PPARalpha Activation Research Articles

Thermogenic and metabolic antiobesity drugs: rationale and opportunities

Antiobesity drugs that target peripheral metabolism may avoid some of the problems that have been encountered with centrally acting anorectic drugs. Moreover, if they cause weight loss by increasing fat oxidation, they not only address a cause of obesity but also should promote loss of fat rather than lean tissue and improve insulin sensitivity. Weight loss may be slow but more sustained than with anorectic drugs, and thermogenesis may be insufficient to cause any discomfort. Some thermogenic approaches are the activation of adrenergic, thyroid hormone or growth hormone receptors and the inhibition of glucocorticoid receptors; the modulation of transcription factors [e.g. peroxisome proliferator-activated receptor delta (PPARdelta) activators] or enzymes [e.g. glutamine fructose-6-phosphate amidotransferase (GFAT) inhibitors] that promote mitochondrial biogenesis, and the modulation of transcription factors (PPAR alpha activators) or enzymes (AMP-activated protein kinase) that promote fatty acid oxidation. More surprisingly, studies on genetically modified animals and with enzyme inhibitors suggest that inhibitors of fatty acid synthesis [e.g. ATP citrate lyase, fatty acid synthase, acetyl-CoA carboxylase (ACC)], fatty acid interconversion [stearoyl-CoA desaturase (SCD)] and triglyceride synthesis (e.g. acyl-CoA : diacylglycerol acyltransferase) may all be thermogenic. Some targets have been validated only by deleting genes in the whole animal. In these cases, it is possible that deletion of the protein in the brain is responsible for the effect on adiposity, and therefore a centrally penetrant drug would be required. Moreover, whilst a genetically modified mouse may display resistance to obesity in response to a high fat diet, it requires a tool compound to demonstrate that a drug might actually cause weight loss. Even then, it is possible that differences between rodents and humans, such as the greater thermogenic capacity of rodents, may give a misleading impression of the potential of a drug.

Differential Activation of Nuclear Receptors by Perfluorinated Fatty Acid Analogs and Natural Fatty Acids: A Comparison of Human, Mouse, and Rat Peroxisome Proliferator-Activated Receptor-α, -β, and -γ, Liver X Receptor-β, and Retinoid X Receptor-α

Administration of ammonium salts of perfluorooctanoate (PFOA) to rats results in peroxisome proliferation and benign liver tumors, events associated with activation of the nuclear receptor (NR) peroxisome proliferator-activated receptor-alpha (PPARalpha). Due to its fatty acid structure, PFOA may activate other NRs, such as PPARbeta, PPARgamma, liver X receptor (LXR), or retinoid X receptor (RXR). In this study, the activation of human, mouse, and rat PPARalpha, PPARbeta, PPARgamma, LXRbeta, and RXRalpha by PFOA (including its linear and branched isomers) and perfluorooctane sulfonate (PFOS) was investigated and compared to several structural classes of natural fatty acids and appropriate positive control ligands. An NR ligand-binding domain/Gal4 DNA-binding domain chimeric reporter system was used. Human, mouse, and rat PPARalpha were activated by PFOA isomers and PFOS. PPARbeta was less sensitive to the agents tested, with only PFOA affecting the mouse receptor. PFOA and PFOS also activated human, mouse, and rat PPARgamma, although the maximum induction of PPARgamma was much less than that seen with rosiglitazone, suggesting that PFOA and PFOS are partial agonists of this receptor. Neither LXRbeta nor the common heterodimerization partner RXRalpha was activated by PFOA in any species examined. Taken together, these data show that of the NRs studied, PPARalpha is the most likely target of PFOA and PFOS, although PPARgamma is also activated to some extent. Compared to naturally occurring long-chain fatty acids, e.g. linoleic and alpha-linolenic acids, these perfluorinated fatty acid analogs were more selective and less potent in their activation of the NRs.

