PPARα/RXRα downregulates amino acid catabolism in the liver via interaction with HNF4α promoting its proteasomal degradation.

The preservation of body proteins is essential to guarantee their functions in organisms. Therefore, the utilization of amino acids as energy substrates is regulated by a precise fine-tuned mechanism. Recent evidence suggests that the transcription factors peroxisome proliferator-activated receptor alpha (PPARα) and hepatocyte nuclear factor 4 alpha (HNF4α) are involved in this regulatory mechanism. Thus, the aim of this study was to determine how these transcription factors interact to regulate the expression of amino acid catabolism genes. In vivo studies using PPARα-knockout mice (Pparα-null) fed different amounts of dietary protein showed that in the absence of PPARα, there was a significant increase in HNF4α abundance in the liver, which corresponded with an increase in amino acid catabolizing enzyme (AACE) expression and the generation of increased amounts of postprandial urea. Moreover, this effect was proportional to the increase in dietary protein consumed. Chromatin immunoprecipitation assays showed that HNF4α can bind to the promoter of AACE serine dehydratase (SDS), an effect that was potentiated by dietary protein in the Pparα-null mice. The mechanistic studies revealed that the presence of retinoid X receptor alpha (RXRα) is essential to repress HNF4α activity in the presence of PPARα, and this interaction accelerates HNF4α degradation via the proteasome pathway. These results showed that PPARα can downregulate liver amino acid catabolism in the presence of RXRα by inhibiting HNF4α activity.

PPARα Inhibition Overcomes Tumor-Derived Exosomal Lipid-Induced Dendritic Cell Dysfunction.

Dendritic cells (DCs) orchestrate the initiation, programming, and regulation of anti-tumor immune responses. Emerging evidence indicates that the tumor microenvironment (TME) induces immune dysfunctional tumor-infiltrating DCs (TIDCs), characterized with both increased intracellular lipid content and mitochondrial respiration. The underlying mechanism, however, remains largely unclear. Here, we report that fatty acid-carrying tumor-derived exosomes (TDEs) induce immune dysfunctional DCs to promote immune evasion. Mechanistically, peroxisome proliferator activated receptor (PPAR) α responds to the fatty acids delivered by TDEs, resulting in excess lipid droplet biogenesis and enhanced fatty acid oxidation (FAO), culminating in a metabolic shift toward mitochondrial oxidative phosphorylation, which drives DC immune dysfunction. Genetic depletion or pharmacologic inhibition of PPARα effectively attenuates TDE-induced DC-based immune dysfunction and enhances the efficacy of immunotherapy. This work uncovers a role for TDE-mediated immune modulation in DCs and reveals that PPARα lies at the center of metabolic-immune regulation of DCs, suggesting a potential immunotherapeutic target.

The critical role of PPARα in the binary switch between life and death induced by endoplasmic reticulum stress.

Endoplasmic reticulum stress (ER stress) just like a double-edged sword depending on different conditions in the development of multiple hepatic diseases. But the molecular mechanisms of functional conversion during ER stress have not been fully elucidated. In this study, we aim to illustrate the role of PPARα and the subtle mechanism in the functional conversion of ER stress. Tunicamycin (TM) and thapsigargin (TG), as ER stress inducers, were used to induce ER stress in AML12 cells. During the ER stress, qRT-PCR and immunoblotting was used to measure the expression levels of GRP78 and CHOP which show a gradually increasing trend, while PPARα and autophagy was significantly activated in the early stage but was inhibited in the late stage. Moreover, PPARα inhibition by siRNA promoted cell injury in the mild-ER stress and PPARα activation by WY-14643 reduced cell apoptosis in the serious ER stress. In the mild-ER stress with PPARα knocked down, activation of autophagy by rapamycin significantly improved cell survival, in the serious ER stress with PPARα activation, inhibition of autophagy by 3-MA aggravate cell injury. In addition, in the mild-ER stress with PPARα knocked down, CHOP knocked down by siRNA reduced cell apoptosis, in the serious ER stress activated PPARα, CHOP over-expression mediated by lentiviral vector contributed to serious cell injury. Furthermore, C57BL/6 mice was used to induce ER stress with TM intraperitoneal injection, PPARα and autophagy was upregulated in the mild-ER stress while downregulated in the serious ER stress, measured by qRT-PCR and immunoblotting, further confirmed the finding in vitro. Our results firstly demonstrated that PPARα is a key molecule in the functional conversion of ER stress: protective effects in the mild ER stress was mediated by PPARα-autophagy pathway and destructive effects in the serious ER stress was mediated by PPARα-CHOP pathway.

