Extracellular vesicle release and uptake by the liver under normo- and hyperlipidemia.

Liver plays a central role in elimination of circulating extracellular vesicles (EVs), and it also significantly contributes to EV release. However, the involvement of the different liver cell populations remains unknown. Here, we investigated EV uptake and release both in normolipemia and hyperlipidemia. C57BL/6 mice were kept on high fat diet for 20-30 weeks before circulating EV profiles were determined. In addition, control mice were intravenously injected with 99mTc-HYNIC-Duramycin labeled EVs, and an hour later, biodistribution was analyzed by SPECT/CT. In vitro, isolated liver cell types were tested for EV release and uptake with/without prior fatty acid treatment. We detected an elevated circulating EV number after the high fat diet. To clarify the differential involvement of liver cell types, we carried out in vitro experiments. We found an increased release of EVs by primary hepatocytes at concentrations of fatty acids comparable to what is characteristic for hyperlipidemia. When investigating EV biodistribution with 99mTc-labeled EVs, we detected EV accumulation primarily in the liver upon intravenous injection of mice with medium (326.3 ± 19.8 nm) and small EVs (130.5 ± 5.8 nm). In vitro, we found that medium and small EVs were preferentially taken up by Kupffer cells, and liver sinusoidal endothelial cells, respectively. Finally, we demonstrated that in hyperlipidemia, there was a decreased EV uptake both by Kupffer cells and liver sinusoidal endothelial cells. Our data suggest that hyperlipidema increases the release and reduces the uptake of EVs by liver cells. We also provide evidence for a size-dependent differential EV uptake by the different cell types of the liver. The EV radiolabeling protocol using 99mTc-Duramycin may provide a fast and simple labeling approach for SPECT/CT imaging of EVs biodistribution.

Molecular Mechanisms Underlying the Elevated Expression of a Potentially Type 2 Diabetes Mellitus Associated SCD1 Variant.

Disturbances in lipid metabolism related to excessive food intake and sedentary lifestyle are among major risk of various metabolic disorders. Stearoyl-CoA desaturase-1 (SCD1) has an essential role in these diseases, as it catalyzes the synthesis of unsaturated fatty acids, both supplying for fat storage and contributing to cellular defense against saturated fatty acid toxicity. Recent studies show that increased activity or over-expression of SCD1 is one of the contributing factors for type 2 diabetes mellitus (T2DM). We aimed to investigate the impact of the common missense rs2234970 (M224L) polymorphism on SCD1 function in transfected cells. We found a higher expression of the minor Leu224 variant, which can be attributed to a combination of mRNA and protein stabilization. The latter was further enhanced by various fatty acids. The increased level of Leu224 variant resulted in an elevated unsaturated: saturated fatty acid ratio, due to higher oleate and palmitoleate contents. Accumulation of Leu224 variant was found in a T2DM patient group, however, the difference was statistically not significant. In conclusion, the minor variant of rs2234970 polymorphism might contribute to the development of obesity-related metabolic disorders, including T2DM, through an increased intracellular level of SCD1.

Different Metabolism and Toxicity of TRANS Fatty Acids, Elaidate and Vaccenate Compared to Cis-Oleate in HepG2 Cells.

Trans fatty acids (TFAs) are not synthesized in the human body but are generally ingested in substantial amounts. The widespread view that TFAs, particularly those of industrial origin, are unhealthy and contribute to obesity, cardiovascular diseases and diabetes is based mostly on in vivo studies, and the underlying molecular mechanisms remain to be elucidated. Here, we used a hepatoma model of palmitate-induced lipotoxicity to compare the metabolism and effects of the representative industrial and ruminant TFAs, elaidate and vaccenate, respectively, with those of cis-oleate. Cellular FAs, triacylglycerols, diacylglycerols and ceramides were quantitated using chromatography, markers of stress and apoptosis were assessed at mRNA and protein levels, ultrastructural changes were examined by electron microscopy and viability was evaluated by MTT assay. While TFAs were just slightly more damaging than oleate when applied alone, they were remarkably less protective against palmitate toxicity in cotreatments. These differences correlated with their diverse incorporation into the accumulating diacylglycerols and ceramides. Our results provide in vitro evidence for the unfavorable metabolic features and potent stress-inducing character of TFAs in comparison with oleate. These findings strengthen the reasoning against dietary trans fat intake, and they can also help us better understand the molecular mechanisms of lipotoxicity.

