Peroxisome Proliferator Response Element Research Articles

Airway epithelial cell PPARγ modulates cigarette smoke-induced chemokine expression and emphysema susceptibility in mice.

Chronic obstructive pulmonary disease (COPD) is a highly prevalent, chronic inflammatory lung disease with limited existing therapeutic options. While modulation of peroxisome proliferator-activating receptor (PPAR)-γ activity can modify inflammatory responses in several models of lung injury, the relevance of the PPARG pathway in COPD pathogenesis has not been previously explored. Mice lacking Pparg specifically in airway epithelial cells displayed increased susceptibility to chronic cigarette smoke (CS)-induced emphysema, with excessive macrophage accumulation associated with increased expression of chemokines, Ccl5, Cxcl10, and Cxcl15. Conversely, treatment of mice with a pharmacological PPARγ activator attenuated Cxcl10 and Cxcl15 expression and macrophage accumulation in response to CS. In vitro, CS increased lung epithelial cell chemokine expression in a PPARγ activation-dependent fashion. The ability of PPARγ to regulate CS-induced chemokine expression in vitro was not specifically associated with peroxisome proliferator response element (PPRE)-mediated transactivation activity but was correlated with PPARγ-mediated transrepression of NF-κB activity. Pharmacological or genetic activation of PPARγ activity abrogated CS-dependent induction of NF-κB activity. Regulation of NF-κB activity involved direct PPARγ-NF-κB interaction and PPARγ-mediated effects on IKK activation, IκBα degradation, and nuclear translocation of p65. Our data indicate that PPARG represents a disease-relevant pathophysiological and pharmacological target in COPD. Its activation state likely contributes to NF-κB-dependent, CS-induced chemokine-mediated regulation of inflammatory cell accumulation.

Peroxisome proliferator-activated receptor γ enhances adiponectin secretion via up-regulating DsbA-L expression

Disulfide-bond A oxidoreductase like-protein (DsbA-L) was identified as a molecular chaperone facilitating the assembly and secretion of adiponectin, an adipokine with multiple beneficial effects. In obesity the level of DsbA-L is reduced with a concomitant decrease of the circulating adiponectin level, especially of the high molecular weight form (HMW). Both rodent and human studies have shown that the nuclear receptor peroxisome proliferator-activated receptor (PPAR)-γ agonists increase adiponectin levels in serum by activating PPARγ, which up-regulates critical endoplasmic reticulum (ER) chaperones thus facilitating protein folding. As shown in the present study, overexpression of PPARγ in human embryonic kidney (HEK) 293 cells elicited the cellular release of HMW adiponectin. PPARγ enhanced expression of DsbA-L by binding directly to peroxisome proliferator response element (PPRE) site within the DsbA-L promoter. Conversely, in differentiated 3T3-L1 cells, PPARγ knockdown resulted in decreased expression of Adiponectin, DsbA-L and ERp44. DsbA-L expression increased after PPARγ agonist treatment and decreased upon treatment with PPARγ antagonist in 3T3-L1 adipocytes. DsbA-L deficiency in differentiated 3T3-L1 cells impaired the secretion of adiponectin. We therefore propose that DsbA-L plays an important role in facilitating HMW adiponectin formation and release from cells under the regulation of PPARγ.

Putative PPAR target genes express highly in skeletal muscle of insulin-resistant MetS model SHR/NDmc-cp rats.

It is known that insulin resistance in skeletal muscle induces subsequent metabolic diseases such as metabolic syndrome (MetS). However, which genes are altered in the skeletal muscle by development of insulin resistance in animal models has not been examined. In this study, we performed microarray and subsequent real-time RT-PCR analyses using total RNA extracted from the gastrocnemius muscle of the MetS model, spontaneously hypertensive corpulent congenic (SHR/NDmc-cp) rats, and control Wistar Kyoto (WKY) rats. SHR/NDmc-cp rats displayed overt insulin resistance relative to WKY rats. The expression of many genes related to fatty acid oxidation was higher in SHR/NDmc-cp rats than in WKY rats. Among 18 upregulated genes, putative peroxisome proliferator responsive elements were found in the upstream region of 15 genes. The protein expression of ACOX2, an upregulated gene, and peroxisome proliferator-activated receptor (PPAR) G1, but not of PPARG2, PPARA or PPARD, was higher in the gastrocnemius muscle of SHR/NDmc-cp rats than that in WKY rats. These results suggest that insulin resistance in the MetS model, SHR/NDmc-cp rats, is positively associated with the expression of fatty acid oxidation-related genes, which are presumably PPARs’ targets, in skeletal muscle.

