ANGPTL3 (Angiopoietin‐Like 3) Preferentially Resides on High‐Density Lipoprotein in the Human Circulation, Affecting Its Activity

Figures

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  Figure 1. ANGPTL3 predominantly resides on HDL but can also be found on LDL. Cholesterol (A) and ANGPTL3 (B) concentrations in plasma fractions of 2 healthy subjects (donor 1 and donor 2). C, ANGPTL3 ELISA results and immunoblot analysis in plasma of a healthy subject (donor 3). ANGPTL3 indicates angiopoietin‐like protein 3; apoA1, apolipoprotein A1; apoE, apolipoprotein E; HDL, high‐density lipoprotein; and LDL, low‐density lipoprotein.

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  Figure 2. ANGPTL3 can be bound to ultracentrifugation‐isolated LDL and HDL. ANGPTL3 (angiopoietin‐like protein 3) ELISA results in fast protein liquid chromatography‐fractions from ultracentrifugation‐isolated low‐density lipoprotein (LDL) and high‐density lipoprotein (HDL) incubated with or without recombinant human ANGPTL3.

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  Figure 3. Subject deficient for HDL has more ANGPTL3 residing on LDL. Cholesterol (A) and ANGPTL3 (B) concentrations in plasma fractions from plasma from a healthy subject and a subject who is homozygous for the C1477R mutation in ABCA1. ABCA1 indicates ATP‐binding cassette‐A1; ANGPTL3, angiopoietin‐like protein 3; HDL, high‐density lipoprotein; and LDL, low‐density lipoprotein.

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  Figure 4. ANGPTL3 has the highest lipase inhibitory activity when residing on LDL. Triglyceride‐rich lipoprotein lipase activity of T37i brown adipocytes incubated with ANGPTL3 in combination with angiopoietin‐like 8 in the absence or presence of LDL or HDL overnight (A) or 1 hour (B). Data are shown as boxplots with results from 2 independent experiments; n=8. C, Results of incubating the T37i cells with 3H‐trioleate for 30 minutes. Data are shown as boxplots with results from 3 independent experiments; n=12. # P<0.05 vs control (no lipoprotein, no ANGPTL3); *P<0.05 vs same lipoprotein condition without ANGPTL3. ANGPTL3 indicates angiopoietin‐like protein 3; FFA, free fatty acid; HDL, high‐density lipoprotein; LDL, low‐density lipoprotein; and O/N, overnight.

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  Figure 5. ANGPTL3 reduces LPL protein on the T37i plasma membrane when residing on HDL or LDL. A, LPL immunoblots of medium of heparin‐treated T37i cells incubated with ANGPTL3 in combination with ANGPTL8 (angiopoietin‐like 8) in the absence or presence of LDL or HDL overnight (O/N) or 1 hour. B, Relative mRNA expression of Lpl, Ucp1, and Angptl4 of T37i cells incubated with ANGPTL3 in combination with ANGPTL8 in the absence or presence of LDL or HDL for 24 hours. Relative mRNA expression was normalized to reference genes Actb and Rplp0 with data from cells not incubated with lipoproteins and ANGPTL3 defined as “1.” Data are shown as boxplots with results from 3 independent experiments; n=12. Actb indicates beta‐actin; ANGPTL3, angiopoietin‐like protein 3; Angptl4, angiopoietin‐like 4; FL, full‐length; HDL, high‐density lipoprotein; LDL, low‐density lipoprotein; LPL, lipoprotein lipase; N‐term, N‐terminal; Rplp0, ribosomal protein lateral stalk subunit P0; and Ucp1, uncoupling protein‐1.

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  Figure 6. Plasma ANGPTL3 concentrations correlate most strongly with HDL cholesterol and large HDL particles in a human cohort study. Correlation coefficients of the associations between plasma ANGPTL3 concentrations and plasma lipids (A) and the amount of varying sizes of lipoproteins (B) in the nested case–cohort EPIC‐Norfolk study. ANGPTL3 indicates angiopoietin‐like protein 3; ApoB, apolipoprotein B; EPIC, European Prospective Investigation Into Cancer; HDL, high‐density lipoprotein; L, large; LDL, low‐density lipoprotein; Lp(a), lipoprotein A; M, medium; S, small; and VLDL, very low‐density lipoprotein.

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  Figure 7. Subjects with the highest plasma ANGPTL3 concentrations have a higher incidence of cardiovascular events. A, Plasma ANGPTL3 concentrations in the nested case–cohort EPIC‐Norfolk study. B, Plasma ANGPTL3 values in controls and cases. C, Odds ratios for incident future cardiovascular events of subjects within the fifth quintile of plasma ANGPTL3 concentrations compared with the other subjects in the nested case–cohort EPIC‐Norfolk study. Upper value is for the age‐ and sex‐adjusted model, lower value is for the multivariable model that also included systolic blood pressure, LDL cholesterol, log‐transformed triglycerides, diabetes, and current smoking status. ANGPTL3 indicates angiopoietin‐like protein 3; CVD, cardiovascular disease; EPIC, European Prospective Investigation Into Cancer; LDL, low‐density lipoprotein; and OR, odds ratio.