Expression and Function of mARC: Roles in Lipogenesis and Metabolic Activation of Ximelagatran

<div><p>Recently two novel enzymes were identified in the outer mitochondrial membrane, mARC1 and mARC2. These molybdenum containing enzymes can reduce a variety of <i>N</i>-hydroxylated compounds, such as N-hydroxy-guanidines and sulfohydroxamic acids, as well as convert nitrite into nitric oxide (NO). However, their endogenous functions remain unknown. Here we demonstrate a specific developmental pattern of expression of these enzymes. mARC1, but not mARC2, was found to be expressed in fetal human liver, whereas both, in particular mARC2, are abundant in adult liver and also expressed in omental and subcutaneous fat. Caloric diet restriction of obese patients caused a decreased expression of mARC2 in liver, similar to that seen in the livers of starved rats. Knock down of mARC2 expression by siRNA in murine adipocytes had statistically significant effect on the level of diglycerides and on the fatty acid composition of some triglycerides, concomitantly a clear trend toward the reduced formation of most of triglyceride and phospholipid species was observed. The involvement of mARC2 in the metabolism of the hepatotoxic drug ximelagatran was evaluated in hepatocytes and adipocytes. Ximelagatran was shown to cause oxidative stress and knock down of mARC2 in adipocytes prevented ximelagatran induced inhibition of mitochondrial respiration. In conclusion, our data indicate that mARC1 and mARC2 have different developmental expression profiles, and that mARC2 is involved in lipogenesis, is regulated by nutritional status and responsible for activation of ximelagatran into a mitotoxic metabolite(s).</p></div

Statistical analysis of the mARC2 downregulation effect on specific lipids in the differentiated adipocytes (TG, triglycerides; DG, diglycerides).

<p>Statistical analysis of the mARC2 downregulation effect on specific lipids in the differentiated adipocytes (TG, triglycerides; DG, diglycerides).</p

Clinical characteristics of the obese patients.

<p>Clinical characteristics of the obese patients.</p

Nutritional status in both human and rats affects the mARC2 levels.

<p>A. mARC2 protein levels in obese patients that were put on a caloric restriction diet (fasted) prior to surgery (n = 7) and control (non-fasted) individuals (n = 7) (patient data are presented in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138487#pone.0138487.t001" target="_blank">Table 1</a>). Equal amounts of protein from the hepatic mitochondrial fractions were analyzed by western blot for mARC2 and loading control, mitochondrial heat shock protein 70 (mHSP70) (lower panels). B. Both bands were quantified by densitometric analysis and mARC2 levels were normalized by mHSP70. The results represent the mean ±S.D. (n = 7 in each group), **p<0.01. C. mARC2 protein levels and the associated amidoxime reductase activity are decreased in the starvation treated rats. The mitochondrial fractions from the livers from control (n = 3) and starvation treated (n = 3) animals were analyzed for amidoxime reductase activity using benzamidoxime as a substrate (left panel). The results are presented as the mean ± S.D. values. ***, p<0.001. The mitochondrial fractions from these animals were also analyzed by western blot for the presence of mARC2 (right panel). mHSP70, mitochondrial chaperone was used as a loading control.</p

EC₅₀ of EP1-4 receptor antagonists on neuroblastoma cell viability <i>in vitro</i>.

<p>Abbreviations: EC₅₀; effective concentration decreasing neuroblastoma cell viability with 50%,</p>a<p>MYCN amplification;</p>b<p>Multidrug-resistant phenotype.</p

dmPGE₂ increases intracellular Ca²⁺ and cAMP concentrations followed by phosphorylation of Akt.

