Fanny Lalloyer

Genome-Wide Profiling of Liver X Receptor, Retinoid X Receptor, and Peroxisome Proliferator-Activated Receptor α in Mouse Liver Reveals Extensive Sharing of Binding Sites

ABSTRACT The liver X receptors (LXRs) are nuclear receptors that form permissive heterodimers with retinoid X receptor (RXR) and are important regulators of lipid metabolism in the liver. We have recently shown that RXR agonist-induced hypertriglyceridemia and hepatic steatosis in mice are dependent on LXRs and correlate with an LXR-dependent hepatic induction of lipogenic genes. To further investigate the roles of RXR and LXR in the regulation of hepatic gene expression, we have mapped the ligand-regulated genome-wide binding of these factors in mouse liver. We find that the RXR agonist bexarotene primarily increases the genomic binding of RXR, whereas the LXR agonist T0901317 greatly increases both LXR and RXR binding. Functional annotation of putative direct LXR target genes revealed a significant association with classical LXR-regulated pathways as well as peroxisome proliferator-activated receptor (PPAR) signaling pathways, and subsequent chromatin immunoprecipitation-sequencing (ChIP-seq) mapping of PPARα binding demonstrated binding of PPARα to 71 to 88% of the identified LXR-RXR binding sites. The combination of sequence analysis of shared binding regions and sequential ChIP on selected sites indicate that LXR-RXR and PPARα-RXR bind to degenerate response elements in a mutually exclusive manner. Together, our findings suggest extensive and unexpected cross talk between hepatic LXR and PPARα at the level of binding to shared genomic sites.

Fibrates, glitazones, and peroxisome proliferator-activated receptors

Several decades ago, fibrates were approved for the treatment of dyslipidemia, whereas thiazolidinediones were screened in animal models to improve glucose homeostasis and were subsequently developed for the treatment of type 2 diabetes mellitus. Relatively recently, these drugs were found to act via peroxisome proliferator–activated receptors, nuclear receptors that control lipid metabolism and glucose homeostasis. In this historical perspective, we discuss the history of discovery of the peroxisome proliferator–activated receptors, from the clinical development of their agonists to the subsequent discovery of these receptors and their mechanisms of action, to finally evoke possibilities of targeted pharmacology for future development of selective peroxisome proliferator–activated receptor modulators.

Peroxisome Proliferator–Activated Receptor α Improves Pancreatic Adaptation to Insulin Resistance in Obese Mice and Reduces Lipotoxicity in Human Islets

Peroxisome proliferator–activated receptor (PPAR) α is a transcription factor controlling lipid and glucose homeostasis. PPARα-deficient (−/−) mice are protected from high-fat diet–induced insulin resistance. However, the impact of PPARα in the pathophysiological setting of obesity-related insulin resistance is unknown. Therefore, PPARα−/− mice in an obese (ob/ob) background were generated. PPARα deficiency did not influence the growth curves of the obese mice but surprisingly resulted in a severe, age-dependent hyperglycemia. PPARα deficiency did not aggravate peripheral insulin resistance. By contrast, PPARα−/− ob/ob mice developed pancreatic β-cell dysfunction characterized by reduced mean islet area and decreased insulin secretion in response to glucose in vitro and in vivo. In primary human pancreatic islets, PPARα agonist treatment prevented fatty acid–induced impairment of glucose-stimulated insulin secretion, apoptosis, and triglyceride accumulation. These results indicate that PPARα improves the adaptative response of the pancreatic β-cell to pathological conditions. PPARα could thus represent a promising target in the prevention of type 2 diabetes.

