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2.
Expert Opin Drug Metab Toxicol ; 15(11): 975-984, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31619082

RESUMO

Objectives: Riociguat is a soluble guanylate cyclase stimulator licensed for the treatment of pulmonary arterial hypertension (PAH), a potentially fatal complication of human immunodeficiency virus infection. This study investigated the inhibitory potency of selected antiretroviral regimens on the metabolic clearance of riociguat.Methods: The inhibitory potential of the components of six antiretroviral combinations (ATRIPLA® (efavirenz/emtricitabine/tenofovir disoproxil), COMPLERA® (rilpivirine/emtricitabine/tenofovir disoproxil), STRIBILD® (elvitegravir/cobicistat/emtricitabine/tenofovir disoproxil), TRIUMEQ® (abacavir/dolutegravir/lamivudine), and two ritonavir-boosted regimens) on riociguat metabolism were evaluated in recombinant human CYP1A1 and CYP3A4 as well as in human hepatocytes exhibiting both CYP1A1 and CYP3A4 activity. In vitro-in vivo correlation was performed between calculated and observed increases in riociguat exposure in vivo.Results: Using both in vitro systems, the predicted increase in exposure of riociguat was highest with components of TRIUMEQ® followed by COMPLERA®, ATRIPLA®, STRIBILD®, and the ritonavir-boosted regimens. Further experiments in human hepatocytes confirmed CYP1A1 to be the predominant enzyme in the metabolic clearance of riociguat.Conclusion: Antiretroviral treatment containing the potent CYP1A1 inhibitor abacavir had the greatest impact on riociguat metabolic clearance. The impact of comedications containing only strong CYP3A4 inhibitors e.g. ritonavir was less pronounced, suggesting a benefit of riociguat over PAH-targeting medications with contraindications for use with strong CYP3A4 inhibitors.


Assuntos
Fármacos Anti-HIV/farmacologia , Citocromo P-450 CYP1A1/metabolismo , Ativadores de Enzimas/metabolismo , Pirazóis/metabolismo , Pirimidinas/metabolismo , Fármacos Anti-HIV/administração & dosagem , Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP3A/efeitos dos fármacos , Citocromo P-450 CYP3A/metabolismo , Inibidores do Citocromo P-450 CYP3A/administração & dosagem , Inibidores do Citocromo P-450 CYP3A/farmacologia , Interações de Medicamentos , Ativadores de Enzimas/administração & dosagem , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Humanos , Técnicas In Vitro , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem
3.
Int J Mol Sci ; 20(18)2019 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-31540136

RESUMO

Insulin resistance and diabetes are both associated with chronic hepatitis C virus (HCV) infection, and the glucagon-like peptide-1(GLP-1) receptor agonist, liraglutide, is a common therapy for diabetes. Our aim was to investigate whether liraglutide treatment can inhibit HCV replication. A cell culture-produced HCV infectious system was generated by transfection of in vitro-transcribed genomic JFH-1 ribonucleic acid (RNA) into Huh-7.5 cells. Total RNA samples were extracted to determine the efficiency of HCV replication. The Ava5 cells were treated with liraglutide and cell viability was calculated. A Western blot analysis of the protein expression was performed. The immunoreactive blot signals were also detected. Liraglutide activated GLP-1 receptors in the HCV infectious system, and inhibited subgenomic HCV RNA replication in the HuH-7.5 cells. The Western blot analysis revealed both HCV protein and replicon RNA were reduced after treatment with liraglutide in a dose-dependent manner. Liraglutide decreased the cell viability of HCV RNA at an optimum concentration of 120 µg/mL, activated the 5' adenosine monophosphate-activated protein kinase (AMPK) and the phosphorylated- transducer of regulated cyclic adenosine monophosphate (CAMP) response element-binding protein 2 (TORC2), thereby decreasing the cell viability of phosphoenolpyruvate carboxykinase (PEPCK) and G6pase RNA Therefore, we conclude that liraglutide can inhibit HCV replication via an AMPK/TORC2-dependent pathway.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Hepacivirus/efeitos dos fármacos , Hepatócitos/enzimologia , Liraglutida/farmacologia , Replicação Viral/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/agonistas , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Hepacivirus/fisiologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
4.
Nutr Metab Cardiovasc Dis ; 29(11): 1245-1253, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31439394

