Regulación de la expresión de caveolina-1 en macrófagos por agonistas PPAR / Regulation of caveolin-1 expression in macrophages by PPAR agonists

Introducción. La activación o la sobreexpresión del receptor activado por proliferadores peroxisómicos * (PPAR*) induce la expresión de caveolina-1 en diferentes tipos celulares. El objetivo de este estudio ha sido evaluar si los agonistas PPAR regulan la expresión de la caveolina-1 en macrófagos, y explorar los posibles mecanismos implicados. Material y métodos. Se diferenciaron monocitos THP-1 por exposición a PMA durante 24 h y, posteriormente, se trataron con rosiglitazona a diferentes dosis y durante distintos períodos. Los valores de ARNm se determinaron por la reacción de la transcriptasa inversa acoplada a la reacción en cadena de la polimerasa (RT-PCR), y la expresión de la proteína mediante western-blot. Resultados. El tratamiento con la rosiglitazona aumentó los valores de ARNm y de proteína de caveolina-1 de forma dependiente de la dosis y el tiempo en macrófagos THP-1. Esta inducción no se observó en presencia de inhibidores de la transcripción o de la síntesis de novo de proteínas. El incremento de la expresión de caveolina-1 producido por rosiglitazona no se relaciona con el estado de diferenciación celular y parece ser dependiente de la activación PPAR, ya que la presencia del antagonista PPAR GW9662 lo anuló por completo. Por último, se ha identificado un elemento de respuesta a proliferadores peroxisómicos (PPRE) funcional en el promotor del gen de la caveolina-1, que se activa por el tratamiento con rosiglitazona en macrófagos THP-1. Conclusiones. La rosiglitazona incrementa la expresión de caveolina-1 en macrófagos, a través de la activación de los PPAR y, probablemente, como consecuencia de la unión al PPRE identificado en la secuencia del promotor del gen de la caveolina-1 (AU)

Introduction. Peroxisome proliferator-activated receptor * (PPAR*) activation or overexpression induce caveolin-1 expression in several cell types. The aim of this study was to ascertain whether PPAR agonists could also regulate the caveolin-1 gene in macrophages, and to investigate the mechanisms involved. Materials and methods. PMA-treated THP-1 monocytes were incubated with rosiglitazone at different concentrations and for different periods of time. MRNA levels were determined by RT-PCR and protein expression by Western blot. Results. Our experiments demonstrated that rosiglitazone dose- and time-dependently increased caveolin-1 mRNA and protein in THP-1 macrophages. This induction was not observed in the presence of transcription inhibitors or de novo protein synthesis. We also showed that the increase in caveolin-1 elicited by rosiglitazone was not related to macrophage differentiation. This inductive effect seems to be dependent on PPAR activation, since the PPAR antagonist GW9662 abolished it. Finally, we identified a functional peroxisome proliferator response element (PPRE) in the caveolin-1 promoter, which was activated upon rosiglitazone treatment in THP-1 macrophages. Conclusions. PPAR activators, such as the PPAR* agonist rosiglitazone, increase caveolin-1 expression in macrophages. This effect appears to be mediated by PPAR activation, possibly by the binding of activated PPAR to the PPRE identified in the caveolin-1 promoter (AU)

Sexual dimorphism in lipid metabolic phenotype associated with old age in Sprague-Dawley rats.

PURPOSE: Aged male rats show a decrease in liver PPARalpha. We aimed to determine if the sexual dimorphism in lipid metabolism observed in the PPARalpha-/- mouse is also present in senescent rats. RESULTS: Eighteen-month old rats were obese and presented high plasma NEFA concentrations. Old male rats were more hypercholesterolemic and hyperleptinemic than females, presenting a higher content in hepatic triglycerides and cholesteryl esters, while 18-month old females were more hypertriglyceridemic than males. Although PPARalpha expression and binding activity was reduced in liver from old male and female rats, the mRNA for a PPARalpha target gene, such as CPT-I, was reduced in old males (-56%), while increased by 286% in old females. LXRalpha protein was increased, and its binding activity was decreased in livers of old males, while livers of old females showed an increase in DGAT1 (2.6-fold) and DGAT2 (4.9-fold) mRNA, with respect to 3-month old animals. The increases in DGAT1 and DGAT2 mRNAs matched in old females those of plasma (3.1-fold) and liver triglycerides (5.0-fold). CONCLUSIONS: These features disclose a marked sexual dimorphism in lipid metabolism associated to old age in rats that can be partially attributed not only to an age-related decrease in liver PPARalpha expression, but also to changes in other hepatic transcription factors and enzymes, such as liver X receptor alpha (LXRalpha) and diacylglycerol acyltransferases (DGAT).

