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1.
Science ; 373(6555): 662-673, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34353949

RESUMO

The functional role of long noncoding RNAs (lncRNAs) in inherited metabolic disorders, including phenylketonuria (PKU), is unknown. Here, we demonstrate that the mouse lncRNA Pair and human HULC associate with phenylalanine hydroxylase (PAH). Pair-knockout mice exhibited excessive blood phenylalanine (Phe), musty odor, hypopigmentation, growth retardation, and progressive neurological symptoms including seizures, which faithfully models human PKU. HULC depletion led to reduced PAH enzymatic activities in human induced pluripotent stem cell-differentiated hepatocytes. Mechanistically, HULC modulated the enzymatic activities of PAH by facilitating PAH-substrate and PAH-cofactor interactions. To develop a therapeutic strategy for restoring liver lncRNAs, we designed GalNAc-tagged lncRNA mimics that exhibit liver enrichment. Treatment with GalNAc-HULC mimics reduced excessive Phe in Pair -/- and Pah R408W/R408W mice and improved the Phe tolerance of these mice.


Assuntos
Fenilalanina Hidroxilase/metabolismo , Fenilalanina/metabolismo , Fenilcetonúrias/genética , RNA Longo não Codificante/genética , Acetilgalactosamina , Animais , Biopterina/análogos & derivados , Biopterina/metabolismo , Biopterina/uso terapêutico , Dieta , Modelos Animais de Doenças , Feminino , Hepatócitos/metabolismo , Humanos , Fígado/embriologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Conformação de Ácido Nucleico , Fenilalanina/administração & dosagem , Fenilalanina Hidroxilase/deficiência , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/tratamento farmacológico , Fenilcetonúrias/metabolismo , Ligação Proteica , RNA Longo não Codificante/química , RNA Longo não Codificante/metabolismo , RNA Longo não Codificante/uso terapêutico
2.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360991

RESUMO

The possibility to reproduce key tissue functions in vitro from induced pluripotent stem cells (iPSCs) is offering an incredible opportunity to gain better insight into biological mechanisms underlying development and disease, and a tool for the rapid screening of drug candidates. This review attempts to summarize recent strategies for specification of iPSCs towards hepatobiliary lineages -hepatocytes and cholangiocytes-and their use as platforms for disease modeling and drug testing. The application of different tissue-engineering methods to promote accurate and reliable readouts is discussed. Space is given to open questions, including to what extent these novel systems can be informative. Potential pathways for improvement are finally suggested.


Assuntos
Técnicas de Reprogramação Celular/métodos , Doenças do Sistema Digestório/terapia , Descoberta de Drogas/métodos , Hepatócitos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Medicina de Precisão/métodos , Animais , Linhagem da Célula , Doenças do Sistema Digestório/metabolismo , Doenças do Sistema Digestório/patologia , Hepatócitos/metabolismo , Humanos , Engenharia Tecidual/métodos
3.
Nat Commun ; 12(1): 5068, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417460

RESUMO

p53 regulates several signaling pathways to maintain the metabolic homeostasis of cells and modulates the cellular response to stress. Deficiency or excess of nutrients causes cellular metabolic stress, and we hypothesized that p53 could be linked to glucose maintenance. We show here that upon starvation hepatic p53 is stabilized by O-GlcNAcylation and plays an essential role in the physiological regulation of glucose homeostasis. More specifically, p53 binds to PCK1 promoter and regulates its transcriptional activation, thereby controlling hepatic glucose production. Mice lacking p53 in the liver show a reduced gluconeogenic response during calorie restriction. Glucagon, adrenaline and glucocorticoids augment protein levels of p53, and administration of these hormones to p53 deficient human hepatocytes and to liver-specific p53 deficient mice fails to increase glucose levels. Moreover, insulin decreases p53 levels, and over-expression of p53 impairs insulin sensitivity. Finally, protein levels of p53, as well as genes responsible of O-GlcNAcylation are elevated in the liver of type 2 diabetic patients and positively correlate with glucose and HOMA-IR. Overall these results indicate that the O-GlcNAcylation of p53 plays an unsuspected key role regulating in vivo glucose homeostasis.


