Depletion of Hepatic SREBP2 Protects Against Hypercholesterolemia and Atherosclerosis through the ANGPTL3‐LPL Axis

Figures

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  Figure 1. Acute depletion of hepatic SREBP2 reduces circulating lipid levels. A–D) Plasma triglyceride (A and C) and cholesterol (B and D) levels of 16 h fasted C57BL/6J male mice injected with AAV8‐scramble control or AAV8‐Srebf2‐shRNA viruses under 5‐week chow diet feeding (A and B, n = 8 mice per group, by student's t test) and 5‐week western diet feeding (C and D, n = 8 mice per group, by two‐way ANOVA). E–H) Plasma triglyceride (E and G) and cholesterol (F and H) levels of 16 h fasted male mice lacking LDLR injected with AAV8‐scramble control or AAV8‐Srebf2‐shRNA viruses under 5‐week chow diet (E and F, n = 8 mice per group, by student's t test) and 5‐week western diet (G and H, n = 6–8 mice per group, by two‐way ANOVA) feeding. I–L) Western blot (I and J) and relative quantification (K and L) of APOB‐100 and APOB‐48 in the plasma of 4 h fasted male C57BL/6J mice (I and K, n = 7 mice per group, by student's t test) and mice lacking LDLR (J and L, n = 6–8 mice per group, by student's t test) after AAV8 injection for 3 weeks. M,N) Quantification of plasma VLDL/LDL and HDL cholesterol levels in 16 h‐fasted C57BL/6J and Ldlr−/− mice injected with AAV8‐scramble control or AAV8‐Srebf2‐shRNA…

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  Figure 2. Knocking down hepatic SREBF2 reduced VLDL secretion both in vitro and in vivo. A–D) Changes of plasma triglyceride after Poloxamer‐407 injection in male C57BL/6J mice (A and C) and male Ldlr−/− (B and D) injected with AAV8‐scramble control or AAV8‐Srebf2‐shRNA viruses under chow or western diet feeding. VLDL secretion levels were estimated and compared using an area‐under‐the‐curve (AUC) approach that subtracts the baseline value (n = 6–8 mice per group, by student's t‐test). E,F) Quantitative polymerase chain reaction (qPCR) expression analysis of de novo lipogenesis and cholesterol synthesis genes in liver of 16h‐fasted male C57BL/6J mice (E) and Ldlr−/− mice (F) injected with AAV8‐scramble control or AAV8‐Srebf2‐shRNA viruses under 5‐week chow followed by 5‐week western diet feeding (n = 6–8 per group, by student's t test). G) Measurement of APOB100 secretion levels in SREBF2 knockout or control Huh7 cells in the presence or absence of LDLR using ELISA (n = 6 replicates per experiment, representative of 3 independent experiments, by two‐way ANOVA). H) Measurement of triglyceride secretion in the culture medium of SREBF2 knockout or control Huh7 cells in the presence or absence…

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  Figure 3. Depletion of hepatic SREBP2 promotes Lipoprotein lipase activity in the absence of LDLR. A,B) Plasma lipoprotein lipase contents (A) and lipoprotein lipase activity (B) measured in heparinized C57BL/6J or Ldlr−/− male mice injected with AAV8‐scramble control or AAV8‐Srebf2‐shRNA viruses under chow diet feeding (n = 8 mice per group, by two‐way ANOVA). C,D) Plasma lipoprotein lipase contents (C) and lipoprotein lipase activity (D) measured in heparinized Ldlr−/− male mice injected with AAV8‐scramble control or AAV8‐Srebf2‐shRNA viruses under 5‐week chow diet feeding followed by 3‐week western diet feeding (n = 6–8 mice per group, by student's t test). E) Relative expression of the genes involved in de novo lipogenesis, cholesterol synthesis and LPL regulation measured by RNA‐seq using different CRISPR/Cas9‐mediated genetically modified AML12 mouse hepatocyte cell line (n = 3 replicates for each cell line). F,G) qPCR expression analysis of genes modulating LPL activity in the liver of C57BL/6J (F) and Ldlr−/− (G) mice injected with AAV8‐scramble control or AAV8‐Srebf2‐shRNA viruses under 5‐week chow followed by 5‐week western diet feeding (n = 5–6 mice per group, by student's t…

