Bezafibrate is a dual ligand for PPARalpha and PPARbeta: studies using null mice

Bezafibrate is a known activator of peroxisome proliferator-activated receptors (PPARs) that can activate both PPARalpha and PPARbeta. To determine the role(s) of these receptors in mediating the biological effects of this chemical, the effect of bezafibrate was examined in PPARalpha-null and PPARbeta-null mice. Wild-type, PPARalpha-null, or PPARbeta-null mice were fed either a control diet or one containing 0.5% bezafibrate for 10 days. Bezafibrate feeding caused a significant increase in liver weight in wild-type and PPARbeta-null mice compared to controls, while liver weight was unchanged in bezafibrate-fed PPARalpha-null mice. Gonadal adipose stores were significantly smaller in wild-type and PPARbeta-null mice fed bezafibrate than in controls, and this effect was not found in similarly fed PPARalpha-null mice. Analysis of liver, white adipose tissue, and intestinal mRNAs showed that bezafibrate caused similar changes of mRNAs encoding lipid metabolizing enzymes in wild-type and PPARbeta-null mice compared to controls. Interestingly, in PPARalpha-null mice, bezafibrate also induced several mRNAs previously thought to be solely controlled by PPARalpha, showing that the effects of this drug are not exclusively modulated by this PPAR isoform. Western blot analysis of liver protein was consistent with changes in mRNA expression showing that the alterations in mRNA expression correlate with protein expression in this tissue. Results from these studies demonstrate that the effect of bezafibrate is mediated in large part by PPARalpha, although some changes in gene expression are dependent on PPARbeta. In contrast to other PPARalpha ligands such as WY-14,643, induction of some target genes by bezafibrate can also be modulated in the absence of a functional PPARalpha.     

Constitutive regulation of cardiac fatty acid metabolism through peroxisome proliferator-activated receptor alpha associated with age-dependent cardiac toxicity

The peroxisome proliferator-activated receptor alpha (PPARalpha) is a member of the nuclear receptor superfamily and mediates the biological effects of peroxisome proliferators. To determine the physiological role of PPARalpha in cardiac fatty acid metabolism, we examined the regulation of expression of cardiac fatty acid-metabolizing proteins using PPARalpha-null mice. The capacity for constitutive myocardial beta-oxidation of the medium and long chain fatty acids, octanoic acid and palmitic acid, was markedly reduced in the PPARalpha-null mice as compared with the wild-type mice, indicating that mitochondrial fatty acid catabolism is impaired in the absence of PPARalpha. In contrast, constitutive beta-oxidation of the very long chain fatty acid, lignoceric acid, did not differ between the mice, suggesting that the constitutive expression of enzymes involved in peroxisomal beta-oxidation is independent of PPARalpha(.) Indeed, PPARalpha-null mice had normal levels of the peroxisomal beta-oxidation enzymes except the D-type bifunctional protein. At least seven mitochondrial fatty acid-metabolizing enzymes were expressed at much lower levels in the PPARalpha-null mice, whereas other fatty acid-metabolizing enzymes were present at similar or slightly lower levels in the PPARalpha-null, as compared with wild-type mice. Additionally, lower constitutive mRNA expression levels of fatty acid transporters were found in the PPARalpha-null mice, suggesting a role for PPARalpha in fatty acid transport and catabolism. Indeed, in fatty acid metabolism experiments in vivo, myocardial uptake of iodophenyl 9-methylpentadecanoic acid and its conversion to 3-methylnonanoic acid were reduced in the PPARalpha-null mice. Interestingly, a decreased ATP concentration after exposure to stress, abnormal cristae of the mitochondria, abnormal caveolae, and fibrosis were observed only in the myocardium of the PPARalpha-null mice. These cardiac abnormalities appeared to proceed in an age-dependent manner. Taken together, the results presented here indicate that PPARalpha controls constitutive fatty acid oxidation, thus establishing a role for the receptor in cardiac fatty acid homeostasis. Furthermore, altered expression of fatty acid-metabolizing proteins seems to lead to myocardial damage and fibrosis, as inflammation and abnormal cell growth control can cause these conditions.     

PPARalpha deficiency increases secretion and serum levels of apolipoprotein B-containing lipoproteins.