Assuntos
Acetilglucosamina/metabolismo , Glucose/metabolismo , Fígado/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Restrição Calórica , Linhagem Celular , Colforsina/farmacologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Epinefrina/metabolismo , Glucagon/metabolismo , Glucocorticoides/metabolismo , Gluconeogênese/efeitos dos fármacos , Glicosilação , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hidrocortisona/metabolismo , Hiperglicemia/complicações , Hiperglicemia/metabolismo , Resistência à Insulina , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/complicações , Obesidade/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Ácido Pirúvico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Genética/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética
4.
Ecotoxicol Environ Saf ; 223: 112570, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34352581

RESUMO

BaP and DBP are ubiquitously and contemporaneously present in the environment. However, Current studies largely concentrate on the effects of a single pollutant (BaP or DBP). The liver is vital for biogenic activities. The effects of BaP and DBP co-exposure on liver remain unclear. Thus, we treated human normal liver cell (L02 cell) with BaP or/and DBP. We found that compared to individual exposure, co-exposure to BaP and DBP induced further increased levels of AST and ALT. BaP and DBP co-exposure caused further increased levels of IL-2, IL-6, and TNF-α, decreased IL-10 level, and a higher percentage of apoptotic cells and S-phase arrest cells. BaP and DBP co-exposure worsen the decrease of miR-122-5p level and chaos of SOCS1/STAT3 signaling. Dual-luciferase reporter gene assays showed that SOCS1 was a validated target of miR-122-5p. miR-122-5p overexpression alleviated the increased SOCS1 expression, decreased phospho-STAT3 expression, decreased IL-10 level, increased TNF-α levels, increased percentage of apoptosis and S-phase arrest, and cytotoxicity induced by BaP and DBP co-exposure in hepatocytes. These results suggested that miR-122-5p negatively regulated the synergistic effects on apoptosis and disorder of inflammatory factor secretion involved in hepatocyte injury caused by BaP and DBP co-exposure through targeting SOCS1/STAT3 signaling.


Assuntos
MicroRNAs , Apoptose , Hepatócitos/metabolismo , Humanos , MicroRNAs/genética , Fator de Transcrição STAT3/genética , Transdução de Sinais , Proteína 1 Supressora da Sinalização de Citocina/genética , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Fator de Necrose Tumoral alfa/genética
5.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361019

RESUMO

Cholestatic liver diseases can progress to end-stage liver disease and reduce patients' quality of life. Although their underlying mechanisms are still incompletely elucidated, oxidative stress is considered to be a key contributor to these diseases. Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that displays antioxidant action. It has been found that this enzyme plays a protective role against various inflammatory diseases. However, the role of HO-1 in cholestatic liver diseases has not yet been investigated. Here, we examined whether pharmacological induction of HO-1 by cobalt protoporphyrin (CoPP) ameliorates cholestatic liver injury. To this end, a murine model of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet feeding was used. Administration of CoPP ameliorated liver damage and cholestasis with HO-1 upregulation in DDC diet-fed mice. Induction of HO-1 by CoPP suppressed the DDC diet-induced oxidative stress and hepatocyte apoptosis. In addition, CoPP attenuated cytokine production and inflammatory cell infiltration. Furthermore, deposition of the extracellular matrix and expression of fibrosis-related genes after DDC feeding were also decreased by CoPP. HO-1 induction decreased the number of myofibroblasts and inhibited the transforming growth factor-ß pathway. Altogether, these data suggest that the pharmacological induction of HO-1 ameliorates cholestatic liver disease by suppressing oxidative stress, hepatocyte apoptosis, and inflammation.


Assuntos
Anti-Inflamatórios/uso terapêutico , Antioxidantes/uso terapêutico , Colestase Intra-Hepática/tratamento farmacológico , Heme Oxigenase-1/metabolismo , Protoporfirinas/uso terapêutico , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Apoptose , Colestase Intra-Hepática/etiologia , Colestase Intra-Hepática/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Protoporfirinas/farmacologia , Piridinas/toxicidade , Xenobióticos/toxicidade
6.
Nutrients ; 13(8)2021 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-34444698

