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1.
Methods Mol Biol ; 2675: 1-13, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37258751

RESUMO

The prevalence of obesity has increased to pandemic levels over the past years. Associated comorbidities linked with the accumulation of lipids in different tissues and blood are responsible for the high mortality in these patients. The increased dietary lipid uptake contributes to these metabolic diseases. Identifying which pathways might be dysregulated in these patients will contribute to find new therapeutic targets. Thus, here, a protocol to follow up the distribution of dietary lipids in blood and tissues is provided. For this, radiolabeled triglyceride in olive oil is administered by oral gavage. To ascertain more precisely the capacity of each tissue for fatty acid uptake, not considering the intestinal barrier, the intravenous (IV) administration of radiolabeled lipids is also described.


Assuntos
Gorduras na Dieta , Oligonucleotídeos Antissenso , Camundongos , Animais , Oligonucleotídeos Antissenso/genética , Triglicerídeos/metabolismo , Ácidos Graxos/metabolismo , Obesidade/genética , Camundongos Endogâmicos C57BL
2.
Methods Mol Biol ; 2675: 15-26, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37258752

RESUMO

The liver is a central organ in regulating the whole body metabolic homeostasis, and, among many other processes, it plays a crucial role in lipoprotein metabolism. The liver controls the secretion of very-low-density lipoproteins (VLDLs), particles specialized in the transport of liver lipids, mainly triglycerides (TGs), to the adipose tissue, heart, and muscle, among other tissues, providing fatty acids to be stored or to be used as an energy source. The analysis of this metabolic process provides relevant information about the crosstalk between the liver and other organs. It also helps to identify how the liver is able to secrete lipids to reduce its accumulation. This protocol shows how to analyze the liver TG secretion rate blocking the VLDL clearance from the blood by the administration of poloxamer 407. In addition, it shows how to isolate the VLDL produced by the liver at the end of the experiment, so that the apolipoprotein and lipid content and size can be measured. Using antisense oligonucleotides (ASOs) for silencing target proteins involved in metabolic diseases has emerged as a new promising therapeutic approach. Thus, the usage of ASOs has also been included in this protocol. As a conclusion, evaluation of TG secretion rate in mice provides key information to understand the organ crosstalk in metabolic diseases and the capacity of the liver to secrete lipids to blood.


Assuntos
Fígado Gorduroso , Camundongos , Animais , Triglicerídeos/metabolismo , Fígado Gorduroso/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Fígado/metabolismo , Lipoproteínas VLDL/metabolismo
3.
Cancers (Basel) ; 14(19)2022 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-36230876

RESUMO

E2F1/E2F2 expression correlates with malignancy in prostate cancer (PCa), but its functional significance remains unresolved. To define the mechanisms governed by E2F in PCa, we analyzed the contribution of E2F target genes to the control of genome integrity, and the impact of modulating E2F activity on PCa progression. We show that silencing or inhibiting E2F1/E2F2 induces DNA damage during S phase and potentiates 5-FU-induced replication stress and cellular toxicity. Inhibition of E2F downregulates the expression of E2F targets involved in nucleotide biosynthesis (TK1, DCK, TYMS), whose expression is upregulated by 5-FU. However, their enzymatic products failed to rescue DNA damage of E2F1/E2F2 knockdown cells, suggesting additional mechanisms for E2F function. Interestingly, targeting E2F1/E2F2 in PCa cells reduced WEE1 expression and resulted in premature CDK1 activation during S phase. Inhibition of CDK1/CDK2 prevented DNA damage induced by E2F loss, suggesting that E2F1/E2F2 safeguard genome integrity by restraining CDK1/CDK2 activity. Importantly, combined inhibition of E2F and ATR boosted replication stress and dramatically reduced tumorigenic capacity of PCa cells in xenografts. Collectively, inhibition of E2F in combination with drugs targeting nucleotide biosynthesis or DNA repair is a promising strategy to provoke catastrophic levels of replication stress that could be applied to PCa treatment.

4.
Nat Commun ; 13(1): 1096, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35232994

RESUMO

Altered methionine metabolism is associated with weight gain in obesity. The methionine adenosyltransferase (MAT), catalyzing the first reaction of the methionine cycle, plays an important role regulating lipid metabolism. However, its role in obesity, when a plethora of metabolic diseases occurs, is still unknown. By using antisense oligonucleotides (ASO) and genetic depletion of Mat1a, here, we demonstrate that Mat1a deficiency in diet-induce obese or genetically obese mice prevented and reversed obesity and obesity-associated insulin resistance and hepatosteatosis by increasing energy expenditure in a hepatocyte FGF21 dependent fashion. The increased NRF2-mediated FGF21 secretion induced by targeting Mat1a, mobilized plasma lipids towards the BAT to be catabolized, induced thermogenesis and reduced body weight, inhibiting hepatic de novo lipogenesis. The beneficial effects of Mat1a ASO were abolished following FGF21 depletion in hepatocytes. Thus, targeting Mat1a activates the liver-BAT axis by increasing NRF2-mediated FGF21 secretion, which prevents obesity, insulin resistance and hepatosteatosis.


