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.

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.

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.

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.

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.

Activation of peroxisome proliferator-activated receptor gamma inhibits interleukin-1beta-induced membrane-associated prostaglandin E2 synthase-1 expression in human synovial fibroblasts by interfering with Egr-1.

Membrane-associated prostaglandin (PG) E(2) synthase-1 (mPGES-1) catalyzes the conversion of PGH(2) to PGE(2), which contributes to many biological processes. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor and plays an important role in growth, differentiation, and inflammation in different tissues. Here, we examined the effect of PPARgamma ligands on interleukin-1beta (IL-1beta)-induced mPGES-1 expression in human synovial fibroblasts. PPARgamma ligands 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)) and the thiazolidinedione troglitazone (TRO), but not PPARalpha ligand Wy14643, dose-dependently suppressed IL-1beta-induced PGE(2) production, as well as mPGES-1 protein and mRNA expression. 15d-PGJ(2) and TRO suppressed IL-1beta-induced activation of the mPGES-1 promoter. Overexpression of wild-type PPARgamma further enhanced, whereas overexpression of a dominant negative PPARgamma alleviated, the suppressive effect of both PPARgamma ligands. Furthermore, pretreatment with an antagonist of PPARgamma, GW9662, relieves the suppressive effect of PPARgamma ligands on mPGES-1 protein expression, suggesting that the inhibition of mPGES-1 expression is mediated by PPARgamma. We demonstrated that PPARgamma ligands suppressed Egr-1-mediated induction of the activities of the mPGES-1 promoter and of a synthetic reporter construct containing three tandem repeats of an Egr-1 binding site. The suppressive effect of PPARgamma ligands was enhanced in the presence of a PPARgamma expression plasmid. Electrophoretic mobility shift and supershift assays for Egr-1 binding sites in the mPGES-1 promoter showed that both 15d-PGJ(2) and TRO suppressed IL-1beta-induced DNA-binding activity of Egr-1. These data define mPGES-1 and Egr-1 as novel targets of PPARgamma and suggest that inhibition of mPGES-1 gene transcription may be one of the mechanisms by which PPARgamma regulates inflammatory responses.

Moderate iron overload enhances lipid peroxidation in livers of rats, but does not affect NF-kappaB activation induced by the peroxisome proliferator, Wy-14,643.

It has been hypothesized that high concentrations of tissue iron may enhance carcinogenesis induced by free radical mechanisms. Wy-14,643 is a peroxisome proliferator that is hepatocarcinogenic in rats. Tumor induction may result in part from excessive production of reactive oxygen species, particularly H(2)O(2). The purpose of this study was to examine the effect of iron status on oxidative stress and NF-kappaB activation in livers of rats treated with Wy-14,643. Forty-eight male Sprague-Dawley rats were fed one of four diets (20, 45, 650, 1500 mg Fe/kg diet) for 28 d. At the time of tissue collection, liver iron ranged from 1.4 to 9.9 micro mol/g wet tissue in the diet groups. Wy-14,643 (0 or 0.1 g/100 g diet) was added to the diet for the final 10 d of the study. Wy-14,643 doubled the liver weight/body weight ratio (P = 0.0001), which was also increased by iron supplementation (P < 0.01). Iron supplementation increased thiobarbituric acid reactive substances and/or conjugated dienes, but there was no synergism between Wy 14,643 and iron on lipid peroxidation measures. The hepatic DNA binding activity of NF-kappaB was increased in rats administered Wy-14,643. However, differences in liver iron concentration did not alter activation of NF-kappaB in untreated rats or in those treated with Wy-14,643. DNA double-strand breakage was not affected by iron or Wy-14,643. In summary, although moderate changes in iron status altered liver lipid peroxidation, iron did not significantly increase oxidative stress induced by a hepatocarcinogenic peroxisome proliferator.

Differential induction of peroxisomal beta-oxidation enzymes by clofibric acid and aspirin in piglet tissues.