RESUMO

Maintaining lipid homeostasis is crucial to liver function, the key organ that governs the whole-body energy metabolism. In contrast, lipid dysregulation has been implicated in mycotoxin-induced liver injury, by which the pathophysiological regulation and the molecular components involved remain elusive. Here we focused on the potential roles of orphan nuclear receptor (NR) RORγ in lipid programming, and aimed to explore its action on cholesterol regulation in the liver of mycotoxin-exposed piglets. We found that liver tissues were damaged in the mycotoxin-exposed piglets compared to the healthy controls, revealed by histological analysis, elevated seral ALT, AST and ALP levels, and increased caspase 3/7 activities. Consistent with the transcriptomic finding of down-regulated cholesterol metabolism, we demonstrated that both cholesterol contents and cholesterol biosynthesis/transformation gene expressions in the mycotoxin-exposed livers were reduced, including HMGCS1, FDPS, SQLE, EBP, FDFT1 and VLDLR. Furthermore, we reported that RORγ binds to the cholesterol metabolic genes in porcine hepatocytes using a genome-wide ChIP-seq analysis, whereas mycotoxin decreased the RORγ binding occupancies genome-wide, especially at the cholesterol metabolic pathway. In addition, we revealed the enrichment of co-factors p300 and SRC, the histone marks H3K27ac and H3K4me2, together with RNA Polymerase II (Pol-II) at the locus of HMGCS1 in hepatocytes, which were reduced by mycotoxin-exposure. Our results provide a deep insight into the cholesterol metabolism regulation during mycotoxin-induced liver injury, and propose NRs as therapeutic targets for anti-mycotoxin treatments.


Assuntos
Doença Hepática Crônica Induzida por Substâncias e Drogas/genética , Colesterol/genética , Regulação da Expressão Gênica/genética , Metabolismo dos Lipídeos/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/fisiologia , Animais , Modelos Animais de Doenças , Hepatócitos/metabolismo , Homeostase/genética , Fígado/metabolismo , Micotoxinas/toxicidade , Suínos
7.
Int J Mol Sci ; 22(16)2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34445270

RESUMO

The opening of the permeability transition pore (mPTP) in mitochondria initiates cell death in numerous diseases. The regulation of mPTP by NAD(H) in the mitochondrial matrix is well established; however, the role of extramitochondrial (cytosolic) NAD(H) is still unclear. We studied the effect of added NADH and NAD+ on: (1) the Ca2+-retention capacity (CRC) of isolated rat liver, heart, and brain mitochondria; (2) the Ca2+-dependent mitochondrial swelling in media whose particles can (KCl) or cannot (sucrose) be extruded from the matrix by mitochondrial carriers; (3) the Ca2+-dependent mitochondrial depolarization and the release of entrapped calcein from mitochondria of permeabilized hepatocytes; and (4) the Ca2+-dependent mitochondrial depolarization and subsequent repolarization. NADH and NAD+ increased the CRC of liver, heart, and brain mitochondria 1.5-2.5 times, insignificantly affecting the rate of Ca2+-uptake and the free Ca2+ concentration in the medium. NAD(H) suppressed the Ca2+-dependent mitochondrial swelling both in KCl- and sucrose-based media but did not induce the contraction and repolarization of swollen mitochondria. By contrast, EGTA caused mitochondrial repolarization in both media and the contraction in KCl-based medium only. NAD(H) delayed the Ca2+-dependent depolarization and the release of calcein from individual mitochondria in hepatocytes. These data unambiguously demonstrate the existence of an external NAD(H)-dependent site of mPTP regulation.


Assuntos
Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Hepáticas/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , NAD/metabolismo , Animais , Cálcio/metabolismo , Fluoresceínas/metabolismo , Hepatócitos/metabolismo , Masculino , Ratos , Ratos Wistar
8.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360670

