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ObjectiveIncreased de novo fatty acid (FA) synthesis and cholesterol biosynthesis have been independently described in many tumour types, including hepatocellular carcinoma (HCC).DesignWe investigated the functional contribution of fatty acid synthase (Fasn)-mediated de novo FA synthesis in a murine HCC model induced by loss of Pten and overexpression of c-Met (sgPten/c-Met) using liver-specific Fasn knockout mice. Expression arrays and lipidomic analysis were performed to characterise the global gene expression and lipid profiles, respectively, of sgPten/c-Met HCC from wild-type and Fasn knockout mice. Human HCC cell lines were used for in vitro studies.ResultsAblation of Fasn significantly delayed sgPten/c-Met-driven hepatocarcinogenesis in mice. However, eventually, HCC emerged in Fasn knockout mice. Comparative genomic and lipidomic analyses revealed the upregulation of genes involved in cholesterol biosynthesis, as well as decreased triglyceride levels and increased cholesterol esters, in HCC from these mice. Mechanistically, loss of Fasn promoted nuclear localisation and activation of sterol regulatory element binding protein 2 (Srebp2), which triggered cholesterogenesis. Blocking cholesterol synthesis via the dominant negative form of Srebp2 (dnSrebp2) completely prevented sgPten/c-Met-driven hepatocarcinogenesis in Fasn knockout mice. Similarly, silencing of FASN resulted in increased SREBP2 activation and hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase (HMGCR) expression in human HCC cell lines. Concomitant inhibition of FASN-mediated FA synthesis and HMGCR-driven cholesterol production was highly detrimental for HCC cell growth in culture.ConclusionOur study uncovers a novel functional crosstalk between aberrant lipogenesis and cholesterol biosynthesis pathways in hepatocarcinogenesis, whose concomitant inhibition might represent a therapeutic option for HCC.

ObjectiveIncreased de novo fatty acid (FA) synthesis and cholesterol biosynthesis have been independently described in many tumour types, including hepatocellular carcinoma (HCC).DesignWe investigated the functional contribution of fatty acid synthase (Fasn)-mediated de novo FA synthesis in a murine HCC model induced by loss of Pten and overexpression of c-Met (sgPten/c-Met) using liver-specific Fasn knockout mice. Expression arrays and lipidomic analysis were performed to characterise the global gene expression and lipid profiles, respectively, of sgPten/c-Met HCC from wild-type and Fasn knockout mice. Human HCC cell lines were used for in vitro studies.ResultsAblation of Fasn significantly delayed sgPten/c-Met-driven hepatocarcinogenesis in mice. However, eventually, HCC emerged in Fasn knockout mice. Comparative genomic and lipidomic analyses revealed the upregulation of genes involved in cholesterol biosynthesis, as well as decreased triglyceride levels and increased cholesterol esters, in HCC from these mice. Mechanistically, loss of Fasn promoted nuclear localisation and activation of sterol regulatory element binding protein 2 (Srebp2), which triggered cholesterogenesis. Blocking cholesterol synthesis via the dominant negative form of Srebp2 (dnSrebp2) completely prevented sgPten/c-Met-driven hepatocarcinogenesis in Fasn knockout mice. Similarly, silencing of FASN resulted in increased SREBP2 activation and hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase (HMGCR) expression in human HCC cell lines. Concomitant inhibition of FASN-mediated FA synthesis and HMGCR-driven cholesterol production was highly detrimental for HCC cell growth in culture.ConclusionOur study uncovers a novel functional crosstalk between aberrant lipogenesis and cholesterol biosynthesis pathways in hepatocarcinogenesis, whose concomitant inhibition might represent a therapeutic option for HCC.

A constellation of metabolic disorders, including obesity, dysregulated lipids, and elevations in blood glucose levels, has been associated with cardiovascular disease and diabetes. Analysis of data from recently published genome-wide association studies (GWAS) demonstrated that reduced-function polymorphisms in the organic cation transporter, OCT1 (SLC22A1), are significantly associated with higher total cholesterol, low-density lipoprotein (LDL) cholesterol, and triglyceride (TG) levels and an increased risk for type 2 diabetes mellitus, yet the mechanism linking OCT1 to these metabolic traits remains puzzling. Here, we show that OCT1, widely characterized as a drug transporter, plays a key role in modulating hepatic glucose and lipid metabolism, potentially by mediating thiamine (vitamin B1) uptake and hence its levels in the liver. Deletion of Oct1 in mice resulted in reduced activity of thiamine-dependent enzymes, including pyruvate dehydrogenase (PDH), which disrupted the hepatic glucose-fatty acid cycle and shifted the source of energy production from glucose to fatty acids, leading to a reduction in glucose utilization, increased gluconeogenesis, and altered lipid metabolism. In turn, these effects resulted in increased total body adiposity and systemic levels of glucose and lipids. Importantly, wild-type mice on thiamine deficient diets (TDs) exhibited impaired glucose metabolism that phenocopied Oct1 deficient mice. Collectively, our study reveals a critical role of hepatic thiamine deficiency through OCT1 deficiency in promoting the metabolic inflexibility that leads to the pathogenesis of cardiometabolic disease.

