Eric Baugé

Regulation of Bile Acid Synthesis by the Nuclear Receptor Rev-erbα

BACKGROUND & AIMS Conversion into bile acids represents an important route to remove excess cholesterol from the body. Rev-erbalpha is a nuclear receptor that participates as one of the clock genes in the control of circadian rhythmicity and plays a regulatory role in lipid metabolism and adipogenesis. Here, we investigate a potential role for Rev-erbalpha in the control of bile acid metabolism via the regulation of the neutral bile acid synthesis pathway. METHODS Bile acid synthesis and CYP7A1 gene expression were studied in vitro and in vivo in mice deficient for or over expressing Rev-erbalpha. RESULTS Rev-erbalpha-deficient mice display a lower synthesis rate and an impaired excretion of bile acids into the bile and feces. Expression of CYP7A1, the rate-limiting enzyme of the neutral pathway, is decreased in livers of Rev-erbalpha-deficient mice, whereas adenovirus-mediated hepatic Rev-erbalpha overexpression induces its expression. Moreover, bile acid feeding resulted in a more pronounced suppression of hepatic CYP7A1 expression in Rev-erbalpha-deficient mice. Hepatic expression of E4BP4 and the orphan nuclear receptor small heterodimer partner (SHP), both negative regulators of CYP7A1 expression, is increased in Rev-erbalpha-deficient mice. Promoter analysis and chromatin immunoprecipitation experiments demonstrated that SHP and E4BP4 are direct Rev-erbalpha target genes. Finally, the circadian rhythms of liver CYP7A1, SHP, and E4BP4 messenger RNA levels were perturbed in Rev-erbalpha-deficient mice. CONCLUSIONS These data identify a role for Rev-erbalpha in the regulatory loop of bile acid synthesis, likely acting by regulating both hepatic SHP and E4BP4 expression.

Antiinflammatory and Antiatherogenic Effects of the NF-κB Inhibitor Acetyl-11-Keto-β-Boswellic Acid in LPS-Challenged ApoE−/− Mice

Objective—In this article, we studied the effect of acetyl-11-keto-β-boswellic acid (AKβBA), a natural inhibitor of the proinflammatory transcription factor NF-&kgr;B on the development of atherosclerotic lesions in apolipoprotein E–deficient (apoE−/−) mice. Methods and Results—Atherosclerotic lesions were induced by weekly LPS injection in apoE−/− mice. LPS alone increased atherosclerotic lesion size by ≈100%, and treatment with AKβBA significantly reduced it by ≈50%. Moreover, the activity of NF-&kgr;B was also reduced in the atherosclerotic plaques of LPS-injected apoE−/− mice treated with AKβBA. As a consequence, AKβBA treatment led to a significant downregulation of several NF-&kgr;B–dependent genes such as MCP-1, MCP-3, IL-1α, MIP-2, VEGF, and TF. By contrast, AKβBA did not affect the plasma concentrations of triglycerides, total cholesterol, antioxidized LDL antibodies, and various subsets of lymphocyte-derived cytokines. Moreover, AKβBA potently inhibited the I&kgr;B kinase (IKK) activity immunoprecipitated from LPS-stimulated mouse macrophages and mononuclear cells leading to decreased phosphorylation of I&kgr;Bα and inhibition of p65/NF-&kgr;B activation. Comparable AKβBA-mediated inhibition was also observed in LPS-stimulated human macrophages. Conclusion—The inhibition of NF-&kgr;B activity by plant resins from species of the Boswellia family might represent an alternative for classical medicine treatments for chronic inflammatory diseases such as atherosclerosis. (Arterioscler Thromb Vasc Biol. 2008;28:272-277)

Effects of Pinus pinaster and Pinus koraiensis seed oil supplementation on lipoprotein metabolism in the rat

The aim of the present study was to assess the effect of vegetal oils obtained from Pinus pinaster and P. koraiensis seeds on plasma lipoprotein levels and apolipoprotein (apo) gene expression in rats. These oils contain two particular fatty acids of the Δ5-unsaturated polymethylene-interrupted fatty acid (Δ5-UPIFA) family: all-cis-5,9,12-18:3 (pinolenic) and/or all-cis-5,11,14-20:3 (sciadonic) acids. Rats were fed for 28 d a diet containing 5% (w/w) oil supplement. Two control diets were prepared to match the fatty acid composition of P. pinaster or P. koraiensis oils with the exception of Δ5-UPIFA, which were replaced by oleic acid. Pinus pinaster seed oil decreased serum triglycerides by 30% (P<0.02), very low density lipoprotein (VLDL)-triglycerides by 40% (P<0.01), and VLDL-cholesterol by 33% (P<0.03). Pinus koraiensis seed oil decreased serum triglycerides by 16% [not statistically significant (ns)] and VLDL-triglycerides by 21% (ns). Gel permeation chromatography and nondenaturating polyacrylamide gel electrophoresis showed a tendency of high density lipoprotein to shift toward larger particles in pine seed oil-supplemented rats. Finally, P. pinaster seed oil treatment was associated with a small decrease of liver apoC-III (P<0.02) but not in apoE, apoA-I, or apoA-II mRNA levels. The levels of circulating apo were not affected by pine seed oil supplementation. In conclusion, P. pinaster seed oil has a triglyceride-lowering effect in rats, an effect that is due to a reduction in circulating VLDL.

