d-2-Hydroxyglutarate is an anti-inflammatory immunometabolite that accumulates in macrophages after TLR4 activation.

Macrophages undergo extensive metabolic rewiring upon activation which assist the cell in roles beyond energy production and synthesis of anabolic building blocks. So-called immunometabolites that accumulate upon immune activation can serve as co-factors for enzymes and can act as signaling molecules to modulate cellular processes. As such, the Krebs-cycle-associated metabolites succinate, itaconate and alpha-ketoglutarate (αKG) have emerged as key regulators of macrophage function. Here, we describe that 2-hydroxyglutarate (2HG), which is structurally similar to αKG and exists as two enantiomers, accumulates during later stages of LPS-induced inflammatory responses in mouse and human macrophages. D-2HG was the most abundant enantiomer in macrophages and its LPS-induced accumulation followed the induction of Hydroxyacid-Oxoacid Transhydrogenase (HOT). HOT interconverts αKG and gamma-hydroxybutyrate into D-2HG and succinic semialdehyde, and we here identified this enzyme as being immune-responsive and regulated during the course of macrophage activation. The buildup of D-2HG may be further explained by reduced expression of D-2HG Dehydrogenase (D2HGDH), which converts D-2HG back into αKG, and showed inverse kinetics with HOT and D-2HG levels. We tested the immunomodulatory effects of D-2HG during LPS-induced inflammatory responses by transcriptomic analyses and functional profiling of D-2HG-pre-treated macrophages in vitro and mice in vivo. Together, these data suggest a role for D-2HG in the negative feedback regulation of inflammatory signaling during late-stage LPS-responses in vitro and as a regulator of local and systemic inflammatory responses in vivo. Finally, we show that D-2HG likely exerts distinct anti-inflammatory effects, which are in part independent of αKG-dependent dioxygenase inhibition. Together, this study reveals an immunometabolic circuit resulting in the accumulation of the immunomodulatory metabolite D-2HG that can inhibit inflammatory macrophage responses.

Improved antiallodynic, antihyperalgesic and anti-inflammatory response achieved through potential prodrug of curcumin, curcumin diethyl diglutarate in a mouse model of neuropathic pain.

Neuropathic pain is a debilitating chronic pain condition, and its treatment remains a clinical challenge. Curcumin, a naturally occurring phenolic compound, possesses diverse biological and pharmacological effects but has not yet been approved as a drug due to its low bioavailability. In order to overcome this limitation, we synthesized a potential ester prodrug of curcumin, curcumin diethyl diglutarate (CurDDG). In this study, we evaluated the pharmacological advantages of CurDDG over curcumin in a mouse model of chronic constriction injury (CCI), and the anti-inflammatory effect of CurDDG in LPS-induced RAW 264.7 macrophage cells was accessed to clarify the underline mechanism. Mice were treated with various oral doses of curcumin (25, 50, 100 and 200 mg/kg/day, daily for 14 days) or equimolar doses of CurDDG. CurDDG at all doses tested significantly attenuated CCI-induced thermal hyperalgesia and mechanical allodynia compared with the CCI-control group. CurDDG at 25, 50 and 100 mg/kg demonstrated significantly greater efficacy on both mechanical and thermal hypersensitivities compared to that of curcumin. The effect of CurDDG correlated well with the inhibition of TNF-α and IL-6 levels in both the sciatic nerve and the spinal cord, as compared to its respective control groups. Similarly, in the in vitro study, CurDDG significantly reduced the LPS-induced expression of TNF-α and IL-6. Moreover, CurDDG significantly decreased COX-2 and iNOS levels and attenuated p38, JNK, and ERK1/2 phosphorylation as compared to the curcumin-treated cells. Altogether, this study demonstrated the improved pharmacological effects of curcumin by its diglutarate conjugate, CurDDG.

The oncometabolite R-2-hydroxyglutarate dysregulates the differentiation of human mesenchymal stromal cells via inducing DNA hypermethylation.