RESUMO

BACKGROUND AND AIMS: Fibroblast growth factor (FGF) 21 has recently been shown to play a potential role in bile acid metabolism. We aimed to investigate the FGF21 response in an ethanol-induced acute-on-chronic liver injury (ACLI) model in Abcb4-/- mice with deficiency of the hepatobiliary phospholipid transporter. METHODS: Total RNA was extracted from wild-type (WT, C57BL/6J) and Abcb4-/- (KO) mice, which were either fed a control diet (WT-Cont and KO-Cont groups; n = 28/group) or ethanol diet, followed by an acute ethanol binge (WT-EtOH and KO-EtOH groups; n = 28/group). A total of 58 human subjects were recruited into the study, including patients with alcohol-associated liver disease (AALD; n = 31) and healthy controls (n = 27). The hepatic and ileal expressions of genes involved in bile acid metabolism, plasma FGF levels, and bile acid and its precursors 7α- and 27-hydroxycholesterol (7α- and 27-OHC) concentrations were determined. Primary mouse hepatocytes were isolated for cell culture experiments. RESULTS: Alcohol feeding significantly induced plasma FGF21 and decreased hepatic Cyp7a1 levels. Hepatic expression levels of Fibroblast growth factor receptor 1 (Fgfr1), Fgfr4, Farnesoid X-activated receptor (Fxr), and Small heterodimer partner (Shp) and plasma FGF15/FGF19 levels did not differ with alcohol challenge. Exogenous FGF21 treatment suppressed Cyp7a1 in a dose-dependent manner in vitro. AALD patients showed markedly higher FGF21 and lower 7α-OHC plasma levels while FGF19 did not differ. CONCLUSIONS: The simultaneous upregulation of FGF21 and downregulation of Cyp7a1 expressions upon chronic plus binge alcohol feeding together with the invariant plasma FGF15 and hepatic Shp and Fxr levels suggest the presence of a direct regulatory mechanism of FGF21 on bile acid homeostasis through inhibition of CYP7A1 by an FGF15-independent pathway in this ACLI model. Lay Summary: Alcohol challenge results in the upregulation of FGF21 and repression of Cyp7a1 expressions while circulating FGF15 and hepatic Shp and Fxr levels remain constant both in healthy and pre-injured livers, suggesting the presence of an alternative FGF15-independent regulatory mechanism of FGF21 on bile acid homeostasis through the inhibition of Cyp7a1.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/fisiologia , Insuficiência Hepática Crônica Agudizada/patologia , Ácidos e Sais Biliares/metabolismo , Colesterol 7-alfa-Hidroxilase/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Hepatócitos/patologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Insuficiência Hepática Crônica Agudizada/metabolismo , Animais , Estudos de Casos e Controles , Colesterol 7-alfa-Hidroxilase/genética , Feminino , Fatores de Crescimento de Fibroblastos/genética , Hepatócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Citoplasmáticos e Nucleares/genética
9.
Int J Mol Sci ; 22(13)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209301

RESUMO

ABCB4 (ATP-binding cassette subfamily B member 4) is an ABC transporter expressed at the canalicular membrane of hepatocytes where it ensures phosphatidylcholine secretion into bile. Genetic variations of ABCB4 are associated with several rare cholestatic diseases. The available treatments are not efficient for a significant proportion of patients with ABCB4-related diseases and liver transplantation is often required. The development of novel therapies requires a deep understanding of the molecular mechanisms regulating ABCB4 expression, intracellular traffic, and function. Using an immunoprecipitation approach combined with mass spectrometry analyses, we have identified the small GTPase RAB10 as a novel molecular partner of ABCB4. Our results indicate that the overexpression of wild type RAB10 or its dominant-active mutant significantly increases the amount of ABCB4 at the plasma membrane expression and its phosphatidylcholine floppase function. Contrariwise, RAB10 silencing induces the intracellular retention of ABCB4 and then indirectly diminishes its secretory function. Taken together, our findings suggest that RAB10 regulates the plasma membrane targeting of ABCB4 and consequently its capacity to mediate phosphatidylcholine secretion.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membrana Celular/metabolismo , Hepatócitos/metabolismo , Fosfatidilcolinas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Transporte Biológico Ativo , Membrana Celular/genética , Células HEK293 , Células HeLa , Humanos , Fosfatidilcolinas/genética , Proteínas rab de Ligação ao GTP/genética
10.
Nat Commun ; 12(1): 4264, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253736

RESUMO

Single-cell RNA-seq reveals the role of pathogenic cell populations in development and progression of chronic diseases. In order to expand our knowledge on cellular heterogeneity, we have developed a single-nucleus RNA-seq2 method tailored for the comprehensive analysis of the nuclear transcriptome from frozen tissues, allowing the dissection of all cell types present in the liver, regardless of cell size or cellular fragility. We use this approach to characterize the transcriptional profile of individual hepatocytes with different levels of ploidy, and have discovered that ploidy states are associated with different metabolic potential, and gene expression in tetraploid mononucleated hepatocytes is conditioned by their position within the hepatic lobule. Our work reveals a remarkable crosstalk between gene dosage and spatial distribution of hepatocytes.