Assuntos
Tecido Adiposo Marrom , Resistência à Insulina , Metionina Adenosiltransferase , Obesidade , Oligonucleotídeos Antissenso , Tecido Adiposo Marrom/metabolismo , Animais , Metabolismo Energético , Fígado/metabolismo , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Obesidade/genética , Obesidade/metabolismo , Obesidade/prevenção & controle , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Antissenso/farmacologia
5.
Hepatology ; 76(6): 1617-1633, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35030285

RESUMO

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) includes a heterogeneous group of biliary cancers with a dismal prognosis. We investigated if lipid metabolism is disrupted in CCA and its role in tumor proliferation. APPROACH AND RESULTS: The in vitro and in vivo tumorigenic capacity of five human CCA cell lines was analyzed. Proteome, lipid content, and metabolic fluxes were evaluated in CCA cells and compared with normal human cholangiocytes (NHC). The Akt1/NOTCH1 intracellular cytoplasmic domain (Nicd1)-driven CCA mouse model was also evaluated. The proteome of CCA cells was enriched in pathways involved in lipid and lipoprotein metabolism. The EGI1 CCA cell line presented the highest tumorigenic capacity. Metabolic studies in high (EGI1) versus low (HUCCT1) proliferative CCA cells in vitro showed that both EGI1 and HUCCT1 incorporated more fatty acids (FA) than NHC, leading to increased triglyceride storage, also observed in Akt1/Nicd1-driven CCA mouse model. The highly proliferative EGI1 CCA cells showed greater uptake of very-low-density and HDLs than NHC and HUCCT1 CCA cells and increased cholesteryl ester content. The FA oxidation (FAO) and related proteome enrichment were specifically up-regulated in EGI1, and consequently, pharmacological blockade of FAO induced more pronounced inhibition of their tumorigenic capacity compared with HUCCT1. The expression of acyl-CoA dehydrogenase ACADM, the first enzyme involved in FAO, was increased in human CCA tissues and correlated with the proliferation marker PCNA. CONCLUSIONS: Highly proliferative human CCA cells rely on lipid and lipoprotein uptake to fuel FA catabolism, suggesting that inhibition of FAO and/or lipid uptake could represent a therapeutic strategy for this CCA subclass.


Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Camundongos , Animais , Humanos , Proteoma , Linhagem Celular Tumoral , Colangiocarcinoma/patologia , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/patologia , Lipídeos/uso terapêutico , Proliferação de Células
6.
Cancer Res ; 81(11): 2874-2887, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33771899

RESUMO

Lipid metabolism rearrangements in nonalcoholic fatty liver disease (NAFLD) contribute to disease progression. NAFLD has emerged as a major risk for hepatocellular carcinoma (HCC), where metabolic reprogramming is a hallmark. Identification of metabolic drivers might reveal therapeutic targets to improve HCC treatment. Here, we investigated the contribution of transcription factors E2F1 and E2F2 to NAFLD-related HCC and their involvement in metabolic rewiring during disease progression. In mice receiving a high-fat diet (HFD) and diethylnitrosamine (DEN) administration, E2f1 and E2f2 expressions were increased in NAFLD-related HCC. In human NAFLD, E2F1 and E2F2 levels were also increased and positively correlated. E2f1 -/- and E2f2 -/- mice were resistant to DEN-HFD-induced hepatocarcinogenesis and associated lipid accumulation. Administration of DEN-HFD in E2f1 -/- and E2f2 -/- mice enhanced fatty acid oxidation (FAO) and increased expression of Cpt2, an enzyme essential for FAO, whose downregulation is linked to NAFLD-related hepatocarcinogenesis. These results were recapitulated following E2f2 knockdown in liver, and overexpression of E2f2 elicited opposing effects. E2F2 binding to the Cpt2 promoter was enhanced in DEN-HFD-administered mouse livers compared with controls, implying a direct role for E2F2 in transcriptional repression. In human HCC, E2F1 and E2F2 expressions inversely correlated with CPT2 expression. Collectively, these results indicate that activation of the E2F1-E2F2-CPT2 axis provides a lipid-rich environment required for hepatocarcinogenesis. SIGNIFICANCE: These findings identify E2F1 and E2F2 transcription factors as metabolic drivers of hepatocellular carcinoma, where deletion of just one is sufficient to prevent disease. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/11/2874/F1.large.jpg.