Moderate aerobic exercise-induced cytokines changes are disturbed in PPARα knockout mice.

The nuclear transcriptional factor peroxisome proliferator activated receptor alpha (PPARα) plays a role in regulating genes involved in lipid metabolism, adipogenesis and inflammation. We aimed to assess the role of PPARα on exercise-mediated locally produced cytokines in adipose fat deposits and skeletal muscle. C57BL/6 (WT) and PPARα knockout (PPARα-/-) mice were examined. Each genotype was randomly subdivided into three groups: non-exercised, and euthanized 2 or 24 h after a moderate aerobic exercise session (run on a treadmill at 60% of maximum speed for 1 h). Fat content in gastrocnemius muscle and lipolytic activity in isolated adipose tissue from mesenteric (MEAT) and retroperitoneal (RPAT) adipose tissue were evaluated. In addition, Interleukin 6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor α (TNF-α) and monocyte chemoattractant protein 1 (MCP-1) content were evaluated by ELISA. WT mice showed a maximum lipolysis rate, as well as higher IL-6, IL-10, and IL10/TNF-α ratio values 2 h post-exercise (RPAT only) compared with PPARα-/- mice. Taken together, our data suggests that PPARα knockout mice exhibited reduced lipolysis and anti-inflammatory response in adipose tissue following exercise, PPARα appears to play an important role in immunomodulatory and lipolysis signaling after acute moderate exercise.

Energy Metabolism in Cellular Regenerative Processes: Focus on PPARα.

Reduced expression of the key regulator of cardiac metabolism, transcription factor PPARα, in surgical samples of the auricles from patients with coronary heart disease and heart failure was detected by real-time quantitative PCR. These changes indicate reduced activity of this factor and a shift of energy metabolism from oxidative phosphorylation to glycolysis typical of dedifferentiated cells. Electron microscopy revealed dedifferentiated cardiomyocytes with disassembled contractile apparatus and disorganized sarcomeres. In the examined specimens from patients with heart failure, severe myocardial fibrosis was revealed.

M3 muscarinic receptor activation reduces hepatocyte lipid accumulation via CaMKKß/AMPK pathway.

Previously, we reported that hepatic muscarinic receptors modulate both acute and chronic liver injury, however, the role of muscarinic receptors in fatty liver disease is unclear. We observed in patients who underwent weight loss surgery, a decrease in hepatic expression of M3 muscarinic receptors (M3R). We also observed that fat loading of hepatocytes, increased M3R expression. Based on these observations, we tested the hypothesis that M3R regulate hepatocyte lipid accumulation. Incubation of AML12 hepatocytes with 1 mM oleic acid resulted in lipid accumulation that was significantly reduced by co-treatment with a muscarinic agonist (pilocarpine or carbachol), an effect blocked by atropine (a muscarinic antagonist). Similar treatment of Hepa 1-6 cells, a mouse hepatoblastoma cell line, showed comparable results. In both, control and fat-loaded AML12 cells, pilocarpine induced time-dependent AMPKα phosphorylation and significantly up-regulated lipolytic genes (ACOX1, CPT1, and PPARα). Compound C, a selective and reversible AMPK inhibitor, significantly blunted pilocarpine-mediated reduction of lipid accumulation and pilocarpine-mediated up-regulation of lipolytic genes. BAPTA-AM, a calcium chelator, and STO-609, a calcium/calmodulin-dependent protein kinase kinase inhibitor, attenuated agonist-induced AMPKα phosphorylation. Finally, M3R siRNA attenuated agonist-induced AMPKα phosphorylation as well as agonist-mediated reduction of hepatocyte steatosis. In conclusion, this proof-of-concept study demonstrates that M3R has protective effects against hepatocyte lipid accumulation by activating AMPK pathway and is a potential therapeutic target for non-alcoholic fatty liver disease.

Chronic oleoylethanolamide treatment attenuates diabetes-induced mice encephalopathy by triggering peroxisome proliferator-activated receptor alpha in the hippocampus.

