ANGPTL3 Decreases Very Low Density Lipoprotein Triglyceride Clearance by Inhibition of Lipoprotein Lipase
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 encodingAngptl3 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 ofAngptl3 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 of3H-triglyceride-labeled VLDL was significantly enhanced in KK/San mice, whereas the clearance of 125I-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. 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 encodingAngptl3 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 ofAngptl3 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 of3H-triglyceride-labeled VLDL was significantly enhanced in KK/San mice, whereas the clearance of 125I-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. non-esterified fatty acid angiopoietin-like protein 3 Chinese hamster ovary Dulbecco's modified Eagle's medium very low density lipoprotein lipoprotein lipase hepatic lipase high performance liquid chromatography 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 (1Rice T. Vogler G.P. Perry T.S. Laskarzewski P.M. Rao D.C. Hum. Hered. 1991; 41: 107-121Crossref PubMed Scopus (41) Google Scholar, 2Perusse L. Despres J.P. Tremblay A. Leblanc C. Talbot J. Allard C. Bouchard C. Arteriosclerosis. 1989; 9: 308-318Crossref PubMed Google Scholar, 3Bucher K.D. Friedlander Y. Kaplan E.B. Namboodiri K.K. Kark J.D. Eisenberg S. Stein Y. Rifkind B.M. Genet. Epidemiol. 1988; 5: 17-33Crossref PubMed Scopus (35) Google Scholar, 4Austin M.A. King M.C. Bawol R.D. Hulley S.B. Friedman G.D. Am. J. Epidemiol. 1987; 125: 308-318Crossref PubMed Scopus (184) Google Scholar). Naturally occurring mutations that affect lipid metabolism in mice have also been reported (5Reue K. Doolittle M.H. J. Lipid Res. 1996; 37: 1387-1405Abstract Full Text PDF PubMed Google Scholar, 6Welch C.L. Xia Y.R. Shechter I. Farese R. Mehrabian M. Mehdizadeh S. Warden C.H. Lusis A.J. J. Lipid Res. 1996; 37: 1406-1421Abstract Full Text PDF PubMed Google Scholar, 7Purcell-Huynh D.A. Weinreb A. Castellani L.W. Mehrabian M. Doolittle M.H. Lusis A.J. J. Clin. Invest. 1995; 96: 1845-1858Crossref PubMed Scopus (81) Google Scholar). 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 (8Kondo K. Nozawa K. Tomita T. Ezaki K. Bull. Exp. Anim. 1957; 6: 107-112Crossref Google Scholar, 9Nakamura M. Yamada K. Diabetologia. 1967; 3: 212-221Crossref PubMed Scopus (136) Google Scholar, 10Nakamura M. Proc. Jpn. Acad. 1962; 38: 348-352Crossref Google Scholar). We have found that KK mice in our laboratory (KK/San) have significantly low plasma lipid levels despite showing signs of hyperinsulinemia and hyperglycemia (11Shiraki T. Yoshioka S. Horikoshi H. Diabetes Frontier. 1993; 4: 641Google Scholar). 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 hyplphenotype 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 (12Koishi R. Ando Y. Ono M. Shimamura M. Yasumo H. Fujiwara T. Horikoshi H. Furukawa H. Nat. Genet. 2002; 30: 151-157Crossref PubMed Scopus (336) Google Scholar). The mRNA of Angptl3is predominantly localized in the liver. The expression ofAngptl3 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, non-esterified fatty acids (NEFAs),1 and especially triglyceride levels (12Koishi R. Ando Y. Ono M. Shimamura M. Yasumo H. Fujiwara T. Horikoshi H. Furukawa H. Nat. Genet. 2002; 30: 151-157Crossref PubMed Scopus (336) Google Scholar). Angptl3 is ∼7 kb long and is composed of seven exons, the last four of which located at the carboxyl terminus end of the protein and encode the fibrinogen-like domain. The amino terminus contains a putative signal sequence and a coiled-coil domain. COS-1 and CHO-K1 cells transfected with SRα promoter-driven mammalian cell expression vectors containing ANGPTL3 cDNA secreted a major ANGPTL3 protein of ∼70 kDa. Deglycosylation reduced the apparent molecular mass of the recombinant ANGPTL3 to 53 kDa as predicted by sequence analysis. Angiopoietins are members of the vascular endothelial growth factor family (13Davis S. Aldrich T.H. Jones P.F. Acheson A. Compton D.L. Jain V. Ryan T.E. Bruno J. Radziejewski C. Maisonpierre P.C. Yancopoulos G.D. Cell. 1996; 87: 1161-1169Abstract Full Text Full Text PDF PubMed Scopus (1703) Google Scholar, 14Maisonpierre P.C. Suri C. Jones P.F. Bartunkova S. Wiegand S.J. Radziejewski C. Compton D. McClain J. Aldrich T.H. Papadopoulos N. Daly T.J. Davis S. Sato T.N. Yancopoulos G.D. Science. 