RESUMO
Epidemiological studies revealed that dietary proteins can contribute to the modulation of the cardiovascular disease risk. Still, direct effects of dietary proteins on serum metabolites and other health-modulating factors have not been fully explored. Here, we compared the effects of dietary lupin protein with the effects of beef protein and casein on the serum metabolite profile, cardiovascular risk markers and the fecal microbiome. Pigs were fed diets containing 15% of the respective proteins for 4 weeks. A classification analysis of the serum metabolites revealed six biomarker sets of two metabolites each that discriminated between the intake of lupin protein, lean beef or casein. These biomarker sets included 1- and 3-methylhistidine, betaine, carnitine, homoarginine and methionine. The study revealed differences in the serum levels of the metabolites 1- and 3- methylhistidine, homoarginine, methionine and homocysteine, which are involved in the one-carbon cycle. However, these changes were not associated with differences in the methylation capacity or the histone methylation pattern. With the exception of serum homocysteine and homoarginine levels, other cardiovascular risk markers, such as the homeostatic model assessment index, trimethylamine-N-oxide and lipids, were not influenced by the dietary protein source. However, the composition of the fecal microorganisms was markedly changed by the dietary protein source. Lupin-protein-fed pigs exhibited more species from the phyla Bacteroidetes and Firmicutes than the other two groups. In conclusion, different dietary protein sources induce distinct serum metabolic fingerprints, have an impact on the cardiovascular risk and modulate the composition of the fecal microbiome.
Assuntos
Aminoácidos/análise , Proteínas Alimentares/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Fígado/metabolismo , Acetilação , Aminoácidos/sangue , Animais , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Caseínas/farmacologia , Fezes/microbiologia , Feminino , Microbioma Gastrointestinal/fisiologia , Histonas/metabolismo , Lipídeos/sangue , Fígado/efeitos dos fármacos , Metilação , Carne Vermelha , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Proteínas de Armazenamento de Sementes/farmacologia , SuínosRESUMO
SCOPE: Fasting leads to a significant downregulation of the hypothalamus-pituitary-thyroid axis, and peroxisome proliferator-activated receptor (PPAR) α is a key transcription factor in mediating a magnitude of adaptive responses to fasting. In this study, we examined the role of PPARα in regulation of the hypothalamus-pituitary-thyroid axis. METHODS AND RESULTS: Thyroid-stimulating hormone ß-subunit (TSHß) mRNA abundance was being reduced in response to treatment of TαT1 cells with PPARα agonists (p < 0.05), indicating an inhibitory transcriptional regulation of TSHß by PPARα. As expected, fasting significantly downregulated TSHß mRNA expression in a two-factorial study with fed or fasted wild-type (WT) and PPARα knockout mice (p < 0.05). In contrast to the in vitro data, fasted PPARα knockout mice revealed lower mRNA concentrations of pituitary TSHß (-64%) and TSH-regulated thyroid genes, and lower plasma concentrations of thyroxine (T4, -25%), triiodothyronine (T3, -25%), free T4 (-60%), and free T3 (-35%) than fasted WT mice (p < 0.05). Those differences were not observed in fed mice. CONCLUSIONS: Data from thyrotrope cells revealed that PPARα could contribute to the fasting-associated downregulation of the TSHß mRNA expression. In a mouse model, fasting led to a significant reduction in TSHß mRNA level, but unexpectedly this effect was stronger in mice lacking PPARα than in WT mice.
