A decreased expression of angiopoietin-like 3 is protective against atherosclerosis in apoE-deficient mice
2003; Elsevier BV; Volume: 44; Issue: 6 Linguagem: Inglês
10.1194/jlr.m300031-jlr200
ISSN1539-7262
AutoresYukio Ando, Tetsuya Shimizugawa, Shigehito Takeshita, Mitsuru Ono, Mitsuru Shimamura, Ryuta Koishi, Hidehiko Furukawa,
Tópico(s)Atherosclerosis and Cardiovascular Diseases
ResumoKK/Snk mice (previously KK/San) possessing a recessive mutation (hypl) of the angiopoietin-like 3 (Angptl3) gene homozygously exhibit a marked reduction of VLDL due to the decreased Angptl3 expression. Recently, we proposed that Angptl3 is a new class of lipid metabolism modulator regulating VLDL triglyceride (TG) levels through the inhibition of lipoprotein lipase (LPL) activity. In this study, to elucidate the role of Angptl3 in atherogenesis, we investigated the effects of hypl mutation against hyperlipidemia and atherosclerosis in apolipoprotein E knockout (apoEKO) mice. ApoEKO mice with hypl mutation (apoEKO-hypl) exhibited a significant reduction of VLDL TG, VLDL cholesterol, and plasma apoB levels compared with apoEKO mice. Hepatic VLDL TG secretion was comparable between both apoE-deficient mice. Turnover studies revealed that the clearance of both [3H]TG-labeled and 125I-labeled VLDL was significantly enhanced in apoEKO-hypl mice. Postprandial plasma TG levels also decreased in apoEKO-hypl mice. Both LPL and hepatic lipase activities in the postheparin plasma increased significantly in apoEKO-hypl mice, explaining the enhanced lipid metabolism. Furthermore, apoEKO-hypl mice developed 3-fold smaller atherogenic lesions in the aortic sinus compared with apoEKO mice.Taken together, the reduction of Angptl3 expression is protective against hyperlipidemia and atherosclerosis, even in the absence of apoE, owing to the enhanced catabolism and clearance of TG-rich lipoproteins. KK/Snk mice (previously KK/San) possessing a recessive mutation (hypl) of the angiopoietin-like 3 (Angptl3) gene homozygously exhibit a marked reduction of VLDL due to the decreased Angptl3 expression. Recently, we proposed that Angptl3 is a new class of lipid metabolism modulator regulating VLDL triglyceride (TG) levels through the inhibition of lipoprotein lipase (LPL) activity. In this study, to elucidate the role of Angptl3 in atherogenesis, we investigated the effects of hypl mutation against hyperlipidemia and atherosclerosis in apolipoprotein E knockout (apoEKO) mice. ApoEKO mice with hypl mutation (apoEKO-hypl) exhibited a significant reduction of VLDL TG, VLDL cholesterol, and plasma apoB levels compared with apoEKO mice. Hepatic VLDL TG secretion was comparable between both apoE-deficient mice. Turnover studies revealed that the clearance of both [3H]TG-labeled and 125I-labeled VLDL was significantly enhanced in apoEKO-hypl mice. Postprandial plasma TG levels also decreased in apoEKO-hypl mice. Both LPL and hepatic lipase activities in the postheparin plasma increased significantly in apoEKO-hypl mice, explaining the enhanced lipid metabolism. Furthermore, apoEKO-hypl mice developed 3-fold smaller atherogenic lesions in the aortic sinus compared with apoEKO mice. Taken together, the reduction of Angptl3 expression is protective against hyperlipidemia and atherosclerosis, even in the absence of apoE, owing to the enhanced catabolism and clearance of TG-rich lipoproteins. The accrued evidence that lipid-lowering therapy limits the progression of atherosclerosis and reduces the events of coronary artery diseases is overwhelming (1Davignon J. Advances in lipid-lowering therapy in atherosclerosis.Adv. Exp. Med. Biol. 2001; 498: 49-58Google Scholar, 2Ballantyne C.M. Herd J.A. Dunn J.K. Jones P.H. Farmer J.A. Gotto Jr., A.M. Effects of lipid lowering therapy on progression of coronary and carotid artery disease.Curr. Opin. Lipidol. 