Gene expression analysis indicates reduced memory and cognitive functions in the hippocampus and increase in synaptic reorganization in the frontal cortex 3 weeks after MDMA administration in Dark Agouti rats.

BACKGROUND: 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") is a widely used entactogenic drug known to impair cognitive functions on the long-run. Both hippocampal and frontal cortical regions have well established roles in behavior, memory formation and other cognitive tasks and damage of these regions is associated with altered behavior and cognitive functions frequently described in otherwise healthy MDMA users. Meanwhile, in post-traumatic stress disorder (PTSD) patients seem to benefit from therapeutic application of the drug, where damage in hippocampal cue extinction may play a role. The aim of this study was to examine the hippocampus, frontal cortex and dorsal raphe of Dark Agouti rats with gene expression arrays (Illumina RatRef bead arrays) looking for possible mechanisms and new candidates contributing to the consequences of a single dose of MDMA (15 mg/kg) 3 weeks earlier. RESULTS: The number of differentially expressed genes in the hippocampus, frontal cortex and the dorsal raphe were 481, 155, and 15, respectively. Gene set enrichment analysis of the microarray data revealed reduced expression of 'memory' and 'cognition', 'dendrite development' and 'regulation of synaptic plasticity' gene sets in the hippocampus, parallel to the downregulation of CaMK II subunits, glutamate-, CB1 cannabinoid- and EphA4, EphA5, EphA6 receptors. Downregulated gene sets in the frontal cortex were related to protein synthesis, chromatin organization, transmembrane transport processes, while 'dendrite development', 'regulation of synaptic plasticity' and 'positive regulation of synapse assembly' gene sets were upregulated besides elevated levels of a CaMK II subunit and NMDA2B glutamate receptor. Changes in the dorsal raphe region were mild and in most cases not significant. CONCLUSION: The present data raise the possibility of new synapse formation / synaptic reorganization in the frontal cortex 3 weeks after a single neurotoxic dose of MDMA. In contrast, a prolonged depression of new neurite formation in the hippocampus is proposed by downregulations of members in long-term potentiation pathway and synaptic plasticity emphasizing the particular vulnerability of this brain region and proposing a mechanism responsible for cognitive problems in healthy individuals. At the same time, these results underpin benefits of MDMA in PTSD, where the drug may help memory extinction.

The emerging role of aryl hydrocarbon receptor in the activation and differentiation of Th17 cells.

The aryl hydrocarbon receptor (AHR) is a cytoplasmic transcription factor, which plays an essential role in the xenobiotic metabolism in a wide variety of cells. The AHR gene is evolutionarily conserved and it has a central role not only in the differentiation and maturation of many tissues, but also in the toxicological metabolism of the cell by the activation of metabolizing enzymes. Several lines of evidence support that both AHR agonists and antagonists have profound immunological effects; and recently, the AHR has been implicated in antibacterial host defense. According to recent studies, the AHR is essential for the differentiation and activation of T helper 17 (Th17) cells. It is well known that Th17 cells have a central role in the development of inflammation, which is crucial in the defense against pathogens. In addition, Th17 cells play a major role in the pathogenesis of several autoimmune diseases such as rheumatoid arthritis. Therefore, the AHR may provide connection between the environmental chemicals, the immune regulation, and autoimmunity. In the present review, we summarize the role of the AHR in the Th17 cell functions.

Differential detergent sensitivity of extracellular vesicle subpopulations.

Extracellular vesicles (including exosomes, microvesicles and apoptotic bodies) are currently attracting rapidly increasing attention from various fields of biology due to their ability to carry complex information and act as autocrine, paracrine and even endocrine intercellular messengers. In the present study we investigated the sensitivity of size-based subpopulations of extracellular vesicles to different concentrations of detergents including sodium dodecyl sulphate, Triton X-100, Tween 20 and deoxycholate. We determined the required detergent concentration that lysed each of the vesicle subpopulations secreted by Jurkat, THP-1, MiaPaCa and U937 human cell lines. We characterized the vesicles by tunable resistive pulse sensing, flow cytometry and transmission electron microscopy. Microvesicles and apoptotic bodies were found to be more sensitive to detergent lysis than exosomes. Furthermore, we found evidence that sodium dodecyl sulphate and Triton X-100 were more effective in vesicle lysis at low concentrations than deoxycholate or Tween 20. Taken together, our data suggest that a combination of differential detergent lysis with tunable resistive pulse sensing or flow cytometry may prove useful for simple and fast differentiation between exosomes and other extracellular vesicle subpopulations as well as between vesicular and non-vesicular structures.

