Effects of coenzyme Q10 on gene expression and inflammation: results from in silico, in vitro and in vivo studies

CoQ10 acts as an obligatory cofactor in the electron transport in the respiratory chain. Additionally, CoQ10 is required for the biosynthesis of pyrimidine nucleotides and the function of uncoupling proteins (UCPs). The reduced form of CoQ10 (Q10H2) serves as a potent antioxidant of lipid membranes. More recently, CoQ10 has been identified as a modulator of gene expression in vitro. This established function of CoQ10 was investigated in the present thesis on the molecular, cellular and physiological level by the use of bioinformatics and cell culture models as well as animal and human studies. Based on text mining analysis, a functional connection of CoQ10-sensitive genes was performed. Through signalling pathways of G-protein coupled receptors, JAK/STAT and integrin, 17 genes were functionally connected as previously published in Caco-2 cells. Moreover, promoter regions of genes related to inflammation revealed binding sites for the pivotal inflammatory transcription factor NFκB. To evaluate the data from the in silico analysis in an experimental context, monocytic cells were either treated with the oxidized (Q10) or reduced (Q10H2) form of CoQ10. Subsequently, the LPS-induced release of NFκB-dependent cytokines and chemokines was determined in cell-free supernatants. Finally, both for Q10- and Q10H2-incubated cells reduced secretion levels of the pro-inflammatory mediators TNFα, RANTES and MIP-1α have been observed. On the basis of the in vitro results, indicating slight differences in the anti-inflammatory properties of Q10 and Q10H2, redox-dependent gene expression patterns were hypothesized. To test this assumption in vivo, a genome-wide expression profiling was performed in various tissues (liver, kidney, heart and brain) of SAMP1 mice. Animals were either supplemented with Q10 or Q10H2 (500 mg/kg BW/d) for 6 (6 M) or 14 (14 M) months, respectively. In doing so, liver seemed to be the main target tissue of CoQ10 intervention, followed by kidney, heart and brain. In comparison to Q10, Q10H2 supplementation was more effective to increase total CoQ10 levels in liver tissues of SAMP1 mice. Evaluation of the array data also indicated a stronger impact on gene expression by Q10H2 when compared to Q10. Gene expression analysis in the liver of 14 M SAMP1 mice identified 11 Q10H2-sensitive genes primarily involved in cholesterol and lipid metabolism as well as inflammation and cell differentiation. Results from text mining revealed a functional connection of these genes in PPARα signalling pathways. Interestingly, these genes were not regulated in liver tissues of Q10-treated mice. Moreover, a key regulator gene in cholesterol metabolism, CYP51, was significantly down-regulated in the Q10H2-treated group, but became up-regulated in Q10-supplemented animals. Hence, the redox sensitivity of the identified genes might be a possible explanation for the observed differences in liver cholesterol levels of Q10H2- and Q10-supplemented mice. For further verification of the results obtained from in vitro experiments, 53 healthy male volunteers were supplemented with Q10H2 (150 mg/d) for 2 weeks. Based on microarray data and stringent selection criteria, 7 Q10H2-sensitive genes related to inflammatory and apoptotic processes were identified in isolated monocytes. For the identified Q10H2-sensitive genes, text mining analysis revealed a functional connection in NFκB and PPAR signalling pathways. As PPARs are known key players in lipid metabolism and cell differentiation, in addition to the transcriptional effects, a putative impact on physiological parameters such as LDL cholesterol and blood cell count was determined. Thereby, Q10H2 supplementation showed a significant reduction of LDL serum cholesterol levels. Additionally, due to the significant differences in the count of maturated red blood cells (erythrocytes) and immature reticulocytes, effects on cell differentiation processes were hypothesized. In summary, the results from the in silico, in vitro and in vivo studies show anti-inflammatory properties of Q10H2 as well as a regulatory role in cholesterol metabolism and cell differentiation processes. These effects could be explained, at least in part, by a modulatory impact of Q10H2 on redox-sensitive NFκB/PPARα dependent gene expression

Ubiquinol affects the expression of genes involved in PPARα signalling and lipid metabolism without changes in methylation of CpG promoter islands in the liver of mice