RESUMO

BACKGROUND AND AIMS: The novel nutraceutical combination containing red yeast rice (monacolin K 3.3 mg), Berberis aristata cortex extract (Berberine 531.25 mg) and Morus alba leaves extract (1-deoxynojirimycin 4 mg) is effective in the management of elevated plasma low-density lipoprotein cholesterol (LDL-C) levels. The aim of the present study was to investigate the effects of the three components on proprotein convertase subtilisin/kexin type 9 (PCSK9), a key regulator of LDL receptor (LDLR) expression, in hepatocyte cell lines and to compare their effects on LDL cellular uptake. METHODS AND RESULTS: HepG2 and Huh7 cells were incubated with B. aristata cortex extract (BCE), red yeast rice (RYR) and M. alba leaves extract (MLE) alone or in combination for 24 h. RYR (50 µg/mL) increased PCSK9 protein expression (Western blot analysis and ELISA), PCSK9 mRNA (qPCR) and its promoter activity (luciferase reporter assay). BCE (40 µg/mL) reduced instead PCSK9 expression, mRNA levels and promoter activity. MLE determined a concentration-dependent reduction of PCSK9 at the mRNA and protein levels, with a maximal reduction at 1 mg/mL, without significant changes of PCSK9 promoter activity. MLE also downregulated the expression of 3-hydroxy-3-methyl-3-glutaryl coenzyme A reductase and fatty acid synthase mRNA levels. The combination of RYR, BCE and MLE reduced the PCSK9 mRNA and protein levels, as well as the promoter activity. Finally, the single components and their combination induced LDL receptor and LDL uptake by the hepatocytes. CONCLUSION: The positive effect of MLE on PCSK9 supports the rationale of using the nutraceutical combination of RYR, BCE and MLE to control hyperlipidemic conditions.


Assuntos
Anticolesterolemiantes/farmacologia , Berberis/química , Produtos Biológicos/farmacologia , LDL-Colesterol/metabolismo , Hepatócitos/efeitos dos fármacos , Lovastatina/farmacologia , Morus/química , Extratos Vegetais/farmacologia , Pró-Proteína Convertase 9/metabolismo , Anticolesterolemiantes/isolamento & purificação , Relação Dose-Resposta a Droga , Regulação para Baixo , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Regulação Enzimológica da Expressão Gênica , Células Hep G2 , Hepatócitos/enzimologia , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Extratos Vegetais/isolamento & purificação , Folhas de Planta/química , Pró-Proteína Convertase 9/genética
5.
Fish Physiol Biochem ; 45(5): 1747-1757, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31297679

RESUMO

An 8-week feeding trial was conducted to investigate effects of dietary protein levels (37, 40, and 43%) on the growth performance, feed utilization, digestive enzyme activity, and gene expressions of target of rapamycin (TOR) signaling pathway in fingerling yellow catfish. One hundred and eighty fingerlings (average weight 0.77 ± 0.03 g) were equally distributed across four replicate tanks for each of the three treatments, with 15 fish per tank. No difference (P > 0.05) was observed in initial body weight, survival rate (SR), hepatosomatic index (HSI), viscera index (VSI), dressing percentage (DP), and condition factor (CF) among all the treatments. The diet containing 40% protein increased significantly (P < 0.05) final body weight, weight gain rate (WGR), specific growth rate (SGR), protein efficiency ratio (PER), nitrogen retention (NRE), and energy retention (ERE) in fish. The highest protease activity in the stomach and intestine was observed in the P40 group (P < 0.05), while amylase and lipase were not significantly different (P > 0.05). The transcriptional levels of IGF-1, IGF-1R, and Akt were significantly (P < 0.05) higher in fish fed P40 or P43 than those of fish fed P37. TOR and S6K1 mRNA expressions were significantly (P < 0.05) increased in the P40 groups. Hence, the diet containing 40% protein would be suitable for the optimum growth and effective protein utilization of fingerling Pelteobagrus fulvidraco. In vitro, the transcriptional levels of IGF-1, IGF-1R, Akt, TOR, and S6K1 in hepatocyte supplemented with a 40-µM mixed amino acids were significantly (P < 0.05) higher compared to other treatments. No difference (P > 0.05) was observed in eukaryotic translation initiation factor 4E-binding protein 1 in vivo and in vitro among all the treatments. Effects of dietary protein level on growth performance likely are involved in the activation of TOR signaling pathway in fingerling Pelteobagrus fulvidraco.


Assuntos
Peixes-Gato/crescimento & desenvolvimento , Proteínas na Dieta/farmacologia , Digestão/efeitos dos fármacos , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Animais , Peixes-Gato/fisiologia , Células Cultivadas , Dieta/veterinária , Digestão/fisiologia , Enzimas/genética , Enzimas/metabolismo , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Defeitos do Tubo Neural , Transdução de Sinais
6.
J Biochem Mol Toxicol ; 33(9): e22372, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31332890

RESUMO

ß-Hydroxybutyrate (BHB), one of ketone body, has been traditionally regarded as an alternative carrier of energy, but recent studies found that BHB plays versatile roles in inflammation. It has been previously reported that the level BHB declined in mice with lipopolysaccharide (LPS)/d-galactosamine (d-Gal)-induced liver damage, but the pathological significance remains unclear. In the present study, the pathophysiological roles of BHB in LPS/d-Gal-induced hepatic damage has been investigated. The results indicated pretreatment with BHB further enhanced LPS/d-Gal-induced elevation of aspartate aminotransferase and alanine aminotransferase, exacerbated the histological abnormalities and increased the mortality. Pretreatment with BHB upregulated the level of tumor necrosis factor α and interleukin-6 in plasma, promoted the activities of caspase-3, caspase-8, and caspase-9 and increased the count of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells. In addition, post-insult supplement with BHB also potentiated LPS/d-Gal-induced apoptotic liver damage. Therefore, BHB might be a detrimental factor in LPS/d-Gal-induced liver injury via enhancing the inflammation and the apoptosis in the liver.


