RESUMO
Cholesterol-derived bile acids (BAs) affect numerous physiological functions such as glucose homeostasis, lipid metabolism and absorption, intestinal inflammation and immunity, as well as intestinal microbiota diversity. Diet influences the composition of the BA pool. In the present study, we analyzed the impact of a dietary supplementation with a freeze-dried blueberry powder (BBP) on the fecal BA pool composition. The diet of 11 men and 13 women at risk of metabolic syndrome was supplemented with 50 g/day of BBP for 8 weeks, and feces were harvested before (pre) and after (post) BBP consumption. BAs were profiled using liquid chromatography coupled with tandem mass spectrometry. No significant changes in total BAs were detected when comparing pre- vs. post-BBP consumption samples. However, post-BBP consumption samples exhibited significant accumulations of glycine-conjugated BAs (p = 0.04), glycochenodeoxycholic (p = 0.01), and glycoursodeoxycholic (p = 0.01) acids, as well as a significant reduction (p = 0.03) in the secondary BA levels compared with pre-BBP feces. In conclusion, the fecal bileacidome is significantly altered after the consumption of BBP for 8 weeks. While additional studies are needed to fully understand the underlying mechanisms and physiological implications of these changes, our data suggest that the consumption of blueberries can modulate toxic BA elimination.
Assuntos
Ácidos e Sais Biliares , Mirtilos Azuis (Planta) , Feminino , Humanos , Masculino , Ácidos e Sais Biliares/análise , Ácido Cólico , Fezes/química , Glucose/análise , Glicina , Projetos Piloto , PósRESUMO
Ursodeoxycholic acid (UDCA) is the first line therapy for the treatment of cholestatic and autoimmune liver diseases. Its clinical use is currently limited by a significant proportion of non-responder patients. Polyunsaturated fatty acids (n-3 PUFAs) possess important anti-inflammatory properties and protect liver cells against bile acid (BA)-induced toxicity. The present study was designed to rapidly evaluate whether combining n-3 PUFAs (i.e., eicosapentaenoic [EPA] and docosahexaenoic [DHA] acids) to UDCA would provide additional benefits when compared to the drug alone. The parameters evaluated were (i) the expression of genes governing BA synthesis, transport, and metabolism; (ii) the prevention of BA-induced apoptosis and endoplasmic reticulum (ER)-stress; and (iii) the control of BA- and LPS-dependent inflammation. In the absence of n-3 PUFAs, most of the parameters investigated were unaffected by UDCA or were only altered by the higher dose (500 µM) of the drug. By contrast, in the presence of EPA/DHA (50/50 µM), all parameters showed a strongly improved response and the lowest UDCA dosage (50 µM) provided equal or better benefits than the highest dose used alone. For example, the combination EPA/DHA + UDCA 50 µM caused comparable down-regulation of the CYP7A1 gene expression and of the BA-induced caspase 3 activity as observed with UDCA 500 µM. In conclusion, these results suggest that the addition of n-3 PUFAs to UDCA may improve the response to the drug, and that such a pharmaco-nutraceutical approach could be used in clinic to open the narrow therapeutic dose of UDCA in cholestatic liver diseases.
Assuntos
Suplementos Nutricionais , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-3/farmacologia , Ácido Ursodesoxicólico/farmacologia , Apoptose/efeitos dos fármacos , Doenças Autoimunes , Ácidos e Sais Biliares/metabolismo , Ácidos e Sais Biliares/toxicidade , Carcinoma Hepatocelular , Caspase 3 , Colangite Esclerosante , Colestanotriol 26-Mono-Oxigenase/genética , Colestase , Colesterol 7-alfa-Hidroxilase/genética , Regulação para Baixo/efeitos dos fármacos , Quimioterapia Combinada , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Inflamação , Fígado/metabolismo , Cirrose Hepática Biliar , Hepatopatias , Células THP-1RESUMO
Diabetic nephropathy (DN) remains the major cause of end-stage renal disease (ESRD). We used high-fat/high-sucrose (HFHS)-fed LDLr-/- /ApoB100/100 mice with transgenic overexpression of IGFII in pancreatic ß-cells (LRKOB100/IGFII) as a model of ESRD to test whether dietary long chain omega-3 polyunsaturated fatty acids LCω3FA-rich fish oil (FO) could prevent ESRD development. We further evaluated the potential of docosahexaenoic acid (DHA)-derived pro-resolving lipid mediators, 17-hydroxy-DHA (17-HDHA) and Protectin DX (PDX), to reverse established ESRD damage. HFHS-fed vehicle-treated LRKOB100/IGFII mice developed severe kidney dysfunction leading to ESRD, as revealed by advanced glomerular fibrosis and mesangial expansion along with reduced percent survival. The kidney failure outcome was associated with cardiac dysfunction, revealed by reduced heart rate and prolonged diastolic and systolic time. Dietary FO prevented kidney damage, lean mass loss, cardiac dysfunction, and death. 17-HDHA reduced podocyte foot process effacement while PDX treatment alleviated kidney fibrosis and mesangial expansion as compared to vehicle treatment. Only PDX therapy was effective at preserving the heart function and survival rate. These results show that dietary LCω3FA intake can prevent ESRD and cardiac dysfunction in LRKOB100/IGFII diabetic mice. Our data further reveals that PDX can protect against renal failure and cardiac dysfunction, offering a potential new therapeutic strategy against ESRD.