Pharmacological Characterization of Hydrolysis-Resistant Analogs of Oleoylethanolamide with Potent Anorexiant Properties

Oleoylethanolamide (OEA) is an endogenous lipid mediator that reduces food intake, promotes lipolysis, and decreases body weight gain in rodents by activating peroxisome proliferator-activated receptor-alpha (PPAR-alpha). The biological effects of OEA are terminated by two intracellular lipid hydrolase enzymes, fatty-acid amide hydrolase and N-acylethanolamine-hydrolyzing acid amidase. In the present study, we describe OEA analogs that resist enzymatic hydrolysis, activate PPAR-alpha with high potency in vitro, and persistently reduce feeding when administered in vivo either parenterally or orally. The most potent of these compounds, (Z)-(R)-9-octadecenamide,N-(2-hydroxyethyl,1-methyl) (KDS-5104), stimulates transcriptional activity of PPAR-alpha with a half-maximal effective concentration (EC50) of 100 +/- 21 nM (n = 11). Parenteral administration of KDS-5104 in rats produces persistent dose-dependent prolongation of feeding latency and postmeal interval (half-maximal effective dose, ED50 = 2.4 +/- 1.8 mg kg(-1) i.p.; n = 18), as well as increased and protracted tissue exposure compared with OEA. Oral administration of the compound also results in a significant tissue exposure and reduction of food intake in free-feeding rats. These results suggest that the endogenous high-affinity PPAR-alpha agonist OEA may provide a scaffold for the discovery of novel orally active PPAR-alpha ligands.

Differential effects of contraction and PPAR agonists on the expression of fatty acid transporters in rat skeletal muscle

We have examined over the course of a 1-week period the independent and combined effects of chronically increased muscle contraction and the peroxisome proliferator-activated receptor (PPAR)alpha and PPARgamma activators, Wy 14,643 and rosiglitazone, on the expression and plasmalemmal content of the fatty acid transporters, FAT/CD36 and FABPpm, as well as on the rate of fatty acid transport. In resting muscle, the activation of either PPARalpha or PPARgamma failed to induce the protein expression of FAT/CD36. PPARalpha activation also failed to induce the protein expression of FABPpm. In contrast, PPARgamma activation induced the expression of FABPpm protein (40%; P < 0.05). Chronic muscle contraction increased the protein expression of FAT/CD36 (approximately 50%; P < 0.05), whereas FABPpm was slightly increased (12%; P < 0.05). Neither PPARalpha nor PPARgamma activation altered the contraction-induced expression of FAT/CD36 or FABPpm. Changes in protein expression of FAT/CD36 or FABPpm, induced by either contractions or by administration of rosiglitazone, were largely attributable to increased transcription. The contraction-induced increments in FAT/CD36 were accompanied by parallel increments in plasmalemmal FAT/CD36 and in rates of fatty acid transport (P < 0.05). Up-regulation of FABPpm expression was, however, accompanied by a reduction in plasmalemmal FABPpm, which did not affect the rates of long chain fatty acid (LCFA) transport. These studies have shown that in skeletal muscle (i) neither PPARalpha nor PPARgamma activation alters FAT/CD36 expression, (ii) PPARgamma activation selectively up-regulates FABPpm expression and (iii) contraction-induced up-regulation of LCFA transport does not appear to occur via activation of either PPARalpha or PPARgamma.

Activation of Peroxisome Proliferator–Activated Receptor Alpha Enhances Apoptosis in the Mouse Liver

Chronic exposure to peroxisome proliferators (PPs) leads to increased incidence of liver tumors in rodents. Liver tumor induction is thought to require increased hepatocyte proliferation and suppression of apoptosis. Transcript profiling showed increased expression of proapoptotic genes and decreased expression of antiapoptotic genes in the livers of mice exposed to the PP WY-14,643 (WY). We tested the hypothesis that prior exposure to WY would increase susceptibility to apoptosis inducers such as Jo2, an antibody which activates the Fas (Apo-1/CD95) death pathway. When compared to their untreated counterparts, wild-type mice pretreated with WY exhibited increased caspase-3 activation and hepatocyte apoptosis following challenge with Jo2. Livers from WY-treated peroxisome proliferator-activated receptor alpha (PPARalpha)-null mice were resistant to the effects of Jo2. In the absence of Jo2 and detectable apoptosis, wild-type mice treated with WY exhibited increases in the activated form of caspase-9. As caspase-9 is a component of the apoptosome, we examined the expression of upstream effectors of apoptosome activity including members of the Bcl-2 family. The levels of the antiapoptotic Mcl-1 transcript and protein were significantly decreased by PPs. PPARalpha-null mice were also resistant to another treatment (concanavalin A) that induces hepatocyte apoptosis. These results (1) indicate that PPARalpha activation increases sensitivity of the liver to apoptosis and (2) identify a mechanism by which PPARalpha could serve as a pharmacological target in diseases where apoptosis is a contributing feature.