Assuntos
Jejum/fisiologia , PPAR alfa/metabolismo , Tireotrofos/fisiologia , Tireotropina Subunidade beta/genética , Animais , Linhagem Celular , Ácidos Graxos não Esterificados/genética , Ácidos Graxos não Esterificados/metabolismo , Ácidos Fíbricos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neurocinina B/análogos & derivados , Neurocinina B/genética , PPAR alfa/agonistas , PPAR alfa/genética , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Pirimidinas/farmacologia , RNA Mensageiro/metabolismo , Receptor X Retinoide alfa/genética , Receptores beta dos Hormônios Tireóideos/genética , Tireotrofos/citologia , Tireotrofos/efeitos dos fármacos , Tiroxina/sangue , Tiroxina/genética , Tri-Iodotironina/sangue , Tri-Iodotironina/genéticaRESUMO
BACKGROUND: Hepatic PPARalpha acts as the primary mediator of the adaptive response to fasting by upregulation of a number of genes involved in fatty acid catabolism. Whether carnitine-acylcarnitine translocase (CACT), which mediates the import of acylcarnitines into the mitochondrial matrix for subsequent beta-oxidation of fatty acid moieties, is also regulated by PPARalpha in the liver has not yet been investigated. METHODS AND RESULTS: Herein, we observed that hepatic mRNA abundance of CACT was increased by both, fasting and treatment with PPARalpha agonist WY-14,643 in wild-type mice but not PPARalpha-knockout mice (P<0.05). Cell culture experiments revealed that CACT mRNA abundance was higher in liver cells treated with either WY-14,643 or PPARdelta agonist GW0742, but not with PPARgamma agonist troglitazone (TGZ) than in control cells (P<0.05). In addition, reporter assays revealed activation of mouse CACT promoter by WY-14,643 and GW0742, but not TGZ. Moreover, deletion and mutation analyses of CACT promoter and 5'-UTR revealed one functional PPRE in the 5'-UTR of mouse CACT. GENERAL SIGNIFICANCE: CACT is upregulated by PPARalpha and PPARdelta, probably by binding to a functional PPRE at position +45 to +57 relative to the transcription start site. The upregulation of CACT by PPARalpha and PPARdelta, which are both important for the regulation of fatty acid oxidation in tissues during fasting, may increase the import of acylcarnitine into the mitochondrial matrix during fasting.
Assuntos
Carnitina Aciltransferases/genética , Fígado/metabolismo , PPAR alfa/genética , PPAR delta/genética , Animais , Sequência de Bases , Peso Corporal/efeitos dos fármacos , Carnitina Aciltransferases/metabolismo , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Linhagem Celular Tumoral , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Jejum , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , Luciferases/genética , Luciferases/metabolismo , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutação , PPAR alfa/agonistas , PPAR alfa/metabolismo , PPAR delta/agonistas , PPAR delta/metabolismo , Regiões Promotoras Genéticas/genética , Pirimidinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tiazóis/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
Fibrates and thiazolidinediones, agonists of PPARalpha and PPARgamma, respectively, reduce triglyceride concentrations in rat liver and plasma. Fatty acid and triacylglycerol synthesis in mammals is regulated by sterol regulatory element-binding protein (SREBP)-1c. Recently, it was shown that insulin-induced gene (Insig)-1, the key regulator of SREBP activity, is up-regulated by both activation of PPARalpha and PPARgamma. In order to elucidate whether inhibition of SREBP-1 activation may contribute to the triacylglycerol lowering effect of PPARalpha and PPARgamma agonists, we incubated rat hepatoma Fao cells with WY 14,643 and troglitazone, strong and selective agonists of PPARalpha and PPARgamma, respectively. Activation of both, PPARalpha and PPARgamma led to increased concentrations of Insig-1 and Insig-2a, with the most prominent effect on Insig-2a after troglitazone incubation. As a result, the amount of nuclear SREBP-1 was reduced in Fao cells by both WY 14,643 and troglitazone treatment. The reduction of nuclear SREBP-1 was associated with decreased mRNA concentrations of its target genes fatty acid synthase and glycerol-3-phosphate acyltransferase, implicated in fatty acid and triacylglycerol synthesis. This was finally reflected in reduced rates of newly synthesized triacylglycerols from de novo-derived fatty acids and decreased intracellular and secreted triacylglycerol concentrations in Fao cells treated with WY 14,643 and troglitazone, respectively. Thus, these data suggest that the triacylglycerol reducing effect of fibrates and thiazolidinediones is partially caused by inhibition of SREBP-1 activation via up-regulation of Insig.
Assuntos
PPAR alfa/agonistas , PPAR gama/agonistas , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Triglicerídeos/biossíntese , Animais , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Cromanos/farmacologia , Ácido Graxo Sintases/metabolismo , Glicerol-3-Fosfato O-Aciltransferase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , PPAR alfa/metabolismo , PPAR gama/metabolismo , Pirimidinas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Tiazolidinedionas/farmacologia , Troglitazona , Regulação para CimaRESUMO
Peroxisome proliferator-activated receptor (PPAR)-alpha mediates an adaptive response to fasting by up-regulation of genes involved in fatty acid oxidation and ketone body synthesis. Ketone bodies are transferred in and out of cells by monocarboxylate transporter (MCT)-1. In this study we observed for the first time that activation of PPARalpha in rats by clofibrate treatment or fasting increased hepatic mRNA concentration of MCT1. In Fao rat hepatoma cells, incubation with the PPARalpha agonist WY 14,643 increased mRNA concentration of MCT1 whereas the PPARgamma agonist troglitazone did not. To elucidate whether up-regulation of MCT1 is indeed mediated by PPARalpha we treated wild-type and PPARalpha-null mice with WY 14,643. In wild-type mice, treatment with WY 14,643 increased mRNA concentrations of MCT1 in liver, kidney and small intestine whereas no up-regulation was observed in PPARalpha-null mice.