Assuntos
Aterosclerose/complicações , Diabetes Mellitus Experimental/fisiopatologia , Nefropatias Diabéticas/tratamento farmacológico , Modelos Animais de Doenças , Ácidos Docosa-Hexaenoicos/administração & dosagem , Óleos de Peixe/administração & dosagem , Falência Renal Crônica/tratamento farmacológico , Animais , Apolipoproteína B-100/fisiologia , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/patologia , Falência Renal Crônica/etiologia , Falência Renal Crônica/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de LDL/fisiologiaRESUMO
OBJECTIVE: The consumption of fruits is strongly associated with better health and higher bacterial diversity in the gut microbiota (GM). Camu camu (Myrciaria dubia) is an Amazonian fruit with a unique phytochemical profile, strong antioxidant potential and purported anti-inflammatory potential. DESIGN: By using metabolic tests coupled with 16S rRNA gene-based taxonomic profiling and faecal microbial transplantation (FMT), we have assessed the effect of a crude extract of camu camu (CC) on obesity and associated immunometabolic disorders in high fat/high sucrose (HFHS)-fed mice. RESULTS: Treatment of HFHS-fed mice with CC prevented weight gain, lowered fat accumulation and blunted metabolic inflammation and endotoxaemia. CC-treated mice displayed improved glucose tolerance and insulin sensitivity and were also fully protected against hepatic steatosis. These effects were linked to increased energy expenditure and upregulation of uncoupling protein 1 mRNA expression in the brown adipose tissue (BAT) of CC-treated mice, which strongly correlated with the mRNA expression of the membrane bile acid (BA) receptor TGR5. Moreover, CC-treated mice showed altered plasma BA pool size and composition and drastic changes in the GM (eg, bloom of Akkermansia muciniphila and a strong reduction of Lactobacillus). Germ-free (GF) mice reconstituted with the GM of CC-treated mice gained less weight and displayed higher energy expenditure than GF-mice colonised with the FM of HFHS controls. CONCLUSION: Our results show that CC prevents visceral and liver fat deposition through BAT activation and increased energy expenditure, a mechanism that is dependent on the GM and linked to major changes in the BA pool size and composition.
Assuntos
Metabolismo Energético/fisiologia , Frutas/química , Microbioma Gastrointestinal/efeitos dos fármacos , Obesidade/prevenção & controle , Animais , Ácido Ascórbico/uso terapêutico , Glicemia/metabolismo , Endotoxemia/prevenção & controle , Fígado Gorduroso/microbiologia , Fígado Gorduroso/fisiopatologia , Fígado Gorduroso/prevenção & controle , Transplante de Microbiota Fecal , Homeostase/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/microbiologia , Obesidade/fisiopatologia , Paniculite/prevenção & controle , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêuticoRESUMO
Cholestasis is characterized by the accumulation of toxic bile acids (BAs) in liver cells. The present study aimed to evaluate the effects of n-3 polyunsaturated fatty acids (n-3 PUFAs), such as docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, on BA homeostasis and toxicity in human cell models. The effects of EPA and/or DHA on the expression of genes involved in the maintenance of BA homeostasis were analyzed in human hepatoma (HepG2) and colon carcinoma (Caco-2) cells, as well as in primary culture of human intestinal (InEpC) and renal (RPTEC) cells. Extracellular BA species were quantified in culture media using LC-MS/MS. BA-induced toxicity was evaluated using caspase-3 and flow cytometry assays. Gene expression analyses of HepG2 cells reveal that n-3 PUFAs reduce the expression of genes involved in BA synthesis (CYP7A1, CYP27A1) and uptake (NTCP), while activating genes encoding metabolic enzymes (SULT2A1) and excretion transporters (MRP2, MRP3). N-3 PUFAs also generate a less toxic BA pool and prevent the BA-dependent activation of apoptosis in HepG2 cells. Conclusion. The present study reveals that n-3 PUFAs stimulate BA detoxification.
