Aerobic exercise alleviates statin-induced PCSK9 upregulation by increasing epoxyeicosatrienoic acid levels through the FoxO3a–Sirt6 axis

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  Fig. 1. Aerobic exercise affects the expression of PCSK9 and LDLR after HFD in vivo without activating the SREBP2 pathway. (A) Three-week-old male ICR mice were continuously fed with HFD for 12 weeks. At Week 4, mice were gavaged with water as control (Ctrl, n = 6) or atorvastatin (Statin, 10 mg/kg/day, n = 6), subjected to aerobic exercise (Ex, n = 8), or administered equivalent atorvastatin combined with aerobic exercise (Statin + Ex, n = 8). (B) Weight, liver weight, liver weight/weight ratio, plasma TG, TC, LDL-C, HDL-C, Glu, ALT, AST and PCSK9. (C) The relative mRNA levels of HMGCR, SREBP2, LDLR, and PCSK9 in the liver. (D–F) Western blot analysis of expression of PCSK9 and LDLR. (E) Quantification of PCSK9. (F) LDLR expression in 4 groups. (G) Morphology of hepatocytes was analyzed by HE staining, lipid deposition in hepatocytes was analyzed by oil red O (ORO) staining, and the expression of PCSK9 and LDLR in hepatocytes was analyzed by immunohistochemical staining (scale bar = 20 μm). Representative data are shown from 3 independent experiments. Data represent mean ± SEM. *p < 0.05. Statistical differences were compared using one-way analysis of variance. ALT = glutamic-pyruvic…

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  Fig. 2. Aerobic exercise increased the levels of EETs in plasma from CHD patients and mice. (A–C) Bulk RNA-sequencing of livers collected from male ICR mice fed HFD for 12 weeks treated with or without exercise (n = 3). (A) volcano plot of each group (padj < 0.05, log2 FC >1). (B) Bubble chart of enriched KEGG pathways. (C) Reactome pathways. (D–F) Patients with CHD were randomly divided into either statin group (Statin, n = 84) or statin combined with aerobic exercise group (Statin + Ex, n = 81). AA metabolomic analysis was performed using LC-MS/MS. DHETs/EETs represent sEH activity. (D)Total DHETs/EETs. (E) change of EETs and DHETs. (F) Change of relative ratio of DHETs/EETs. (G–I) Three week-old male ICR mice were fed HFD for 12 weeks. At Week 4, mice were gavaged with water as control (Ctrl) or atorvastatin (Statin, 10 mg/kg/day), subjected to aerobic exercise (Ex), or administered equivalent atorvastatin combined with aerobic exercise (Statin+Ex). (G) Concentration of EETs and (H) DHETs. (I) Relative ratio of total DHETs/EETs in plasma of 4 groups (n = 6–8 per group). Data represent as mean ± SEM, * p < 0.05. Statistical differences were compared using Student's t test for A, B, C,…

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  Fig. 3. Effects of low EETs concentration on PCSK9. Three-week-old male ICR mice were fed a HFD for 12 weeks. At week 1, AAV-GFP or AAV-sEH was injected into male ICR mice. At Week 4, mice were gavaged with atorvastatin (Statin, 10 mg/kg/day, n = 6), subjected to aerobic exercise (Ex, n = 8), or administered an equivalent amount of atorvastatin combined with aerobic exercise (Statin + Ex, n = 8). (A) Concentration of EETs and DHETs, and relative ratio of total DHETs/EETs in plasma from the AAV-GFP group and AAV-sEH group. (B) HE and ORO staining of livers. (C) Plasma TG, TC, LDL-C, HDL-C, and PCSK9. (D) Protein was extracted from the liver of mice in 4 groups. Western blot and quantification analysis of the expression of sEH, PCSK9, LDLR, HMGCR, and SREBP2. (E) DAPI fluorescence, PCSK9, and LDLR immunofluorescence in these mouse livers from the 4 groups (scale bar = 50 μm). Data represent as mean ± SEM. Representative data are shown from 3 independent experiments. *p < 0.05. Statistical differences were compared using one-way analysis of variance (ANOVA). AAV = adeno-associated virus; DAPI = 4,6-diamidino-2-phenylindole; DHETs = dihydroxyeicosatrienoic acids; EETs = epoxyeicosatrienoic…