1997; 8: 354-361Google Scholar). The focus has been on the reduction of LDL cholesterol (3Foody J.M. Nissen S.E. Effectiveness of statins in acute coronary syndromes.Am. J. Cardiol. 2001; 88: 31F-35FGoogle Scholar, 4The Long-Term Intervention with Pravastatin in Ischaemic Disease (LIPID) Study GroupPrevention of cardiovascular events and death with pravastatin in patients with coronary heart disease and a broad range of initial cholesterol levels.N. Engl. J. Med. 1998; 339: 1349-1357Google Scholar). Recent studies have also pointed out the importance of reducing triglyceride (TG)-rich lipoproteins such as chylomicrons, VLDL, and their remnants, and of raising HDL cholesterol (5Ginsberg H.N. New perspectives on atherogenesis: role of abnormal triglyceride-rich lipoprotein metabolism.Circulation. 2002; 106: 2137-2142Google Scholar, 6Assmann G. Nofer J.R. Atheroprotective effects of high-density lipoproteins.Annu. Rev. Med. 2003; 54: 321-341Google Scholar). In addition, postprandial hypertriglyceridemia is mentioned as an independent risk factor for atherogenesis (7Teno S. Uto Y. Nagashima H. Endoh Y. Iwamoto Y. Omori Y. Takizawa T. Association of postprandial hypertriglyceridemia and carotid intima-media thickness in patients with type 2 diabetes.Diabetes Care. 2000; 23: 1401-1406Google Scholar, 8Roche H.M. Gibney M.J. The impact of postprandial lipemia in accelerating atherothrombosis.J. Cardiovasc. Risk. 2000; 7: 317-324Google Scholar). In a colony of KK mice with mild obesity, hyperlipidemia, and diabetes, we found mutant mice (KK/Snk, previously KK/San) that were characterized by a significant decrease in plasma lipid levels, mainly due to the reduction of TG-rich lipoproteins, despite their obesity and diabetes. Genetic studies for the mutation, named hypolipidemia (hypl), in KK/Snk mice identified a 4 bp nucleotide insertion in exon 6 of a gene encoding angiopoietin-like 3 (Angptl3), which causes a premature stop codon after a frameshift (9Koishi R. Ando Y. Ono M. Shimamura M. Yasumo H. Fujiwara T. Horikoshi H. Furukawa H. Angptl3 regulates lipid metabolism in mice.Nat. Genet. 2002; 30: 151-157Google Scholar). Angptl3 is a secretory protein of 70 kDa expressed predominantly in the liver, and has a signal sequence, coiled-coil domain, and fibrinogen-like domain similar to those of other angiopoietin families (10Conklin D. Gilbertson D. Taft D.W. Maurer M.F. Whitmore T.E. Smith D.L. Walker K.M. Chen L.H. Wattler S. Nehls M. Lewis K.B. Identification of a mammalian angiopoietin-related protein expressed specifically in liver.Genomics. 1999; 62: 477-482Google Scholar, 11Davis 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. Isolation of angiopoietin-1, a ligand for the Tie2 receptor, by secretion-trap expression cloning.Cell. 1996; 87: 1161-1169Google Scholar). In KK/Snk mice with the homozygous hypl mutation, Angptl3 expression was markedly decreased, probably due to the instability of mutant mRNA, resulting in a hypolipidemic trait (9Koishi R. Ando Y. Ono M. Shimamura M. Yasumo H. Fujiwara T. Horikoshi H. Furukawa H. Angptl3 regulates lipid metabolism in mice.Nat. Genet. 2002; 30: 151-157Google Scholar). In contrast, adenovirus-mediated overexpression of Angptl3 or intravenous injection of the purified protein elicited a marked elevation in circulating plasma lipid levels (9Koishi R. Ando Y. Ono M. Shimamura M. Yasumo H. Fujiwara T. Horikoshi H. Furukawa H. Angptl3 regulates lipid metabolism in mice.Nat. Genet. 