Assuntos
Ácidos e Sais Biliares/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Expressão Gênica/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Células CACO-2 , Regulação para Baixo/efeitos dos fármacos , Células Epiteliais , Células Hep G2 , Homeostase/genética , Humanos , Mucosa Intestinal/citologia , Túbulos Renais Proximais/citologia , Necrose , Cultura Primária de Células , Regulação para Cima/efeitos dos fármacosRESUMO
AIMS/HYPOTHESIS: There is growing evidence that fruit polyphenols exert beneficial effects on the metabolic syndrome, but the underlying mechanisms remain poorly understood. In the present study, we aimed to analyse the effects of polyphenolic extracts from five types of Arctic berries in a model of diet-induced obesity. METHODS: Male C57BL/6 J mice were fed a high-fat/high-sucrose (HFHS) diet and orally treated with extracts of bog blueberry (BBE), cloudberry (CLE), crowberry (CRE), alpine bearberry (ABE), lingonberry (LGE) or vehicle (HFHS) for 8 weeks. An additional group of standard-chow-fed, vehicle-treated mice was included as a reference control for diet-induced obesity. OGTTs and insulin tolerance tests were conducted, and both plasma insulin and C-peptide were assessed throughout the OGTT. Quantitative PCR, western blot analysis and ELISAs were used to assess enterohepatic immunometabolic features. Faecal DNA was extracted and 16S rRNA gene-based analysis was used to profile the gut microbiota. RESULTS: Treatment with CLE, ABE and LGE, but not with BBE or CRE, prevented both fasting hyperinsulinaemia (mean ± SEM [pmol/l]: chow 67.2 ± 12.3, HFHS 153.9 ± 19.3, BBE 114.4 ± 14.3, CLE 82.5 ± 13.0, CRE 152.3 ± 24.4, ABE 90.6 ± 18.0, LGE 95.4 ± 10.5) and postprandial hyperinsulinaemia (mean ± SEM AUC [pmol/l × min]: chow 14.3 ± 1.4, HFHS 31.4 ± 3.1, BBE 27.2 ± 4.0, CLE 17.7 ± 2.2, CRE 32.6 ± 6.3, ABE 22.7 ± 18.0, LGE 23.9 ± 2.5). None of the berry extracts affected C-peptide levels or body weight gain. Levels of hepatic serine phosphorylated Akt were 1.6-, 1.5- and 1.2-fold higher with CLE, ABE and LGE treatment, respectively, and hepatic carcinoembryonic antigen-related cell adhesion molecule (CEACAM)-1 tyrosine phosphorylation was 0.6-, 0.7- and 0.9-fold increased in these mice vs vehicle-treated, HFHS-fed mice. These changes were associated with reduced liver triacylglycerol deposition, lower circulating endotoxins, alleviated hepatic and intestinal inflammation, and major gut microbial alterations (e.g. bloom of Akkermansia muciniphila, Turicibacter and Oscillibacter) in CLE-, ABE- and LGE-treated mice. CONCLUSIONS/INTERPRETATION: Our findings reveal novel mechanisms by which polyphenolic extracts from ABE, LGE and especially CLE target the gut-liver axis to protect diet-induced obese mice against metabolic endotoxaemia, insulin resistance and hepatic steatosis, which importantly improves hepatic insulin clearance. These results support the potential benefits of these Arctic berries and their integration into health programmes to help attenuate obesity-related chronic inflammation and metabolic disorders. DATA AVAILABILITY: All raw sequences have been deposited in the public European Nucleotide Archive server under accession number PRJEB19783 ( https://www.ebi.ac.uk/ena/data/view/PRJEB19783 ).
Assuntos
Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/metabolismo , Resistência à Insulina , Intestinos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Peptídeo C/sangue , Dieta Hiperlipídica , Endotoxemia/metabolismo , Frutas/química , Glucose/metabolismo , Homeostase , Insulina/sangue , Insulina/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/metabolismo , RNA Ribossômico 16S/genética , Fatores de TempoRESUMO
The gastrointestinal tract is enriched with xenobiotic processing proteins that play important roles in xenobiotic bioactivation, metabolism, and detoxification. The application of genetically modified mouse models has been instrumental in characterizing the function of xenobiotic processing genes (XPG) and their proteins in drug metabolism. Here, we report the utilization of three-dimensional crypt organoid cultures from these animal models to study intestinal drug metabolism and toxicity. With the successful culturing of crypt organoids, we profiled the abundance of Phase I and Phase II XPG expression, drug transporter gene expression, and xenobiotic nuclear receptor (XNR) gene expression. Functions of XNRs were examined by treating crypt cells with XNR prototypical agonists. Real-time quantitative polymerase chain reaction demonstrated that the representative downstream target genes were induced. These findings were validated from cultures developed from XNR-null mice. In crypt cultures isolated from Pxr-/- mice, pregnenolone 16α-carbonitrile failed to induce Cyp3a11 gene expression; similarly, WY14643 failed to induce Cyp4a10 in the Pparα-/- crypts. Crypt cultures from control (Ugt1F/F ) and intestinal epithelial cell (IEC) specific Ugt1 null mice (Ugt1ΔIEC ) were treated with camptothecin-11, an anticancer prodrug with severe intestinal toxicity that originates from insufficient UGT1A1-dependent glucuronidation of its active metabolite SN-38. In the absence of Ugt1 gene expression, Ugt1ΔIEC crypt cultures exhibit very limited production of SN-38 glucuronide, concordant with increased apoptosis in comparison with Ugt1F/F crypt cultures. This study suggests crypt organoid cultures as an effective in vitro model for studying intestinal drug metabolism and toxicity.
