Fibrates downregulate apolipoprotein C-III expression independent of induction of peroxisomal acyl coenzyme A oxidase. A potential mechanism for the hypolipidemic action of fibrates.

Epidemiological and transgenic animal studies have implicated apo C-III as a major determinant of plasma triglyceride metabolism. Since fibrates are very efficient in lowering triglycerides, it was investigated whether fibrates regulate apo C-III gene expression. Different fibrates lowered rat liver apo C-III mRNA levels up to 90% in a dose- and time-dependent manner, whereas intestinal apo C-III mRNA remained constant. This decrease in liver apo C-III mRNA was rapid (1 d) and reversible, since it was restored to control levels within 1 wk after cessation of treatment. In addition, fenofibrate treatment abolished the developmental rise of hepatic apo C-III mRNA observed during the suckling-weaning period. Administration of fibrates to rats induced liver and intestinal expression of the acyl CoA oxidase gene, the rate-limiting enzyme for peroxisomal beta-oxidation of fatty acids. In primary cultures of rat and human hepatocytes, fenofibric acid lowered apo C-III mRNA in a time- and dose-dependent manner. This reduction in apo C-III mRNA levels was accompanied by a decreased secretion of apo C-III in the culture medium of human hepatocytes. In rat hepatocytes fenofibric acid induced acyl CoA oxidase gene expression, whereas acyl CoA oxidase mRNA remained unchanged in human hepatocytes. Nuclear run-on and transient transfection experiments of a reporter construct driven by the human apo C-III gene promoter indicated that fibrates downregulate apo C-III gene expression at the transcriptional level. In conclusion, these studies demonstrate that fibrates decrease rat and human liver apo C-III gene expression. In humans the mechanisms appears to be independent of the induction of peroxisomal enzymes. This downregulation of liver apo C-III gene expression by fibrates may contribute to the hypotriglyceridemic action of these drugs.

Thiazolidinediones repress ob gene expression in rodents via activation of peroxisome proliferator-activated receptor gamma.

The ob gene product, leptin, is a signaling factor regulating body weight and energy balance. ob gene expression in rodents is increased in obesity and is regulated by feeding patterns and hormones, such as insulin and glucocorticoids. In humans with gross obesity, ob mRNA levels are higher, but other modulators of human ob expression are unknown. In view of the importance of peroxisome proliferator-activated receptor gamma (PPARgamma) in adipocyte differentiation, we analyzed whether ob gene expression is subject to regulation by factors activating PPARs. Treatment of rats with the PPARalpha activator fenofibrate did not change adipose tissue and body weight and had no significant effect on ob mRNA levels. However, administration of the thiazolidinedione BRL49653, a PPARgamma ligand, increased food intake and adipose tissue weight while reducing ob mRNA levels in rats in a dose-dependent manner. The inhibitory action of the thiazolidinedione BRL49653 on ob mRNA levels was also observed in vitro. Thiazolidinediones reduced the expression of the human ob promoter in primary adipocytes, however, in undifferentiated 3T3-L1 preadipocytes lacking endogenous PPARgamma, cotransfection of PPARgamma was required to observe the decrease. In conclusion, these data suggest that PPARgamma activators reduce ob mRNA levels through an effect of PPARgamma on the ob promoter.

Circadian and glucocorticoid regulation of Rev-erbalpha expression in liver.

Rev-erbalpha [NR1D1], a member of the nuclear receptor superfamily, is an orphan receptor that constitutively represses gene transcription. Rev-erbalpha has been shown to play a role in myocyte differentiation and to be induced during adipogenesis. Furthermore, Rev-erbalpha is a regulator of lipoprotein metabolism. It was recently shown that Rev-erbalpha messenger RNA (mRNA) levels oscillate diurnally in rat liver. Here, we report that the circadian rhythm of Rev-erbalpha in liver is maintained in primary cultures of rat hepatocytes. Because glucocorticoids have been shown to regulate other transcription factors with circadian expression, it was furthermore examined whether hepatic Rev-erbalpha expression is also regulated by glucocorticoids. Treatment of rats with dexamethasone resulted in a decrease of Rev-erbalpha mRNA levels by 70% after 6 h. Furthermore, dexamethasone decreased Rev-erbalpha expression in rat primary hepatocytes in a dose-dependent fashion. This effect was mediated by the glucocorticoid receptor because simultaneous addition of the glucocorticoid antagonist RU486 prevented the decrease in Rev-erbalpha mRNA levels by dexamethasone. Protein synthesis inhibition with cycloheximide markedly induced Rev-erbalpha mRNA levels; however, this induction was reduced by dexamethasone supplementation in both rat and human primary hepatocytes. Treatment with actinomycin D blocked the repression of Rev-erbalpha expression by dexamethasone in rat hepatocytes, suggesting that glucocorticoids regulate Rev-erbalpha expression at the transcriptional level. Transient transfection experiments further indicated that Rev-erbalpha promoter activity is repressed by dexamethasone in the presence of cotransfected glucocorticoid receptor. Taken together, these data demonstrate that Rev-erbalpha expression is under the control of both the circadian clock and glucocorticoids in the liver.

cDNA cloning and characterization of the transcriptional activities of the hamster peroxisome proliferator-activated receptor haPPAR gamma.