Assuntos
Fígado/metabolismo , Ploidias , Análise de Sequência de RNA , Análise de Célula Única , Animais , Biomarcadores/metabolismo , Núcleo Celular/metabolismo , Doença Crônica , Secções Congeladas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Hepatócitos/metabolismo , Fígado/patologia , Hepatopatias/patologia , Camundongos Endogâmicos C57BL , Regeneração , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética
11.
Int J Mol Sci ; 22(14)2021 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-34298998

RESUMO

The purpose of the present study was to investigate molecular compositions of lipid droplets changing in live hepatic cells stimulated with major fatty acids in the human body, i.e., palmitic, stearic, oleic, and linoleic acids. HepG2 cells were used as the model hepatic cells. Morphological changes of lipid droplets were observed by optical microscopy and transmission electron microscopy (TEM) during co-cultivation with fatty acids up to 5 days. The compositional changes in the fatty chains included in the lipid droplets were analyzed via Raman spectroscopy and chemometrics. The growth curves of the cells indicated that palmitic, stearic, and linoleic acids induced cell death in HepG2 cells, but oleic acid did not. Microscopic observations suggested that the rates of fat accumulation were high for oleic and linoleic acids, but low for palmitic and stearic acids. Raman analysis indicated that linoleic fatty chains taken into the cells are modified into oleic fatty chains. These results suggest that the signaling pathway of cell death is independent of fat stimulations. Moreover, these results suggest that hepatic cells have a high affinity for linoleic acid, but linoleic acid induces cell death in these cells. This may be one of the causes of inflammation in nonalcoholic fatty liver disease (NAFLD).


Assuntos
Morte Celular/efeitos dos fármacos , Meios de Cultura/química , Ácidos Graxos/efeitos adversos , Hepatócitos/metabolismo , Gotículas Lipídicas/química , Análise Espectral Raman , Ácidos Graxos/metabolismo , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Ácido Linoleico/farmacologia , Ácido Linoleico/toxicidade , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Microscopia Eletrônica de Transmissão , Ácido Oleico/farmacologia , Ácido Palmítico/farmacologia , Ácido Palmítico/toxicidade , Transdução de Sinais/efeitos dos fármacos , Ácidos Esteáricos/farmacologia , Ácidos Esteáricos/toxicidade
12.
Int J Mol Sci ; 22(14)2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34299321

RESUMO

The liver plays a key role in systemic metabolic processes, which include detoxification, synthesis, storage, and export of carbohydrates, lipids, and proteins. The raising trends of obesity and metabolic disorders worldwide is often associated with the nonalcoholic fatty liver disease (NAFLD), which has become the most frequent type of chronic liver disorder with risk of progression to cirrhosis and hepatocellular carcinoma. Liver mitochondria play a key role in degrading the pathways of carbohydrates, proteins, lipids, and xenobiotics, and to provide energy for the body cells. The morphological and functional integrity of mitochondria guarantee the proper functioning of ß-oxidation of free fatty acids and of the tricarboxylic acid cycle. Evaluation of the liver in clinical medicine needs to be accurate in NAFLD patients and includes history, physical exam, imaging, and laboratory assays. Evaluation of mitochondrial function in chronic liver disease and NAFLD is now possible by novel diagnostic tools. "Dynamic" liver function tests include the breath test (BT) based on the use of substrates marked with the non-radioactive, naturally occurring stable isotope 13C. Hepatocellular metabolization of the substrate will generate 13CO2, which is excreted in breath and measured by mass spectrometry or infrared spectroscopy. Breath levels of 13CO2 are biomarkers of specific metabolic processes occurring in the hepatocyte cytosol, microsomes, and mitochondria. 13C-BTs explore distinct chronic liver diseases including simple liver steatosis, non-alcoholic steatohepatitis, liver fibrosis, cirrhosis, hepatocellular carcinoma, drug, and alcohol effects. In NAFLD, 13C-BT use substrates such as α-ketoisocaproic acid, methionine, and octanoic acid to assess mitochondrial oxidation capacity which can be impaired at an early stage of disease. 13C-BTs represent an indirect, cost-effective, and easy method to evaluate dynamic liver function. Further applications are expected in clinical medicine. In this review, we discuss the involvement of liver mitochondria in the progression of NAFLD, together with the role of 13C-BT in assessing mitochondrial function and its potential use in the prevention and management of NAFLD.