BACKGROUND: Isocitrate dehydrogenase (IDH1/2) gene mutations are the most frequently observed mutations in cartilaginous tumors. The mutant IDH causes elevation in the levels of R-enantiomer of 2-hydroxylglutarate (R-2HG). Mesenchymal stromal cells (MSCs) are reasonable precursor cell candidates of cartilaginous tumors. This study aimed to investigate the effect of oncometabolite R-2HG on MSCs. METHODS: Human bone marrow MSCs treated with or without R-2HG at concentrations 0.1 to 1.5 mM were used for experiments. Cell Counting Kit-8 was used to detect the proliferation of MSCs. To determine the effects of R-2HG on MSC differentiation, cells were cultured in osteogenic, chondrogenic and adipogenic medium. Specific staining approaches were performed and differentiation-related genes were quantified. Furthermore, DNA methylation status was explored by Illumina array-based arrays. Real-time PCR was applied to examine the signaling component mRNAs involved in. RESULTS: R-2HG showed no influence on the proliferation of human MSCs. R-2HG blocked osteogenic differentiation, whereas promoted adipogenic differentiation of MSCs in a dose-dependent manner. R-2HG inhibited chondrogenic differentiation of MSCs, but increased the expression of genes related to chondrocyte hypertrophy in a lower concentration (1.0 mM). Moreover, R-2HG induced a pronounced DNA hypermethylation state of MSC. R-2HG also improved promotor methylation of lineage-specific genes during osteogenic and chondrogenic differentiation. In addition, R-2HG induced hypermethylation and decreased the mRNA levels of SHH, GLI1and GLI2, indicating Sonic Hedgehog (Shh) signaling inhibition. CONCLUSIONS: The oncometabolite R-2HG dysregulated the chondrogenic and osteogenic differentiation of MSCs possibly via induction of DNA hypermethylation, improving the role of R-2HG in cartilaginous tumor development.

R-2-hydroxyglutarate attenuates aerobic glycolysis in leukemia by targeting the FTO/m6A/PFKP/LDHB axis.

R-2-hydroxyglutarate (R-2HG), a metabolite produced by mutant isocitrate dehydrogenases (IDHs), was recently reported to exhibit anti-tumor activity. However, its effect on cancer metabolism remains largely elusive. Here we show that R-2HG effectively attenuates aerobic glycolysis, a hallmark of cancer metabolism, in (R-2HG-sensitive) leukemia cells. Mechanistically, R-2HG abrogates fat-mass- and obesity-associated protein (FTO)/N6-methyladenosine (m6A)/YTH N6-methyladenosine RNA binding protein 2 (YTHDF2)-mediated post-transcriptional upregulation of phosphofructokinase platelet (PFKP) and lactate dehydrogenase B (LDHB) (two critical glycolytic genes) expression and thereby suppresses aerobic glycolysis. Knockdown of FTO, PFKP, or LDHB recapitulates R-2HG-induced glycolytic inhibition in (R-2HG-sensitive) leukemia cells, but not in normal CD34+ hematopoietic stem/progenitor cells, and inhibits leukemogenesis in vivo; conversely, their overexpression reverses R-2HG-induced effects. R-2HG also suppresses glycolysis and downregulates FTO/PFKP/LDHB expression in human primary IDH-wild-type acute myeloid leukemia (AML) cells, demonstrating the clinical relevance. Collectively, our study reveals previously unrecognized effects of R-2HG and RNA modification on aerobic glycolysis in leukemia, highlighting the therapeutic potential of targeting cancer epitranscriptomics and metabolism.

Inhibition of 2-hydroxyglutarate elicits metabolic reprogramming and mutant IDH1 glioma immunity in mice.