We have isolated a cDNA corresponding to the hamster peroxisome proliferator-activated receptor haPPAR gamma, a member of the steroid nuclear hormone receptor superfamily of transcription factors. haPPAR gamma mRNA is highly expressed in adipose tissue, and is expressed in lung, heart, kidney, liver and spleen to a lower extent. Thus, haPPAR gamma may function in activating the transcription of target genes in a variety of tissues, including those not particularly subjected to peroxisomal beta-oxidation. haPPAR gamma binds efficiently in the presence of retinoid X receptor alpha (RXR alpha) to a peroxisome proliferator response element (PPRE) first identified in the acyl-CoA oxidase (ACO) promoter, the rate-limiting enzyme of peroxisomal beta-oxidation. The gene (ACO) encoding this enzyme has been previously shown to be under the transcriptional control of mouse PPAR (mPPAR). Although binding of haPPAR gamma/RXR alpha on the PPRE of the ACO promoter in vitro is similar to that observed for mPPAR/RXR alpha, we show that the transcriptional activities of mPPAR and haPPAR gamma are regulated differently in vivo in response to peroxisome proliferators and heterodimerization with RXR.

Induction of ob gene expression by corticosteroids is accompanied by body weight loss and reduced food intake.

Genetic studies in mice have identified the ob gene product as a potential signaling factor regulating body weight homeostasis and energy balance. It is suggested that modulation of ob gene expression results in changes in body weight and food intake. Glucocorticoids are shown to have important metabolic effects and to modulate food intake and body weight. In order to test the hypothesis that these metabolic effects of glucocorticoids are linked to changes in the expression of the ob gene, ob mRNA levels were evaluated in rats treated with different glucocorticosteroids at catabolic doses and correlated to the kinetics of changes in body weight gain and food intake. Results from time course experiments demonstrate that adipose tissue ob gene expression is rapidly induced by glucocorticosteroids. This induction is followed by a concordant decrease in body weight gain and food consumption. These data suggest that the catabolic effects of corticosteroids on body weight mass and food intake might be mediated by changes in ob expression. Modulation of ob expression may therefore constitute a mechanism through which hormonal, pharmacological, or other factors control body weight homeostasis.

Fenofibrate and rosiglitazone lower serum triglycerides with opposing effects on body weight.

Activators of peroxisome proliferator activated receptors (PPARs) are effective drugs to improve the metabolic abnormalities linking hypertriglyceridemia to diabetes, hyperglycemia, insulin-resistance, and atherosclerosis. We compared the pharmacological profile of a PPARalpha activator, fenofibrate, and a PPARgamma activator, rosiglitazone, on serum parameters, target gene expression, and body weight gain in (fa/fa) fatty Zucker rats and db/db mice as well as their association in db/db mice. Fenofibrate faithfully modified the expression of PPARalpha responsive genes. Rosiglitazone increased adipose tissue aP2 mRNA in both models while increasing liver acyl CoA oxidase mRNA in db/db mice but not in fatty Zucker rats. Both drugs lowered serum triglycerides yet rosiglitazone markedly increased body weight gain while fenofibrate decreased body weight gain in fatty Zucker rats. KRP 297, which has been reported to be a PPARalpha and gamma co-activator, also affected serum triglycerides and insulin in fatty Zucker rats although no change in body weight gain was noted. These results serve to clearly differentiate the metabolic finality of two distinct classes of drugs, as well as their corresponding nuclear receptors, having similar effects on serum triglycerides.

Effects of pargyline and diethyldithiocarbamate in vivo treatment on aldehyde dehydrogenase activities of submitochondrial fractions.

1) Aldehyde dehydrogenase with a Km for acetaldehyde in the micromolar range (referred to as low Km-AldDH) is located in the matrix and that with a Km in the millimolar range (referred to as high Km-AldDH) is located in both the matrix and the outer membrane of mitochondria. 2) Pargyline in vivo treatment (100 mg/kg i.p.) caused a complete inhibition of the low Km-AldDH activity of the matrix fraction but caused only a 10% inhibition of the high Km-AldDH activities of the matrix and the outer membrane fractions. Diethyldithiocarbamate (DDC) treatment (700 mg/kg i.p.) caused less than 80% inhibition of the low Km-AldDH activity of the matrix fraction and caused a 20% inhibition of the high Km-AldDH activity of the matrix fraction. DDC treatment had no effect on the enzyme activity of the outer membrane fraction. The difference in the degree of inhibition of the low Km-AldDH activity of the matrix fraction by pargyline and DDC treatment may cause different blood acetaldehyde levels during ethanol oxidation.

