Inactivation of ANGPTL3 reduces hepatic VLDL-triglyceride secretion

Abstract

Humans and mice lacking angiopoietin-like protein 3 (ANGPTL3) have pan-hypolipidemia. ANGPTL3 inhibits two intravascular lipases, LPL and endothelial lipase, and the low plasma TG and HDL-cholesterol levels in ANGPTL3 deficiency reflect increased activity of these enzymes. The mechanism responsible for the low LDL-cholesterol levels associated with ANGPTL3 deficiency is not known. Here we used an anti-ANGPTL3 monoclonal antibody (REGN1500) to inactivate ANGPTL3 in mice with genetic deficiencies in key proteins involved in clearance of ApoB-containing lipoproteins. REGN1500 treatment consistently reduced plasma cholesterol levels in mice in which Apoe, Ldlr, Lrp1, and Sdc1 were inactivated singly or in combination, but did not alter clearance of rabbit (125)I-βVLDL or mouse (125)I-LDL. Despite a 61% reduction in VLDL-TG production, VLDL-ApoB-100 production was unchanged in REGN1500-treated animals. Hepatic TG content, fatty acid synthesis, and fatty acid oxidation were similar in REGN1500 and control antibody-treated animals. Taken together, our findings indicate that inactivation of ANGPTL3 does not affect the number of ApoB-containing lipoproteins secreted by the liver but alters the particles that are made such that they are cleared more rapidly from the circulation via a noncanonical pathway(s). The increased clearance of lipolytic remnants results in decreased production of LDL in ANGPTL3-deficient animals.

Keywords: angiopoietin-like protein 3; cholesterol; dyslipidemias; lipase/endothelial; lipase/lipoprotein; very low density lipoprotein.

Fig. 1.

Effects of anti-ANGPTL3 antibody (REGN1500) on levels and distribution of plasma cholesterol and TG in Ldlr^−/− and Ldlr^−/−;Lrp1^−/− (liver-specific Lrp1 KO) mice. A: Ldlr^−/− and Ldlr^−/−;Lrp1^−/− mice (n = 4–6 males per group, age 10–15 weeks) were injected with control IgG or REGN1500 (10 mg/kg) via the tail vein. Four days later, at the end of the dark cycle, the mice were fasted for 2 h prior to blood sampling. Plasma lipid levels were measured as described in the Materials and Methods. B: Distribution of plasma lipids. Pooled plasma from each group of mice was fractionated by FPLC. Cholesterol and TG levels were measured enzymatically in each fraction. The experiment was repeated and the results were similar. *P < 0.05, **P < 0.01.

Fig. 2.

REGN1500 reduces plasma levels of cholesterol and TG in Apoe^−/− and Apoe^−/−;Ldlr^−/− mice. A: Apoe^−/− and Apoe^−/−;Ldlr^−/− mice were injected with control IgG or REGN1500 (10 mg/kg) by tail vein (n = 4–5 males per group, 11 weeks old). Four days after the injection, mice were fasted for 2 h at the end of the dark cycle, and blood was collected. Plasma lipid levels were measured enzymatically as described in the Materials and Methods. B: Pooled plasma from each group of mice was fractionated by FPLC and both cholesterol and TG levels were measured in each fraction. *P < 0.05, **P < 0.01.

Fig. 3.

REGN1500 reduces circulating levels of cholesterol and TG in Sdc1^−/− mice. A: Sdc1^−/− and WT C57BL/6J mice were injected with control IgG or REGN1500 (10 mg/kg) by tail vein (n = 6 males per group, 10–12 weeks old). Four days later mice were fasted for 2 h at the end of the dark cycle and blood was collected and plasma lipid levels were measured. B: Pooled plasma from each group of mice was fractionated by FPLC and the cholesterol and TG levels were measured enzymatically in each fraction. The experiment was repeated and the results were similar. *P < 0.05, **P < 0.01.

Fig. 4.