Mutant isocitrate dehydrogenase 1 (IDH1-R132H; mIDH1) is a hallmark of adult gliomas. Lower grade mIDH1 gliomas are classified into 2 molecular subgroups: 1p/19q codeletion/TERT-promoter mutations or inactivating mutations in α-thalassemia/mental retardation syndrome X-linked (ATRX) and TP53. This work focuses on glioma subtypes harboring mIDH1, TP53, and ATRX inactivation. IDH1-R132H is a gain-of-function mutation that converts α-ketoglutarate into 2-hydroxyglutarate (D-2HG). The role of D-2HG within the tumor microenvironment of mIDH1/mATRX/mTP53 gliomas remains unexplored. Inhibition of D-2HG, when used as monotherapy or in combination with radiation and temozolomide (IR/TMZ), led to increased median survival (MS) of mIDH1 glioma-bearing mice. Also, D-2HG inhibition elicited anti-mIDH1 glioma immunological memory. In response to D-2HG inhibition, PD-L1 expression levels on mIDH1-glioma cells increased to similar levels as observed in WT-IDH gliomas. Thus, we combined D-2HG inhibition/IR/TMZ with anti-PDL1 immune checkpoint blockade and observed complete tumor regression in 60% of mIDH1 glioma-bearing mice. This combination strategy reduced T cell exhaustion and favored the generation of memory CD8+ T cells. Our findings demonstrate that metabolic reprogramming elicits anti-mIDH1 glioma immunity, leading to increased MS and immunological memory. Our preclinical data support the testing of IDH-R132H inhibitors in combination with IR/TMZ and anti-PDL1 as targeted therapy for mIDH1/mATRX/mTP53 glioma patients.

Novel amide derivatives of 3-phenylglutaric acid as potent soluble epoxide hydrolase inhibitors.

Soluble epoxide hydrolase (sEH) enzyme plays an important role in the metabolism of endogenous chemical mediators, epoxyeicosatrienoic acids, which are involved in the regulation of blood pressure and inflammation. According to the pharmacophoric model suggested for sEH inhibitors, some new amide-based derivatives of 3-phenylglutaric acid were designed, synthesized and biologically evaluated. Docking study illustrated that the amide group as a primary pharmacophore had a suitable distance from the three amino acids of Tyr383, Tyr466 and Asp335 for effective hydrogen binding. Most of the compounds showed moderate to high sEH inhibitory activities in in vitro test in comparison with 12-(3-Adamantan-1-yl-ureido)-dodecanoic acid, as a potent urea-based sEH inhibitor. Compound 6o with phenethyl in R position exhibited the highest activity with IC50 value of 0.5 nM. In this study, some new amide-based derivatives of 3-phenylglutaric acid were designed, synthesized and biologically evaluated. Most of the synthesized compounds provided nanomolar range inhibition against sEH enzyme. The best observed IC50 value was 0.5 nM. Incorporating a carboxylic moiety into these structures by forming carboxylate salts would increase the solubility and improving physicochemical properties.

Neuroprotective effects of a dendrimer-based glutamate carboxypeptidase inhibitor on superoxide dismutase transgenic mice after neonatal hypoxic-ischemic brain injury.

The result of a deprivation of oxygen and glucose to the brain, hypoxic-ischemic encephalopathy (HIE), remains the most common cause of death and disability in human neonates globally and is mediated by glutamate toxicity and inflammation. We have previously shown that the enzyme glutamate carboxypeptidase (GCPII) is overexpressed in activated microglia in the presence of inflammation in fetal/newborn rabbit brain. We assessed the therapeutic utility of a GCPII enzyme inhibitor called 2-(3-Mercaptopropyl) pentanedioic acid (2MPPA) attached to a dendrimer (D-2MPPA), in order to target activated microglia in an experimental neonatal hypoxia-ischemia (HI) model using superoxide dismutase transgenic (SOD) mice that are often more injured after hypoxia-ischemia than wildtype animals. SOD overexpressing and wild type (WT) mice underwent permanent ligation of the left common carotid artery followed by 50 min of asphyxiation (10% O2) to induce HI injury on postnatal day 9 (P9). Cy5-labeled dendrimers were administered to the mice at 6 h, 24 h or 72 h after HI and brains were evaluated by immunofluorescence analysis 24 h after the injection to visualize microglial localization and uptake over time. Expression of GCPII enzyme was analyzed in microglia 24 h after the HI injury. The expression of pro- and anti-inflammatory cytokines were analyzed 24 h and 72 h post-HI. Brain damage was analyzed histologically 7 days post-HI in the three randomly assigned groups: control (C); hypoxic-ischemic (HI); and HI mice who received a single dose of D-2MPPA 6 h post-HI (HI+D-2MPPA). First, we found that GCPII was overexpressed in activated microglia 24 h after HI in the SOD overexpressing mice. Also, there was an increase in microglial activation 24 h after HI in the ipsilateral hippocampus which was most visible in the SOD+HI group. Dendrimers were mostly taken up by microglia by 24 h post-HI; uptake was more prominent in the SOD+HI mice than in the WT+HI. The inflammatory profile showed significant increase in expression of KC/GRO following injury in SOD mice compared to WT at 24 and 72 h. A greater and significant decrease in KC/GRO was seen in the SOD mice following treatment with D-2MPPA. Seven days after HI, D-2MPPA treatment decreased brain injury in the SOD+HI group, but not in WT+HI. This reduced damage was mainly seen in hippocampus and cortex. Our data indicate that the best time point to administer D-2MPPA is 6 h post-HI in order to suppress the expression of GCPII by 24 h after the damage since dendrimer localization in microglia is seen as early as 6 h with the peak of GCPII upregulation in activated microglia seen at 24 h post-HI. Ultimately, treatment with D-2MPPA at 6 h post-HI leads to a decrease in inflammatory profiles by 24 h and reduction in brain injury in the SOD overexpressing mice.

