Lupeol alleviates autoimmune myocarditis by suppressing macrophage pyroptosis and polarization via PPARα/LACC1/NF-κB signaling pathway.

BACKGROUND: Autoimmune myocarditis, with increasing incidence and limited therapeutic strategies, is in urgent need to explore its underlying mechanisms and effective drugs. Pyroptosis is a programmed cell death that may contribute to the pathogenesis of myocarditis. Nonetheless, no direct evidence validated the role of pyroptosis in autoimmune myocarditis. Lupeol (Lup), a pentacyclic triterpene, possesses various biological activities such as antidiabetic properties. However, the effects of Lup on autoimmune myocarditis and pyroptosis remain unelucidated. PURPOSE: This study aimed to reveal the role of pyroptosis in autoimmune myocarditis and explore the protective effects of Lup, and its engaged mechanisms. METHODS: The experimental autoimmune myocarditis (EAM) mouse model was established by immunization with a fragment of cardiac myosin in Balb/c mice. Lup and MCC950 were administered after EAM induction. The protective effects were assessed by inflammation score, cardiac injury, chronic fibrosis, and cardiac function. Mechanistically, the effects of Lup on the M1 polarization and pyroptosis of macrophages were evaluated. Transcriptome sequencing and molecular docking were subsequently employed, and the underlying mechanisms of Lup were further explored in vitro with small interfering RNA and adenovirus. RESULTS: Administration of Lup and MCC950 alleviated EAM progression. Western blotting and immunofluorescence staining identified macrophages as the primary cells undergoing pyroptosis. Lup inhibited the expression of pyroptosis-associated proteins in macrophages during EAM in a dose-dependent manner. Furthermore, Lup suppressed pyroptosis in both bone marrow-derived macrophages (BMDMs) and THP-1-derived macrophages in vitro. In addition, Lup inhibited the M1 polarization of macrophages both in vivo and in vitro. Mechanistically, the protective effects of Lup were demonstrated via the suppression of the nuclear factor-κΒ (NF-κB) signaling pathway. Transcriptome sequencing and molecular docking revealed the potential involvement of peroxisome proliferator-associated receptor α (PPARα). Subsequently, we demonstrated that Lup activated PPARα to reduce the expression level of LACC1, thereby inhibiting the NF-κB pathway and pyroptosis. CONCLUSION: Our findings indicated the crucial role of macrophage pyroptosis in the pathogenesis of EAM. Lup ameliorated EAM by inhibiting the M1 polarization and pyroptosis of macrophages through the PPARα/LACC1/NF-κB signaling pathway. Thus, our results provided a novel therapeutic target and agent for myocarditis.

Oxidized corn oil changes the liver lipid metabolism of broilers by upregulating peroxisome proliferators activate receptor-α.

The objective of the following study was to investigate the effects of naturally oxidized corn oil on the antioxidant capacity and lipid metabolism of broilers. A total of 450, 1-day-old Arbor Acres male broilers were randomly divided into 5 treatments with 6 replicate cages and 15 birds/cage. The dietary treatment array consisted of ratios of naturally oxidized corn oil to non-oxidized corn oil from 0:100, 25:75, 50:50, 75:25, and 100:0, respectively. Serum, liver, and abdominal fat samples were taken at 42 d. The results showed that the liver organ index, liver catalase (CAT) activity, malondialdehyde (MDA) content, and the serum aspartate aminotransferase (AST) content had significant quadratic relationships with the ratio of naturally oxidized corn oil (P < 0.05). Inflammatory infiltrating cells appeared in the liver of the 50% and 75% oxidized corn oil group. The percentage of abdominal fat, and serum free fatty acids (FFA) content increased linearly with the increased proportion of oxidized corn oil (P < 0.05). The mRNA expression of NADH quinone oxidoreductase 1 (NQO-1), nuclear factor kappa B (NF-κB), toll-like receptor-4 (TLR-4), peroxisome proliferators activate receptor-α (PPARα), carnitine acyltransferase (CPT1), and acyl-coenzyme oxidase (ACO) of the liver increased linearly while oxidized corn oil increased in the diet (P < 0.05). Diets containing 100% oxidized corn oil significantly changed the mRNA expression of liver Caveolin compared with other treatment groups (P < 0.05). Taken together, this study demonstrated that naturally oxidized corn oil could change liver lipid metabolism and accelerate lipid deposition of broilers by upregulating PPARα.