PPARα agonist fenofibrate improves diabetic nephropathy in db/db mice

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a member of the ligand-activated nuclear receptor superfamily, and plays an important role in lipid metabolism and glucose homeostasis. The purpose of this study is to determine whether the activation of PPARalpha by fenofbrate would improve diabetes and its renal complications in type II diabetes mellitus. Male C57 BLKS db/db mice and db/m controls at 8 weeks of age were divided to receive either a regular diet chow (db/db, n=8; db/m, n=6) or a diet containing fenofibrate (db/db, n=8; db/m, n=7). Mice were followed for 8 weeks. Fenofibrate treatment dramatically reduced fasting blood glucose (P<0.001) and HbA1c levels (P<0.001), and was associated with decreased food intake (P<0.01) and slightly reduced body weight. Fenofibrate also ameliorated insulin resistance (P<0.001) and reduced plasma insulin levels (P<0.05) in db/db mice. Hypertrophy of pancreatic islets was decreased and insulin content markedly increased (P<0.05) in fenofibrate-treated diabetic animals. In addition, fenofibrate treatment significantly reduced urinary albumin excretion (P<0.001). This was accompanied by dramatically reduced glomerular hypertrophy and mesangial matrix expansion. Furthermore, the addition of fenofibrate to cultured mesangial cells, which possess functional active PPARalpha, decreased type I collagen production. Taken together, the PPARalpha agonist fenofibrate dramatically improves hyperglycemia, insulin resistance, albuminuria, and glomerular lesions in db/db mice. The activation of PPARalpha by fenofibrate in mesangial cells may partially contribute to its renal protection. Thus, fenofibrate may serve as a therapeutic agent for type II diabetes and diabetic nephropathy.

The role of PPAR‐alpha: in the regulation of blood pressure and renal function in salt‐loaded animals

PPAR-alpha, a member of the super family of nuclear receptors is highly expressed in several tissues including the vasculature and the kidney. Its role in the regulation of renal function and blood pressure (BP) is suggested but there are no definitive studies. However, in our previous study, PPAR-alpha activators increased nitric oxide (NO) production. In this study, we evaluated the contribution of PPAR-alpha to renal function and BP in PPAR-alpha null (KO) mice and wild type (WT) littermates placed on normal (NS; 0.3% NaCI) or high salt (HS; 8% NaCI) diet for 3 weeks. Systolic BP (plethysmography) was greater (124 mmHg) in WT mice placed on HS diet as did nitrite excretion (UNOxV), urinary volume (UV) and sodium excretion (UNaV). However, SBP, UNOxV, UNaV was similar in KO mice placed on NS or HS diet. In KO and WT mice placed on acute sodium load (2.5 ml/100g i.p), 5-hour UV and UNaV was similar (WT, 0.64 ml; KO, 0.50 ml). In amiloride-treated mice, UV and UNaV was greater in KO mice (0.26 ml vs 1.26 ml). These data suggest that PPAR-alpha contributes to the regulation of renal function affecting UNaV via amiloride-insensitive transport mechanisms.