Assuntos
Camptotecina/análogos & derivados , Inativação Metabólica/fisiologia , Organoides/metabolismo , Animais , Apoptose/fisiologia , Camptotecina/metabolismo , Técnicas de Cultura de Células/métodos , Citocromo P-450 CYP3A/metabolismo , Expressão Gênica/fisiologia , Mucosa Intestinal/metabolismo , Irinotecano , Taxa de Depuração Metabólica/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Xenobióticos/metabolismoRESUMO
Based on our recent finding that disruption of bile acid (BA) homeostasis in mice results in the induction of hepatic long noncoding RNA H19 expression, we sought to elucidate the role of H19 in cholestatic liver fibrosis. Hepatic overexpression of H19RNA augmented bile duct ligation (BDL)-induced liver fibrosis, which was accompanied by the elevation of serum alanine aminotransferase, aspartate aminotransferase, bilirubin, and BA levels. Multiple genes related to liver fibrosis, inflammation, and biliary hyperplasia were increased in H19-BDL versus null-BDL mice, whereas genes in BA synthesis were decreased. Livers and spleens of H19-BDL mice showed significant enrichment of CD3+γδ+, interleukin-4, and interleukin-17 producing CD4+ and CD8+ immune cell populations. H19 down-regulated hepatic zinc finger E-box-binding homeobox 1 (ZEB1) but up-regulated epithelial cell adhesion molecule (EpCAM) and SRY (sex determining region Y)-box 9 expression. Mechanistically, ZEB1 repressed EpCAM promoter activity and gene transcription. H19RNA impeded ZEB1's inhibitory action by interacting with ZEB1 protein to prevent its binding to the EpCAM promoter. Hepatic overexpression of ZEB1 or knockdown of EpCAM diminished H19-induced fibrosis; the latter was also prevented in H19-/- mice. H19RNA was markedly induced by bile acids in mouse small cholangiocytes and to a lesser extent in mouse large cholangiocytes. The up-regulation of H19RNA and EpCAM correlated positively with the down-regulation of ZEB1 in primary sclerosing cholangitis and primary biliary cirrhosis liver specimens. CONCLUSION: The activation of hepatic H19RNA promoted cholestatic liver fibrosis in mice through the ZEB1/EpCAM signaling pathway. (Hepatology 2017;66:1183-1196).
Assuntos
Colestase/metabolismo , Molécula de Adesão da Célula Epitelial/metabolismo , Cirrose Hepática/etiologia , RNA Longo não Codificante/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Adulto , Idoso , Animais , Ácidos e Sais Biliares , Colestase/complicações , Feminino , Células HEK293 , Células Hep G2 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Regulação para Cima , Adulto JovemRESUMO
Bile acids (BAs) play critical physiological functions in cholesterol homeostasis, and deregulation of BA metabolism causes cholestatic liver injury. The long noncoding RNA maternally expressed gene 3 (MEG3) was recently shown as a potential tumor suppressor; however, its basic hepatic function remains elusive. Using RNA pull-down with biotin-labeled sense or anti-sense MEG 3RNA followed by mass spectrometry, we identified RNA-binding protein polypyrimidine tract-binding protein 1 (PTBP1) as a MEG3 interacting protein and validated their interaction by RNA immunoprecipitation (RIP). Bioinformatics analysis revealed putative binding sites for PTBP1 within the coding region (CDS) of small heterodimer partner (SHP), a key repressor of BA biosynthesis. Forced expression of MEG3 in hepatocellular carcinoma cells guided and facilitated PTBP1 binding to the Shp CDS, resulting in Shp mRNA decay. Transient overexpression of MEG3 RNA in vivo in mouse liver caused rapid Shp mRNA degradation and cholestatic liver injury, which was accompanied by the disruption of BA homeostasis, elevation of liver enzymes, as well as dysregulation of BA synthetic enzymes and metabolic genes. Interestingly, RNA sequencing coupled with quantitative PCR (qPCR) revealed a drastic induction of MEG3 RNA in Shp-/- liver. SHP inhibited MEG3 gene transcription by repressing cAMP response element-binding protein (CREB) transactivation of the MEG3 promoter. In addition, the expression of MEG3 and PTBP1 was activated in human fibrotic and cirrhotic livers. CONCLUSION: MEG3 causes cholestasis by serving as a guide RNA scaffold to recruit PTBP1 to destabilize Shp mRNA. SHP in turn represses CREB-mediated activation of MEG3 expression in a feedback-regulatory fashion. (Hepatology 2017;65:604-615).
