Peroxisome proliferator‐activated receptor‐δ as emerging target in liver disease

Liver fibrosis is characterized by an excessive deposition of extracellular matrix (ECM) proteins that occurs in chronic liver disease of any origin, including nonalcoholic steatohepatitis (NASH), alcohol abuse, and viral hepatitis. Cirrhosis occurs with the development of regenerating nodules of hepatocytes and is a major health burden worldwide. Patients with decompensated liver cirrhosis have a poor prognosis, with liver transplantation often being necessary. The current treatment paradigm for patients with hepatic fibrosis is to treat the underlying liver disease. However, if this cannot be achieved, there are currently no effective antifibrotic treatments for patients with chronic liver diseases. With the advent of basic molecular technology providing insight into the mechanisms of the development of hepatic fibrosis, there is now an opportunity to develop therapeutic interventions for human clinical use. In this review, the function of peroxisome proliferator‐activated receptor‐δ (PPAR δ) will be summarized with a special emphasis on ligand activation as potential use in liver disease. Drug Dev Res 2009. © 2009 Wiley‐Liss, Inc.     

Telmisartan attenuates peritoneal fibrosis via peroxisome proliferator‐activated receptor‐γ activation in rats

Peritoneal dialysis (PD) is an effective treatment for patients with end‐stage renal diseases, but long‐term continuous PD causes peritoneal fibrosis (PF). This study aims to evaluate the anti‐fibrotic effect of telmisartan on a rat model of PF and to investigate the underlying mechanisms. Five‐sixths kidney nephrectomy and PD were used to establish the PF rat model. Glucose (2.5%) was used to establish an in vitro model in rat peritoneal mesothelial cells (PMC). Haematoxylin–eosin staining was used to examine the structural alterations. Masson's trichrome staining was used to observe the tissue fibrosis in peritoneal membrane of rats. Real‐time polymerase chain reaction was used to measure messenger RNA expressions of profibrotic factors. Western blotting was used to determine protein expressions of profibrotic factors, peroxisome proliferator‐activated receptor‐γ, and mitogen‐activated protein kinases (MAPK). Results demonstrated that administration of telmisartan dose‐dependently attenuated the thickening of the peritoneal membrane and the fibrosis induced by long‐term PD fluid exposure in rats. In addition, telmisartan treatment inhibited the upregulation of profibrotic factors induced by PD in the peritoneum of rats and by high‐concentration glucose in PMC. Telmisartan was also effective in inhibiting PD and high‐concentration, glucose‐induced phosphorylation of MAPK in the peritoneum and PMC. Furthermore, peroxisome proliferator‐activated receptor‐γ (PPARγ) inhibitor GW9662 blocked these protective effects of telmisartan in PMC. The results suggest that telmisartan is effective in attenuating PD‐induced PF, and this effect may be associated with the inhibition of profibrotic factor expression and MAPK phosphorylation via PPARγ activation.     

The peroxisome proliferator‐activated receptor‐γ agonist pioglitazone protects against cisplatin‐induced renal damage in mice

Peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) agonists not only improve metabolic abnormalities of diabetes and consequent diabetic nephropathy, but they also protect against non‐diabetic kidney disease in experimental models. Here, we investigated the effect of PPAR‐γ agonist pioglitazone against acute renal injury on a cisplatin model in mice. Nephrotoxicity was induced by a single intraperitoneal (i.p.) injection of cisplatin (10 mg kg^–1). Pioglitazone was administered for six consecutive days in doses of 15 or 30 mg kg^–1 day^–1, per os (p.o.), starting 3 days before cisplatin injection. Cisplatin treatment to mice induced a marked renal failure, characterized by a significant increase in serum urea and creatinine levels and alterations in renal tissue architecture. Cisplatin exposure induced oxidative stress as indicated by decreased levels of non‐enzymatic antioxidant defenses [glutathione (GSH) and ascorbic acid levels] and components of the enzymatic antioxidant defenses [superoxide dismutase (SOD), catalase (CAT) glutathione peroxidase (GPx), glutathione reductase (GR) and and glutathione S‐transferase(GST) activities)] in renal tissue. Administration of pioglitazone markedly protected against the increase in urea and creatinine levels and histological alterations in kidney induced by cisplatin treatment. Pioglitazone administration ameliorated GSH and ascorbic acid levels decreased by cisplatin exposure in mice. Pioglitazone protected against the inhibition of CAT, SOD, GPx, GR and GST activities induced by cisplatin in the kidneys of mice. These results indicated that pioglitazone has a protective effect against cisplatin‐induced renal damage in mice. The protection is mediated by preventing the decline of antioxidant status. The results have implications in use of PPAR‐γ agonists in human application for protecting against drugs‐induced nephrotoxicity. Copyright © 2012 John Wiley & Sons, Ltd.     