Combined therapy with PPARα agonist and l-carnitine rescues lipotoxic cardiomyopathy due to systemic carnitine deficiency

Peroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily and are key regulators of fatty acid oxidation (FAO) in the heart. Systemic carnitine deficiency (SCD) causes disorders of FAO and induces hypertrophic cardiomyopathy with lipid accumulation. We hypothesized that activation of PPARalpha by fenofibrate, a PPARalpha agonist, in addition to conventional L-carnitine supplementation may exert beneficial effects on the lipotoxic cardiomyopathy in juvenile visceral steatosis (JVS) mouse, a murine model of SCD. Both wild-type (WT) and JVS mice were fed a normal chow, 0.2% fenofibrate containing chow (FE), a 0.1% L-carnitine containing chow (CA) or a 0.1% L-carnitine + 0.2% fenofibrate containing chow (CA + FE) from 4 weeks of age. Four to 8 animals per group were used for each experiment and 9 to 11 animals per group were used for survival analysis. At 8 weeks of age, JVS mice exhibited marked ventricular hypertrophy, which was more attenuated by CA + FE than by CA or FE alone. CA + FE markedly reduced the high plasma and myocardial triglyceride levels and increased the low myocardial ATP content to control levels in JVS mice. In JVS mice, myocardial 1,2-diacylglycerol (DAG) was significantly increased and showed a distinct fatty acid composition with elevation of 18:1(n-7,9) and 18:2(n-6) fatty acids compared with that in WT mice. CA + FE significantly altered the fatty acid composition of DAG and inhibited the membrane translocation of cardiac protein kinase C beta2 in JVS mice. Furthermore, CA + FE prevented the progressive left ventricular dysfunction and dramatically improved the survival rate in JVS mice (survival rate at 400 days after birth: 89 vs. 0%, P < 0.0001). PPARalpha activation, in addition to l-carnitine supplementation, may rescue the detrimental lipotoxic cardiomyopathy in SCD by improving cardiac energy and lipid metabolism as well as systemic lipid metabolism.

Peroxisome proliferator-activated receptor-α selective ligand reduces adiposity, improves insulin sensitivity and inhibits atherosclerosis in LDL receptor-deficient mice

Fenofibrate, a selective (1)PPAR-alpha activator, is prescribed to treat human dyslipidemia. The aim of this study was to delineate the mechanism of fenofibrate-mediated reductions in adiposity, improvements in insulin sensitivity, and lowering of triglycerides (TG) and free fatty acids (FFA) and to investigate if these favorable changes are related to the inhibition of lipid deposition in the aorta. To test this hypothesis we used male LDLr deficient mice that exhibit the clinical features of metabolic syndrome X when fed a high fat high cholesterol (HF) diet. LDLr deficient mice fed HF diet and simultaneously treated with fenofibrate (100 mg/kg body weight) prevented development of obesity, lowered serum triglycerides and cholesterol, improved insulin sensitivity, and prevented accumulation of lipids in the aorta. Lowering of circulating lipids occurred via down-regulation of lipogenic genes, including fatty acid synthase, acetyl CoA carboxylase and diacyl glycerol acyl transferase-2, concomitant with decreased liver TG and cholesterol, and TG output rate. Fenofibrate also suppressed liver apoCIII mRNA levels and markedly increased lipoprotein lipase mRNA levels, known to enhance serum TG catabolism. In addition, fenofibrate profoundly reduced epididymal fat and mesenteric fat mass to the levels seen in lean mice. The reductions in body weight were associated with elevation of hepatic uncoupling protein 2 (UCP2) mRNA, a concomitant increase in the ketone body formation, and improved insulin sensitivity associated with tumor necrosis factor-alpha reductions and phosphoenol pyruvate carboxykinase down-regulation. These results demonstrate that fenofibrate improves lipid abnormalities partly via inhibition of TG production and partly via clearance of TG-rich apoB particles by elevating LPL and reduced apoCIII. The prevention of obesity development occurred via energy expenditure. Fenofibrate-mediated hypolipidemic effects together with improved insulin sensitivity and loss of adiposity led to the reductions in the aortic lipid deposition by inhibiting early stages of atherosclerosis possibly via vascular cell adhesion molecule-1 (VCAM-1) modulation. These results suggest that potent PPAR-alpha activators may be useful in the treatment of syndrome X.