Assuntos
Carcinoma Hepatocelular/patologia , Carnitina O-Palmitoiltransferase/antagonistas & inibidores , Fator de Transcrição E2F1/metabolismo , Fator de Transcrição E2F2/metabolismo , Lipídeos/análise , Neoplasias Hepáticas/patologia , Hepatopatia Gordurosa não Alcoólica/complicações , Animais , Carcinógenos , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/metabolismo , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Dieta Hiperlipídica/efeitos adversos , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F2/genética , Regulação da Expressão Gênica , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Prognóstico , Regiões Promotoras Genéticas
7.
Aging Cell ; 19(8): e13183, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32638492

RESUMO

Osteopontin (OPN), a senescence-associated secretory phenotype factor, is increased in patients with nonalcoholic fatty liver disease (NAFLD). Cellular senescence has been associated with age-dependent hepatosteatosis. Thus, we investigated the role of OPN in the age-related hepatosteatosis. For this, human serum samples, animal models of aging, and cell lines in which senescence was induced were used. Metabolic fluxes, lipid, and protein concentration were determined. Among individuals with a normal liver, we observed a positive correlation between serum OPN levels and increasing age. This correlation with age, however, was absent in patients with NAFLD. In wild-type (WT) mice, serum and liver OPN were increased at 10 months old (m) along with liver p53 levels and remained elevated at 20m. Markers of liver senescence increased in association with synthesis and concentration of triglycerides (TG) in 10m OPN-deficient (KO) hepatocytes when compared to WT hepatocytes. These changes in senescence and lipid metabolism in 10m OPN-KO mice liver were associated with the decrease of 78 kDa glucose-regulated protein (GRP78), induction of ER stress, and the increase in fatty acid synthase and CD36 levels. OPN deficiency in senescent cells also diminished GRP78, the accumulation of intracellular TG, and the increase in CD36 levels. In 20m mice, OPN loss led to increased liver fibrosis. Finally, we showed that OPN expression in vitro and in vivo was regulated by p53. In conclusion, OPN deficiency leads to earlier cellular senescence, ER stress, and TG accumulation during aging. The p53-OPN axis is required to inhibit the onset of age-related hepatosteatosis.


Assuntos
Fígado/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Osteopontina/uso terapêutico , Idoso , Animais , Progressão da Doença , Chaperona BiP do Retículo Endoplasmático , Feminino , Humanos , Fígado/fisiopatologia , Masculino , Camundongos , Pessoa de Meia-Idade , Osteopontina/farmacologia
8.
Hepatol Commun ; 1(9): 911-927, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29159325

RESUMO

Nonalcoholic steatohepatitis (NASH) is the advanced form of nonalcoholic fatty liver disease (NAFLD) which sets the stage for further liver damage. The mechanism for the progression of NASH involves multiple parallel hits including oxidative stress, mitochondrial dysfunction, inflammation and others. Manipulation of any of these pathways may be an approach to prevent NASH development and progression. Aramchol (arachidyl-amido cholanoic acid) is presently in a phase IIb NASH study. The aim of this study was to investigate Aramchol's mechanism of action and its effect on fibrosis using the methionine- and choline-deficient (MCD) diet model of NASH. We collected liver and serum from mice fed a MCD diet containing 0.1% methionine (0.1MCD) for four weeks, which developed steatohepatitis and fibrosis, as well as mice receiving a control diet; the metabolomes and proteomes were determined. 0.1MCD fed mice were given Aramchol (5mg/kg/day for the last 2 weeks); liver samples were analyzed histologically. Aramchol administration reduced features of steatohepatitis and fibrosis in 0.1MCD fed mice. Aramchol downregulated stearoyl-CoA desaturase 1 (SCD1), a key enzyme involved in triglyceride biosynthesis whose loss enhances fatty acid ß-oxidation. Aramchol increased the flux through the transsulfuration pathway, leading to a rise in glutathione (GSH) and GSH/GSSG ratio, the main cellular antioxidant that maintains intracellular redox status. Comparison of serum metabolomic pattern between 0.1MCD fed mice and NAFLD patients showed a substantial overlap. CONCLUSIONS: Aramchol treatment improved steatohepatitis and fibrosis by 1) decreasing SCD1, and 2) increasing the flux through the transsulfuration pathway maintaining cellular redox homeostasis. We also demonstrated that the 0.1MCD model resembles the metabolic phenotype observed in about 50% of NAFLD patients, which supports the potential use of Aramchol in NASH treatment.

9.
Cell Metab ; 26(1): 212-229.e12, 2017 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-28683288

RESUMO

Thyroid hormones (THs) act in the brain to modulate energy balance. We show that central triiodothyronine (T3) regulates de novo lipogenesis in liver and lipid oxidation in brown adipose tissue (BAT) through the parasympathetic (PSNS) and sympathetic nervous system (SNS), respectively. Central T3 promotes hepatic lipogenesis with parallel stimulation of the thermogenic program in BAT. The action of T3 depends on AMP-activated protein kinase (AMPK)-induced regulation of two signaling pathways in the ventromedial nucleus of the hypothalamus (VMH): decreased ceramide-induced endoplasmic reticulum (ER) stress, which promotes BAT thermogenesis, and increased c-Jun N-terminal kinase (JNK) activation, which controls hepatic lipid metabolism. Of note, ablation of AMPKα1 in steroidogenic factor 1 (SF1) neurons of the VMH fully recapitulated the effect of central T3, pointing to this population in mediating the effect of central THs on metabolism. Overall, these findings uncover the underlying pathways through which central T3 modulates peripheral metabolism.