REGN1500 does not alter LDL and β-VLDL turnover in WT mice. A: LDL was isolated from Ldlr^−/− mice and radiolabeled with iodine as described in the Materials and Methods. The labeled LDL (15 μg, specific activity: 170 cpm/ng) was injected into WT mice 4 days after injection of REGN1500, as described in the legend to Fig. 1 (n = 6 males per group, 10 weeks old). Blood was obtained from the tail vein at the indicated times after LDL injection. ApoB was precipitated from plasma using isopropanol and the radioactivity was quantitated in a scintillation counter. The percentage of injected label remaining at each time point was determined using the value obtained at 2 min as the starting value. The half-life was calculated from the decay curve of ApoB activity plotted against time. B: Rabbit β-VLDL was isolated and labeled with iodine as described in the Materials and Methods. The labeled β-VLDL (15 μg, specific activity: 567 cpm/ng) was injected into WT mice 4 days after treatment with REGN1500, as described in the legend to Fig. 1 (n = 5 males per group, 10 weeks old). Blood was collected and processed as described in (A). C: Two hours after the β-VLDL was injected into the mice, the mice were euthanized and tissues were collected. Tissue total uptake and acetone precipitated counts were measured as described in the Materials and Methods. The experiments were repeated once with similar results. BAT, brown adipose tissue; epiWAT, epidydimal white adipose tissue (WAT); scWAT, subcutaneous WAT.

Fig. 5.

Decreased VLDL-TG production in REGN1500-treated WT mice. A: WT mice were synchronized for 3 days (fasting: 5:00 PM to 7:00 AM and feeding: 7:00 AM to 5:00 PM). On day 4, mice were refed at 7:00 AM and injected at 9:00 AM with either a control antibody or REGN1500 (10 mg/kg) (n = 5 male mice per group, 10 weeks old). Two hours after the injection, Triton WR1339 (500 mg/kg) was injected into the tail vein. Blood samples were drawn from the tail veins at the indicated time points and then the mice were euthanized and blood was collected. Plasma TG levels were measured enzymatically. Plasma collected at the 90 min time point was pooled and fractionated by FPLC as described in the Materials and Methods, and TG and cholesterol were measured enzymatically in each fraction. The total TG and cholesterol content of the pooled VLDL fractions (7, 8, and 9) is shown below. B: ApoB-100 and ApoB-48 protein levels in the pooled VLDL fractions (7, 8, and 9) were assayed by immunoblotting (top) and quantified using ImageJ. ***, P < 0.001.

Fig. 6.

ApoB secretion in REGN1500-treated WT mice. A: WT mice were synchronized for 3 days (fasting: 5:00 PM to 7:00 AM and feeding: 7:00 AM to 5:00 PM). On day 4, mice were refed at 7:00 AM and injected at 9:00 AM with either a control antibody or REGN1500 (10 mg/kg) (n = 6 male mice per group, 10 weeks old). Two hours after the injection, Triton WR1339 (500 mg/kg) and 200 μCi of [³⁵S]methionine were injected into the tail vein. Blood samples were drawn from the tail veins before and 45 min after the injection. The mice were euthanized at 90 min and blood was collected. Plasma TG levels were measured at the indicated time after Triton WR1339 injection. B: Plasma collected at the 90 min time point was delipidated and size fractionated by SDS-PAGE (5%). Gels were dried and exposed to X-ray film (BIOMAX XAR; Kodak, catalog number 1651579) for 4 days at −80°C. The films were scanned using a HP Scanjet 5590 and quantified using ImageJ. The intensity of each band was corrected for background using a blank from the same film. C: The same plasma samples were subjected to immunoblot analysis with an anti-ApoB antibody and the bands were quantified using an Odyssey Image Analyzer (Li-Cor). *P < 0.05, ***P < 0.001.

Fig. 7.

Hepatic de novo lipogenesis and fatty acid oxidation did not change after ANGPTL3 depletion. A: mRNA levels of genes involved in fatty acid synthesis or oxidation. Livers were collected from refed mice used in the experiment shown in supplementary Fig. 2A. Total RNA was extracted and subjected to whole transcriptome shotgun sequencing using an Illumina 2500 Hi-Seq as described previously (27). B: Rates of de novo lipogenesis in livers and kidneys of Angptl3^−/− mice and littermate controls (n = 8 males per group, 12–16 weeks old). Mice were synchronized for 5 days. De novo lipogenesis was measured at the end of the refeeding cycle as described in the Materials and Methods. C: Fatty acid oxidation in primary hepatocytes isolated from REGN1500- or control antibody-treated WT mice (n = 4 males per group, 12 weeks old). Mice were treated with REGN1500 or control antibody (10 mg/kg) for 4 days. Primary hepatocytes were isolated and oxygen consumption rates were measured using a Seahorse XF analyzer as described in the Materials and Methods.