1997; 277: 55-60Crossref PubMed Scopus (2995) Google Scholar). They have fibrinogen-like domains that are conserved and are predicted to come into direct contact with receptors (15Valenzuela D.M. Griffiths J.A. Rojas J. Aldrich T.H. Jones P.F. Zhou H. McClain J. Copeland N.G. Gilbert D.J. Jenkins N.A. Huang T. Papadopoulos N. Maisonpierre P.C. Davis S. Yancopoulos G.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1904-1909Crossref PubMed Scopus (400) Google Scholar, 16Procopio W.N. Pelavin P.I. Lee W.M. Yeilding N.M. J. Biol. Chem. 1999; 274: 30196-30201Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Within the fibrinogen-like domain, angiopoietins have a cystein-based motif (15Valenzuela D.M. Griffiths J.A. Rojas J. Aldrich T.H. Jones P.F. Zhou H. McClain J. Copeland N.G. Gilbert D.J. Jenkins N.A. Huang T. Papadopoulos N. Maisonpierre P.C. Davis S. Yancopoulos G.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1904-1909Crossref PubMed Scopus (400) Google Scholar). In contrast with other members of the angiopoietin family, however, ANGPTL3 lacks this motif, and preliminary data obtained from a BIAcore assay indicate that ANGPTL3 does not bind Tie2, an angiopoietin receptor. ANGPTL3 is involved primarily in the regulation of lipid metabolism rather than in mediating the growth of vascular epithelium. Although ANGPTL3 regulates lipid metabolism, the direct effect of ANGPTL3 on VLDL triglyceride metabolism has not yet been investigated. Therefore, in the present study, the effect of a mutation in a gene encoding Angptl3 on VLDL metabolism was investigated by performing VLDL turnover studies in KK/San and wild-type KK mice. We found that KK/San mice showed enhanced VLDL clearance compared with wild-type KK mice due to enhanced lipolysis of VLDL triglycerides, whereas VLDL triglyceride production was not affected. We also found that recombinant ANGPTL3 protein directly inhibited LPL activity. Our results suggest that AGNPTL3 affects VLDL triglyceride clearance by interfering with LPL activity. KK mice were obtained from Nagoya University and BALB/c mice from Charles River. The KK mice used were males between 3 and 5 months old, which were housed in a room under controlled temperature (23 ± 1 °C) with free access to water and mouse chow (Oriental Yeast). Total plasma cholesterol and triglyceride levels were measured enzymatically using assay kits from Wako Pure Chemical Industries. The distribution of cholesterol within the plasma lipoproteins was determined by high performance liquid chromatography (HPLC) (17Okazaki M. Komoriya N. Tomoike H. Inoue N. Usui S. Itoh S. Hosaki S. J. Chromatogr. B Biomed. Sci. Appl. 1998; 709: 179-187Crossref PubMed Scopus (32) Google Scholar). Samples of 40 μl of the diluted plasma were applied onto two columns of TSKgel Lipopropak XL (Tosoh). The elution was performed at a flow rate of 0.7 ml/min for the TSK eluent LP-2 (Tosoh) and 0.35 ml/min for the enzyme solution (Determiner LTC, Kyowa Medex Co., Tokyo). The detection of cholesterol in the eluted fractions was carried out at a wavelength of 550 nm following the enzymatic reaction using a computerized system. We isolated full-length mouse Angptl3 cDNA from a KK mouse liver λ-ZAP cDNA library constructed using the ZAP Express cDNA Gigapack III Gold cloning kit (Stratagene). We used a 200-bp PCR-amplified fragment of Angptl3 cDNA (nucleotides 62–260 from the GenBankTM library) as a probe. We obtained full-length human ANGPTL3 cDNA from a human liver cDNA library (CLONTECH) by plaque hybridization. We digested this cDNA with EcoRI andXbaI and subcloned the resulting fragment into theEcoRI/XbaI sites of pME18S (pMEh55-1). We determined the nucleotide sequences of these cloned fragments for both strands using the ABI BigDye Terminator kit (Applied Biosystems). We generated recombinant adenoviruses as