RESUMEN
Increased leptin levels are associated with cardiovascular disease in obesity although the mechanism is unknown. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a key regulator of macrophage lipid metabolism and its activation by thiazolidinediones protects against atherosclerosis. The aim of this study was to assess the effects of human recombinant leptin on PPARgamma mRNA levels in primary human macrophages and macrophage-derived foam cells. Leptin treatment (100 ng/ml) for 24 h caused a 41% reduction (p < 0.01) in PPARgamma transcript levels in human-derived macrophages. This fall was accompanied by a reduction in the mRNA expression of carnitine palmitoyltransferase (CPT-I) (36%, p < 0.05) and ABCA1 (62%, p < 0.05), whereas CD36 mRNA reduction (34%) was not significant. In macrophage-derived foam cells, leptin at 20 ng/ml reduced PPARgamma mRNA levels by 33% (p < 0.01) and CPT-I by 27% (p < 0.05). At this concentration, leptin did not modify the expression of either ABCA1 or CD36. In agreement with these results, intracellular cholesterol ester accumulation was not altered in macrophage-derived foam cells by leptin at 20 ng/ml. We propose that the reduction in PPARgamma expression in both macrophages and foam cells may be one of the factors linking high leptin levels and cardiovascular disease.
Asunto(s)
Regulación hacia Abajo/efectos de los fármacos , Leptina/farmacología , Macrófagos/efectos de los fármacos , Monocitos/citología , PPAR gamma/metabolismo , ARN Mensajero/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Leptina/genética , Macrófagos/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
Type 2 diabetes has been related to a decrease of mitochondrial DNA (mtDNA) content. In this study, we show increased expression of the peroxisome proliferator-activated receptor-alpha (PPARalpha) and its target genes involved in fatty acid metabolism in skeletal muscle of Zucker Diabetic Fatty (ZDF) (fa/fa) rats. In contrast, the mRNA levels of genes involved in glucose transport and utilization (GLUT4 and phosphofructokinase) were decreased, whereas the expression of pyruvate dehydrogenase kinase 4 (PDK-4), which suppresses glucose oxidation, was increased. The shift from glucose to fatty acids as the source of energy in skeletal muscle of ZDF rats was accompanied by a reduction of subunit 1 of complex I (NADH dehydrogenase subunit 1, ND1) and subunit II of complex IV (cytochrome c oxidase II, COII), two genes of the electronic transport chain encoded by mtDNA. The transcript levels of PPARgamma Coactivator 1 (PGC-1) showed a significant reduction. Treatment with troglitazone (30 mg/kg/day) for 15 days reduced insulin values and reversed the increase in PDK-4 mRNA levels, suggesting improved insulin sensitivity. In addition, troglitazone treatment restored ND1 and PGC-1 expression in skeletal muscle. These results suggest that troglitazone may avoid mitochondrial metabolic derangement during the development of diabetes mellitus 2 in skeletal muscle.
Asunto(s)
Cromanos/farmacología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , NADH Deshidrogenasa/genética , Tiazolidinedionas/farmacología , Factores de Transcripción/genética , Animales , Glucemia/metabolismo , ADN Mitocondrial/genética , Regulación hacia Abajo/efectos de los fármacos , Canales Iónicos , Masculino , Proteínas de Transporte de Membrana/genética , Proteínas Mitocondriales/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Zucker , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Troglitazona , Proteína Desacopladora 2RESUMEN
Cardiac expression of genes involved in fatty acid metabolism may suffer alterations depending on the substrate availability. We studied how troglitazone, an antidiabetic drug that selectively activates peroxisome proliferator-activated receptor gamma (PPARgamma), affected the expression of several of these genes. A single-day troglitazone administration (100 mg/kg/day) did not significantly alter plasma free fatty acids or triglyceride levels. In contrast, a 10-day period of troglitazone treatment significantly reduced plasma free fatty acids and triglyceride levels by 74% (P < 0.001) and 56% (P < 0.01), respectively. Cardiac mRNA expression of acyl-CoA oxidase (ACO) increased (8.3-fold induction) after 1-day troglitazone treatment, whereas after 10 days of treatment ACO mRNA levels were dramatically reduced (98% reduction, P < 0.02), as well as those of uncoupling protein 3 (41% reduction, P = 0.05). The mRNA expression of PPARalpha and several PPAR target genes, such as medium chain acyl-CoA dehydrogenase or fatty acid translocase were not altered after 10 days of troglitazone treatment, whereas muscle-type carnitine palmitoyltransferase I increased 1.7-fold (P < 0.05). The reduction in ACO expression in the hearts of 10-day troglitazone-treated mice was accompanied by an increase in the protein levels of the transcriptional repressor chicken ovalbumin upstream promoter transcription factor II (COUP-TF II). Electrophoretic mobility shift assays performed with COUP-TF II antibody to examine its interaction with a labeled peroxisome proliferator response element probe showed enhanced binding of COUP-TFII in cardiac nuclear extracts from troglitazone-treated mice for 10 days but not in the control nuclear extracts. Overall, the findings presented here show that 10 days of troglitazone treatment decreased expression of the ACO gene through a mechanism involving the transcriptional repressor COUP-TF II.