This study investigates the importance of peroxisome proliferator activated receptor alpha (PPARalpha) for serum apolipoprotein B (apoB) levels and hepatic secretion of apoB-containing lipoproteins. Total serum apoB and VLDL-apoB levels were higher in female PPARalpha-null mice compared with female wild-type mice, but no difference was seen in male mice. Furthermore, hepatic triglyceride secretion rate, determined in vivo after Triton WR1339 injection, was 2.4-fold higher in female PPARalpha-null mice compared with female wild-type mice, but no difference was observed in male mice. However, when fed a high fat diet, male PPARalpha-null mice displayed 2-fold higher serum levels of apoB and LDL cholesterol compared with male wild-type mice, but triglyceride levels were not affected. Hepatic LDL receptor protein levels were not influenced by PPARalpha deficiency, gender, or the fat diet. Hepatocyte cultures from female PPARalpha-null mice (cultured for 4 days in serum free medium) showed 2-fold higher total apoB secretion and increased secretion of apoB-48 VLDL, as well as 2.7-fold larger accumulation of VLDL-triglycerides in the medium compared with wild-type cultures. In conclusion, PPARalpha-deficient female mice, but not males, display high serum apoB associated with VLDL and increased hepatic triglyceride secretion. Moreover, male PPARalpha-null mice show increased susceptibility to high fat diet in terms of serum apoB levels.     

Fenofibrate improves lipid metabolism and obesity in ovariectomized LDL receptor-null mice

We investigated whether fenofibrate improves lipid metabolism and obesity in female ovariectomized (OVX) or sham-operated (SO) low density lipoprotein receptor-null (LDLR-null) mice. All mice fed a high-fat diet exhibited increases in serum triglycerides and cholesterol as well as in body weight and white adipose tissue (WAT) mass compared to mice fed a low fat control diet. However, fenofibrate prevented high-fat diet-induced increases in body weight and WAT mass in female OVX LDLR-null mice, but not in SO mice. In addition, administration of fenofibrate reduced serum lipids and hepatic apolipoprotein C-III mRNA while increasing the mRNA of acyl-CoA oxidase in both groups of mice, however, these effects were more pronounced in OVX LDLR-null mice. The results of this study provide first evidence that fenofibrate improves both lipid metabolism and obesity, in part through PPARalpha activation, in female OVX LDLR-null mice.     

Ginsenoside Rf, a component of ginseng, regulates lipoprotein metabolism through peroxisome proliferator-activated receptor alpha

We investigated whether ginseng regulates lipoprotein metabolism by altering peroxisome proliferator-activated receptor alpha (PPARalpha)-mediated pathways, using a PPARalpha-null mouse model. Administration of ginseng extract, ginsenosides, and ginsenoside Rf (Rf) to wild-type mice not only significantly increased basal levels of hepatic apolipoprotein (apo) A-I and C-III mRNA compared with wild-type controls, but also substantially reversed the reductions in mRNA levels of apo A-I and C-III expected following treatment with the potent PPARalpha ligand Wy14,643. In contrast, no effect was detected in the PPARalpha-null mice. Testing of eight main ginsenosides on PPARalpha reporter gene expression indicated that Rf was responsible for the effects of ginseng on lipoprotein metabolism. Furthermore, the inhibition of PPARalpha-dependent transactivation by Rf seems to occur at the level of DNA binding. These results demonstrate that ginseng component Rf regulates apo A-I and C-III mRNA and the actions of Rf on lipoprotein metabolism are mediated via interactions with PPARalpha.     

Peroxisome proliferator-activated receptor alpha is required for feedback regulation of highly unsaturated fatty acid synthesis

Delta6 desaturase (D6D), the rate-limiting enzyme for highly unsaturated fatty acid (HUFA) synthesis, is induced by essential fatty acid-deficient diets. Sterol regulatory element-binding protein-1c (SREBP-1c) in part mediates this induction. Paradoxically, D6D is also induced by ligands of peroxisome proliferator-activated receptor alpha (PPARalpha). Here, we report a novel physiological role of PPARalpha in the induction of genes specific for HUFA synthesis by essential fatty acid-deficient diets. D6D mRNA induction by essential fatty acid-deficient diets in wild-type mice was diminished in PPARalpha-null mice. This impaired D6D induction in PPARalpha-null mice was not attributable to feedback suppression by tissue HUFAs because PPARalpha-null mice had lower HUFAs in liver phospholipids than did wild-type mice. Furthermore, PPARalpha-responsive genes were induced in wild-type mice under essential fatty acid deficiency, suggesting the generation of endogenous PPARalpha ligand(s). Contrary to genes for HUFA synthesis, the induction of other lipogenic genes under essential fatty acid deficiency was higher in PPARalpha-null mice than in wild-type mice even though mature SREBP-1c protein did not differ between the genotypes. The expression of PPARgamma was markedly increased in PPARalpha-null mice and might have contributed to the induction of genes for de novo lipogenesis. Our study suggests that PPARalpha, together with SREBP-1c, senses HUFA status and confers pathway-specific induction of HUFA synthesis by essential fatty acid-deficient diets.     