[Sichuan Dark Tea-Based Medicated Dietary Formula Improves Obesity-Induced Renal Lipid Metabolism Disorder in Mice by Remodeling Gut Microbiota and Short-Chain Fatty Acid Metabolism]. / å››å·é»‘èŒ¶è¯è†³é æ–¹é€šè¿‡é‡å¡‘è‚ é“èŒç¾¤å’ŒçŸ­é“¾è„‚è‚ªé ¸ä»£è°¢æ”¹å–„è‚¥èƒ–å°é¼ è‚¾è„è„‚è´¨ç´Šä¹±.

Objective: To investigate the renoprotective effects of a Sichuan dark tea-based medicated dietary formula (alternatively referred to as Qing, or clarity in Chinese) on mice with diet-induced obesity (DIO) and to explore the specific mechanisms involved. Methods: Male C57BL/6 mice were randomly assigned to three groups, a control group, a DIO group, and a Qing treatment group, or the Qing group, with 8 mice in each group. The mice in the control group were given normal maintenance feed and purified water, and the other two groups were fed a high-fat diet for 12 weeks to establish the DIO model. After that, high-fat diet continued in the DIO group, while the Qing group was given Qing at the same time for 12 weeks, during which period the weight of the mice was monitored and recorded every week. The mice were sacrificed after 12 weeks. Serum samples were collected and the levels of triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin were measured to evaluate liver function. In addition, renal lipids were extracted to determine the levels of TG and TC in the kidney and periodic acid-Schiff (PAS) and oil red O stainings were performed to evaluate kidney pathological injury. Western blot was performed to determine the phosphorylated AMPK (pAMPK)/AMPK ratio in the kidney tissue. RT-qPCR and Western blot were used to determine the expression of proteins related to fatty acid oxidation, including acetyl-CoA carboxylase 1 (ACC1), carnitine acyltransferase 1 (CTP1), peroxisome proliferators-activated receptor γ (PPARγ), peroxisome proliferators-activated receptor-1 α (PPAR1α), sterol-regulatory element binding proteins (SREBP-1), and key proteins related to lipid synthesis, including fatty acid synthase (FASN) and stearoyl-coenzyme A desaturase 1 (stearoyl-CoA desaturase) in the kidney tissue. 16SrRNA and metabolomics were applied to analyze the gut microbiota in the intestinal contents and its metabolites. Results: Compared with those of the control group, the levels of liver mass (P=0.0003), serum ALT (P<0.0001) and AST (P=0.0001), and kidney TC (P=0.0191) and TG (P=0.0101) of the DIO group were significantly increased and there was lipid deposition in the kidney. Compared with those of the DIO group, mice in the Qing group showed effective reduction in liver mass (P=0.0316) and improvements in the abnormal serum levels of AST (P=0.0012) and ALT (P=0.0027) and kidney TC (P=0.0200) and TG (P=0.0499). In addition, mice in the Qing group showed significant improvement in lipid deposition in the kidney. Qing group showed increased pAMPK/AMPK ratio in comparison with that of the DIO group. In comparison with those of the control group, mice in the DIO group had upregulated expression of lipid synthesis-related genes and proteins (SREBP-1, FASN, and SCD1). As for the fatty acid oxidation-related genes and proteins, DIO mice showed upregulated expression of ACC1 and downregulated expression of CPT1A, PPARγ, and PGC1α in comparison with those of the control group. In the Qing goup, improvements in regard to all these changes were observed. The Qing group demonstrated improvement in the disrupted homeostasis of the gut microbiota. Short-chain fatty acids in the cecal contents, especially isovaleric acid and propionic acid, were also restored. Conclusion: Sichuan dark tea-based medicated dietary formula may improve renal lipid metabolism by regulating gut microbiota and the levels of intestinal short-chain fatty acids, thereby protecting obesity-related kidney injury. Isovaleric acid and propionic acid may be the metabolites key to its regulation of gut microbiota.

Auraptene, a citrus peel-derived natural product, prevents myocardial infarction-induced heart failure by activating PPARα in rats.

