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Severe Hypercholesterolemia, Impaired Fat Tolerance, and Advanced Atherosclerosis in Mice Lacking Both Low Density Lipoprotein Receptor-related Protein 5 and Apolipoprotein E*

      LDL receptor-related protein 5 (LRP5) plays multiple roles, including embryonic development and bone accrual development. Recently, we demonstrated that LRP5 is also required for normal cholesterol metabolism and glucose-induced insulin secretion. To further define the role of LRP5 in the lipoprotein metabolism, we compared plasma lipoproteins in mice lacking LRP5, apolipoprotein E (apoE), or both (apoE;LRP5 double knockout). On a normal chow diet, the apoE;LRP5 double knockout mice (older than 4 months of age) had ∼60% higher plasma cholesterol levels compared with the age-matched apoE knockout mice. In contrast, LRP5 deficiency alone had no significant effects on the plasma cholesterol levels. High performance liquid chromatography analysis of plasma lipoproteins revealed that cholesterol levels in the very low density lipoprotein and low density lipoprotein fractions were markedly increased in the apoE;LRP5 double knockout mice. There were no apparent differences in the pattern of apoproteins between the apoE knockout mice and the apoE;LRP5 double knockout mice. The plasma clearance of intragastrically loaded triglyceride was markedly impaired by LRP5 deficiency. The atherosclerotic lesions of the apoE;LRP5 double knockout mice aged 6 months were ∼3-fold greater than those in the age-matched apoE-knockout mice. Furthermore, histological examination revealed highly advanced arthrosclerosis, with remarkable accumulation of foam cells and destruction of the internal elastic lamina in the apoE;LRP5 double knockout mice. These data suggest that LRP5 mediates both apoE-dependent and apoE-independent catabolism of plasma lipoproteins.
      LDL
      low density lipoprotein
      LDLR
      LDL receptor
      apoE
      apolipoprotein E
      CM
      chylomicron
      Dkk
      Dickkopf
      HDL
      high density lipoprotein
      HPLC
      high performance liquid chromatography
      LRP
      LDL receptor-related protein
      VLDL
      very low density lipoprotein, EMSE,N-ethyl-N-(3-methylphenyl)-N′-succinylethylendiamine
      MOPS
      4-morpholinepropanesulfonic acid
      PIPES
      1,4-piperazinediethanesulfonic acid
      Genetic defects in the catabolism of plasma lipoproteins are important causes of hypercholesterolemia and atherosclerosis in humans. The prototypic diseases are familial hypercholesterolemia, caused by a defect in the LDL1 receptor (LDLR) (
      • Brown M.S.
      • Goldstein J.L.
      ), and familial type III hyperlipoproteinemia, caused by a defect in one of the ligands for LDLR, apolipoprotein E (apoE) (
      • Mahley R.W.
      • Weisgraber K.H.
      • Innerarity T.L.
      • Rall S.J.
      ).
      ApoE is hypothesized to mediate lipoprotein clearance by binding two receptors: (i) LDLR and (ii) a hepatic chylomicron remnant receptor. ApoE-deficient mice (
      • Piedrahita J.A.
      • Zhang S.H.
      • Hagaman J.R.
      • Oliver P.M.
      • Maeda N.
      ,
      • Zhang S.H.
      • Reddick R.L.
      • Piedrahita J.A.
      • Maeda N.
      ,
      • Kashyap V.S.
      • Santamarina F.S.
      • Brown D.R.
      • Parrott C.L.
      • Applebaum B.D.
      • Meyn S.
      • Talley G.
      • Paigen B.
      • Maeda N.
      • Brewer H.J.
      ) and LDLR-deficient mice (
      • Ishibashi S.
      • Brown M.S.
      • Goldstein J.L.
      • Gerard R.D.
      • Hammer R.E.
      • Herz J.