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  Figure 4. Hepatic SREBP2 regulates peripheral LPL activity mainly through ANGPTL3 in the absence of LDLR. A,B) Plasma triglyceride (A) and cholesterol (B) levels of 16 h fasted Ldlr−/−,Srebf2fl/fl male mice injected with AAV8‐TBG‐GFP or AAV8‐TBG‐Cre together with AAV8‐shRNA‐scramble or AAV8‐shRNA‐Angptl3 to achieve hepatocyte‐specific knockout of Srebf2 (Srebf2 HKO), hepatic depletion of ANGPTL3 (Angptl3 KD), and SREBP2 and ANGPTL3 double depletion (Srebf2 HKO+ Angptl3 KD) under chow diet feeding (n = 5 mice per group, by two‐way ANOVA). C) Changes of plasma triglyceride after Poloxamer‐407 injection in Ldlr−/−,Srebf2fl/fl male mice injected with different AAVs to achieve hepatic depletion of SREBP2 and/or ANGPTL3 under chow diet feeding (n = 5 mice per group). VLDL secretion levels were estimated and compared using an area‐under‐the‐curve (AUC) approach that subtracts the baseline value (n = 5 mice per group, by two‐way ANOVA). D,E) Plasma total lipoprotein lipase contents (D) and lipoprotein lipase activity (E) measured in heparinized Ldlr−/−,Srebf2fl/fl male mice injected with different AAVs to achieve hepatic depletion of SREBP2 and/or ANGPTL3 under chow diet feeding (n = 5 mice per…

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  Figure 5. SREBP2 promotes proteasome‐mediated degradation of ANGPTL3 in the presence of LDLR. A–C) Western Blot and quantification of cellular and secreted ANGPTL3 in SREBF2 knockout or control LDLR+/+ Huh7 cells upon 1 um MG‐132 or DMSO treatment for 4 h. (n = 3 replicates from individual wells, representative of 3 independent experiments, by two‐way ANOVA). D) Western Blot of cellular and secreted ANGPTL3 in SREBF2 knockout or control LDLR+/+ Huh7 cells upon 1 um MG‐132 treatment for 0, 2, 4, and 6 h (representative of 3 independent experiments). E–G) Western Blot and quantification of cellular and secreted ANGPTL3 in SREBF2 knockout or control LDLR−/− Huh7 cells upon 1 um MG‐132 or DMSO treatment for 4 h. (n = 3 replicates from individual wells, representative of 3 independent experiments, by two‐way ANOVA). H) Western Blot of cellular and secreted ANGPTL3 in SREBF2 knockout or control LDLR−/− Huh7 cells upon 1uM MG‐132 treatment for 0, 2, 4, and 6 h (representative of 3 independent experiments). *p < 0.05, **p < 0.01, and ***p < 0.001; Error bars indicate mean ± SD.

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  Figure 6. Acute depletion of hepatic SREBP2 synergizes with PCSK9 inhibition to reduce plasma cholesterol levels. A,B) Plasma triglyceride (A) and cholesterol (B) levels of Srebf2fl/fl and Srebf2 hepatocyte‐specific knockout (Srebf2 HKO) mice administrated with single‐dose Alirocumab or saline (n = 5 mice per group, by two‐way ANOVA). *p < 0.05, **p < 0.01, and ***p < 0.001; Error bars indicate mean ± SD.

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  Figure 7. Acute depletion of hepatic SREBP2 protects against atherosclerosis. A,B) Representative images (A) and quantification of aortic lesion areas (B) from cross sectional aortic root sections stained with Oil Red O and hematoxylin for lesion area from LDLR‐Deficient mice injected with AAV8‐scramble control or AAV8‐Srebf2‐shRNA and fed western diet for 14 weeks (n = 4–5 mice per group, each mouse has at least 3 sections for analysis, by student's t test). C,D) Representative images (C) and quantification of necrosis level (D) from cross sectional aortic root sections stained with hematoxylin and eosin for lesion area from LDLR‐Deficient mice injected with AAV8‐scramble control or AAV8‐Srebf2‐shRNA and fed western diet for 14 weeks (n = 4–5 mice per group, each mouse has at least 3 sections for analysis, by student's t test). E,F) Representative images (E) and quantification of aortic lesion areas (F) from en face analysis of aortas before and after Oil Red O staining in the male Ldlr−/− mice injected with AAV8‐scramble control or AAV8‐Srebf2‐shRNA and fed with western diet for 14 weeks (n = 4–5 mice per group, by student's t test). G) Model of the mechanism by which hepatic SREBP2…