Regulation of ob gene expression in rodents and humans.

The discovery of the obese gene in the mouse and its conserved homologue in humans has led to important discoveries in energy metabolism. One of the chief findings was the fact that the expression of the leptin gene was regulated and that it, in turn, could regulate metabolism and behavior. Much of the literature has focused on the physiological role of leptin in driving processes as diverse as reproduction, starvation defence, feeding behavior or body weight, all dependent on expression levels of the ob gene. Here, we will describe our work, in which we have begun to elucidate the regulatory processes controlling obese gene expression.

Regulation of the peroxisome proliferator-activated receptor alpha gene by glucocorticoids.

This study demonstrates that the expression of the peroxisome proliferator-activated receptor alpha (PPAR alpha) is regulated by glucocorticoid hormones in hepatocytes. Hydrocortisone, dexamethasone, and triamcinolone stimulated PPAR alpha mRNA synthesis in a dose-dependent manner in primary rat hepatocyte cultures. This glucocorticoid stimulation was inhibited by RU 486, a specific glucocorticoid antagonist. Moreover, in contrast to glucocorticoid hormones, the mineralocorticoid aldosterone had only a weak effect, suggesting that the hormonal stimulation of PPAR alpha was mediated by the glucocorticoid receptor. The induction was not prevented by cycloheximide treatment of the hepatocytes, indicating that it was mediated by preexisting glucocorticoid receptor. Finally, the RNA synthesis inhibitor actinomycin D abolished the stimulatory effect of dexamethasone, and nuclear run-on analysis showed an increase of PPAR alpha transcripts after hormonal induction. Thus, the PPAR alpha gene is an early response gene of glucocorticoids that control its expression at the transcriptional level.

Genetic control of responsiveness of rat liver supernatant aldehyde dehydrogenase to phenobarbital and 3-methylcholanthrene.

The responsiveness of the hepatic supernatant NAD+-dependent aldehyde dehydrogenase with a high Km value (high Km-AldDH) to phenobarbital (PB) and 3-methylcholanthrene (3-MC) treatment was studied in male rats of three strains; Wistar, Long-Evans, and Sprague-Dawley. A remarkable strain difference in the response of the enzyme to PB or 3-MC was observed. In rats of the Wistar strain the enzyme activity remained unchanged ("non-responsive") in all rats after treatment with PB while it increased ("responsive") 5- to 19-fo-d in all rats after treatment with 3-MC. The enzyme activity increased 8- to 20-fold and 2- to 8-fold respectively after treatment with PB and 3-MC in all rats of the Long-Evans strain. In rats of the Sprague-Dawley strain the enzyme activity remained unchanged in half of all the rats treated with PB or 3-MC and increased 2- to 7-fold over the basal level in half of the treated rats. The non-responsive rats to PB were all responsive to 3-MC treatment while the responsive rats to PB were responsive in 65% and non-responsive in 35% to 3-MC treatment.

Differential regulation of peroxisome proliferator activated receptor gamma1 (PPARgamma1) and PPARgamma2 messenger RNA expression in the early stages of adipogenesis.

Adipocyte differentiation is driven by the expression and activation of three transcription factor families: the differentially expressed CAAT/enhancer binding proteins (C/EBPs) alpha, beta, and delta; the helix-loop-helix adipocyte differentiation and determination factor-1; and peroxisome proliferator activated receptor gamma (PPARgamma), expressed as two isoforms, PPARgamma1 and the adipocyte-specific PPARgamma2. Overexpression of PPARgamma can induce adipocyte differentiation; therefore, we analyzed the expression of the two PPARgamma isoforms during early stages of differentiation to determine whether one was preferentially induced as an early determining event. Surprisingly, in the first 24 h, a 3-6-fold increase of PPARgamma2 mRNA was observed, whereas PPARgamma1 mRNA remained unchanged. PPARgamma1 was induced 1 day later. Overexpression of C/EBPbeta has also been shown to induce adipocyte differentiation. A C/EBP site was identified only in the human PPARgamma2 promoter. Its deletion blunted the response of PPARgamma2 promoter to cotransfected C/EBPbeta or methylisobutylxanthine treatment. We hypothesize that PPARgamma2 initiates adipocyte differentiation.

Transcriptional induction of rat liver apolipoprotein A-I gene expression by glucocorticoids requires the glucocorticoid receptor and a labile cell-specific protein.