Peroxisome proliferator-activated receptors and the control of inflammation.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors which form a subfamily of the nuclear receptor gene family. This subfamily consists of three isotypes, alpha (NR1C1), gamma (NR1C3), and beta/delta (NRC1C2) with a differential tissue distribution. PPARalpha is expressed primarily in tissues with a high level of fatty acid catabolism such as liver, brown fat, kidney, heart and skeletal muscle. PPARbeta is ubiquitously expressed, and PPARgamma has a restricted pattern of expression, mainly in white and brown adipose tissues, whereas other tissues such as skeletal muscle and heart contain limited amounts. Furthermore, PPARalpha and gamma isotypes are expressed in vascular cells including endothelial and smooth muscle cells and macrophages/foam cells. PPARs are activated by ligands, such as naturally occurring fatty acids, which are activators of all three PPAR isotypes. In addition to fatty acids, several synthetic compounds, such as fibrates and thiazolidinediones, bind and activate PPARalpha and PPARgamma, respectively. In order to be transcriptionally active, PPARs need to heterodimerize with the retinoid-X-receptor (RXR). Upon activation, PPAR-RXR heterodimers bind to DNA specific sequences called peroxisome proliferator-response elements (PPRE) and stimulate transcription of target genes. PPARs play a critical role in lipid and glucose homeostasis, but lately they have been implicated as regulators of inflammatory responses. The first evidence of the involvement of PPARs in the control of inflammation came from the PPARalpha null mice, which showed a prolonged inflammatory response. PPARalpha activation results in the repression of NF-kappaB signaling and inflammatory cytokine production in different cell-types. A role for PPARgamma in inflammation has also been reported in monocyte/macrophages, where ligands of this receptor inhibited the activation of macrophages and the production of inflammatory cytokines (TNFalpha, interleukin 6 and 1beta), although part of the anti-inflammatory effects of these ligands seems to be mediated by a mechanism not involving PPARgamma. All these findings suggest a role of PPARs in the control of the inflammatory response with potential therapeutic applications in inflammation-related diseases.

Walnut-enriched diet increases the association of LDL from hypercholesterolemic men with human HepG2 cells.

In a randomized, cross-over feeding trial involving 10 men with polygenic hypercholesterolemia, a control, Mediterranean-type cholesterol-lowering diet, and a diet of similar composition in which walnuts replaced approximately 35% of energy from unsaturated fat, were given for 6 weeks each. Compared with the control diet, the walnut diet reduced serum total and LDL cholesterol by 4.2% (P = 0.176), and 6.0% (P = 0.087), respectively. No changes were observed in HDL cholesterol, triglycerides, and apolipoprotein A-I levels or in the relative proportion of protein, triglycerides, phospholipids, and cholesteryl esters in LDL particles. The apolipoprotein B level declined in parallel with LDL cholesterol (6.0% reduction). Whole LDL, particularly the triglyceride fraction, was enriched in polyunsaturated fatty acids from walnuts (linoleic and alpha-linolenic acids). In comparison with LDL obtained during the control diet, LDL obtained during the walnut diet showed a 50% increase in association rates to the LDL receptor in human hepatoma HepG2 cells. LDL uptake by HepG2 cells was correlated with alpha-linolenic acid content of the triglyceride plus cholesteryl ester fractions of LDL particles (r(2) = 0.42, P < 0.05). Changes in the quantity and quality of LDL lipid fatty acids after a walnut-enriched diet facilitate receptor-mediated LDL clearance and may contribute to the cholesterol-lowering effect of walnut consumption.

Increase in hepatic expression of SREBP-2 by gemfibrozil administration to rats.