The synthesis of three isotopomers of 2‐methyl‐2‐(4‐[3‐[1‐(4‐methylbenzyl)‐5‐oxo‐4,5‐dihydro‐1H‐[1,2,4]triazol‐3‐yl]propyl]phenoxy)propionic acid,a potent and selective peroxisome proliferator‐activated receptor alpha agonist

Although fenofibrate ( 1a ) is commercially available and clinically effective in lowering serum triglycerides, its activity and sub‐type selectivity at the PPARα receptors are only moderate; therefore, there exists a need for more potent and sub‐type selective PPARα agonists. To that end, discovery efforts have identified 2‐methyl‐2‐(4‐[3‐[1‐(4‐methylbenzyl)‐5‐oxo‐4,5‐dihydro‐1H‐[1,2,4]triazol‐3‐yl]propyl]phenoxy)propionic acid ( 2 ), a potent and selective human PPARα receptor agonist. In support of pre‐clinical ADME studies and bioanalysis, three isotopomers of 2 have been synthesized. The results of these efforts are described below. Copyright © 2007 John Wiley & Sons, Ltd.     

Review of the expression of peroxisome proliferator-activated receptors alpha (PPARα), beta (PPARβ), and gamma (PPARγ) in rodent and human development

The peroxisome proliferator-activated receptors (PPAR) belong to the nuclear hormone receptor superfamily and there are three primary subtypes, PPARα, β, and γ. These receptors regulate important physiological processes that impact lipid homeostasis, inflammation, adipogenesis, reproduction, wound healing, and carcinogenesis. These nuclear receptors have important roles in reproduction and development and their expression may influence the responses of an embryo exposed to PPAR agonists. PPARs are relevant to the study of the biological effects of the perfluorinated alkyl acids as these compounds, including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), activate PPARα. Exposure of the rodent to PFOA or PFOS during gestation results in neonatal deaths, developmental delay and growth deficits. Studies in PPARα knockout mice demonstrate that the developmental effects of PFOA, but not PFOS, depend on expression of PPARα. This review provides an overview of PPARα, β, and γ protein and mRNA expression during mouse, rat, and human development. The review presents the results from many published studies and the information is organized by organ system and collated to show patterns of expression at comparable developmental stages for human, mouse, and rat. The features of the PPAR nuclear receptor family are introduced and what is known or inferred about their roles in development is discussed relative to insights from genetically modified mice and studies in the adult.     

Leptin induces hypertrophy through activating the peroxisome proliferator‐activated receptor α pathway in cultured neonatal rat cardiomyocytes