Activation of PPARγ by 12/15-lipoxygenase during cerebral ischemia-reperfusion injury.

Peroxisome proliferator-activated receptor γ (PPARγ) expression and activity are increased in brain ischemic injury and its agonists have shown potential for brain injury protection. The influence of 12/15-lipoxygenase (12/15-LOX) on the activity of PPARγ in oxygen-glucose deprivation (OGD) and ischemia-reperfusion (I/R) was investigated. A middle cerebral artery occlusion/reperfusion model with Sprague Dawley (SD) rats was established. For I/R intervention, the rats were treated with the 12/15-LOX-derived product 12-hydroxyeicosatetraenoic acid (12-HETE) for 30 min before cerebral artery occlusion. Primary cortical neurons from SD rats were used to establish an OGD cell model. 12-HETE or a 12/15-LOX antisense oligonucleotide (asON-12/15-LOX) was added to OGD-treated neurons. Western blots, immunofluorescence and enzyme-linked immunosorbent assays detected protein. Reverse transcription-polymerase chain reaction analyzed the expression of the PPARγ target genes. PPARγ-DNA binding activity was determined by peroxisome proliferator responsive element luciferase reporter vectors. 12/15-LOX total protein increased significantly with I/R, and expression of 12-HETE was also upregulated. 12-HETE treatment increased PPARγ protein expression and inhibited inducible nitric oxide synthase protein expression, which was upregulated with I/R. PPARγ nuclear protein and 12/15-LOX total protein expression in OGD-treated neurons increased significantly. 12-HETE treatment increased the expression of PPARγ nuclear protein, upregulated the mRNA levels of PPARγ target genes (lipoprotein lipase and acyl-CoA oxidase) and enhanced PPARγ-DNA binding activity. asON-12/15-LOX treatment inhibited 12/15-LOX and PPARγ protein expression and lipoprotein lipase mRNA. Cerebral I/R injury in rats and OGD treatment in neurons promoted 12/15-LOX expression, and 12-HETE activated PPARγ. Therefore, PPARγ can be activated by the 12/15-LOX pathway during cerebral I/R injury.

L-carnitine protects against carboplatin-mediated renal injury: AMPK- and PPARα-dependent inactivation of NFAT3.

We have previously shown that carboplatin induces inflammation and apoptosis in renal tubular cells (RTCs) through the activation of the nuclear factor of activated T cells-3 (NFAT3) protein by reactive oxygen species (ROS), and that the ROS-mediated activation of NFAT3 is prevented by N-acetyl cysteine and heme oxygenase-1 treatment. In the current study, we investigated the underlying molecular mechanisms of the protective effect of L-carnitine on carboplatin-mediated renal injury. Balb/c mice and RTCs were used as model systems. Carboplatin-induced apoptosis in RTCs was examined using terminal-deoxynucleotidyl-transferase-mediated dUTP nick end labeling. We evaluated the effects of the overexpression of the peroxisome-proliferator-activated receptor alpha (PPARα) protein, the knockdown of PPARα gene, and the blockade of AMPK activation and PPARα to investigate the underlying mechanisms of the protective effect of L-carnitine on carboplatin-mediated renal injury. Carboplatin reduced the nuclear translocation, phosphorylation, and peroxisome proliferator responsive element transactivational activity of PPARα. These carboplatin-mediated effects were prevented by L-carnitine through a mechanism dependent on AMPK phosphorylation and subsequent PPARα activation. The activation of PPARα induced cyclooxygenase 2 (COX-2) and prostacyclin (PGI2) synthase expression that formed a positive feedback loop to further activate PPARα. The coimmunoprecipitation of the nuclear factor (NF) κB proteins increased following the induction of PPARα by L-carnitine, which reduced NFκB transactivational activity and cytokine expression. The in vivo study showed that the inactivation of AMPK suppressed the protective effect of L-carnitine in carboplatin-treated mice, indicating that AMPK phosphorylation is required for PPARα activation in the L-carnitine-mediated protection of RTC apoptosis caused by carboplatin. The results of our study provide molecular evidence that L-carnitine prevents carboplatin-mediated apoptosis through AMPK-mediated PPARα activation.