previously described (18Miyake S. Makimura M. Kanegae Y. Harada S. Sato Y. Takamori K. Tokuda C. Saito I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1320-1324Crossref PubMed Scopus (789) Google Scholar) using an Adenovirus Expression Vector Kit (Takara Shuzo). We prepared Ad/lacZ and Ad/Angptl3 by inserting the β-galactosidase and Angptl3 cDNAs, respectively, into the Ad E1-deleted region under the control of the CAG promoter. Recombinant adenoviruses were propagated and purified as described previously (19Kanegae Y. Makimura M. Saito I. Jpn. J. Med. Sci. Biol. 1994; 47: 157-166Crossref PubMed Scopus (431) Google Scholar). We injected 2 ×109 plaque-forming units of each recombinant adenovirus intravenously to KK/San mice via the tail vein. We obtained blood from the retro-orbital plexus 3 days after injection. Fasted KK/San and wild-type KK mice were injected intravenously with Triton WR1339 (400 mg/kg body weight) using 20% (w/v) Triton solution in 0.9% NaCl (20Otway S. Robinson D.S. J. Physiol. 1967; 190: 321-332Crossref PubMed Scopus (217) Google Scholar). Blood samples were drawn from the tail vein at 0, 60, 120, and 180 min after the Triton injection and analyzed for triglycerides as described above. Plasma samples (1 μl/lane) were separated on 2–15% gradient gels (Daiichi Pure Chemicals), and the proteins were transferred onto The were with was used as a and were by detection The of the was using an In VLDL turnover studies were on a previously described M.C. M.H. J. Clin. Invest. 1996; PubMed Scopus Google Scholar). acid in was under and in 0.9% NaCl containing 2 to a of 1 BALB/c mice were injected intravenously via the tail vein with of the prepared and from the min after injection. VLDL for in the clearance studies was isolated from the plasma of mice by To the in of VLDL triglycerides, KK/San and wild-type KK mice were injected intravenously with of The clearance rate of the radiolabeled VLDL was determined from blood samples from mice, drawn at the after the injection. Total plasma was used to VLDL triglyceride Blood was from BALB/c mice. Plasma samples were and VLDL was obtained by VLDL was with by the Eisenberg S. 212-221Crossref PubMed Scopus Google Scholar). The of was of the VLDL samples were a containing NaCl and KK/San and wild-type KK mice were injected into the tail vein with 125I-labeled VLDL of in μl of 0.9% NaCl containing 2 of Blood samples of μl were from the retro-orbital plexus at the after the injection. The plasma of 125I-labeled was determined by the in the obtained after L. K. PubMed Scopus Google Scholar, T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). We digested full-length human ANGPTL3 cDNA with EcoRI andXbaI and subcloned the resulting fragment into theEcoRI/XbaI sites of pME18S (pMEh55-1). We transfected the expression into CHO-K1 cells using The medium of CHO-K1 cells transfected with the expression was from to 1 We applied to a and eluted with We applied the to a flow and eluted with NaCl in We applied the to a and eluted with 1 NaCl in The was diluted to NaCl with We applied the diluted to a and eluted with in we the we applied the to a and eluted with NaCl in LPL was obtained from The medium containing LPL was obtained from cells were from The cells were in modified medium containing 1 and medium is to as the cells the medium was with the medium containing and days after the medium was with the medium containing 2 days of the cells were for min with of and the was for LPL LPL were on a previously described M.C. J. Lipid Res. Full Text PDF PubMed Google Scholar). The were carried out in a total of with of assay and of enzyme The assay solution 2 and The was at for min with recombinant ANGPTL3 at a of 0, or The enzyme reaction was by the of 1 of and of The was for and at for in 1 of the was using a of enzymatic was as the of 1 of at To elucidate the between and human was of as the LPL the mouse plasma was used as the of and the reaction was performed in the as in the LPL assay that the NaCl used was We previously reported (12Koishi R. Ando Y. Ono M. Shimamura M. Yasumo H. Fujiwara T. Horikoshi H. Furukawa H. Nat. Genet. 