Induction of Hepatic Transporters Multidrug Resistance-Associated Proteins (Mrp) 3 and 4 by Clofibrate Is Regulated by Peroxisome Proliferator-Activated Receptor α

Hepatic transporters play a vital role in the disposition of endogenous compounds and xenobiotics in the liver. The current study investigates the expression and regulation of hepatic efflux transporters in response to treatment with the peroxisome proliferator-activated receptor (PPAR)alpha agonist clofibrate (CFB). Changes in mRNA and protein levels for several hepatic transporters were assessed in male CD-1 mice after 10 days of CFB dosing (500 mg/kg i.p.). Administration of CFB up-regulated mRNA levels for breast cancer resistance protein (Bcrp) and multidrug resistance-associated proteins 3 and 4 (Mrp3 and Mrp4, respectively). Western blot analysis confirmed that CFB enhances protein expression of liver Bcrp, Mrp3, and Mrp4 in CD-1 mice. To further characterize the regulation of these hepatic transporters, CFB-mediated changes in transporter mRNA levels were assessed in wild-type (sv/129) and PPARalpha-null male mice. Wild-type mice treated with CFB showed similar changes in mRNA levels for all of these transporters, whereas the PPARalpha-null mice did not. Although protein expression of Mrp3 and Mrp4 in the wild-type mice correlated well with changes in mRNA levels, Bcrp protein was not up-regulated by CFB treatment. These results show that PPARalpha activation by CFB coordinately regulates the hepatic efflux transporters Mrp3 and Mrp4. Induction of Mrp3 and Mrp4 by CFB may alter the disposition of toxicants and xenobiotics that are substrates for these transporters.

Basis for Improved Permeability Barrier Homeostasis Induced by PPAR and LXR Activators: Liposensors Stimulate Lipid Synthesis, Lamellar Body Secretion, and Post-Secretory Lipid Processing

Previously, we demonstrated that topical applications of peroxisome proliferator-activated receptors (PPARs) and liver X receptor (LXR) activators improve permeability barrier homeostasis. We showed further that stimulation of epidermal differentiation provides one mechanism that could account for such improvement. Here, we studied the effects of these agents on the lipid matrix of the stratum corneum. Hairless mice were treated topically with activators of PPARalpha (WY14643), PPARdelta (GW1514), PPARgamma (ciglitazone), and LXR (22(R)-cholesterol or TO901317) or vehicle twice daily for 3 days. All activators significantly increased epidermal cholesterol, fatty acid, and sphingolipid synthesis, including the production of barrier-specific ceramide species. In addition, lamellar body (LB) formation, secretion, and post-secretory processing accelerated significantly following acute barrier disruption in PPAR/LXR-activator-treated animals. Finally, the activity of epidermal beta-glucocerebrosidase, a key lipid-processing enzyme, increased in PPAR/LXR-activator-treated animals. Thus, topical PPAR and LXR activators stimulate epidermal lipid synthesis, increase LB secretion, and accelerate extracellular lipid processing, providing additional mechanisms that further account for their ability to improve epidermal permeability barrier homeostasis. Since the liposensors are activated by endogenous lipid metabolites, they may serve as unique regulators of barrier homeostasis.

(P)PARsing Epidermal Development

Overexpression of PPAR-alpha, a developmental transcription factor important in epidermal embryogenesis, in basal keratinocytes causes epidermal thinning when activated constitutively during development, but not if activated in adults; and lack of PPAR-alpha transiently delays stratum corneum formation within a window late in epidermal development (day 18.5 to birth). In contrast, pharmacologic activation of PPAR-alpha inhibits proliferation and induces differentiation in mouse epidermis regardless of developmental stage. Thus, PPAR-alpha is an important regulator of epidermal homeostasis.

Protection against myocardial ischaemia/reperfusion injury by PPAR–α activation is related to production of nitric oxide and endothelin–1