FKBP51 Reciprocally Regulates GRα and PPARγ Activation via the Akt-p38 Pathway

FK506-binding protein 51 (FKBP51) is a negative regulator of glucocorticoid receptor-α (GRα), although the mechanism is unknown. We show here that FKBP51 is also a chaperone to peroxisome proliferator-activated receptor-γ (PPARγ), which is essential for activity, and uncover the mechanism underlying this differential regulation. In COS-7 cells, FKBP51 overexpression reduced GRα activity at a glucocorticoid response element-luciferase reporter, while increasing PPARγ activity at a peroxisome proliferator response element reporter. Conversely, FKBP51-deficient (knockout) (51KO) mouse embryonic fibroblasts (MEFs) showed elevated GRα but reduced PPARγ activities compared with those in wild-type MEFs. Phosphorylation is known to exert a similar pattern of reciprocal modulation of GRα and PPARγ. Knockdown of FKBP51 in 3T3-L1 preadipocytes increased phosphorylation of PPARγ at serine 112, a phospho-residue that inhibits activity. In 51KO cells, elevated phosphorylation of GRα at serines 220 and 234, phospho-residues that promote activity, was observed. Because FKBP51 is an essential chaperone to the Akt-specific phosphatase PH domain leucine-rich repeat protein phosphatase, Akt signaling was investigated. Elevated Akt activation and increased activation of p38 kinase, a downstream target of Akt that phosphorylates GRα and PPARγ, were seen in 51KO MEFs, causing activation and inhibition, respectively. Inactivation of p38 with PD169316 reversed the effects of FKBP51 deficiency on GRα and PPARγ activities and reduced PPARγ phosphorylation. Last, loss of FKBP51 caused a shift of PPARγ from cytoplasm to nucleus, as previously shown for GRα. A model is proposed in which FKBP51 loss reciprocally regulates GRα and PPARγ via 2 complementary mechanisms: activation of Akt-p38-mediated phosphorylation and redistribution of the receptors to the nucleus for direct targeting by p38.

Short communication: Effect of trans-10,cis-12 conjugated linoleic acid on activation of lipogenic transcription factors in bovine mammary epithelial cells

The objective of this study was to examine the effect of trans-10,cis-12 conjugated linoleic acid (t10c12CLA) on the activation of transcription factors that potentially regulate lipid synthesis in a bovine mammary epithelial cell line (MAC-T). Cells were transfected with luciferase reporter constructs containing sterol response element (SRE and SRE complex) for sterol regulatory element binding protein-1, peroxisome proliferator response element for peroxisome proliferator-activated receptor γ, or liver X receptor response element for liver X receptor. Different concentrations of t10c12CLA (0, 25, 50, 75, or 100μM) were applied to cells to determine the activation of transcription factors. The influence of t10c12CLA bond structure on transcription factor activation was also investigated by treating cells with different 18:1 fatty acid isomers (trans-10 18:1 or cis-12 18:1) at 100μM. Cells were harvested for luciferase assay after 24h of treatment. Compared with linoleic acid and cis-9,trans-11 CLA controls, the SRE reporters had significantly lower activity in t10c12CLA-treated cells at 50 and 75μM for SRE complex and SRE, respectively. Lower SRE and SRE complex activation was observed in t10c12CLA treatment at 25, 50, and 75μM compared with 0μM. The peroxisome proliferator response element and liver X receptor response element reporters did not respond differently between the t10c12CLA treatment and controls. Compared with t10c12CLA, both trans-10 18:1 and cis-12 18:1 increased the activities of SRE and SRE complex reporters by 1.3- to 4.2-fold. In conclusion, t10c12CLA has an inhibitory role in lipogenic transcription factor activation of SRE, and this negative effect is due to the conjugation of trans-10 and cis-12 double bonds in the fatty acid. Furthermore, we found no support for a regulatory role of response elements for peroxisome proliferator-activated receptor γ or liver X receptor in the t10c12CLA inhibition of mammary lipid synthesis.

