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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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Aspirin Promotes RCT(A) In mice, AA can be metabolized via three main pathways: (1) Cox I/II-mediated generation of prostaglandins and thromboxane (PG2, TXA2); (2) Alox5-mediated generation of leukotriene B4 (LTB4) and cysteinyl-leukotrienes; and (3) Alox12/15- and Alox5-mediated generation of lipoxins A4 and B4 (LXA4, LXB4). To inhibit Cox I/II, thereby shifting the AA metabolism to Alox5 and Alox12/15 pathways, C57BL/6 mice were treated with aspirin in their drinking water for 7 days.(B) For macrophage-to-feces RCT studies, control and aspirin-treated mice were injected intraperitoneally with cholesterol-loaded, [3H]-labeled J774 macrophages (Φ). The tracer was measured in plasma at indicated time points and in fecal sterols collected for 48 hr.(C and D) (C) Plasma [3H]-cholesterol levels and (D) fecal [3H]-sterol levels (n = 6–10, data representative of three independent macrophage-to-feces RCT experiments).(E) FPLC analysis of plasma pooled from control and aspirin mice (n = 6).(F) Enzymatic measurement of bile acids in feces collected for 48 hr (n = 7).(G) qRT-PCR measurement of neutral sterol transporters Abcg5 and Abcg8, and bile acid converting enzyme Cyp7a1 in livers of mice (n = 7).(H and I) (H) Immunoblot analysis of bile acid secreting pump Abcb11 and (I) Abcc2 protein expression in livers of mice (n = 4–5, bars represent densitometric quantification normalized to actin).(J) [14C]-glycocholic acid was injected into the tail vein of mice, and after 30 min the tracer was quantified in total bile (n = 5).(K) Plasma [3H]-cholesterol levels at indicated time-points and (L) fecal [3H]-sterol levels (0–48 hr) from a macrophage-to-feces RCT study performed in Abcb11−/− mice (n = 4–5).(M) Atheroregression in LDLr−/− mice treated with aspirin. Graphs show mean ± SEM (n = 6), #p = 0.062, ∗p < 0.05, ∗∗∗p < 0.001.
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fig2: Aspirin Promotes RCT(A) In mice, AA can be metabolized via three main pathways: (1) Cox I/II-mediated generation of prostaglandins and thromboxane (PG2, TXA2); (2) Alox5-mediated generation of leukotriene B4 (LTB4) and cysteinyl-leukotrienes; and (3) Alox12/15- and Alox5-mediated generation of lipoxins A4 and B4 (LXA4, LXB4). To inhibit Cox I/II, thereby shifting the AA metabolism to Alox5 and Alox12/15 pathways, C57BL/6 mice were treated with aspirin in their drinking water for 7 days.(B) For macrophage-to-feces RCT studies, control and aspirin-treated mice were injected intraperitoneally with cholesterol-loaded, [3H]-labeled J774 macrophages (Φ). The tracer was measured in plasma at indicated time points and in fecal sterols collected for 48 hr.(C and D) (C) Plasma [3H]-cholesterol levels and (D) fecal [3H]-sterol levels (n = 6–10, data representative of three independent macrophage-to-feces RCT experiments).(E) FPLC analysis of plasma pooled from control and aspirin mice (n = 6).(F) Enzymatic measurement of bile acids in feces collected for 48 hr (n = 7).(G) qRT-PCR measurement of neutral sterol transporters Abcg5 and Abcg8, and bile acid converting enzyme Cyp7a1 in livers of mice (n = 7).(H and I) (H) Immunoblot analysis of bile acid secreting pump Abcb11 and (I) Abcc2 protein expression in livers of mice (n = 4–5, bars represent densitometric quantification normalized to actin).(J) [14C]-glycocholic acid was injected into the tail vein of mice, and after 30 min the tracer was quantified in total bile (n = 5).(K) Plasma [3H]-cholesterol levels at indicated time-points and (L) fecal [3H]-sterol levels (0–48 hr) from a macrophage-to-feces RCT study performed in Abcb11−/− mice (n = 4–5).(M) Atheroregression in LDLr−/− mice treated with aspirin. Graphs show mean ± SEM (n = 6), #p = 0.062, ∗p < 0.05, ∗∗∗p < 0.001.