2002; 30: 151-157Google Scholar). We also investigated the regulatory mechanism of Angptl3 on the metabolism of TG-rich lipoproteins (12Shimizugawa T. Ono M. Shimamura M. Yoshida K. Ando Y. Koishi R. Ueda K. Inaba T. Minekura H. Kohama T. Furukawa H. ANGPTL3 decreases very low density lipoprotein triglyceride clearance by inhibition of lipoprotein lipase.J. Biol. Chem. 2002; 277: 33742-33748Google Scholar). VLDL turnover studies revealed that KK/Snk mice exhibited enhanced VLDL TG clearance compared with wild-type KK mice. Moreover, addition of recombinant human ANGPTL3 protein directly inhibited lipoprotein lipase (LPL) and hepatic lipase (HL) activities in in vitro studies (12Shimizugawa T. Ono M. Shimamura M. Yoshida K. Ando Y. Koishi R. Ueda K. Inaba T. Minekura H. Kohama T. Furukawa H. ANGPTL3 decreases very low density lipoprotein triglyceride clearance by inhibition of lipoprotein lipase.J. Biol. Chem. 2002; 277: 33742-33748Google Scholar). Taken together, we consider that Angptl3 is a new class of metabolic modulator affecting lipid homeostasis. Over the past several years, significant advances have been made in our understanding of new, alternative mechanisms by which LPL and HL modulate lipoprotein metabolism and the development of atherosclerosis (13Goldberg I.J. Lipoprotein lipase and lipolysis: central roles in lipoprotein metabolism and atherogenesis.J. Lipid Res. 1996; 37: 693-707Google Scholar, 14Santamarina-Fojo S. Haudenschild C. Amar M. The role of hepatic lipase in lipoprotein metabolism and atherosclerosis.Curr. Opin. Lipidol. 1998; 9: 211-219Google Scholar). Studies using transgenic and knockout animal models have shown that plasma LPL and HL are involved in the susceptibility to atherosclerosis in addition to regulating plasma lipid levels (15Yagyu H. Ishibashi S. Chen Z. Osuga J. Okazaki M. Perrey S. Kitamine T. Shimada M. Ohashi K. Harada K. Shionoiri F. Yahagi N. Gotoda T. Yazaki Y. Yamada N. Overexpressed lipoprotein lipase protects against atherosclerosis in apolipoprotein E knockout mice.J. Lipid Res. 1999; 40: 1677-1685Google Scholar, 16Shimada M. Ishibashi S. Inaba T. Yagyu H. Harada K. Osuga J. Ohashi K. Yazaki Y. Yamada N. Suppression of diet-induced atherosclerosis in low density lipoprotein receptor knockout mice overexpressing lipoprotein lipase.Proc. Natl. Acad. Sci. USA. 1996; 93: 7242-7246Google Scholar, 17Mezdour H. Jones R. Dengremont C. Castro G. Maeda N. Hepatic lipase deficiency increases plasma cholesterol but reduces susceptibility to atherosclerosis in apolipoprotein E-deficient mice.J. Biol. Chem. 1997; 272: 13570-13575Google Scholar, 18Amar M.J. Dugi K.A. Haudenschild C.C. Shamburek R.D. Foger B. Chase M. Bensadoun A. Hoyt Jr., R.F. Brewer Jr., H.B. Santamarina-Fojo S. Hepatic lipase facilitates the selective uptake of cholesteryl esters from remnant lipoproteins in apoE-deficient mice.J. Lipid Res. 1998; 39: 2436-2442Google Scholar). In our previous in vitro study, it was also predicted that the hypl mutation, which markedly reduces Angptl3 expression, would increase the plasma LPL and HL activities (12Shimizugawa T. Ono M. Shimamura M. Yoshida K. Ando Y. Koishi R. Ueda K. Inaba T. Minekura H. Kohama T. Furukawa H. ANGPTL3 decreases very low density lipoprotein triglyceride clearance by inhibition of lipoprotein lipase.J. Biol. Chem. 2002; 277: 33742-33748Google Scholar). Thus, in the present study, to elucidate the role of Angptl3 in atherogenesis, we investigated the effects of the hypl mutation on hyperlipidemia and atherosclerosis, which are developed due to the accumulation of TG-rich lipoproteins in the circulation in apolipoprotein (apo) E-deficient mice (19Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E.Science. 