      ) exhibit hypercholesterolemia, but the severity and manifestations differ markedly. On a normal laboratory chow diet, the apoE knockout mice have much more profound hypercholesterolemia and develop spontaneous atherosclerosis (
      • Zhang S.H.
      • Reddick R.L.
      • Piedrahita J.A.
      • Maeda N.
      ).
      LDL receptor-related protein 5 (LRP5) is a member of the LDL receptor family that are characterized by the presence of cysteine-rich complement type ligand binding domains. LRP5 binds apoE-containing lipoproteins in vitro and is widely expressed in many tissues including hepatocytes, adrenal gland, and pancreas (
      • Kim D.H.
      • Inagaki Y.
      • Suzuki T.
      • Ioka R.X.
      • Yoshioka S.Z.
      • Magoori K.
      • Kang M.J.
      • Cho Y.
      • Nakano A.Z.
      • Liu Q.
      • Fujino T.
      • Suzuki H.
      • Sasano H.
      • Yamamoto T.T.
      ).
      LRP5 and its homologue, LRP6, are postulated to play as co-receptors for Wnt receptors, Frizzled (
      • Wehrli M.
      • Dougan S.T.
      • Caldwell K.
      • O'Keefe L.
      • Schwartz S.
      • Vaizel-Ohayon D.
      • Schejter E.
      • Tomlinson A.
      • DiNardo S.
      ,
      • Tamai K.
      • Semenov M.
      • Kato Y.
      • Spokony R.
      • Liu C.
      • Katsuyama Y.
      • Hess F.
      • Saint-Jeannet J.P.
      • He X.
      ,
      • Pinson K.I.
      • Brennan J.
      • Monkley S.
      • Avery B.J.
      • Skarnes W.C.
      ,
      • Bafico A.
      • Liu G.
      • Yaniv A.
      • Gazit A.
      • Aaronson S.A.
      ,
      • Mao B.
      • Wu W.
      • Li Y.
      • Hoppe D.
      • Stannek P.
      • Glinka A.
      • Niehrs C.
      ,
      • Mao J.
      • Wang J.
      • Liu B.
      • Pan W.
      • Farr G.H.R.
      • Flynn C.
      • Yuan H.
      • Takada S.
      • Kimelman D.
      • Li L.
      • Wu D.
      ). The Wnt signaling pathway plays an essential role in embryonic development (
      • Nusse R.
      • Varmus H.E.
      ,
      • Wodarz A.
      • Nusse R.
      ) and oncogenesis (
      • Sparks A.B.
      • Morin P.J.
      • Vogelstein B.
      • Kinzler K.W.
      ) through various signaling molecules including Frizzled receptors (
      • Bhanot P.
      • Brink M.
      • Samos C.H.
      • Hsieh J.C.
      • Wang Y.
      • Macke J.P.
      • Andrew D.
      • Nathans J.
      • Nusse R.
      ), LRP5 and LRP6 (
      • Wehrli M.
      • Dougan S.T.
      • Caldwell K.
      • O'Keefe L.
      • Schwartz S.
      • Vaizel-Ohayon D.
      • Schejter E.
      • Tomlinson A.
      • DiNardo S.
      ,
      • Tamai K.
      • Semenov M.
      • Kato Y.
      • Spokony R.
      • Liu C.
      • Katsuyama Y.
      • Hess F.
      • Saint-Jeannet J.P.
      • He X.
      ,
      • Pinson K.I.
      • Brennan J.
      • Monkley S.
      • Avery B.J.
      • Skarnes W.C.
      ,
      • Bafico A.
      • Liu G.
      • Yaniv A.
      • Gazit A.
      • Aaronson S.A.
      ,
      • Mao B.
      • Wu W.
      • Li Y.
      • Hoppe D.
      • Stannek P.
      • Glinka A.
      • Niehrs C.
      ,
      • Mao J.
      • Wang J.
      • Liu B.
      • Pan W.
      • Farr G.H.R.
      • Flynn C.
      • Yuan H.
      • Takada S.
      • Kimelman D.
      • Li L.
      • Wu D.
      ), and Dickkopf proteins (
      • Bafico A.
      • Liu G.
      • Yaniv A.
      • Gazit A.
      • Aaronson S.A.
      ,
      • Mao B.
      • Wu W.
      • Li Y.
      • Hoppe D.
      • Stannek P.
      • Glinka A.
      • Niehrs C.
      ,
      • Zorn A.M.
      ). The Wnt signaling is also involved in adipogenesis by negatively regulating adipogenic transcription factors (
      • Ross S.E.