1. Our previous study has shown that leptin induces cardiomyocyte hypertrophy; however, the mechanisms are poorly understood. Recent studies have shown that peroxisome proliferator‐activated receptor α (PPARα) activation might be responsible for pathological remodeling and severe cardiomyopathy. Leptin, as an endogenous activator of PPARα, regulates energy metabolism through activating PPARα in many cells. Therefore, we hypothesized that leptin induces cardiomyocyte hypertrophy through activating the cardiac PPARα pathway. 2. Cultured neonatal rat cardiomyocytes were used to evaluate the effects of PPARα on hypertrophy. The selective PPARα antagonist GW6471 concentration‐dependently decreased atrial natriuretic factor mRNA expression by 23%, 36%, 44% and 59%, and significantly decreased total RNA levels, protein synthesis and cell surface areas, all of which were elevated by 72 h of leptin treatment. The augmentation of reactive oxygen species levels in leptin treated cardiomyocytes was reversed by 0.1–10 μmol/L GW6471 (40%, 52% and 58%). After 24 h of treatment, leptin concentration‐dependently enhanced mRNA expression by 7%, 93%, 100% and 256%, and protein expression by 31.2%, 64.2%, 143% and 199%, and the activity of PPARα. Meanwhile, cardiomycytes receiving 72 h of treatment with the PPARα agonist, fenofibrate, concentration‐dependently increased total RNA levels, atrial natriuretic factor mRNA expression, protein synthesis and cell surface area. Treatment of fenofibrate for 4 h also elevated oxygen species levels in a concentration‐dependent manner. 3. In conclusion, these findings show that leptin induces hypertrophy through the activation of the PPARα pathway in cultured neonatal rat cardiomyocytes.     

Peroxisome proliferator‐activated receptor‐γ coactivator‐1α in muscle links metabolism to inflammation

1. In higher eukaryotes, metabolism and immunity are tightly coupled. However, whereas in evolutionary terms a compromised immune response due to undernourishment has been the predominant problem, the inflammatory response to obesity and other lifestyle‐associated diseases has increased in relevance in Western societies in the past 100 years. 2. Traditionally, fat tissue has been considered as the major source of pro‐inflammatory secreted factors in these pathologies. However, in recent years the contribution of other tissues to disease‐causing chronic inflammation has been increasingly appreciated. 3. Peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α) is one of the key regulatory factors in active skeletal muscle. Aberrant expression of PGC‐1α in inactive muscle fibres could be linked to a sedentary lifestyle, persistent systemic inflammation and a higher risk for many chronic diseases. Accordingly, modulation of PGC‐1α activity in skeletal muscle may have a broad range of therapeutic effects. Here, recent advances in the understanding of the role of muscle PGC‐1α in health and disease are reviewed.     

Relationship between peroxisome proliferator‐activated receptor alpha activity and cellular concentration of 14 perfluoroalkyl substances in HepG2 cells

Peroxisome proliferator‐activated receptor alpha (PPARα) is a molecular target for perfluoroalkyl substances (PFASs). Little is known about the cellular uptake of PFASs and how it affects the PPARα activity. We investigated the relationship between PPARα activity and cellular concentration in HepG2 cells of 14 PFASs, including perfluoroalkyl carboxylates (PFCAs), perfluoroalkyl sulfonates and perfluorooctane sulfonamide (FOSA). Cellular concentrations were determined by high‐performance liquid chromatography–tandem mass spectrometry and PPARα activity was determined in transiently transfected cells by reporter gene assay. Cellular uptake of the PFASs was low (0.04–4.1%) with absolute cellular concentrations in the range 4–2500 ng mg^?1 protein. Cellular concentration of PFCAs increased with perfluorocarbon chain length up to perfluorododecanoate. PPARα activity of PFCAs increased with chain length up to perfluorooctanoate. The maximum induction of PPARα activity was similar for short‐chain (perfluorobutanoate and perfluoropentanoate) and long‐chain PFCAs (perfluorododecanoate and perfluorotetradecanoate) (approximately twofold). However, PPARα activities were induced at lower cellular concentrations for the short‐chain homologs compared to the long‐chain homologs. Perfluorohexanoate, perfluoroheptanoate, perfluorooctanoate, perfluorononanoate (PFNA) and perfluorodecanoate induced PPARα activities >2.5‐fold compared to controls. The concentration–response relationships were positive for all the tested compounds, except perfluorooctane sulfonate PFOS and FOSA, and were compound‐specific, as demonstrated by differences in the estimated slopes. The relationships were steeper for PFCAs with chain lengths up to and including PFNA than for the other studied PFASs. To our knowledge, this is the first report establishing relationships between PPARα activity and cellular concentration of a broad range of PFASs.     