Assuntos
Jejum/metabolismo , Corpos Cetônicos/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , PPAR alfa/metabolismo , Simportadores/metabolismo , Animais , Clofibrato/farmacologia , Ácidos Graxos/metabolismo , Hipolipemiantes/farmacologia , Masculino , Camundongos , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/fisiologia , Oxirredução/efeitos dos fármacos , PPAR alfa/agonistas , Proliferadores de Peroxissomos/farmacologia , Pirimidinas/farmacologia , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
We tested the hypothesis that transcription of novel organic cation transporters (OCTNs) is directly regulated by peroxisome proliferator-activated receptor (PPAR)-alpha. Therefore, wild-type mice and mice deficient in PPAR alpha (PPAR alpha-/-) were treated with the PPAR alpha agonist WY 14,643. Wild-type mice treated with WY 14,643 had a greater abundance of OCTN2 mRNA in their liver, muscle, kidney, and small intestine and a greater abundance of OCTN3 mRNA in kidney and small intestine than did untreated wild-type mice (P < 0.05). Moreover, wild-type mice treated with WY 14,643 had greater mRNA abundances of enzymes involved in hepatic carnitine synthesis (4-N-trimethylaminobutyraldehyde dehydrogenase, gamma-butyrobetaine dioxygenase) and increased carnitine concentrations in liver and muscle than did untreated wild-type mice (P < 0.05). Untreated PPAR alpha-/- mice had a lower abundance of OCTN2 mRNA in liver, kidney, and small intestine and lower carnitine concentrations in plasma, liver, and kidney than did untreated wild-type mice (P < 0.05). In PPAR alpha-/- mice, treatment with WY 14,643 did not influence mRNA abundance of OCTN2 and OCTN3 and carnitine concentrations in all tissues analyzed. The abundance of OCTN1 mRNA in all the tissues analyzed was not changed by treatment with WY 14,643 in wild-type or PPAR alpha-/- mice. In conclusion, this study shows that transcriptional upregulation of OCTN2 and OCTN3 in tissues and of enzymes involved in hepatic carnitine biosynthesis are mediated by PPAR alpha. It also shows that PPAR alpha mediates changes of whole-body carnitine homeostasis in mice by upregulation of carnitine transporters and enzymes involved in carnitine synthesis.
Assuntos
Carnitina/biossíntese , Fígado/enzimologia , Fígado/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , PPAR alfa/metabolismo , Transcrição Gênica/genética , Animais , Betaína/análogos & derivados , Betaína/metabolismo , Peso Corporal , Carnitina/metabolismo , Lisina/análogos & derivados , Lisina/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Tamanho do Órgão , Especificidade de Órgãos , Proteínas de Transporte de Cátions Orgânicos/genética , PPAR alfa/deficiência , PPAR alfa/genética , RNA Mensageiro/genética , Membro 5 da Família 22 de Carreadores de Soluto , Regulação para Cima/genéticaRESUMO
In mammals, (n-3) PUFA and conjugated linoleic acids (CLA) act as activators of PPAR alpha and alter nuclear concentrations of sterol regulatory element-binding proteins (SREBP) in the liver, and thereby influence hepatic lipid catabolism and synthesis. In this study, we investigated the hypothesis that (n-3) PUFA and CLA exert similar effects in the liver of laying hens. Thirty hens (64 weeks old) were fed diets containing 30 g/kg of sunflower oil (control), fish oil (salmon oil) or CLA in TAG form (containing predominantly cis-9, trans-11 CLA and trans-10, cis-12 CLA) for 5 weeks. Hens fed fish oil had a higher expression of some PPAR alpha target genes and a lower nuclear concentration of SREBP-2 in the liver and lower concentrations of cholesterol and TAG in plasma than control hens. Nuclear concentration of SREBP-1 and its target genes involved in lipogenesis were not altered in hens fed fish oil. Hens fed CLA had increased concentrations of TAG and cholesterol in the liver. However, their mRNA levels of PPAR alpha target genes and nuclear concentrations of SREBP-1 and SREBP-2 as well as mRNA levels of their target genes in the liver were largely unchanged compared to control hens. The results of this study suggest that (n-3) PUFA cause a moderate activation of PPAR alpha and lower cholesterol synthesis but do not impair fatty acid synthesis in the liver of laying hens. CLA lead to an accumulation of TAG and cholesterol in the liver of hens by mechanisms to be elucidated in further studies.