Asunto(s)
Cromanos/farmacología , Proteínas de Unión al ADN/fisiología , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Miocardio/enzimología , Oxidorreductasas/biosíntesis , Oxidorreductasas/genética , Receptores de Esteroides , Proteínas Represoras/fisiología , Tiazoles/farmacología , Tiazolidinedionas , Factores de Transcripción/fisiología , Acil-CoA Oxidasa , Animales , Factor de Transcripción COUP II , Factores de Transcripción COUP , Pollos , Cromanos/administración & dosificación , Proteínas de Unión al ADN/genética , Represión Enzimática/efectos de los fármacos , Represión Enzimática/genética , Masculino , Ratones , Miocardio/metabolismo , Proteínas Represoras/genética , Tiazoles/administración & dosificación , Factores de Transcripción/genética , TroglitazonaRESUMEN
Peroxisome proliferator-activated receptors (PPARs) are key regulators of macrophage lipid metabolism. We compared the effects of three PPAR activators (bezafibrate, fenofibrate, and troglitazone) on the mRNA levels of genes involved in lipid metabolism in primary human macrophages and macrophage-derived foam cells. Treatment of human macrophages for 24 hours with 100 micro mol/L bezafibrate, a nonselective drug that activates the 3 PPAR subtypes (PPARalpha, PPARbeta/delta, and PPARgamma), caused an 87% (P <.01) and a 230% rise in CD36 and adipocyte fatty acid-binding protein (aP2) mRNA levels, respectively, whereas the expressions of PPARgamma, PPARalpha, acyl-CoA oxidase, carnitine palmitoyltransferase I (CPT-I), adenosine triphosphate (ATP)-binding cassette transporter 1 (ABCA1), neutral cholesteryl ester hydrolase, and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) were not modified. However, treatment with selective PPARalpha (fenofibrate at 100 micro mol/L) and PPARgamma (troglitazone at 5 micro mol/L) activators had different effects. Fenofibrate increased PPARalpha (62%, P <.05) and LOX-1 (180%, P <.05) mRNA levels; and troglitazone upregulated CPT-I expression (75%, P <.05). When the effects of these drugs were assessed in macrophage-derived foam cells, we found that troglitazone caused a 134% (P <.05) and a 66% (P <.01) rise in ABCA1 and CPT-I mRNA levels, respectively, whereas the 3 drugs significantly increased aP2 transcripts (about 100% induction). Given that troglitazone treatment resulted in the upregulation of genes involved in the mitochondrial beta-oxidation of fatty acids (CPT-I) and in the reverse-cholesterol-transport pathway (ABCA1), we subsequently determined whether these changes affected intracellular cholesterol ester accumulation. In macrophage-derived foam cells a significant reduction (32%, P <.01) was observed in intracellular cholesterol accumulation after troglitazone, but not after bezafibrate or fenofibrate treatment. Since CPT-I inhibition promotes cholesterol incorporation into cholesteryl esters in macrophages, study is now needed on whether CPT-I induction by troglitazone may reduce the availability of fatty acids for synthesizing cholesterol esters, leading to less foam cell formation.
Asunto(s)
Metabolismo de los Lípidos , Macrófagos/metabolismo , Monocitos/metabolismo , ARN Mensajero/biosíntesis , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Tiazolidinedionas , Factores de Transcripción/efectos de los fármacos , Bezafibrato/farmacología , Colesterol/sangre , Cromanos/farmacología , Fenofibrato/farmacología , Humanos , Hipoglucemiantes/farmacología , Hipolipemiantes/farmacología , Lipoproteínas LDL/sangre , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Monocitos/efectos de los fármacos , Monocitos/enzimología , Receptores Citoplasmáticos y Nucleares/metabolismo , Tiazoles/farmacología , Factores de Transcripción/metabolismo , TroglitazonaRESUMEN
The adipocyte-derived cytokine, resistin, has been proposed as the link between obesity and type 2 diabetes mellitus in murine models. In humans, resistin is identical to FIZZ3 (found in inflammatory zone 3), which belongs to a family of proteins that appears to be involved in inflammatory processes. To study the mechanisms by which fibrates improve glucose homeostasis, we determined resistin mRNA levels by using relative quantitative reverse-transcriptase-polymerase chain reaction (RT-PCR) in omental white adipose tissue samples obtained from patients treated with placebo or fenofibrate (200 mg/d) for 8 weeks before elective cholecystectomy. Fenofibrate treatment reduced total plasma cholesterol and low-density lipoprotein (LDL)-cholesterol levels by 24% and 35%, respectively. Compared with placebo values, a 2.4-fold induction in resistin mRNA levels was observed in white adipose tissue of fenofibrate-treated patients, whereas no changes were observed in the mRNA levels of the well-known perosixome proliferator-activated receptor (PPAR) target genes CD36, acyl-CoA oxidase, and carnitine palmitoyltransferase. These findings indicate that resistin changes were not related to PPAR activation by fenofibrate. Interestingly, resistin mRNA levels showed a negative correlation with plasma cholesterol levels (r2 =.53, P =.039, n = 8), but not with triglyceride levels (r2 =.02, P =.73, n = 8). These results suggest that cholesterol regulates resistin expression in human white adipose tissue.