Assuntos
Colestase/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Fígado/lesões , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Estabilidade de RNA/genética , RNA Longo não Codificante/genética , Animais , Sítios de Ligação , Células Cultivadas , Colestase/patologia , Modelos Animais de Doenças , Regulação para Baixo , Células Hep G2 , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas , Distribuição Aleatória , Sensibilidade e EspecificidadeRESUMO
AIMS: To compare the therapeutic potential of TP-113, a unique molecular entity linking DHA with metformin, for alleviating insulin resistance in obese diabetic mice through the PDX/IL-6 pathway. MATERIAL AND METHODS: We utilized the generically obese diabetic db/db mouse model for all experiments. Initial studies investigated both a dose and time course response. These results were then utilized to design a long-term (5 week) treatment protocol. Mice were gavaged twice daily with 1 of 3 treatments: 200 mg/kg BW TP113, an equivalent dose of metformin alone (70 mg/kg BW) or water. Whole-body insulin sensitivity was measured using the hyperinsulinaemic-isoglycaemic clamp procedure in awake unrestrained mice. RESULTS: We first confirmed that acute TP-113 treatment raises PDX and IL-6 levels in skeletal muscle. We next tested the long-term glucoregulatory effect of oral TP-113 in obese diabetic db/db mice and compared its effect to an equivalent dose of metformin. A 5-week oral treatment with TP-113 reduced insulin resistance compared to both vehicle treatment and metformin alone, revealed by the determination of whole-body insulin sensitivity for glucose disposal using the clamp technique. This insulin-sensitizing effect was explained primarily by improvement of insulin action to suppress hepatic glucose production in TP-113-treated mice. These effects of TP-113 were greater than that of an equivalent dose of metformin, indicating that TP-113 increases metformin efficacy for reducing insulin resistance. CONCLUSION: We conclude that TP-113 improves insulin sensitivity in obese diabetic mice through activation of the PDX/IL-6 signaling axis in skeletal muscle and improved glucoregulatory action in the liver.
Assuntos
Glicemia/efeitos dos fármacos , Diabetes Mellitus Tipo 2/metabolismo , Ácidos Docosa-Hexaenoicos/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Glutamatos/farmacologia , Hipoglicemiantes/farmacologia , Resistência à Insulina , Interleucina-6/metabolismo , Fígado/efeitos dos fármacos , Metformina/farmacologia , Músculo Esquelético/efeitos dos fármacos , Obesidade/metabolismo , Animais , Glicemia/metabolismo , Modelos Animais de Doenças , Combinação de Medicamentos , Glucose/metabolismo , Técnica Clamp de Glucose , Fígado/metabolismo , Camundongos , Camundongos Obesos , Músculo Esquelético/metabolismoRESUMO
Biliary obstruction, a severe cholestatic condition, results in a huge accumulation of toxic bile acids (BA) in the liver. Glucuronidation, a conjugation reaction, is thought to protect the liver by both reducing hepatic BA toxicity and increasing their urinary elimination. The present study evaluates the contribution of each process in the overall BA detoxification by glucuronidation. Glucuronide (G), glycine, taurine conjugates, and unconjugated BAs were quantified in pre- and post-biliary stenting urine samples from 12 patients with biliary obstruction, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The same LC-MS/MS procedure was used to quantify intra- and extracellular BA-G in Hepatoma HepG2 cells. Bile acid-induced toxicity in HepG2 cells was evaluated using MTS reduction, caspase-3 and flow cytometry assays. When compared to post-treatment samples, pre-stenting urines were enriched in glucuronide-, taurine- and glycine-conjugated BAs. Biliary stenting increased the relative BA-G abundance in the urinary BA pool, and reduced the proportion of taurine- and glycine-conjugates. Lithocholic, deoxycholic and chenodeoxycholic acids were the most cytotoxic and pro-apoptotic/necrotic BAs for HepG2 cells. Other species, such as the cholic, hyocholic and hyodeoxycholic acids were nontoxic. All BA-G assayed were less toxic and displayed lower pro-apoptotic/necrotic effects than their unconjugated precursors, even if they were able to penetrate into HepG2 cells. Under severe cholestatic conditions, urinary excretion favors the elimination of amidated BAs, while glucuronidation allows the conversion of cytotoxic BAs into nontoxic derivatives.