It is well known that gemfibrozil increases the biliary output of cholesterol and phospholipids, but we have little knowledge about the impact these changes have on liver cholesterol and phospholipid biosynthetic pathways. In the present study, no changes were detected in liver lipids and CTP:phosphocholine cytidylyltransferase after gemfibrozil administration to rats. On the contrary, 3-hydroxy-3-methylglutaryl-CoA reductase mRNA (9.9-fold) and Rd activity (16.7-fold) and phosphatidate phosphohydrolase activity (1.7-fold) increased, while plasma apo B-cholesterol (40%) and triglyceride (43%) levels decreased. As a part of a compensatory homeostatic response, we report for the first time that gemfibrozil administration to rats increased the hepatic sterol regulatory element binding protein-2 (SREBP-2) mRNA (2.9-fold) and mature protein (2.2-fold) levels. An early increase in the transcriptional activity of SREBP-2 elicited by gemfibrozil administration might be responsible for the observed changes in HMG-CoA reductase, phosphatidate phosphohydrolase, and SREBP-2 expression.

Bezafibrate reduces mRNA levels of adipocyte markers and increases fatty acid oxidation in primary culture of adipocytes.

The molecular mechanisms by which peroxisome proliferator-activated receptor (PPAR) activation by fibrates reduces fat deposition and improves insulin sensitivity are not completely understood. We report that exposure of a rat primary culture of adipocytes for 24 h to the PPAR activator bezafibrate increased the mRNA levels of crucial genes involved in peroxisomal and mitochondrial beta-oxidation. The mRNA levels of the peroxisomal beta-oxidation rate-limiting enzyme acyl-CoA oxidase and of the muscle-type carnitine palmitoyl transferase I (M-CPT-I), which determines the flux of mitochondrial beta-oxidation, increased by 1.6-fold (P < 0.02) and 4.5-fold (P = 0.001), respectively. These changes were accompanied by an increase in the transcript levels of the uncoupling protein-2 (UCP-2; 1.5-fold induction; P < 0.05) and UCP-3 (3.7-fold induction; P < 0.001), mitochondrial proteins that reduce ATP yield and may facilitate the oxidation of fatty acids. Furthermore, bezafibrate increased the mRNA levels of the fatty acid translocase (2-fold induction; P < 0.01), suggesting a higher fatty acid uptake into adipocytes. In agreement with these changes, bezafibrate caused a 1.9-fold induction (P < 0.02) in 9,10-[(3)H]palmitate oxidation. Moreover, bezafibrate reduced the mRNA expression of several adipocyte markers, including PPARgamma (30% reduction; P = 0.05), tumor necrosis factor-alpha (33% reduction; P < 0.05), and the ob gene (26% reduction). In contrast, adipocyte fatty acid binding protein mRNA levels increased (1.5-fold induction; P < 0.01), pointing to a mobilization of fatty acids to mitochondria and peroxisomes. The reduction of the adipocyte markers caused by bezafibrate was accompanied by an increase in the mRNA levels of the preadipocyte marker Pref-1 (1.6-fold induction; P < 0.01). Some of the changes observed in the primary culture of rat adipocytes also were studied in the epididymal white adipose tissue of bezafibrate-treated rats for 7 days. In vivo, M-CPT-I mRNA levels increased (4.5-fold induction; P = 0.001) in epididymal white adipose tissue of bezafibrate-treated rats. Similarly, fatty acid translocase (2.6-fold induction; P = 0.002) and Pref-1 (5.6-fold induction) mRNA levels increased, although differences in the latter were not significant because of huge individual variations. These results indicate that exposure of adipocytes to bezafibrate, independent of its hepatic effects, increases the degradation of fatty acids, reducing their availability to synthesize triglycerides. As a result, some degree of dedifferentiation of adipocytes to preadipocyte-like cells is achieved. These changes may be involved in the reduction in fat depots and in the improvement of insulin sensitivity observed after bezafibrate 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.

Bezafibrate induces acyl-CoA oxidase mRNA levels and fatty acid peroxisomal beta-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 beta-oxidation in white adipose tissue. Further, bezafibrate significantly reduced plasma triglyceride and leptin concentrations, without modifying the levels of PPARgamma 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.