1992; 258: 468-471Google Scholar). The hypl mutation enhanced lipolysis of TG-rich lipoproteins and their clearance in the liver, and resulted in a marked reduction of plasma lipid and apoB levels in apoE-deficient mice. Both LPL and HL activities in the postheparin plasma were increased significantly by the hypl mutation, explaining such an enhanced lipid metabolism. Atherogenic lesions in the aortic valves observed in the absence of apoE were also significantly decreased in mice carrying the hypl mutation. These findings revealed that a reduction in Angptl3 expression has protective effects against hyperlipidemia and atherosclerosis even in the absence of apoE. Therefore, it was considered that Angptl3 plays an important role in atherogenesis and that Angptl3 might be a useful target in the development of new treatments for atherosclerosis. Hypolipidemic KK/Snk mice from Nagoya University were bred in our laboratory. ApoE knockout (apoEKO) mice (B6;129-Apoetm1Unc) were obtained from Jackson Laboratory. C57BL/6J mice were obtained from Hamamatsu University School of Medicine. KK/Snk mice were backcrossed to C57BL/6J mice for ten generations by selecting heterozygous mice that possessed KK-type alleles for the D4Mit15 and D4Mit219 loci mapped to 3.2 cM proximal and 1.5 cM distal of the Angptl3 locus, respectively (9Koishi R. Ando Y. Ono M. Shimamura M. Yasumo H. Fujiwara T. Horikoshi H. Furukawa H. Angptl3 regulates lipid metabolism in mice.Nat. Genet. 2002; 30: 151-157Google Scholar), and then crossbred with apoEKO mice. Wild-type (Apoe+/+ and Angptl3+/+), hypl (Apoe+/+ and Angptl3hypl/hypl), homozygous for the apoEKO allele (Apoe−/− and Angptl3+/+), and homozygous for both the apoEKO and the hypl allele (apoEKO-hypl) (Apoe−/− and Angptl3hypl/hypl) mice were selected by genotyping with PCR using specific primers. For the Apoe locus, two sets of primers for the apoE gene (sense: 5′-TCCCAAGTCACACAAGAACTGAC-3′, antisense: 5′-CATCCAGAAGGCTAAAGAAGGCA-3′, GenBank accession number D00466) and the neomycin-resistant gene (Neo1285: 5′-AGGATCTCGTCGTGACCCATGGCGA-3′, Neo1485: 5′-GAGCGGCGATACCGTAAAGCACGAGG-3′) (20Gaw A. Mancini F.P. Ishibashi S. Rapid genotyping of low density lipoprotein receptor knockout mice using a polymerase chain reaction technique.Lab. Anim. 1995; 29: 447-449Google Scholar) were used. For the Angptl3 locus, a set of primers distinguishing the wild-type from the hypl allele (sense: 5′-GGCTAAATAGTAAAACCCTGGCG-3′, antisense: 5′-GTGCTTGCTGTCTTTCCAGTCTT-3′) was used. Of these four groups of mice used in this study, 75% and 25% of the genetic background was derived from C57BL/6 and 129P2 strain, respectively. All mice were housed under a controlled temperature (23 ± 1°C) with free access to water and mouse chow (CMF; Oriental Yeast), and male littermates were used at 6 to 31 weeks of age for this study. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Sankyo Co., Ltd. Total RNA was extracted from the liver using Trizol Reagent (Invitrogen) and subjected to reverse transcription and amplification according to the protocol supplied with the TaqMan Gold RT-PCR kit (Perkin-Elmer) for Angptl3 and 36B4 gene. Primers and probes were designed as follows: Angptl3 primers (sense: 5′-ACATGTGGCTGAGATTGCTGG-3′, antisense: 5′-CCTTTGCTCTGTGATTCCATGTAG-3′), Angptl3 probe (5′-CCTCCCAGAGCACACAGACCTGATGTTT-3′), 36B4 primers (sense: 5′-GCTCCAAGCAGATGCAGCA-3′, antisense: 5′-CCGGATGTGAGGCAGCAG-3′), and 36B4 probe (5′-CAAGAACACCATGATGCGCAAGGC-5′). The amount of Angptl3 mRNA was corrected by dividing the amount of 36B4 mRNA in each sample. Plasma lipids were measured enzymatically using assay kits [Wako Pure Chemical Industries for TG, total cholesterol, and nonesterified fatty acids (NEFAs); Azwell, Inc. for phospholipids]. To determine the plasma lipoprotein distribution, 50 μl of pooled plasma was analyzed by fast protein liquid chromatography on a Superose 6 PC 3.2/30 