BACKGROUND: Auraptene derived from the peel of Citrus hassaku possesses anti-tumor, anti-inflammatory, and neuroprotective activities. Thus, it could be a valuable pharmacological alternative to treat some diseases. However, the therapeutic value of auraptene for heart failure (HF) is unknown. STUDY DESIGN/METHODS: In cultured cardiomyocytes from neonatal rats, the effect of auraptene on phenylephrine-induced hypertrophic responses and peroxisome proliferator-activated receptor-alpha (PPARα)-dependent gene transcriptions. To investigate whether auraptene prevents the development of heart failure after myocardial infarction (MI) in vivo, Sprague-Dawley rats with moderate MI (fractional shortening < 40%) were randomly assigned for treatment with low- or high-dose auraptene (5 or 50 mg/kg/day, respectively) or vehicle for 6 weeks. The effects of auraptene were evaluated by echocardiography, histological analysis, and the measurement of mRNA levels of hypertrophy, fibrosis, and PPARα-associated genes. RESULTS: In cultured cardiomyocytes, auraptene repressed phenylephrine-induced hypertrophic responses, such as increases in cell size and activities of atrial natriuretic factor and endothelin-1 promoters. Auraptene induced PPARα-dependent gene activation by enhancing cardiomyocyte peroxisome proliferator-responsive element reporter activity. The inhibition of PPARα abrogated the protective effect of auraptene on phenylephrine-induced hypertrophic responses. In rats with MI, auraptene significantly improved MI-induced systolic dysfunction and increased posterior wall thickness compared to the vehicle. Auraptene treatment also suppressed MI-induced increases in myocardial cell diameter, perivascular fibrosis, and expression of hypertrophy and fibrosis response markers at the mRNA level compared with vehicle treatment. MI-induced decreases in the expression of PPARα-dependent genes were improved by auraptene treatment. CONCLUSIONS: Auraptene has beneficial effects on MI-induced cardiac hypertrophy and left ventricular systolic dysfunction in rats, at least partly due to PPARα activation. Further clinical studies are required to evaluate the efficacy of auraptene in patients with HF.

Brassica rapa L. (Tibetan turnip) prevents sleep-deprivation induced cognitive deficits via the inhibition of neuroinflammation and mitochondrial depolarization.

Brassica rapa L., an edible, feeding and medicinal plant cultivated on the Tibetan plateau with altitudes above 3800 m, has several pharmacological effects. However, its therapeutic effects against memory impairment and central fatigue have yet to be conclusively established. In this study, the Y-maze and Morris water maze tasks revealed that Brassica rapa L. aqueous extract (BE) significantly ameliorated cognitive deficits of sleep deprivation (SD)-treated mice. Moreover, BE treatment partially alleviated SD-induced reductions in the levels of peripheral energy metabolism, and significantly decreased inflammatory factor levels in serum and hippocampus. In addition, BE treatment significantly relieved central fatigue and stabilized the excitability as well as activities of neurons by regulating the levels of hypothalamus tryptophan metabolites and striatum neurotransmitters. The neuroprotective effects of BE were also confirmed using glutamate-treated HT22 cells, whereby BE pretreatment significantly attenuated intracellular ROS production and mitochondrial depolarization via adenosine 5'-monophosphate activated protein kinase/peroxisome proliferators-activated receptors (AMPK/PPAR-γ) signaling pathways. Thus, BE might probably prevent SD-induced learning and memory deficits by inhibiting neuroinflammation and restoring mitochondrial energy metabolism in the hippocampus. These findings imply that BE is a potential complementary therapy for those suffering from deficient sleep or neurometabolic disorders, although this needs verification by prospective clinical studies.

Omega-6 DPA and its 12-lipoxygenase-oxidized lipids regulate platelet reactivity in a nongenomic PPARα-dependent manner.

Arterial thrombosis is the underlying cause for a number of cardiovascular-related events. Although dietary supplementation that includes polyunsaturated fatty acids (PUFAs) has been proposed to elicit cardiovascular protection, a mechanism for antithrombotic protection has not been well established. The current study sought to investigate whether an omega-6 essential fatty acid, docosapentaenoic acid (DPAn-6), and its oxidized lipid metabolites (oxylipins) provide direct cardiovascular protection through inhibition of platelet reactivity. Human and mouse blood and isolated platelets were treated with DPAn-6 and its 12-lipoxygenase (12-LOX)-derived oxylipins, 11-hydroxy-docosapentaenoic acid and 14-hydroxy-docosapentaenoic acid, to assess their ability to inhibit platelet activation. Pharmacological and genetic approaches were used to elucidate a role for DPA and its oxylipins in preventing platelet activation. DPAn-6 was found to be significantly increased in platelets following fatty acid supplementation, and it potently inhibited platelet activation through its 12-LOX-derived oxylipins. The inhibitory effects were selectively reversed through inhibition of the nuclear receptor peroxisome proliferator activator receptor-α (PPARα). PPARα binding was confirmed using a PPARα transcription reporter assay, as well as PPARα-/- mice. These approaches confirmed that selectivity of platelet inhibition was due to effects of DPA oxylipins acting through PPARα. Mice administered DPAn-6 or its oxylipins exhibited reduced thrombus formation following vessel injury, which was prevented in PPARα-/- mice. Hence, the current study demonstrates that DPAn-6 and its oxylipins potently and effectively inhibit platelet activation and thrombosis following a vascular injury. Platelet function is regulated, in part, through an oxylipin-induced PPARα-dependent manner, suggesting that targeting PPARα may represent an alternative strategy to treat thrombotic-related diseases.