Asunto(s)
Tejido Adiposo/metabolismo , Colesterol/sangre , Fenofibrato/uso terapéutico , Expresión Génica , Hormonas Ectópicas/genética , Hipolipemiantes/uso terapéutico , Péptidos y Proteínas de Señalización Intercelular , Acil-CoA Oxidasa , Anciano , Antígenos CD36/genética , Carnitina O-Palmitoiltransferasa/genética , Colecistectomía , HDL-Colesterol/sangre , LDL-Colesterol/sangre , Humanos , Persona de Mediana Edad , Oxidorreductasas/genética , Placebos , ARN Mensajero/análisis , Receptores Citoplasmáticos y Nucleares/metabolismo , Resistina , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/metabolismo , Triglicéridos/sangreRESUMEN
Activation of nuclear factor-kappaB (NF-kappaB) is required for hypertrophic growth of cardiomyocytes. Etomoxir is an irreversible inhibitor of carnitine palmitoyltransferase I (CPT-I) that activates peroxisome proliferator-activated receptor alpha (PPARalpha) and induces cardiac hypertrophy through an unknown mechanism. We studied the mRNA expression of genes involved in fatty acid oxidation in the heart of mice treated for 1 or 10 days with etomoxir (100 mg/kg/day). Etomoxir administration for 1 day significantly increased (4.4-fold induction) the mRNA expression of acyl-CoA oxidase (ACO), which catalyzes the rate-limiting step in peroxisomal beta-oxidation. In contrast, etomoxir treatment for 10 days dramatically decreased ACO mRNA levels by 96%. The reduction in ACO expression in the hearts of 10-day etomoxir-treated mice was accompanied by an increase in the mRNA expression of the antioxidant enzyme glutathione peroxidase and the cardiac marker of oxidative stress bax. Moreover, the activity of the redox-regulated transcription factor NF-kappaB was increased in heart after 10 days of etomoxir treatment. Overall, the findings here presented show that etomoxir treatment may induce cardiac hypertrophy via increased cellular oxidative stress and NF-kappaB activation.
Asunto(s)
Acil-CoA Oxidasa/genética , Acil-CoA Oxidasa/metabolismo , Cardiomegalia/metabolismo , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Compuestos Epoxi/farmacología , Corazón/efectos de los fármacos , FN-kappa B/metabolismo , Receptores de Esteroides , Animales , Glucemia/metabolismo , Factores de Transcripción COUP , Cardiomegalia/inducido químicamente , Cardiomegalia/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Masculino , Ratones , Miocardio/citología , Miocardio/enzimología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estrés Oxidativo , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Receptores X Retinoide , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND AND OBJECTIVES: Fibrates induce hepatic peroxisome proliferation and carcinogenesis in rodents by activating peroxisome proliferator-activated receptor alpha (PPAR(alpha)). There is no conclusive evidence that humans are unresponsive to peroxisome proliferation, and concern exists about the long-term safety of fibrate treatment. METHODS: In a university hospital setting, 48 patients with uncomplicated gallstones and a serum level of low-density lipoprotein cholesterol greater than 130 mg/dL were randomly assigned to open-label treatment with bezafibrate (400 mg/d), fenofibrate (200 mg/d), gemfibrozil (900 mg/d), or placebo for 8 weeks before elective cholecystectomy. Serum samples for lipid determinations were obtained at baseline and before surgery. A liver specimen was obtained at operation, and the relative levels of messenger ribonucleic acid (mRNA) for the wild and truncated forms of PPAR(alpha), acyl coenzyme A oxidase, liver carnitine palmitoyltransferase I, apolipoprotein A-I, and stearoyl coenzyme A desaturase were determined. RESULTS: Fenofibrate, bezafibrate, and gemfibrozil reduced plasma low-density lipoprotein cholesterol levels by 22% (P =.009), 14% (P =.042), and 11% (not significant), respectively. Plasma triglyceride levels decreased significantly (24%-36%; P <.05), whereas high-density lipoprotein cholesterol levels rose nonsignificantly after treatment with the 3 fibrates. Except for a 35% increase of apolipoprotein A-I mRNA after fenofibrate administration (P <.05), none of the individual fibrates induced significant changes in the mRNAs tested, although as a group they increased the mRNA for liver carnitine palmitoyltransferase I by 40%(P =.08; marginally significant). CONCLUSIONS: Fibrate administration to humans at pharmacologic doses able to activate PPAR(alpha) and to induce a hypolipidemic effect does not increase the hepatic expression of acyl coenzyme A oxidase, a well-known marker of peroxisome proliferation in rodents.