column (SMART system; Amersham Biosciences), and eluted at a constant flow rate of 50 μl/min with PBS (pH 7.4, containing 1 mM EDTA). Fractions of 25 μl were collected and assayed for total cholesterol and TG levels as described above. Plasma samples (1 μl per lane) were separated on 2–15% gradient gels (Daiichi Pure Chemicals), and the proteins were transferred onto nitrocellulose membranes (Bio-Rad). The membranes were incubated with goat anti-mouse apoB antibody (Santa Cruz Biotechnology). Horseradish peroxidase-labeled anti-goat immunoglobulin G (Chemicon) was used as a secondary antibody, and apoB bands were detected by enhanced chemiluminescence Western blotting detection reagents (Amersham Biosciences). The intensity of the bands was estimated by an imaging analyzer. Mice were fasted for 16 h. After taking a basal blood sample by tail bleeding at t = 0, animals received an intragastric load of 400 μl of olive oil. Additional blood samples were drawn at 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h after the oral administration of olive oil. Plasma TG levels were measured at the time points as described above. Mice were fasted for 16 h, and injected intravenously via the tail vein with Triton WR1339 (400 mg/kg body weight) using 20% (w/v) Triton solution in 0.9% NaCl. Blood samples were drawn from the tail vein at 0 min, 30 min, 60 min, and 120 min after the Triton injection and analyzed for TG as described above. In vivo [3H]TG-labeled VLDL turnover studies were based on a previously described method (21Jong M.C. Dahlmans V.E. van Gorp P.J. van Dijk K.W. Breuer M.L. Hofker M.H. Havekes L.M. In the absence of the low density lipoprotein receptor, human apolipoprotein C1 overexpression in transgenic mice inhibits the hepatic uptake of very low density lipoproteins via a receptor-associated protein-sensitive pathway.J. Clin. Invest. 1996; 98: 2259-2267Google Scholar). [3H]palmitic acid (Amersham Biosciences) in toluene was evaporated under nitrogen gas and redissolved in 0.9% NaCl containing 2 mg/ml BSA to a final concentration of 1 mCi/ml. The apoEKO mice were injected intravenously via the tail vein with 100 μCi of the prepared [3H]palmitate and bled from the abdominal aorta 25 min after injection. Radiolabeled VLDL to be analyzed in the clearance studies was isolated from the plasma of 20 mice by ultracentrifugation (d < 1.006 g/ml). To study the in vivo clearance of labeled VLDL TG, apoEKO, and apoEKO-hypl, mice were injected intravenously with 150,000 dpm of [3H]TG-labeled VLDL. The disappearance rate of the radiolabeled VLDL was determined in 70 μl blood samples of mice drawn at the indicated time points after the injection. Total plasma radioactivity was used to represent VLDL TG radioactivity. Blood was collected from 19 apoEKO mice. Plasma samples were pooled, and VLDL (d < 1.006 g/ml) was obtained by ultracentrifugation. VLDL was labeled with 125I by the ICl method (22Bilheimer D.W. Eisenberg S. Levy R.I. The metabolism of very low density lipoproteins. I. Preliminary in vitro and in vivo observations.Biochim. Biophys. Acta. 1972; 260: 212-221Google Scholar). The specific radioactivity of 125I-VLDL was ∼86.8 cpm/ng of protein. After iodination, the VLDL samples were dialyzed extensively against a buffer containing 0.15 M NaCl and 0.3 mM EDTA (pH 7.4). 125I-labeled VLDL (10 μg of tracer in 200 μl of 0.9% NaCl containing 2 mg/ml of BSA) was injected into the tail vein of the apoEKO and apoEKO-hypl mice. Blood samples of 70 μl were collected from the retro-orbital plexus at the indicated time points after the injection. The plasma content of 125I-labeled apoB was determined by measuring the 125I content in the pellet after propan-2-ol precipitation (23Holmquist L. Carlson K. Carlson L.A. Comparison between the use of isopropanol and tetramethylurea for the solubilisation and quantitation of human serum very low density apolipoproteins.Anal. Biochem. 