Receptores activados por proliferadores peroxisómicos (PPAR), metabolismo energético y aterosclerosis / Peroxysomel proliferator actived receptors (PPARs), energy metabolism and atherosclerosis

Los receptores activados por proliferadores peroxisómicos (PPAR) son una familia de factores de transcripción que pertenecen a la superfamilia de los receptores esteroides y que incluye tres subtipos: PPAR*, PPARß/* y PPAR*. Estos receptores se unen a repeticiones directas hexaméricas en forma de heterodímeros con el receptor del retinoide X*. Los PPAR regulan la expresión de una amplia variedad de genes que codifican proteínas implicadas en el metabolismo lipídico, la homeostasis energética, la diferenciación celular y la formación de tumores. En esta revisión se describen las características, regulación y los genes diana de los PPAR y se destacan sus implicaciones fisiopatológicas sobre el metabolismo de los lípidos y la glucosa, la homeostasis energética, la aterosclerosis y la diferenciación celular (AU)

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 PPARalpha 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 PPARgamma, strongly decreased UCP-3 and UCP-2 mRNA levels. Another thiazolidinedione, ciglitazone, had the same effect, but to a lower extent, suggesting that PPARgamma 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 PPARalpha mRNA levels produced by this drug, linking the reduction in PPARalpha mRNA levels with the down-regulation of UCP mRNA in C2C12 myotubes after thiazolidinedione treatment.

Peroxisome proliferator-activated receptor alpha (PPARalpha) activators, bezafibrate and Wy-14,643, increase uncoupling protein-3 mRNA levels without modifying the mitochondrial membrane potential in primary culture of rat preadipocytes.

Uncoupling proteins (UCPs) are inner mitochondrial membrane transporters which act as pores for H(+) ions, dissipating the electrochemical gradient that develops during mitochondrial respiration at the expense of ATP synthesis. We have studied the effects of two fibrates, bezafibrate and Wy-14,643, on UCP-3 and UCP-2 mRNA levels in primary monolayer cultures of rat adipocytes and undifferentiated preadipocytes. Treatment with both PPARalpha activators for 24 h up-regulated UCP-3 mRNA levels. Thus, bezafibrate treatment resulted in an 8-fold induction in UCP-3 mRNA levels in preadipocytes compared with the 3.5-fold induction observed in adipocytes. Differences in the induction of UCP-3 between these cells correlated well with the higher expression of PPARalpha and RXRalpha mRNA values in preadipocytes compared to adipocytes. Wy-14,643 caused similar effects on UCP-3 mRNA expression. In contrast to UCP-3, UCP-2 mRNA levels were only slightly modified by bezafibrate in adipocytes. The induction in UCP-3 expression was not accompanied by changes in the mitochondrial membrane potential of rat primary preadipocytes after bezafibrate or Wy-14,643 treatment. Since it has been proposed that UCP-3 could be involved in the regulation of the use of fatty acids as fuel substrates, the UCP-3 induction achieved after bezafibrate and Wy-14, 643 treatment may indicate a higher oxidation of fatty acids, limiting their availability to be stored as triglycerides. This change may result in a reduced rate of conversion of preadipocytes to adipocytes, which directly affects fat depots.

Differences in the formation of PPARalpha-RXR/acoPPRE complexes between responsive and nonresponsive species upon fibrate administration.

Peroxisome proliferator-activated receptor-alpha (PPARalpha) is responsible for the hypolipidemic, peroxisome proliferation and carcinogenic effects of fibrates. Rats and mice are responsive, but guinea pigs and primates are resistant to the proliferative and carcinogenic effects of these drugs, but the hypolipidemic effect is still manifest. It is not yet clear whether humans should be considered unresponsive, and there is concern about the long-term safety of fibrates. We present molecular evidence for the reported resistance of human cells to peroxisome proliferation by describing a deficient interaction of nuclear extracts from human cells with an acyl-CoA oxidase (ACO)-peroxisome proliferator response element probe upon fibrate addition. Electrophoretic mobility shift assay analysis showed that ciprofibrate elicited a concentration-dependent increase in the binding of nuclear extracts from cells of rat (Morris) and human (HepG2) origin to an ACO-peroxisome proliferator response element probe, although in HepG2 cells the increase was of marginal statistical significance. In Morris cells, the increase was more marked than in HepG2 cells (4-fold versus 1.5-fold at 0.2 mM ciprofibrate), and maximal binding was achieved earlier in Morris (30 min) than in HepG2 cells (3 h). Morris cells responded to the addition of ciprofibrate by increasing the levels of ACO mRNA, whereas HepG2 did not. The ratio between PPARbeta/PPARalpha mRNAs was higher in HepG2 cells than in Morris cells (3.2 versus 1.9), pointing to an antagonizing effect of PPARbeta on PPARalpha activity. These results were obtained in untransfected cells expressing their own basal set of receptors. We also provide evidence of the translocation of PPARalpha from the cytosol to the nucleus upon activation by ciprofibrate.