Treatment with glucocorticoids increases the concentration of plasma high-density lipoprotein (HDL), which is inversely correlated to the development of atherosclerosis. Previously, we demonstrated that repeated administration of glucocorticoids increases apolipoprotein (apo) A-I gene expression and decreases apoA-II gene expression in rat liver. In the present study, the mechanism of glucocorticoid action on hepatic apoA-I and apoA-II expression was studied. A single injection of rats with dexamethasone increased hepatic apoA-I mRNA levels within 6 h and further increases were observed after 12 h and 24 h. In contrast, liver apoA-II mRNA levels gradually decreased after dexamethasone treatment to less than 25% control levels after 24 h. In rat primary hepatocytes and McARH8994 hepatoma cells, addition of dexamethasone increased apoA-I mRNA levels in a time-dependent and dose-dependent manner, whereas apoA-II mRNA levels were unchanged. Simultaneous addition of the glucocorticoid antagonist RU486 prevented the increase in apoA-I mRNA levels after dexamethasone treatment, which suggests that the effects of dexamethasone are mediated through the glucocorticoid receptor. Inhibition of transcription by actinomycin D and nuclear-run-on experiments in McARH8994 cells and primary hepatocytes showed that dexamethasone induced apoA-I, but not apoA-II, gene transcription. Transient-transfection assays in McARH8994 cells with a chloramphenicol acetyl transferase vector driven by the rat-apoA-I-gene promoter demonstrated that the proximal apoA-I promoter could be induced by dexamethasone, and this effect could be abolished by simultaneous treatment with RU486. However, in COS-1 cells, apoA-I promoter transcription was not induced by dexamethasone or cotransfected glucocorticoid receptor. In addition, the induction of apoA-I gene transcription by dexamethasone was blocked by the protein-synthesis inhibitor cycloheximide, which suggests the presence of a labile protein involved in apoA-I gene activation by dexamethasone. In conclusion, our results demonstrate that dexamethasone regulates rat apoA-I, but not apoA-II, gene expression through direct action on the hepatocyte. The induction of apoA-I gene transcription by dexamethasone requires the glucocorticoid receptor and a labile cell-specific protein.

Transient increase in obese gene expression after food intake or insulin administration.

Obesity is a disorder of energy balance, indicating a chronic disequilibrium between energy intake and expenditure. Recently, the mouse ob gene, and subsequently its human and rat homologues, have been cloned. The ob gene product, leptin, is expressed exclusively in adipose tissue, and appears to be a signalling factor regulating body-weight homeostasis and energy balance. Because the level of ob gene expression might indicate the size of the adipose depot, we suggest that it is regulated by factors modulating adipose tissue size. Here we show that ob gene exhibits diurnal variation, increasing during the night, after rats start eating. This variation was linked to changes in food intake, as fasting prevented the cyclic variation and decreased ob messenger RNA. Furthermore, refeeding fasted rats restored ob mRNA within 4 hours to levels of fed animals. A single insulin injection in fasted animals increased ob mRNA to levels of fed controls. Experiments to control glucose and insulin independently in animals, and studies in primary adipocytes, showed that insulin regulates ob gene expression directly in rats, regardless of its glucose-lowering effects. Whereas the ob gene product, leptin, has been shown to reduce food intake and increase energy expenditure, our data demonstrate that ob gene expression is increased after food ingestion in rats, perhaps through a direct action of insulin on the adipocyte.

Expression of the peroxisome proliferator-activated receptor alpha gene is stimulated by stress and follows a diurnal rhythm.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that can be activated by fatty acids and peroxisome proliferators. The PPAR alpha subtype mediates the pleiotropic effects of these activators in liver and regulates several target genes involved in fatty acid catabolism. In primary hepatocytes cultured in vitro, the PPAR alpha gene is regulated at the transcriptional level by glucocorticoids. We investigated if this hormonal regulation also occurs in the whole animal in physiological situations leading to increased plasma corticosterone levels in rats. We show here that an immobilization stress is a potent and rapid stimulator of PPAR alpha expression in liver but not in hippocampus. The injection of the synthetic glucocorticoid dexamethasone into adult rats produces a similar increase in PPAR alpha expression in liver, whereas the administration of the antiglucocorticoid RU 486 inhibits the stress-dependent stimulation. We conclude that glucocorticoids are major mediators of the stress response. Consistent with this hormonal regulation, hepatic PPAR alpha mRNA and protein levels follow a diurnal rhythm, which parallels that of circulating corticosterone. To test the effects of variations in PPAR alpha expression on PPAR alpha target gene activity, high glucocorticoid-dependent PPAR alpha expression was mimicked in cultured primary hepatocytes. Under these conditions, hormonal stimulation of receptor expression synergizes with receptor activation by WY-14,643 to induce the expression of the PPAR alpha target gene acyl-CoA oxidase. Together, these results show that regulation of the PPAR alpha expression levels efficiently modulates PPAR activator signaling and thus may affect downstream metabolic pathways involved in lipid homeostasis.