Assuntos
Galinhas/metabolismo , Óleos de Peixe/farmacologia , Ácidos Linoleicos Conjugados/farmacologia , Fígado/efeitos dos fármacos , PPAR alfa/biossíntese , Proteínas de Ligação a Elemento Regulador de Esterol/biossíntese , Fenômenos Fisiológicos da Nutrição Animal , Animais , Peso Corporal/efeitos dos fármacos , Peso Corporal/fisiologia , Galinhas/fisiologia , Colesterol/metabolismo , Ingestão de Alimentos/fisiologia , Gema de Ovo/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/anatomia & histologia , Fígado/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Tamanho do Órgão/fisiologia , Oviposição/fisiologia , PPAR alfa/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Proteínas de Ligação a Elemento Regulador de Esterol/genética , Triglicerídeos/metabolismoRESUMO
Atopic/allergic diseases are characterized by T helper 2 (Th2)-dominated immune responses resulting in immunoglobulin E (IgE) production. DNA-based immunotherapies have been shown to shift the immune response towards Th1 in animal models. In further studies we showed that human dendritic cells (DC) transfected with allergen-DNA are able to stimulate autologous CD4(+) T cells from atopic individuals to produce Th1 instead of Th2 cytokines and to activate interferon-gamma (IFN-gamma)-producing CD8(+) T cells. The aim of this study was to analyse whether DC transfected with allergen-DNA are also able to influence immunoglobulin production of B cells from atopic donors. For this purpose, human monocyte-derived DC from grass-pollen allergic donors were transfected with an adenovirus encoding the allergen Phleum pratense 1 and cocultured with B cells, autologous CD4(+) T cells, and CD40 ligand-transfected L-cells. B cells receiving help from CD4(+) T cells stimulated with allergen-transfected dendritic cells produced more allergen-specific IgG4 compared to stimulation with allergen protein pulsed DC or medium, while total IgG4 production was not affected. In contrast, specific IgE production was not enhanced by stimulation with allergen-DNA transfected DC compared to medium and inhibited compared to allergen protein-pulsed DC with similar effects on total IgE production in vitro. Allergen-DNA transfected dendritic cells are able to direct the human allergic immune response from Th2-dominance towards Th1 and Tc1 also resulting in decreased IgE and increased IgG4 production.
Assuntos
Linfócitos B/imunologia , Células Dendríticas/imunologia , Hipersensibilidade Imediata/imunologia , Imunoglobulina E/biossíntese , Imunoglobulina G/biossíntese , Alérgenos/genética , Alérgenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Citocinas/biossíntese , Humanos , Ativação Linfocitária/imunologia , Proteínas de Plantas/imunologia , Rinite/imunologia , Linfócitos T Citotóxicos/imunologia , Células Th1/imunologia , TransfecçãoRESUMO
Transrectal ultrasound (TRUS) of the prostate is a specific urological examination. This morphological imaging technique is often capable of identifying the cause for raised values of prostate-specific antigen (PSA) or of clarifying hard tissue regions found during rectal palpation. Particulary in view of constantly increasing number of patients undergoing PSA tests, there is a rising need for further prostate diagnostics in otherwise asymptomatic men. Especially in the gray zone between 4 and 10 ng/ml the tissue marker PSA is frequently influenced by benign alterations, so that it is not possible--on the basis of the PSA value alone--to differentiate between benign and malignant causes. Only a clearly increased serum PSA value (>20 ng/ml) indicates the presence of a prostate carcinoma at a very high probability. However, it is necessary that all patients whose PSA is elevated, undergo a bioptical tissue sample procedure in order to try to diagnose prostate cancer. Today, we regard the technique of TRUS-based transrectal prostate biopsy, carried out with a semi-automatic coil spring device and an 18-gauge needle, as the gold standard. The core problem of visual TRUS assessment lies in its lack of specificity, especially if the examiner has only limited experience. There can be low-echo, cancer-suspicious areas that may be histologically either benign or malignant. Benign prostatic hyperplasia (BPH), vessels, centers of prostatitis as well as shadows and artefacts can often also be low in echo-density. Only adequate application of this technology and experience with this method can lead to satisfying biopsy and diagnostic results.