Brain is a site of diabetic end-organ damage. Diabetes-associated cognitive dysfunction, referred as "diabetic encephalopathy" (DE) has been coined for the patients with type 2 diabetes mellitus showing decline in their cognitive function, especially weak episodic memory, cognitive inflexibility and poor psychomotor performance leading towards Alzheimer's disease. Current evidence supported that aberrant synapses, energy metabolism imbalance, advanced glycation end products (AGEs) accumulation and Tau hyperphosphorylation are associated with cognition deficits induced by diabetes. Oleoylethanolamide (OEA), an endogenous peroxisome proliferator-activated receptor alpha (PPARα) agonist, has anti-hyperlipidemia, anti-inflammatory and neuroprotective activities. However, the effect of OEA on DE is unknown. Therefore, we tested its influence against cognitive dysfunction in high fat diet and streptozotocin (HFD + STZ)-induced diabetic C57BL/6J and PPARα--/- mice using Morris water maze (MWM) test. Neuron staining, dementia markers and neuroplasticity in the hippocampus were assessed to evaluate the neuropathological changes. The results showed that chronic OEA treatment significantly lowered hyperglycemia, recovered cognitive performance, reduced dementia markers, and inhibited hippocampal neuron loss and neuroplasticity impairments in diabetic mice. In contrast, the changes in MWM performance and neuron loss were not observed in PPARα knockout mice via OEA administration. These results indicated that OEA may provide a potential alternative therapeutic for DE by activating PPARα signaling.

Effects of PPARs/20-HETE on the renal impairment under diabetic conditions.

Diabetic nephropathy (DN) is one of the most severe complications of diabetes mellitus. The pathomolecular events behind DN remain uncertain. Peroxisome proliferator-activated receptors (PPARs) play essential functions in the development of DN. Meanwhile, 20-hydroxyeicosatetraenoic acid (20-HETE) also plays central roles in the regulation of renal function. However, the relationship between PPARs and 20-HETE is rarely studied in DN. It was revealed in our study that both PPARs expression and CYP4A-20-HETE level were decreased under DN conditions in vivo and in vitro. Supplementation with bezafibrate, a PPAR pan-agonist, improved the damage of kidney in DN mice and in high glucose-induced NRK-52E cells, following the up-regulation of PPARs and the increase of CYP4A-20-HETE. PPARα antagonist (MK886), PPARß antagonist (GSK0660), and PPARγ antagonist (GW9662) reversed the protection of bezafibrate in NRK-52E, and abrogated the up-regulation of CYP4A-20-HETE produced by bezafibrate. Noteworthily, 20-HETE synthetase inhibitor, HET0016, also blocked the bezafibrate-mediated improvement of NRK-52E, and abolished the up-regulation of PPARs expression. Collectively, our data suggest that the concurrent down-regulation and interaction of PPARs and 20-HETE play crucial roles in the pathogenesis process of DN, and we provide a novel evidence that PPARs/20-HETE signaling may be served as a therapeutic target for DN patients.

Hepatic PPARα is critical in the metabolic adaptation to sepsis.

BACKGROUND & AIMS: Although the role of inflammation to combat infection is known, the contribution of metabolic changes in response to sepsis is poorly understood. Sepsis induces the release of lipid mediators, many of which activate nuclear receptors such as the peroxisome proliferator-activated receptor (PPAR)α, which controls both lipid metabolism and inflammation. We aimed to elucidate the previously unknown role of hepatic PPARα in the response to sepsis. METHODS: Sepsis was induced by intraperitoneal injection of Escherichia coli in different models of cell-specific Ppara-deficiency and their controls. The systemic and hepatic metabolic response was analyzed using biochemical, transcriptomic and functional assays. PPARα expression was analyzed in livers from elective surgery and critically ill patients and correlated with hepatic gene expression and blood parameters. RESULTS: Both whole body and non-hematopoietic Ppara-deficiency in mice decreased survival upon bacterial infection. Livers of septic Ppara-deficient mice displayed an impaired metabolic shift from glucose to lipid utilization resulting in more severe hypoglycemia, impaired induction of hyperketonemia and increased steatosis due to lower expression of genes involved in fatty acid catabolism and ketogenesis. Hepatocyte-specific deletion of PPARα impaired the metabolic response to sepsis and was sufficient to decrease survival upon bacterial infection. Hepatic PPARA expression was lower in critically ill patients and correlated positively with expression of lipid metabolism genes, but not with systemic inflammatory markers. CONCLUSION: During sepsis, Ppara-deficiency in hepatocytes is deleterious as it impairs the adaptive metabolic shift from glucose to FA utilization. Metabolic control by PPARα in hepatocytes plays a key role in the host defense against infection. LAY SUMMARY: As the main cause of death in critically ill patients, sepsis remains a major health issue lacking efficacious therapies. While current clinical literature suggests an important role for inflammation, metabolic aspects of sepsis have mostly been overlooked. Here, we show that mice with an impaired metabolic response, due to deficiency of the nuclear receptor PPARα in the liver, exhibit enhanced mortality upon bacterial infection despite a similar inflammatory response, suggesting that metabolic interventions may be a viable strategy for improving sepsis outcomes.