Ligands of peroxisome proliferator-activated receptor alpha (PPAR-alpha) have been shown to reduce ischaemia/reperfusion injury. The mechanisms behind this effect are not well known. We hypothesized that activation of PPAR-alpha exerts cardioprotection via a mechanism related to nitric oxide (NO) and endothelin-1 (ET-1). Five groups of anaesthetized open-chest Sprague-Dawley rats were given the PPAR-alpha agonist WY 14643 1 mg/kg (WY; n = 7), dimethyl sulfoxide (DMSO, vehicle for WY; n = 6), the combination of WY and the NO synthase inhibitor N-nitro-L-arginine (L-NNA, 2 mg/kg) (n = 7), L-NNA only (n = 8) or 0.9% sodium chloride (NaCl, vehicle for DMSO and L-NNA; n = 8) i.v. before a 30 min period of coronary artery occlusion followed by 2 h of reperfusion. Infarct size (IS), eNOS and iNOS protein and ET-1 mRNA expression were determined. There were no haemodynamic differences between the groups during the experiment. The IS was 78 +/- 3% of the area at risk in the DMSO group and 77 +/- 2% in the NaCl group (P = NS). WY reduced IS to 56 +/- 3% (P < 0.001 vs. DMSO group). When WY was administered in combination with L-NNA the cardioprotective effect was abolished (IS 73 +/- 3%, P < 0.01 vs. WY 14643). L-NNA did not affect IS per se (78 +/- 2%, P = NS). The expression of eNOS but not iNOS protein in ischaemic myocardium from rats was increased in the group given WY (P < 0.05). ET-1 mRNA levels were lower in the ischaemic myocardium following WY administration. The results suggest that the PPAR-alpha activation protects the rat myocardium against ischaemia/ reperfusion injury via a mechanism related to production of NO, and possibly ET-1.

Thioredoxin-mediated negative autoregulation of peroxisome proliferator-activated receptor alpha transcriptional activity.

PPARalpha, a member of the nuclear receptor superfamily, and thioredoxin, a critical redox-regulator in cells, were found to form a negative feedback loop, which autoregulates transcriptional activity of PPARalpha. Thioredoxin was identified as a target gene of PPARalpha. Activation of PPARalpha leads to increase of thioredoxin expression as well as its translocation from cytoplasm to nucleus, whereas ectopic overexpression of thioredoxin in the nucleus dramatically inhibited both constitutive and ligand-dependent PPARalpha activation. As PPARalpha-target genes, the expression of muscle carnitine palmitoyltransferase I, medium chain acyl CoA dehydrogenase, and apolipoprotein A-I were significantly down-regulated by nucleus-targeted thioredoxin at transcriptional or protein level. The suppression of PPARalpha transcriptional activity by Trx could be enhanced by overexpression of thioredoxin reductase or knockdown of thioredoxin-interacting protein, but abrogated by mutating the redox-active sites of thioredoxin. Mammalian one-hybrid assays showed that thioredoxin inhibited PPARalpha activity by modulating its AF-1 transactivation domain. It was also demonstrated by electrophoretic mobility-shift assay that thioredoxin inhibited the binding of PPARalpha to the PPAR-response element. Together, it is speculated that the reported negative-feedback loop may be essential for maintaining the homeostasis of PPARalpha activity.

Mode of action of fibrates in the regulation of triglyceride and HDL-cholesterol metabolism

Epidemiological studies have shown that hypertriglyceridemia and low HDL-cholesterol were both associated with an increased risk of coronary heart disease. Furthermore, hypertriglyceridemia is now recognized as an independent risk factor for coronary artery disease. Secondary prevention trials (e.g., LOCAT, BECAIT, BIP and DAIS) in coronary artery disease with drugs acting primarily on triglycerides (e.g., the PPAR-alpha activators bezafibrate, fenofibrate and gemfibrozil) have shown that reducing triglycerides and increasing HDL-cholesterol, without significantly affecting LDL-cholesterol, slows down coronary artery luminal narrowing. Furthermore, the VA-HIT study recently showed that gemfibrozil decreased coronary artery disease mortality in secondary prevention trials, partly by increasing HDL-cholesterol. The peroxisome proliferator-activated receptors (PPARs) (i.e., PPAR-alpha, -beta(delta) and -gamma) form a subfamily of the nuclear receptor gene family. Whereas all PPARs are, albeit to differing extents, activated by fatty acids and derivatives, PPAR-alpha binds the hypolipidemic fibrates. PPAR-alpha activation mediates changes in lipoprotein metabolism. Moreover, PPAR-alpha activators increase hepatic uptake and esterification of free fatty acids, in addition to increasing mitochondrial free fatty acid uptake and the resulting free fatty acid oxidation. The effect of fibrates on the metabolism of triglyceride-rich lipoproteins is due to a PPAR-alpha-dependent stimulation of lipoprotein lipase and of apolipoprotein (apo)A-V and to an inhibition of apoC-III expression, whereas the increase in plasma HDL-cholesterol depends partly on an overexpression of apoA-I and apoA-II. PPARs are also expressed in atherosclerotic lesions.