Allele-specific effect of various dietary fatty acids and ETS1 transcription factor on SCD1 expression.

Overnutrition and genetic predisposition are major risk factors for various metabolic disorders. Stearoyl-CoA desaturase-1 (SCD1) plays a key role in these conditions by synthesizing unsaturated fatty acids (FAs), thereby promoting fat storage and alleviating lipotoxicity. Expression of SCD1 is influenced by various saturated and cis-unsaturated FAs, but the possible role of dietary trans FAs (TFAs) and SCD1 promoter polymorphisms in its regulations has not been addressed. Therefore, we aimed to investigate the impact of the two main TFAs, vaccenate and elaidate, and four common promoter polymorphisms (rs1054411, rs670213, rs2275657, rs2275656) on SCD1 expression in HEK293T and HepG2 cell cultures using luciferase reporter assay, qPCR and immunoblotting. We found that SCD1 protein and mRNA levels as well as SCD1 promoter activity are markedly elevated by elaidate, but not altered by vaccenate. The promoter polymorphisms did not affect the basal transcriptional activity of SCD1. However, the minor allele of rs1054411 increased SCD1 expression in the presence of various FAs. Moreover, this variant was predicted in silico and verified in vitro to reduce the binding of ETS1 transcription factor to SCD1 promoter. Although we could not confirm an association with type 2 diabetes mellitus, the FA-dependent and ETS1-mediated effect of rs1054411 polymorphism deserves further investigation as it may modulate the development of lipid metabolism-related conditions.

Gene expression analysis indicates CB1 receptor upregulation in the hippocampus and neurotoxic effects in the frontal cortex 3 weeks after single-dose MDMA administration in Dark Agouti rats.

BACKGROUND: 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") is a widely used recreational drug known to impair cognitive functions on the long-run. Both hippocampal and frontal cortical regions have well established roles in behavior, memory formation and other cognitive tasks and damage of these regions is associated with altered behavior and cognitive functions, impairments frequently described in heavy MDMA users. The aim of this study was to examine the hippocampus, frontal cortex and dorsal raphe of Dark Agouti rats with gene expression arrays (Illumina RatRef bead arrays) looking for possible mechanisms and new candidates contributing to the effects of a single dose of MDMA (15 mg/kg) 3 weeks earlier. RESULTS: The number of differentially expressed genes in the hippocampus, frontal cortex and the dorsal raphe were 481, 155, and 15, respectively. Gene set enrichment analysis of the microarray data revealed reduced expression of 'memory' and 'cognition', 'dendrite development' and 'regulation of synaptic plasticity' gene sets in the hippocampus, parallel to the upregulation of the CB1 cannabinoid- and Epha4, Epha5, Epha6 ephrin receptors. Downregulated gene sets in the frontal cortex were related to protein synthesis, chromatin organization, transmembrane transport processes, while 'dendrite development', 'regulation of synaptic plasticity' and 'positive regulation of synapse assembly' gene sets were upregulated. Changes in the dorsal raphe region were mild and in most cases not significant. CONCLUSION: The present data raise the possibility of new synapse formation/synaptic reorganization in the frontal cortex three weeks after a single neurotoxic dose of MDMA. In contrast, a prolonged depression of new neurite formation in the hippocampus is suggested by the data, which underlines the particular vulnerability of this brain region after the drug treatment. Finally, our results also suggest the substantial contribution of CB1 receptor and endocannabinoid mediated pathways in the hippocampal impairments. Taken together the present study provides evidence for the participation of new molecular candidates in the long-term effects of MDMA.

Role of Extracellular Vesicles in Liver Diseases.