Identification of 2-PMPA as a novel inhibitor of cytosolic carboxypeptidases.

Cytosolic carboxypeptidases (CCPs) comprise a unique subfamily of M14 carboxypeptidases and are erasers of the reversible protein posttranslational modification- polyglutamylation. Potent inhibitors for CCPs may serve as leading compounds targeting imbalanced polyglutamylation. However, no efficient CCP inhibitor has yet been reported. Here, we showed that 2-phosphonomethylpentanedioic acid (2-PMPA), a potent inhibitor of the distant M28 family member glutamate carboxypeptidase II (GCPII), rather than the typical M14 inhibitor 2-benzylsuccinic acid, could efficiently inhibit CCP activities. 2-PMPA inhibited the recombinant Nna1 (a.k.a. CCP1) for hydrolyzing a synthetic peptide in a mixed manner, with Ki and Ki' being 0.11 µM and 0.24 µM respectively. It inhibited Nna1 for deglutamylating tubulin, the best-known polyglutamylated protein, with an IC50 of 0.21 mM. Homology modeling predicted that the R-form of 2-PMPA is more favorable to bind Nna1, unlike that GCPII prefers to S-form. This work for the first time identified a potent inhibitor for CCP family.

Sustained glial reactivity induced by glutaric acid may be the trigger to learning delay in early and late phases of development: Involvement of p75NTR receptor and protection by N-acetylcysteine.

Degeneration of striatal neurons and cortical atrophy are pathological characteristics of glutaric acidemia type I (GA-I), a disease characterized by accumulation of glutaric acid (GA). The mechanisms that lead to neuronal loss and cognitive impairment are still unclear. The purpose of this study was to verify if acute exposure to GA during the neonatal period is sufficient to trigger apoptotic processes and lead to learning delay in early and late period. Besides, whether N-acetylcysteine (NAC) would protect against impairment induced by GA. Pups mice received a dose of GA (2.5 µmol/ g) or saline, 12 hs after birth, and were treated with NAC (250 mg/kg) or saline, up to 21th day of life. Although GA exhibited deficits in the procedural and working memories in 21 and 40-day-old mice, NAC protected against cognitive impairment. In striatum and cortex, NAC prevented glial cells activation (GFAP and Iba-1), decreased NGF, Bcl-2 and NeuN, the increase of lipid peroxidation and PARP induced by GA in both ages. NAC protected against increased p75NTR induced by GA, but not in cortex of 21-day-old mice. Thus, we showed that the integrity of striatal and cortical pathways has an important role for learning and suggested that sustained glial reactivity in neonatal period can be an initial trigger for delay of cognitive development. Furthermore, NAC protected against cognitive impairment induced by GA. This work shows that early identification of the alterations induced by GA is important to avoid future clinical complications and suggest that NAC could be an adjuvant treatment for this acidemia.

Glycogen phosphorylase inhibitor, 2,3-bis[(2E)-3-(4-hydroxyphenyl)prop-2-enamido] butanedioic acid (BF142), improves baseline insulin secretion of MIN6 insulinoma cells.