ß-catenin-activated hepatocellular carcinomas are addicted to fatty acids.

OBJECTIVES: CTNNB1-mutated hepatocellular carcinomas (HCCs) constitute a major part of human HCC and are largely inaccessible to target therapy. Yet, little is known about the metabolic reprogramming induced by ß-catenin oncogenic activation in the liver. We aimed to decipher such reprogramming and assess whether it may represent a new avenue for targeted therapy of CTNNB1-mutated HCC. DESIGN: We used mice with hepatocyte-specific oncogenic activation of ß-catenin to evaluate metabolic reprogramming using metabolic fluxes on tumourous explants and primary hepatocytes. We assess the role of Pparα in knock-out mice and analysed the consequences of fatty acid oxidation (FAO) using etomoxir. We explored the expression of the FAO pathway in an annotated human HCC dataset. RESULTS: ß-catenin-activated HCC were not glycolytic but intensively oxidised fatty acids. We found that Pparα is a ß-catenin target involved in FAO metabolic reprograming. Deletion of Pparα was sufficient to block the initiation and progression of ß-catenin-dependent HCC development. FAO was also enriched in human CTNNB1-mutated HCC, under the control of the transcription factor PPARα. CONCLUSIONS: FAO induced by ß-catenin oncogenic activation in the liver is the driving force of the ß-catenin-induced HCC. Inhibiting FAO by genetic and pharmacological approaches blocks HCC development, showing that inhibition of FAO is a suitable therapeutic approach for CTNNB1-mutated HCC.

Pparα deficiency inhibits the proliferation of neuronal and glial precursors in the zebrafish central nervous system.

BACKGROUND: Many molecules and signaling pathways involved in neural development play a role in neurodegenerative diseases and brain tumor progression. Peroxisome proliferator-activated receptor (PPAR) proteins regulate the differentiation of tissues and the progression of many diseases. However, the role of these proteins in neural development is unclear. RESULTS: We examined the function of Pparα in the neural development of zebrafish. Two duplicate paralogs for mammalian PPARA/Ppara, namely pparaa and pparab, are present in the zebrafish genome. Both pparaa and pparab are expressed in the developing central nervous system in zebrafish embryos. Inhibiting the function of Pparα by using either the PPARα/Pparα antagonist GW6471 or pparaa or pparab truncated constructs produced identical phenotypes, which were sufficient to reduce the proliferation of neuronal and glial precursor cells without affecting the formation of neural progenitors. CONCLUSIONS: We demonstrated that both Pparαa and Pparαb proteins are essential regulators of the proliferation of neuronal and glial precursors. This study provides a better understanding of the functions of PPARα/Pparα in neural development and further expands our knowledge of the potential role of PPARα/Pparα in neurological disorders and brain tumors. Developmental Dynamics 247:1264-1275, 2018. © 2018 Wiley Periodicals, Inc.

PPARα Is Necessary for Radiation-Induced Activation of Noncanonical TGFß Signaling in the Heart.