Extracellular vesicles (EVs) are cell-derived membrane structures that are formed by budding from the plasma membrane or originate from the endosomal system. These microparticles (100 nm-100 µm) or nanoparticles (>100 nm) can transport complex cargos to other cells and, thus, provide communication and intercellular regulation. Various cells, such as hepatocytes, liver sinusoidal endothelial cells (LSECs) or hepatic stellate cells (HSCs), secrete and take up EVs in the healthy liver, and the amount, size and content of these vesicles are markedly altered under pathophysiological conditions. A comprehensive knowledge of the modified EV-related processes is very important, as they are of great value as biomarkers or therapeutic targets. In this review, we summarize the latest knowledge on hepatic EVs and the role they play in the homeostatic processes in the healthy liver. In addition, we discuss the characteristic changes of EVs and their potential exacerbating or ameliorating effects in certain liver diseases, such as non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease (AFLD), drug induced liver injury (DILI), autoimmune hepatitis (AIH), hepatocarcinoma (HCC) and viral hepatitis.

Decreasing effects of protein kinase inhibitors on the expression of NOS2 and inflammatory cytokines and on phagocytosis in rat peritoneal macrophages is partly related to repolarization.

The JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) pathway plays a pivotal role in macrophage polarization, but other signaling routes may also be involved. The aim of this study was to reveal the relationship of activation between rat peritoneal macrophages and their polarization, to detect the signaling routes involved, and find selective protein kinase inhibitors decreasing the production of inflammatory proteins in activated peritoneal macrophages. Rat macrophages were elicited with i.p. casein injection. CD80 and CD206 markers, NOS2 (Nitric oxide synthase 2), arginase, cytokines and phagocytosis were investigated by ELISA (Enzyme Linked Immunosorbent Assay), Western Blot, fluorescent microscopic and flow cytometry. Statistical methods were ANOVA (Analysis Of Variance) and Student t-tests. Resident and elicited cells expressed both CD80 and CD206 polarization markers. The involvement of MAPK (mitogen-activated protein kinases) and JAK/STAT pathways in the polarization was evidenced by a phosphorylation array, supported by Western blotting, by cytokine markers and by the inhibitory effects of kinase inhibitors. The expression of NOS2 and inflammatory cytokines was higher in elicited cells suggesting their M1 polarization. This effect was reduced by the inhibitors of MAPK and JAK/STAT pathways. Phagocytosis was also higher in elicited macrophages and decreased by these inhibitors. Nevertheless, they cannot change macrophage polarization unambiguously, as levels of CD80 and CD206 markers were not changed. For comparison, human blood macrophages were also studied. Similar effects and several differences were observed between the two types of macrophages, suggesting the role of the previous differentiation in defining their characteristics. Selected anti-cancer protein kinase inhibitors of p38, MAPK and JAK/STAT pathways are possible candidates for the therapy of inflammatory diseases.

High fat diet and PCSK9 knockout modulates lipid profile of the liver and changes the expression of lipid homeostasis related genes.

BACKGROUND: High fat diet (HFD) increases the likelihood of dyslipidemia, which can be a serious risk factor for atherosclerosis, diabetes or hepatosteatosis. Although changes in different blood lipid levels were broadly investigated, such alterations in the liver tissue have not been studied before. The aim of the current study was to investigate the effect of HFD on hepatic triglyceride (TG), diglyceride (DG) and ceramide (CER) levels and on the expression of four key genes involved in lipid homeostasis (Pcsk9, Ldlr, Cd36 and Anxa2) in the liver. In addition, the potential role of PCSK9 in the observed changes was further investigated by using PCSK9 deficient mice. METHODS: We used two in vivo models: mice kept on HFD for 20 weeks and PCSK9-/- mice. The amount of the major TGs, DGs and CERs was measured by using HPLC-MS/MS analysis. The expression profiles of four lipid related genes, namely Pcsk9, Ldlr, Cd36 and Anxa2 were assessed. Co-localization studies were performed by confocal microscopy. RESULTS: In HFD mice, hepatic PCSK9 expression was decreased and ANXA2 expression was increased both on mRNA and protein levels, and the amount of LDLR and CD36 receptor proteins was increased. While LDLR protein level was also elevated in the livers of PCSK9-/- mice, there was no significant change in the expression of ANXA2 and CD36 in these animals. HFD induced a significant elevation in the hepatic levels of all measured TG and DG but not of CER types, and increased the proportion of monounsaturated vs. saturated TGs and DGs. Similar changes were detected in the hepatic lipid profiles of HFD and PCSK9-/- mice. Co-localization of PCSK9 with LDLR, CD36 and ANXA2 was verified in HepG2 cells. CONCLUSIONS: Our results show that obesogenic HFD downregulates PCSK9 expression in the liver and causes alterations in the hepatic lipid accumulation, which resemble those observed in PCSK9 deficiency. These findings suggest that PCSK9-mediated modulation of LDLR and CD36 expression might contribute to the HFD-induced changes in lipid homeostasis.