Assuntos
Metabolismo Energético , Hipotálamo/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Transdução de Sinais , Hormônios Tireóideos/metabolismo , Tecido Adiposo Marrom/metabolismo , Animais , Metabolismo dos Lipídeos , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Termogênese , Tri-Iodotironina/metabolismo
10.
Hepatology ; 65(2): 694-709, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28035772

RESUMO

Hepatic fibrosis is a global health problem currently without effective therapeutic approaches. Even though the ubiquitin-like posttranslational modification of neddylation, that conjugates Nedd8 (neural precursor cell expressed developmentally downregulated) to specific targets, is aberrant in many pathologies, its relevance in liver fibrosis (LF) remained unexplored. Our results show deregulated neddylation in clinical fibrosis and both in mouse bileductligation- and CCl4 -induced fibrosis. Importantly, neddylation inhibition, by using the pharmacological inhibitor, MLN4924, reduced liver injury, apoptosis, inflammation, and fibrosis by targeting different hepatic cell types. On one hand, increased neddylation was associated with augmented caspase 3 activity in bile-acid-induced apoptosis in mouse hepatocytes whereas neddylation inhibition ameliorated apoptosis through reduction of expression of the Cxcl1 and Ccl2 chemokines. On the other hand, chemokine receptors and cytokines, usually induced in activated macrophages, were reduced after neddylation inhibition in mouse Kupffer cells. Under these circumstances, decreased hepatocyte cell death and inflammation after neddylation inhibition could partly account for reduction of hepatic stellate cell (HSC) activation. We provide evidence that augmented neddylation characterizes activated HSCs, suggesting that neddylation inhibition could be important for resolving LF by directly targeting these fibrogenic cells. Indeed, neddylation inhibition in activated HSCs induces apoptosis in a process partly mediated by accumulation of c-Jun, whose cullin-mediated degradation is impaired under these circumstances. CONCLUSION: Neddylation inhibition reduces fibrosis, suggesting neddylation as a potential and attractive therapeutic target in liver fibrosis. (Hepatology 2017;65:694-709).


Assuntos
Apoptose/genética , Quimiocinas/metabolismo , Ciclopentanos/farmacologia , Cirrose Hepática/genética , Cirrose Hepática/patologia , Pirimidinas/farmacologia , Ubiquitinas/genética , Envelhecimento/efeitos dos fármacos , Análise de Variância , Animais , Biópsia por Agulha , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Quimiocina CCL4/farmacologia , Quimiocinas/efeitos dos fármacos , Modelos Animais de Doenças , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína NEDD8 , Distribuição Aleatória , Transdução de Sinais
11.
J Cell Biol ; 210(1): 153-68, 2015 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-26150392

RESUMO

Although Schwann cell myelin breakdown is the universal outcome of a remarkably wide range of conditions that cause disease or injury to peripheral nerves, the cellular and molecular mechanisms that make Schwann cell-mediated myelin digestion possible have not been established. We report that Schwann cells degrade myelin after injury by a novel form of selective autophagy, myelinophagy. Autophagy was up-regulated by myelinating Schwann cells after nerve injury, myelin debris was present in autophagosomes, and pharmacological and genetic inhibition of autophagy impaired myelin clearance. Myelinophagy was positively regulated by the Schwann cell JNK/c-Jun pathway, a central regulator of the Schwann cell reprogramming induced by nerve injury. We also present evidence that myelinophagy is defective in the injured central nervous system. These results reveal an important role for inductive autophagy during Wallerian degeneration, and point to potential mechanistic targets for accelerating myelin clearance and improving demyelinating disease.


Assuntos
Autofagia , Bainha de Mielina/patologia , Traumatismos dos Nervos Periféricos/patologia , Animais , Células Cultivadas , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Metabolismo dos Lipídeos , Camundongos Transgênicos , Bainha de Mielina/fisiologia , Traumatismos dos Nervos Periféricos/enzimologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Nervo Isquiático/patologia , Serina-Treonina Quinases TOR/metabolismo , Degeneração Walleriana/patologia
12.
J Hepatol ; 62(3): 673-81, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25457203

