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The arachidonic acid metabolome serves as a conserved regulator of cholesterol metabolism.

Demetz E, Schroll A, Auer K, Heim C, Patsch JR, Eller P, Theurl M, Theurl I, Theurl M, Seifert M, Lener D, Stanzl U, Haschka D, Asshoff M, Dichtl S, Nairz M, Huber E, Stadlinger M, Moschen AR, Li X, Pallweber P, Scharnagl H, Stojakovic T, März W, Kleber ME, Garlaschelli K, Uboldi P, Catapano AL, Stellaard F, Rudling M, Kuba K, Imai Y, Arita M, Schuetz JD, Pramstaller PP, Tietge UJ, Trauner M, Norata GD, Claudel T, Hicks AA, Weiss G, Tancevski I - Cell Metab. (2014)

Bottom Line: Pharmacological modulation of AA metabolism by aspirin induced hepatic generation of leukotrienes (LTs) and lipoxins (LXs), thereby increasing hepatic expression of the bile salt export pump Abcb11.Induction of Abcb11 translated in enhanced reverse cholesterol transport, one key function of HDL.Further characterization of the bioactive AA-derivatives identified LX mimetics to lower plasma LDL-C.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine VI, Innsbruck Medical University, Anichstrasse 35, 6020 Innsbruck, Austria

ABSTRACT
Cholesterol metabolism is closely interrelated with cardiovascular disease in humans. Dietary supplementation with omega-6 polyunsaturated fatty acids including arachidonic acid (AA) was shown to favorably affect plasma LDL-C and HDL-C. However, the underlying mechanisms are poorly understood. By combining data from a GWAS screening in >100,000 individuals of European ancestry, mediator lipidomics, and functional validation studies in mice, we identify the AA metabolome as an important regulator of cholesterol homeostasis. Pharmacological modulation of AA metabolism by aspirin induced hepatic generation of leukotrienes (LTs) and lipoxins (LXs), thereby increasing hepatic expression of the bile salt export pump Abcb11. Induction of Abcb11 translated in enhanced reverse cholesterol transport, one key function of HDL. Further characterization of the bioactive AA-derivatives identified LX mimetics to lower plasma LDL-C. Our results define the AA metabolomeasconserved regulator of cholesterol metabolism, and identify AA derivatives as promising therapeutics to treat cardiovascular disease in humans.

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Related in: MedlinePlus

Leukotrienes and Lipoxins Regulate Abcb11 in a Posttranslational FashionTo investigate the mechanisms underlying Abcb11 regulation by aspirin, studies in primary murine hepatocytes derived from C57BL/6 mice were performed.(A and C–E) Hepatocytes were incubated with indicated compounds at given concentrations for 24 hr, after which Abcb11 protein expression was measured by immunoblot analysis. Additionally, protein expression of SR-BI and LDLr was analyzed; actin served as loading control.(B) Heatmap showing relative changes of TXB2, LTB4, and LXA4 levels in livers and hepatocytes measured by means of mediator lipidomics (aspirin/control).(F) FXR reporter assay with positive control (GW4064), LXB4, and LTB4 used at indicated concentrations.(G) qRT-PCR analysis of Abcb11 in primary murine hepatocytes treated with vehicle or aspirin (100 μM) for 6 and 12 hr.(H) To measure Abcb11 mRNA degradation, murine hepatocytes were pretreated with actinomycin D (Act. D), after which they were treated with vehicle or aspirin (100 μM) for 24 hr. RNA levels were measured by qRT-PCR, ∗∗∗p < 0.001.(I) Protein translation in murine hepatocytes was inhibited by preincubation with cycloheximide (10 μg ml−1) for 6 hr, after which cells were treated with vehicle, LTB4 (50 nM), and LXB4 (10 nM) for another 18 hr. Immunoblot showing Abcb11 expression; actin served as loading control.(J) Primary murine hepatocytes were coincubated with MAPK p38 ihibitor SB 203580 (10 μM) and vehicle, LTB4 (50 nM), and LXB4 (10 nM) for 24 hr, after which immunoblot analysis of Abcb11 was performed. Actin served as loading control.
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fig4: Leukotrienes and Lipoxins Regulate Abcb11 in a Posttranslational FashionTo investigate the mechanisms underlying Abcb11 regulation by aspirin, studies in primary murine hepatocytes derived from C57BL/6 mice were performed.(A and C–E) Hepatocytes were incubated with indicated compounds at given concentrations for 24 hr, after which Abcb11 protein expression was measured by immunoblot analysis. Additionally, protein expression of SR-BI and LDLr was analyzed; actin served as loading control.(B) Heatmap showing relative changes of TXB2, LTB4, and LXA4 levels in livers and hepatocytes measured by means of mediator lipidomics (aspirin/control).(F) FXR reporter assay with positive control (GW4064), LXB4, and LTB4 used at indicated concentrations.(G) qRT-PCR analysis of Abcb11 in primary murine hepatocytes treated with vehicle or aspirin (100 μM) for 6 and 12 hr.(H) To measure Abcb11 mRNA degradation, murine hepatocytes were pretreated with actinomycin D (Act. D), after which they were treated with vehicle or aspirin (100 μM) for 24 hr. RNA levels were measured by qRT-PCR, ∗∗∗p < 0.001.(I) Protein translation in murine hepatocytes was inhibited by preincubation with cycloheximide (10 μg ml−1) for 6 hr, after which cells were treated with vehicle, LTB4 (50 nM), and LXB4 (10 nM) for another 18 hr. Immunoblot showing Abcb11 expression; actin served as loading control.(J) Primary murine hepatocytes were coincubated with MAPK p38 ihibitor SB 203580 (10 μM) and vehicle, LTB4 (50 nM), and LXB4 (10 nM) for 24 hr, after which immunoblot analysis of Abcb11 was performed. Actin served as loading control.