Role of xenobiotic metabolism in cancer: involvement of transcriptional and miRNA regulation of P450s.

Cytochrome P450 enzymes (P450s) are important targets in cancer, due to their role in xenobiotic metabolism. Since P450s are the "bridges" between the environment and our body, their function can be linked in many ways to carcinogenesis: they activate dietary and environmental components to ultimate carcinogens (i), the cancer tissue maintains its drug resistance with altered expression of P450s (ii), P450s metabolize (sometimes activate) drugs used for cancer treatment (iii) and they are potential targets for anticancer therapy (iiii). These highly polymorphic enzymes are regulated at multiple molecular levels. Regulation is as important as genetic difference in the existing individual variability in P450 activity. In this review, examples of the transcriptional (DNA methylation, histone modification, modulation by xenosensors) and post-transcriptional (miRNA) regulation will be presented and thereby introduce potential molecular targets at which the metabolism of anticancer drugs, the elimination of cancerogenes or the progress of carcinogenesis could be affected.

Chronic Exposure to the Food Additive tBHQ Modulates Expression of Genes Related to SARS-CoV-2 and Influenza Viruses.

BACKGROUND: tert-butylhydroquinone (tBHQ) is an antioxidant commonly used as a food additive. Studies suggest that tBHQ could modulate immune responses to influenza and SARS-CoV-2 infection. In our transcriptomic analysis we explored the molecular mechanisms behind tBHQ's modulatory properties and the relationships to respiratory viral infections. METHODS: tBHQ was administered per os to BALB/c mice (1.5% [w/w]) for 20 days. Splenic T cells were isolated with magnetic separation and subjected to transcriptomic analysis. Gene-set enrichment analysis and g:Profiler was conducted to provide a functional interpretation of significantly changed genes. Further analysis for AHR/NRF2 binding sites was performed with GeneHancer. RESULTS: In CD4+ cells, we found significantly altered expression of 269 genes by tBHQ. Of them, many had relevance in influenza infection such as genes responsible for virus entry (Anxa1/2, Cd14), interferon signaling (Dusp10, Tnfsf13), or prostaglandin synthesis (Ptgs1/2). In SARS-CoV-2 infections, interferon signaling (Ifitm1), proteolytic enzymes (CtsB), and also cell-surface proteins (Cd14, Cd151) were among the prominent alterations after tBHQ exposure. Of these genes, many had one or more binding sites for AHR and NRF2, two major xenosensors triggered by tBHQ. CONCLUSIONS: Our results strongly suggest that a common food additive, tBHQ, can modulate virus-dependent processes in both influenza and SARS-CoV-2 infections.

Cytochromes P450 catalyze the reduction of α,ß-unsaturated aldehydes.