Assuntos
Ácido 3-Hidroxibutírico/farmacologia , Apoptose/efeitos dos fármacos , Galactosamina/toxicidade , Hepatócitos/efeitos dos fármacos , Inflamação/induzido quimicamente , Lipopolissacarídeos/toxicidade , Animais , Caspases/metabolismo , Ativação Enzimática , Hepatócitos/citologia , Hepatócitos/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C
7.
Chemosphere ; 234: 513-519, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31229712

RESUMO

Given the ubiquity of iodinated disinfection by-products (I-DBPs) in drinking water and their prominent toxicity, it is of vital significance to evaluate I-DBPs toxicity and explore the underlying mechanism. The toxicity of iodoacetic acid (IAA), a typical type of I-DBPs, might be linked with oxidative stress. However, it remains unknown for the response of antioxidant enzyme superoxide dismutase (SOD) in the mouse primary hepatocytes when exposed to IAA and the underlying mechanism. This study explored SOD response to IAA and the underlying mechanisms at the molecular and cellular levels. Under IAA exposure, the observed increase of SOD activity in the hepatocytes was caused by the increase of SOD production via ROS stimulation and the increase of SOD molecular activity. Molecular experiments showed that IAA binds to SOD molecule mainly via electrostatic forces with one binding site around the active site and six binding sites in the surface of protein. The binding interaction leads to the conformational changes of SOD and the disruption of protein aggregates. This work could offer basic data for the comprehensive understanding of the adverse effects of IAA and references for assessing the harmful effects of DBPs.


Assuntos
Desinfecção/métodos , Ácido Iodoacético/química , Superóxido Dismutase/metabolismo , Animais , Antioxidantes/farmacologia , Água Potável/química , Hepatócitos/enzimologia , Ácido Iodoacético/metabolismo , Ácido Iodoacético/toxicidade , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Agregados Proteicos/efeitos dos fármacos , Ligação Proteica , Conformação Proteica/efeitos dos fármacos
8.
Ecotoxicol Environ Saf ; 181: 388-394, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31212187

RESUMO

With the wider application of cadmium-containing quantum dots (Cd-QDs) in biomedical fields, it is easier for people to be exposed. Studies have suggested that Cd-QDs could release cadmium ion and induce oxidative effects due to the disruption of redox equilibrium. Antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD), play an important role in organisms to resist the negative impact of exogenous substances. Molecular mechanisms of antioxidant enzymes with Cd-QDs remain unclear, however. In this study, structural and functional changes of CAT and SOD have been investigated under low dose Cd-QDs exposure. Cell viability, malondialdehyde (MDA) level, CAT and SOD activities were influenced by Cd-QDs in hepatocytes of mice. To further investigate the responses of CAT and SOD to Cd-QDs, multiple spectroscopic, calorimetric and activity measurements were carried out. Similar interaction patterns were observed that result in interaction force, structural and functional changes: Cd-QDs combine with CAT and SOD through hydrophobic forces; Intrinsic fluorescence of proteins was statically quenched by Cd-QDs and new complexes were formed; Also, the skeleton and secondary structure (with α-helix decrease) of CAT and SOD was influenced. Taken together, we suggest that Cd-QDs chosen in this study induce oxidative stress effects to hepatocytes but have not caused serious oxidative stress damage at concentrations below 10 µg/mL. MPA-CdSe/ZnS QDs caused the lowest level of oxidative stress which is associated with the induction of antioxidant proteins. This paper presents responses of CAT and SOD to low-dose Cd-QDs, and provides a reference for evaluating health damages caused by Cd-QDs.