<p>(A) Intracellular calcium mobilization in response to dmPGE₂. SK-N-SH cells were loaded with the calcium fluorescent dye Fluo-4/AM before the addition of 1 µM dmPGE₂ or (B) pre-treatment with 2 mM EGTA before exposure to 1 µM dmPGE₂. The fluorescence intensity was followed using a confocal laser scanning microscope and representative single-cell recordings are shown. The arrows indicate when dmPGE₂ is added. (C) Intracellular accumulation of cAMP in response to dmPGE₂. SK-N-SH cells were incubated overnight in a medium without serum before the addition of 1 µM of dmPGE₂. Pretreatment with 10 µM NF 449, which is a Gαs inhibitor, before the incubation in dmPGE₂ for 10 min inhibited the production of cAMP. Forskolin, 10 µM for 10 min, was used as a positive control. The graph shows mean (±SD) in % of untreated control of three independent experiments. A statistical analysis was performed using 2-sided t-test, P<0.05. (D) PGE₂ induces phosphorlyation of Akt. SK-N-BE(2) and SK-N-SH cells were grown in the presence of serum (Ctr) before 24 h of culturing in the absence of serum (0 h) prior to the addition of 1 µM of dmPGE₂. Cells were further incubated in dmPGE₂ for 1, 2, 4, 6, 12 or 24 h and protein extracts were subjected to western blotting to detect phosphorylated Akt(ser473). An antibody detecting unphosphorylated Akt was used to exclude differences in total protein expression. β-actin was used to control for equal protein loading. The western blots are representative of three independent experiments.</p

Neuroblastoma cells produce PGE₂ and dmPGE₂ increases cell viability.

<p>(A) Neuroblastoma cells produce PGE₂. SK-N-BE(2) and SK-N-SH cells were cultured with or without 40 µM of arachidonic acid (AA) for 48 h and 10 ng/mL IL-1β for 12 h. Cell homogenates were incubated with 80 µM of arachidonic acid and the concentration of produced PGE₂ was measured using LC-MS/MS. (B) PGE₂ increases neuroblastoma cell viability. SK-N-BE(2) and SK-N-SH cells were incubated in a serum-free medium for 24 h before adding different concentrations of dmPGE₂. Cell viability was measured using MTT-assay after 24, 48, 72 or 96 h. Values are representative of two independent experiments and data are expressed as mean (±SD) in percentage of control at 24 h. A statistical analysis was performed using 2-way ANOVA p<0.0001 for both concentration and incubation time. (C) PGE₂ rescues neuroblastoma cells from celecoxib induced apoptosis. SK-N-BE(2) cells were incubated in 35 µM celecoxib alone or in combination with 5 µM dmPGE₂. After 48 h cell viability was assessed using MTT-assay. Mean (±SD) of six replicate wells is shown; values are representative of three independent experiments. Statistical analysis was performed using 2-sided t test P<0.0001.</p

Microsomal Glutathione Transferase 1 Protects Against Toxicity Induced by Silica Nanoparticles but Not by Zinc Oxide Nanoparticles

Microsomal glutathione transferase 1 (MGST1) is an antioxidant enzyme located predominantly in the mitochondrial outer membrane and endoplasmic reticulum and has been shown to protect cells from lipid peroxidation induced by a variety of cytostatic drugs and pro-oxidant stimuli. We hypothesized that MGST1 may also protect against nanomaterial-induced cytotoxicity through a specific effect on lipid peroxidation. We evaluated the induction of cytotoxicity and oxidative stress by TiO₂, CeO₂, SiO₂, and ZnO in the human MCF-7 cell line with or without overexpression of MGST1. SiO₂ and ZnO nanoparticles caused dose- and time-dependent toxicity, whereas no obvious cytotoxic effects were induced by nanoparticles of TiO₂ and CeO₂. We also noted pronounced cytotoxicity for three out of four additional SiO₂ nanoparticles tested. Overexpression of MGST1 reversed the cytotoxicity of the main SiO₂ nanoparticles tested and for one of the supplementary SiO₂ nanoparticles but did not protect cells against ZnO-induced cytotoxic effects. The data point toward a role of lipid peroxidation in SiO₂ nanoparticle-induced cell death. For ZnO nanoparticles, rapid dissolution was observed, and the subsequent interaction of Zn²⁺ with cellular targets is likely to contribute to the cytotoxic effects. A direct inhibition of MGST1 by Zn²⁺ could provide a possible explanation for the lack of protection against ZnO nanoparticles in this model. Our data also showed that SiO₂ nanoparticle-induced cytotoxicity is mitigated in the presence of serum, potentially through masking of reactive surface groups by serum proteins, whereas ZnO nanoparticles were cytotoxic both in the presence and in the absence of serum