1978; 88: 457-460Google Scholar, 24Kita T. Brown M.S. Bilheimer D.W. Goldstein J.L. Delayed clearance of very low density and intermediate density lipoproteins with enhanced conversion to low density lipoprotein in WHHL rabbits.Proc. Natl. Acad. Sci. USA. 1982; 79: 5693-5697Google Scholar). Postheparin plasma was prepared from blood taken 10 min after intravenous injection of heparin at a dose of 100 U/kg body weight into male mice fasted for 5 h. LPL and HL activities in postheparin plasma were determined on 5 μl of plasma. LPL activity assays were based on the method of Nilsson-Ehle and Schotz (25Nilsson-Ehle P. Schotz M.C. A stable, radioactive substrate emulsion for assay of lipoprotein lipase.J. Lipid Res. 1976; 17: 536-541Google Scholar). The assays were carried out in a total volume of 0.2 ml with 0.1 ml of assay substrate and 0.1 ml of enzyme source. The assay substrate solution contained 2 mM glycerol-tri [9,10 (n)-3H]oleate, 189 ng/ml l-α-phosphatidylcholine, 14 mg/ml BSA, 140 mM Tris-HCl (pH 8.0), 15% glycerol, and 10% FBS. The mixture was incubated at 37°C for 120 min and the enzyme reaction was terminated by the addition of 1 ml of 0.1 M potassium carbonate-borate buffer (pH 10.5) and 3.25 ml of methanol-chloroform-hexane, 1.41:1.25:1 (v/v/v). The mixture was vortexed vigorously for 15 s and centrifuged at 3,000 g for 15 min. Then, radioactivity in 1 ml of the supernatant was counted using a scintillation counter. HL assay was performed in the same manner as the LPL assay except that the NaCl concentration used was 1 M. LPL activity was calculated by the subtraction of HL activity from the lipase activity in the absence of 1 M NaCl. LPL and HL activities were expressed as micromoles of FFA/h/ml. The cross-sectional lesion area was evaluated according to a modified method of Paigen et al. (26Paigen B. Morrow A. Holmes P.A. Mitchell D. Williams R.A. Quantitative assessment of atherosclerotic lesions in mice.Atherosclerosis. 1987; 68: 231-240Google Scholar). In brief, the heart, including aorta, was perfused with saline containing 4% formalin, and fixed for more than 48 h in the same solution. The basal half of the hearts was embedded in paraffin, and 5 μm thick serial sections were obtained from the aortic sinus. Ten sections, sliced 50 μm apart, from each mouse were subjected to Elastica Masson staining, and the sum of the stained lesion areas was calculated using the IPAP-WIN system (Sumika Technoservice Corporation, Japan). Student's t-test was used to compare mean values between the wild-type and hypl mice and between the apoEKO and apoEKO-hypl mice. Expression levels of Angptl3 mRNA in the liver of the hypl and apoEKO-hypl mice were markedly lower than those in the wild-type and apoEKO mice, respectively (Fig. 1A, B). Plasma lipid levels in fasted hypl mice in the presence and absence of apoE are summarized in Table 1. The levels of all lipids in the hypl and apoEKO-hypl mice were significantly lower than those in the wild-type and apoEKO mice, respectively. In particular, in the apoEKO-hypl mice, the TG levels were markedly reduced, and the levels of TG and NEFA were comparable to those in the wild-type mice. The reduction in the plasma TG levels was primarily due to the scarcity of VLDL-sized particles with and without apoE (Fig. 2A, B). Plasma cholesterol was mainly found in HDL-sized particles with apoE, and the peak of HDL cholesterol in the hypl mice was reduced to 68.7% compared with that in the wild-type mice (Fig. 2A). In the absence of apoE, VLDL- and IDL/LDL-sized particles