MRS) scan to visualize glutamate and glutamine content. Afterward, results were verified by mGluR5 immunohistochemistry (IHC). PET/CT analysis revealed that animals receiving chronic MK-801 or ebselen had a significant (P < 0.05) higher binding potential (2.90 ± 0.47 and 2.87 ± 0.46, respectively) when compared with saline (1.97 ± 0.39) in the caudate putamen. This was confirmed by mGluR5 IHC, with 60.83% ± 6.30% of the area being highlighted for ebselen and 57.14% ± 9.23% for MK-801 versus 50.21% ± 5.71% for the saline group. MRS displayed significant changes on the glutamine level when comparing chronic ebselen (2.20 ± 0.40 µmol/g) to control (2.72 ± 0.34 µmol/g). Therefore, although no direct effects on glutamate were visualized, the changes in glutamine suggest changes in the total glutamate-glutamine pool. This highlights the potential of both drugs to modulate glutamatergic pathologies.

Novel 1,3,4-Selenadiazole-Containing Kidney-Type Glutaminase Inhibitors Showed Improved Cellular Uptake and Antitumor Activity.

Kidney-type glutaminase [KGA/isoenzyme glutaminase C (GAC)] is becoming an important tumor metabolism target in cancer chemotherapy. Its allosteric inhibitor, CB839, showed early promise in cancer therapeutics but limited efficacy in in vivo cancer models. To improve the in vivo activity, we explored a bioisostere replacement of the sulfur atom in bis-2-(5-phenylacetamido-1,2,4-thiadiazol)ethyl sulfide and CB839 analogues with selenium using a novel synthesis of the selenadiazole moiety from carboxylic acids or nitriles. The resulting selenadiazole compounds showed enhanced KGA inhibition, more potent induction of reactive oxygen species, improved inhibition of cancer cells, and higher cellular and tumor accumulation than the corresponding sulfur-containing molecules. However, both CB839 and its selenium analogues show incomplete inhibition of the tested cancer cells, and a partial reduction in tumor size was observed in both the glutamine-dependent HCT116 and aggressive H22 liver cancer xenograft models. Despite this, tumor tissue damage and prolonged survival were observed in animals treated with the selenium analogue of CB839.

Metabolic Changes Associated with a Rat Model of Diabetic Depression Detected by Ex Vivo 1H Nuclear Magnetic Resonance Spectroscopy in the Prefrontal Cortex, Hippocampus, and Hypothalamus.

Diabetic patients often present with comorbid depression. However, the pathogenetic mechanisms underlying diabetic depression (DD) remain unclear. To explore the mechanisms underpinning the pathogenesis of the disease, we used ex vivo 1H nuclear magnetic resonance spectroscopy and immunohistochemistry to investigate the main metabolic and pathological changes in various rat brain areas in an animal model of DD. Compared with the control group, rats in the DD group showed significant decreases in neurotransmitter concentrations of glutamate (Glu) and glutamine (Gln) in the prefrontal cortex (PFC), hippocampus, and hypothalamus and aspartate and glycine in the PFC and hypothalamus. Gamma-aminobutyric acid (GABA) was decreased only in the hypothalamus. Levels of the energy product, lactate, were higher in the PFC, hippocampus, and hypothalamus of rats with DD than those in control rats, while creatine was lower in the PFC and hippocampus, and alanine was lower in the hypothalamus. The levels of other brain metabolites were altered, including N-acetyl aspartate, taurine, and choline. Immunohistochemistry analysis revealed that expressions of both glutamine synthetase and glutaminase were decreased in the PFC, hippocampus, and hypothalamus of rats with DD. The metabolic changes in levels of Glu, Gln, and GABA indicate an imbalance of the Glu-Gln metabolic cycle between astrocytes and neurons. Our results suggest that the development of DD in rats may be linked to brain metabolic changes, including inhibition of the Glu-Gln cycle, increases in anaerobic glycolysis, and disturbances in the lactate-alanine shuttle, and associated with dysfunction of neurons and astrocytes.