Peroxisomal beta-oxidation (POX) of fatty acids is important in lipid catabolism and thermogenesis. To investigate the effects of peroxisome proliferators on peroxisomal and mitochondrial beta-oxidation in piglet tissues, newborn pigs (1-2 days old) were allowed ad libitum access to milk replacer supplemented with 0.5% clofibric acid (CA) or 1% aspirin for 14 days. CA increased ratios of liver weight to body weight (P < 0.07), kidney weight to body weight (P < 0.05), and heart weight to body weight (P < 0.001). Aspirin decreased daily food intake and final body weight but increased the ratio of heart weight to body weight (P < 0.01). In liver, activities of POX, fatty acyl-CoA oxidase (FAO), total carnitine palmitoyltransferase (CPT), and catalase were 2.7-, 2.2-, 1.5-fold, and 33% greater, respectively, for pigs given CA than for control pigs. In heart, these variables were 2.2-, 4.1-, 1.9-, and 1.8-fold greater, respectively, for pigs given CA than for control pigs. CA did not change these variables in either kidney or muscle, except that CPT activity was increased approximately 110% (P < 0.01) in kidney. Aspirin increased only hepatic FAO and CPT activities. Northern blot analysis revealed that CA increased the abundance of catalase mRNA in heart by approximately 2.2-fold. We conclude that 1) POX and CPT in newborn pigs can be induced by peroxisomal proliferators with tissue specificity and 2) the relatively smaller induction of POX in piglets (compared with that in young or adult rodents) may be related to either age or species differences.

Cross-talk between fatty acid and cholesterol metabolism mediated by liver X receptor-alpha.

LXR alpha (liver X receptor, also called RLD-1) is a nuclear receptor, highly expressed in tissues that play a role in lipid homeostasis. In this report we show that fatty acids are positive regulators of LXR alpha gene expression and we investigate the molecular mechanisms underlying this regulation. In cultured rat hepatoma and primary hepatocyte cells, fatty acids and the sulfur-substituted fatty acid analog, tetradecylthioacetic acid, robustly induce LXR alpha (up to 3.5- and 7-fold, respectively) but not LXR beta (also called OR-1) mRNA steady state levels, with unsaturated fatty acids being more effective than saturated fatty acids. RNA stability and nuclear run-on studies demonstrate that changes in the transcription rate of the LXR alpha gene account for the major part of the induction of LXR alpha mRNA levels. A similar induction of protein level was also seen after treatment of primary hepatocytes with the same fatty acids. Consistent with such a transcriptional effect, transient transfection studies with a luciferase reporter gene, driven by 1.5 kb of the 5'-flanking region of the mouse (m)LXR alpha gene, show a peroxisome proliferator-activated receptor-alpha-dependent increase in luciferase activity upon treatment with tetradecylthioacetic acid and the synthetic peroxisome proliferator-activated receptor-alpha activator, Wy 14.643, suggesting that the mLXR alpha 5'-flanking region contains the necessary sequence elements for fatty acid responsiveness. In addition, in vivo LXR alpha expression was induced by fatty acids, consistent with the in vitro cell culture data. These observations demonstrate that LXR alpha expression is controlled by fatty acid signaling pathways and suggest an important cross-talk between fatty acid and cholesterol regulation of lipid metabolism.

Evidence against the peroxisome proliferator-activated receptor alpha (PPARalpha) as the mediator for polyunsaturated fatty acid suppression of hepatic L-pyruvate kinase gene transcription.