High-dose ionizing radiation is known to induce adverse effects such as inflammation and fibrosis in the heart. Transcriptional regulators PPARα and TGFß are known to be involved in this radiation response. PPARα, an anti-inflammatory transcription factor controlling cardiac energy metabolism, is inactivated by irradiation. The pro-inflammatory and pro-fibrotic TGFß is activated by irradiation via SMAD-dependent and SMAD-independent pathways. The goal of this study was to investigate how altering the level of PPARα influences the radiation response of these signaling pathways. For this purpose, we used genetically modified C57Bl/6 mice with wild type (+/+), heterozygous (+/-) or homozygous (-/-) PPARα genotype. Mice were locally irradiated to the heart using doses of 8 or 16 Gy; the controls were sham-irradiated. The heart tissue was investigated using label-free proteomics 20 weeks after the irradiation and the predicted pathways were validated using immunoblotting, ELISA, and immunohistochemistry. The heterozygous PPARα mice showed most radiation-induced changes in the cardiac proteome, whereas the homozygous PPARα mice showed the least changes. Irradiation induced SMAD-dependent TGFß signaling independently of the PPARα status, but the presence of PPARα was necessary for the activation of the SMAD-independent pathway. These data indicate a central role of PPARα in cardiac response to ionizing radiation.

The role and regulation of the peroxisome proliferator activated receptor alpha in human liver.

The peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated transcription factor that is abundantly expressed in liver. PPARα is activated by fatty acids and various other lipid species, as well as by a class of chemicals referred to as peroxisome proliferators. Studies in mice have shown that PPARα serves as the master regulator of hepatic lipid metabolism during fasting. In addition, PPARα suppresses inflammation and the acute phase response. Comparatively little is known about PPARα in human liver. Here, an overview is provided of the role and regulation of PPARα in human liver. The main outcomes are: 1) the level of PPARA mRNA expression in human and mouse liver is similar. 2) Expression of PPARA in human liver is reduced in patients with non-alcoholic steatohepatitis or infected with the hepatitis C virus. 3) PPARα in human liver is able to effectively induce the expression of numerous genes involved in numerous lipid metabolic pathways, including microsomal, peroxisomal and mitochondrial fatty acid oxidation, fatty acid binding and activation, fatty acid elongation and desaturation, synthesis and breakdown of triglycerides and lipid droplets, lipoprotein metabolism, gluconeogenesis, bile acid metabolism, and various other metabolic pathways and genes. 4) PPARα activation in human liver causes the down-regulation of a large number of genes involved in various immunity-related pathways. 5) Peroxisome proliferators do not promote tumour formation in human liver as opposed to mouse liver because of structural and functional differences between human and mouse PPARα. 6) In addition to helping to correct dyslipidemia, PPARα agonists may hold promise as a therapy for patients with cholestatic liver diseases, non-alcoholic fatty liver disease, and/or type 2 diabetes.

PPAR-α, a lipid-sensing transcription factor, regulates blood-brain barrier efflux transporter expression.

Lipid sensor peroxisome proliferator-activated receptor alpha (PPAR- α) is the master regulator of lipid metabolism. Dietary release of endogenous free fatty acids, fibrates, and certain persistent environmental pollutants, e.g. perfluoroalkyl fire-fighting foam components, are peroxisome proliferator-activated receptor alpha ligands. Here, we define a role for peroxisome proliferator-activated receptor alpha in regulating the expression of three ATP-driven drug efflux transporters at the rat and mouse blood-brain barriers: P-glycoprotein (Abcb1), breast cancer resistance protein (Bcrp/Abcg2), and multidrug resistance-associated protein 2 (Mrp2/Abcc2). Exposing isolated rat brain capillaries to linoleic acid, clofibrate, or PKAs increased the transport activity and protein expression of the three ABC transporters. These effects were blocked by the PPAR- α antagonist, GW6471. Dosing rats with 20 mg/kg or 200 mg/kg of clofibrate decreased the brain accumulation of the P-glycoprotein substrate, verapamil, by 50% (in situ brain perfusion; effects blocked by GW6471) and increased P-glycoprotein expression and activity in capillaries ex vivo. Fasting C57Bl/6 wild-type mice for 24 h increased both serum lipids and brain capillary P-glycoprotein transport activity. Fasting did not alter P-glycoprotein activity in PPAR- α knockout mice. These results indicate that hyperlipidemia, lipid-lowering fibrates and exposure to certain fire-fighting foam components activate blood-brain barrier peroxisome proliferator-activated receptor alpha, increase drug efflux transporter expression and reduce drug delivery to the brain.

Roles of Peroxisome Proliferator-Activated Receptor α in Bitter Melon Seed Oil-Corrected Lipid Disorders and Conversion of α-Eleostearic Acid into Rumenic Acid in C57BL/6J Mice.