Assuntos
Cádmio/toxicidade , Catalase/metabolismo , Estresse Oxidativo , Pontos Quânticos/toxicidade , Superóxido Dismutase/metabolismo , Animais , Antioxidantes/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Malondialdeído/metabolismo , Camundongos
9.
J Dairy Sci ; 102(8): 7536-7547, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31178189

RESUMO

High blood concentrations of nonesterified fatty acids (NEFA) and altered lipid metabolism are key characteristics of fatty liver in dairy cows. In nonruminants, the mitochondrial membrane protein mitofusin 2 (MFN2) plays important roles in regulating mitochondrial function and intrahepatic lipid metabolism. Whether MFN2 is associated with hepatic lipid metabolism in dairy cows with moderate fatty liver is unknown. Therefore, to investigate changes in MFN2 expression and lipid metabolic status in dairy cows with moderate fatty liver, blood and liver samples were collected from healthy dairy cows (n = 10) and cows with moderate fatty liver (n = 10). To determine the effects of MFN2 on lipid metabolism in vitro, hepatocytes isolated from healthy calves were used for small interfering RNA-mediated silencing of MFN2 or adenovirus-mediated overexpression of MFN2 for 48 h, or treated with 0, 0.6, 1.2, or 2.4 mM NEFA for 12 h. Milk production and plasma glucose concentrations in dairy cows with moderate fatty liver were lower, but concentrations of NEFA and ß-hydroxybutyrate (BHB) were greater in dairy cows with moderate fatty liver. Dairy cows with moderate fatty liver displayed hepatic lipid accumulation and lower abundance of hepatic MFN2, peroxisome proliferator-activated receptor-α (PPARα), and carnitine palmitoyltransferase 1A (CPT1A). However, sterol regulatory element-binding protein 1c (SREBP-1c), acetyl CoA carboxylase 1 (ACACA), fatty acid synthase (FASN), and diacylglycerol acyltransferase 1 (DGAT1) were more abundant in the livers of dairy cows with moderate fatty liver. In vitro, exogenous NEFA treatment upregulated abundance of SREBP-1c, ACACA, FASN, and DGAT1, and downregulated the abundance of PPARα and CPT1A. These changes were associated with greater lipid accumulation in calf hepatocytes, and MFN2 silencing aggravated this effect. In contrast, overexpression of MFN2-ameliorated exogenous NEFA-induced lipid accumulation by downregulating the abundance of SREBP-1c, ACACA, FASN, and DGAT1, and upregulating the abundance of PPARα and CPT1A in calf hepatocytes. Overall, these data suggest that one cause for the negative effect of excessive NEFA on hepatic lipid accumulation is the inhibition of MFN2. As such, these mechanisms partly explain the development of hepatic steatosis in dairy cows.


Assuntos
Doenças dos Bovinos/metabolismo , Bovinos/metabolismo , Fígado Gorduroso/veterinária , GTP Fosfo-Hidrolases/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Ácido 3-Hidroxibutírico/metabolismo , Animais , Bovinos/genética , Doenças dos Bovinos/enzimologia , Doenças dos Bovinos/genética , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Fígado Gorduroso/enzimologia , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Feminino , GTP Fosfo-Hidrolases/genética , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Mitocôndrias/metabolismo , PPAR alfa/genética , PPAR alfa/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo
10.
Toxicol Lett ; 313: 66-76, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31201936

RESUMO

Mono-methylindoles (MMI) were described as agonists and/or antagonists of the human aryl hydrocarbon receptor (AhR). Here, we investigated the effects of MMI on AhR-CYP1A pathway in human hepatocytes and HepaRG cells derived from human progenitor hepatic cells. All MMI, except of 2-methylindole, strongly induced CYP1A1 and CYP1A2 mRNAs in HepaRG cells. Induction of CYP1A genes was absent in AhR-knock-out HepaRG cells. Consistently, CYP1A1 and CYP1A2 mRNAs and proteins were induced by all MMIs (except 2-methylindole), in human hepatocytes. The enzyme activity of CYP1A1 was inhibited by MMIs in human hepatocytes and LS180 colon cancer cells in a concentration-dependent manner (IC50 values from 1.2 µM to 23.8 µM and from 3.4 µM to 11.4 µM, respectively). Inhibition of CYP1A1 activity by MMI in human liver microsomes was much weaker as compared to that in intact cells. Incubation of parental MMI with human hepatocytes either diminished (4-methylindole, 6-methylindole) or enhanced (7-methylindole) their agonist effects on AhR in AZ-AHR reporter cells. In conclusion, overall effects of MMI on AhR-CYP1A pathway in human cells comprise the induction of CYP1A genes through AhR, the inhibition of CYP1A catalytic activity and possibly the metabolic transformation causing loss or gain of AhR agonist activity of parental compounds.