Deficiency of PPARalpha disturbs the response of lipogenic flux and of lipogenic and cholesterogenic gene expression to dietary cholesterol in mouse white adipose tissue

PPARalpha-deficiency in mice fed a high-carbohydrate, low-cholesterol diet was associated with a decreased weight of epididymal adipose tissue and an increased concentration of adipose tissue cholesterol. Consumption of a high (2% w/w) cholesterol diet resulted in a further increase in the concentration of cholesterol and a further decrease in epididymal fat pad weight in PPARalpha-null mice, but had no effect in the wild-type. These reductions in fat pad weight were associated with an increase in hepatic triacylglycerol content, indicating that both PPARalpha-deficiency and cholesterol altered the distribution of triacylglycerol in the body. Adipose tissue de novo lipogenesis was increased in PPARalpha-null mice and was further enhanced when they were fed a cholesterol-rich diet; no such effect was observed in the wild-type mice. The increased lipogenesis in the chow-fed PPARalpha-null mice was accompanied paradoxically by lower mRNA expression of SREBP-1c and its target genes, acetyl-CoA carboxylase and fatty acid synthase. Consumption of a high-cholesterol diet increased the mRNA expression of these genes in the PPARalpha-deficient mice but not in the wild-type. De novo cholesterol synthesis was not detectable in the adipose tissue of either genotype despite a relatively high expression of the mRNA's encoding SREBP-2 and 3-hydroxy-3-methylglutaryl Coenzyme A reductase. The mRNA expression of these genes and of the LDL-receptor in adipose tissue of the PPARalpha-deficient mice was lower than that of the wild-type and was not downregulated by cholesterol feeding. The results suggest that PPARalpha plays a role in adipose tissue cholesterol and triacylglycerol homeostasis and prevents cholesterol-mediated changes in de novo lipogenesis.     

Pancreatic islet response to hyperglycemia is dependent on peroxisome proliferator-activated receptor alpha (PPARalpha)

This study tests the hypothesis that islet peroxisome proliferator-activated receptor alpha (PPARalpha) influences insulin secretion. Freshly isolated islets of normoglycemic PPARalpha-null mice display no major alteration of glucose-stimulated insulin release. However, after 24 h of culture in high glucose, PPARalpha-null islets exhibit elevated basal insulin secretion and fail to increase insulin mRNA. 24-h culture with palmitate replicates this phenotype in wild-type islets. The data suggest that PPARalpha is needed to ensure appropriate insulin secretory response in situation of short-term hyperglycemia, likely by maintaining islet lipid homeostasis. As such, islet PPARalpha could contribute to delay the progression of type 2 diabetes.     

Deletion of peroxisome proliferator-activated receptor-alpha induces an alteration of cardiac functions

The peroxisome proliferator-activated receptor-alpha (PPARalpha) plays a major role in the control of cardiac energy metabolism. The role of PPARalpha on cardiac functions was evaluated by using PPARalpha knockout (PPARalpha -/-) mice. Hemodynamic parameters by sphygmomanometric measurements show that deletion of PPARalpha did not affect systolic blood pressure and heart rate. Echocardiographic measurements demonstrated reduced systolic performance as shown by the decrease of left ventricular fractional shortening in PPARalpha -/- mice. Telemetric electrocardiography revealed neither atrio- nor intraventricular conduction defects in PPARalpha -/- mice. Also, heart rate, P-wave duration and amplitude, and QT interval were not affected. However, the amplitude of T wave from PPARalpha -/- mice was lower compared with wild-type (PPARalpha +/+) mice. When the myocardial function was measured by ex vivo Langendorff's heart preparation, basal and beta-adrenergic agonist-induced developed forces were significantly reduced in PPARalpha-null mice. In addition, Western blot analysis shows that the protein expression of beta1-adrenergic receptor is reduced in hearts from PPARalpha -/- mice. Histological analysis showed that hearts from PPARalpha -/- but not PPARalpha +/+ mice displayed myocardial fibrosis. These results suggest that PPARalpha-null mice have an alteration of cardiac contractile performance under basal and under stimulation of beta1-adrenergic receptors. These effects are associated with myocardial fibrosis. The data shed light on the role of PPARalpha in maintaining cardiac functions.     