Medium-chain dicarboxylic acylcarnitines as markers of n-3 PUFA-induced peroxisomal oxidation of fatty acids.

SCOPE: Omega-3 polyunsaturated fatty acids (n-3 PUFA) found in fish oil activate PPAR-α, stimulate peroxisomal fatty acid (FA) ß-oxidation and prevent impairments on glucose homeostasis. METHODS AND RESULTS: Glucose metabolism and FA oxidation were studied in C57/Bl6 mice fed with diets containing either 3.6 and 31.5% fish oil or lard. To assess the effects of peroxisomal proliferation on FA oxidation independent of n-3 PUFA intake, mice were treated with the PPAR-α agonist WY-14643. n-3 PUFA-fed mice were protected from glucose intolerance and dyslipidemia compared to animals fed a lard-based high-fat diet. Most importantly, mice fed on the hyperlipidic diet based on fish oil as well as the WY-14643 treated mice showed twofold increase of odd, medium-chain, dicarboxylic acylcarnitines in the liver suggesting that not only ß-oxidation, but also α- and ω-oxidation of FA were increased. Finally, an oxidation assay using liver homogenates and palmitic acid as substrate revealed an over tenfold increased production of similar acylcarnitines, indicating that FA are their precursors. CONCLUSION: This study shows at the metabolite level that peroxisome proliferation induced either by fish oil or WY-14643 is associated with increased α- and ω-oxidation of FA producing specific acylcarnitines that can be utilized as biomarkers of peroxisomal FA oxidation.

Fine metabolic regulation in ruminants via nutrient-gene interactions: saturated long-chain fatty acids increase expression of genes involved in lipid metabolism and immune response partly through PPAR-α activation.

Madin-Darby Bovine Kidney cells cultured with 150 µm of Wy-14 643 (WY, PPARα agonist) or twelve long-chain fatty acids (LCFA; 16 : 0, 18 : 0, cis-9-18 : 1, trans-10-18 : 1, trans-11-18 : 1, 18 : 2n-6, 18 : 3n-3, cis-9, trans-11-18 : 2, trans-10, cis-12-18 : 2, 20 : 0, 20 : 5n-3 and 22 : 6n-3) were used to uncover PPAR-α target genes and determine the effects of LCFA on expression of thirty genes with key functions in lipid metabolism and inflammation. Among fifteen known PPAR-α targets in non-ruminants, ten had greater expression with WY, suggesting that they are bovine PPAR-α targets. The expression of SPP1 and LPIN3 was increased by WY, with no evidence of a similar effect in the published literature, suggesting that both represent bovine-specific PPAR-α targets. We observed the strongest effect on the expression of PPAR-α targets with 16 : 0, 18 : 0 and 20 : 5n-3.When considering the overall effect on expression of the thirty selected genes 20 : 5n-3, 16 : 0 and 18 : 0 had the greatest effect followed by 20 : 0 and c9t11-18 : 2. Gene network analysis indicated an overall increase in lipid metabolism by WY and all LCFA with a central role of PPAR-α but also additional putative transcription factors. A greater increase in the expression of inflammatory genes was observed with 16 : 0 and 18 : 0. Among LCFA, 20 : 5n-3, 16 : 0 and 18 : 0 were the most potent PPAR-α agonists. They also affected the expression of non-PPAR-α targets, eliciting an overall increase in the expression of genes related to lipid metabolism, signalling and inflammatory response. Data appear to highlight a teleological evolutionary adaptation of PPAR in ruminants to cope with the greater availability of saturated rather than unsaturated LCFA.

Ursolic acid is a PPAR-α agonist that regulates hepatic lipid metabolism.

In this study, we confirmed that ursolic acid, a plant triterpenoid, activates peroxisome proliferator-activated receptor (PPAR)-α in vitro. Surface plasmon resonance and time-resolved fluorescence resonance energy transfer analyses do not show direct binding of ursolic acid to the ligand-binding domain of PPAR-α; however, ursolic acid enhances the binding of PPAR-α to the peroxisome proliferator response element in PPAR-α-responsive genes, alters the expression of key genes in lipid metabolism, significantly reducing intracellular triglyceride and cholesterol concentrations in hepatocytes. Thus, ursolic acid is a PPAR-α agonist that regulates the expression of lipid metabolism genes, but it is not a direct ligand of PPAR-α.

Differentiated CaCo-2 cells as an in-vitro model to evaluate de-novo apolipoprotein A-I production in the small intestine.