Mentions: Using primary murine hepatocytes derived from C57BL/6 mice, we confirmed our in vivo studies by demonstrating that both aspirin and its active metabolite salicylic acid induce Abcb11 protein expression. Moreover, aspirin and salicylic acid induced SR-BI protein expression, whereas they downregulated the expression of the LDLr in vitro (Figure 4A). Presently, little is known about lipoxygenase pathways within hepatocytes. One main difference between hepatocytes and other cell types is that hepatocytes do not express Alox5. In this regard, Claría’s lab showed that Kupffer cells, which are of myeloid origin and express high levels of Alox5, are the major source of LTB4 and LXs in rat liver exposed to aspirin (Planagumà et al., 2002). Accordingly, when comparing mediator lipidomic profiles of mouse liver and of isolated murine hepatocytes, we found that (1) in both settings, aspirin dramatically reduced TXB2 levels as result of efficient Cox I inhibition, (2) in liver extracts, aspirin treatment increased both LTs and LXs, and (3) in isolated mouse hepatocytes, aspirin treatment increased only LX formation (Figure 4B). When analyzing the relative effects of different LX isomers on Abcb11 expression, we found that LXA4, 15-epi-LXA4, and LXB4 increased Abcb11 at 10 nM, with LXB4 showing the strongest induction (Figure 4C). Dose-titration studies as well as coincubation experiments with LTB4 and LXB4 in primary hepatocytes derived from C57BL/6 mice revealed that both lipid mediators increase Abcb11 expression. Intriguingly, LXB4 decreased Abcb11 protein expression at high dosages. On the other hand, SR-BI and LDLr protein expression was induced mainly by LTB4 (Figures 4D and 4E).


The arachidonic acid metabolome serves as a conserved regulator of cholesterol metabolism.

Demetz E, Schroll A, Auer K, Heim C, Patsch JR, Eller P, Theurl M, Theurl I, Theurl M, Seifert M, Lener D, Stanzl U, Haschka D, Asshoff M, Dichtl S, Nairz M, Huber E, Stadlinger M, Moschen AR, Li X, Pallweber P, Scharnagl H, Stojakovic T, März W, Kleber ME, Garlaschelli K, Uboldi P, Catapano AL, Stellaard F, Rudling M, Kuba K, Imai Y, Arita M, Schuetz JD, Pramstaller PP, Tietge UJ, Trauner M, Norata GD, Claudel T, Hicks AA, Weiss G, Tancevski I - Cell Metab. (2014)

Leukotrienes and Lipoxins Regulate Abcb11 in a Posttranslational FashionTo investigate the mechanisms underlying Abcb11 regulation by aspirin, studies in primary murine hepatocytes derived from C57BL/6 mice were performed.(A and C–E) Hepatocytes were incubated with indicated compounds at given concentrations for 24 hr, after which Abcb11 protein expression was measured by immunoblot analysis. Additionally, protein expression of SR-BI and LDLr was analyzed; actin served as loading control.(B) Heatmap showing relative changes of TXB2, LTB4, and LXA4 levels in livers and hepatocytes measured by means of mediator lipidomics (aspirin/control).(F) FXR reporter assay with positive control (GW4064), LXB4, and LTB4 used at indicated concentrations.(G) qRT-PCR analysis of Abcb11 in primary murine hepatocytes treated with vehicle or aspirin (100 μM) for 6 and 12 hr.(H) To measure Abcb11 mRNA degradation, murine hepatocytes were pretreated with actinomycin D (Act. D), after which they were treated with vehicle or aspirin (100 μM) for 24 hr. RNA levels were measured by qRT-PCR, ∗∗∗p < 0.001.(I) Protein translation in murine hepatocytes was inhibited by preincubation with cycloheximide (10 μg ml−1) for 6 hr, after which cells were treated with vehicle, LTB4 (50 nM), and LXB4 (10 nM) for another 18 hr. Immunoblot showing Abcb11 expression; actin served as loading control.(J) Primary murine hepatocytes were coincubated with MAPK p38 ihibitor SB 203580 (10 μM) and vehicle, LTB4 (50 nM), and LXB4 (10 nM) for 24 hr, after which immunoblot analysis of Abcb11 was performed. Actin served as loading control.
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Related In: Results  -  Collection