Asunto(s)
Hipolipemiantes/farmacología , Hígado/efectos de los fármacos , Oxidorreductasas/efectos de los fármacos , Acil-CoA Oxidasa , Apolipoproteína A-I/sangre , Apolipoproteína A-I/efectos de los fármacos , Apolipoproteínas B/sangre , Apolipoproteínas B/efectos de los fármacos , Bezafibrato/farmacología , Carnitina O-Palmitoiltransferasa/efectos de los fármacos , Colecistectomía , Colelitiasis/cirugía , HDL-Colesterol/sangre , HDL-Colesterol/efectos de los fármacos , LDL-Colesterol/sangre , LDL-Colesterol/efectos de los fármacos , Cartilla de ADN , Femenino , Fenofibrato/farmacología , Gemfibrozilo/farmacología , Humanos , Hígado/enzimología , Masculino , Persona de Mediana Edad , Oxidorreductasas/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Estearoil-CoA Desaturasa/efectos de los fármacos , Factores de Transcripción/efectos de los fármacos , Triglicéridos/sangreRESUMEN
Previous reports showed that recombinant fragments of adiponectin (adipo) displayed pharmacological effects when injected into rodents, but the relevance of these observations to the physiological function of adipo is unclear. We generated Adipo(-/-) mice by gene targeting. Adipo(-/-) mice are fertile with normal body and fat pad weights. Plasma glucose and insulin levels of Adipo(-/-) and Adipo(+/+) mice are similar under fasting conditions and during an intraperitoneal glucose tolerance test (GTT). Insulin tolerance test (ITT) also produces similar plasma glucose and insulin levels in the two groups of mice. Hyperinsulinemic-euglycemic clamp analysis showed that Adipo(-/-) and Adipo(+/+) mice have similar glucose infusion rates to maintain a similar serum glucose. High-fat diet feeding for 7 months led to similar weight gain and similar GTT and ITT responses. We next measured beta-oxidation and found it to be significantly increased in muscle and liver of Adipo(-/-) mice. In conclusion, our study indicates that absence of adipo causes increased beta-oxidation but does not cause glucose intolerance or insulin resistance in mice.
Asunto(s)
Prueba de Tolerancia a la Glucosa , Resistencia a la Insulina , Péptidos y Proteínas de Señalización Intercelular , Proteínas/fisiología , Adiponectina , Animales , Glucemia/análisis , Insulina/sangre , Ratones , Ratones Noqueados , Oxidación-Reducción , Proteínas/genéticaRESUMEN
Generation of reactive oxygen species may contribute to the pathogenesis of diseases involving intracellular lipid accumulation. To explore the mechanisms leading to these pathologies we tested the effects of etomoxir, an inhibitor of carnitine palmitoyltransferase I which contains a fatty acid-derived structure, in C2C12 skeletal muscle cells. Etomoxir treatment for 24 h resulted in a down-regulation of peroxisome proliferator-activated receptor alpha (PPARalpha) mRNA expression, achieving an 87% reduction at 80 microm etomoxir. The mRNA levels of most of the PPARalpha target genes studied were reduced at 100 microm etomoxir. By using several inhibitors of de novo ceramide synthesis and C(2)-ceramide we showed that they were not involved in the effects of etomoxir. Interestingly, the addition of triacsin C, a potent inhibitor of acyl-CoA synthetase, to etomoxir-treated C2C12 skeletal muscle cells did not prevent the down-regulation in PPARalpha mRNA levels, suggesting that the active form of the drug, etomoxir-CoA, was not involved. Given that saturated fatty acids may generate reactive oxygen species (ROS), we determined whether the addition of etomoxir resulted in ROS generation. Etomoxir increased ROS production and the activity of the well known redox transcription factor NF-kappaB. In the presence of the pyrrolidine dithiocarbamate, a potent antioxidant and inhibitor of NF-kappaB activity, etomoxir did not down-regulate PPARalpha mRNA in C2C12 skeletal muscle cells. These results indicate that ROS generation and NF-kappaB activation are responsible for the down-regulation of PPARalpha and may provide a new mechanism by which intracellular lipid accumulation occurs in skeletal muscle cells.