Transcriptional activation of PIK3R1 by PPARγ in adipocytes.

Phosphatidylinositol 3-kinase (PI3K) plays an important role in the metabolic actions of insulin and is required for adipogenesis. Regulatory subunit 1 of PI3K (PIK3R1) is a critical component of the PI3K signaling pathway. Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of adipogenesis. Although the PPARγ agonist rosiglitazone induces the expression of PIK3R1, the transcriptional regulation of PIK3R1 in adipocytes remains unknown. In this study, we investigated whether PIK3R1 is a direct target of PPARγ. The level of PIK3R1 expression in 3T3-L1 cells was increased after the induction of adipocyte differentiation and was also induced by overexpression of PPARγ. Furthermore, the upregulation of PPARγ-mediated PIK3R1 expression enhanced the insulin-stimulated AKT activation in 3T3-L1 cells. Two putative peroxisome proliferator response elements (PPREs) in the PIK3R1 promoter were identified as PPARγ binding sites. By chromatin immunoprecipitation, we observed that PPARγ interacts with the two PPRE regions of the PIK3R1 promoter in mature adipocytes. In addition, luciferase reporter assays showed that the -1183/-1161 and -573/-551 regions of the PIK3R1 promoter contain essential elements for PPARγ binding. Taken together, these results suggest that PPARγ is essential for the transcriptional activity of PIK3R1 during adipogenesis.

지방분화시 PPARγ에 의한 microRNA-17의 발현 조절

MicroRNAs comprise a family of small noncoding RNAs that modulate physiological processes, including adipogenesis. MicroRNA-17 (miR-17) promotes adipocyte differentiation and enhances lipid accumulation. The transcriptional regulation of miR-17 during adipogenesis remains unknown. In this study, we investigated whether miR-17 is a target of peroxisome proliferator-activated receptor γ (PPARγ), which is a key regulator of adipogenesis. The levels of miR-17 and the expression of PPARγ increased after the induction of adipocyte differentiation. Three putative peroxisome proliferator response elements (PPREs) were identified in the miR-17 promoter region. Using chromatin immunoprecipitation and luciferase reporter assays, we observed the interaction of PPARγ with the miR-17 promoter. Mutagenesis experiments showed that the -677/-655 region of the miR-17 promoter could function as a PPRE site. These results suggest that PPARγ is essential for transcriptional activation of the miR-17 gene, thereby contributing to understanding the molecular mechanism of adipogenesis in adipocytes.

PPARα Activation Sensitizes Cancer Cells to Epigallocatechin-3-Gallate (EGCG) Treatment via Suppressing Heme Oxygenase-1

Epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent of green tea, is a potent antioxidant that may have potential therapeutic applications for the treatment of many disorders, including cancer. Peroxisome proliferator-activated receptor-α (PPARα) has been shown to play a key role in diverse metabolic and cellular functions. PPARα modulates target gene expression by binding to specific regions on the DNA of target genes. The effects and mechanisms of PPARα activation on EGCG efficacy have not yet been analyzed in cancer cells. We found that when cancer cells were exposed to EGCG, the expression of PPARα was increased at the protein level in a dose-dependent manner. The PPARα agonist clofibrate blocked cytoprotective heme oxygenase-1 (HO-1) induction and sensitized multiple types of cancer cells to EGCG-induced cell death. Conversely, the PPARα inhibitor G6471 and PPARα siRNA increased HO-1 expression. Electro-mobility shift assays (EMSA) and in vivo chromatin immunoprecipitation (ChIP) confirmed that PPARα interacts with the peroxisome proliferator-responsive element of the HO-1 promoter. Moreover, cell death induced by EGCG plus clofibrate was partially reversed by HO-1 overexpression in PANC1 cells. These results indicate that PPARα is a direct and negative regulator of HO-1 induced by EGCG and confers cell susceptibility to EGCG.