contained the largest amounts of plasma cholesterol, and the peak was reduced to 39.4% compared with that in the apoEKO mice (Fig. 2B). We also investigated the difference of apoB-100/apoB-48 composition in the lipoproteins of the apoEKO and apoEKO-hypl mice. Contents of apoB-100 and apoB-48 in the apoEKO-hypl mice were decreased by 53.6% and 36.7%, respectively, compared with those in the apoEKO mice (Fig. 3). These results indicate that the hypl mutation of the Angptl3 gene resulted in a reduction of apoB-containing lipoproteins in the absence of apoE.TABLE 1Plasma lipid levels in hypl mutant mice in the presence and absence of apolipoprotein EPresence of ApoEAbsence of ApoEWild-type (n = 8)hypl (n = 9)apoEKO (n = 9)apoEKO-hypl (n = 10)Triglyceride (mg/dl) 160 ± 43.1 85 ± 18.7aP < 0.01, indicating the difference between the wild-type and hypl mice.660.8 ± 216.5132.3 ± 34.4bP < 0.001, indicating the difference between apoEKO and apoEKO-hypl mice.Total cholesterol (mg/dl) 95 ± 1558.3 ± 19.7aP < 0.01, indicating the difference between the wild-type and hypl mice.1,308.8 ± 357.1558.6 ± 100.6bP < 0.001, indicating the difference between apoEKO and apoEKO-hypl mice.NEFA (mEq/l)1.537 ± 0.1671.147 ± 0.182aP < 0.01, indicating the difference between the wild-type and hypl mice.2.549 ± 0.6031.265 ± 0.138bP < 0.001, indicating the difference between apoEKO and apoEKO-hypl mice.Phospholipid (mg/dl)179.6 ± 19.4134.3 ± 29.9aP < 0.01, indicating the difference between the wild-type and hypl mice.720.3 ± 204.5308.4 ± 48bP < 0.001, indicating the difference between apoEKO and apoEKO-hypl mice.Body weight (g) 30.1 ± 1.929.3 ± 2.3 29 ± 2.628.8 ± 1.4ApoEKO, homozygous for apoE gene knockout allele; apoEKO-hypl, homozygous for both apoE gene knockout and hypl allele; hypl, a recessive mutation in Angptl3 gene causing hypolipidemia; NEFA, nonesterified fatty acids. Data were measured using plasma samples of 24- to 25-week-old male mice fasted for 16 h. Values are depicted as mean ± SD.a P < 0.01, indicating the difference between the wild-type and hypl mice.b P < 0.001, indicating the difference between apoEKO and apoEKO-hypl mice. Open table in a new tab Fig. 2Lipoprotein profiles in hypl-mutated mice in the presence and absence of apoE. A: Plasma of the wild-type (open squares) and hypl (closed squares) mice and (B) plasma of the homozygous mice for apoE gene knockout allele (apoEKO) (open circles) and homozygous mice for both apoE gene knockout and hypl allele (apoEKO-hypl) (closed circles) were obtained after 16 h of fasting. The pooled plasma samples (n = 5 per group) were fractionated by fast protein liquid chromatography. Total cholesterol and triglyceride (TG) contents in the individual fractions were determined enzymatically. The relative positions of VLDL, IDL/LDL, and HDL are indicated.View Large Image Figure ViewerDownload (PPT)Fig. 3Immunoblot analysis of plasma apoB in the apoEKO and apoEKO-hypl mice. Plasma was obtained from 17-week-old apoEKO (n = 4) and apoEKO-hypl (n = 5) mice fasted for 16 h. Plasma samples (1 μl per lane) were separated on 2–15% gradient gels and immunoblotted with the polyclonal antibody against mouse apoB. The intensity of the bands was estimated by an imaging analyzer, and the percentage to that of apoEKO was calculated. The values are depicted as means ± SD.View Large Image Figure ViewerDownload (PPT) ApoEKO, homozygous for apoE gene knockout allele; apoEKO-hypl, homozygous for both apoE gene knockout and hypl allele; hypl, a recessive mutation in Angptl3 gene causing hypolipidemia; NEFA, nonesterified fatty acids. Data were measured using plasma samples of 24- to 25-week-old male mice fasted for 16 h. Values are depicted as mean ± SD. To determine whether the hypl