Grossamide attenuates inflammation by balancing macrophage polarization through metabolic reprogramming of macrophages in mice.

Macrophages exhibited different phenotypes in response to environmental cues. To meet the needs of rapid response to stimuli, M1-activated macrophages preferred glycolysis to oxidative phosphorylation (OXPHOS) in mitochondria to quickly produce energy and obtain ample raw materials to support cell activation at the same time. Activated macrophages produced free radicals and cytokines to eradicate pathogens but also induced oxidative damage and enhanced inflammation. Grossamide (GSE), a lignanamide from Polygonum multiflorum Thunb., exhibited notable anti-inflammatory effects. In this study, the potential of GSE on macrophage polarization was explored. GSE significantly down-regulated the levels of M1 macrophage biomarkers (Cd32a, Cd80 and Cd86) while increased the levels of M2 indicators (Cd163, Mrc1 and Socs1), showing its potential to inhibit LPS-induced M1 polarization of macrophages. This ability has close a link to its effect on metabolic reprogramming of macrophage. GSE shunted nitric oxide (NO) production from arginine by up-regulation of arginase and down-regulation of inducible nitric oxide synthase, thus attenuated the inhibition of NO on OXPHOS. LPS created three breakpoints in the tricarboxylic acid cycle (TCA) cycle of macrophage as evidenced by down-regulated isocitrate dehydrogenase, accumulation of succinate and the inhibited SDH activity, significantly decreased level of oxoglutarate dehydrogenase expression and its substrate α-ketoglutarate. Thus GSE reduced oxidative stress and amended fragmented TCA cycle. As a result, GSE maintained redox (NAD+/NADH) and energy (ATP/ADP) state, reduced extracellular acidification rate and enhanced the oxygen consumption rate. In addition, GSE decreased the release of inflammatory cytokines by inhibiting the activation of the LPS/TLR4/NF-κB pathway. These findings highlighted the central role of immunometabolism of macrophages in its functional plasticity, which invited future study of mode of action of anti-inflammatory drugs from viewpoint of metabolic reprogramming.

Effects of microplastics alone or with sorbed oil compounds from the water accommodated fraction of a North Sea crude oil on marine mussels (Mytilus galloprovincialis).

Microplastics (MPs) can adsorb persistent organic pollutants such as oil hydrocarbons and may facilitate their transfer to organisms (Trojan horse effect). The aim of this study was to examine the effects of a 21 day dietary exposure to polystyrene MPs of 4.5 µm at 1000 particles/mL, alone and with sorbed oil compounds from the water accommodated fraction (WAF) of a naphthenic North Sea crude oil at two dilutions (25 % and 100 %), on marine mussels. An additional group of mussels was exposed to 25 % WAF for comparison. PAHs were accumulated in mussels exposed to WAF but not in those exposed to MPs with sorbed oil compounds from WAF (MPs-WAF), partly due to the low concentration of PAHs in the studied crude oil. Exposure to MPs or to WAF alone altered the activity of enzymes involved in aerobic (isocitrate dehydrogenase) and biotransformation metabolism (glutathione S-transferase). Prevalence of oocyte atresia and volume density of basophilic cells were higher and absorption efficiency lower in mussels exposed to MPs and to WAF than in controls. After 21 days MPs caused DNA damage (Comet assay) in mussel hemocytes. In conclusion, a Trojan horse effect was not observed but both MPs and oil WAF caused an array of deleterious effects on marine mussels at different levels of biological organization.

2-Hydroxyglutarate produced by neomorphic IDH mutations suppresses homologous recombination and induces PARP inhibitor sensitivity.

2-Hydroxyglutarate (2HG) exists as two enantiomers, (R)-2HG and (S)-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations, whereas the latter is produced under pathologic processes such as hypoxia. We report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitors. This "BRCAness" phenotype of IDH mutant cells can be completely reversed by treatment with small-molecule inhibitors of the mutant IDH1 enzyme, and conversely, it can be entirely recapitulated by treatment with either of the 2HG enantiomers in cells with intact IDH1/2 proteins. We demonstrate mutant IDH1-dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH, rather than attempting to block 2HG production, by targeting the 2HG-dependent HR deficiency with PARP inhibition. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair, and genetic instability.

IDH1(R132H) mutation causes a less aggressive phenotype and radiosensitizes human malignant glioma cells independent of the oxygenation status.

BACKGROUND AND PURPOSE: In malignant glioma the presence of the IDH1 mutation (IDH1(R132H)) is associated with better clinical outcome. However, it is unclear whether IDH1 mutation is associated with a less aggressive phenotype or directly linked to increased sensitivity to radiotherapy. MATERIAL AND METHODS: We determined the influence of IDH1(R132H) mutant protein on proliferation and growth in 3D culture, migration, cell survival and radiosensitivity in vitro under normoxia (21% O2) and hypoxia (<1% O2) in a panel of human malignant glioma cell lines (U-251MG, U-343MG, LN-229) with stable overexpression of wild-type (IDH1(wt)) and mutated IDH1 (IDH1(R132H)). RESULTS: Overexpression of IDH1(R132H) in glioma cells resulted in slightly decreased cell proliferation, considerably reduced cell migration and caused differences in growth properties in 3D spheroid cultures. Furthermore, IDH1(R132H)-positive cells consistently demonstrated an increased radiosensitivity in human malignant glioma cells U-251MG (DMF10: 1.52, p<0.01 and 1.42, p<0.01), U-343MG (DMF10: 1.78, p<0.01 and 1.75, p<0.01) and LN-229 (DMF10: 1.41, p<0.05 and 1.68, p<0.01) under normoxia and hypoxia, respectively. CONCLUSION: Our data indicate that IDH1(R132H) mutation causes both a less aggressive biological behavior and direct radiosensitization of human malignant glioma cells. Targeting IDH1 appears to be an attractive approach in combination with radiotherapy.