Type 2 diabetes mellitus (T2DM), one of the most common metabolic diseases, is characterized by insulin resistance and inadequate insulin secretion of ß cells. Glycogen phosphorylase (GP) is the key enzyme in glycogen breakdown, and contributes to hepatic glucose production during fasting or during insulin resistance. Pharmacological GP inhibitors are potential glucose lowering agents, which may be used in T2DM therapy. A natural product isolated from the cultured broth of the fungal strain No. 138354, called 2,3-bis(4-hydroxycinnamoyloxy)glutaric acid (FR258900), was discovered a decade ago. In vivo studies showed that FR258900 significantly reduced blood glucose levels in diabetic mice. We previously showed that GP inhibitors can potently enhance the function of ß cells. The purpose of this study was to assess whether an analogue of FR258900 can influence ß cell function. BF142 (Meso-Dimethyl 2,3-bis[(E)-3-(4-acetoxyphenyl)prop-2-enamido]butanedioate) treatment activated the glucose-stimulated insulin secretion pathway, as indicated by enhanced glycolysis, increased mitochondrial oxidation, significantly increased ATP production, and elevated calcium influx in MIN6 cells. Furthermore, BF142 induced mTORC1-specific phosphorylation of S6K, increased levels of PDX1 and insulin protein, and increased insulin secretion. Our data suggest that BF142 can influence ß cell function and can support the insulin producing ability of ß cells.

Material-Sparing and Expedited Development of a Tablet Formulation of Carbamazepine Glutaric Acid Cocrystal- a QbD Approach.

PURPOSE: To efficiently develop a tablet formulation of carbamazepine using a soluble cocrystal with excess coformer to maintain phase stability during dissolution. METHODS: The carbamazepine - glutaric acid cocrystal (CBZ-GLA, 1:1) and excess glutaric acid (GLA) were mixed with suitable tablet excipients, which were selected to address powder flowability and tabletability deficiencies specifically. Tablet friability and dissolution profiles were evaluated to guide formulation optimization. Dry granules were prepared by milling simulated ribbons. RESULTS: A binary blend of CBZ-GLA and GLA had poor flowability and marginal tabletability. Therefore, silica coated Avicel PH-102 (sMCC) was applied as a binder to improve the flow property and tabletability. A formulation consisting of sMCC, CBZ-GLA, and GLA exhibited good manufacturability but did not show improved dissolution because of rapid precipitation of CBZ dihydrate when CBZ-GLA came in contact with water. Dry granulation of CBZ-GLA and GLA dramatically improved dissolution profile due to the intimate contact between CBZ-GLA and GLA. Such cocrystal - coformer granules also led to much improved tablet manufacturability and dissolution. CONCLUSION: The successful tablet development of CBZ-GLA, using < 3 g of the cocrystal in <3 weeks, demonstrates an efficient workflow for tablet formulation development based on material-sparing and predictive powder characterization techniques. This workflow is useful for early tablet development using enabling solid form, such as cocrystal, when only a small amount of material is available.

Novel Metal-Organic Framework-Based Photocrosslinked Hydrogel System for Efficient Antibacterial Applications.

Metal-organic frameworks (MOFs) can be applied in biology and medicine as drug delivery systems by carrying drugs on their surfaces or releasing bioactive ligands. To investigate the therapeutic potential of hydrogels that contain MOFs, three MOFs containing glutarate and 1,2-bis(4-pyridyl)ethylene ligands were synthesized by the previously reported hydrothermal or solvothermal reactions: Cu-MOF 1, Co-MOF 2, and Zn-MOF 3. Bioactive MOF-embedded hydrogels (hydrogel@Cu-MOF 1, hydrogel@Co-MOF 2, and hydrogel@Zn-MOF 3) were prepared by UV light-mediated thiol-ene photopolymerization using diacrylated polyethylene glycol (PEG), 4-arm-thiolated PEG, and MOFs. The activities of the MOF-embedded hydrogels were tested against the Gram-negative bacterium Escherichia coli and the Gram-positive bacterium Staphylococcus aureus. These MOF-embedded hydrogels were observed to be very stable, based on the release test of MII ions, and both hydrogel@Cu-MOF 1 and hydrogel@Co-MOF 2 showed excellent antibacterial activity. Although, in human dermal fibroblasts, hydrogel@Cu-MOF 1 showed no cytotoxic effects, it exhibited 99.9% antibacterial effects at the minimum bactericidal concentration. Physical properties such as the surface area and dimension of MOFs with different central metals appeared to be more important than the chemical properties of the ligands in determining the effects on bacteria. These MOF-embedded hydrogels may be useful in antibacterial applications such as cosmetics, treatment of skin diseases, and drug delivery owing to their low cytotoxicity and high bactericidal activity.