The metabolism of α,ß-unsaturated aldehydes, e.g., 4-hydroxynonenal, involves oxidation to carboxylic acids, reduction to alcohols, and glutathionylation to eventually form mercapturide conjugates. Recently, we demonstrated that P450s can oxidize aldehydes to carboxylic acids, a reaction previously thought to involve aldehyde dehydrogenase. When recombinant cytochrome P450 3A4 was incubated with 4-hydroxynonenal, O(2), and NADPH, several products were produced, including 1,4-dihydroxynonene (DHN), 4-hydroxy-2-nonenoic acid (HNA), and an unknown metabolite. Several P450s catalyzed the reduction reaction in the order (human) P450 2B6 ≅ P450 3A4 > P450 1A2 > P450 2J2 > (mouse) P450 2c29. Other P450s did not catalyze the reduction reaction (human P450 2E1 and rabbit P450 2B4). Metabolism by isolated rat hepatocytes showed that HNA formation was inhibited by cyanamide, while DHN formation was not affected. Troleandomycin increased HNA production 1.6-fold while inhibiting DHN formation, suggesting that P450 3A11 is a major enzyme involved in rat hepatic clearance of 4-HNE. A fluorescent assay was developed using 9-anthracenealdehyde to measure both reactions. Feeding mice a diet containing t-butylated hydroxyanisole increased the level of both activities with hepatic microsomal fractions but not proportionally. Miconazole (0.5 mM) was a potent inhibitor of these microsomal reduction reactions, while phenytoin and α-naphthoflavone (both at 0.5 mM) were partial inhibitors, suggesting the role of multiple P450 enzymes. The oxidative metabolism of these aldehydes was inhibited >90% in an Ar or CO atmosphere, while the reductive reactions were not greatly affected. These results suggest that P450s are significant catalysts of the reduction of α,ß-unsaturated aldehydes in the liver.

A novel pregnane X receptor and S14-mediated lipogenic pathway in human hepatocyte.

The pregnane X receptor (PXR) initially isolated as a nuclear receptor regulating xenobiotic and drug metabolism and elimination, seems to play an endobiotic role by affecting lipid homeostasis. In mice, PXR affects lipid homeostasis and increases hepatic deposit of triglycerides. In this study, we show that, in human hepatocyte, PXR activation induces an increase of de novo lipogenesis through the up-regulation of S14. S14 was first identified as a thyroid-responsive gene and is known to transduce hormone-related and nutrient-related signals to genes involved in lipogenesis through a molecular mechanism not yet elucidated. We demonstrate that S14 is a novel transcriptional target of PXR. In addition, we report an increase of fatty acid synthase (FASN) and adenosine triphosphate citrate lyase genes expression after PXR activation in human hepatocyte, leading to an increase of fatty acids accumulation and de novo lipogenesis. RNA interference of the expression of S14 proportionally decreases the FASN induction, whereas S14 overexpression in human hepatic cells provokes an increase of fatty acids accumulation and lipogenesis. These results demonstrate for the first time that xenobiotic or drug-activated PXR promote aberrant hepatic de novo lipogenesis via activation of the nonclassical S14 pathway. In addition, these data suggest that the up-regulation of S14 by PXR may promote aberrant hepatic lipogenesis and hepatic steatosis in human hepatocytes.

Changes in miRNA expression in solid tumors: an miRNA profiling in melanomas.

In this review, we briefly described microRNA biogenesis, function and the principal approaches for studying the function of microRNAs (miRNA) in solid cancers. There are currently hundreds of confirmed miRNAs in humans, and computational predictions suggest that the total count might be more than thousand. The regulatory nature of miRNAs combined with the large number of presumptive target genes suggests that they are essential regulators of a wide range of cellular processes. To illustrate the importance of miRNA-mediated regulation in solid cancer some confirmed interactions were collected. Their relevance is described in detail in melanomas from the aspect of diagnosis, the potential application of miRNAs as biomarkers and as potential therapeutic tools.

Effect of CAR activation on selected metabolic pathways in normal and hyperlipidemic mouse livers.

BACKGROUND: Detoxification in the liver involves activation of nuclear receptors, such as the constitutive androstane receptor (CAR), which regulate downstream genes of xenobiotic metabolism. Frequently, the metabolism of endobiotics is also modulated, resulting in potentially harmful effects. We therefore used 1,4-Bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) to study the effect of CAR activation on mouse hepatic transcriptome and lipid metabolome under conditions of diet-induced hyperlipidemia. RESULTS: Using gene expression profiling with a dedicated microarray, we show that xenobiotic metabolism, PPARalpha and adipocytokine signaling, and steroid synthesis are the pathways most affected by TCPOBOP in normal and hyperlipidemic mice. TCPOBOP-induced CAR activation prevented the increased hepatic and serum cholesterol caused by feeding mice a diet containing 1% cholesterol. We show that this is due to increased bile acid metabolism and up-regulated removal of LDL, even though TCPOBOP increased cholesterol synthesis under conditions of hyperlipidemia. Up-regulation of cholesterol synthesis was not accompanied by an increase in mature SREBP2 protein. As determined by studies in CAR -/- mice, up-regulation of cholesterol synthesis is however CAR-dependent; and no obvious CAR binding sites were detected in promoters of cholesterogenic genes. TCPOBOP also affected serum glucose and triglyceride levels and other metabolic processes in the liver, irrespective of the diet. CONCLUSION: Our data show that CAR activation modulates hepatic metabolism by lowering cholesterol and glucose levels, through effects on PPARalpha and adiponectin signaling pathways, and by compromising liver adaptations to hyperlipidemia.