RESUMO

BACKGROUND & AIMS: Very-low-density lipoproteins (VLDLs) export lipids from the liver to peripheral tissues and are the precursors of low-density-lipoproteins. Low levels of hepatic S-adenosylmethionine (SAMe) decrease triglyceride (TG) secretion in VLDLs, contributing to hepatosteatosis in methionine adenosyltransferase 1A knockout mice but nothing is known about the effect of SAMe on the circulating VLDL metabolism. We wanted to investigate whether excess SAMe could disrupt VLDL plasma metabolism and unravel the mechanisms involved. METHODS: Glycine N-methyltransferase (GNMT) knockout (KO) mice, GNMT and perilipin-2 (PLIN2) double KO (GNMT-PLIN2-KO) and their respective wild type (WT) controls were used. A high fat diet (HFD) or a methionine deficient diet (MDD) was administrated to exacerbate or recover VLDL metabolism, respectively. Finally, 33 patients with non-alcoholic fatty-liver disease (NAFLD); 11 with hypertriglyceridemia and 22 with normal lipidemia were used in this study. RESULTS: We found that excess SAMe increases the turnover of hepatic TG stores for secretion in VLDL in GNMT-KO mice, a model of NAFLD with high SAMe levels. The disrupted VLDL assembly resulted in the secretion of enlarged, phosphatidylethanolamine-poor, TG- and apoE-enriched VLDL-particles; special features that lead to increased VLDL clearance and decreased serum TG levels. Re-establishing normal SAMe levels restored VLDL secretion, features and metabolism. In NAFLD patients, serum TG levels were lower when hepatic GNMT-protein expression was decreased. CONCLUSIONS: Excess hepatic SAMe levels disrupt VLDL assembly and features and increase circulating VLDL clearance, which will cause increased VLDL-lipid supply to tissues and might contribute to the extrahepatic complications of NAFLD.


Assuntos
Lipoproteínas VLDL/sangue , Hepatopatia Gordurosa não Alcoólica/metabolismo , S-Adenosilmetionina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Feminino , Glicina N-Metiltransferase/deficiência , Glicina N-Metiltransferase/genética , Glicina N-Metiltransferase/metabolismo , Humanos , Lipoproteínas VLDL/metabolismo , Fígado/metabolismo , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Modelos Biológicos , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/patologia , Perilipina-2 , S-Adenosilmetionina/deficiência , Triglicerídeos/metabolismo , Adulto Jovem
13.
Hepatology ; 58(4): 1296-305, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23505042

RESUMO

UNLABELLED: Methionine adenosyltransferase 1A (MAT1A) and glycine N-methyltransferase (GNMT) are the primary genes involved in hepatic S-adenosylmethionine (SAMe) synthesis and degradation, respectively. Mat1a ablation in mice induces a decrease in hepatic SAMe, activation of lipogenesis, inhibition of triglyceride (TG) release, and steatosis. Gnmt-deficient mice, despite showing a large increase in hepatic SAMe, also develop steatosis. We hypothesized that as an adaptive response to hepatic SAMe accumulation, phosphatidylcholine (PC) synthesis by way of the phosphatidylethanolamine (PE) N-methyltransferase (PEMT) pathway is stimulated in Gnmt(-/-) mice. We also propose that the excess PC thus generated is catabolized, leading to TG synthesis and steatosis by way of diglyceride (DG) generation. We observed that Gnmt(-/-) mice present with normal hepatic lipogenesis and increased TG release. We also observed that the flux from PE to PC is stimulated in the liver of Gnmt(-/-) mice and that this results in a reduction in PE content and a marked increase in DG and TG. Conversely, reduction of hepatic SAMe following the administration of a methionine-deficient diet reverted the flux from PE to PC of Gnmt(-/-) mice to that of wildtype animals and normalized DG and TG content preventing the development of steatosis. Gnmt(-/-) mice with an additional deletion of perilipin2, the predominant lipid droplet protein, maintain high SAMe levels, with a concurrent increased flux from PE to PC, but do not develop liver steatosis. CONCLUSION: These findings indicate that excess SAMe reroutes PE towards PC and TG synthesis and lipid sequestration.


Assuntos
Fígado/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , S-Adenosilmetionina/metabolismo , Triglicerídeos/metabolismo , Animais , Modelos Animais de Doenças , Fígado Gorduroso/metabolismo , Fígado Gorduroso/fisiopatologia , Feminino , Glicina N-Metiltransferase/deficiência , Glicina N-Metiltransferase/genética , Homeostase/fisiologia , Metabolismo dos Lipídeos/fisiologia , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Perilipina-2
14.
Hepatology ; 54(6): 1975-86, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21837751