Assuntos
Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP1A1/biossíntese , Indutores das Enzimas do Citocromo P-450/farmacologia , Inibidores das Enzimas do Citocromo P-450/farmacologia , Hepatócitos/efeitos dos fármacos , Indóis/farmacologia , Idoso , Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/enzimologia , Citocromo P-450 CYP1A1/genética , Relação Dose-Resposta a Droga , Indução Enzimática , Feminino , Hepatócitos/enzimologia , Humanos , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Pessoa de Meia-Idade , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Adulto Jovem
11.
Hepatobiliary Pancreat Dis Int ; 18(4): 360-365, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31126802

RESUMO

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent chronic liver diseases. However, the exact pathogenesis of NAFLD remains to be elucidated. Despite the association with tumors and cardiovascular diseases, the role of miR-222 in NAFLD remains unclear. The present study was to investigate the role of miR-222 in NAFLD. METHODS: Wild-type C57BL/6 mice were fed a high-fat diet for 12 weeks to induce NAFLD. Normal human liver cell line (L02) was cultured with free fatty acid (FFA)-containing medium to stimulate cell steatosis. The mRNA levels of miR-222 and acyl Coenzyme A xidase 1 (ACOX1) were detected by quantitative-PCR (Q-PCR). The prediction of ACOX1 as the target gene for miR-222 was conducted via TargetScan. The overexpression or inhibition of miR-222 was mediated by miR-222 mimics or antagomir, and intracellular triglyceride levels were measured using a triglyceride kit. Luciferase reporter assays verified ACOX1 as the target gene for miR-222. RESULTS: miR-222 was significantly elevated in both the in vivo and in vitro NAFLD models. Overexpression of miR-222 significantly increased triglyceride content in the L02 cells, while inhibition of miR-222 expression restricted the accumulation of triglyceride. Overexpression of miR-222 significantly inhibited ACOX1 expression. Transient transfection assays verified that ACOX1 3'-UTR luciferase reporter activity could be inhibited by miR-222 overexpression. CONCLUSIONS: The present study suggested that miR-222 promotes the accumulation of triglycerides by inhibiting ACOX1.


Assuntos
Acil-CoA Oxidase/metabolismo , Hepatócitos/enzimologia , MicroRNAs/metabolismo , Hepatopatia Gordurosa não Alcoólica/enzimologia , Oxirredutases/metabolismo , Triglicerídeos/metabolismo , Regiões 3' não Traduzidas , Acil-CoA Oxidase/genética , Animais , Sítios de Ligação , Linhagem Celular , Dieta Hiperlipídica , Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica , Hepatócitos/patologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Oxirredutases/genética , Regulação para Cima
12.
Nutrients ; 11(5)2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31137828

RESUMO

Sulforaphane (SFA), a naturally active isothiocyanate compound from cruciferous vegetables used in clinical trials for cancer treatment, was found to possess potency to alleviate insulin resistance. But its underlying molecular mechanisms are still incompletely understood. In this study, we assessed whether SFA could improve insulin sensitivity and glucose homeostasis both in vitro and in vivo by regulating ceramide production. The effects of SFA on glucose metabolism and expression levels of key proteins in the hepatic insulin signaling pathway were evaluated in insulin-resistant human hepatic carcinoma HepG2 cells. The results showed that SFA dose-dependently increased glucose uptake and intracellular glycogen content by regulating the insulin receptor substrate 1 (IRS-1)/protein kinase B (Akt) signaling pathway in insulin-resistant HepG2 cells. SFA also reduced ceramide contents and downregulated transcription of ceramide-related genes. In addition, knockdown of serine palmitoyltransferase 3 (SPTLC3) in HepG2 cells prevented ceramide accumulation and alleviated insulin resistance. Moreover, SFA treatment improved glucose tolerance and insulin sensitivity, inhibited SPTLC3 expression and hepatic ceramide production and reduced hepatic triglyceride content in vivo. We conclude that SFA recovers glucose homeostasis and improves insulin sensitivity by blocking ceramide biosynthesis through modulating SPTLC3, indicating that SFA may be a potential candidate for prevention and amelioration of hepatic insulin resistance via a ceramide-dependent mechanism.


Assuntos
Ceramidas/biossíntese , Inibidores Enzimáticos/farmacologia , Glucose/metabolismo , Hepatócitos/efeitos dos fármacos , Resistência à Insulina , Isotiocianatos/farmacologia , Fígado/efeitos dos fármacos , Serina C-Palmitoiltransferase/antagonistas & inibidores , Animais , Glicogênio/metabolismo , Células Hep G2 , Hepatócitos/enzimologia , Humanos , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina/metabolismo , Fígado/enzimologia , Masculino , Camundongos Endogâmicos C57BL , Ácido Palmítico/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Transdução de Sinais , Triglicerídeos/metabolismo
13.
Sensors (Basel) ; 19(10)2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31096615

