Àgatha Cabrero

Impaired expression of NADH dehydrogenase subunit 1 and PPARγ coactivator-1 in skeletal muscle of ZDF rats restoration by troglitazone

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-α (PPARα) 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 pyruvatedehydrogenase 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 PPARγ 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.

Increased reactive oxygen species production down-regulates peroxisome proliferator-activated alpha pathway in C2C12 skeletal muscle cells.

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 α (PPARα) mRNA expression, achieving an 87% reduction at 80 μm etomoxir. The mRNA levels of most of the PPARα target genes studied were reduced at 100 μm etomoxir. By using several inhibitors of de novo ceramide synthesis and C2-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 PPARα 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-κB. In the presence of the pyrrolidine dithiocarbamate, a potent antioxidant and inhibitor of NF-κB activity, etomoxir did not down-regulate PPARα mRNA in C2C12 skeletal muscle cells. These results indicate that ROS generation and NF-κB activation are responsible for the down-regulation of PPARα and may provide a new mechanism by which intracellular lipid accumulation occurs in skeletal muscle cells.

Fibrate treatment does not modify the expression of acyl coenzyme A oxidase in human liver

Fibrates induce hepatic peroxisome proliferation and carcinogenesis in rodents by activating peroxisome proliferator‐activated receptor α (PPARα). There is no conclusive evidence that humans are unresponsive to peroxisome proliferation, and concern exists about the long‐term safety of fibrate treatment.

Down-regulation of acyl-CoA oxidase gene expression and increased NF-κB activity in etomoxir-induced cardiac hypertrophy

Activation of nuclear factor-κB (NF-κB) is required for hypertrophic growth of cardiomyocytes. Etomoxir is an irreversible inhibitor of carnitine palmitoyltransferase I (CPT-I) that activates peroxisome proliferator-activated receptor α (PPARα) 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 β-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-κB 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-κB activation.

Benzafibrate induces acyl-CoA oxidase mRNA levels and fatty acid peroxisomal β-oxidation in rat white adipose tissue

Rats treated with bezafibrate, a PPAR activator, gain less body weight and increase daily food intake. Previously, we have related these changes to a shift of thermogenesis from brown adipose tissue to white adipose tissue attributable to bezafibrate, which induces uncoupling proteins (UCP), UCP-1 and UCP-3, in rat white adipocytes. Nevertheless, UCP induction was weak, implying additional mechanisms in the change of energy homeostasis produced by bezafibrate. Here we show that bezafibrate, in addition to inducing UCPs, modifies energy homeostasis by directly inducing aco gene expression and peroxisomal fatty acid β-oxidation in white adipose tissue. Further, bezafibrate significantly reduced plasma triglyceride and leptin concentrations, without modifying the levels of PPARγ or ob gene in white adipose tissue. These results indicate that bezafibrate reduces the amount of fatty acids available for triglyceride synthesis in white adipose tissue.

Leptin down-regulates peroxisome proliferator-activated receptor γ (PPARγ) mRNA levels in primary human monocyte-derived macrophages

Increased leptin levels are associated with cardiovascular disease in obesity although the mechanism is unknown. Peroxisome proliferator-activated receptor γ (PPARγ) 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 PPARγ 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 PPARγ 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 PPARγ 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 PPARγ expression in both macrophages and foam cells may be one of the factors linking high leptin levels and cardiovascular disease.

Down-regulation of uncoupling protein-3 and -2 by thiazolidinediones in C2C12 myotubes.

Uncoupling proteins (UCPs) are mitochondrial membrane proton transporters that uncouple respiration from oxidative phosphorylation by dissipating the proton gradient across the membrane. We studied the direct effect of several peroxisome proliferator‐activated receptor (PPAR) ligands on UCP‐3 and UCP‐2 mRNA expression in C2C12 myotubes for 24 h. In the absence of exogenous fatty acids, treatment of C2C12 cells with a selective PPARα activator (Wy‐14,643) or a non‐selective PPAR activator (bezafibrate) did not affect the expression of UCP‐3 mRNA levels, whereas UCP‐2 expression was slightly increased. In contrast, troglitazone, a thiazolidinedione which selectively activates PPARγ, strongly decreased UCP‐3 and UCP‐2 mRNA levels. Another thiazolidinedione, ciglitazone, had the same effect, but to a lower extent, suggesting that PPARγ activation is involved. Further, the presence of 0.5 mM oleic acid strongly increased UCP‐3 mRNA levels and troglitazone addition failed to block the effect of this fatty acid. The drop in UCP expression after thiazolidinedione treatment correlated well with a reduction in PPARα mRNA levels produced by this drug, linking the reduction in PPARα mRNA levels with the down‐regulation of UCP mRNA in C2C12 myotubes after thiazolidinedione treatment.

Uncoupling protein-3 mRNA up-regulation in C2C12 myotubes after etomoxir treatment.

Uncoupling proteins (UCPs) are mitochondrial membrane proton transporters that uncouple respiration from oxidative phosphorylation by dissipating the proton gradient across the membrane. Treatment of C2C12 myotubes for 24 h with 40 microM etomoxir, an irreversible inhibitor of carnitine palmitoyltransferase I (CPT-I), up-regulated uncoupling protein 3 (UCP-3) mRNA levels (2-fold induction), whereas UCP-2 mRNA levels were not modified. Etomoxir treatment also caused a 2.5-fold induction in M-CPT-I (muscle-type CPT-I) mRNA levels. In contrast, other well-known peroxisome proliferator-activated receptor alpha (PPAR alpha) target genes, such as acyl-CoA oxidase and medium-chain acyl-CoA dehydrogenase, were not affected, suggesting that this transcription factor was not involved in the effects of etomoxir. Since it has been reported that CPT-I inhibition by etomoxir leads to a further increase in ceramide synthesis, we test the possibility that ceramides were involved in the changes reported. Similarly to etomoxir, addition of 20 microM C(2)-ceramide to C2C12 myotubes for 3, 6 and 9 h resulted in increased UCP-3 and M-CPT-I mRNA levels. These results indicate that the effects on UCP-3 mRNA levels could be mediated by increased ceramide synthesis.