RESUMO

UNLABELLED: Very low-density lipoprotein (VLDL) secretion provides a mechanism to export triglycerides (TG) from the liver to peripheral tissues, maintaining lipid homeostasis. In nonalcoholic fatty liver disease (NAFLD), VLDL secretion disturbances are unclear. Methionine adenosyltransferase (MAT) is responsible for S-adenosylmethionine (SAMe) synthesis and MAT I and III are the products of the MAT1A gene. Deficient MAT I and III activities and SAMe content in the liver have been associated with NAFLD, but whether MAT1A is required for normal VLDL assembly remains unknown. We investigated the role of MAT1A on VLDL assembly in two metabolic contexts: in 3-month-old MAT1A-knockout mice (3-KO), with no signs of liver injury, and in 8-month-old MAT1A-knockout mice (8-KO), harboring nonalcoholic steatohepatitis. In 3-KO mouse liver, there is a potent effect of MAT1A deletion on lipid handling, decreasing mobilization of TG stores, TG secretion in VLDL and phosphatidylcholine synthesis via phosphatidylethanolamine N-methyltransferase. MAT1A deletion also increased VLDL-apolipoprotein B secretion, leading to small, lipid-poor VLDL particles. Administration of SAMe to 3-KO mice for 7 days recovered crucial altered processes in VLDL assembly and features of the secreted lipoproteins. The unfolded protein response was activated in 8-KO mouse liver, in which TG accumulated and the phosphatidylcholine-to-phosphatidylethanolamine ratio was reduced in the endoplasmic reticulum, whereas secretion of TG and apolipoprotein B in VLDL was increased and the VLDL physical characteristics resembled that in 3-KO mice. MAT1A deletion also altered plasma lipid homeostasis, with an increase in lipid transport in low-density lipoprotein subclasses and decrease in high-density lipoprotein subclasses. CONCLUSION: MAT1A is required for normal VLDL assembly and plasma lipid homeostasis in mice. Impaired VLDL synthesis, mainly due to SAMe deficiency, contributes to NAFLD development in MAT1A-KO mice.


Assuntos
Fígado Gorduroso/metabolismo , Lipoproteínas VLDL/metabolismo , Fígado/metabolismo , Metionina Adenosiltransferase/genética , Animais , Apolipoproteínas B/metabolismo , Deleção de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microssomos Hepáticos/química , Hepatopatia Gordurosa não Alcoólica , S-Adenosilmetionina/metabolismo , Triglicerídeos/sangue , Resposta a Proteínas não Dobradas/fisiologia
15.
J Exp Clin Cancer Res ; 30: 45, 2011 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-21521500

RESUMO

BACKGROUND: Many factors influence breast cancer progression, including the ability of progenitor cells to sustain or increase net tumour cell numbers. Our aim was to define whether alterations in putative progenitor populations could predict clinicopathological factors of prognostic importance for cancer progression. METHODS: Primary cultures were established from human breast tumour and adjacent non-tumour tissue. Putative progenitor cell populations were isolated based on co-expression or concomitant absence of the epithelial and myoepithelial markers EPCAM and CALLA respectively. RESULTS: Significant reductions in cellular senescence were observed in tumour versus non-tumour cultures, accompanied by a stepwise increase in proliferation:senescence ratios. A novel correlation between tumour aggressiveness and an imbalance of putative progenitor subpopulations was also observed. Specifically, an increased double-negative (DN) to double-positive (DP) ratio distinguished aggressive tumours of high grade, estrogen receptor-negativity or HER2-positivity. The DN:DP ratio was also higher in malignant MDA-MB-231 cells relative to non-tumorigenic MCF-10A cells. Ultrastructural analysis of the DN subpopulation in an invasive tumour culture revealed enrichment in lipofuscin bodies, markers of ageing or senescent cells. CONCLUSIONS: Our results suggest that an imbalance in tumour progenitor subpopulations imbalances the functional relationship between proliferation and senescence, creating a microenvironment favouring tumour progression.


Assuntos
Neoplasias da Mama/patologia , Células-Tronco Neoplásicas/patologia , Actinas/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/ultraestrutura , Técnicas de Cultura de Células , Proliferação de Células , Forma Celular , Senescência Celular , Feminino , Humanos , Queratinas Tipo I/metabolismo , Proteínas de Membrana/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/ultraestrutura , Células Tumorais Cultivadas , Vimentina/metabolismo
16.
Menopause ; 18(5): 575-81, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21242819

RESUMO

OBJECTIVE: The aims of this work were to determine (1) the effects of estrogen plus progestogen therapy (EPT) and raloxifene on oxidative stress and cardiovascular risk biomarkers in postmenopausal women and (2) the involvement of the functional G-463A polymorphism of the myeloperoxidase (MPO) gene in the therapy responses. METHODS: Postmenopausal women (45-55 y old) were assigned to three groups receiving (1) EPT (continuous 50 µg transdermal estradiol daily and 200 mg/d micronized progesterone orally the first 14 d of each month; n = 21), (2) raloxifene (60 mg daily; n = 17), and (3) no treatment (control; n = 21). Blood and urine samples were taken before and after 6 months of therapy. Measurements were serum lipid profile, C-reactive intercellular adhesion molecule 1 (ICAM-1), α-tocopherol, γ-tocopherol, uric acid, total antioxidant activity (TAA), malondialdehyde, and urinary 1,4-dihydroxynonane-mercapturic acid (the major urinary 4-hydroxynonenal metabolite). The G-463A MPO polymorphism was analyzed by polymerase chain reaction and restriction fragment length polymorphism. RESULTS: EPT significantly decreased TAA and the levels of ICAM-1, not modifying other cardiovascular risk or oxidative stress markers. The raloxifene and control groups experienced no modifications in oxidative stress or endothelial dysfunction markers. The MPO genotype specifically influenced the outcomes in the EPT group. Thus, TAA decreased significantly in GG (high-expression genotype) homozygotes, whereas ICAM-1 levels were reduced in A allele carriers. CONCLUSIONS: EPT exerted a negative action on the serum oxidant/antioxidant balance in the MPO GG homozygotes and a positive effect on the ICAM-1 endothelial dysfunction marker in carriers of the low-expression A allele. This observation provides evidence of the importance of this polymorphism in the response to EPT.