Assuntos
Ácidos e Sais Biliares/toxicidade , Ácidos e Sais Biliares/urina , Colestase/metabolismo , Colestase/urina , Fígado/metabolismo , Apoptose/efeitos dos fármacos , Ácido Quenodesoxicólico/toxicidade , Ácido Quenodesoxicólico/urina , Ácido Desoxicólico/toxicidade , Ácido Desoxicólico/urina , Feminino , Células Hep G2 , Humanos , Ácido Litocólico/toxicidade , Ácido Litocólico/urina , MasculinoRESUMO
Bicalutamide (Casodex(®) ) is a non-steroidal pure anti-androgen used in the treatment of localized prostate cancer. It is a racemate drug, and its activity resides in the (R)-enantiomer, with little in the (S)-enantiomer. A major metabolic pathway for bicalutamide is glucuronidation catalysed by UDP-glucuronosyltransferase (UGT) enzymes. While (S)bicalutamide is directly glucuronidated, (R)bicalutamide requires hydroxylation prior to glucuronidation. The contribution of human tissues and UGT isoforms in the metabolism of these enantiomers has not been extensively investigated. In this study, both (R) and/or (S)bicalutamide were converted into glucuronide (-G) derivatives after incubation of pure and racemic solutions with microsomal extracts from human liver and kidney. Intestinal microsomes exhibited only low reactivity with these substrates. Km values of liver and kidney samples for (S)bicalutamide glucuronidation were similar, and lower than values obtained with the (R)-enantiomer. Among the 16 human UGTs tested, UGT1A8 and UGT1A9 were able to form both (S) and (R)bicalutamide-G from pure or racemic substrates. UGT2B7 was also able to form (R)bicalutamide-G. Kinetic parameters of the recombinant UGT2B7, UGT1A8 and UGT1A9 enzymes support a predominant role of the UGT1A9 isoform in bicalutamide metabolism. Accordingly, (S)bicalutamide inhibited the ability of human liver and kidney microsomes to glucuronidate the UGT1A9 probe substrate, propofol. In conclusion, the present study provides the first comprehensive analysis of in vitro bicalutamide glucuronidation by human tissues and UGTs and identifies UGT1A9 as a major contributor for (R) and (S) glucuronidation in the human liver and kidney.
Assuntos
Antagonistas de Androgênios/farmacologia , Anilidas/farmacologia , Glucuronosiltransferase/metabolismo , Rim/enzimologia , Fígado/enzimologia , Nitrilas/farmacologia , Compostos de Tosil/farmacologia , Cromatografia Líquida , Humanos , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Masculino , Microssomos/enzimologia , Neoplasias da Próstata/tratamento farmacológico , Estereoisomerismo , Espectrometria de Massas em Tandem , UDP-Glucuronosiltransferase 1ARESUMO
Recent progresses in molecular pharmacology approaches have allowed the identification and characterization of a series of nuclear receptors (NR) which efficiently control the level UDP-glucuronosyltransferase (UGT) genes expression. These regulatory processes ensure optimized UGT expression in response to specific endogenous and/or exogenous stimuli. Interestingly, numerous endogenous activators of these NRs are conjugated by the UGT enzymes they regulate. In such a case, the NR-dependent regulation of UGT genes corresponds to a feedforward/feedback mechanism by which a bioactive molecule controls its own concentrations. In the present review, we will discuss i) how bilirubin reduces its circulating levels by activating AhR in the liver; ii) how bile acids modulate their hepatic glucuronidation via PXR- and FXR-dependent processes in enterohepatic tissues; and iii) how androgens inhibit their cellular metabolism in prostate cancer cells through an AR-dependent mechanism. Subsequently, with further discussion of the same examples (bilirubin and bile acids), we will illustrate how NR-dependent regulation of UGT enzymes may contribute to the beneficial effects of pharmacological activators of nuclear receptors, such as CAR and PPARa.
Assuntos
Fatores Ativadores da Transcrição/fisiologia , Ácidos e Sais Biliares/metabolismo , Bilirrubina/sangue , Fígado/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Fatores Ativadores da Transcrição/metabolismo , Células Cultivadas , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais/fisiologiaRESUMO
Bile acids subserve important physiological functions in the control of cholesterol homeostasis. Indeed, hepatic bile acid synthesis and biliary excretion constitute the main route for cholesterol removal from the human body. On the other hand, bile acids serve as natural detergents for the intestinal absorption of dietary cholesterol. However, due to their detergent properties, bile acids are inherently cytotoxic, and their cellular level may be tightly controlled to avoid pathological situations such as cholestasis. Recent investigations have illustrated the crucial roles that a series of ligand-activated transcription factors has in the control of hepatic bile acids synthesis, transport and metabolism. Thus, the lipid-activated nuclear receptors, farnesoid X-receptor (FXR), liver X-receptor (LXR), pregnane X-receptor (PXR) and peroxisome proliferator-activated receptor alpha (PPAR alpha), modulate the expression and activity of genes controlling bile acid homeostasis in the liver. Several members of the UDP-glucuronosyltransferase (UGT) enzymes family are among the bile acid metabolizing enzymes regulated by these receptors. UGTs catalyze glucuronidation, a major phase II metabolic reaction, which converts hydrophobic bile acids into polar and urinary excretable metabolites. This article summarizes our recent observations on the regulation of bile acid conjugating UGTs upon pharmacological activation of lipid-activated receptors, with a particular interest for the role of PPAR alpha and LXRalpha in controlling human UGT1A3 expression.