      • Hemati N.
      • Longo K.A.
      • Bennett C.N.
      • Lucas P.C.
      • Erickson R.L.
      • MacDougald O.A.
      ). Recent studies have revealed that loss of function mutations in the LRP5 gene cause the autosomal recessive disorder osteoporosis-pseudoglioma syndrome (
      • Gong Y.
      • Slee R.B.
      • Fukai N.
      • Rawadi G.
      • Roman-Roman S.
      • Reginato A.M.
      • Wang H.
      • Cundy T.
      • Glorieux F.H.
      • Lev D.
      • Zacharin M.
      • Oexle K.
      • Marcelino J.
      • Suwairi W.
      • Heeger S.
      • Sabatakos G.
      • Apte S.
      • Adkins W.N.
      • Allgrove J.
      • Arslan-Kirchner M.
      • Batch J.A.
      • Beighton P.
      • Black G.C.
      • Boles R.G.
      • Boon L.M.
      • Borrone C.
      • Brunner H.G.
      • Carle G.F.
      • Dallapiccola B.
      • De Paepe A.
      • Floege B.
      • Halfhide M.L.
      • Hall B.
      • Hennekam R.C.
      • Hirose T.
      • Jans A.
      • Juppner H.
      • Kim C.A.
      • Keppler-Noreuil K.
      • Kohlschuetter A.
      • LaCombe D.
      • Lambert M.
      • Lemyre E.
      • Letteboer T.
      • Peltonen L.
      • Ramesar R.S.
      • Romanengo M.
      • Somer H.
      • Steichen-Gersdorf E.
      • Steinmann B.
      • Sullivan B.
      • Superti-Furga A.
      • Swoboda W.
      • van den Boogaard M.J.
      • Van Hul W.
      • Vikkula M.
      • Votruba M.
      • Zabel B.
      • Garcia T.
      • Baron R.
      • Olsen B.R.
      • Warman M.L.
      ). Consistent with human osteoporosis-pseudoglioma syndrome, LRP5 knockout mice generated by Kato et al. (
      • Kato M.
      • Patel M.S.
      • Levasseur R.
      • Lobov I.
      • Chang B.H.
      • Glass 2nd, D.A.
      • Hartmann C.
      • Li L.
      • Hwang T.H.
      • Brayton C.F.
      • Lang R.A.
      • Karsenty G.
      • Chan L.
      ) exhibit a severe low bone mass phenotype.
      Recently, we demonstrated that LRP5-deficient mice develop high plasma cholesterol levels after feeding a high fat diet (
      • Fujino T.
      • Asaba H.
      • Kang M.J.
      • Ikeda Y.
      • Sone H.
      • Takada S.
      • Kim D.H.
      • Ioka R.X.
      • Ono M.
      • Tomoyori H.
      • Okubo M.
      • Murase T.
      • Kamataki A.
      • Yamamoto J.
      • Magoori K.
      • Takahashi S.
      • Miyamoto Y.
      • Oishi H.
      • Nose M.
      • Okazaki M.
      • Usui S.
      • Imaizumi K.
      • Yanagisawa M.
      • Sakai J.
      • Yamamoto T.T.
      ). The hepatic clearance of apoE-rich chylomicron remnants was also markedly decreased in the LRP5 knockout mice. These data suggested that LRP5 plays a role in the hepatic clearance of chylomicron remnants. In addition, we showed that the LRP5-deficient mice fed a normal diet showed marked impaired glucose tolerance. The LRP5-deficient islets had a marked reduction in the levels of intracellular ATP and Ca2+ in response to glucose; thereby, glucose-induced insulin secretion was decreased (
      • Fujino T.
      • Asaba H.
      • Kang M.J.
      • Ikeda Y.
      • Sone H.
      • Takada S.
      • Kim D.H.
      • Ioka R.X.
      • Ono M.
      • Tomoyori H.
      • Okubo M.
      • Murase T.
      • Kamataki A.
      • Yamamoto J.
      • Magoori K.
      • Takahashi S.
      • Miyamoto Y.
      • Oishi H.
      • Nose M.
      • Okazaki M.
      • Usui S.
      • Imaizumi K.
      • Yanagisawa M.
      • Sakai J.
      • Yamamoto T.T.
      ). Together with the roles of LRP5 in the bone accrual development (
      • Gong Y.
      • Slee R.B.
      • Fukai N.
      • Rawadi G.
      • Roman-Roman S.
      • Reginato A.M.
