Lipid-lowering effects of anti-angiopoietin-like 4 antibody recapitulate the lipid phenotype found in angiopoietin-like 4 knockout mice

Abstract

We used gene knockout mice to explore the role of Angiopoietin-like-4 (Angptl4) in lipid metabolism as well as to generate anti-Angptl4 mAbs with pharmacological activity. Angptl4 -/- mice had lower triglyceride (TG) levels resulting both from increased very low-density lipoprotein (VLDL) clearance and decreased VLDL production and had modestly lower cholesterol levels. Also, both Angptl4 -/- suckling mice and adult mice fed a high-fat diet showed reduced viability associated with lipogranulomatous lesions of the intestines and their draining lymphatics and mesenteric lymph nodes. Treating C57BL/6J, ApoE -/-, LDLr -/-, and db/db mice with the anti-Angptl4 mAb 14D12 recapitulated the lipid and histopathologic phenotypes noted in Angptl4 -/- mice. This demonstrates that the knockout phenotype reflects not only the physiologic function of the Angptl4 gene but also predicts the pharmacologic consequences of Angptl4 protein inhibition with a neutralizing antibody in relevant models of human disease.

Fig. 1.

Lower TG levels in Angptl4 −/− mice and mice treated with 14D12 mAb. (A) Fasting total TG levels in Angptl4 −/− and +/+ mice maintained on HFD (TG measured by COBAS) or chow and 4 days after C57BL/6J mice maintained on HFD or chow received 14D12 or control mAb. (B) Fasting total TG levels 4 days after LDLr −/− mice received their 10th weekly dose of 14D12 mAb, control mAb, or vehicle; 4 days after ApoE −/− mice received their 9th weekly dose of 14D12 mAb, control mAb, or vehicle; and 6 days after db/db mice received a single dose of 14D12 or control mAb. The number (n) of mice in each group is shown. a, P < 0.0001; b, P = 0.0003.

Fig. 2.

Increased serum TG clearance and decreased VLDL production in Angptl4 −/− mice and mice treated with 14D12 mAb. (A) Intragastric lipid loading test. (Left) Serum TG measured at baseline (0) and 2, 4, and 6 h after a 15 ml/kg olive oil gavage in overnight-fasted Angptl4 −/− mice (n = 6) and +/+ mice (n = 7). (Right) Four days after receiving either 14D12 (n = 10) or control (n = 10) mAb, overnight-fasted C57BL/6J mice had serum TG measured at baseline (0) and 1, 3, and 6 h after a 15 ml/kg olive oil gavage. (B) i.v. lipid loading test. (Left) Serum TG measured at baseline (0) and 3, 15, and 30 min after Intralipid was given i.v. to overnight-fasted Angptl4 −/− mice (n = 8) and +/+ mice (n = 10). (Right) Four days after receiving either 14D12 (n = 7) or control (n = 9) mAb, overnight-fasted C57BL/6J mice had serum TG measured at baseline (0) and 3, 15, and 30 min after i.v. Intralipid delivery. (C) NEFA levels measured in the same samples presented in B. (D) VLDL production test. (Left) Serum TG measured at baseline (0) and 2, 4, and 6 h after Triton WR1339 was delivered i.v. to overnight-fasted Angptl4 −/− mice (n = 5) and +/+ mice (n = 6). (Right) Four days after receiving either 14D12 (n = 15) or control (n = 15) mAb, overnight-fasted C57BL/6J mice had serum TG measured at baseline (0) and 2, 4, and 6 h after i.v. delivery of Triton WR1339. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Fig. 3.

Decreased survival of Angptl4 −/− mice maintained on HFD. Shown is the percentage of Angptl4 −/− mice fed HFD (n = 69) or chow (n = 76) from weaning and Angptl4 +/+ mice fed HFD (n = 140) or chow (n = 93) from weaning that remained alive at various time points up to 45 weeks of age. Because gender did not affect survival, male and female mice were combined.

Fig. 4.

14D12 mAb lowers lipid levels in Angptl4 −/− mice expressing hAngptl4. (A) Fasting TG and cholesterol (chol) levels 4 days after Angptl4 +/+ and −/− mice received either 14D12 or control mAb. (B) Fasting TG levels measured in Angptl4 −/− mice at baseline on day 0 (d0) and then on day 4 (d4) and day 6 (d6). On d0, Angptl4 −/− mice were infected with Ad5-hAngptl4. After 4 days, serum TG levels were used to divide these mice into two groups with comparable TG levels. These groups then received 90 mg/kg of either 14D12 (n = 5) or control (n = 6) mAb on days 4 and 5.