RESUMO

Human hepatoma HepaRG cells express most drug metabolizing enzymes and constitute a pertinent in vitro alternative cell system to primary cultures of human hepatocytes in order to determine drug metabolism and evaluate the toxicity of xenobiotics. In this work, we established novel transgenic HepaRG cells transduced with lentiviruses encoding the reporter green fluorescent protein (GFP) transcriptionally regulated by promoter sequences of cytochromes P450 (CYP) 1A1/2, 2B6 and 3A4 genes. Here, we demonstrated that GFP-biosensor transgenes shared similar expression patterns with the corresponding endogenous CYP genes during proliferation and differentiation in HepaRG cells. Interestingly, differentiated hepatocyte-like HepaRG cells expressed GFP at higher levels than cholangiocyte-like cells. Despite weaker inductions of GFP expression compared to the strong increases in mRNA levels of endogenous genes, we also demonstrated that the biosensor transgenes were induced by prototypical drug inducers benzo(a)pyrene and phenobarbital. In addition, we used the differentiated biosensor HepaRG cells to evidence that pesticide mancozeb triggered selective cytotoxicity of hepatocyte-like cells. Our data demonstrate that these new biosensor HepaRG cells have potential applications in the field of chemicals safety evaluation and the assessment of drug hepatotoxicity.


Assuntos
Técnicas Biossensoriais , Citocromo P-450 CYP1A1/isolamento & purificação , Citocromo P-450 CYP2B6/isolamento & purificação , Citocromo P-450 CYP3A/isolamento & purificação , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP3A/genética , Proteínas de Fluorescência Verde/genética , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Humanos , Lentivirus/genética , Taxa de Depuração Metabólica , Transgenes/genética
14.
Yakugaku Zasshi ; 139(5): 699-704, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31061338

RESUMO

Human hepatocytes possess a wider range of phase I and II drug-metabolizing enzyme activities than other liver tissue-derived products, such as human liver microsomes. Thus, hepatocytes may be useful for predicting the in vivo metabolic fate of new drugs of abuse in humans. Recently, new types of human hepatocytes have been made commercially available for use in drug metabolism studies, such as a liver tumor-derived cell line (HepaRG), and a human induced pluripotent stem cell-derived hepatocyte (h-iPS-HEP). In our laboratory, HepaRG has been used to elucidate the metabolic pathways of XLR-11, a synthetic cannabinoid, and its thermal degradant. In addition, the potential of h-iPS-HEP to metabolize drugs was assessed using fentanyl as a model drug, and indeed, h-iPS-HEP exhibited a pattern for fentanyl metabolite formation similar to that observed in vivo. In addition, the phase I and II drug-metabolizing enzyme activities of HepaRG, h-iPS-HEP, liver-humanized mouse-derived hepatocytes (PXB-cellsTM), and human primary hepatocytes were evaluated and compared. HepaRG showed high phase I and II drug metabolism activities; however, the CYP2D6 activity in these cells was quite low, and therefore h-iPS-HEP lacked O-methylation and conjugation activities. PXB-cells provided optimal results, i.e., these cells are extremely easy to use, and they possess higher phase I and II drug-metabolizing enzyme activities than the other cells tested. Although PXB-cells are contaminated with mouse-derived cells up to a concentration of several percent, this cell system appears to be promising for the prediction of in vivo human metabolism of new drugs of abuse.


Assuntos
Canabinoides/metabolismo , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Transtornos Relacionados ao Uso de Substâncias/metabolismo , Animais , Linhagem Celular , Citocromo P-450 CYP2D6/metabolismo , Fentanila/metabolismo , Humanos , Metilação , Camundongos
15.
Nihon Yakurigaku Zasshi ; 153(5): 235-241, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31092757

RESUMO

Three-dimensional (3D) cultured hepatocyte capable of maintaining liver-specific function in an in vivo state over a relatively long period of time have drawn attention as a new method for evaluating the metabolic process, hepatotoxicity and enzyme induction potential of drugs. When human hepatocytes were seeded on a plate for spheroid formation, and cell morphology and albumin secretion were examined, hepatocyte spheroid was stably maintained for at least 21 days after seeding. As a result of drug exposure to this spheroid, sequential metabolic reactions by Phase I and Phase II enzymes and metabolic reactions peculiar to only humans were observed. Moreover, when several drugs were exposed to spheroids and hepatotoxicity was evaluated, stable values were obtained for the 50% inhibitory concentration (IC50) of albumin secretion at 14 and 21 days. The IC50 values of most of the tested drugs were lower than in conventional assays, suggesting that the reported evaluation methods might underestimate hepatotoxicity. Furthermore, examination of mRNA expression level and activity of various cytochrome P450 (CYP) after exposure of typical inducers of CYPs to hepatocyte spheroid resulted in a significant increase in the expression level and activity of each. From these results, it was shown that this 3D hepatocyte spheroid system is suitable for follow-up of metabolic processes, long-term tests of hepatotoxicity and enzyme activity induction potential of drugs.