Microgram-order ammonium perfluorooctanoate may activate mouse peroxisome proliferator-activated receptor α, but not human PPARα

Perfluorooctanoic acid (PFOA) is a ligand for peroxisome proliferator-activated receptor (PPAR) α, which exhibits marked species differences in expression and function, especially between rodents and humans. We investigated the functional difference in PFOA response between mice and humans, using a humanized PPARα transgenic mouse line. Three genotyped mice, 129/Sv wild-type (mPPARα), Pparα-null mice and humanized PPARα (hPPARα) mice (8-week-old males) were divided into three groups: the first was treated with water daily for 2 weeks by gavage (control group), and the remaining two groups were treated with 0.1 and 0.3 mg/kg ammonium perflurooctanate (APFO), respectively, for 2 weeks by gavage. The APFO dosages used did not influence the plasma triglyceride or total cholesterol levels in any mouse line, but the high dose increased both hepatic lipid levels only in mPPARα mice. APFO increased mRNA and/or protein levels of PPARα target genes cytochrome P450 Cyp4a10, peroxisomal thiolase and bifunctional protein only in the liver of mPPARα mice, but not in Pparα-null or hPPARα mice. This chemical also increased expression of mitochondrial very long chain acyl-CoA dehydrogenase only in the liver of mPPARα mice. Taken together, human PPARα may be less responsive to PFOA than that of mice when a relatively low dose is applied. This information may be very valuable in considering whether PFOA influences the lipid metabolism in humans.     

Synthesis of tritium labelled L783483 ‐ a PPARδ ligand

The potent peroxisome proliferator‐activated receptor (PPAR) δ ligand L783483 (3‐chloro‐4‐(3‐(7‐propyl‐3‐trifluoromethyl‐benzisoxazol‐6‐oxy)propylsulfanyl)phenylacetic acid) has been labelled with tritium via selective tritium/bromine exchange of 5‐bromo‐6‐(3‐bromopropyloxy)‐7‐propyl‐3‐trifluoromethyl‐benzisoxazole. [³H]‐L783483 had a specific activity of 529 GBq/mmol (14.3 Ci/mmol) and a radiochemical purity of 98%. Copyright © 2003 John Wiley & Sons, Ltd.     

Peroxisome proliferator-activated receptor δ (PPARδ), a novel target site for drug discovery in metabolic syndrome

The development of new treatments for metabolic syndrome is urgent project for decreasing the prevalence of coronary heart disease and diabetes mellitus in the advanced countries. Peroxisome proliferator-activated receptor (PPAR)α and γ agonists have shed light on the treatment of hypertriglyceridemia and type 2 diabetes mellitus, respectively. Among PPARs, analysis of the PPARδ functions is lagging behind because specific PPARδ agonists have not been developed. The appearance of new PPARδ agonists is brightening the prospects for elucidating the physiological role of PPARδ. PPARδ is a new target for the treatment of metabolic syndrome. In particular, the fact that fatty acid oxidation and energy dissipation in skeletal muscle and adipose tissue by PPARδ agonists lead to improved lipid profile, reduced adiposity and insulin sensitivity is a breakthrough. It seems that treatment of PPARδ agonists operate similarly to the caloric restriction and prolonged exercise. We suggest that the physiological role of PPARδ may be an indicator for switching from glucose metabolism to fatty acid metabolism. To receive new benefits of PPARδ agonists against metabolic syndrome by increasing fatty acid consumption in skeletal muscle and adipose tissue, we need to unveil more details on the functions of PPARδ itself and its agonists in the future.     

Synthesis of tritium and carbon‐14 labelled NNC 61‐4655: a potent dual‐acting PPARα and PPARγ agonist

The synthesis of the potent dual‐acting PPARα and PPARγ agonist NNC 61‐4655 labelled with tritium and carbon‐14 is reported. Tritium labelled NNC 61‐4655 was obtained in three steps with introduction of tritium through catalytic tritium‐halogen exchange of an aryl bromide precursor. This provided [³H]NNC 61‐4655 in 39% overall radiochemical yield with a specific activity of 49 Ci/mmol. Carbon‐14 labelled NNC 61‐4655 was obtained in five steps starting from bromo[1‐¹⁴C]acetic acid. The synthetic sequence, which included a Horner–Wadsworth–Emmons olefination and a Mitsunobu alkylation, provided [¹⁴C]NNC 61‐4655 in 33% overall radiochemical yield with a specific activity of 57.4 mCi/mmol. Copyright © 2003 John Wiley & Sons, Ltd.     