Differential induction of stearoyl-CoA desaturase and acyl-CoA oxidase genes by fibrates in HepG2 cells

We studied whether two typical effects of fibrates, induction of stearoyl-CoA desaturase (EC 1.14.99.5) and peroxisome proliferation, are related. The effect of bezafibrate on the activity and mRNA of stearoyl-CoA desaturase and acyl-CoA oxidase in the liver and epididymal white adipose tissue of male Sprague-Dawley rats was determined. The same parameters were measured in HepG2 cells, a cell line resistant to peroxisome proliferation, following incubation with ciprofibrate. Bezafibrate increased the hepatic mRNA levels (14.5-fold on day 7) and activity (9.3-fold on day 15) of acyl-CoA oxidase. Stearoyl-CoA desaturase mRNA levels were transiently increased (2.7-fold on day 7), while its activity remained increased at the end of the treatment (2.4-fold). In white adipose tissue, bezafibrate increased the mRNA (5-fold) and activity (1.9-fold) of acyl-CoA oxidase, while stearoyl-CoA desaturase was not modified. Ciprofibrate addition to HepG2 cells cultured in 7% fetal bovine serum (FBS) only increased the stearoyl-CoA desaturase mRNA (1.9-fold). When cells were cultured in 0.5% FBS, ciprofibrate increased acyl-CoA oxidase mRNA (2.2-fold), while the increase in stearoyl-CoA desaturase mRNA was identical (1.9-fold). Further, its activity was also increased (1.5-fold). Incubation of HepG2 cells in the presence of cycloheximide did not alter the capacity of ciprofibrate to induce stearoyl-CoA desaturase mRNA, whereas the presence of actinomycin abolished the induction. In addition, preincubation of HepG2 cells with ciprofibrate increased the rate of stearoyl-CoA desaturase mRNA degradation. The results presented in this study suggest that fibrates induce stearoyl-CoA desaturase activity and mRNA levels independently of peroxisome proliferation.

Efecto de los agonistas PPAR sobre los valores de ARNm de genes implicados en el metabolismo lipídico de macrófagos humanos

Fundamento Los receptores activados por proliferadores peroxisomicos (peroxisome proliferator-activated receptors [PPAR]) desempenan un papel fundamental en el control del metabolismo lipidico de los macrofagos. El objetivo del presente estudio ha sido determinar los efectos de 3 activadores de PPAR (bezafibrato, fenofibrato y troglitazona) sobre las concentraciones de ARNm de genes implicados en el metabolismo lipidico de macrofagos humanos y celulas espumosas. Material y metodos Se han utilizado cultivos primarios de monocitos humanos separados por centrifugacion en gradiente de densidad a partir de buffy coats de donantes. Los monocitos asi obtenidos se cultivan en suero humano inactivado durante 10 dias para permitir su maduracion a macrofagos y, posteriormente, se convierten en celulas espumosas por exposicion a lipoproteinas de baja densidad acetiladas (150 ag/ml) durante 48 h. Los valores de ARNm se determinaron mediante reaccion en cadena de la polimerasa de la transcriptasa inversa (RT-PCR). Los resultados se expresan como la media ± desviacion estandar de 3 experimentos. Resultados Los macrofagos humanos tratados durante 24 h con bezafibrato 100 dM, un farmaco que activa los 3 subtipos de PPAR (a,///y y), mostraron un incremento en los valores de ARNm de cd36 y ap2 del 87% (p 0,01) y el 230%, respectivamente, mientras que las expresiones de PPAR, PPAR, acil- CoA oxidasa, carnitina palmitoiltransferasa I (CPT-I), ATP-binding cassette transporter 1 (abcd1), colesteril ester hidrolasa neutra y lectin-like oxidized low density receptor-1 (lox-1) no resultaron modificadas. Por el contrario, el tratamiento con agonistas selectivos pparpp(100 (M de fenofibrato) y mm(5 (M de troglitazona) provoco diferentes efectos. El fenofibrato incremento los valores de ARNm de PPARii(el 62%; p 0,05) y lox-1 (el 180%; p 0,05), mientras que la troglitazona aumento la expresion de CPT-I (el 75%; P 0,05). Cuando se estudio el efecto de estos farmacos en las celulas espumosas derivadas de macrofagos, se observo que la troglitazona incrementaba un 134% (P 0,05) y un 66% (P 0,01) los valores de ARNm de abca1 y CPT-I, respectivamente, mientras que los 3 farmacos estudiados incrementaron de forma significativa los valores de ap2 (aproximadamente, un 100%). Puesto que la troglitazona incrementaba la expresion de genes implicados en la e-oxidacion mitocondrial de acidos grasos (CPT-I), asi como en el transporte reverso de colesterol (abca1), se determino si estos cambios afectaban a la acumulacion intracelular de esteres de colesterol. En celulas espumosas derivadas de macrofagos se observo una reduccion (el 32%; P 0,01) en la acumulacion intracelular de colesterol tras el tratamiento con troglitazona, pero no tras el tratamiento con bezafibrato o fenofibrato. Conclusion Se necesitan estudios complementarios para establecer si la induccion en la expresion de CPT-I por la troglitazona reduce la disponibilidad de acidos grasos necesarios para la sintesis de esteres de colesterol y la formacion de las celulas espumosas.