Interethnic differences of CYP2C9 alleles in healthy Hungarian and Roma population samples: relationship to worldwide allelic frequencies.

CYP2C9 gene polymorphisms are widely studied in several ethnic groups, however they are less known in the Roma population. The aim of this work was to study the ethnic differences of the CYP2C9 allele distribution in a healthy Roma population in order to compare them with a healthy Hungarian population. A total of 535 Hungarian and 465 Roma volunteers were genotyped for the CYP2C9*2 (Arg144Cys) and CYP2C9*3 (Ile359Leu) allelic variants by PCR-RFLP assay. The frequencies of the CYP2C9*1, *2 and *3 alleles in the Hungarian population were 0.787, 0.125, and 0.088 and in Roma 0.727, 0.118, and 0.155, respectively. We found a significant difference in CYP2C9*3 prevalence between the Hungarian and Roma populations, which have therapeutic consequences (p<0.005). The distribution of *1/*1, *1/*2, *1/*3, *2/*2, *2/*3, and *3/*3 genotypes in Hungarians were 0.620, 0.195, 0.139, 0.021, 0.015, and 0.011, while in Roma were 0.533, 0.168, 0.219, 0.011, 0.047, and 0.022, respectively. A significant difference was found between the Hungarian and Roma populations regarding the *1/*1, *1/*3 and the *2/*3 (p<0.005) genotypes. This is the first study to investigate the polymorphisms of CYP2C9 gene in the two largest populations in Hungary, healthy Hungarians and Roma. The prevalence of variant CYP2C9 alleles in the Hungarian population is similar to that observed in other European populations. In contrast, the Roma population differs from Hungarians, from most of other Caucasian groups, and from Indians in the incidence of CYP2C9 common variants. The difference in allele distribution patterns between the two populations studied has therapeutic implications as it influences the optimization of therapies.

Modulation of receptor phosphorylation contributes to activation of peroxisome proliferator activated receptor alpha by dehydroepiandrosterone and other peroxisome proliferators.

Dehydroepiandrosterone (DHEA), a C19 human adrenal steroid, activates peroxisome proliferator-activated receptor alpha (PPARalpha) in vivo but does not ligand-activate PPARalpha in transient transfection experiments. We demonstrate that DHEA regulates PPARalpha action by altering both the levels and phosphorylation status of the receptor. Human hepatoma cells (HepG2) were transiently transfected with the expression plasmid encoding PPARalpha and a plasmid containing two copies of fatty acyl coenzyme oxidase (FACO) peroxisome-proliferator activated receptor responsive element consensus oligonucleotide in a luciferase reporter gene. Nafenopin treatment increased reporter gene activity in this system, whereas DHEA treatment did not. Okadaic acid significantly decreased nafenopin-induced reporter activity in a concentration-dependent manner. Okadaic acid treatment of primary rat hepatocytes decreased both DHEA- and nafenopin-induced FACO activity in primary rat hepatocytes. DHEA induced both PPARalpha mRNA and protein levels, as well as PP2A message in primary rat hepatocytes. Western blot analysis showed that the serines at positions 12 and 21 were rapidly dephosphorylated upon treatment with DHEA and nafenopin. Results using specific protein phosphatase inhibitors suggested that protein phosphatase 2A (PP2A) is responsible for DHEA action, and protein phosphatase 1 might be involved in nafenopin induction. Mutation of serines at position 6, 12, and 21 to an uncharged alanine residue significantly increased transcriptional activity, whereas mutation to negative charged aspartate residues (mimicking receptor phosphorylation) decreased transcriptional activity. DHEA action involves induction of PPARalpha mRNA and protein levels as well as increased PPARalpha transcriptional activity through decreasing receptor phosphorylation at serines in the AF1 region.