A constellation of metabolic disorders, including obesity, dysregulated lipids, and elevations in blood glucose levels, has been associated with cardiovascular disease and diabetes. Analysis of data from recently published genome-wide association studies (GWAS) demonstrated that reduced-function polymorphisms in the organic cation transporter, OCT1 (SLC22A1), are significantly associated with higher total cholesterol, low-density lipoprotein (LDL) cholesterol, and triglyceride (TG) levels and an increased risk for type 2 diabetes mellitus, yet the mechanism linking OCT1 to these metabolic traits remains puzzling. Here, we show that OCT1, widely characterized as a drug transporter, plays a key role in modulating hepatic glucose and lipid metabolism, potentially by mediating thiamine (vitamin B1) uptake and hence its levels in the liver. Deletion of Oct1 in mice resulted in reduced activity of thiamine-dependent enzymes, including pyruvate dehydrogenase (PDH), which disrupted the hepatic glucose-fatty acid cycle and shifted the source of energy production from glucose to fatty acids, leading to a reduction in glucose utilization, increased gluconeogenesis, and altered lipid metabolism. In turn, these effects resulted in increased total body adiposity and systemic levels of glucose and lipids. Importantly, wild-type mice on thiamine deficient diets (TDs) exhibited impaired glucose metabolism that phenocopied Oct1 deficient mice. Collectively, our study reveals a critical role of hepatic thiamine deficiency through OCT1 deficiency in promoting the metabolic inflexibility that leads to the pathogenesis of cardiometabolic disease.

Organic cation transporter 1, OCT1 (SLC22A1), is the major hepatic uptake transporter for metformin, the most prescribed antidiabetic drug. However, its endogenous role is poorly understood. Here we show that similar to metformin treatment, loss of Oct1 caused an increase in the ratio of AMP to ATP, activated the energy sensor AMP-activated kinase (AMPK), and substantially reduced triglyceride (TG) levels in livers from healthy and leptin-deficient mice. Conversely, livers of human OCT1 transgenic mice fed high-fat diets were enlarged with high TG levels. Metabolomic and isotopic uptake methods identified thiamine as a principal endogenous substrate of OCT1. Thiamine deficiency enhanced the phosphorylation of AMPK and its downstream target, acetyl-CoA carboxylase. Metformin and the biguanide analog, phenformin, competitively inhibited OCT1-mediated thiamine uptake. Acute administration of metformin to wild-type mice reduced intestinal accumulation of thiamine. These findings suggest that OCT1 plays a role in hepatic steatosis through modulation of energy status. The studies implicate OCT1 as well as metformin in thiamine disposition, suggesting an intriguing and parallel mechanism for metformin and its major hepatic transporter in metabolic function.

Amplification and/or activation of the c-Myc proto-oncogene is one of the leading genetic events along hepatocarcinogenesis. The oncogenic potential of c-Myc has been proven experimentally by the finding that its overexpression in the mouse liver triggers tumor formation. However, the molecular mechanism whereby c-Myc exerts its oncogenic activity in the liver remains poorly understood. Here, we demonstrate that the mammalian target of rapamycin complex 1 (mTORC1) cascade is activated and necessary for c-Myc-dependent hepatocarcinogenesis. Specifically, we found that ablation of Raptor, the unique member of mTORC1, strongly inhibits c-Myc liver tumor formation. Also, the p70 ribosomal S6 kinase/ribosomal protein S6 and eukaryotic translation initiation factor 4E-binding protein 1/eukaryotic translation initiation factor 4E signaling cascades downstream of mTORC1 are required for c-Myc-driven tumorigenesis. Intriguingly, microarray expression analysis revealed up-regulation of multiple amino acid transporters, including solute carrier family 1 member A5 (SLC1A5) and SLC7A6, leading to robust uptake of amino acids, including glutamine, into c-Myc tumor cells. Subsequent functional studies showed that amino acids are critical for activation of mTORC1 as their inhibition suppressed mTORC1 in c-Myc tumor cells. In human hepatocellular carcinoma specimens, levels of c-Myc directly correlate with those of mTORC1 activation as well as of SLC1A5 and SLC7A6.ConclusionOur current study indicates that an intact mTORC1 axis is required for c-Myc-driven hepatocarcinogenesis; thus, targeting the mTOR pathway or amino acid transporters may be an effective and novel therapeutic option for the treatment of hepatocellular carcinoma with activated c-Myc signaling. (Hepatology 2017;66:167-181).