Assuntos
Estradiol/uso terapêutico , Terapia de Reposição Hormonal/métodos , Peroxidase/genética , Polimorfismo Genético , Pós-Menopausa/efeitos dos fármacos , Progesterona/uso terapêutico , Acetilcisteína/análogos & derivados , Acetilcisteína/urina , Antioxidantes/análise , Feminino , Humanos , Molécula 1 de Adesão Intercelular/sangue , Lipídeos/sangue , Malondialdeído/sangue , Pessoa de Meia-Idade , Pós-Menopausa/genética , Cloridrato de Raloxifeno/uso terapêutico , Moduladores Seletivos de Receptor Estrogênico/uso terapêutico , Ácido Úrico/sangue , alfa-Tocoferol/sangue
17.
Fertil Steril ; 95(5): 1601-5, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21130430

RESUMO

OBJECTIVE: To investigate whether the Ala16Val polymorphism in the SOD2 gene, encoding for mitochondrial manganese superoxide dismutase (SOD), is associated with [1] infertility and [2] the pregnancy rate (PR) in IVF cycles. DESIGN: Prospective case-control study. SETTING: Public university and public university hospital. PATIENT(S): A total of 362 newborns (nonselected population) and 148 infertile women undergoing an IVF cycle, from which 44 became pregnant and 104 did not. INTERVENTION(S): Blood samples extracted from the patients and newborn umbilical cord. MAIN OUTCOME MEASURE(S): Genotype and allele distribution of the Ala16Val polymorphism in the SOD2 gene using the tetra-primer amplification refractory mutation system-polymerase chain reaction (PCR). RESULT(S): The polymorphism distribution of the subfertile women was similar to that of a nonselected population. The SOD2 Ala allele frequency was 49% both in controls and IVF patients. In IVF population the Ala/Ala SOD2 genotype was 25%, with a 28% Val/Val homozygous. In contrast, the Ala/Ala genotype was associated with higher PRs in IVF (47% in Ala/Ala vs. 23% in no Ala/Ala). A multivariate logistic regression analysis revealed that the Ala/Ala genotype was an independent predictor of pregnancy (odds ratio [OR] = 3.29), followed by the number of transferred embryos (OR = 2.37) and age (OR = 0.84). CONCLUSION(S): The Ala/Ala SOD2 genotype is a significant independent predictor of the occurrence of pregnancy in IVF. Data also support a role for antioxidant defense, particularly in the mitochondria, in conception in IVF.


Assuntos
Fertilização in vitro/métodos , Infertilidade/genética , Infertilidade/terapia , Polimorfismo de Nucleotídeo Único , Taxa de Gravidez , Superóxido Dismutase/genética , Adulto , Alanina/genética , Substituição de Aminoácidos/genética , Sequência de Bases , Estudos de Casos e Controles , Feminino , Estudo de Associação Genômica Ampla , Humanos , Recém-Nascido , Infertilidade/diagnóstico , Masculino , Dados de Sequência Molecular , Periodicidade , Polimorfismo de Nucleotídeo Único/fisiologia , Gravidez , Prognóstico , Resultado do Tratamento , Valina/genética
18.
Fertil Steril ; 94(4): 1279-1286, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19540482

RESUMO

OBJECTIVE: To evaluate the serum oxidizability and antioxidant status in women undergoing an in vitro fertilization (IVF) cycle and to assess the possible relationship of the oxidizability indexes with the pregnancy rate. DESIGN: Prospective, longitudinal study. SETTING: Public university and public university hospital. PATIENT(S): Systematically recruited cohort of 125 women undergoing either IVF or intracytoplasmic sperm injection (ICSI). INTERVENTION(S): Serum samples were collected before the beginning of the use of gonadotropins (basal) and the day of human chorionic gonadotropin (hCG) administration (final) during an IVF cycle. MAIN OUTCOME MEASURE(S): The Cu2+-induced serum oxidation in terms of the oxidation rate in the lag (Vlag) and propagation (Vmax) phases and the time at which the oxidation rate is maximal (tmax), and measurements of serum total antioxidant activity (TAA), tocopherol, hydrophilic antioxidants, malondialdehyde, and nitric oxide. RESULT(S): Albumin, urate, bilirubin, alpha-tocopherol and gamma-tocopherol, TAA, and tmax statistically significantly decreased after the IVF cycle. Conception cycles were associated with a serum more prone to oxidation compared with nonconception cycles. In multivariate logistic regression analysis, the difference (final-basal) of the oxidation index Vlag (OR 1.394) and the body mass index (OR 0.785) were independent predictors of pregnancy. CONCLUSION(S): Treatment with IVF induces the production of reactive oxygen species (ROS), which is reflected in a serum less protected against oxidation. The results also suggest a role for ROS in the occurrence of conception in IVF.