Assuntos
Sistema Enzimático do Citocromo P-450/fisiologia , Hepatócitos/citologia , Esferoides Celulares/citologia , Células Cultivadas , Hepatócitos/enzimologia , Humanos
16.
Elife ; 82019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30990169

RESUMO

Hepatocellular carcinoma (HCC) is a deadly human cancer associated with chronic inflammation. The cytosolic pathogen sensor NLRP12 has emerged as a negative regulator of inflammation, but its role in HCC is unknown. Here we investigated the role of NLRP12 in HCC using mouse models of HCC induced by carcinogen diethylnitrosamine (DEN). Nlrp12-/- mice were highly susceptible to DEN-induced HCC with increased inflammation, hepatocyte proliferation, and tumor burden. Consistently, Nlrp12-/- tumors showed higher expression of proto-oncogenes cJun and cMyc and downregulation of tumor suppressor p21. Interestingly, antibiotics treatment dramatically diminished tumorigenesis in Nlrp12-/- mouse livers. Signaling analyses demonstrated higher JNK activation in Nlrp12-/- HCC and cultured hepatocytes during stimulation with microbial pattern molecules. JNK inhibition or NLRP12 overexpression reduced proliferative and inflammatory responses of Nlrp12-/- hepatocytes. In summary, NLRP12 negatively regulates HCC pathogenesis via downregulation of JNK-dependent inflammation and proliferation of hepatocytes.


Assuntos
Carcinoma Hepatocelular/fisiopatologia , Regulação para Baixo , Hepatócitos/enzimologia , Hepatócitos/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/biossíntese , Neoplasias Hepáticas/fisiopatologia , Animais , Carcinógenos/administração & dosagem , Carcinoma Hepatocelular/induzido quimicamente , Proliferação de Células , Dietilnitrosamina/administração & dosagem , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Neoplasias Hepáticas/induzido quimicamente , Camundongos Knockout , Proteínas Proto-Oncogênicas c-myc/metabolismo
17.
Clin Exp Nephrol ; 23(7): 890-897, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30852714

RESUMO

BACKGROUND: Dysfunction of oxalate synthesis can cause calcium oxalate stone disease and inherited primary hyperoxaluria (PH) disorders. PH type I (PH1) is one of the most severe hyperoxaluria disorders, which results in urolithiasis, nephrocalcinosis, and end-stage renal disease. Here, we sought to determine the role of microRNAs in regulating AGXT to contribute to the pathogenesis of mutation-negative idiopathic oxalosis. METHODS: We conducted bioinformatics to search for microRNAs binding to AGXT, and examined the expression of the highest hit (miR-4660) in serum samples of patients with oxalosis, liver tissue samples, and determined the correlation and regulation between the microRNA and AGXT in vitro. RESULTS: MiR-4660 expression was downregulated in patients with oxalosis compared with healthy controls (84.03 copies/µL vs 33.02 copies/µL, P < 0.0001). Moreover, miR-4660 epigenetically decreased the expression of AGT in human liver tissues (Rho = - 0543, P = 0.037). Overexpression of miR-4660 in HepG2 and L02 cell lines led to dysregulation of AGXT at both the mRNA (by 71% and 81%, respectively; P < 0.001) and protein (by 49% and 42%, respectively; P < 0.0001) levels. We confirmed the direct target site of miR-4660 binding to the 3'UTR of AGXT by a luciferase assay. CONCLUSION: MiR-4660 is probably a new biomarker for mutation-negative idiopathic oxalosis by regulating the post-transcription of AGXT, providing a potential treatment target of mutation-negative idiopathic oxalosis.


Assuntos
Hepatócitos/enzimologia , Hiperoxalúria Primária/genética , MicroRNAs/genética , Transaminases/genética , Regiões 3' não Traduzidas , Sítios de Ligação , Estudos de Casos e Controles , Epigênese Genética , Regulação Enzimológica da Expressão Gênica , Marcadores Genéticos , Predisposição Genética para Doença , Células HeLa , Células Hep G2 , Humanos , Hiperoxalúria Primária/diagnóstico , Hiperoxalúria Primária/enzimologia , MicroRNAs/metabolismo , Fenótipo , Transaminases/metabolismo
18.
J Dairy Res ; 86(1): 68-72, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30732670

RESUMO

Dairy cows with ketosis display severe oxidative stress as well as high blood concentrations of non-esterified fatty acids (NEFA) and ß-hydroxybutyrate (BHB). Cytochrome P4502E1 (CYP2E1) plays an important role in the induction of oxidative stress. The aim of this study was to investigate CYP2E1 expression and activity in the liver of clinically ketotic cows (in vivo) and the effects of NEFA and BHB on CYP2E1 expression and activity in hepatocytes (in vitro). Dairy cows with clinical ketosis exhibited a low blood concentration of glucose but high concentrations of NEFA and BHB. Hepatic mRNA, protein expression, and activity of CYP2E1 were significantly higher in cows with clinical ketosis than in control cows. In vitro, both NEFA and BHB treatment markedly up-regulated the mRNA and protein expressions as well as activity of CYP2E1 in cow hepatocytes. Taken together, these results indicate that high levels of NEFA and BHB significantly up-regulate the expression and activity of hepatic CYP2E1, and may be influential in the induction of oxidative stress in cows with clinical ketosis.