      • Wang H.
      • Cundy T.
      • Glorieux F.H.
      • Lev D.
      • Zacharin M.
      • Oexle K.
      • Marcelino J.
      • Suwairi W.
      • Heeger S.
      • Sabatakos G.
      • Apte S.
      • Adkins W.N.
      • Allgrove J.
      • Arslan-Kirchner M.
      • Batch J.A.
      • Beighton P.
      • Black G.C.
      • Boles R.G.
      • Boon L.M.
      • Borrone C.
      • Brunner H.G.
      • Carle G.F.
      • Dallapiccola B.
      • De Paepe A.
      • Floege B.
      • Halfhide M.L.
      • Hall B.
      • Hennekam R.C.
      • Hirose T.
      • Jans A.
      • Juppner H.
      • Kim C.A.
      • Keppler-Noreuil K.
      • Kohlschuetter A.
      • LaCombe D.
      • Lambert M.
      • Lemyre E.
      • Letteboer T.
      • Peltonen L.
      • Ramesar R.S.
      • Romanengo M.
      • Somer H.
      • Steichen-Gersdorf E.
      • Steinmann B.
      • Sullivan B.
      • Superti-Furga A.
      • Swoboda W.
      • van den Boogaard M.J.
      • Van Hul W.
      • Vikkula M.
      • Votruba M.
      • Zabel B.
      • Garcia T.
      • Baron R.
      • Olsen B.R.
      • Warman M.L.
      ,
      • Little R.D.
      • Carulli J.P.
      • Del Mastro R.G.
      • Dupuis J.
      • Osborne M.
      • Folz C.
      • Manning S.P.
      • Swain P.M.
      • Zhao S.C.
      • Eustace B.
      • Lappe M.M.
      • Spitzer L.
      • Zweier S.
      • Braunschweiger K.
      • Benchekroun Y.
      • Hu X.
      • Adair R.
      • Chee L.
      • FitzGerald M.G.
      • Tulig C.
      • Caruso A.
      • Tzellas N.
      • Bawa A.
      • Franklin B.
      • McGuire S.
      • Nogues X.
      • Gong G.
      • Allen K.M.
      • Anisowicz A.
      • Morales A.J.
      • Lomedico P.T.
      • Recker S.M.
      • Van Eerdewegh P.
      • Recker R.R.
      • Johnson M.L.
      ,
      • Boyden L.M.
      • Mao J.
      • Belsky J.
      • Mitzner L.
      • Farhi A.
      • Mitnick M.A.
      • Wu D.
      • Insogna K.
      • Lifton R.P.
      ) as well as in the Wnt signaling pathways (
      • Wehrli M.
      • Dougan S.T.
      • Caldwell K.
      • O'Keefe L.
      • Schwartz S.
      • Vaizel-Ohayon D.
      • Schejter E.
      • Tomlinson A.
      • DiNardo S.
      ,
      • Tamai K.
      • Semenov M.
      • Kato Y.
      • Spokony R.
      • Liu C.
      • Katsuyama Y.
      • Hess F.
      • Saint-Jeannet J.P.
      • He X.
      ,
      • Pinson K.I.
      • Brennan J.
      • Monkley S.
      • Avery B.J.
      • Skarnes W.C.
      ,
      • Bafico A.
      • Liu G.
      • Yaniv A.
      • Gazit A.
      • Aaronson S.A.
      ,
      • Mao J.
      • Wang J.
      • Liu B.
      • Pan W.
      • Farr G.H.R.
      • Flynn C.
      • Yuan H.
      • Takada S.
      • Kimelman D.
      • Li L.
      • Wu D.
      ), our data indicated that LRP5 is a multifunctional receptor physiologically linked to common human disorders, including hypercholesterolemia and impaired glucose tolerance.
      To further define the role of LRP5 in lipoprotein metabolism, we produced double knockout mice that are deficient in apoE as well as in LRP5 (apoE;LRP5 double knockout mice). In the current paper, we describe that superimposition of an LRP5 deficiency onto apoE deficiency increased plasma cholesterol beyond the level observed with apoE deficiency alone. We also show that fat tolerance was markedly impaired in the LRP5 knockout mice as well as in the apoE;LRP5 double knockout mice. Consistent with extreme hypercholesterolemia, severe atherosclerosis developed in the apoE;LRP5 double knockout mice. These results provide further evidence for the role of LRP5 in the catabolism of plasma lipoproteins.

