ANGPTL3 Decreases Very Low Density Lipoprotein Triglyceride Clearance by Inhibition of Lipoprotein Lipase

Tetsuya Shimizugawa,Mitsuru Ono,Mitsuru Shimamura,Kenichi Yoshida,Yosuke Ando,Ryuta Koishi,Kenjiro Ueda,Toshimori Inaba,Hiroyuki Minekura,Takafumi Kohama,Hidehiko Furukawa

doi:10.1074/jbc.m203215200

Abstract

KK/San is a mutant mouse strain established in our laboratory from KK obese mice. KK/San mice show low plasma lipid levels compared with wild-type KK mice despite showing signs of hyperglycemia and hyperinsulinemia. Recently, we identified a mutation in the gene encoding angiopoietin-like protein 3 (Angptl3) in KK/San mice, and injection of adenoviruses encoding Angptl3 or recombinant ANGPTL3 protein to mutant KK/San mice raised plasma lipid levels. To elucidate the regulatory mechanism of ANGPTL3 on lipid metabolism, we focused on the metabolic pathways of triglyceride in the present study. Overexpression of Angptl3 in KK/San mice resulted in a marked increase of triglyceride-enriched very low density lipoprotein (VLDL). In vivo studies using Triton WR1339 revealed that there is no significant difference between mutant and wild-type KK mice in the hepatic VLDL triglyceride secretion rate. However, turnover studies using radiolabeled VLDL revealed that the clearance of (3)H-triglyceride-labeled VLDL was significantly enhanced in KK/San mice, whereas the clearance of (125)I-labeled VLDL was only slightly enhanced. In vitro analysis of recombinant protein revealed that ANGPTL3 directly inhibits LPL activity. These data strongly support the hypothesis that ANGPTL3 is a new class of lipid metabolism modulator, which regulates VLDL triglyceride levels through the inhibition of LPL activity.

Highlights

EXPERIMENTAL PROCEDURESAnimals—KK mice were obtained from Nagoya University and BALB/c mice from Charles River
Hyperlipidemia is a major risk factor of coronary heart disease
Plasma Lipid Levels and Lipoprotein Profiles—We previously reported (12) that KK/San mice exhibit extremely low plasma triglyceride levels compared with wild-type KK mice and that a low level expression of angiopoietin-like protein 3 (Angptl3) causes hypolipidemia

Summary

EXPERIMENTAL PROCEDURES

Animals—KK mice were obtained from Nagoya University and BALB/c mice from Charles River. KK/San and wild-type KK mice were injected into the tail vein with 125I-labeled VLDL (10 ␮g of tracer in 200 ␮l of 0.9% NaCl containing 2 mg/ml of bovine serum albumin). The serum-free culture medium of CHO-K1 cells transfected with the pMEh55-1 expression plasmid DNA was concentrated from 10 to 1 liters We applied it to a Sephadex 25 column (Amersham Biosciences) and eluted it with 20 mM Tris-HCl, pH 7.5, buffer. We applied the sample to a Q Sepharose fast flow column (Amersham Biosciences) and eluted it with 0.5 M NaCl in 20 mM Tris-HCl, pH 7.5, buffer. We applied the diluted sample to a lentil-lectin-Sepharose column (Amersham Biosciences) and eluted it with 0.5 M NaCl, 0.3 M methylmannopyranoside in 20 mM Tris-HCl, pH 7.5, buffer. For the HL assay, mouse plasma was used as the source of HL, and the reaction was performed in the same manner as in the LPL assay except that the NaCl concentration used was 1 M

RESULTS

Body weight

Immediately after adenovirus n injection

DISCUSSION