In vivo 2-hydroxyglutarate-proton magnetic resonance spectroscopy (3 T, PRESS technique) in treatment-naïve suspect lower-grade gliomas: feasibility and accuracy in a clinical setting.

Isocitrate dehydrogenase 1/2 (IDH1/2) mutations are often detected in lower-grade gliomas (LGG) and result into 2-hydroxyglutarate (2HG) synthesis. Prior studies showed that 2HG can be detected in vivo using magnetic resonance spectroscopy (MRS), but its accuracy and translational impact are still under investigation. PURPOSE: To investigate the clinical feasibility of MRS for in vivo detection and quantification of 2HG on consecutive treatment-naïve suspect LGG patients and to compare MRS accuracy with tissue IDH1/2 analysis. METHODS: MRS spectra at 3 T were acquired with 1H-MRS single-voxel PRESS 2HG-tailored sequences with TE 30 (group 1) or TE 97 (groups 2A and B). Voxel sizes were 1.5 × 1.5 × 1.5 cm3 for group 1 (n = 13) and group 2A (n = 14) and 2 × 2 × 2 cm3 for group 2B (n = 32). Multiple metabolites' concentrations were analyzed with LCModel. Tumors were assessed for IDH status and main molecular markers. 2HG levels in urine/blood were measured by liquid chromatography-mass spectrometry. RESULTS: The larger voxel TE 97 sequence resulted in highest specificity (100%), sensitivity (79%), and accuracy (87%). Urine and blood 2HG did not result predictive. CONCLUSION: Our data confirm that 2 × 2 × 2-cm3 voxel TE 97 MRS shows high accuracy for 2HG detection, with good sensitivity and 100% specificity in distinguishing IDH mutant gliomas. Main limits of the technique are small tumor volume and low cellularity. Integrating 2HG-MRS with other metabolites may help non-invasive diagnosis of glioma, prognostic assessment, and treatment planning in clinical setting.

2-hydroxyglutarate inhibits MyoD-mediated differentiation by preventing H3K9 demethylation.

Oncogenic IDH1/2 mutations produce 2-hydroxyglutarate (2HG), resulting in competitive inhibition of DNA and protein demethylation. IDH-mutant cancer cells show an inability to differentiate but whether 2HG accumulation is sufficient to perturb differentiation directed by lineage-specifying transcription factors is unknown. A MyoD-driven model was used to study the role of IDH mutations in the differentiation of mesenchymal cells. The presence of 2HG produced by oncogenic IDH2 blocks the ability of MyoD to drive differentiation into myotubes. DNA 5mC hypermethylation is dispensable while H3K9 hypermethylation is required for this differentiation block. IDH2-R172K mutation results in H3K9 hypermethylation and impaired accessibility at myogenic chromatin regions but does not result in genome-wide decrease in accessibility. The results demonstrate the ability of the oncometabolite 2HG to block transcription factor-mediated differentiation in a molecularly defined system.

Glutaric Acid Affects Pericyte Contractility and Migration: Possible Implications for GA-I Pathogenesis.

Glutaric acidemia I (GA-I) is an inherited neurometabolic childhood disease characterized by bilateral striatal neurodegeneration upon brain accumulation of millimolar concentrations of glutaric acid (GA) and related metabolites. Vascular dysfunction, including abnormal cerebral blood flow and blood-brain barrier damage, is an early pathological feature in GA-I, although the affected cellular targets and underlying mechanisms remain unknown. In the present study, we have assessed the effects of GA on capillary pericyte contractility in cerebral cortical slices and pericyte cultures, as well as on the survival, proliferation, and migration of cultured pericytes. GA induced a significant reduction in capillary diameter at distances up to ~ 10 µm from the center of pericyte somata. However, GA did not affect the contractility of cultured pericytes, suggesting that the response elicited in slices may involve GA evoking pericyte contraction by acting on other cellular components of the neurovascular unit. Moreover, GA indirectly inhibited migration of cultured pericytes, an effect that was dependent on soluble glial factors since it was observed upon application of conditioned media from GA-treated astrocytes (CM-GA), but not upon direct GA addition to the medium. Remarkably, CM-GA showed increased expression of cytokines and growth factors that might mediate the effects of increased GA levels not only on pericyte migration but also on vascular permeability and angiogenesis. These data suggest that some effects elicited by GA might be produced by altering astrocyte-pericyte communication, rather than directly acting on pericytes. Importantly, GA-evoked alteration of capillary pericyte contractility may account for the reduced cerebral blood flow observed in GA-I patients.

