Hypertriglyceridemia in Apoa5–/– mice results from reduced amounts of lipoprotein lipase in the capillary lumen

Figures

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  Figure 1. Reduced amounts of mLPL in the heart and BAT of Apoa5–/– mice. Heart and BAT cryosections from Apoa5–/– and Apoa5+/+ mice were stained with the mLPL-specific Ab Ab3174, the GPIHBP1-specific mAb 11A12, and the CD31-specific mAb 2H8. Sections from ΔLPL mice, which lack mLPL but express hLPL in endothelial cells, were also examined. (A and B) Confocal micrographs show mLPL, GPIHBP1, and CD31 staining in heart (A) and BAT (B). Scale bars: 20 μm.

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  Figure 2. Reduced amounts of mLPL, relative to GPIHBP1 or CD31, in heart and BAT capillaries of Apoa5–/– mice. Heart and BAT sections from Apoa5–/–, Apoa5+/+, and ΔLPL mice were stained with Ab3174, 11A12, and 2H8 (n = 4 independent experiments; 2–4 micrographs/tissue section). ΔLPL mice lack mLPL but express hLPL in endothelial cells. Ab3174, 11A12, and 2H8 fluorescence intensities were recorded in individual capillaries; the number of capillaries examined ranged from 323 to 1,870 per group. (A and B) LPL/GPIHBP1 and LPL/CD31 fluorescence intensity ratios in capillaries of heart (A) and BAT (B). Each dot represents the signal intensity ratio in a single capillary; ratio data were normalized to the mean ratio in capillaries of Apoa5+/+ mice (set as 1.0). Data represent the mean ± SD. ***P < 0.001, by 1-way ANOVA.

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  Figure 3. Amounts of LPL along the luminal surface of heart and BAT capillaries, relative to GPIHBP1 or CD31, are lower in Apoa5–/– mice. Apoa5+/+, Apoa5–/–, and ΔLPL mice were given an intravenous injection of Alexa Fluor–labeled mAbs against mLPL (27A7), GPIHBP1 (11A12), and CD31 (2H8). Ten minutes later, the mice were euthanized; perfused with PBS; and tissue sections were prepared for fluorescence microscopy. (A and B) Confocal micrographs of LPL, GPIHBP1, and CD31 along the luminal surface of capillaries in heart (A) and BAT (B). Scale bars: 20 μm.

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  Figure 4. Amounts of LPL in heart and BAT capillaries, relative to GPIHBP1 or CD31, are lower in Apoa5–/– mice. Apoa5–/–, Apoa5+/+, and ΔLPL mice were given an intravenous injection of Alexa Fluor–labeled 27A7 (against mLPL), 11A12 (against GPIHBP1), and 2H8 (against CD31). Following perfusion of the vasculature, tissue sections were prepared for fluorescence microscopy (n = 4 independent experiments; 2–4 micrographs/tissue section). 27A7, 11A12, and 2H8 fluorescence intensities in individual capillaries were recorded; the number of heart and BAT capillaries examined ranged from 256 to 2,031. (A and B) LPL/GPIHBP1 and LPL/CD31 fluorescence intensity ratios in heart (A) and BAT (B) capillaries of Apoa5–/–, Apoa5+/+, and ΔLPL mice. Each dot represents the mean signal intensity ratio in a single capillary, normalized to the mean ratio in capillaries of Apoa5+/+ mice (set as 1.0). Data represent the mean ± SD. ***P < 0.001, by 1-way ANOVA.

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  Figure 5. Reduced amounts of LPL in capillaries of Apoa5–/– mice, as judged by infrared scanning of sections spanning the entire heart or an entire BAT pad. Apoa5+/+, Apoa5–/–, and ΔLPL mice were given an intravenous injection of IRDye 680-27A7 and IRDye 800-11A12. Ten minutes later, the mice were euthanized, and the vasculature was perfused with PBS. (A and B) Infrared scans of heart (A) and BAT (B) sections revealing reduced amounts of intracapillary LPL, relative to GPIHBP1, in the heart and BAT of Apoa5–/– mice. Scale bars: 5 mm. (C and D) LPL/GPIHBP1 ratios in the hearts (C) and BAT (D) of Apoa5–/– (n = 4) and Apoa5+/+ (n = 3) mice. Signal intensities were measured in 10 tissue sections per mouse. Each dot represents the mean LPL/GPIHBP1 signal intensity ratio in 10 sections from 1 mouse; data in Apoa5–/– and ΔLPL mice were normalized to the mean ratio in Apoa5+/+ mice (set at 1.0). Data show the mean ± SEM. *P < 0.05 and **P < 0.01, by unpaired, 2-tailed Student’s t test for differences between Apoa5–/– and Apoa5+/+ mice.