      EXPERIMENTAL PROCEDURES

       Materials

      For the lipoprotein analysis, blood was collected from the retroorbital plexus after 4 h of fasting. Plasma total cholesterol levels were determined in individual mice at each time point by enzymatic assay kits (Wako Pure Chemicals Co., Osaka, Japan).
      For the detection of cholesterol and triglycerides with the high performance liquid chromatography (HPLC) method (see below), we obtained enzymatic reagents from Kyowa Medex Co. (Tokyo, Japan). The reagent system for cholesterol detection consists of reagent 1 (R1-C) and reagent 2 (R2-C) (R1-C: 20 mm MOPS, pH 7.0, 1.1 mm EMSE, 10 units/ml peroxidase, detergents, and stabilizer; R2-C: 20 mm MOPS, pH 7.0, 1.5 mm4-aminoantipyrine, 0.68 mm CaCl2, 0.3 units/ml cholesterol esterase, 2 units/ml cholesterol oxidase, 10 units/ml peroxidase, detergents, and stabilizer). The triglyceride reagent system includes reagent 1 (R1-TG) and reagent 2 (R2-TG) (R1-TG: 50 mm PIPES, pH 6.2, 1.1 mm EMSE, 2 mmMgSO4, 4.9 mm ATP, 3 units/ml glycerol kinase, 1.5 units/ml glycerol-3-phosphate oxidase, 5 units/ml peroxidase, detergents, and stabilizer; R2-TG: 50 mm PIPES, pH 6.2, 1.5 mm 4-aminoantipyrine, 2 mm MgSO4, 3 units/ml lipoprotein lipase, 5 units/ml peroxidase, detergents, and stabilizer. Equal amounts of R1 and R2 were mixed before use. After mixing, the cholesterol reagent was used within 4 weeks, and the triglyceride reagent was used within 2 weeks.