Assuntos
Testes Respiratórios/métodos , Mitocôndrias/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Biomarcadores/metabolismo , Carcinoma Hepatocelular/metabolismo , Hepatócitos/metabolismo , Humanos , Fígado/patologia , Fígado/fisiopatologia , Cirrose Hepática/metabolismo , Testes de Função Hepática , Neoplasias Hepáticas/metabolismo , Mitocôndrias/patologia , Mitocôndrias Hepáticas/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Obesidade/metabolismo
13.
Methods Mol Biol ; 2350: 31-41, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331277

RESUMO

Posttranslational histone modifications are associated with the regulation of genome function. Some modifications are quite stable to maintain epigenome states of chromatin, and others can exhibit dynamic changes in response to internal and external stimuli. To track the local and global changes in histone modifications, multiplexed imaging in living cells is beneficial. Among live cell probes for detecting histone modifications, genetically encoded modification-specific intracellular antibodies, or mintbodies, are convenient and suitable tools for this purpose. We here describe the mintbody-based methods to monitor the changes in histone modification levels induced by histone methyltransferase and deacetylase inhibitors. By measuring the nuclear to cytoplasmic intensity ratios of mintbodies in living cells, changes in histone H4 lysine 20 methylation states and the increase in histone H3 acetylation were detected.


Assuntos
Hepatócitos/metabolismo , Imagem Molecular/métodos , Processamento de Proteína Pós-Traducional , Animais , Linhagem Celular , Epigenômica/métodos , Expressão Gênica , Regulação da Expressão Gênica , Genes Reporter , Código das Histonas , Histonas/metabolismo , Humanos , Metilação , Camundongos
14.
Biomolecules ; 11(6)2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207346

RESUMO

BACKGROUND: Underage drinking is associated with health risk behaviors. Serum keratin-18 (CK18) levels are increased in liver diseases and may be biomarkers of outcome. The purpose of this study was to determine if the total CK18 (M65) or caspase-cleaved CK18 (M30) levels were different in adolescents admitted to hospital because of alcohol intoxication and controls with excluded liver diseases. METHODS: A prospective study included 57 adolescents after alcohol use and 23 control subjects. The concentrations of M30 and M65 in the serum samples were evaluated using an enzyme-linked immunosorbent assay. RESULTS: The median age was 15 (14-17) years and 49% were male. There were significant differences in M65 levels between the study and control groups (p = 0.03). The concentrations of M30 and M65 were insignificant in adolescents divided into subgroups according to blood alcohol concentrations (BAC). Significant positive correlations were found between BAC and M65 levels (p = 0.038; r = 0.3). In receiver operating characteristic (ROC) analysis M65 (cut-off = 125.966 IU/l, Se = 70.2%, Sp = 43.5%) allowed to differentiate between patients with and without alcohol intoxication (AUC = 0.66, p = 0.03). CONCLUSION: M65 appears to be a promising non-invasive biomarker of hepatocyte injury during alcohol intoxication in adolescents. Moreover, a higher concentration of M65 may indicate early organ injury before the increase in the activity of liver enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST).


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Hepatócitos/patologia , Queratina-18/análise , Adolescente , Alanina Transaminase/sangue , Intoxicação Alcoólica/sangue , Intoxicação Alcoólica/fisiopatologia , Apoptose , Aspartato Aminotransferases/sangue , Biomarcadores/sangue , Doença Hepática Induzida por Substâncias e Drogas/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Hepatócitos/metabolismo , Humanos , Queratina-18/sangue , Masculino , Fragmentos de Peptídeos/sangue , Polônia , Dados Preliminares , Estudos Prospectivos , Curva ROC , Consumo de Álcool por Menores
15.
Nat Methods ; 18(7): 799-805, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34226721