Assuntos
Ácido 3-Hidroxibutírico/farmacologia , Doenças dos Bovinos/enzimologia , Citocromo P-450 CYP2E1/efeitos dos fármacos , Ácidos Graxos não Esterificados/farmacologia , Cetose/enzimologia , Fígado/enzimologia , Ácido 3-Hidroxibutírico/sangue , Animais , Bovinos , Doenças dos Bovinos/sangue , China , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP2E1/metabolismo , Indústria de Laticínios , Ácidos Graxos não Esterificados/sangue , Feminino , Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Cetose/sangue , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/análise , Regulação para Cima/efeitos dos fármacos
19.
Biochim Biophys Acta Gene Regul Mech ; 1862(5): 567-581, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30753902

RESUMO

MiR-15/16 play an important role in liver development and hepatocyte differentiation, but the mechanisms by which these miRNAs regulate their targets and downstream genes to influence cell fate are poorly understood. In this study, we showed up-regulation of miR-15/16 during HGF- and FGF4-induced hepatocyte differentiation from amniotic epithelial cells (AECs). To elucidate the role of miR-15/16 and their targets in hepatocyte differentiation, we investigated the roles of miR-15/16 in both the MAPK and Wnt/ß-catenin pathways, which were predicted to be involved in miR-15/16 signaling. Our results demonstrated that the transcription of miR-15/16 was enhanced by c-Fos, c-Jun, and CREB, important elements of the MAPK pathway, and miR-15/16 in turn directly targeted adenomatous polyposis coli (APC) protein, a major member of the ß-catenin degradation complex. MiR-15/16 destroyed these degradation complexes to activate ß-catenin, and the activated ß-catenin combined with LEF/TCF7L1 to form a transcriptional complex that enhanced transcription of hepatocyte nuclear factor 4 alpha (HNF4α). HNF4α also bound the promoter region of miR-15/16 and promoted its transcription, thereby forming a regulatory circuit to promote the differentiation of AECs into hepatocytes. Endogenous miRNAs are, therefore, involved in hepatocyte differentiation from AECs and should be considered during the development of an effective hepatocyte transplant therapy for liver damage.


Assuntos
Âmnio/citologia , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Sistema de Sinalização das MAP Quinases , MicroRNAs/fisiologia , Via de Sinalização Wnt , Diferenciação Celular , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células HEK293 , Fator 4 Nuclear de Hepatócito/biossíntese , Fator 4 Nuclear de Hepatócito/genética , Hepatócitos/citologia , Hepatócitos/enzimologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , MicroRNAs/biossíntese , MicroRNAs/genética , Transcrição Genética , Via de Sinalização Wnt/efeitos dos fármacos
20.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1864(4): 500-511, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30639734

RESUMO

Lysosomal acid lipase (LAL) hydrolyzes cholesteryl esters (CE) and triglycerides (TG) to generate fatty acids (FA) and cholesterol. LAL deficiency (LAL-D) in both humans and mice leads to hepatomegaly, hypercholesterolemia, and shortened life span. Despite its essential role in lysosomal neutral lipid catabolism, the cell type-specific contribution of LAL to disease progression is still elusive. To investigate the role of LAL in the liver in more detail and to exclude the contribution of LAL in macrophages, we generated hepatocyte-specific LAL-deficient mice (Liv-Lipa-/-) and fed them either chow or high fat/high cholesterol diets (HF/HCD). Comparable to systemic LAL-D, Liv-Lipa-/- mice were resistant to diet-induced obesity independent of food intake, movement, and energy expenditure. Reduced body weight gain was mainly due to reduced white adipose tissue depots. Furthermore, Liv-Lipa-/- mice exhibited improved glucose clearance during glucose and insulin tolerance tests compared to control mice. Analysis of hepatic lipid content revealed a massive reduction of TG, whereas CE concentrations were markedly increased, leading to CE crystal formation in the livers of Liv-Lipa-/- mice. Elevated plasma transaminase activities, increased pro-inflammatory cytokines and chemokines as well as hepatic macrophage infiltration indicated liver inflammation. Our data provide evidence that hepatocyte-specific LAL deficiency is sufficient to alter whole-body lipid and energy homeostasis in mice. We conclude that hepatic LAL plays a pivotal role by preventing liver damage and maintaining lipid and energy homeostasis, especially during high lipid availability.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Hepatite/genética , Hepatócitos/enzimologia , Obesidade/prevenção & controle , Esterol Esterase/deficiência , Animais , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Hepatócitos/imunologia , Homeostase , Metabolismo dos Lipídeos , Masculino , Camundongos , Obesidade/induzido quimicamente , Obesidade/genética , Esterol Esterase/genética , Esterol Esterase/metabolismo
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