PPAR-alpha and insulin sensitivity

Peroxisome proliferator-activated receptors (PPAR) belong to the nuclear receptor superfamily of ligand-activated transcription factors. PPAR-alpha, first of its three subtypes (alpha, beta, gamma) has traditionally been considered an important regulator of lipid metabolism while its role in the regulation of insulin sensitivity has not been recognized until recently. Here we summarize the experimental and clinical studies focusing on the role of PPAR- alpha in the regulation of insulin sensitivity. In most of the experimental studies the activation of PPAR-alpha in rodents leads to improvement of insulin sensitivity by multiple mechanisms including improvement of insulin signaling due to a decrease of ectopic lipids in non-adipose tissues and decrease of circulating fatty acids and triglycerides. In contrast, the effect of PPAR-alpha agonist in humans is much less pronounced probably due to a lower expression of PPAR-alpha relative to rodents and possibly other mechanisms. Further clinical studies using more potent PPAR-alpha agonists on a larger population need to be performed to evaluate the possible role of PPAR- alpha in the regulation of insulin sensitivity in humans.

Differential susceptibility of mice humanized for peroxisome proliferator-activated receptor alpha to Wy-14,643-induced liver tumorigenesis.

Peroxisome proliferators, such as lipid-lowering fibrate drugs, are agonists for the peroxisome proliferator-activated receptor alpha (PPARalpha). Sustained activation of PPARalpha leads to the development of liver tumors in rodents. Paradoxically, humans appear to be resistant to the induction of peroxisome proliferation and development of liver tumors by peroxisome proliferators. To examine the species differences in response to peroxisome proliferators, a PPARalpha humanized mouse (hPPARalpha) was generated, in which the human PPARalpha was expressed in liver under control of the Tet-OFF system. To evaluate the susceptibility of hPPARalpha mice to peroxisome proliferator-induced hepatocarcinogenesis, a long-term feeding study of Wy-14,643 was carried out. hPPARalpha and wild-type (mPPARalpha) mice were fed either a control diet or one containing 0.1% Wy-14,643 for 44 and 38 weeks, respectively. Gene expression analysis for peroxisomal and mitochondrial fatty acid metabolizing enzymes revealed that both hPPARalpha and mPPARalpha were functional. However, the incidence of liver tumors including hepatocellular carcinoma was 71% in Wy-14,643-treated mPPARalpha mice, and 5% in Wy-14,643-treated hPPARalpha mice. Upregulation of cell cycle regulated genes such as cd1 and Cdks were observed in non-tumorous liver tissue of Wy-14,643-treated mPPARalpha mice, whereas p53 gene expression was increased only in the livers of Wy-14,643-treated hPPARalpha mice. These findings suggest that structural differences between human and mouse PPARalpha are responsible for the differential susceptibility to the peroxisome proliferator-induced hepatocarcinogenesis. This mouse model will be useful for human cancer risk assessment of PPARalpha ligands.

Phytocomponents of triterpenoids, oleanolic acid and ursolic acid, regulated differently the processing of epidermal keratinocytes via PPAR‐α pathway

Naturally occurring triterpenoids such as oleanolic acid (OA) and ursolic acid (UA) are known to have anti-inflammatory and anticarcinogenic activities in some types of cells. Although it has been reported that UA increases the amount of ceramide in keratinocytes, there is little study on the mechanism of triterpenoids involved in the differentiation of keratinocytes as well as their effects on epidermal permeability barrier. A study was therefore conducted to determine whether OA and UA could stimulate the differentiation of epidermal keratinocytes through peroxisome proliferator-activated receptor (PPAR)-alpha activation. This work was then extended to investigate the rate of formation of cornified envelope as a marker in the terminal differentiation of keratinocytes and the amount of transglutaminase in human keratinocytes treated with OA and UA. It was shown that OA induced the differentiation of keratinocytes, whereas UA had little effect. In addition, reporter gene assay using PPAR response element activity demonstrated that OA might be related to the increase of PPAR-alpha activity in CV-1 cells. Moreover, it enhanced the recovery of epidermal permeability barrier function as well as increased ceramides in epidermis after topical application. We therefore propose that the effect of OA on the stimulation of differentiation in epidermal keratinocytes seems to be highly related to activation of PPAR-alpha.