       Lipoprotein Analysis by a Dual Detection HPLC System

      Plasma lipoproteins were analyzed by an improved HPLC analysis according to the procedure as described by Usui et al. (
      • Usui S.
      • Hara Y.
      • Hosaki S.
      • Okazaki M.
      ). The HPLC system consisted of an AS-8020 autoinjector, CCPS and CCPM-II pumps, and two UV-8020 detectors (Tosoh, Japan) (
      • Okazaki M.
      • Usui S.
      • Hosaki S.
      ). An SC-8020 system controller (Tosoh) was used for instrument regulation and data collection. Lipoproteins were fractionated on two tandem connected TSKgel LipopropakXL columns (300 × 7.8-mm; Tosoh) with 50 mm Tris acetate, pH 8.0, containing 0.3 msodium acetate, 0.05% sodium azide, and 0.005% Brij-35 at a flow rate of 0.7 ml/min. The TSK column medium is composed of porous polymermatrices with a nominal bead size of 10 μm and a pore size of 100 nm, which is expected to exclude most of chylomicron (CM) to the void volume. Two TSK columns were connected in tandem and used to obtain higher resolution within a relatively short analytical time. The running buffer was filtered through a 0.22-μm filter (Millipore Corp.) before use and continuously degassed with an SD-8022 on-line degasser (Tosoh) during analysis. The column effluent was split equally into two lines by a Micro-Splitter P-460 (Upchurch Scientific Inc., Oak Harbor, WA), one mixing with cholesterol reagent and the other with triglyceride reagent, in order to achieve simultaneous profiles from a single injection. The two enzymatic reagents were each pumped at a flow rate of 0.35 ml/min for the TSK column. Both enzymatic reactions proceeded at 37 °C in a reactor coil (Teflon, 15 m × 0.4 mm, inner diameter). 10-μl samples diluted with saline were injected by an AS-8020 autoinjector with a presuction volume of 25 μl at intervals of 24 min. The enzymatic determination of cholesterol and triglycerides involved the detection of hydrogen peroxide produced by cholesterol oxidase and glycerol-3-phosphate oxidase, respectively. Total cholesterol and triglyceride concentrations (in mg/dl) were calculated by comparison with total area under the chromatographic curves of a calibration material of known concentration.

       SDS-Polyacrylamide Gel Electrophoresis

      Total lipoprotein fractions (d < 1.215 g/ml) from pooled plasma of the mice were isolated by ultracentrifugation, and the delipidated apolipoproteins were boiled for 3 min in SDS sample buffer containing 2-mercaptoethanol and subjected to electrophoresis on an SDS/5–15% polyacrylamide gel. Proteins were stained with Coomassie Blue.

       Fat Tolerance Test

      6-month-old male mice were fasted for 16 h, and olive oil (1 ml/30 g body weight; Wako Pure Chemicals Co.) was administered intragastrically as a bolus. Approximately 50 μl of blood was taken from the tail vein at the indicated times for the measurement of triglyceride levels and HPLC analysis.

       Mice

      LRP5 “knockout” mice (originally C57BL/6J-CBA hybrids (
      • Fujino T.
      • Asaba H.
      • Kang M.J.
      • Ikeda Y.
      • Sone H.
      • Takada S.
      • Kim D.H.
      • Ioka R.X.
      • Ono M.
      • Tomoyori H.
      • Okubo M.
      • Murase T.
      • Kamataki A.
      • Yamamoto J.
      • Magoori K.
      • Takahashi S.
      • Miyamoto Y.
      • Oishi H.
      • Nose M.
      • Okazaki M.
      • Usui S.
      • Imaizumi K.
      • Yanagisawa M.
      • Sakai J.
      • Yamamoto T.T.
      )), LRP5−/−, have been continually mated with C57BL/6J; N6 and N7 generation descendents from this cross into the C57BL/6J background were used. ApoE−/− mice (
      • Piedrahita J.A.
      • Zhang S.H.
      • Hagaman J.R.
      • Oliver P.M.
      • Maeda N.
      ) backcrossed 10 times on the C57BL/6J background were obtained from the Jackson Laboratory (Bar Harbor, ME). To obtain knockout mice that are homozygous for disruption of both the LRP5 and apoE loci, male apoE−/− mice were mated to female LRP5−/− mice. The resulting apoE+/−;LRP5+/− mice were identified by PCR analysis and bred each other to produce apoE−/−;LRP5−/− mice. Experiments were performed with those mice or those with the same genotype from the next generation by breeding apoE−/−;LRP5−/− with each other. Mice were maintained on 12-h dark/12-h light cycles and had free access to a normal laboratory chow diet (4.5% fat, 0% cholesterol, CE-2; CLEA, Tokyo, Japan) and water.