mutation affects the production of TG by the liver, we injected Triton WR1339 into mice and monitored the secretion of endogenous VLDL TG over time. As shown in Fig. 4B, the VLDL TG secretion rate was comparable between the apoEKO and apoEKO-hypl mice, although a 25% decrease was observed in the hypl mice compared with the wild-type mice (Fig. 4A); however, there were no changes in weight or pathology of the liver between the wild-type and hypl mice or between the apoEKO and apoEKO-hypl mice (unpublished observations). Therefore, these results indicate that the hypl mutation has little effect on hepatic VLDL TG production, particularly in the absence of apoE. Next, to investigate whether the plasma TG decrease in the apoEKO-hypl mice is due to enhanced TG clearance, the apoEKO and apoEKO-hypl mice were injected with [3H]TG-labeled VLDL. As shown in Fig. 5A, the 3H-labeled TGs were more rapidly cleared from the circulation in the apoEKO-hypl mice. To determine the metabolic pathway of the lipoproteins containing apoB, we also performed a turnover study using 125I-labeled VLDL as a marker for whole-particle clearance. As a result, the injected 125I-labeled VLDL apoB disappeared significantly faster from the plasma in the apoEKO-hypl mice compared with the apoEKO mice (Fig. 5B). These data indicate that the reduction of plasma lipid levels by hypl mutation in the absence of apoE is due to an enhancement of TG hydrolysis and whole-particle clearance of VLDLs and their remnants. To investigate the postprandial response of plasma TG levels, mice received an intragastric load of olive oil. Plasma TG levels after an intragastric fat load in the apoEKO-hypl mice were significantly lower than those in the apoEKO mice (Fig. 6B), and showed a gradual increase similar to that seen in wild-type mice (Fig. 6A). By contrast, no such postprandial response was observed in the hypl mice (Fig. 6A). These results indicate that postprandial hypertriglyceridemia was prevented from developing by the hypl mutation even in the absence of apoE. To elucidate whether the enhanced lipid metabolism by the hypl mutation results from the increase of lipase activity, we determined LPL and HL activities of postheparin plasma in the apoEKO and apoEKO-hypl mice. As shown in Fig. 7A and B, activities of both LPL and HL increased significantly in the apoEKO-hypl mice compared with the apoEKO mice. These results suggest that the reduction of Angptl3 increases lipase activity in vivo, resulting in the enhancement of lipolysis and clearance of TG-rich lipoproteins in the hypl mutant mice. To examine the effect of the hypl mutation on atherogenesis in apoE-deficient mice, a histological study was conducted. As shown in Fig. 8A, the apoEKO mice exhibited typical fatty streak lesions with foam cells and cholesterol crystals in the aortic sinus, whereas the apoEKO-hypl mice had only small fatty streak lesions. The cross-sectional lesion area of the apoEKO-hypl mice was significantly smaller than that of the apoEKO mice by 69% (3.608 ± 1.921 × 105 μm2 vs. 11.559 ± 6.204 × 105 μm2, P < 0.01) (Fig. 8B). These results indicate that the hypl mutation has protective effects against atherosclerosis even when associated with apoE deficiency. In the present study, we clarified that the hypl mutation, which reduces Angptl3 expression, enhanced lipolysis and the clearance of TG-rich lipoproteins, and prevented hypertriglyceridemia, hypercholesterolemia, and atherosclerosis, often observed in apoE-deficient mice, from developing. We reported previously that adenovirus-mediated overexpression of Angptl3 gene or intravenous injection of human ANGPTL3 protein increased the plasma TG levels in mice
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