Mentions: In our first in vivo experiments, we simultaneously induced the processing of AA by Alox5 and Alox12/15 through pharmacological inhibition of Cox I/II, which shifts the biosynthetic pathways of the AA metabolome toward the formation of LXs and LTs in murine systems (Figure 2A) (Brink et al., 2003; Serhan, 2007; Spite and Serhan, 2010). Inhibition of Cox I/II in mice was achieved by systemic treatment with aspirin (Tancevski et al., 2006), and in vivo macrophage-to-feces RCT studies were performed as described (Tancevski et al., 2010; Zhang et al., 2003): after intraperitoneal injection of [3H]-cholesterol-labeled J774 macrophages, the tracer was measured in plasma and feces (Figure 2B). Aspirin-treated mice had significantly decreased plasma [3H]-cholesterol levels 24 hr postinjection (Figure 2C), which was associated with significantly increased [3H]-sterol levels in feces (Figure 2D). These findings suggested that the increase in fecal tracer content has been caused either by enhanced uptake of [3H]-HDL-C into liver and/or by increased biliary transport of sterols. Hepatic protein expression of the HDL receptor (scavenger receptor BI, SR-BI) and of the LDL receptor (LDLr) were unaffected in aspirin-treated mice (Figure S3A), making the hypothesis of enhanced cholesterol clearance from plasma rather unlikely. Accordingly, plasma total cholesterol levels as well as HDL-C levels were unchanged in aspirin-treated mice (Figures S3B and S3C), which was further confirmed by lipoprotein separation analysis via fast protein liquid chromatography (FPLC) (Figure 2E).


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)

Aspirin Promotes RCT(A) In mice, AA can be metabolized via three main pathways: (1) Cox I/II-mediated generation of prostaglandins and thromboxane (PG2, TXA2); (2) Alox5-mediated generation of leukotriene B4 (LTB4) and cysteinyl-leukotrienes; and (3) Alox12/15- and Alox5-mediated generation of lipoxins A4 and B4 (LXA4, LXB4). To inhibit Cox I/II, thereby shifting the AA metabolism to Alox5 and Alox12/15 pathways, C57BL/6 mice were treated with aspirin in their drinking water for 7 days.(B) For macrophage-to-feces RCT studies, control and aspirin-treated mice were injected intraperitoneally with cholesterol-loaded, [3H]-labeled J774 macrophages (Φ). The tracer was measured in plasma at indicated time points and in fecal sterols collected for 48 hr.(C and D) (C) Plasma [3H]-cholesterol levels and (D) fecal [3H]-sterol levels (n = 6–10, data representative of three independent macrophage-to-feces RCT experiments).(E) FPLC analysis of plasma pooled from control and aspirin mice (n = 6).(F) Enzymatic measurement of bile acids in feces collected for 48 hr (n = 7).(G) qRT-PCR measurement of neutral sterol transporters Abcg5 and Abcg8, and bile acid converting enzyme Cyp7a1 in livers of mice (n = 7).(H and I) (H) Immunoblot analysis of bile acid secreting pump Abcb11 and (I) Abcc2 protein expression in livers of mice (n = 4–5, bars represent densitometric quantification normalized to actin).(J) [14C]-glycocholic acid was injected into the tail vein of mice, and after 30 min the tracer was quantified in total bile (n = 5).(K) Plasma [3H]-cholesterol levels at indicated time-points and (L) fecal [3H]-sterol levels (0–48 hr) from a macrophage-to-feces RCT study performed in Abcb11−/− mice (n = 4–5).(M) Atheroregression in LDLr−/− mice treated with aspirin. Graphs show mean ± SEM (n = 6), #p = 0.062, ∗p < 0.05, ∗∗∗p < 0.001.