Acute lysine overload provokes marked striatum injury involving oxidative stress signaling pathways in glutaryl-CoA dehydrogenase deficient mice.

Glutaric acidemia type I (GA I) is a neurometabolic disorder of lysine (Lys) catabolism caused by glutaryl-CoA dehydrogenase (GCDH) deficiency. Patients are susceptible to develop acute striatum degeneration during catabolic stress situations whose underlying mechanisms are not fully established. Thus, in the present work we investigated the effects of a single intrastriatal Lys administration (1.5-4 µmol) to 30-day-old wild type (WT) and GCDH deficient (Gcdh-/-) mice on brain morphology, neuronal injury, astrocyte reactivity and myelin structure, as well as signaling pathways of redox homeostasis. We observed a marked vacuolation/edema in striatum and at higher doses also in cerebral cortex of Gcdh-/-, but not of WT mice. Lys also provoked a reduction of NeuN and synaptophysin, as well as an increase of astrocytic GFAP, in the striatum of Gcdh-/- mice, indicating neuronal loss and astrocyte reactivity. Furthermore, we verified an increase of Nrf2 and NF-κB expression in the nuclear fraction, and a decrease of heme oxygenase-1 (HO-1) content in the striatum of Lys-injected Gcdh-/- mice, implying disruption of redox homeostasis. Finally, it was found that Lys provoked alterations of myelin structure reflected by decreased myelin basic protein (MBP) in the cerebral cortex of Gcdh-/- mice. Taken together, the present data demonstrate neuronal loss, gliosis, altered redox homeostasis and demyelination caused by acute Lys overload in brain of Gcdh-/- mice, supporting the hypothesis that increased brain concentrations of glutaric and 3-hydroxyglutaric acids formed from Lys may be responsible for the acute brain degeneration observed in GA I patients during episodes of metabolic decompensation.

The oncometabolite 2-hydroxyglutarate inhibits microglial activation via the AMPK/mTOR/NF-κB pathway.

Microglia, the brain-resident macrophage, is known as the innate immune cell type in the central nervous system. Microglia is also the major cellular component of tumor mass of gliomas that plays a key role in glioma development. Mutations of isocitrate dehydrogenases 1 and 2 (IDH1/2) frequently occur in gliomas, which leads to accumulation of oncometabolic product 2-hydroxyglutarate (2HG). Moreover, IDH1/2 mutations were found to correlate with better prognosis in glioma patients. In the present study, we investigated the effects of the 2HG on microglial inflammatory activation. We showed that the conditioned media (CM) from GL261 glioma cells stimulated the activation of BV-2 microglia cells, evidenced by markedly increased expression of interleukin-6 (IL-6), IL-1ß, tumor necrosis factor-α (TNF-α), CCL2 (C-C motif chemokine ligand 2) and CXCL10 (C-X-C motif chemokine 10). CM-induced expression of proinflammatory genes was significantly suppressed by pretreatment with a synthetic cell-permeable 2HG (1 mM) or a nuclear factor-κB (NF-κB) inhibitor BAY11-7082 (10 µM). In lipopolysaccharide (LPS)- or TNF-α-stimulated BV-2 microglia cells and primary microglia, pretreatment with 2HG (0.25-1 mM) dose-dependently suppressed the expression of proinflammatory genes. We further demonstrated that 2HG significantly suppressed LPS-induced phosphorylation of IκB kinase α/ß (IKKα/ß), IκBα and p65, IκB degradation, and nuclear translocation of p65 subunit of NF-κB, as well as NF-κB transcriptional activity. Similarly, ectopic expression of mutant isocitrate dehydrogenase 1 (IDH1) (R132H) significantly decreased TNF-α-induced activation of NF-κB signaling pathway. Finally, we revealed that activation of adenosine 5'-monophosphate-activated protein kinase (AMPK) and subsequent inhibition of mammalian target of rapamycin (mTOR) signaling contributed to the inhibitory effect of 2HG on NF-κB signaling pathway in BV-2 cells. Taken together, these results, for the first time, show that oncometabolite 2HG inhibits microglial activation through affecting AMPK/mTOR/NF-κB signaling pathway and provide evidence that oncometabolite 2HG may regulate glioma development via modulating microglial activation in tumor microenvironment.