We previously reported that bitter melon seed oil (BMSO) was an effective anti-steatosis and antiobesity agent. Since the major fatty acid α-eleostearic acid (α-ESA) in BMSO is a peroxisome proliferator-activated receptor α (PPARα) activator, the objective was to investigate the role of PPARα in BMSO-modulated lipid disorders and α-ESA metabolism. C57BL/6J wild (WD) and PPARα knockout (KO) mice were fed a high-fat diet containing BMSO (15% soybean oil + 15% BMSO, HB) or not (30% soybean oil, HS) for 5 weeks. The HB diet significantly reduced hepatic triglyceride concentrations and increased acyl-CoA oxidase activity in WD, but not in KO mice. However, regardless of genotype, body fat percentage was lowered along with upregulated protein levels of uncoupling protein 1 (UCP1) and tyrosine hydroxylase, as well as signaling pathway of cAMP-dependent protein kinase and AMP-activated protein kinase in the white adipose tissue of HB-treated groups compared to HS cohorts. In WD-HB and KO-HB groups, white adipose tissue had autophagy, apoptosis, inflammation, and browning characteristics. Without PPARα, in vivo reduction of α-ESA into rumenic acid was slightly but significantly lowered, along with remarkable reduction of hepatic retinol saturase (RetSat) expression. We concluded that BMSO-mediated anti-steatosis depended on PPARα, whereas the anti-adiposity effect was PPARα-independent. In addition, PPARα-dependent enzymes may participate in α-ESA conversion, but only have a minor role.

Pathway Based Toxicology and Fit-for-Purpose Assays.

The field of toxicity testing for non-pharmaceutical chemicals is in flux with multiple initiatives in North America and the EU to move away from animal testing to mode-of-action based in vitro assays. In this arena, there are still obstacles to overcome, such as developing appropriate cellular assays, creating pathway-based dose-response models and refining in vitro-in vivo extrapolation (IVIVE) tools. Overall, it is necessary to provide assurances that these new approaches are adequately protective of human and ecological health. Another major challenge for individual scientists and regulatory agencies is developing a cultural willingness to shed old biases developed around animal tests and become more comfortable with mode-of-action based assays in human cells. At present, most initiatives focus on developing in vitro alternatives and assessing how well these alternative methods reproduce past results related to predicting organism level toxicity in intact animals. The path forward requires looking beyond benchmarking against high dose animal studies. We need to develop targeted cellular assays, new cell biology-based extrapolation models for assessing regions of safety for chemical exposures in human populations, and mode-of-action-based approaches which are constructed on an understanding of human biology. Furthermore, it is essential that assay developers have the flexibility to 'validate' against the most appropriate mode-of-action data rather than against apical endpoints in high dose animal studies. This chapter demonstrates the principles of fit-for-purpose assay development using pathway-targeted case studies. The projects include p53-mdm2-mediated DNA-repair, estrogen receptor-mediated cell proliferation and PPARα receptor-mediated liver responses.

PPAR Agonists: I. Role of Receptor Subunits in Alcohol Consumption in Male and Female Mice.

BACKGROUND: Several peroxisome proliferator-activated receptor (PPAR) agonists reduce voluntary alcohol consumption in rodent models, and evidence suggests that PPARα and γ subunits play an important role in this effect. To define the subunit dependence of this action, we tested selective PPARα and α/γ agonists and antagonists in addition to null mutant mice lacking PPARα. METHODS: The effects of fenofibrate (PPARα agonist) and tesaglitazar (PPARα/γ agonist) on continuous and intermittent 2-bottle choice drinking tests were examined in male and female wild-type mice and in male mice lacking PPARα. We compared the ability of MK886 (PPARα antagonist) and GW9662 (PPARγ antagonist) to inhibit the effects of fenofibrate and tesaglitazar in wild-type mice. The estrogen receptor antagonist, tamoxifen, can inhibit PPARγ-dependent transcription and was also studied in male and female mice. RESULTS: Fenofibrate and tesaglitazar reduced ethanol (EtOH) consumption and preference in wild-type mice, but these effects were not observed in mice lacking PPARα. MK886 inhibited the action of fenofibrate, but not tesaglitazer, while GW9662 did not inhibit either agonist. The PPAR agonists were more effective in male mice compared to females, and drinking in the continuous 2-bottle choice test was more sensitive to fenofibrate and tesaglitazar compared to drinking in the intermittent access test. Tamoxifen also reduced EtOH consumption in male mice and this action was inhibited by GW9662, but not MK886, suggesting that it acts by activation of PPARγ. CONCLUSIONS: Our study using selective PPAR agonists, antagonists, and null mutant mice indicates a key role for PPARα in mediating reduced EtOH consumption by fenofibrate and tesaglitazar.