[14C] and [3H]‐labelling of Ragaglitazar: A dual acting PPARα and PPARγ agonist with hypolipidemic and anti‐diabetic activity

Currently, Ragaglitazar is being developed as a drug for the treatment of hyperglycaemia and hyperlipidemia in patients with type 2 diabetes. Here, we report the labelling of Ragaglitazar with carbon‐14 and tritium for in vivo and in vitro investigations. Two different carbon‐14 labelled as well as two different tritium labelled tracers of Ragaglitazar were synthesised. The carbon‐14 label was introduced from either ethyl bromo[2‐¹⁴C]acetate (5 steps/33% overall yield) or [U‐¹⁴C]phenoxazine (4 steps/48% overall yield). Tritium was incorporated either by catalytic tritiation of an alkene precursor followed by chiral HPLC separation (2 steps/17% overall yield) or by catalytic tritium–halogen exchange of an aryl bromide precursor (2 steps/68% overall yield). Copyright © 2003 John Wiley & Sons, Ltd.     

Estrogen and ERα enhanced β‐catenin degradation and suppressed its downstream target genes to block the metastatic function of HA22T hepatocellular carcinoma cells via modulating GSK‐3β and β‐TrCP expression

In our previous experiments, we found β‐catenin was highly expressed in the tumor area with high invasive ability and poor prognosis. In this study, we have examined the mechanism by which ERα regulates β‐catenin expression as well as the metastasis ability of hepatocellular cancer HA22T cells. To identify whether the anticancer effect of estrogen and ERα is mediated through suppression of β‐catenin expression, we co‐transfected pCMV‐β‐catenin and ERα into HA22T cells, and determined the cell motility by wound healing, invasion, and migration assays. Results showed that estrogen and/or ERα inhibited β‐catenin gene expression and repressed HA22T cell motility demonstrated that similar data was observed in cells expressing the ERα stable clone. Moreover, we examined the protein‐protein interaction between ERα and β‐catenin by immunostain, co‐immunoprecipitation, and Western blotting. E2 enhanced the binding of ERα with β‐catenin and then triggered β‐catenin to bind with E3 ligase (βTrCP) to promote β‐catenin degradation. Finally by employing systematic ChIP studies, we showed ERα can interact directly with the β‐catenin promoter region following E2 treatment. All our results reveal that estrogen and ERα blocked metastatic function of HA22T cells by modulating GSK3β and βTrCP expression and further enhanced β‐catenin degradation and suppressed its downstream target genes. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 519–529, 2017.     

Deleterious effects of non‐synonymous single nucleotide variants of human IL‐1β gene

The IL‐1β gene is currently topic of interest for its important role in the pathogenesis of intervertebral disk degeneration. The new sequencing technology makes it crucial to study the effects of variants in IL‐1β. Thus, 714 IL‐1β variants with evidence supporting were collected from the EMBL database. Among them, 62 were non‐synonymous single nucleotide variants (nsSNVs). Furthermore, six common nsSNVs were predicted to have damaging effects by SIFT, PolyPhen, PROVEAN and SNPs&GO. Based on the constructed three‐dimensional structure of pro‐IL‐1β, rs375479974 with a mutation of Phe to Ser was proposed to reduce the stability of the pro‐IL‐1β protein. The rs375479974 variant was found to cause least common stabilizing amino acid residues, decrease hydrophilic and increase hydrophobic surface areas in the greatest degree, and have the lowest free energy alterations in I‐Mutant 2.0 sequence analysis. When analyzing the interaction between the experimental 3D structure of mature IL‐1β and its neutralizing McAb canakinumab complex, the rs775174784 substitution of Leu with Phe was found to attenuate this interaction by reducing binding energy, while rs375479974 not. Molecular dynamics simulation results in intervertebral disk environment supported rs775174784's effects. These results suggest that both rs375479974 and rs775174784 may have potential clinical and drug target implications.