Peroxisome Proliferator Activated Receptor-δ

Adaptation to changes in energy balance is critical for survival of all organisms. Successful adaptation during mammalian evolution was driven by starvation, but energy excess leading to insulin resistance and atherosclerosis1 presents challenges for adaptation in the current era. The highly orchestrated response to nutritional status is mediated, in part, by peroxisome proliferator activated receptors (PPARs), which control lipid and glucose utilization as well as inflammatory responses in many tissues. The 3 members of the PPAR family, PPARα, PPARδ, and PPARγ, form heterodimers with the retinoid X receptor and bind to peroxisome proliferator response elements.2 PPAR expression patterns overlap, but PPARα is present in liver and muscle, PPARγ is mostly in adipocytes, PPARδ is present in many tissues, and all 3 are in macrophages. See accompanying article on page 52 PPARδ, the least characterized family member, is induced in skeletal muscle after exercise.3 In mice, PPARδ activation increased muscle fatty acid oxidation and improved exercise tolerance.4 In healthy humans, a synthetic PPARδ agonist, GW501516, reduced postprandial plasma triglycerides, increased high-density lipoprotein, and increased muscle expression of enzymes involved in fatty acid oxidation.5 Similar effects were observed when PPARδ …

The hypolipidemic effect of cilostazol can be mediated by regulation of hepatic low-density lipoprotein receptor-related protein 1 (LRP1) expression

ObjectivesCilostazol, a selective phosphodiesterase 3 (PDE3) inhibitor, is a vasodilator and an anti-thrombotic agent. The mechanism whereby cilostazol reduces plasma triglyceride is not completely understood. Here we investigated the effect of cilostazol on a remnant lipoprotein receptor, low-density lipoprotein receptor-related protein 1 (LRP1), which has been reported to play an essential role in clearance of circulating triglyceride in the liver. Materials/MethodsTotal cellular expression, and functional and transcriptional regulation of LRP1 were analyzed in human hepatocarcinoma cell lines incubated with cilostazol. Also, C57BL/6 mice were subjected to high-fat diet (60% kcal) and cilostazol (30mg/kg) treatment for 10weeks. ResultsCilostazol increased both mRNA and protein expression of LRP1 in HepG2 and Hep3B cells. In addition, enhanced transcriptional activity of the LRP1 promoter containing a peroxisome proliferator response element (PPRE) was observed after cilostazol exposure. Cilostazol treatment enhanced the uptake of lipidated apoE3, and this effect was abolished when LRP1 was silenced by siRNA knockdown. High-fat diet induced hyperglycemia with high level of plasma triglycerides, and reduced hepatic LRP1 expression in mice. Treatment with cilostazol for the same period of time, however, successfully prevented this down-regulation of LRP1 expression and reduced plasma triglycerides. ConclusionTaken together, our results demonstrated that cilostazol enhances LRP1 expression in liver by activating PPARγ through the PPRE in the LRP1 promoter. Increased hepatic LRP1 may be essential for the reduction of circulating triglycerides brought about by cilostazol.

Transcriptional activation of melanocortin 2 receptor accessory protein by PPARγ in adipocytes

Adrenocorticotropic hormone (ACTH) in rodents decreases lipid accumulation and body weight. Melanocortin receptor 2 (MC2R) and MC2R accessory protein (MRAP) are specific receptors for ACTH in adipocytes. Peroxisome proliferator-activated receptor γ (PPARγ) plays a role in the transcriptional regulation of metabolic pathways such as adipogenesis and β-oxidation of fatty acids. In this study we investigated the transcriptional regulation of MRAP expression during differentiation of 3T3-L1 cells. Stimulation with ACTH affected lipolysis in murine mature adipocytes via MRAP. Putative peroxisome proliferator response element (PPRE) was identified in the MRAP promoter region. In chromatin immunoprecipitation and reporter assays, we observed binding of PPARγ to the MRAP promoter. The mutagenesis experiments showed that the −1209/−1198 region of the MRAP promoter could function as a PPRE site. These results suggest that PPARγ is required for transcriptional activation of the MRAP gene during adipogenesis, which contributes to understanding of the molecular mechanism of lipolysis in adipocytes.