Metformin, the most widely prescribed antidiabetic drug, requires transporters to enter tissues involved in its pharmacologic action, including liver, kidney, and peripheral tissues. Organic cation transporter 3 (OCT3, SLC22A3), expressed ubiquitously, transports metformin, but its in vivo role in metformin response is not known. Using Oct3 knockout mice, the role of the transporter in metformin pharmacokinetics and pharmacodynamics was determined. After an intravenous dose of metformin, a 2-fold decrease in the apparent volume of distribution and clearance was observed in knockout compared with wild-type mice (P < 0.001), indicating an important role of OCT3 in tissue distribution and elimination of the drug. After oral doses, a significantly lower bioavailability was observed in knockout compared with wild-type mice (0.27 versus 0.58, P < 0.001). Importantly, metformin's effect on the plasma glucose concentration-time curve was reduced in knockout compared with wild-type mice (12 versus 30% reduction, respectively, P < 0.05) along with its accumulation in skeletal muscle and adipose tissue (P < 0.05). Furthermore, the effect of metformin on phosphorylation of AMP activated protein kinase, and expression of glucose transporter type 4 was absent in the adipose tissue of Oct3(-/-) mice. Additional analysis revealed that an OCT3 3' untranslated region variant was associated with reduced activity in luciferase assays and reduced response to metformin in 57 healthy volunteers. These findings suggest that OCT3 plays an important role in the absorption and elimination of metformin and that the transporter is a critical determinant of metformin bioavailability, clearance, and pharmacologic action.

Metformin, the most widely prescribed antidiabetic drug, requires transporters to enter tissues involved in its pharmacologic action, including liver, kidney, and peripheral tissues. Organic cation transporter 3 (OCT3, SLC22A3), expressed ubiquitously, transports metformin, but its in vivo role in metformin response is not known. Using Oct3 knockout mice, the role of the transporter in metformin pharmacokinetics and pharmacodynamics was determined. After an intravenous dose of metformin, a 2-fold decrease in the apparent volume of distribution and clearance was observed in knockout compared with wild-type mice (P < 0.001), indicating an important role of OCT3 in tissue distribution and elimination of the drug. After oral doses, a significantly lower bioavailability was observed in knockout compared with wild-type mice (0.27 versus 0.58, P < 0.001). Importantly, metformin's effect on the plasma glucose concentration-time curve was reduced in knockout compared with wild-type mice (12 versus 30% reduction, respectively, P < 0.05) along with its accumulation in skeletal muscle and adipose tissue (P < 0.05). Furthermore, the effect of metformin on phosphorylation of AMP activated protein kinase, and expression of glucose transporter type 4 was absent in the adipose tissue of Oct3(-/-) mice. Additional analysis revealed that an OCT3 3' untranslated region variant was associated with reduced activity in luciferase assays and reduced response to metformin in 57 healthy volunteers. These findings suggest that OCT3 plays an important role in the absorption and elimination of metformin and that the transporter is a critical determinant of metformin bioavailability, clearance, and pharmacologic action.

Background: The underlying molecular mechanisms of PTSD are largely unknown. Distinctexpression signatures for PTSD have been found, in particular for immune activation transcripts. DNA methylation may be significant in the pathophysiology of PTSD, since the process is intrinsically linked to gene expression.We evaluated temporal changes in DNA methylation in select promoter regions of immune system-related genes in U.S. military service members with a PTSD diagnosis, pre- and post-diagnosis, and in controls. Methods: Cases (nD75) had a post-deployment diagnosis of PTSD in their medical record. Controls (nD75) were randomly selected service members with no PTSD diagnosis. DNA was extracted from pre- and post-deployment sera. DNA methylation (%5-mC) was quantified at specific CpG sites in promoter regions of insulin-like growth factor 2 (IGF2), long non-coding RNA transcript H19, interleukin-8 (IL8), IL16, and IL18 via pyrosequencing.We used multivariate analysis of variance and generalized linear models to calculate adjusted means (adjusted for age, gender, and race) to make temporal comparisons of %5-mC for cases (pre- to post-deployment) versus controls (pre- to post-deployment). Results: There were significant differences in the change of %5-mC pre- to postdeployment between cases and controls for H19 (cases: C0.57%, controls: 1.97%; p D0.04) and IL18 (cases: C1.39%, controls: 3.83%; p D0.01). For H19 the difference was driven by a significant reduction in %5-mC among controls; for IL18 the difference was driven by both a reduction in %5-mC among controls and an increase in %5-mC among cases. Stratified analyses revealed more pronounced differences in the adjusted means of pre-post H19 and IL18 methylation differences for cases versus controls among older service members, males, service members of white race, and those with shorter deployments (6 12 months)., Published in Frontiers in Psychiatry Molecular Psychiatry, v4 article56 p1-12, June 2013. The original document contains color images.