2002; 30: 151-157Crossref PubMed Scopus (336) Google Scholar) that KK/San mice low plasma triglyceride levels compared with wild-type KK mice and also that a low expression of Angptl3 To the mechanism for the triglyceride levels in KK/San mice, we analyzed lipoprotein in in plasma triglyceride and total cholesterol levels in KK/San mice were than in wild-type KK mice at the of 5 significant in body were observed between the wild-type KK and KK/San mice. To the lipoprotein lipoprotein plasma samples of wild-type KK mice and KK/San mice were to analysis The levels of triglycerides and total cholesterol in KK/San mice were due to a in the fractions of cholesterol and triglyceride levels in KK/San and wild-type KK ± ± significantly from wild-type KK mice, using ± significantly from wild-type KK mice, using ± ± ± cholesterol and triglyceride levels were measured in the plasma of KK/San and wild-type KK mice. The ± of significantly from wild-type KK mice, using in a new Total cholesterol and triglyceride levels were measured in the plasma of KK/San and wild-type KK mice. The ± of shows the plasma lipid levels after the gene of Angptl3 in KK/San mice. Overexpression of Angptl3 in KK/San mice resulted in a marked increase in plasma triglyceride levels as compared with KK/San mice. Although cholesterol levels were only slightly KK/San mice showed a marked increase in the VLDL cholesterol and triglyceride levels KK/San mice in after of after adenovirus 3 after adenovirus ± ± ± mice were injected intravenously with 2 units of or and 3 days after adenovirus plasma was obtained and analyzed for cholesterol and The ± that were not in a new KK/San mice were injected intravenously with 2 units of or and 3 days after adenovirus plasma was obtained and analyzed for cholesterol and The ± that were not We also compared the levels of KK/San and wild-type KK mice. significant were observed between the KK/San and wild-type KK mice for plasma and levels The in plasma triglycerides rather than cholesterol levels in KK/San mice that ANGPTL3 with triglyceride ANGPTL3 the VLDL triglyceride secretion or VLDL triglyceride to the low plasma triglyceride levels in KK/San mice were due to VLDL triglyceride we measured the VLDL triglyceride secretion rate by the Triton WR1339 in the increase in plasma triglycerides was for wild-type KK and KK/San mice. The VLDL triglyceride secretion on the was not found to be significantly between KK/San and wild-type KK mice. We investigated the low plasma triglyceride levels in KK/San mice were due to enhanced triglyceride To triglyceride KK/San and wild-type KK mice were injected with in triglycerides were more from the in KK/San mice. To this enhanced clearance of triglycerides in KK/San mice was due to enhanced lipolysis or hepatic VLDL turnover studies were performed as a for the injection of VLDL was to be at a slightly rate in KK/San mice the low plasma triglyceride levels in KK/San mice compared with wild-type KK mice were primarily due to enhanced lipolysis rather than to enhanced We the effect of ANGPTL3 on VLDL triglyceride clearance resulted from a direct effect of ANGPTL3 on the of LPL and Recombinant ANGPTL3 protein was to purified LPL or to a medium of and the effect on LPL was in ANGPTL3 inhibited not only LPL but also purified The inhibition of LPL by ANGPTL3 was at a of was also measured using plasma from mice mouse is found in free in the inhibition of by ANGPTL3 was very compared with that of ANGPTL3 inhibited by only at a of of ANGPTL3 on enzyme in mouse plasma was determined in the of recombinant ANGPTL3 at the The ± as a of the control determined in the of recombinant we the between ANGPTL3 and a of human was used as a of and the of was to the of purified in ANGPTL3 inhibited purified LPL in a both in the and of However, the LPL inhibition by ANGPTL3 was at of in a In the present study, we have that ANGPTL3 is involved in the metabolism of is by the that of ANGPTL3 in KK/San mice plasma total cholesterol, and especially plasma increase in plasma was to the VLDL Plasma VLDL triglyceride levels are to be by the between secretion and in the hepatic VLDL triglyceride secretion rate was not significantly between KK/San and wild-type KK mice. using of KK/San mice, we observed a in the hepatic VLDL triglyceride secretion rate compared with wild-type KK mice (11Shiraki T. Yoshioka S. Horikoshi H. Diabetes Frontier. 