In vivo actions of peroxisome proliferator-activated receptors: glycemic control, insulin sensitivity, and insulin secretion.

Peroxisome proliferator–activated receptors (PPARs) form a family of nuclear hormone receptors involved in energy hemostasis and lipid metabolism (1,2) and include three isotypes encoded by different genes: PPARα (chromosome 22q12–13.1), PPARβ/δ (chromosome 6p21.2–21.1), and PPARγ (chromosome 3p25). PPARα was the first discovered and causes cellular peroxisome proliferation in rodent livers (3), giving this receptor family its name. Upon activation, PPARs interact with retinoid X receptor to create heterodimers, which bind to a specific DNA sequence motif termed peroxisome proliferator response element (4). Peroxisome proliferator response element usually appears in promoter regions and is constructed from repeats of nucleotide sequence AGGTCA separated by a single nucleotide. PPARα is widely expressed in tissues with high fatty acid catabolic activity: brown fat, heart, liver, kidney, and intestine (5). Upon activation by endogenous fatty acids and their derivatives, PPARα mediates fatty acid catabolism, gluconeogenesis, and ketone body synthesis, mainly in liver (6–9). In rodents, PPARα activation also influences immune modulation (10,11) and amino acid metabolism (12), reduces plasma triglyceride, reduces muscle and liver steatosis, and ameliorates insulin resistance (IR) (13,14). Pharmacologic PPARα activation is achieved by fibrates (7) and results in reduced (30–50%) triglyceride and VLDL levels by increasing lipid uptake, lipoprotein lipase–mediated lipolysis, and β-oxidation (15). This is accompanied by a modest increase in HDL cholesterol (5–20%), secondary to transcriptional induction of apolipoprotein A-I/A-II synthesis in liver (15). In man, the primary effect of PPARα is to reduce plasma triglyceride concentration; effects on plasma free fatty acid (FFA) concentration/FFA oxidation, muscle/liver fat content, and muscle/hepatic insulin sensitivity have not been demonstrated with current PPARα agonists such as fenofibrate (16,17). Fibrates are used to treat severe hypertriglyceridemia and combined hyperlipidemia (18–20). Clinical trials to establish a role for PPARα agonists (fenofibrate, gemfibrozil) …

Insulin Receptor-Overexpressing β-Cells Ameliorate Hyperglycemia in Diabetic Rats through Wnt Signaling Activation

To investigate the therapeutic efficacy and mechanism of β-cells with insulin receptor (IR) overexpression on diabetes mellitus (DM), rat insulinoma (INS-1) cells were engineered to stably express human insulin receptor (INS-IR cells), and subsequently transplanted into streptozotocin- induced diabetic rats. Compared with INS-1 cells, INS-IR cells showed improved β-cell function, including the increase in glucose utilization, calcium mobilization, and insulin secretion, and exhibited a higher rate of cell proliferation, and maintained lower levels of blood glucose in diabetic rats. These results were attributed to the increase of β-catenin/PPARγ complex bindings to peroxisome proliferator response elements in rat glucokinase (GK) promoter and the prolongation of S-phase of cell cycle by cyclin D1. These events resulted from more rapid and higher phosphorylation levels of insulin-signaling intermediates, including insulin receptor substrate (IRS)-1/IRS-2/phosphotylinositol 3 kinase/v-akt murine thymoma viral oncogene homolog (AKT) 1, and the consequent enhancement of β-catenin nuclear translocation and Wnt responsive genes including GK and cyclin D1. Indeed, the higher functionality and proliferation shown in INS-IR cells were offset by β-catenin, cyclin D1, GK, AKT1, and IRS-2 gene depletion. In addition, the promotion of cell proliferation and insulin secretion by Wnt signaling activation was shown by 100 nM insulin treatment, and to a similar degree, was shown in INS-IR cells. In this regard, this study suggests that transferring INS-IR cells into diabetic animals is an effective and feasible DM treatment. Accordingly, the method might be a promising alternative strategy for treatment of DM given the adverse effects of insulin among patients, including the increased risk of modest weight gain and hypoglycemia. Additionally, this study demonstrates that the novel mechanism of cross-talk between insulin and Wnt signaling plays a primary role in the higher therapeutic efficacy of IR-overexpressing β-cells.