RESUMO

A growing appreciation of the importance of cellular metabolism and revelations concerning the extent of cell-cell heterogeneity demand metabolic characterization of individual cells. We present SpaceM, an open-source method for in situ single-cell metabolomics that detects >100 metabolites from >1,000 individual cells per hour, together with a fluorescence-based readout and retention of morpho-spatial features. We validated SpaceM by predicting the cell types of cocultured human epithelial cells and mouse fibroblasts. We used SpaceM to show that stimulating human hepatocytes with fatty acids leads to the emergence of two coexisting subpopulations outlined by distinct cellular metabolic states. Inducing inflammation with the cytokine interleukin-17A perturbs the balance of these states in a process dependent on NF-κB signaling. The metabolic state markers were reproduced in a murine model of nonalcoholic steatohepatitis. We anticipate SpaceM to be broadly applicable for investigations of diverse cellular models and to democratize single-cell metabolomics.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Metabolômica/métodos , Análise de Célula Única/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Técnicas de Cocultura , Células Epiteliais , Ácidos Graxos/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Inflamação/metabolismo , Interleucina-17/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Células NIH 3T3 , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Transdução de Sinais , Estresse Fisiológico
16.
Am J Physiol Endocrinol Metab ; 321(2): E292-E304, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34229476

RESUMO

We have generated the transgenic mouse line LTCFDN in which dominant negative TCF7L2 (TCF7L2DN) is specifically expressed in the liver during adulthood. Male but not female LTCFDN mice showed elevated hepatic and plasma triglyceride (TG) levels, indicating the existence of estrogen-ß-cat/TCF signaling cascade that regulates hepatic lipid homeostasis. We show here that hepatic fibroblast growth factor 21 (FGF21) expression was reduced in male but not in female LTCFDN mice. The reduction was not associated with altered hepatic expression of peroxisome proliferator-activated receptor α (PPARα). In mouse primary hepatocytes (MPH), Wnt-3a treatment increased FGF21 expression in the presence of PPARα inhibitor. Results from our luciferase-reporter assay and chromatin immunoprecipitation suggest that evolutionarily conserved TCF binding motifs (TCFBs) on Fgf21 promoter mediate Wnt-3a-induced Fgf21 transactivation. Female mice showed reduced hepatic FGF21 production and circulating FGF21 level following ovariectomy (OVX), associated with reduced hepatic TCF expression and ß-catenin S675 phosphorylation. Finally, in MPH, estradiol (E2) treatment enhanced FGF21 expression, as well as binding of TCF7L2 and ribonucleic acid (RNA) polymerase II to the Fgf21 promoter; and the enhancement can be attenuated by the G-protein-coupled estrogen receptor 1 (GPER) antagonist G15. Our observations hence indicate that hepatic FGF21 is among the effectors of the newly recognized E2-ß-cat/TCF signaling cascade.NEW & NOTEWORTHY FGF21 is mainly produced in the liver. Therapeutic effect of FGF21 analogues has been demonstrated in clinical trials on reducing hyperlipidemia. We show here that Fgf21 transcription is positively regulated by Wnt pathway effector ß-cat/TCF. Importantly, hepatic ß-cat/TCF activity can be regulated by the female hormone estradiol, involving GPER. The investigation enriched our understanding on hepatic FGF21 hormone production, and expanded our view on metabolic functions of the Wnt pathway in the liver.


Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Fígado/metabolismo , Via de Sinalização Wnt , Animais , Células Cultivadas , Estrogênios/metabolismo , Feminino , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , PPAR alfa/metabolismo
17.
Toxicology ; 459: 152854, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34271081

RESUMO

A methylimidizolium ionic liquid (M8OI) was recently found to be contaminating the environment and to be related to and/or potentially a component of an environmental trigger for the autoimmune liver disease primary biliary cholangitis (PBC). The aims of this study were to investigate human exposure to M8OI, hepatic metabolism and excretion. PBC patient and control sera were screened for the presence of M8OI. Human livers were perfused with 50µM M8OI in a closed circuit and its hepatic disposition examined. Metabolism was examined in cultured human hepatocytes and differentiated HepaRG cells by the addition of M8OI and metabolites in the range 10-100 µM. M8OI was detected in the sera from 5/20 PBC patients and 1/10 controls. In perfused livers, M8OI was cleared from the plasma with its appearance - primarily in the form of its hydroxylated (HO8IM) and carboxylated (COOH7IM) products - in the bile. Metabolism was reflected in cultured hepatocytes with HO8IM production inhibited by the cytochrome P450 inhibitor ketoconazole. Further oxidation of HO8IM to COOH7IM was sequentially inhibited by the alcohol and acetaldehyde dehydrogenase inhibitors 4-methyl pyrazole and disulfiram respectively. Hepatocytes from 1 donor failed to metabolise M8OI to COOH7IM over a 24 h period. These results demonstrate exposure to M8OI in the human population, monooxygenation by cytochromes P450 followed by alcohol and acetaldehyde dehydrogenase oxidation to a carboxylic acid that are excreted, in part, via the bile in human liver.