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  Figure 6. Amounts of LPL in the postheparin plasma are lower in Apoa5–/– mice than in Apoa5+/+ mice. Apoa5–/– and Apoa5+/+ mice were given an intravenous injection of heparin (500 U/kg), and plasma samples were collected 2 minutes later. (A and B) LPL mass and activity levels in the postheparin plasma of individual Apoa5–/– and Apoa5+/+ mice during ad libitum feeding (A) and during fasting (B). Apoa5–/– mice, n = 5 in A and n = 6 in B; Apoa5+/+ mice, n = 7 in A and n = 9 in B. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by unpaired, 2-tailed Student’s t test. (C–E) Amounts of LPL in postheparin plasma from Apoa5–/– and Apoa5+/+ mice (n = 3 mice/group), as assessed by HS chromatography. LPL appeared in the “high-salt” fractions (fractions 21–27; 1.13–1.38 M NaCl). (C) LPL mass and activity levels in the HS fractions from the postheparin plasma of Apoa5–/– and Apoa5+/+ mice during ad libitum feeding. (D) Western blots (WB) of LPL in fractions 21–27 from fasted Apoa5–/– and Apoa5+/+ mice. Band intensity was measured with an infrared scanner. (E) Levels of LPL mass and activity in fractions 21–27 from fasted Apoa5–/– and Apoa5+/+ mice. The reported results were…

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  Figure 7. IBA490, an inhibitory ANGPTL3/8-specific mAb, lowers plasma TG levels in Apoa5–/– mice. Apoa5–/– and Apoa5+/+ mice were given a subcutaneous injection of IBA490 or a control mAb (10 mg/kg; n = 5/group). Plasma samples were obtained at baseline (t0) and 4, 24, 72, and 168 hours after administration of the mAbs. Data represent the mean ± SEM. **P < 0.01 and ***P < 0.001, by 2-way ANOVA, for comparisons of plasma TG levels in IBA490- and control mAb–treated Apoa5–/– mice; #P < 0.05 and ##P < 0.01, for comparisons of TG levels in IBA490- and control mAb–treated Apoa5+/+ mice. There were no significant differences in plasma TG levels in IBA490-treated Apoa5–/– or IBA490-treated Apoa5+/+ mice at 4, 24, and 72 hours.

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  Figure 8. IBA490, an inhibitory ANGPTL3/8-specific mAb, increases intracapillary LPL levels, relative to GPIHBP1 or CD31, in Apoa5–/– mice. Apoa5–/– and Apoa5+/+ mice were given a subcutaneous injection of IBA490 or a control mAb (10 mg/kg). Twenty-four hours later, the mice were given an intravenous injection of Alexa Fluor–labeled mAbs against mLPL (27A7), GPIHBP1 (11A12), and CD31 (2H8). After 10 minutes, mice were euthanized and then perfused with PBS, and tissue sections were prepared for fluorescence microscopy. (A and B) Confocal micrographs of LPL, GPIHBP1, and CD31 staining along the luminal surface of capillaries in heart tissue (A) and BAT (B) of IBA490- or control mAb–treated Apoa5–/– and Apoa5+/+ mice. Scale bars: 50 μm. The amounts of intracapillary LPL relative to the amounts of intracapillary GPIHBP1 or CD31 were quantified in 3 independent experiments, and the data are shown in Supplemental Figure 7.

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  Figure 9. Increased amounts of intracapillary LPL, relative to GPIHBP1 or CD31, in Apoa5–/– mice after injection of recombinant APOA5. Apoa5+/+ and Apoa5–/– mice were given an intravenous injection of recombinant APOA5 (10 mg/kg) or PBS alone. After 4 hours, mice were given an intravenous injection of Alexa Fluor–labeled mAbs against LPL (27A7), GPIHBP1 (11A12), and CD31 (2H8). After 10 minutes, the mice were euthanized, the vasculature was perfused with PBS, and tissue sections were examined by fluorescence microscopy. (A and B) Confocal micrographs of LPL, GPIHBP1, and CD31 staining along the luminal surface of capillaries in the heart (A) and BAT (B) of Apoa5–/– and Apoa5+/+ mice that had been treated with APOA5 or PBS alone. Scale bars: 50 μm. The amounts of intracapillary LPL relative to the amounts of intracapillary GPIHBP1 or CD31 were quantified in 3 independent experiments, and data are shown in Supplemental Figure 11.

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  Figure 10. ANGPTL3/8 releases hLPL from the surface of cells. (A and B) CHO-K1 cells (A) and CHO pgsA-745 cells that had been transiently transfected with a mouse GPIHBP1 vector (B) were incubated with 50 nM hLPL at 37°C for 10 minutes. After washing the cells with PBS/Ca/Mg, the cells were incubated with 0.1 U/mL heparin or with 100 nM ANGPTL3/8 in the presence or absence of 1 μM control mAb, 1 μM IBA490, or 1.4 μM APOA5 at 37°C for 15 minutes. The cells were washed and cooled on ice for 15 minutes. The amounts of hLPL remaining on the surface of cells were assessed by fluorescence microscopy with an Alexa Fluor–labeled mAb against hLPL (5D2) in A or with Alexa Fluor-labeled mAbs 5D2 and 11A12 in B. These results were observed in 2 independent experiments. Scale bars: 10 μm.