Nutrient-dependent phosphorylation channels lipid synthesis to regulate PPARα

Peroxisome proliferator-activated receptor (PPAR)α is a nuclear receptor that coordinates liver metabolism during fasting. Fatty acid synthase (FAS) is an enzyme that stores excess calories as fat during feeding, but it also activates hepatic PPARα by promoting synthesis of an endogenous ligand. Here we show that the mechanism underlying this paradoxical relationship involves the differential regulation of FAS in at least two distinct subcellular pools: cytoplasmic and membrane-associated. In mouse liver and cultured hepatoma cells, the ratio of cytoplasmic to membrane FAS-specific activity was increased with fasting, indicating higher cytoplasmic FAS activity under conditions associated with PPARα activation. This effect was due to a nutrient-dependent and compartment-selective covalent modification of FAS. Cytoplasmic FAS was preferentially phosphorylated during feeding or insulin treatment at Thr-1029 and Thr-1033, which flank a dehydratase domain catalytic residue. Mutating these sites to alanines promoted PPARα target gene expression. Rapamycin-induced inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1), a mediator of the feeding/insulin signal to induce lipogenesis, reduced FAS phosphorylation, increased cytoplasmic FAS enzyme activity, and increased PPARα target gene expression. Rapamycin-mediated induction of the same gene was abrogated with FAS knockdown. These findings suggest that hepatic FAS channels lipid synthesis through specific subcellular compartments that allow differential gene expression based on nutritional status.

Human mannose-binding lectin 2 is directly regulated by peroxisome proliferator-activated receptors via a peroxisome proliferator responsive element

Human mannose-binding lectin (MBL) is encoded by the MBL2 gene and is a key player in innate immunity. However, the mechanism of the transcriptional regulation of MBL2 is largely unknown. The peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that play an important role in a number of biological responses, including lipid homeostasis, immune function and adipogenesis. In this study, we showed that PPARα and PPARγ up-regulate the expression of human MBL2. Using a luciferase assay, electrophoretic mobility-shift assay and chromatin immunoprecipitation assay, we demonstrated that PPARs regulate the expression of human MBL2 via the peroxisome proliferator responsive element (PPRE). On the other hand, MBL2 mRNA expression was not affected by the PPARα ligand both in vivo in rat liver and in vitro in rat H4IIE hepatoma cells. Thus, there is a species difference in regulation of MBL2 gene expression by PPARs between humans and rodents. We also show that the species differences in response to PPAR could be due in part to sequence-specific differences in the PPRE in the promoter region of MBL2. These results indicate that human, but not rat, MBL2 expression is regulated by PPARs via a PPRE.

FAM3A is a target gene of peroxisome proliferator-activated receptor gamma

BackgroundTo date, the biological function of FAM3A, the first member of FAM3 gene family, remains unknown. We aimed to investigate whether the expression of FAM3A in liver cells is regulated by peroxisome proliferator-activated receptors (PPARs). Methods and resultsThe transcriptional activity of human and mouse FAM3A gene promoters was determined by luciferase reporter assay system. PPARγ agonist rosiglitazone induced FAM3A expression in primary cultured mouse hepatocytes and human HepG2 cells. PPARγ antagonism blocked rosiglitazone-induced FAM3A expression, whereas PPARγ overexpression stimulated FAM3A expression in HepG2 cells. In contrast, PPARα agonist fenofibrate or PPARβ agonist GW0742 failed to affect FAM3A expression in HepG2 cells. The transcriptional activities of human and mouse FAM3A promoters were markedly stimulated by PPARγ activation, but not by PPARα and PPARβ activation. Chromatin immunoprecipitation (ChIP) assay revealed a direct binding of PPARγ to the putative peroxisome proliferator response element (PPRE) located at −1258/−1246 in the human FAM3A promoter. Site-directed mutagenesis of this PPRE-like motif abolished PPARγ's stimulatory effect on the transcriptional activity of human FAM3A promoter. In vivo, oral rosiglitazone treatment upregulated FAM3A expression in the livers of C57BL/6 mice and db/db mice. Moreover, upregulation of FAM3A by PPARγ activation was correlated with increased level of phosphorylated Akt (pAkt) in liver cells. ConclusionsFAM3A as a novel target gene of PPARγ. Upregulation of FAM3A by PPARγ activation is correlated with increased pAkt level in liver cells. General significanceUpregulation of FAM3A might contribute to PPARγ's metabolic effects in the liver.