Substituting walnuts for monounsaturated fat improves the serum lipid profile of hypercholesterolemic men and women. A randomized crossover trial.

BACKGROUND: It has been reported that walnuts reduce serum cholesterol levels in normal young men. OBJECTIVE: To assess the acceptability of walnuts and their effects on serum lipid levels and low-density lipoprotein (LDL) oxidizability in free-living hypercholesterolemic persons. DESIGN: Randomized, crossover feeding trial. SETTING: Lipid clinic at a university hospital. PATIENTS: 55 men and women (mean age, 56 years) with polygenic hypercholesterolemia. INTERVENTION: A cholesterol-lowering Mediterranean diet and a diet of similar energy and fat content in which walnuts replaced approximately 35% of the energy obtained from monounsaturated fat. Patients followed each diet for 6 weeks. MEASUREMENTS: Low-density lipoprotein fatty acids (to assess compliance), serum lipid levels, lipoprotein(a) levels, and LDL resistance to in vitro oxidative stress. RESULTS: 49 persons completed the trial. The walnut diet was well tolerated. Planned and observed diets were closely matched. Compared with the Mediterranean diet, the walnut diet produced mean changes of -4.1% in total cholesterol level, -5.9% in LDL cholesterol level, and -6.2% in lipoprotein(a) level. The mean differences in the changes in serum lipid levels were -0.28 mmol/L (95% CI, -0.43 to -0.12 mmol/L) (-10.8 mg/dL [-16.8 to -4.8 mg/dL]) (P<0.001) for total cholesterol level, -0.29 mmol/L (CI, -0.41 to -0.15 mmol/L) (-11.2 mg/dL [-16.3 to -6.1 mg/dL]) (P<0.001) for LDL cholesterol level, and -0.021 g/L (CI, -0.042 to -0.001 g/L) (P = 0.042) for lipoprotein(a) level. Lipid changes were similar in men and women except for lipoprotein(a) levels, which decreased only in men. Low-density lipoprotein particles were enriched with polyunsaturated fatty acids from walnuts, but their resistance to oxidation was preserved. CONCLUSION: Substituting walnuts for part of the mono-unsaturated fat in a cholesterol-lowering Mediterranean diet further reduced total and LDL cholesterol levels in men and women with hypercholesterolemia.

Etomoxir, sodium 2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate, up-regulates uncoupling protein-3 mRNA levels in primary culture of rat preadipocytes.

Uncoupling proteins (UCPs) are mitochondrial membrane proton transporters that uncouple respiration from oxidative phosphorylation by dissipating the proton gradient across the membrane. Treatment of primary culture of rat preadipocytes for 24 h with 40 microM etomoxir, an irreversible inhibitor of carnitine palmitoyltransferase I (CPT-I), up-regulated UCP-3 mRNA levels (3. 6-fold induction), whereas changes in UCP-2 mRNA levels were not significant. As a consequence of increased UCP-3 expression, a fall in the mitochondrial membrane potential was detected by flow cytometry. Etomoxir treatment modified neither L-CPT-I (liver-type) nor PPARalpha mRNA levels in preadipocytes. In contrast, mRNA expression of acyl-CoA oxidase (ACO), the rate-limiting enzyme of peroxisomal fatty acid beta-oxidation, whose transcription is controlled by PPARalpha, was significantly induced (1.3-fold induction, P = 0.015). These findings suggest that the effects of etomoxir were mediated by PPARalpha. Since it has been reported that the intracellular accumulation of lipids following the inhibition of CPT-I by etomoxir leads to a PPARalpha-mediated metabolic response that increases the expression of genes involved in alternate fatty acid oxidation pathways, these results seem to implicate UCP-3 in this protective metabolic response. It remains to be studied whether reductions in the expression of UCP-3 could compromise this response, giving rise to lipotoxic effects on cells.