Peroxisome Proliferator–Activated Receptor-α Gene Level Differently Affects Lipid Metabolism and Inflammation in Apolipoprotein E2 Knock-In Mice

Objective—Peroxisome proliferator–activated receptor-&agr; (PPAR&agr;) is a ligand-activated transcription factor that controls lipid metabolism and inflammation. PPAR&agr; is activated by fibrates, hypolipidemic drugs used in the treatment of dyslipidemia. Previous studies assessing the influence of PPAR&agr; agonists on atherosclerosis in mice yielded conflicting results, and the implication of PPAR&agr; therein has not been assessed. The human apolipoprotein E2 knock-in (apoE2-KI) mouse is a model of mixed dyslipidemia, atherosclerosis, and nonalcoholic steatohepatitis (NASH). The aim of this study was to analyze, using homo- and heterozygous PPAR&agr;-deficient mice, the consequences of quantitative variations of PPAR&agr; gene levels and their response to the synthetic PPAR&agr; agonist fenofibrate on NASH and atherosclerosis in apoE2-KI mice. Methods and Results—Wild-type (+/+), heterozygous (+/−), and homozygous (−/−) PPAR&agr;-deficient mice in the apoE2-KI background were generated and subjected to a Western diet supplemented with fenofibrate or not supplemented. Western diet–fed PPAR&agr;−/− apoE2-KI mice displayed an aggravation of liver steatosis and inflammation compared with PPAR&agr;+/+ and PPAR&agr;+/− apoE2-KI mice, indicating a role of PPAR&agr; in liver protection. Moreover, PPAR&agr; expression was required for the fenofibrate-induced protection against NASH. Interestingly, fenofibrate treatment induced a similar response on hepatic lipid metabolism in PPAR&agr;+/+ and PPAR&agr;+/− apoE2-KI mice, whereas, for a maximal antiinflammatory response, both alleles of the PPAR&agr; gene were required. Surprisingly, atherosclerosis development was not significantly different among PPAR&agr;+/+, PPAR&agr;+/−, and PPAR&agr;−/− apoE2-KI mice. However, PPAR&agr; gene level determined both the antiatherosclerotic and vascular antiinflammatory responses to fenofibrate in a dose-dependent manner. Conclusion—These results demonstrate a necessary but quantitatively different role of PPAR&agr; in the modulation of liver metabolism, inflammation, and atherogenesis.

The RXR Agonist Bexarotene Improves Cholesterol Homeostasis and Inhibits Atherosclerosis Progression in a Mouse Model of Mixed Dyslipidemia

Objective—The activity of the antitumoral agent bexarotene (Targretin, Bexarotene) depends on its binding to the nuclear retinoid-X receptor (RXR) and subsequent transcriptional regulation of target genes. Through RXR activation, bexarotene may modulate numerous metabolic pathways involved in atherosclerosis. Here, we investigated the effect of bexarotene on atherosclerosis progression in a dyslipidemic murine model, the human apolipoprotein E2 knockin mouse, that develops essentially macrophage-laden lesions. Methods and Results—Atherosclerotic lesions together with different metabolic pathways involved in atherosclerosis were investigated in mice treated or not with bexarotene. Bexarotene protects from atherosclerosis development in mice, at least in part by improving the circulating cholesterol distribution profile likely via a marked decrease of dietary cholesterol absorption caused by modulation of intestinal expression of genes recently identified as major players in this process, Niemann-Pick-C1-Like1 (NPC1L1) and CD13. This atheroprotection appears despite a strong hypertriglyceridemia. Moreover, bexarotene treatment only modestly modulates inflammatory gene expression in the vascular wall, but markedly enhanced the capacity of macrophages to efflux cellular lipids. Conclusion—These data provide evidence of a favorable pharmacological effect of bexarotene on atherosclerosis despite the induction of hypertriglyceridemia, likely via a beneficial action on intestinal absorption and macrophage efflux.

The transrepressive activity of peroxisome proliferator‐activated receptor alpha is necessary and sufficient to prevent liver fibrosis in mice