Assuntos
Endossonografia/métodos , Próstata/diagnóstico por imagem , Doenças Prostáticas/diagnóstico por imagem , Diagnóstico Diferencial , Endossonografia/instrumentação , Desenho de Equipamento , Humanos , Masculino , Reto , Reprodutibilidade dos TestesRESUMO
Administration of oxidized oils to rats or pigs causes a reduction of their cholesterol concentrations in liver and plasma. The reason for this effect is unknown. We tested the hypothesis that oxidized oils lower cholesterol concentrations by inhibiting the proteolytic activation of sterol regulatory element-binding protein (SREBP)-2 in the liver and transcription of its target genes involved in cholesterol synthesis and uptake through an upregulation of gene expression of insulin-induced genes (Insig). For 6 d, 18 rats were orally administered either sunflower oil (control group) or an oxidized oil prepared by heating sunflower oil. Rats administered the oxidized oil had higher messenger RNA (mRNA) concentrations of acyl-CoA oxidase and cytochrome P450 4A1 in the liver than control rats (P < 0.05), indicative of activation of PPARalpha. Furthermore, rats administered the oxidized oil had higher mRNA concentrations of Insig-1 and Insig-2a, a lower concentration of the mature SREBP-2 in the nucleus, lower mRNA concentrations of the SREBP-2 target genes 3-hydroxy-3-methylglutaryl CoA reductase and LDL receptor in their livers, and a lower concentration of cholesterol in liver, plasma, VLDL, and HDL than control rats (P < 0.05). In conclusion, this study shows that reduced cholesterol concentrations in liver and plasma of rats administered an oxidized oil were due to an inhibition of the activation of SREBP-2 by an upregulation of Insig, which in turn inhibited transcription of proteins involved in hepatic cholesterol synthesis and uptake.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Insulina/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Óleos/farmacologia , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Temperatura , Acil Coenzima A/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Núcleo Celular/metabolismo , Colesterol/metabolismo , Lipoproteínas/metabolismo , Masculino , Óleos/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Oxirredução , PPAR alfa/genética , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Receptores de LDL/metabolismoRESUMO
To elucidate the mechanisms underlying the cholesterol lowering effects of PPARalpha agonists we investigated key regulators of cholesterol synthesis and uptake in rats and in the rat hepatoma cell line Fao after treatment with the PPARalpha agonists clofibrate and WY 14,643, respectively. In rat liver as well as in Fao cells, PPARalpha activation led to a decrease of transcriptionally active nuclear SREBP-2. mRNA concentrations of the key regulators of SREBP processing, Insig-1 in rat liver and Insig-1 and Insig-2a in Fao cells, were increased upon PPARalpha activation. Thus we suggest, that the observed reduction of the amount of nuclear SREBP-2 was due to an inhibition of the processing of the precursor protein. Both, in rat liver and in Fao cells, mRNA concentrations of the SREBP-2 target genes HMG-CoA reductase (EC1.1.1.34) and LDL receptor were reduced after treatment with the PPARalpha agonists. Furthermore, treatment of Fao cells with WY 14,643 reduced cholesterol synthesis. As a result, the amount of total cholesterol in liver, plasma and lipoproteins of clofibrate treated rats and in WY 14,643 treated Fao cells was decreased compared to control animals and cells, respectively. In conclusion, we could show a novel link between PPARalpha and cholesterol metabolism by demonstrating that PPARalpha activation lowers cholesterol concentration by reducing the abundance of nuclear SREBP-2.