BACKGROUND: Increasing HDL cholesterol concentrations by stimulating de-novo apolipoprotein A-I (apoA-I) production in the liver and/or in the small intestine is a potential strategy to reduce coronary heart disease risk. Although there is quite some knowledge concerning regulatory effects in the liver, less is known concerning potential agents that could elevate de-novo apoA-I production in the small intestine. METHODS: Therefore, we compared side-by-side effects of various peroxisome proliferator-activated receptor (PPAR)alpha, PPARgamma, retinoid-X-receptor alpha, and farnesoid-X-receptor agonists on de-novo apoA-I production in differentiated CaCo-2 and HepG2 cells. RESULTS: For PPARa agonists, we showed that GW7647 elevated apoA-I concentrations in the medium of both cell models, whereas WY14643 elevated only de-novo apoA-I concentrations in differentiated CaCo-2 cells. Unexpectedly, fenofibric acid lowered apoA-I medium concentrations in both cell lines, which could not be explained by a lack of PPAR transactivation or a lack of retinoid-X-receptor a activation. For farnesoid-X-receptor agonists, chenodeoxycholic acid strongly reduced apoA-I concentrations both in differentiated CaCo-2 and HepG2 cells, whereas GW4064 and taurocholate only lowered apoA-I in CaCo-2 cells (GW4064) or in HepG2 cells (taurocholate). However, overall effects of all individual components on apoA-I production in differentiated CaCo-2 and HepG2 cells were highly correlated (r = 0.68; P = 0.037; N=9). CONCLUSION: We conclude that differentiated CaCo-2 cells are suitable models to study de-novo small intestinal apoA-I production in vitro enabling the possibility to screen for potential bioactive dietary components. This cell model may also determine small-intestinal-specific effects, as some discrepancy was found between both cell models.

Gypenoside XLIX, a naturally occurring gynosaponin, PPAR-alpha dependently inhibits LPS-induced tissue factor expression and activity in human THP-1 monocytic cells.

Tissue factor (TF) is involved not only in the progression of atherosclerosis and other cardiovascular diseases, but is also associated with tumor growth, metastasis, and angiogenesis and hence may be an attractive target for directed cancer therapeutics. Gynostemma pentaphyllum (GP) is widely used in the treatment of various cardiovascular diseases including atherosclerosis, as well as cancers. Gypenoside (Gyp) XLIX, a dammarane-type glycoside, is one of the prominent components in GP. We have recently reported Gyp XLIX to be a potent peroxisome proliferator-activated receptor (PPAR)-alpha activator. Here we demonstrate that Gyp XLIX (0-300 microM) concentration dependently inhibited TF promoter activity after induction by the inflammatory stimulus lipopolysaccharide (LPS) in human monocytic THP-1 cells transfected with promoter reporter constructs pTF-LUC. Furthermore, Gyp XLIX inhibited LPS-induced TF mRNA and protein overexpression in THP-1 monocyte cells. Its inhibition of LPS-induced TF hyperactivity was further confirmed by chromogenic enzyme activity assay. The activities of Gyp XLIX reported in this study were similar to those of Wy-14643, a potent synthetic PPAR-alpha activator. Furthermore, the Gyp XLIX-induced inhibitory effect on TF luciferase activity was completely abolished in the presence of the PPAR-alpha selective antagonist MK-886. The present findings suggest that Gyp XLIX inhibits LPS-induced TF overexpression and enhancement of its activity in human THP-1 monocytic cells via PPAR-alpha-dependent pathways. The data provide new insights into the basis of the use of the traditional Chinese herbal medicine G. pentaphyllum for the treatment of cardiovascular and inflammatory diseases, as well as cancers.

Subchronic urinary bladder effects of muraglitazar in male rats.

Muraglitazar, a PPARalpha/gamma dual agonist, was dosed orally to rats once daily for 13 weeks to evaluate urinary and urothelial changes of potential relevance to urinary bladder tumorigenesis. Groups of 17 young or aged rats per sex were fed a normal or 1% NH4Cl-supplemented diet and were dosed with 0, 1, or 50 mg/kg muraglitazar. Lithogenic ions and sediment were profiled from freshly voided urine samples collected 24 h after dosing, and drug exposures were measured. Urinary citrate, oxalate, and epidermal growth factor (EGF) were assayed from 18-h urine collections. Urothelium was assessed by light microscopy, scanning electron microscopy, and BrdU and TUNEL immunohistochemistry. When fed a normal diet, urine pH was higher in males (above 6.5). Urine volume/body weight was greater in females. Urine soluble/total calcium and magnesium and phosphorus/creatinine ratios were lower in male rats fed a normal diet. Urine citrate levels were decreased and oxalate was increased in young male rats treated with 50 mg/kg muraglitazar compared to age/sex/diet-matched controls. No changes in urine sediment were detected 24 h after dosing. In young male rats treated with 50 mg/kg on normal diet, multifocal urothelial necrosis and proliferation were observed, whereas urothelial apoptosis and urine EGF levels were unchanged compared to age/sex/diet-matched controls. Urothelial necrosis and proliferation were not correlated to systemic or urinary drug exposures and were prevented by dietary acidification. These data suggest that muraglitazar-associated changes in urine composition predispose to urothelial cytotoxicity and proliferation in the urinary bladder of young male rats and that urine sediment must be profiled at multiple daily timepoints to fully qualify drug-induced changes in urine composition.