A multi-assay screening approach for assessment of endocrine-active contaminants in wastewater effluent samples

Environmental agencies must monitor an ever increasing range of contaminants of emerging concern, including endocrine disrupting compounds (EDCs). An alternative to using ultra-trace chemical analysis of samples for EDCs is to test for biological activity using in vitro screening assays, then use these assay results to direct analytical chemistry approaches. In this study, we used both analytical approaches and in vitro bioassays to characterize the EDCs present in treated wastewater from four wastewater treatment plants (WWTPs) in Ontario, Canada. Estrogen-mediated activity was assessed using a yeast estrogenicity screening (YES) assay. An in vitro competitive binding assay was used to assess capacity to interfere with binding of the thyroid hormone, thyroxine (T4) to the recombinant human thyroid hormone transport protein, transthyretin (i.e. hTTR). An in vitro binding assay with a rat peroxisome proliferator responsive element transfected into a rainbow trout gill cell line was used to evaluate binding and subsequent gene expression via the peroxisome proliferator activated receptor (PPAR). Analyses of a suite of contaminants known to be EDCs in extracts from treated wastewater were conducted using either gas chromatography with mass spectrometry (GC-MS) or liquid chromatography with tandem mass spectrometry (LC-MS/MS). Estrogenic activity was detected in the YES assay only in those extracts that contained detectable amounts of estradiol (E2). There was a positive relationship between the degree of response in the T4-hTTR assay and the amounts of polybrominated diphenyl ether (PBDE) congeners 47 and 99, triclosan and the PBDE metabolite, 4-OH-BDE17. Several wastewater extracts gave a positive response in the PPAR assay, but these responses were not correlated with the amounts of any of the EDCs analyzed by LC-MS/MS. Overall, these data indicate that a step-wise approach is feasible using a combination of in vitro testing and instrumental analysis to monitor for EDCs in wastewater and other environmental matrixes.

Tissue-specific transcriptional modulation of fatty acid-binding protein genes, fabp2, fabp3 and fabp6, by fatty acids and the peroxisome proliferator, clofibrate, in zebrafish (Danio rerio)

All fabp genes, except fabp2, fabp3 and fabp6, exist as duplicates in the zebrafish genome owing to a whole genome duplication event ~230–400million years ago. Transcription of some duplicated fabp genes is modulated by fatty acids (FAs) and/or clofibrate, a peroxisome proliferator-activated receptor (PPAR) agonist. We had also shown previously that the steady-state level of acyl-CoA oxidase 1 (acox1) mRNA, a marker of PPARα activation, was elevated in liver, intestine, heart and muscle of fish fed clofibrate demonstrating that zebrafish, unlike some fishes, is responsive to this drug. acox1 transcripts were not induced in the brain of fish fed clofibrate, which suggests this drug may not cross the blood brain barrier. Here, we investigated the effect of dietary FAs and clofibrate on the transcription of single copy fabp genes, fabp2, fabp3 and fabp6, in five tissues of inbred zebrafish. The steady-state level of fabp2 transcripts increased in intestine, while fabp3 mRNA increased in liver of fish fed diets differing in FA content. In fish fed clofibrate, fabp3 mRNA in intestine, and fabp6 mRNA in intestine and heart, were elevated. Based on these findings, modulation of fabp2, fabp3 and fabp6 transcription by FAs and/or clofibrate in zebrafish implicates control of these genes by PPAR interaction with peroxisome proliferator response elements (PPRE) most likely in fabp promoters. Moreover, transcriptional induction of these fabp genes by dietary FAs and/or clofibrate is over-ridden by a tissue-specific mechanism(s), e.g., transcriptional activator or repressor proteins.