Mitochondrial isocitrate dehydrogenase protects human neuroblastoma SH-SY5Y cells against oxidative stress.

The neuroprotective effect of mitochondrial isocitrate dehydrogenase (IDPm), an enzyme involved in the reduction of NADP(+) to NADPH and the supply of glutathione (GSH) in mitochondria, was examined using SH-SY5Y cells overexpressing IDPm (S1). S1 cells showed higher NADPH and GSH levels than vector transfectant (V) cells and were more resistant to staurosporine-induced cell death than controls. Staurosporine-induced cytochrome c release, caspase-3 activation, and production of reactive oxygen species (ROS) were significantly attenuated in S1 cells as compared to V cells and reduced by antioxidants, trolox and GSH-ethyl ester (GSH-EE). Staurosporine-induced the release of Mcl-1 from mitochondria that formed a complex with Bim. Mcl-1 was then cleaved to a shortened form in a caspase-3 dependent manner; its release was attenuated far more in S1 than in V cells after staurosporine treatment. Finally, the staurosporine-induced decrease in mitochondrial membrane potential (Deltapsi(m)) was correlated with the time of mitochondrial Mcl-1 release; the loss of Deltapsi(m) was attenuated significantly in S1 cells as compared to that in V cells. These results suggest that the neuroprotective effect of IDPm may result from increases in NADPH and GSH levels in the mitochondria. This, in turn, inhibits mitochondrial ROS production after cytochrome c release, which seems to be mediated through Mcl-1 release.

Anti-diabetic effects of DA-11004, a synthetic IDPc inhibitor in high fat high sucrose diet-fed C57BL/6J mice.

DA-11004 is a synthetic, potent NADP-dependent isocitrate dehydrogenase (IDPc) inhibitor where IC50 for IDPc is 1.49 microM. The purpose of this study was to evaluate the effects of DA-11004 on the high fat high sucrose (HF)-induced obesity in male C57BL/6J mice. After completing a 8-week period of experimentation, the mice were sacrificed 1 hr after the last DA-11004 treatment and their blood, liver, and adipose tissues (epididymal and retroperitoneal fat) were collected. There was a significant difference in the pattern of increasing body weight between the HF control and the DA-11004 group. In the DA-11004 (100 mg/kg) treated group the increase in body weight significantly declined and a content of epididymal fat and retroperitoneal fat was also significantly decreased as opposed to the HF control. DA-11004 (100 mg/ kg) inhibited the IDPc activity, and thus, NADPH levels in plasma and the levels of free fatty acid (FFA) or glucose in plasma were less than the levels of the HF control group. In conclusion, DA-11004 inhibited the fatty acid synthesis in adipose tissues via IDPc inhibition, and it decreased the plasma glucose levels and FFA in HF diet-induced obesity of C57BL/6J mice.

Isocitric and citric acid in human prostatic and seminal fluid: implications for prostatic metabolism and secretion.

Human prostatic secretion is remarkably rich in citric acid but the mechanisms to account for this accumulation are not well understood. One factor may be the extent of citrate oxidation to isocitrate, catalyzed by aconitase. The citrate-to-isocitrate ratio will help characterize the relative significance of this reaction in prostatic production and secretion of citrate. Isocitric acid and citric acid were measured in samples of seminal fluid and expressed prostatic secretion (EPS). A constant ratio between citrate and isocitrate of about 33:1 was found (r = 0.93, P < 0.0001) despite the wide variation in concentrations. Citrate ranged from 1 to 180 mM in EPS and from 13 to 50 mM in seminal fluid while isocitrate varied between 0 to 4.8 mM in EPS and from 0.4 to 1.5 mM in seminal fluid. Isocitrate is present in EPS and semen at much higher levels than found in most other animal or plant tissues or fluids and may be actively secreted by the same mechanism as citrate. The high citrate to isocitrate ratio of about 33:1, compared to the expected value of about 10:1, supports suggestions that citrate to isocitrate oxidation by aconitase is a rate limiting step in prostatic citrate metabolism. A low aconitase activity will therefore play a significant role in enabling accumulation of high citrate levels in prostatic epithelia and acini.

Stimulation of NADH oxidation by xanthine oxidase and polyvanadate in presence of some dehydrogenases and flavin compounds.

The rates of NADH oxidation in presence of xanthine oxidase increase to a small and variable extent on addition of high concentrations of lactate dehydrogenase and other dehydrogenases. This heat stable activity is similar to polyvanadate-stimulation with respect to pH profile and SOD sensitivity. Isocitric dehydrogenase (NADP-specific) showed heat labile, SOD-sensitive polyvanadate-stimulated NADH oxidation activity. Polyvanadate-stimulated SOD-sensitive NADH oxidation was also found to occur with riboflavin, FMN and FAD in presence of a non-specific protein, BSA, suggesting that some flavoproteins may possess this activity.

Wound healing in children as assessed by the CELLSTIC method.

Assessment of the quality and speed of wound healing in man has been rather difficult until the present time. By the use of CEELSTIC, a tissue-sensitive test drain composed of a standardized piece of viscose cellulose sponge inside a thin Silastic tube, cells existing between wound edges can be analyzed by histologic, cytologic, and enzyme histochemical means. The 166 pediatric surgical patients studied showed that wound healing is an age-dependent process reflected by the increased time needed for cellular transformation and maximal activation of hexokinase and isocitrate dehydrogenase with increasing age.