Peroxisome proliferator-activated receptor-alpha-null mice have increased white adipose tissue glucose utilization, GLUT4, and fat mass: Role in liver and brain.

Activation of the peroxisome proliferator-activated receptor (PPAR)-alpha increases lipid catabolism and lowers the concentration of circulating lipid, but its role in the control of glucose metabolism is not as clearly established. Here we compared PPARalpha knockout mice with wild type and confirmed that the former developed hypoglycemia during fasting. This was associated with only a slight increase in insulin sensitivity but a dramatic increase in whole-body and adipose tissue glucose use rates in the fasting state. The white sc and visceral fat depots were larger due to an increase in the size and number of adipocytes, and their level of GLUT4 expression was higher and no longer regulated by the fed-to-fast transition. To evaluate whether these adipocyte deregulations were secondary to the absence of PPARalpha from liver, we reexpresssed this transcription factor in the liver of knockout mice using recombinant adenoviruses. Whereas more than 90% of the hepatocytes were infected and PPARalpha expression was restored to normal levels, the whole-body glucose use rate remained elevated. Next, to evaluate whether brain PPARalpha could affect glucose homeostasis, we activated brain PPARalpha in wild-type mice by infusing WY14643 into the lateral ventricle and showed that whole-body glucose use was reduced. Hence, our data show that PPARalpha is involved in the regulation of glucose homeostasis, insulin sensitivity, fat accumulation, and adipose tissue glucose use by a mechanism that does not require PPARalpha expression in the liver. By contrast, activation of PPARalpha in the brain stimulates peripheral glucose use. This suggests that the alteration in adipocyte glucose metabolism in the knockout mice may result from the absence of PPARalpha in the brain.

Effects of fatty acids on skeletal muscle cell differentiation in vitro.

Previous studies have shown stimulatory effects of linoleic acid (LA, C18:2) on differentiation of rat muscle cells in culture (Allen et al. 1985), but there appears to be little investigation of the effects of other fatty acids. The present study therefore compared the effects of different fatty acids on muscle cell differentiation in vitro. L6 myoblasts were cultured (Dulbecco's Modified Eagles Medium + 10 % fetal calf serum) in six-well plates until 80 % confluent (day 0). Cells were then either harvested or the medium switched to differentiation medium (Dulbecco's Modified Eagles Medium+2 % horse serum), supplemented with fatty acid or drug treatments. Cells were harvested on days 0-5 and assayed for creatine kinase (CK), protein and DNA contents, to give a measure of differentiation (CK/DNA). Initial studies indicated a stimulatory effect of the cis9,trans11 (c9,t11) isomer of conjugated linoleic acid (CLA) relative to control. By contrast, the trans10,cis12 (t10,c12) isomer of CLA inhibited differentiation. Further experiments indicated that inhibition of differentiation by the t10,c12 CLA isomer was dose-dependent (up to 200 microm) and may be via increased cell proliferation. LA and c9,t11 CLA stimulated differentiation at low concentrations (up to 50 microm), but inhibited differentiation at high concentrations (200 microm). In contrast, oleic acid stimulated differentiation at all concentrations, whereas the saturated fatty acid, palmitic acid, had no effect. The mechanism appeared not to involve either peroxisome proliferator-activated receptors alpha or gamma. The data suggest that only unsaturated fatty acids have an effect and the presence or absence of a cis-9 double bond may be important.

A teleost in vitro reporter gene assay to screen for agonists of the peroxisome proliferator-activated receptors.