Coenzyme Q10 is an essential cofactor in the respiratory chain and serves as a potent antioxidant in biological membranes. Recent studies in vitro and in vivo provide evidence that Coenzyme Q10 is involved in inflammatory processes and lipid metabolism via gene expression. To study these effects at the epigenomic level, C57BL6J mice were supplemented for one week with reduced Coenzyme Q10 (ubiquinol). Afterwards, gene expression signatures and DNA promoter methylation patterns of selected genes were analysed. Genome-wide transcript profiling in the liver identified 1112 up-regulated and 571 down-regulated transcripts as differentially regulated between ubiquinol-treated and control animals. Text mining and GeneOntology analysis revealed that the ”top 20” ubiquinol-regulated genes play a role in lipid metabolism and are functionally connected by the PPARα signalling pathway. With regard to the ubiquinol-induced changes in gene expression of about +3.14-fold (p≤0.05), +2.18-fold (p≤0.01), and −2.13-fold (p≤0.05) for ABCA1, ACYP1, and ACSL1 genes, respectively, hepatic DNA methylation analysis of 282 (sense orientation) and 271 (antisense) CpG units in the respective promoter islands revealed no significant effect of ubiquinol. In conclusion, ubiquinol affects the expression of genes involved in PPARα signalling and lipid metabolism without changing the promoter DNA methylation status in the liver of mice

Paraoxonase 1 polymorphism Q192R affects the pro-inflammatory cytokine TNF-alpha in healthy males

<p>Abstract</p> <p>Background</p> <p>Human paraoxonase 1 (PON1) is an HDL-associated enzyme with anti-oxidant/anti-inflammatory properties that has been suggested to play an important protective role against coronary heart diseases and underlying atherogenesis. The common <it>PON1 </it>Q192R polymorphism (<it>rs662</it>, A>G), a glutamine to arginine substitution at amino acid residue 192, has been analyzed in numerous association studies as a genetic marker for coronary heart diseases, however, with controversial results.</p> <p>Findings</p> <p>To get a better understanding about the pathophysiological function of PON1, we analyzed the relationships between the Q192R polymorphism, serum paraoxonase activity and serum biomarkers important for atherogenesis. Genotyping a cohort of 49 healthy German males for the Q192R polymorphism revealed an allele distribution of 0.74 and 0.26 for the Q and R allele, respectively, typical for Caucasian populations. Presence of the R192 allele was found to be associated with a significantly increased paraoxonase enzyme activity of 187.8 ± 11.4 U/l in comparison to the QQ192 genotype with 60.5 ± 4.9 U/l. No significant differences among the genotypes were found for blood pressure, asymmetric dimethylarginine, LDL, HDL, triglycerides, and cholesterol. As expected, MIP-2 alpha a cytokine rather not related to atherosclerosis is not affected by the <it>PON1 </it>polymorphism. In contrast to that, the pro-inflammatory cytokine TNF-alpha is enhanced in R192 carriers (163.8 ± 24.7 pg/ml vs 94.7 ± 3.2 pg/ml in QQ192 carriers).</p> <p>Conclusions</p> <p>Our findings support the hypothesis that the common <it>PON1 </it>R192 allele may be a genetic risk factor for atherogenesis by inducing chronic low-grade inflammation.</p

Effects of Ubiquinol-10 on MicroRNA-146a Expression In Vitro and In Vivo

MicroRNAs (miRs) are involved in key biological processes via suppression of gene expression at posttranscriptional levels. According to their superior functions, subtle modulation of miR expression by certain compounds or nutrients is desirable under particular conditions. Bacterial lipopolysaccharide (LPS) induces a reactive oxygen species-/NF-κB-dependent pathway which increases the expression of the anti-inflammatory miR-146a. We hypothesized that this induction could be modulated by the antioxidant ubiquinol-10. Preincubation of human monocytic THP-1 cells with ubiquinol-10 reduced the LPS-induced expression level of miR-146a to 78.9 ± 13.22%. In liver samples of mice injected with LPS, supplementation with ubiquinol-10 leads to a reduction of LPS-induced miR-146a expression to 78.12 ± 21.25%. From these consistent in vitro and in vivo data, we conclude that ubiquinol-10 may fine-tune the inflammatory response via moderate reduction of miR-146a expression

Association between genetic variants in the Coenzyme Q10 metabolism and Coenzyme Q10 status in humans