Assuntos
Ácidos e Sais Biliares/metabolismo , Proteínas de Ligação a DNA/metabolismo , Glucuronosiltransferase/metabolismo , PPAR alfa/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Humanos , Metabolismo dos Lipídeos , Lipídeos/farmacologia , Fígado/metabolismo , Receptores X do Fígado , Modelos Biológicos , Receptores Nucleares Órfãos , Receptor de Pregnano X , Receptores de Esteroides/metabolismoRESUMO
The UDP-glucuronosyltransferase (UGT) 1A genes in humans have been shown to be differentially regulated in a tissue-specific fashion. Transgenic mice carrying the human UGT1 locus (Tg-UGT1) were recently created, demonstrating that expression of the nine UGT1A genes closely resembles the patterns of expression observed in human tissues. In the present study, UGT1A1, UGT1A3, UGT1A4, and UGT1A6 have been identified as targets of the peroxisome proliferator-activated receptor (PPAR) alpha in human hepatocytes and Tg-UGT1 mice. Oral administration of the PPARalpha agonist 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (pirinixic acid, WY-14643) to Tg-UGT1 mice led to induction of these proteins in either the liver, gastrointestinal tract, or kidney. The levels of induced UGT1A3 gene transcripts in liver and UGT1A4 protein in small intestine correlated with induced lamotrigine glucuronidation activity in these tissues. With UGT1A3 previously identified as the major human enzyme involved in human C24-glucuronidation of lithocholic acid (LCA), the dramatic induction of liver UGT1A3 RNA in Tg-UGT1 mice was consistent with the formation of LCA-24G in plasma. Furthermore, PPAR-responsive elements (PPREs) were identified flanking the UGT1A1, UGT1A3, and UGT1A6 genes by a combination of site-directed mutagenesis, specific binding to PPARalpha and retinoic acid X receptor alpha, and functional response of the concatenated PPREs in HepG2 cells overexpressing PPARalpha. In conclusion, these results suggest that oral fibrate treatment in humans will induce the UGT1A family of proteins in the gastrointestinal tract and liver, influencing bile acid glucuronidation and first-pass metabolism of other drugs that are taken concurrently with hypolipidemic therapy.
Assuntos
Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , PPAR alfa/agonistas , Proliferadores de Peroxissomos/farmacologia , Pirimidinas/farmacologia , Animais , Anticonvulsivantes/metabolismo , Linhagem Celular , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Humanos , Lamotrigina , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Camundongos , Camundongos Transgênicos , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Triazinas/metabolismoRESUMO
Chenodeoxycholic acid (CDCA) is a liver-formed detergent and plays an important role in the control of cholesterol homeostasis. During cholestasis, toxic bile acids (BA) accumulate in hepatocytes causing damage and consequent impairment of their function. Glucuronidation, a conjugation reaction catalyzed by UDP-glucuronosyltransferase (UGT) enzymes, is considered an important metabolic pathway for hepatic BA. This study identifies the human UGT1A3 enzyme as the major enzyme responsible for the hepatic formation of the acyl CDCA-24glucuronide (CDCA-24G). Kinetic analyses revealed that human liver and UGT1A3 catalyze the formation of CDCA-24G with similar K(m) values of 10.6 to 18.6 mumol/L, respectively. In addition, electrophoretic mobility shift assays and transient transfection experiments revealed that glucuronidation reduces the ability of CDCA to act as an activator of the nuclear farnesoid X-receptor (FXR). Finally, we observed that treatment of human hepatocytes with fibrates increases the expression and activity of UGT1A3, whereas CDCA has no effect. In conclusion, UGT1A3 is the main UGT enzyme for the hepatic formation of CDCA-24G and glucuronidation inhibits the ability of CDCA to act as an FXR activator. In vitro data also suggest that fibrates may favor the formation of bile acid glucuronides in cholestatic patients.