Analysis of Apolipoprotein A5, C3, and Plasma Triglyceride Concentrations in Genetically Engineered Mice

Objective—Both the apolipoprotein A5 and C3 genes have repeatedly been shown to play an important role in determining plasma triglyceride concentrations in humans and mice. In mice, transgenic and knockout experiments indicate that plasma triglyceride levels are strongly altered by changes in the expression of either of these 2 genes. In humans, common polymorphisms in both genes have also been associated with plasma triglyceride concentrations. These similar findings raised the issue of the relationship between these 2 genes and altered triglycerides. Methods and Results—To address this issue, we generated independent lines of mice that either overexpressed (“double transgenic”) or completely lacked (“double knockout”) both apolipoprotein genes. We report that both “double transgenic” and “double knockout” mice display normal triglyceride concentrations compared with overexpression or deletion of either gene alone. Furthermore, we find that human ApoAV plasma protein levels in the “double transgenic” mice are ≈500-fold lower than human ApoCIII levels, supporting ApoAV as a potent triglyceride modulator despite its low concentration. Conclusions—Together, these data support that APOA5 and APOC3 independently influence plasma triglyceride concentrations but in an opposing manner.

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 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)

Liver microRNA-21 is overexpressed in non-alcoholic steatohepatitis and contributes to the disease in experimental models by inhibiting PPARα expression

Objective Previous studies suggested that microRNA-21 may be upregulated in the liver in non-alcoholic steatohepatitis (NASH), but its role in the development of this disease remains unknown. This study aimed to determine the role of microRNA-21 in NASH. Design We inhibited or suppressed microRNA-21 in different mouse models of NASH: (a) low-density lipoprotein receptor-deficient (Ldlr−/−) mice fed a high-fat diet and treated with antagomir-21 or antagomir control; (b) microRNA-21-deficient and wild-type mice fed a methionine-choline-deficient (MCD) diet; (c) peroxisome proliferation-activator receptor α (PPARα)-deficient mice fed an MCD diet and treated with antagomir-21 or antagomir control. We assessed features of NASH and determined liver microRNA-21 levels and cell localisation. MicroRNA-21 levels were also quantified in the liver of patients with NASH, bland steatosis or normal liver and localisation was determined. Results Inhibiting or suppressing liver microRNA-21 expression reduced liver cell injury, inflammation and fibrogenesis without affecting liver lipid accumulation in Ldlr−/− fed a high-fat diet and in wild-type mice fed an MCD diet. Liver microRNA-21 was overexpressed, primarily in biliary and inflammatory cells, in mouse models as well as in patients with NASH, but not in patients with bland steatosis. PPARα, a known microRNA-21 target, implicated in NASH, was decreased in the liver of mice with NASH and restored following microRNA-21 inhibition or suppression. The effect of antagomir-21 was lost in PPARα-deficient mice. Conclusions MicroRNA-21 inhibition or suppression decreases liver injury, inflammation and fibrosis, by restoring PPARα expression. Antagomir-21 might be a future therapeutic strategy for NASH.

LEPROT and LEPROTL1 cooperatively decrease hepatic growth hormone action in mice

Growth hormone (GH) is a major metabolic regulator that functions by stimulating lipolysis, preventing protein catabolism, and decreasing insulin-dependent glucose disposal. Modulation of hepatic sensitivity to GH and the downstream effects on the GH/IGF1 axis are important events in the regulation of metabolism in response to variations in food availability. For example, during periods of reduced nutrient availability, the liver becomes resistant to GH actions. However, the mechanisms controlling hepatic GH resistance are currently unknown. Here, we investigated the role of 2 tetraspanning membrane proteins, leptin receptor overlapping transcript (LEPROT; also known as OB-RGRP) and LEPROT-like 1 (LEPROTL1), in controlling GH sensitivity. Transgenic mice expressing either human LEPROT or human LEPROTL1 displayed growth retardation, reduced plasma IGF1 levels, and impaired hepatic sensitivity to GH, as measured by STAT5 phosphorylation and Socs2 mRNA expression. These phenotypes were accentuated in transgenic mice expressing both proteins. Moreover, gene silencing of either endogenous Leprot or Leprotl1 in H4IIE hepatocytes increased GH signaling and enhanced cell-surface GH receptor. Importantly, we found that both LEPROT and LEPROTL1 expression were regulated in the mouse liver by physiologic and pathologic changes in glucose homeostasis. Together, these data provide evidence that LEPROT and LEPROTL1 influence liver GH signaling and that regulation of the genes encoding these proteins may constitute a molecular link between nutritional signals and GH actions on body growth and metabolism.

Atheroprotective Effect of Human Apolipoprotein A5 in a Mouse Model of Mixed Dyslipidemia

Hypertriglyceridemia is an independent risk factor for coronary artery disease. Because apolipoprotein (Apo)A5 regulates plasma triglyceride levels, we investigated the impact of human (h)ApoA5 on atherogenesis. The influence of hApoA5 transgenic expression was studied in the ApoE2 knock-in mouse model of mixed dyslipidemia. Our results demonstrate that hApoA5 lowers plasma triglyceride levels in Western diet–fed ApoE2 knock-in mice. Moreover, atherosclerotic lesion development was significantly decreased in the hApoA5 transgenic mice. Finally, pharmacologic activation of hApoA5 expression by the peroxisome proliferator-activated receptor-&agr; agonist fenofibrate resulted in an enhanced atheroprotection. These results identify an atheroprotective role of hApoA5 in a mouse model of mixed dyslipidemia.

PPARalpha regulates the production of serum Vanin-1 by liver.

The membrane‐bound Vanin‐1 pantetheinase regulates tissue adaptation to stress. We investigated Vnn1 expression and its regulation in liver. Vnn1 is expressed by centrolobular hepatocytes. Using novel tools, we identify a soluble form of Vnn1 in mouse and human serum and show the contribution of a cysteine to its catalytic activity. We show that liver contributes to Vanin‐1 secretion in serum and that PPARalpha is a limiting factor in serum Vnn1 production. Functional PPRE sites are identified in the Vnn1 promoter. These results indicate that serum Vnn1 might be a reliable reporter of PPARalpha activity in liver.