Several contaminants detected in aquatic ecosystems are agonists of peroxisome proliferator-activated receptors (PPARs). Peroxisome proliferator-activated receptors interact with the retinoid X receptor (RXR) to activate the transcription of genes that control a variety of physiological functions. We cloned and sequenced partial cDNA fragments of rainbow trout (Oncorhynchus mykiss) PPARalpha and PPARbeta from rainbow trout (rt) gill-W1 cells, a cell line derived from rainbow trout gills; predicted amino acid identities are 77% and 82% compared with their respective human homologs and 83 to 88% and 91 to 98% identical to fish homologs. A reporter gene assay was developed by transfecting rt-gill-W1 cells with a reporter gene construct containing the peroxisome proliferator response element (PPRE) of the rat liver 3-ketoacyl-CoA thiolase B (TB) gene, which drives luciferase expression. Agonists of both PPARalpha (WY14,643 and gemfibrozil) and PPARbeta (bezafibrate) induced luciferase activity, while rosiglitazone, a PPARgamma agonist, was not effective. The fibrate drug, bezafibrate increased luciferase activity in a dose-dependent manner, but addition of 50 nM 9-cis-retinoic acid to the transfected rt-gill-W1 cell culture maximized the sensitivity of the assay so that bezafibrate could be detected at concentrations as low as 6 nM. Extracts from treated domestic wastewater containing fibrate drugs induced luciferase activity in the transfected gill cells. This in vitro reporter gene assay shows promise as a rapid and sensitive technique for screening environmental samples for PPAR-active substances.

A peroxisome proliferator response elements regulatory system in xenopus oocytes and its application.

BACKGROUND: Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a kind of ligand-activated transcription factors binding to peroxisome proliferator response element (PPRE), a specific recognition site. It is thought to play a critical role in glucose and lipid metabolism and in inflammation control. The aim of this study was to establish a new cellular model for the quick screening of lipid-lowering drugs, which may be effective as PPAR-gamma ligands on the PPRE-mediated pathway regulatory system. METHODS: Two plasmids were constructed: pXOE-PPARgamma, in which the human PPARgamma gene was in the downstream of TFIIIA gene promoter, and pLXRN-PPRE-d2EGFP, in which the enhanced green fluorescent protein (EGFP) gene was subcloned into PPRE. The xenopus oocytes were injected with these two plasmids, and consequently treated with prostaglandin E1, pioglitazone, and different kinds of lipid-lowering drugs. After 3 days, the oocytes were observed under a fluorescence microscope. To confirm the drug action,we injected pXOE-PPARgamma plasmid into the oocytes, which then treated with prostaglandin E1 and Hawthorn flavonoids. The mass of expressed lipoprotein lipase (LPL) in the cells was determined by enzyme labeling linked immunosorbent assay (ELISA). RESULTS: The expression of EGFP was only induced by prostagalandin E1, pioglitazone, Hawthorn flavonoids. A concentration-response relationship was seen between expressed EGFP and Hawthorn flavonoids. The levels of LPL in both Hawthorn flavonoids groups and PPARgamma ligand prostagalandin E1 group injected with pXOE-PPARgamma plasmid increased significantly (< 0.001) compared with controls, and a concentration-response relationship was observed between LPL mass and Hawthorn flavonoids. CONCLUSIONS: It is possible to establish a PPRE regulatory EGFP reporter system in xenopus oocytes to monitor the activity of PPARgamma ligand. Hawthorn flavonoids can increase the expression of gene downsteam of PPRE by effect on the PPRE pathway regulatory system.

Peroxisome proliferation in liver of rats fed oxidized frying oil.

Oxidized frying oil (OFO) activates peroxisome proliferator-activated receptor a (PPAR alpha) in vitro and in vivo. As most PPARalpha activators are also peroxisome proliferators (PP), this study was aimed at exploring whether OFO induces peroxisome proliferation in the liver of rats. Four groups of male weanling Sprague-Dawley rats were fed the following diets for 6 wk: a basal diet containing 5 g/100 g fresh soybean oil (LSB), high-fat diets containing 20 g/100 g of fresh soybean oil (HSB as a control). OFO (HO) or fish oil (HF, as a positive control). Hepatomegaly and peroxisome proliferation in the liver of the HO group of rats were higher than those of the HF group. In addition, the acyl-CoA oxidase (ACO) activity, as well as cytochrome P450 4A (CYP4A) protein content in the livers of the HO group were 6 fold those of the HSB group, but were 2.5 fold in those of the HF group. These results indicated that dietary OFO induced typical responses to PPARalpha signaling. Moreover. as a dietary source, the OFO prepared under our frying conditions appears to be a more potent peroxisome proliferator than fish oil.