Nonalcoholic fatty liver disease (NAFLD) is increasingly prevalent and strongly associated with central obesity, dyslipidemia, and insulin resistance. According to the multiple‐hit model of NAFLD pathogenesis, lipid accumulation drives nonalcoholic steatohepatitis (NASH) initiation by triggering oxidative stress, lipotoxicity, and subsequent activation of hepatic inflammatory responses that may progress, in predisposed individuals, to fibrosis and cirrhosis. While there is an unmet therapeutical need for NASH and fibrosis, recent preclinical studies showed that peroxisome proliferator‐activated receptor (PPAR)‐α agonism can efficiently oppose these symptoms. To dissect the relative contribution of antisteatotic versus anti‐inflammatory PPAR‐α activities in counteracting dietary‐induced liver fibrosis, we used a PPAR‐α mutant lacking its DNA‐binding‐dependent activity on fatty acid metabolism. Liver‐specific expression of wild‐type or a DNA‐binding‐deficient PPAR‐α in acute and chronic models of inflammation were used to study PPAR‐αs anti‐inflammatory versus metabolic activities in NASH and fibrosis. Pharmacologically activated PPAR‐α inhibited hepatic inflammatory responses and the transition from steatosis toward NASH and fibrosis through a direct, anti‐inflammatory mechanism independent of its lipid handling properties. Conclusion: The transrepression activity of PPAR‐α on chronic liver inflammation is sufficient to prevent progression of NASH to liver fibrosis. Dissociated PPAR‐α agonists, selectively modulating PPAR‐α transrepression activity, could thus be an option to prevent NASH and fibrosis progression. (Hepatology 2014;60:1593–1606)

PPARα activation differently affects microparticle content in atherosclerotic lesions and liver of a mouse model of atherosclerosis and NASH

BACKGROUND Atherosclerosis and non-alcoholic fatty liver disease (NAFLD) are complex pathologies characterized by lipid accumulation, chronic inflammation and extensive tissue remodelling. Microparticles (MPs), small membrane vesicles produced by activated and apoptotic cells, might not only be biomarkers, but also functional actors in these pathologies. The apoE2-KI mouse is a model of atherosclerosis and NAFLD. Activation of the nuclear receptor PPARα decreases atherosclerosis and components of non-alcoholic steatohepatitis (NASH) in the apoE2-KI mouse. OBJECTIVES (1) To determine whether MPs are present in atherosclerotic lesions, liver and plasma during atherosclerosis and NASH progression in apoE2-KI mice, and (2) to study whether PPARα activation modulates MP concentrations. METHODS ApoE2-KI mice were fed a Western diet to induce atherosclerosis and NASH. MPs were isolated from atherosclerotic lesions, liver and blood and quantified by flow cytometry. RESULTS An increase of MPs was observed in the atherosclerotic lesions and in the liver of apoE2-KI mice upon Western diet feeding. PPARα activation with fenofibrate decreased MP levels in the atherosclerotic lesions in a PPARα-dependent manner, but did not influence MP concentrations in the liver. CONCLUSION Here we report that MPs are present in atherosclerotic lesions and in the liver of apoE2-KI mice. Their concentration increased during atherosclerosis and NASH development. PPARα activation differentially modulates MP levels in a tissue-specific manner.

Interspecies NASH disease activity whole-genome profiling identifies a fibrogenic role of PPAR α -regulated dermatopontin

Nonalcoholic fatty liver disease prevalence is soaring with the obesity pandemic, but the pathogenic mechanisms leading to the progression toward active nonalcoholic steatohepatitis (NASH) and fibrosis, major causes of liver-related death, are poorly defined. To identify key components during the progression toward NASH and fibrosis, we investigated the liver transcriptome in a human cohort of NASH patients. The transition from histologically proven fatty liver to NASH and fibrosis was characterized by gene expression patterns that successively reflected altered functions in metabolism, inflammation, and epithelial-mesenchymal transition. A meta-analysis combining our and public human transcriptomic datasets with murine models of NASH and fibrosis defined a molecular signature characterizing NASH and fibrosis and evidencing abnormal inflammation and extracellular matrix (ECM) homeostasis. Dermatopontin expression was found increased in fibrosis, and reversal of fibrosis after gastric bypass correlated with decreased dermatopontin expression. Functional studies in mice identified an active role for dermatopontin in collagen deposition and fibrosis. PPARα activation lowered dermatopontin expression through a transrepressive mechanism affecting the Klf6/TGFβ1 pathway. Liver fibrotic histological damages are thus characterized by the deregulated expression of a restricted set of inflammation- and ECM-related genes. Among them, dermatopontin may be a valuable target to reverse the hepatic fibrotic process.