The glycolytic enzyme, L-pyruvate kinase (L-PK), plays an important role in hepatic glucose metabolism. Insulin and glucose induce L-PK gene expression, while glucagon and polyunsaturated fatty acids (PUFA) inhibit L-PK gene expression. We have been interested in defining the PUFA regulation of L-PK. The cis-regulatory target for PUFA action includes an imperfect direct repeat (DR1) that binds HNF-4. HNF4 plays an ancillary role in the insulin/glucose-mediated transactivation of the L-PK gene. Because the fatty acid-activated nuclear receptor, peroxisome proliferator-activated receptor (PPARalpha), binds DR1-like elements and has been reported to interfere with HNF4 action, we examined the role PPARalpha plays in the regulation of L-PK gene transcription. Feeding rats either fish oil or the potent PPARalpha activator, WY14,643, suppressed rat hepatic L-PK mRNA and gene transcription. The PPARalpha-null mouse was used to evaluate the role of the PPARalpha in hepatic transcriptional control of L-PK. While WY14,643 control of L-PK gene expression required the PPARalpha, PUFA regulation of L-PK gene expression was independent of the PPARalpha. Transfection studies in cultured primary hepatocytes localized the cis-regulatory target for WY14,643/PPARalpha action to the L-PK HNF4 binding site. However, PPARalpha/RXRalpha heterodimers did not bind this region. Although both WY14,643 and PUFA suppress L-PK gene transcription through the same element, PUFA regulation of L-PK does not require the PPARalpha and PPARalpha/RXRalpha does not bind the L-PK promoter. These studies suggest that other intermediary factors are involved in both the PUFA and PPARalpha regulation of L-PK gene transcription.

Proliferation of hepatic peroxisomes in rats following the intake of green or black tea.

Rats maintained on green, black or decaffeinated black tea (2.5%, w/v) as their sole drinking fluid displayed higher hepatic CN- insensitive palmitoyl CoA oxidase activity than controls; the extent of increase was similar with the three types of tea. Morphological examination of the liver using electron microscopy revealed an increase in the number of peroxisomes in the tea-treated animals. The same treatment of the animals with green and black tea resulted in a similar rise in hepatic microsomal lauric acid hydroxylation. Analysis by HPLC of the aqueous tea extracts employed in the current study showed that the total flavanol content of the green variety was much higher than the black varieties, and confirmed the absence of caffeine in the decaffeinated black tea. It may be concluded from the present studies that neither caffeine nor flavanoids are likely to be responsible for the proliferation of peroxisomes observed in rats treated with tea.

Sterol regulatory element binding protein-1 expression is suppressed by dietary polyunsaturated fatty acids. A mechanism for the coordinate suppression of lipogenic genes by polyunsaturated fats.

Polyunsaturated fatty acids (PUFA) coordinately suppress the transcription of a wide array of hepatic lipogenic genes including fatty acid synthase (FAS) and acetyl-CoA carboxylase. Interestingly, the over-expression of sterol regulatory element binding protein-1 (SREBP-1) induces the expression of all of the enzymes suppressed by PUFA. This observation led us to hypothesize that PUFA coordinately inhibit lipogenic gene transcription by suppressing the expression of SREBP-1. Our initial studies revealed that the SREBP-1 and FAS mRNA contents of HepG2 cells were reduced by 20:4(n-6) in a dose-dependent manner (i.e. EC(50) approximately 10 microM), whereas 18:1(n-9) had no effect. Similarly, supplementing a fat-free, high glucose diet with oils rich in (n-6) or (n-3) PUFA reduced the hepatic content of precursor and nuclear SREBP-1 60 and 85%, respectively; however, PUFA had no effect on the nuclear content of upstream stimulatory factor (USF)-1. The PUFA-dependent decrease in nuclear content of mature SREBP-1 was paralleled by a 70-90% suppression in FAS gene transcription. In contrast, dietary 18:1(n-9), i.e. triolein, had no inhibitory influence on the expression of SREBP-1 or FAS. The decrease in hepatic expression of SREBP-1 and FAS associated with PUFA ingestion was mimicked by supplementing the fat-free diet with the PPARalpha-activator, WY 14, 643. Interestingly, nuclear run-on assays revealed that changes in SREBP-1 mRNA abundance were not accompanied by changes in SREBP-1 gene transcription. These results support the concept that PUFA coordinately inhibit lipogenic gene transcription by suppressing the expression of SREBP-1 and that the PUFA regulation of SREBP-1 appears to occur at the post-transcriptional level.