1993; 4: 641Google Scholar). the difference between the two mice was very the low triglyceride levels in KK/San mice is not due to a secretion rate. Therefore, we that ANGPTL3 does not affect VLDL triglyceride we analyzed VLDL triglyceride using two of VLDL VLDL to VLDL triglyceride clearance and VLDL by the liver. on the clearance of VLDL and only a of the clearance rate in KK/San mice, is strongly that the low plasma triglyceride levels in KK/San mice were primarily due to enhanced lipolysis of VLDL triglycerides rather than to enhanced is also by the that plasma and levels of KK/San mice were to of wild-type KK mice. H. Lee D. J. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) have previously reported that plasma VLDL cholesterol and triglyceride levels. These on VLDL metabolism in mice to be due to triglyceride in the whereas lipid and hepatic VLDL triglyceride secretion were not affected. to a very phenotype in KK/San mice with to VLDL is to be of the of VLDL triglyceride metabolism through the inhibition of both and of VLDL triglycerides J. Clin. Invest. PubMed Scopus Google Scholar, H. A. J. Lipid Res. Full Text PDF PubMed Google Scholar, C. PubMed Scopus Google Scholar). Our in revealed that ANGPTL3 inhibited LPL in a Therefore, ANGPTL3 regulates VLDL metabolism via the inhibition of in a to is also by the increase in plasma triglyceride levels after of recombinant ANGPTL3 (12Koishi R. Ando Y. Ono M. Shimamura M. Yasumo H. Fujiwara T. Horikoshi H. Furukawa H. Nat. Genet. 2002; 30: 151-157Crossref PubMed Scopus (336) Google Scholar, L. Nat. Med. 2002; PubMed Scopus Google Scholar, D.J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The of plasma of ANGPTL3 is very in the between the in vivo and in vitro inhibition of We the ANGPTL3 protein in the circulating blood of injected KK/San mice by analysis. of plasma ANGPTL3 levels in the mice was not ANGPTL3 inhibited LPL at of we that lipase inhibition is the mechanism by which ANGPTL3 regulates VLDL in To the mechanism of LPL inhibition by we have the between a and ANGPTL3 to LPL directly purified LPL was inhibited by ANGPTL3 in the of both and LPL showed very by ANGPTL3 ANGPTL3 to a on the LPL from the and inhibits LPL of However, ANGPTL3 have with or bind to a on LPL the high of the inhibition by ANGPTL3 are to the mechanism of the inhibition by We also found a increase in total cholesterol and levels by the of ANGPTL3 in KK/San mice. is that the of from lipoproteins R.D. M. A. S. J. Lipid Res. 1998; Full Text Full Text PDF PubMed Google Scholar). The inhibition of by ANGPTL3 is not but is ANGPTL3 the of from In mice LPL in both the and S. H. A. R. R. J. Clin. Invest. 1995; 96: PubMed Scopus Google Scholar) have that LPL in the Therefore, the increase in plasma levels by the of ANGPTL3 have been due to inhibition of in the The of the is ANGPTL3 or Our preliminary showed that the plasma ANGPTL3 of human were not is very to the ANGPTL3 to LPL in Therefore, ANGPTL3 VLDL triglyceride metabolism the other there are LPL factors in The plasma from has been reported to have an effect on LPL PubMed Scopus Google Scholar, T. D.C. Full Text PDF PubMed Scopus Google Scholar). T. D.C. Full Text PDF PubMed Scopus Google Scholar) have reported that an factor was observed in the separated by Our preliminary data suggest that ANGPTL3 is present in the as were the factors not Therefore, ANGPTL3 be of the LPL factors reported is to elucidate the of In this we that ANGPTL3 is a new class of lipid metabolism that regulates VLDL triglyceride levels through the inhibition of LPL both and of or a in VLDL is the apparent for the triglyceride in type and the of LPL for the triglyceride levels in type These the that ANGPTL3 affects the triglyceride levels in of ANGPTL3 be a in the of new for and other human We N. for with the T. for with the and K. for with protein We are to J. T. and H. Horikoshi for and