Differential regulation of cytosolic and peroxisomal bile acid amidation by PPAR alpha activation favors the formation of unconjugated bile acids

In human liver, unconjugated bile acids can be formed by the action of bile acid-CoA thioesterases (BACTEs), whereas bile acid conjugation with taurine or glycine (amidation) is catalyzed by bile acid-CoA:amino acid N-acyltransferases (BACATs). Both pathways exist in peroxisomes and cytosol. Bile acid amidation facilitates biliary excretion, whereas the accumulation of unconjugated bile acids may become hepatotoxic. We hypothesized that the formation of unconjugated and conjugated bile acids from their common substrate bile acid-CoA thioesters by BACTE and BACAT is regulated via the peroxisome proliferator-activated receptor alpha (PPARalpha). Livers from wild-type and PPARalpha-null mice either untreated or treated with the PPARalpha activator WY-14,643 were analyzed for BACTE and BACAT expression. The total liver capacity of taurochenodeoxycholate and taurocholate formation was decreased in WY-14,643-treated wild-type mice by 60% and 40%, respectively, but not in PPARalpha-null mice. Suppression of the peroxisomal BACAT activity was responsible for the decrease in liver capacity, whereas cytosolic BACAT activity was essentially unchanged by the treatment. In both cytosol and peroxisomes, the BACTE activities and protein levels were upregulated 5- to 10-fold by the treatment. These effects caused by WY-14,643 treatment were abolished in PPARalpha-null mice. The results from this study suggest that an increased formation of unconjugated bile acids occurs during PPARalpha activation.     

Adaptive increase in pyruvate dehydrogenase kinase 4 during starvation is mediated by peroxisome proliferator-activated receptor alpha

Pyruvate dehydrogenase kinase isoform 4 (PDK4) is upregulated by starvation in many tissues of the body during starvation. This causes inactivation of the pyruvate dehydrogenase complex which blocks pyruvate oxidation and conserves lactate and alanine for gluconeogenesis. Enhanced PDK4 expression may be caused by the increase in free fatty acids that occurs during starvation. Free fatty acids can activate peroxisome proliferator-activated receptor alpha (PPARalpha), and activation of PPARalpha can promote PDK4 expression. This model is supported by the findings reported here that WY-14,643, a synthetic PPARalpha activator, increases PDK4 expression in wild-type mice but not in PPARalpha-null mice. Starvation likewise increases the expression of PDK4 in tissues of wild-type mice but not in tissues of PPARalpha-null mice. These findings document the functional importance of PPARalpha for PDK4 expression during starvation and suggest an important role for elevated free fatty acids in the induction.     

Induction of intestinal peptide transporter 1 expression during fasting is mediated via peroxisome proliferator-activated receptor alpha

We previously demonstrated that starvation markedly increased the amount of mRNA and protein levels of the intestinal H+/peptide cotransporter (PEPT1) in rats, leading to altered pharmacokinetics of the PEPT1 substrates. In the present study, the mechanism underlying this augmentation was investigated. We focused on peroxisome proliferator-activated receptor alpha (PPARalpha), which plays a pivotal role in the adaptive response to fasting in the liver and other tissues. In 48-h fasted rats, the expression level of PPARalpha mRNA in the small intestine markedly increased, accompanied by the elevation of serum free fatty acids, which are endogenous PPARalpha ligands. Oral administration of the synthetic PPARalpha ligand WY-14643 to fed rats increased the mRNA level of intestinal PEPT1. Furthermore, treatment of the human intestinal model, Caco-2 cells, with WY-14643 resulted in enhanced PEPT1 mRNA expression and uptake activity of glycylsarcosine. In the small intestine of PPARalpha-null mice, augmentation of PEPT1 mRNA during fasting was completely abolished. In the kidney, fasting did not induce PEPT1 expression in either PPARalpha-null or wild-type mice. Together, these results indicate that PPARalpha plays critical roles in fasting-induced intestinal PEPT1 expression. In addition to the well-established roles of PPARalpha, we propose a novel function of PPARalpha in the small intestine, that is, the regulation of nitrogen absorption through PEPT1 during fasting.     

Influence of peroxisome proliferator-activated receptor alpha on ubiquinone biosynthesis

The control of ubiquinone biosynthesis by peroxisome proliferators was investigated using peroxisome proliferator activated receptor alpha (PPARalpha)-null mice. Administration of 2-(diethylhexyl)phthalate to control mice resulted in elevated ubiquinone levels in the liver, while dolichol, dolichyl-P and cholesterol concentrations remained unchanged. In PPARalpha-null mice, the level of these lipids were similar to control levels and administration of the peroxisome proliferator did not increase the levels of ubiquinone. The increase in ubiquinone levels was the result of increased synthesis. Induction was most pronounced in liver, kidney and heart, which have relatively high levels of PPARalpha. When the tissue concentration of hydrogen peroxide was elevated by inhibition of catalase activity with aminotriazole, the amount of ubiquinone was not increased, suggesting that the induction of ubiquinone synthesis occured through a direct mechanism. The activities of branch-point enzymes FPP-synthase, squalene synthase, cis-prenyltransferase, trans-prenyltransferase and NPHB-transferase were substantially increased in control but not in PPARalpha-null mice after treatment with peroxisome proliferators. These data suggest that the induction of ubiquinone biosynthesis after administration of peroxisome proliferators is dependent on the PPARalpha through regulation of some of the mevalonate pathway enzymes.     

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.