In a case-control study of OEF/OIF service members with both pre- and post-deployment/injury serum samples, we investigated DNA methylation patterns and serum cytokine expression for mild, moderate, and severe TBI. We found differential methylation (%5-mC) of IL 1beta for mild cases, TNFbeta for moderate cases, and IL8 for severe cases, as well as differential serum cytokine expression of IL6 in mild and moderate cases, IL8 in mild, moderate, and severe cases, and MMP3 in severe cases. This is the first study, to our knowledge, of chronic epigenetic and protein-based alterations in TBI. These patterns are based on a small sample size, should be interpreted with caution, and should be examined in larger studies., The original document contains color images.

A continuous process was established for the production of 2,2,6,6-tetramethylpiperidin-4-ol over Cu–Cr/γ-Al2O3 in a fixed-bed reactor. The catalyst was characterized by X-ray diffraction, X-ray photoelectron spectroscopy and temperature-programmed reduction. Cu0 was believed to be the active site for the hydrogenation, and the doped chromium was supposed to exert a positive impact on the dispersion of active species. The catalyst and parameters of hydrogenation were optimized. Thus, 2,2,6,6-tetramethylpiperidin-4-ol was obtained in the yield of 90% from 2,2,6,6-tetramethylpiperidin-4-one (purity, 95%) under the optimum reaction conditions.

Membrane transporters play crucial roles in the cellular uptake and efflux of an array of small molecules including nutrients, environmental toxins, and many clinically used drugs. We hypothesized that common genetic variation in the proximal promoter regions of transporter genes contribute to observed variation in drug response. A total of 579 polymorphisms were identified in the proximal promoters (-250 to +50 bp) and flanking 5' sequence of 107 transporters in the ATP Binding Cassette (ABC) and Solute Carrier (SLC) superfamilies in 272 DNA samples from ethnically diverse populations. Many transporter promoters contained multiple common polymorphisms. Using a sliding window analysis, we observed that, on average, nucleotide diversity (pi) was lowest at approximately 300 bp upstream of the transcription start site, suggesting that this region may harbor important functional elements. The proximal promoters of transporters that were highly expressed in the liver had greater nucleotide diversity than those that were highly expressed in the kidney consistent with greater negative selective pressure on the promoters of kidney transporters. Twenty-one promoters were evaluated for activity using reporter assays. Greater nucleotide diversity was observed in promoters with strong activity compared to promoters with weak activity, suggesting that weak promoters are under more negative selective pressure than promoters with high activity. Collectively, these results suggest that the proximal promoter region of membrane transporters is rich in variation and that variants in these regions may play a role in interindividual variation in drug disposition and response.

Membrane transporters play crucial roles in the cellular uptake and efflux of an array of small molecules including nutrients, environmental toxins, and many clinically used drugs. We hypothesized that common genetic variation in the proximal promoter regions of transporter genes contribute to observed variation in drug response. A total of 579 polymorphisms were identified in the proximal promoters (-250 to +50 bp) and flanking 5' sequence of 107 transporters in the ATP Binding Cassette (ABC) and Solute Carrier (SLC) superfamilies in 272 DNA samples from ethnically diverse populations. Many transporter promoters contained multiple common polymorphisms. Using a sliding window analysis, we observed that, on average, nucleotide diversity (pi) was lowest at approximately 300 bp upstream of the transcription start site, suggesting that this region may harbor important functional elements. The proximal promoters of transporters that were highly expressed in the liver had greater nucleotide diversity than those that were highly expressed in the kidney consistent with greater negative selective pressure on the promoters of kidney transporters. Twenty-one promoters were evaluated for activity using reporter assays. Greater nucleotide diversity was observed in promoters with strong activity compared to promoters with weak activity, suggesting that weak promoters are under more negative selective pressure than promoters with high activity. Collectively, these results suggest that the proximal promoter region of membrane transporters is rich in variation and that variants in these regions may play a role in interindividual variation in drug disposition and response., De fyra första författarna delar förstaförfattarskapet