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fig2: Aspirin Promotes RCT(A) In mice, AA can be metabolized via three main pathways: (1) Cox I/II-mediated generation of prostaglandins and thromboxane (PG2, TXA2); (2) Alox5-mediated generation of leukotriene B4 (LTB4) and cysteinyl-leukotrienes; and (3) Alox12/15- and Alox5-mediated generation of lipoxins A4 and B4 (LXA4, LXB4). To inhibit Cox I/II, thereby shifting the AA metabolism to Alox5 and Alox12/15 pathways, C57BL/6 mice were treated with aspirin in their drinking water for 7 days.(B) For macrophage-to-feces RCT studies, control and aspirin-treated mice were injected intraperitoneally with cholesterol-loaded, [3H]-labeled J774 macrophages (Φ). The tracer was measured in plasma at indicated time points and in fecal sterols collected for 48 hr.(C and D) (C) Plasma [3H]-cholesterol levels and (D) fecal [3H]-sterol levels (n = 6–10, data representative of three independent macrophage-to-feces RCT experiments).(E) FPLC analysis of plasma pooled from control and aspirin mice (n = 6).(F) Enzymatic measurement of bile acids in feces collected for 48 hr (n = 7).(G) qRT-PCR measurement of neutral sterol transporters Abcg5 and Abcg8, and bile acid converting enzyme Cyp7a1 in livers of mice (n = 7).(H and I) (H) Immunoblot analysis of bile acid secreting pump Abcb11 and (I) Abcc2 protein expression in livers of mice (n = 4–5, bars represent densitometric quantification normalized to actin).(J) [14C]-glycocholic acid was injected into the tail vein of mice, and after 30 min the tracer was quantified in total bile (n = 5).(K) Plasma [3H]-cholesterol levels at indicated time-points and (L) fecal [3H]-sterol levels (0–48 hr) from a macrophage-to-feces RCT study performed in Abcb11−/− mice (n = 4–5).(M) Atheroregression in LDLr−/− mice treated with aspirin. Graphs show mean ± SEM (n = 6), #p = 0.062, ∗p < 0.05, ∗∗∗p < 0.001.
Mentions: In our first in vivo experiments, we simultaneously induced the processing of AA by Alox5 and Alox12/15 through pharmacological inhibition of Cox I/II, which shifts the biosynthetic pathways of the AA metabolome toward the formation of LXs and LTs in murine systems (Figure 2A) (Brink et al., 2003; Serhan, 2007; Spite and Serhan, 2010). Inhibition of Cox I/II in mice was achieved by systemic treatment with aspirin (Tancevski et al., 2006), and in vivo macrophage-to-feces RCT studies were performed as described (Tancevski et al., 2010; Zhang et al., 2003): after intraperitoneal injection of [3H]-cholesterol-labeled J774 macrophages, the tracer was measured in plasma and feces (Figure 2B). Aspirin-treated mice had significantly decreased plasma [3H]-cholesterol levels 24 hr postinjection (Figure 2C), which was associated with significantly increased [3H]-sterol levels in feces (Figure 2D). These findings suggested that the increase in fecal tracer content has been caused either by enhanced uptake of [3H]-HDL-C into liver and/or by increased biliary transport of sterols. Hepatic protein expression of the HDL receptor (scavenger receptor BI, SR-BI) and of the LDL receptor (LDLr) were unaffected in aspirin-treated mice (Figure S3A), making the hypothesis of enhanced cholesterol clearance from plasma rather unlikely. Accordingly, plasma total cholesterol levels as well as HDL-C levels were unchanged in aspirin-treated mice (Figures S3B and S3C), which was further confirmed by lipoprotein separation analysis via fast protein liquid chromatography (FPLC) (Figure 2E).

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