D-2-Hydroxyglutarate and L-2-Hydroxyglutarate Inhibit IL-12 Secretion by Human Monocyte-Derived Dendritic Cells.

Mutations in isocitrate dehydrogenase (IDH) or a reduced expression of L-2-hydroxyglutarate (HG)-dehydrogenase result in accumulation of D-2-HG or L-2-HG, respectively, in tumor tissues. D-2-HG and L-2-HG have been shown to affect T-cell differentiation and activation; however, effects on human myeloid cells have not been investigated so far. In this study we analyzed the impact of D-2-HG and L-2-HG on activation and maturation of human monocyte-derived dendritic cells (DCs). 2-HG was taken up by DCs and had no impact on cell viability but diminished CD83 expression after Lipopolysaccharides (LPS) stimulation. Furthermore, D-2-HG and L-2-HG significantly reduced IL-12 secretion but had no impact on other cytokines such as IL-6, IL-10 or TNF. Gene expression analyses of the IL-12 subunits p35/IL-12A and p40/IL-12B in DCs revealed decreased expression of both subunits. Signaling pathways involved in LPS-induced cytokine expression (NFkB, Akt, p38) were not altered by D-2-HG. However, 2-HG reprogrammed LPS-induced metabolic changes in DCs and increased oxygen consumption. Addition of the ATP synthase inhibitor oligomycin to DC cultures increased IL-12 secretion and was able to partially revert the effect of 2-HG. Our data show that both enantiomers of 2-HG can limit activation of DCs in the tumor environment.

The oncometabolite d­2­hydroxyglutarate induces angiogenic activity through the vascular endothelial growth factor receptor 2 signaling pathway.

The mutation of isocitrate dehydrogenase (IDH)1 (R132H) and IDH2 (R172K) and the induction of hypoxia in various solid tumors results in alterations in metabolic profiles, including the production of the d­ or l­forms of 2­hydroxyglutarate (2HG) from α­ketoglutarate in aerobic metabolism in the tricarboxylic acid (TCA) cycle. However, it is unclear whether the oncometabolite d­2HG increases angiogenesis in endothelial cells. Therefore, in this study, we analyzed the levels of various metabolites, including d­2HG, under hypoxic conditions and in IDH2R172K mutant breast cancer cells by mass spectrometry. We then further evaluated the effects of this metabolite on angiogenesis in breast cancer cells. The results revealed that treatment with d­2HG increased the levels of secreted vascular endothelial growth factor (VEGF) in cancer cells and enhanced endothelial cell proliferation in a concentration­dependent manner. Wound healing and cell migration (examined by Transwell assay) were significantly increased by d­2HG to a level similar to that induced by VEGF. Tube formation was significantly stimulated by d­2HG, and chick chorioallantoic membrane angiogenesis was also enhanced by d­2HG. d­2HG activated VEGF receptor (VEGFR)2 and VEGFR2 downstream signaling, extracellular signal­regulated kinase 1/2, focal adhesion kinase, AKT and matrix metalloproteinase (MMP)2. Taken together, the findings of this study suggested that d­2HG induced angiogenic activity via VEGFR2 signaling and increased MMP2 activity.

Chronopharmacological Characteristics of the Effect of Hepatoprotectors in Experiment?

We studied circadian rhythms of activity of hepatoprotectors (Antral, Carsil, and glutargin) under conditions of acute paracetamol-induced hepatitis simulated in the morning, afternoon, evening, and at night (09.00, 15.00, 21.00, and 03.00). Antral and Carsil exhibited similar chronoprofiles with the maximum hepatoprotective activity at 09.00 and 21.00, while glutargin exhibited circadian pattern opposite and its activity was maximum at 15.00 and 03.00.