Peroxisome Proliferator-Activated Receptor ?? (PPAR??) and Atherosclerosis

The peroxisome proliferator-activated receptors (PPARs) alpha, beta/delta and gamma are ligand-activated transcription factors belonging to the nuclear receptor superfamily. PPARs heterodimerize with the retinoid X receptor (RXR) and modulate the function of many target genes. They were originally described as regulators of various metabolic, pathways, but have been recently found to also exert modulating actions in the vascular wall. PPARalpha is activated by endogenous ligands, such as polyunsaturated fatty acids and by synthetic agonists such as the fibrates. PPARalpha is expressed mainly in the liver, kidney and skeletal muscle and is involved in fatty acid oxidation. However, it is also expressed in vascular cells such as the endothelial cells, vascular smooth muscle cells and macrophages, where it exerts anti-inflammatory and antioxidant effects. Since atherosclerosis is both a chronic inflammatory and a lipid disorder and since PPARalpha is expressed in vascular cells and regulates the expression of genes involved in lipid metabolism and inflammation, PPARalpha activators may constitute useful agents for the prevention of atherosclerosis, beyond their effects on lipid metabolism. This review will focus on the functions of PPARalpha on lipid metabolism, on vascular inflammation and its relationship to atherosclerosis. Furthermore, the currently available preclinical and clinical data on PPARalpha activators as well as their future perspectives will be discussed.

Fibrate prevents cisplatin-induced proximal tubule cell death

In previous studies we have shown that cisplatin inhibits peroxisome proliferator-activated receptor-alpha (PPAR-alpha) activity and consequently fatty acid oxidation, and these events precede proximal tubule cell death. In addition the use of fibrate class of PPAR-alpha ligands ameliorate renal function by preventing both inhibition of fatty acid oxidation and proximal tubule cell death. LLC-PK1 cells were treated with cisplatin and apoptosis was established by the presence of nuclear fragmentation and by cell cycle analysis. Proximal tubular cells treated with cisplatin and bezafibrate were subjected to sub cellular fractionation and the presence of Bax, Bcl-2, cytochrome c, and active caspase-3 in the cytosolic and mitochondrial membrane fractions was determined by Western blot analysis. PPAR-alpha activity was measured by determining luciferase activity after transfection of LLC-PK1 cells with TK-Luc 3x PPAR response elements (PPRE), and the accumulation of nonesterified free fatty acids was measured in lysates obtained from cells treated with cisplatin and bezafibrate. Incubation of LLC-PK1 cells with 25 micromol/L cisplatin for 18 hours induced 41.5% apoptosis measured by cell cycle analysis. Cisplatin-induced apoptosis was significantly suppressed by bezafibrate, a fibrate class of PPAR-alpha ligand. Bezafibrate treatment of LLC-PK1 cells prevented cisplatin-induced translocation of proapoptotic Bax from the cytosol to the mitochondrial fraction, and increased the expression of antiapoptotic molecule Bcl-2. Cisplatin-induced inhibition of PPAR-alpha activity was accompanied by increased accumulation of nonesterified free fatty acids. Pretreatment with bezafibrate prevented both the inhibition of PPAR-alpha activity and the accumulation of nonesterified free fatty acids induced by cisplatin. Finally, bezafibrate prevented cisplatin-induced release of cytochrome c from the mitochondria to the cytosol, and the cleavage of procaspase-3 to active caspase-3. Bezafibrate treatment inhibits cisplatin-mediated tubular injury by preventing the activation of various cellular mechanisms that lead to proximal tubule cell death. These findings support our previous observations where the use of fibrates represents a novel strategy to ameliorate proximal tubule cell death in cisplatin-induced acute renal failure.