Assuntos
Ácido Quenodesoxicólico/metabolismo , Glucuronosiltransferase/metabolismo , Fígado/enzimologia , Adulto , Linhagem Celular , Ácido Clofíbrico/farmacologia , Proteínas de Ligação a DNA/metabolismo , Feminino , Hepatócitos/metabolismo , Humanos , Masculino , Microcorpos , Microssomos Hepáticos/metabolismo , Pessoa de Meia-Idade , PPAR alfa/metabolismo , Pirimidinas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Glucuronidation, an important bile acid detoxification pathway, is catalyzed by enzymes belonging to the UDP-glucuronosyltransferase (UGT) family. Among UGT enzymes, UGT1A3 is considered the major human enzyme for the hepatic C24-glucuronidation of the primary chenodeoxycholic (CDCA) and secondary lithocholic (LCA) bile acids. We identify UGT1A3 as a positively regulated target gene of the oxysterol-activated nuclear receptor liver X-receptor alpha (LXRalpha). In human hepatic cells and human UGT1A transgenic mice, LXRalpha activators induce UGT1A3 mRNA levels and the formation of CDCA-24glucuronide (24G) and LCA-24G. Furthermore, a functional LXR response element (LXRE) was identified in the UGT1A3 promoter by site-directed mutagenesis, electrophoretic mobility shift assays and chromatin immunoprecipitation experiment. In addition, LXRalpha is found to interact with the SRC-1alpha and NCoR cofactors to regulate the UGT1A3 gene, but not with PGC-1beta. In conclusion, these observations establish LXRalpha as a crucial regulator of bile acid glucuronidation in humans and suggest that accumulation of oxysterols in hepatocytes during cholestasis favors bile acid detoxification as glucuronide conjugates. LXR agonists may be useful for stimulating both bile acid detoxification and cholesterol removal in cholestatic or hypercholesterolemic patients, respectively.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Glucuronosiltransferase/genética , Hepatócitos/metabolismo , RNA Mensageiro/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Western Blotting , Células Cultivadas , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/efeitos dos fármacos , Glucuronosiltransferase/efeitos dos fármacos , Glucuronosiltransferase/metabolismo , Hepatócitos/citologia , Humanos , Hidrocarbonetos Fluorados , Técnicas In Vitro , Receptores X do Fígado , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores Nucleares Órfãos , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfonamidas/farmacologiaRESUMO
The present study reports a novel method for the production and purification of analytical standards of glucuronide conjugates of bile acids, chenodeoxycholic (CDCA), lithocholic, (LCA) and hyodeoxycholic (HDCA) acids. CDCA-3G (CDCA-3-glucuronide) and -24G, LCA-3G and -24G, and HDCA-6G and -24G were enzymatically formed by using microsomes from human liver, purified by liquid chromatography, digested with recombinant beta-glucuronidase, and quantified by liquid chromatography/electrospray ionization coupled to mass spectrometry (LC-ESI/MS). The position of the glucuronosyl moiety on the bile acids was determined by analyzing the susceptibility to hydrolysis under elevated pH and temperature conditions of the standards. By using the purified analytical standards, a LC-ESI/MS/MS method was developed for the determination of these glucuronide conjugates in in vitro assays. The linearity of the assay ranged from 0.5 to 40 ng/mL for the six glucuronides, and the limit of quantification (LOQ) was 0.5 ng/mL. Intra- and interday precisions and accuracy values were all lower than 10.2%. Furthermore, processed sample stability analyses revealed that the six standards were stable at 4 degrees C for more than 24 h. This method was successfully used for the quantification of CDCA, LCA, and HDCA glucuronides formed by human liver or hepatoma HepG2 cells. In conclusion, such a method allows the purification of high-quality analytical standards of glucuronide derivatives and may easily be used for the quantification of other endo- and xenobiotics that are glucuronidated.
Assuntos
Ácidos e Sais Biliares/normas , Cromatografia Líquida/métodos , Glucuronídeos/normas , Espectrometria de Massas por Ionização por Electrospray/métodos , Ácidos e Sais Biliares/química , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Ácido Desoxicólico/química , Ácido Desoxicólico/normas , Glucuronídeos/química , Humanos , Ácido Litocólico/química , Ácido Litocólico/normas , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estatística como AssuntoRESUMO
The human UDP-glucuronosyltransferase 1 (UGT1) locus spans nearly 200 kb on chromosome 2 and encodes nine UGT1A proteins that play a prominent role in drug and xenobiotic metabolism. Transgenic UGT1 (Tg-UGT1) mice have been created, and it has been demonstrated that tissue-specific and xenobiotic receptor control of the UGT1A genes is influenced through circulating humoral factors. In Tg-UGT1 mice, the UGT1A proteins are differentially expressed in the liver and gastrointestinal tract. Gene expression profiles confirmed that all of the UGT1A genes can be targeted for regulation by the pregnane X receptor activator pregnenolone-16alpha-carbonitrile (PCN) or the Ah receptor ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In addition, the selective induction of glucuronidation activity toward lamotrigine, ethinyl estradiol, chenodeoxycholic acid, and lithocholic acid by either PCN or TCDD in small intestine from Tg-UGT1 mice corresponded to expression of the locus in this tissue. Induction of UGT1A1 by PCN and TCDD is believed to be highly dependent upon glucocorticoids, because submicromolar concentrations of dexamethasone actively promote PCN and TCDD induction of UGT1A1 in Tg-UGT1 primary hepatocytes. The role of hormonal control of the UGT1 locus was further verified in pregnant and nursing Tg-UGT1 mice. In maternal 14-day post-conception Tg-UGT1mice, liver UGT1A1, UGT1A4, and UGT1A6 were induced, with the levels returning to near normal by birth. However, maternal liver UGT1A4 and UGT1A6 were dramatically elevated and maintained after birth, indicating that these proteins may play a critical role in maternal metabolism during lactation. With expression of the UGT1 locus confirmed in a variety of mouse tissues, these results suggested that the Tg-UGT1 mice will be a useful model to examine the regulatory and functional properties of human glucuronidation.