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fig4: Leukotrienes and Lipoxins Regulate Abcb11 in a Posttranslational FashionTo investigate the mechanisms underlying Abcb11 regulation by aspirin, studies in primary murine hepatocytes derived from C57BL/6 mice were performed.(A and C–E) Hepatocytes were incubated with indicated compounds at given concentrations for 24 hr, after which Abcb11 protein expression was measured by immunoblot analysis. Additionally, protein expression of SR-BI and LDLr was analyzed; actin served as loading control.(B) Heatmap showing relative changes of TXB2, LTB4, and LXA4 levels in livers and hepatocytes measured by means of mediator lipidomics (aspirin/control).(F) FXR reporter assay with positive control (GW4064), LXB4, and LTB4 used at indicated concentrations.(G) qRT-PCR analysis of Abcb11 in primary murine hepatocytes treated with vehicle or aspirin (100 μM) for 6 and 12 hr.(H) To measure Abcb11 mRNA degradation, murine hepatocytes were pretreated with actinomycin D (Act. D), after which they were treated with vehicle or aspirin (100 μM) for 24 hr. RNA levels were measured by qRT-PCR, ∗∗∗p < 0.001.(I) Protein translation in murine hepatocytes was inhibited by preincubation with cycloheximide (10 μg ml−1) for 6 hr, after which cells were treated with vehicle, LTB4 (50 nM), and LXB4 (10 nM) for another 18 hr. Immunoblot showing Abcb11 expression; actin served as loading control.(J) Primary murine hepatocytes were coincubated with MAPK p38 ihibitor SB 203580 (10 μM) and vehicle, LTB4 (50 nM), and LXB4 (10 nM) for 24 hr, after which immunoblot analysis of Abcb11 was performed. Actin served as loading control.
Mentions: Using primary murine hepatocytes derived from C57BL/6 mice, we confirmed our in vivo studies by demonstrating that both aspirin and its active metabolite salicylic acid induce Abcb11 protein expression. Moreover, aspirin and salicylic acid induced SR-BI protein expression, whereas they downregulated the expression of the LDLr in vitro (Figure 4A). Presently, little is known about lipoxygenase pathways within hepatocytes. One main difference between hepatocytes and other cell types is that hepatocytes do not express Alox5. In this regard, Claría’s lab showed that Kupffer cells, which are of myeloid origin and express high levels of Alox5, are the major source of LTB4 and LXs in rat liver exposed to aspirin (Planagumà et al., 2002). Accordingly, when comparing mediator lipidomic profiles of mouse liver and of isolated murine hepatocytes, we found that (1) in both settings, aspirin dramatically reduced TXB2 levels as result of efficient Cox I inhibition, (2) in liver extracts, aspirin treatment increased both LTs and LXs, and (3) in isolated mouse hepatocytes, aspirin treatment increased only LX formation (Figure 4B). When analyzing the relative effects of different LX isomers on Abcb11 expression, we found that LXA4, 15-epi-LXA4, and LXB4 increased Abcb11 at 10 nM, with LXB4 showing the strongest induction (Figure 4C). Dose-titration studies as well as coincubation experiments with LTB4 and LXB4 in primary hepatocytes derived from C57BL/6 mice revealed that both lipid mediators increase Abcb11 expression. Intriguingly, LXB4 decreased Abcb11 protein expression at high dosages. On the other hand, SR-BI and LDLr protein expression was induced mainly by LTB4 (Figures 4D and 4E).

Bottom Line: Pharmacological modulation of AA metabolism by aspirin induced hepatic generation of leukotrienes (LTs) and lipoxins (LXs), thereby increasing hepatic expression of the bile salt export pump Abcb11.Induction of Abcb11 translated in enhanced reverse cholesterol transport, one key function of HDL.Further characterization of the bioactive AA-derivatives identified LX mimetics to lower plasma LDL-C.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine VI, Innsbruck Medical University, Anichstrasse 35, 6020 Innsbruck, Austria

ABSTRACT
Cholesterol metabolism is closely interrelated with cardiovascular disease in humans. Dietary supplementation with omega-6 polyunsaturated fatty acids including arachidonic acid (AA) was shown to favorably affect plasma LDL-C and HDL-C. However, the underlying mechanisms are poorly understood. By combining data from a GWAS screening in >100,000 individuals of European ancestry, mediator lipidomics, and functional validation studies in mice, we identify the AA metabolome as an important regulator of cholesterol homeostasis. Pharmacological modulation of AA metabolism by aspirin induced hepatic generation of leukotrienes (LTs) and lipoxins (LXs), thereby increasing hepatic expression of the bile salt export pump Abcb11. Induction of Abcb11 translated in enhanced reverse cholesterol transport, one key function of HDL. Further characterization of the bioactive AA-derivatives identified LX mimetics to lower plasma LDL-C. Our results define the AA metabolomeasconserved regulator of cholesterol metabolism, and identify AA derivatives as promising therapeutics to treat cardiovascular disease in humans.

Show MeSH
Related in: MedlinePlus