The KK obese mouse is moderately obese and has abnormally high levels of plasma insulin (hyperinsulinemia), glucose (hyperglycemia) and lipids (hyperlipidemia). In one strain (KK/San), we observed abnormally low plasma lipid levels (hypolipidemia). This mutant phenotype is inherited recessively as a mendelian trait. Here we report the mapping of the hypolipidemia (hypl) locus to the middle of chromosome 4 and positional cloning of the autosomal recessive mutation responsible for the hypolipidemia. The hypl locus encodes a unique angiopoietin-like lipoprotein modulator, which we named Allm1. It is identical to angiopoietin-like protein 3, encoded by Angptl3, and has a highly conserved counterpart in humans. Overexpression of Angptl3 or intravenous injection of the purified protein in KK/San mice elicited an increase in circulating plasma lipid levels. This increase was also observed in C57BL/6J normal mice. Taken together, these data suggest that Angptl3 regulates lipid metabolism in animals.

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.Hyperlipidemia is a major risk factor of coronary heart disease. Variations in human plasma lipid levels result from both genetic and environmental factors. Genetic factors account for more than 50% of the variation in plasma lipid levels in the human population (1-4). Naturally occurring mutations that affect lipid metabolism in mice have also been reported (5-7). In most cases, the mutated genes have not yet been identified, but elucidating the mutations could lead to the identification of the relevant genes.KK obese mice have a multigenic syndrome of moderate obesity and a diabetic phenotype that resembles human hereditary type 2 diabetes; they show signs of hyperinsulinemia, hyperglycemia, and hyperlipidemia (8 -10). We have found that KK mice in our laboratory (KK/San) have significantly low plasma lipid levels despite showing signs of hyperinsulinemia and hyperglycemia (11). Genetic analysis shows that the mutant phenotype of KK/San mice is inherited recessively as a Mendelian trait. We therefore named this locus hypl (for hypolipidemia). We observed the autosomal recessive hypl phenotype in the progeny of the KK/San strain and mapped the locus to the middle of chromosome 4. We identified a mutation in the gene encoding angiopoietin-like protein 3 (Angptl3) as the cause of the hypl trait (12). The mRNA of Angptl3 is predominantly localized in the liver. The expression of Angptl3 in KK/San mice was found to be 1/30 to 1/40 that of wild-type mice. Overexpression of Angptl3 using adenoviruses or by an intravenous injection of the recombinant protein in KK/San mice elicited a marked increase in circulating plasma total cholesterol...

Objectives-A low level of high-density lipoprotein (HDL) in plasma has been recognized as an aspect of metabolic syndrome and as a crucial risk factor of cardiovascular events. However, the physiological regulation of plasma HDL levels has not been completely defined. Current studies aim to reveal the contribution of angiopoietin-like protein3 (angptl3), previously known as a plasma suppressor of lipoprotein lipase, to HDL metabolism. Methods and Results-Angptl3-deficient mice showed low plasma HDL cholesterol and HDL phospholipid (PL), and which were increased by ANGPTL3 supplementation via adenovirus. In vitro, ANGPTL3 inhibited the phospholipase activity of endothelial lipase (EL), which hydrolyzes HDL-PL and hence decreases plasma HDL levels, through a putative heparin-binding site in the N-terminal domain of ANGPTL3. Post-heparin plasma in Angptl3-knockout mice had higher phospholipase activity than did that in wild-type mice, suggesting that the activity of endogenous EL is elevated in Angptl3-deficient mice. Furthermore, we established an ELISA system for human ANGPTL3 and found that plasma ANGPTL3 levels significantly correlated with plasma HDL cholesterol and HDL-PL levels in human subjects. Key Words: angptl3 Ⅲ high density lipoprotein Ⅲ endothelial lipase Ⅲ phospholipase Ⅲ triglyceride P lasma concentrations of high-density lipoprotein (HDL) cholesterol are inversely correlated with the risk of atherosclerotic cardiovascular disease. 1 HDL cholesterol levels are low in patients with metabolic disorders, such as obesity, insulin resistance, and diabetes. 2,3 However, the genetic and metabolic factors that regulate HDL metabolism remain to be elucidated. Recently, endothelial lipase (EL) has been recognized as one factor that influences HDL metabolism. EL was originally discovered as a member of the family of triglyceride (TG)-lipases together with lipoprotein lipase (LPL) and hepatic lipase (HL). In contrast to LPL or HL, however, EL has relatively lower triglyceride lipase activity and substantially higher phospholipid lipase activity and can hydrolyze HDL phospholipids (PL). 4 Overexpression of EL in mice resulted in reduced plasma HDL levels and EL knockout mice showed significant increase in HDL levels, [5][6][7] indicating that EL regulates HDL metabolism. Conclusions-Angptl3In the colony of KK mice, characterized by obesity, diabetes mellitus, and hypertriglyceridemia, we recently identified a mutant subgroup of KK/Snk mice with low plasma TG levels despite maintaining the phenotype of obesity and diabetes. Genetic mapping and positional cloning identified the gene of angiopoietin-like protein 3 (Angptl3), which was mutated in the KK/Snk mice. The Angptl3 gene in KK/Snk mice contained a 4-bp nucleotide insertion in exon 6, which caused a premature stop codon attributable to a frameshift, leading to a lack of production of the protein. 8 Angptl3 mRNA is expressed exclusively in the livers of humans and mice. ANGPTL3 protein contains a signal sequence of 18 amino acids at the N terminus, followed b...