A peroxisome proliferator response elements regulatory system in xenopus oocytes and its application / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a kind of ligand-activated transcription factors binding to peroxisome proliferator response element (PPRE), a specific recognition site. It is thought to play a critical role in glucose and lipid metabolism and in inflammation control. The aim of this study was to establish a new cellular model for the quick screening of lipid-lowering drugs, which may be effective as PPAR-gamma ligands on the PPRE-mediated pathway regulatory system.</p><p><b>METHODS</b>Two plasmids were constructed: pXOE-PPARgamma, in which the human PPARgamma gene was in the downstream of TFIIIA gene promoter, and pLXRN-PPRE-d2EGFP, in which the enhanced green fluorescent protein (EGFP) gene was subcloned into PPRE. The xenopus oocytes were injected with these two plasmids, and consequently treated with prostaglandin E1, pioglitazone, and different kinds of lipid-lowering drugs. After 3 days, the oocytes were observed under a fluorescence microscope. To confirm the drug action,we injected pXOE-PPARgamma plasmid into the oocytes, which then treated with prostaglandin E1 and Hawthorn flavonoids. The mass of expressed lipoprotein lipase (LPL) in the cells was determined by enzyme labeling linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>The expression of EGFP was only induced by prostagalandin E1, pioglitazone, Hawthorn flavonoids. A concentration-response relationship was seen between expressed EGFP and Hawthorn flavonoids. The levels of LPL in both Hawthorn flavonoids groups and PPARgamma ligand prostagalandin E1 group injected with pXOE-PPARgamma plasmid increased significantly (< 0.001) compared with controls, and a concentration-response relationship was observed between LPL mass and Hawthorn flavonoids.</p><p><b>CONCLUSIONS</b>It is possible to establish a PPRE regulatory EGFP reporter system in xenopus oocytes to monitor the activity of PPARgamma ligand. Hawthorn flavonoids can increase the expression of gene downsteam of PPRE by effect on the PPRE pathway regulatory system.</p>

Vitamin A prevents high fat diet-induced ACF development and modifies the pattern of expression of peroxisome proliferator and retinoic acid receptor m-RNA.

Some dietary compounds, among them fats, are modulators of colon cancer risk. This study reports the modulating effects of n-6, with or without vitamin A, on promotion of colon preneoplasic lesions induced by 1,2-dimethylhydrazine (DMH) and on the expression of nuclear receptors (PPARgamma, RXRalpha, and RARbeta). One group of male Fisher rats was fed a basic diet (5% safflower oil) and two groups were fed a high-fat diet (HFD, 25% safflower oil). Of these, one was supplemented with 200 IU vitamin A for 5 mo. The safflower oil contained polyunsaturated fatty acids, mainly linoleic acid (73%). The data showed an increasing effect of safflower oil-enriched diet on aberrant crypt foci occurrence and multiplicity. This effect was impaired by vitamin A supplementation. In addition, an HFD-related up-regulation of PPARgamma and a concomitant down-regulation of RARbeta mRNA expression were observed with or without chemical initiation and were prevented by vitamin A. Moreover, when treated with DMH, HFD rats exhibited a dramatically decreased expression of RXRalpha mRNA (-49%). It was hypothesized that HFD, leading to hyperexpression of PPARgamma, would produce an alteration of retinoic acid signaling and, in this way, create a background modulating colon cancer risk.

Peroxisome proliferator-activated receptors and atherogenesis: regulators of gene expression in vascular cells.

A large body of data gathered over the past couple of years has identified the peroxisome proliferator-activated receptors (PPAR) alpha, gamma, and beta/delta as transcription factors exerting modulatory actions in vascular cells. PPARs, which belong to the nuclear receptor family of ligand-activated transcription factors, were originally described as gene regulators of various metabolic pathways. Although the PPARalpha, gamma, and beta/delta subtypes are approximately 60% to 80% homologous in their ligand- and DNA-binding domains, significant differences in ligand and target gene specificities are observed. PPARalpha is activated by polyunsaturated fatty acids and oxidized derivatives and by lipid-modifying drugs of the fibrate family, including fenofibrate or gemfibrozil. PPARalpha controls expression of genes implicated in lipid metabolism. PPARgamma, in contrast, is a key regulator of glucose homeostasis and adipogenesis. Ligands of PPARgamma include naturally occurring FA derivatives, such as hydroxyoctadecadienoic acids (HODEs), prostaglandin derivatives such as 15-deoxyDelta12,14-prostaglandin J2, and glitazones, insulin-sensitizing drugs presently used to treat patients with type 2 diabetes. Ligands for PPARbeta/delta are polyunsaturated fatty acids, prostaglandins, and synthetic compounds, some of which are presently in clinical development. PPARbeta/delta stimulates fatty acid oxidation predominantly acting in muscle. All PPARs are expressed in vascular cells, where they exhibit antiinflammatory and antiatherogenic properties. In addition, studies in various animal models as well as clinical data suggest that PPARalpha and PPARgamma activators can modulate atherogenesis in vivo. At present, no data are available relating to possible effects of PPARbeta/delta agonists on atherogenesis. Given the widespread use of PPARalpha and PPARgamma agonists in patients at high risk for cardiovascular disease, the understanding of their function in the vasculature is not only of basic interest but also has important clinical implications. This review will focus on the role of PPARs in the vasculature and summarize the present understanding of their effects on atherogenesis and its cardiovascular complications.