Assuntos
Antioxidantes/metabolismo , Fertilização in vitro , Infertilidade/sangue , Infertilidade/terapia , Soro/metabolismo , Adulto , Proteínas Sanguíneas/metabolismo , Feminino , Humanos , Infertilidade/metabolismo , Estudos Longitudinais , Oxirredução , Estresse Oxidativo/fisiologia , Gravidez , Taxa de Gravidez , Estabilidade Proteica
19.
J Virol ; 83(6): 2663-74, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19116251

RESUMO

Chronic hepatitis B is a major cause of liver-related death worldwide. Interleukin-12 (IL-12) induction accompanies viral clearance in chronic hepatitis B virus infection. Here, we tested the therapeutic potential of IL-12 gene therapy in woodchucks chronically infected with woodchuck hepatitis virus (WHV), an infection that closely resembles chronic hepatitis B. The woodchucks were treated by intrahepatic injection of a helper-dependent adenoviral vector encoding IL-12 under the control of a liver-specific RU486-responsive promoter. All woodchucks with viral loads below 10(10) viral genomes (vg)/ml showed a marked and sustained reduction of viremia that was accompanied by a reduction in hepatic WHV DNA, a loss of e antigen and surface antigen, and improved liver histology. In contrast, none of the woodchucks with higher viremia levels responded to therapy. The antiviral effect was associated with the induction of T-cell immunity against viral antigens and a reduction of hepatic expression of Foxp3 in the responsive animals. Studies were performed in vitro to elucidate the resistance to therapy in highly viremic woodchucks. These studies showed that lymphocytes from healthy woodchucks or from animals with low viremia levels produced gamma interferon (IFN-gamma) upon IL-12 stimulation, while lymphocytes from woodchucks with high viremia failed to upregulate IFN-gamma in response to IL-12. In conclusion, IL-12-based gene therapy is an efficient approach to treat chronic hepadnavirus infection in woodchucks with viral loads below 10(10) vg/ml. Interestingly, this therapy is able to break immunological tolerance to viral antigens in chronic WHV carriers.


Assuntos
Terapia Genética/métodos , Vírus da Hepatite B da Marmota/imunologia , Hepatite B/veterinária , Interleucina-12/biossíntese , Interleucina-12/imunologia , Marmota/virologia , Adenoviridae/genética , Animais , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/genética , Vetores Genéticos , Hepatite B/imunologia , Interferon gama/biossíntese , Fígado/patologia , Fígado/virologia , Dados de Sequência Molecular , Análise de Sequência de DNA , Linfócitos T/imunologia , Viremia
20.
J Gene Med ; 10(10): 1092-101, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18642400

RESUMO

BACKGROUND: High-capacity adenoviruses (HC-Ad) hold great promise for the treatment of many diseases. The major drawbacks for the clinical application of this vector concern difficulties with respect to large-scale production, and the absence of standardized methods for production and titration. In the present study, we compare the different methods found in the literature for characterizing HC-Ad production. METHODS: Two productions of the HC-Ad carrying murine IL-12 gene were obtained. The viral titer and adenovirus-helper contamination as well as viral particle concentration of both productions were determined using different methods: (i) quantification of total viral particles by spectrophotometry and plaque assay to estimate first-generation (FG)-helper-Ad contamination; (ii) quantification of HC-Ad and FG-helper-Ad genomes by the quantitative polymerase chain reaction (qPCR) directly from viral stock; (iii) quantification of viral genomes after cell infection by the slot-blot hybridization assay and (iv) qPCR. RESULTS: Dramatic differences with respect to viral titer were found depending on the method used. The first method overestimates HC-Ad titer and underestimates FG-helper-Ad contamination and no information on the infectivity of the HC-Ad is obtained. qPCR analysis of viral stock is more sensitive and accurate, but information about infectivity remains unknown and FG-helper-Ad contamination is overestimated. Quantification of HC-Ad and FG-helper-Ad infectious units by-slot blot DNA hybridization and qPCR assay are found to be equally sensitive and accurate. CONCLUSIONS: The results of the present study demonstrate that a standardized method should be developed for HC-Ad characterization for future clinical applications of this vector. Quantification of HC-Ad production by qPCR is a fast, safe and reliable method for determining HC-Ad and FG-helper-Ad particles and infectious units.


Assuntos
Adenoviridae/genética , Reação em Cadeia da Polimerase/normas , Adenoviridae/metabolismo , Animais , DNA Viral/análise , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Genoma Viral , Células HeLa , Humanos , Interleucina-12/genética , Interleucina-12/metabolismo , Camundongos
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