Angiopoietin-like 3 (ANGPTL3) is a secreted protein that is mainly expressed in the liver and regulates lipid metabolism by inhibiting the lipolysis of triglyceriderich lipoproteins. Using deletion mutants of human ANGPTL3, we demonstrated that the N-terminal coiledcoil domain-containing fragment-(17-207) and not the C-terminal fibrinogen-like domain-containing fragment-(207-460) increased the plasma triglyceride levels in mice. We also found that the N-terminal region 17-165 was required to increase plasma triglyceride levels in mice and that a substitution of basic amino acid residues in the region 61-66 of the fragment showed no increase in the plasma triglyceride levels and no inhibition of lipolysis by lipoprotein lipase. In addition, when we analyzed ANGPTL3 in human plasma, we detected cleaved fragments of ANGPTL3. By analyzing recombinant ANGPTL3 in mouse plasma, we found that it was cleaved at two sites, Arg 221 2Ala 222 and Arg 224 2Thr 225 , which are located in the linker region between the coiled-coil domain and the fibrinogen-like domain. Furthermore, a cleavage-resistant mutant of ANGPTL3 was determined to be less active than wild-type ANGPTL3 in increasing mouse plasma triglyceride levels but not in inhibiting lipoprotein lipase activity. These findings suggest that the cleavage of ANGPTL3 is important for the activation of ANGPTL3 in vivo. Angiopoietin-like 3 (ANGPTL3)1 is a secreted protein that is expressed predominantly in the liver (1). ANGPTL3 is structurally similar to angiopoietins (2-4), which are vascular endothelial growth factors. It also has a signal peptide for secretion, a helical domain predicted to form coiled-coils (CCD), and a C-terminal globular fibrinogen-like domain (FLD). Angiopoietins are ligands that bind to the receptor tyrosine kinase Tie2. The FLDs of angiopoietins bind to Tie2, and the CCDs of angiopoietins mediate ligand homo-oligomerization (5). Although ANGPTL3 does not bind to Tie2, it may also induce angiogenesis by binding to integrin ␣ V ␤ 3 (6).We reported that KK/Snk mice (previously, KK/San) had abnormally low plasma lipid levels and identified a mutation in the Angptl3 gene as the cause of the hypolipidemic phenotype of KK/Snk mice (7,8). Injection of recombinant ANGPTL3 or adenoviruses encoding ANGPTL3 resulted in an increase of plasma lipid levels in mice. It has been speculated that ANGPTL3 was capable of inhibiting lipolysis of triglycerides, because an injection of recombinant ANGPTL3 induced a rapid increase in mouse plasma triglyceride levels (9, 10). We further reported that very low density lipoprotein clearance was enhanced in KK/Snk mice and that recombinant ANGPTL3 directly inhibited lipoprotein lipase (LPL) activity in vitro (11). These observations suggest that ANGPTL3 could be involved in the regulation of lipid metabolism by inhibiting LPL activity.Thus, ANGPTL3 seems to be a multifunctional factor that may regulate both lipid metabolism and angiogenesis. The C-terminal FLD of ANGPTL3 was reported to be involved in angiogenesis via binding t...