Assuntos
Eliminação Hepatobiliar , Imidazóis/sangue , Imidazóis/farmacocinética , Adulto , Idoso , Álcool Desidrogenase/antagonistas & inibidores , Aldeído Oxirredutases/antagonistas & inibidores , Células Cultivadas , Inibidores das Enzimas do Citocromo P-450/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hidroxilação , Técnicas In Vitro , Cetoconazol/farmacologia , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células , Adulto Jovem
18.
Methods Mol Biol ; 2342: 369-417, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272702

RESUMO

Accurate estimation of in vivo clearance in human is pivotal to determine the dose and dosing regimen for drug development. In vitro-in vivo extrapolation (IVIVE) has been performed to predict drug clearance using empirical and physiological scalars. Multiple in vitro systems and mathematical modeling techniques have been employed to estimate in vivo clearance. The models for predicting clearance have significantly improved and have evolved to become more complex by integrating multiple processes such as drug metabolism and transport as well as passive diffusion. This chapter covers the use of conventional as well as recently developed methods to predict metabolic and transporter-mediated clearance along with the advantages and disadvantages of using these methods and the associated experimental considerations. The general approaches to improve IVIVE by use of appropriate scalars, incorporation of extrahepatic metabolism and transport and application of physiologically based pharmacokinetic (PBPK) models with proteomics data are also discussed. The chapter also provides an overview of the advantages of using such dynamic mechanistic models over static models for clearance predictions to improve IVIVE.


Assuntos
Hepatócitos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Cálculos da Dosagem de Medicamento , Vias de Eliminação de Fármacos , Hepatócitos/química , Humanos , Técnicas In Vitro , Cinética , Taxa de Depuração Metabólica , Modelos Teóricos , Proteômica
19.
Methods Mol Biol ; 2342: 443-479, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272704

RESUMO

There are many factors which are known to cause variability in human in vitro enzyme kinetic data. Factors such as the source of enzyme and how it was prepared, the genetics and background of the donor, how the in vitro studies are designed, and how the data are analyzed contribute to variability in the resulting kinetic parameters. It is important to consider not only the factors which cause variability within an experiment, such as selection of a probe substrate, but also those that cause variability when comparing kinetic data across studies and laboratories. For example, the artificial nature of the microsomal lipid membrane and microenvironment in some recombinantly expressed enzymes, relative to those found in native tissue microsomes, has been shown to influence enzyme activity and thus can be a source of variability when comparing across the two different systems. All of these factors, and several others, are discussed in detail in the chapter below. In addition, approaches which can be used to visualize the uncertainty arising from the use of enzyme kinetic data within the context of predicting human pharmacokinetics are discussed.


Assuntos
Enzimas/metabolismo , Glucuronosiltransferase/metabolismo , Hepatócitos/citologia , Microssomos Hepáticos/enzimologia , Técnicas de Cultura de Células , Células Cultivadas , Enzimas/genética , Glucuronosiltransferase/genética , Hepatócitos/metabolismo , Humanos , Cinética , Variantes Farmacogenômicos , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
20.
Methods Mol Biol ; 2342: 633-642, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272708

RESUMO

This chapter deals with practical considerations on key issues such as choosing an enzyme source, determining linear conditions, and choosing appropriate substrate and organic solvent concentrations. Practical solutions for working with limited resources and carrying out inhibition experiments are also addressed. Thus, after reading this chapter, the novice reader should have a better idea of how to design, develop, and interpret basic experiments using drug metabolism enzymes.


Assuntos
Enzimas/metabolismo , Hepatócitos/metabolismo , Preparações Farmacêuticas/metabolismo , Animais , Hepatócitos/enzimologia , Humanos , Cinética , Lisossomos/enzimologia , Projetos de Pesquisa
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