Conjugated linoleic acids influence fatty acid metabolism in ovine ruminal epithelial cells.

Conjugated linoleic acids (CLA), particularly cis-9,trans-11 (c9t11) and trans-10,cis-12 (t10c12), are used as feed additives to adapt to constantly increasing demands on the performance of lactating cows. Under these feeding conditions, the rumen wall, and the rumen epithelial cells (REC) in particular, are directly exposed to high amounts of CLA. This study determined the effect of CLA on the fatty acid (FA) metabolism of REC and expression of genes known to be modulated by FA. Cultured REC were incubated with c9t11, t10c12, and the structurally similar FA linoleic acid (LA), oleic acid (OA), and trans-vaccenic acid (TVA) for 48 h at a concentration of 100 µM. Cellular FA levels were determined by gas chromatography. Messenger RNA expression levels of stearoyl-CoA desaturase (SCD) and monocarboxylate transporter (MCT) 1 and 4 were quantified by reverse transcription-quantitative PCR. Fatty acid evaluation revealed significant effects of CLA, LA, OA, and TVA on the amount of FA metabolites of ß-oxidation and elongation and of metabolites related to desaturation by SCD. The observed changes in FA content point (among others) to the ability of REC to synthesize c9t11 from TVA endogenously. The mRNA expression levels of SCD identified a decrease after CLA, LA, OA, or TVA treatment. In line with the changes in mRNA expression, we found reduced amounts of C16:1n-7 cis-9 and C18:1n-9 cis-9, the main products of SCD. The expression of MCT1 mRNA increased after c9t11 and t10c12 treatment, and CLA c9t11 induced an upregulation of MCT4. Application of peroxisome proliferator-activated receptor (PPAR) α antagonist suggested that activation of PPARα is involved in the changes of MCT1, MCT4, and SCD mRNA expression induced by c9t11. Participation of PPARγ in the changes of MCT1 and SCD mRNA expression was shown by the application of the respective antagonist. The study demonstrates that exposure to CLA affects both FA metabolism and regulatory pathways within REC.

Dose and Effect Thresholds for Early Key Events in a PPARα-Mediated Mode of Action.

Current strategies for predicting adverse health outcomes of environmental chemicals are centered on early key events in toxicity pathways. However, quantitative relationships between early molecular changes in a given pathway and later health effects are often poorly defined. The goal of this study was to evaluate short-term key event indicators using qualitative and quantitative methods in an established pathway of mouse liver tumorigenesis mediated by peroxisome proliferator-activated receptor alpha (PPARα). Male B6C3F1 mice were exposed for 7 days to di (2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DNOP), and n-butyl benzyl phthalate (BBP), which vary in PPARα activity and liver tumorigenicity. Each phthalate increased expression of select PPARα target genes at 7 days, while only DEHP significantly increased liver cell proliferation labeling index (LI). Transcriptional benchmark dose (BMDT) estimates for dose-related genomic markers stratified phthalates according to hypothetical tumorigenic potencies, unlike BMDs for non-genomic endpoints (relative liver weights or proliferation). The 7-day BMDT values for Acot1 as a surrogate measure for PPARα activation were 29, 370, and 676 mg/kg/day for DEHP, DNOP, and BBP, respectively, distinguishing DEHP (liver tumor BMD of 35 mg/kg/day) from non-tumorigenic DNOP and BBP. Effect thresholds were generated using linear regression of DEHP effects at 7 days and 2-year tumor incidence values to anchor early response molecular indicators and a later phenotypic outcome. Thresholds varied widely by marker, from 2-fold (Pdk4 and proliferation LI) to 30-fold (Acot1) induction to reach hypothetical tumorigenic expression levels. These findings highlight key issues in defining thresholds for biological adversity based on molecular changes.