Assuntos
Anticolesterolemiantes/farmacologia , Núcleo Celular/efeitos dos fármacos , Colesterol/metabolismo , PPAR alfa/agonistas , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Acil-CoA Oxidase/genética , Acil-CoA Oxidase/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Colesterol/biossíntese , VLDL-Colesterol/metabolismo , Clofibrato/administração & dosagem , Clofibrato/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Família 4 do Citocromo P450 , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Proteínas de Membrana/metabolismo , PPAR alfa/metabolismo , Pirimidinas/administração & dosagem , Pirimidinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de LDL/genética , Receptores de LDL/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Triglicerídeos/metabolismoRESUMO
It has been shown that clofibrate treatment increases the carnitine concentration in the liver of rats. However, the molecular mechanism is still unknown. In this study, we observed for the first time that treatment of rats with the peroxisome proliferator activated receptor (PPAR)-alpha agonist clofibrate increases hepatic mRNA concentrations of organic cation transporters (OCTNs)-1 and -2 which act as transporters of carnitine into the cell. In rat hepatoma (Fao) cells, treatment with WY-14,643 also increased the mRNA concentration of OCTN-2. mRNA concentrations of enzymes involved in carnitine biosynthesis were not altered by treatment with the PPARalpha agonists in livers of rats and in Fao cells. We conclude that PPARalpha agonists increase carnitine concentrations in livers of rats and cells by an increased uptake of carnitine into the cell but not by an increased carnitine biosynthesis.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proteínas da Membrana Plasmática de Transporte de Catecolaminas/metabolismo , Clofibrato/administração & dosagem , Hepatócitos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , PPAR alfa/antagonistas & inibidores , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Masculino , Transportador 2 de Cátion Orgânico , Ratos , Ratos Sprague-Dawley , Regulação para Cima/efeitos dos fármacosRESUMO
Conjugated linoleic acids (CLA) have attracted scientific interest due to their potential beneficial effects on atherosclerosis. Recently, a mixture of CLA isomers was demonstrated to upregulate LDL receptor expression in the human hepatoma cell line HepG2. However, the underlying mechanisms remain to be resolved. Thus, the aim of this study was to elucidate how CLA mediates upregulation of LDL receptor in HepG2 cells and whether this upregulation is isomer-specific. The results revealed that LDL receptor promoter activity and mRNA expression were strongly induced upon treatment with t10c12-CLA (P<0.05), whereas c9t11-CLA and linoleic acid (LA) had no effect. In addition, only treatment with t10c12-CLA markedly induced mRNA expression of SREBP-2 and HMG-CoA reductase and slightly induced that of SREBP-1 (P<0.05). Using SREBP-2 knockdown cells, we could demonstrate that the effect of t10c12-CLA on LDL receptor gene transcription was significantly reduced when compared to control cells (P<0.05). When using SREBP-1 knockdown cells the effect of t10c12-CLA on LDL receptor mRNA only slightly decreased compared to control cells. In addition, using different deletion constructs of the LDL receptor gene promoter we showed that the induction of the LDL receptor by t10c12-CLA is independent of the AP-1 motif in the LDL receptor promoter. In conclusion, the present study revealed that transcriptional activation of the LDL receptor gene by t10c12-CLA is dependent on the upregulation of SREBP-2 and is probably due to the activation of the SRE-1 in the LDL receptor gene promoter in HepG2 cells. Thus, the decreased plasma cholesterol levels in response to CLA as observed in a limited number of animal and human studies might be explained by an enhanced uptake of VLDL and LDL cholesterol via hepatic LDL receptors. However, it provides no explanation for the outcome of most human studies reporting unaltered or even increased plasma and LDL cholesterol concentrations in response to supplementation with CLA.
Assuntos
Ácidos Linoleicos Conjugados/farmacologia , Receptores de LDL/genética , Linhagem Celular Tumoral , Colesterol/biossíntese , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Ácido Linoleico/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores de LDL/biossíntese , Transdução de Sinais/efeitos dos fármacos , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Fator de Transcrição AP-1/metabolismo , Regulação para CimaRESUMO
In rats, oxidized fats activate the peroxisome proliferator-activated receptor alpha (PPARalpha), leading to reduced triglyceride concentrations in liver, plasma and very low density lipoproteins. Oxidation products of linoleic acid constitute an important portion of oxidized dietary fats. This study was conducted to check whether the primary lipid peroxidation product of linoleic acid, 13-hydroperoxy-9,11-octadecadienoic acid (13-HPODE), might be involved in the PPARalpha-activating effect of oxidized fats. Therefore, we examined the effect of 13-HPODE on the expression of PPARalpha target genes in the rat Fao and the human HepG2 hepatoma cell lines. In Fao cells, 13-HPODE increased the mRNA concentration of the PPARalpha target genes acyl-CoA oxidase (ACO), cytochrome P450 4A1 and carnitine-palmitoyltransferase 1A (CPT1A). Furthermore, the concentration of cellular and secreted triglycerides was reduced in Fao cells treated with 13-HPODE. Because PPARalpha mRNA was not influenced, we conclude that these effects are due to an activation of PPARalpha by 13-HPODE. In contrast, HepG2 cells seemed to be resistant to PPARalpha activation by 13-HPODE because no remarkable induction of the PPARalpha target genes ACO, CPT1A, mitochondrial HMG-CoA synthase and delta9-desaturase was observed. Consequently, cellular and secreted triglyceride levels were not changed after incubation of HepG2 cells with 13-HPODE. In conclusion, this study shows that 13-HPODE activates PPARalpha in rat Fao but not in human HepG2 hepatoma cells.