Membrane transporters play crucial roles in the cellular uptake and efflux of an array of small molecules including nutrients, environmental toxins, and many clinically used drugs. We hypothesized that common genetic variation in the proximal promoter regions of transporter genes contribute to observed variation in drug response. A total of 579 polymorphisms were identified in the proximal promoters (-250 to +50 bp) and flanking 5' sequence of 107 transporters in the ATP Binding Cassette (ABC) and Solute Carrier (SLC) superfamilies in 272 DNA samples from ethnically diverse populations. Many transporter promoters contained multiple common polymorphisms. Using a sliding window analysis, we observed that, on average, nucleotide diversity (pi) was lowest at approximately 300 bp upstream of the transcription start site, suggesting that this region may harbor important functional elements. The proximal promoters of transporters that were highly expressed in the liver had greater nucleotide diversity than those that were highly expressed in the kidney consistent with greater negative selective pressure on the promoters of kidney transporters. Twenty-one promoters were evaluated for activity using reporter assays. Greater nucleotide diversity was observed in promoters with strong activity compared to promoters with weak activity, suggesting that weak promoters are under more negative selective pressure than promoters with high activity. Collectively, these results suggest that the proximal promoter region of membrane transporters is rich in variation and that variants in these regions may play a role in interindividual variation in drug disposition and response.

Udgivelsesdato: 2002-Sep-1, A partial syndecan-2 sequence (147 bp) was obtained from chicken embryonic fibroblast poly(A)+ RNA by reverse transcription-PCR. This partial sequence was used to produce a 5'-end-labelled probe. A chicken liver cDNA library was screened with this probe, and overlapping clones were obtained encompassing the entire cDNA of 3 kb. The open reading frame encodes a protein of 201 amino acids. The cytoplasmic domain is identical with that of mammalian syndecan-2, and highly similar to those of Xenopus laevis and zebrafish syndecan-2. The transmembrane domain is identical with that of mammalian and zebrafish syndecan-2, and highly similar to that of Xenopus laevis syndecan-2. The ectodomain is 45-62% identical with that of zebrafish, Xenopus laevis and mammalian syndecan-2. Two coding single nucleotide polymorphisms were observed. In vitro transcription and translation yielded a product of 30 kDa. Western blotting of chicken embryonic fibroblast cell lysates with species-specific monoclonal antibody mAb 8.1 showed that chicken syndecan-2 is substituted with heparan sulphate, and that the major form of chicken syndecan-2 isolated from chicken fibroblasts is consistent with the formation of SDS-resistant dimers, which is common for syndecans. A 5'-end-labelled probe hybridized to two mRNA species in chicken embryonic fibroblasts, while Northern analysis with poly(A)+ RNAs from different tissues of chicken embryos showed wide and distinct distributions of chicken syndecan-2 during embryonic development. This pattern was different from that reported for syndecan-4, but consistent with the roles of syndecan-2 in neural maturation and in mesenchymal-matrix interactions.

Udgivelsesdato: 2002-Sep-1, A partial syndecan-2 sequence (147 bp) was obtained from chicken embryonic fibroblast poly(A)+ RNA by reverse transcription-PCR. This partial sequence was used to produce a 5'-end-labelled probe. A chicken liver cDNA library was screened with this probe, and overlapping clones were obtained encompassing the entire cDNA of 3 kb. The open reading frame encodes a protein of 201 amino acids. The cytoplasmic domain is identical with that of mammalian syndecan-2, and highly similar to those of Xenopus laevis and zebrafish syndecan-2. The transmembrane domain is identical with that of mammalian and zebrafish syndecan-2, and highly similar to that of Xenopus laevis syndecan-2. The ectodomain is 45-62% identical with that of zebrafish, Xenopus laevis and mammalian syndecan-2. Two coding single nucleotide polymorphisms were observed. In vitro transcription and translation yielded a product of 30 kDa. Western blotting of chicken embryonic fibroblast cell lysates with species-specific monoclonal antibody mAb 8.1 showed that chicken syndecan-2 is substituted with heparan sulphate, and that the major form of chicken syndecan-2 isolated from chicken fibroblasts is consistent with the formation of SDS-resistant dimers, which is common for syndecans. A 5'-end-labelled probe hybridized to two mRNA species in chicken embryonic fibroblasts, while Northern analysis with poly(A)+ RNAs from different tissues of chicken embryos showed wide and distinct distributions of chicken syndecan-2 during embryonic development. This pattern was different from that reported for syndecan-4, but consistent with the roles of syndecan-2 in neural maturation and in mesenchymal-matrix interactions.