<p>Abstract</p> <p>Background</p> <p>Coenzyme Q₁₀(CoQ₁₀) is essential for mitochondrial energy production and serves as an antioxidants in extra mitochondrial membranes. The genetics of primary CoQ₁₀deficiency has been described in several studies, whereas the influence of common genetic variants on CoQ₁₀status is largely unknown. Here we tested for non-synonymous single-nucleotidepolymorphisms (SNP) in genes involved in the biosynthesis (CoQ3^G272S, CoQ6^M406V, CoQ7^M103T), reduction (NQO1^P187S, NQO2^L47F) and metabolism (apoE3/4) of CoQ₁₀and their association with CoQ₁₀status. For this purpose, CoQ₁₀serum levels of 54 healthy male volunteers were determined before (T₀) and after a 14 days supplementation (T₁₄) with 150 mg/d of the reduced form of CoQ₁₀.</p> <p>Findings</p> <p>At T₀, the CoQ₁₀level of heterozygous NQO1^P187Scarriers were significantly lower than homozygous S/S carriers (0.93 ± 0.25 μM versus 1.34 ± 0.42 μM, p = 0.044). For this polymorphism a structure homology-based method (PolyPhen) revealed a possibly damaging effect on NQO1 protein activity. Furthermore, CoQ₁₀plasma levels were significantly increased in apoE4/E4 genotype after supplementation in comparison to apoE2/E3 genotype (5.93 ± 0.151 μM versus 4.38 ± 0.792 μM, p = 0.034). Likewise heterozygous CoQ3^G272Scarriers had higher CoQ₁₀plasma levels at T₁₄compared to G/G carriers but this difference did not reach significance (5.30 ± 0.96 μM versus 4.42 ± 1.67 μM, p = 0.082).</p> <p>Conclusions</p> <p>In conclusion, our pilot study provides evidence that NQO1^P187Sand apoE polymorphisms influence CoQ₁₀status in humans.</p

Ascorbic acid partly antagonizes resveratrol mediated heme oxygenase-1 but not paraoxonase-1 induction in cultured hepatocytes - role of the redox-regulated transcription factor Nrf2

<p>Abstract</p> <p>Background</p> <p>Both resveratrol and vitamin C (ascorbic acid) are frequently used in complementary and alternative medicine. However, little is known about the underlying mechanisms for potential health benefits of resveratrol and its interactions with ascorbic acid.</p> <p>Methods</p> <p>The antioxidant enzymes heme oxygenase-1 and paraoxonase-1 were analysed for their mRNA and protein levels in HUH7 liver cells treated with 10 and 25 μmol/l resveratrol in the absence and presence of 100 and 1000 μmol/l ascorbic acid. Additionally the transactivation of the transcription factor Nrf2 and paraoxonase-1 were determined by reporter gene assays.</p> <p>Results</p> <p>Here, we demonstrate that resveratrol induces the antioxidant enzymes heme oxygenase-1 and paraoxonase-1 in cultured hepatocytes. Heme oxygenase-1 induction by resveratrol was accompanied by an increase in Nrf2 transactivation. Resveratrol mediated Nrf2 transactivation as well as heme oxygenase-1 induction were partly antagonized by 1000 μmol/l ascorbic acid.</p> <p>Conclusions</p> <p>Unlike heme oxygenase-1 (which is highly regulated by Nrf2) paraoxonase-1 (which exhibits fewer ARE/Nrf2 binding sites in its promoter) induction by resveratrol was not counteracted by ascorbic acid. Addition of resveratrol to the cell culture medium produced relatively low levels of hydrogen peroxide which may be a positive hormetic redox-signal for Nrf2 dependent gene expression thereby driving heme oxygenase-1 induction. However, high concentrations of ascorbic acid manifold increased hydrogen peroxide production in the cell culture medium which may be a stress signal thereby disrupting the Nrf2 signalling pathway.</p

Ubiquinol decreases monocytic expression and DNA methylation of the pro-inflammatory chemokine ligand 2 gene in humans

<p>Abstract</p> <p>Background</p> <p>Coenzyme Q₁₀ is an essential cofactor in the respiratory chain and serves in its reduced form, ubiquinol, as a potent antioxidant. Studies <it>in vitro</it> and <it>in vivo</it> provide evidence that ubiquinol reduces inflammatory processes via gene expression. Here we investigate the putative link between expression and DNA methylation of ubiquinol sensitive genes in monocytes obtained from human volunteers supplemented with 150 mg/ day ubiquinol for 14 days.</p> <p>Findings</p> <p>Ubiquinol decreases the expression of the pro-inflammatory chemokine (C-X-C motif) ligand 2 gene (CXCL2) more than 10-fold. Bisulfite-/ MALDI-TOF-based analysis of regulatory regions of the CXCL2 gene identified six adjacent CpG islands which showed a 3.4-fold decrease of methylation status after ubiquinol supplementation. This effect seems to be rather gene specific, because ubiquinol reduced the expression of two other pro-inflammatory genes (PMAIP1, MMD) without changing the methylation pattern of the respective gene.</p> <p>Conclusion</p> <p>In conclusion, ubiquinol decreases monocytic expression and DNA methylation of the pro-inflammatory CXCL2 gene in humans. Current Controlled Trials ISRCTN26780329.</p