Assuntos
Expressão Gênica/efeitos dos fármacos , Ácidos Linoleicos/farmacologia , Peróxidos Lipídicos/farmacologia , Fígado/metabolismo , PPAR alfa/genética , Acil-CoA Oxidase/genética , Animais , Carcinoma Hepatocelular , Carnitina O-Palmitoiltransferase/genética , Linhagem Celular Tumoral , Citocromo P-450 CYP4A/genética , Humanos , Ácido Linoleico/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/análise , Fígado/química , Neoplasias Hepáticas , Neoplasias Hepáticas Experimentais , RNA Mensageiro/análise , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Triglicerídeos/análise , Triglicerídeos/metabolismoRESUMO
Regulatory CD4+ CD25+ FoxP3-positive T cells (Treg) are functional in most atopic patients with allergic rhinitis and are able to inhibit T helper type 1 (Th1) and Th2 cytokine production of CD4+ CD25- T cells. This study was designed to analyse the following additional aspects: influence of allergen concentration, influence of the type of allergen, and influence of the atopy status of the donor on the strength of the regulatory activity. CD4+ CD25- T cells from healthy non-atopic controls or from grass-pollen-allergic or wasp-venom-allergic donors were stimulated alone or in the presence of Treg with autologous mature monocyte-derived dendritic cells which were pulsed with different concentrations of the respective allergens. Treg from grass-pollen-allergic donors failed to inhibit proliferation but not cytokine production of CD4+ CD25- T cells at high antigen doses while Treg from non-atopic donors did not fail at these allergen concentrations. Proliferative responses and cytokine production of CD4+ CD25- T cells from most of the examined wasp-venom-allergic patients were not inhibited at any concentration of wasp venom. The use of wasp venom- or phospholipase A2-pulsed dendritic cells for stimulation of CD4+ CD25- T cells from donors who were not allergic to wasp stings only resulted in an inhibited proliferation and Th2 cytokine production by Treg at 10-fold lower than the optimal concentration, while interferon-gamma production was inhibited at all concentrations investigated. These data demonstrate that in allergic diseases the function of Treg is dependent on the concentration and the type of the respective allergen with different thresholds for individual allergens and patients.
Assuntos
Alérgenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Hipersensibilidade Imediata/imunologia , Receptores de Interleucina-2/análise , Subpopulações de Linfócitos T/imunologia , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Citocinas/biossíntese , Relação Dose-Resposta Imunológica , Fatores de Transcrição Forkhead/metabolismo , Proteína Relacionada a TNFR Induzida por Glucocorticoide , Humanos , Poaceae/imunologia , Pólen/imunologia , Receptores de Fator de Crescimento Neural/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Células Th1/imunologia , Células Th2/imunologia , Venenos de Vespas/imunologiaRESUMO
We analysed eight monoclonal antibodies (mAbs) directed against the Mip (macrophage infectivity potentiator) protein, a virulence factor of the intracellular pathogen Legionella pneumophila. Mip belongs to the FK506-binding proteins (FKBPs) and exhibits peptidyl prolyl cis/trans isomerase (PPIase) activity. Five of the mAbs recognised epitopes in the C-terminal, FKBP-homologous domain of Mip, which is highly conserved among all Legionella species. Upon immunological binding to Mip, all but one of these mAbs caused inhibition of the PPIase activity in vitro. mAb binding to the N-terminal domain of Mip did not influence its enzymatic activity. All but one of the PPIase inhibiting mAbs were able to significantly inhibit the early establishment and initiation of an intracellular infection of the bacteria in Acanthamoeba castellanii, the natural host, and in the human phagocytic cell line U937. These data demonstrate for the first time that for the virulence-enhancing property of the L. pneumophila Mip protein, an intact active site of the enzyme is an essential requirement.