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  Fig. 4. Effects of high EETs concentrations on PCSK9. (A)AML-12 hepatocytes were incubated with different concentrations of TPPU (0–10 μM) and subjected to western blot analysis of the expression of PCSK9 and LDLR. (B–D) AML-12 hepatocytes treated with atorvastatin (0.5 μM), TPPU (10 μM), and/or 14,15-EEZE (0.5 μM) with DMSO control for 24 h. (B) Western blot analysis of the expression of PCSK9, LDLR, HMGCR, and SREBP2 in whole cell lysate and PCSK9 in culture medium. (C) Relative mRNA levels of HMGCR, SREBP2, LDLR, and PCSK9 in AML-12 hepatocytes. (D) Uptake of BODIPY-LDL (green) and DAPI (blue) in different groups. (E–I) Three-week-old male ICR mice were fed HFD for 12 weeks. At Week 4, mice were gavaged with water as control (Ctrl, n = 6) or atorvastatin (Statin, 10 mg/kg/day, n = 8), TPPU (TPPU, 10 mg/kg/day, n = 8), or equivalent amount of atorvastatin combined with TPPU (Statin+TPPU). (F) Relative mRNA levels of HMGCR, SREBP2, LDLR, and PCSK9 in the livers. (G) Concentration of EETs and DHETs, and relative ratio of total DHETs/EETs in plasma from 4 groups. DHETs/EETs represent sEH activity. (H) Weight, liver weight, liver weight/weight ratio, and plasma TG, TC, LDL-C, HDL-C, Glu,…

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  Fig. 5. Aerobic exercise activates the FoxO3a–Sirt6 axis via EETs.Bulk RNA-sequencing of livers collected from male ICR mice fed HFD for 12 weeks. At Week 4, mice were gavaged with atorvastatin (Statin, 10 mg/kg/day, n = 6) or equivalent amount of atorvastatin combined with TPPU (10 mg/kg/day, n = 8) (Statin+TPPU). (A) Volcano plot of each group (padj < 0.05, log2 FC > 1). (B) Bubble chart of enriched KEGG pathways. (C) The relative mRNA levels of FoxO1, FoxO3, and FoxO4 in mouse liver. (D–K) AML-12 hepatocytes treated with atorvastatin (0.5 μM), TPPU (10 μM), and/or 14,15-EEZE (0.5 μM) for 24 h. (D–G) Western blot and quantification of the expression of Sirt6, FoxO3a, pFoxO3a (pSer253), and H3 in the Ctrl, Statin, Ex, and Statin+Ex groups. (H–K) Western blot and quantification of Sirt6, FoxO3a, pFoxO3a (pSer253), and H3 expression in the Ctrl, Statin, TPPU, and Statin+TPPU groups. (L) Western blot analysis of Sirt6, FoxO3a, pFoxO3a (pSer253), and H3 expression. (M) Laser confocal analysis of FoxO3a and Sirt6 expression in AM-12 cells in 4 groups (scale bar = 10 μm). (N) Quantitative ChIP assay using AML-12 hepatocytes treated with atorvastatin (0.5 μM) and/or TPPU (10 μM), with DMSO as…

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  Fig. 6. Changes in blood lipid parameters and plasma PCSK9 levels in CHD patients after aerobic exercise. Patients with CHD were randomly divided into either statin group (Statin, n = 84) or statin combined with aerobic exercise group (Statin+Ex, n = 81). Changes in the levels of (A) plasma LDL-C, (B) TG, (C) TC, (D) HDL-C, (E) Lp(a), (F) FFA, (G) ApoA1, (H) ApoB, and (I) PCSK9. The differences were detected after 7 days of exercise treatment in the Statin+Ex groups. All points and P values are shown. Statistical differences were compared using Student's t test. ApoA1 = apolipoprotein A1; ApoB = apolipoprotein B; CHD = coronary heart disease; FFA = free fatty acid; HDL-C = high density lipoprotein cholesterol; LDL-C = low density lipoprotein cholesterol; Lp(a) = lipoprotein(a); PCSK9 = proprotein convertase subtilisin/kexin type 9; TC = total cholesterol; TG = triglycerides.