Ketone Body Therapy Protects From Lipotoxicity and Acute Liver Failure Upon Pparα Deficiency

Acute liver failure (ALF) is a severe and rapid liver injury, often occurring without any preexisting liver disease, which may precipitate multiorgan failure and death. ALF is often associated with impaired β-oxidation and increased oxidative stress (OS), characterized by elevated levels of hepatic reactive oxygen species (ROS) and lipid peroxidation (LPO) products. Peroxisome proliferator-activated receptor (PPAR)α has been shown to confer hepatoprotection in acute and chronic liver injury, at least in part, related to its ability to control peroxisomal and mitochondrial β-oxidation. To study the pathophysiological role of PPARα in hepatic response to high OS, we induced a pronounced LPO by treating wild-type and Pparα-deficient mice with high doses of fish oil (FO), containing n-3 polyunsaturated fatty acids. FO feeding of Pparα-deficient mice, in contrast to control sunflower oil, surprisingly induced coma and death due to ALF as indicated by elevated serum alanine aminotransferase, aspartate aminotransferase, ammonia, and a liver-specific increase of ROS and LPO-derived malondialdehyde. Reconstitution of PPARα specifically in the liver using adeno-associated serotype 8 virus-PPARα in Pparα-deficient mice restored β-oxidation and ketogenesis and protected mice from FO-induced lipotoxicity and death. Interestingly, administration of the ketone body β-hydroxybutyrate prevented FO-induced ALF in Pparα-deficient mice, and normalized liver ROS and malondialdehyde levels. Therefore, PPARα protects the liver from FO-induced OS through its regulatory actions on ketone body levels. β-Hydroxybutyrate treatment could thus be an option to prevent LPO-induced liver damage.

PPARA GENE LEVEL DIFFERENTLY AFFECTS LIPID METABOLISM AND INFLAMMATION IN APOLIPOPROTEIN E2 KNOCK-IN MICE

Abstract Objective : Peroxisome Proliferator -Activated Receptor D (PPAR D ) is a ligand -activated transcription factor which controls lipid metabolism and inflammation. PPAR D is activated by fibrates, hypolipidemic drugs used in the treatment of dyslipidemia. Previous studies assessing the influence of PPAR D agonists on atherosclerosis in mice yielded conflicting results and the implication of PPAR D therein has not been assessed. The human apoE2 knock -in (apoE2 -KI) mouse is a model of mixed dyslipidemia, atherosclerosis and non -alcoholic steatohepatitis ( NASH). T he aim of this study was, using homo - and heterozygous PPAR D -deficient mice, to analyze the consequences of quantitative variations of PPAR D gene levels and its response to the synthetic PPAR D agonist fenofibrate, o n NASH and atherosclerosis in apoE2 -KI mice. Metho ds and results : W ildtype (+/+), heterozygous (+/ -) and homozygous ( -/-) PPAR D -deficient mice in the apoE2 -KI background were generated and submitted to a western diet supplemented or not with fenofibrate. Western diet -fed PPAR D -/- apoE2 -KI mice displayed a n aggravation of liver steatosis and inflammation compared to PPAR D +/+ and PPAR D +/ - apoE2 -KI mice, indicating a role of PPAR D in liver protection. Moreover, PPAR D expression was required for t he fenofibrate -induced protection against NASH . Interestingly , fenofibrate treatment induced a similar response on hepatic lipid metabolism in PPAR D +/+ and PPAR D +/ - apoE2 -KI mice, whereas, for a maximal anti -inflammatory response, both alleles of the PPAR D gene were required. Surprisingly, atherosclerosis development was not significantly different between PPAR D +/+, PPAR D +/ - and PPAR D -/- apoE2 -KI mice. However, PPAR D gene level determined both the anti -atherosclerotic and vascular anti -inflammatory responses to fenofibrate in a dose -dependent manner. Conclusions: These results demonstrate a necessary, but quantitatively different role of PPAR D in the modulation of liver metabolism, inflammation and atherogenesis. Keywords: PPAR D , fatty liver disease, atherosclerosis, inflammation, lipid metabolism, murine model