       Measurement of Atherosclerotic Lesions

      Mice were euthanized, and thoracic and abdominal aorta were used for en face staining with Oil Red O to visualize neutral lipid (cholesteryl ester and triglycerides) accumulation. In brief, the aorta was removed, cleaned, and cut open with the luminal surface facing up and then immersion-fixed in 10% formalin in 10 mmphosphate-buffered saline. After rinsing with phosphate-buffered saline, the aorta was thoroughly cleaned of adventitial fat using microforceps and spring iris scissors under a stereoscopic microscope. The inner aortic surface was stained with Oil Red O for 25 min at room temperature. After rinsing with 60% isopropyl alcohol and distilled water, the Oil Red O-stained area was quantified by NIH Image 1.62f software analysis of the digitized microscopic images. Results are expressed as percentage of lipid-accumulating lesion area of the total aortic area analyzed.
      For light microscopy, the aortic tissue samples were fixed with 10% formalin in 10 mm phosphate buffer (pH 7.2) and embedded in paraffin. Sections 2–3 μm thick were taken longitudinally through the aortic lumen and stained with hematoxylin and eosin or elastica-Masson. For Oil Red O staining, aortic tissue samples were frozen in OCT compound (Miles Inc., Elkhart, IN). Cryostat tissue sections were cut to a thickness of 5 μm and stained with Oil Red O. Nuclei were counterstained with hematoxylin.

      RESULTS

       Plasma Cholesterol and Lipoprotein Profile

      Fig.1 compares the levels of total cholesterol of mice of four different genotypes at the indicated ages. Mice were fed a normal laboratory chow diet containing 4.5% (w/w) fat and 0% cholesterol. Although there were no significant differences in the total plasma cholesterol levels between the apoE knockout mice (apoE−/−;LRP5+/+) and the apoE;LRP5 double knockout mice (apoE−/−;LRP5−/−) at 2 months of age, the cholesterol levels of the double knockout mice older than 4 months were greatly increased (by approximately 60%) beyond the levels observed with apoE deficiency alone. In contrast, LRP5 deficiency alone had no significant effects on the plasma cholesterol levels.
      Figure thumbnail gr1
      Figure 1Age-dependent changes in plasma cholesterol concentrations in mice with different genotypes fed a normal diet. A, plasma levels of total cholesterol of mice of each genotype at the indicated age were determined enzymatically after 4 h of fasting. Data are mean ± S.D. of six mice. *, p < 0.01; Student'st test. B, HPLC analysis of plasma lipoproteins. Plasma samples from mice of each genotype at 4 months of age were separated by HPLC, and cholesterol (red line) and triglyceride (blue line) contents were determined as described under “Experimental Procedures.” Representative data from six animals with the indicated genotype is shown. The CM, VLDL, LDL, and HDL fractions are labeled C, V,L, and H, respectively. Free glycerol is indicated by an arrowhead. The cholesterol levels in the CM, VLDL, LDL, and HDL fractions are shown in .
      High resolution HPLC analysis (
      • Usui S.
      • Hara Y.
      • Hosaki S.
      • Okazaki M.
      ) of plasma lipoprotein of 4-month-old mice revealed that cholesterol levels in the VLDL and LDL fractions were markedly increased in the apoE;LRP5 double knockout mice compared with the apoE knockout mice (Fig. 1 B and Table I): the cholesterol levels in the VLDL and LDL fractions in the apoE knockout were 180 ± 35 and 145 ± 7 mg/dl, respectively, and those in the apoE;LRP5 double knockout mice were 244 ± 24 and 171 ± 21 mg/dl, respectively (Table I). There were no significant differences in the levels of CM- and HDL-cholesterol between the apoE knockout mice and the apoE;LRP5 double knockout mice, although HDL-cholesterol levels in these mice were ∼50% of those in the LRP5 knockout mice and normal controls. Despite the severe hypercholesterolemia in the apoE knockout and apoE;LRP5 double knockout mice, there were no significant differences in the total triglyceride levels among mice with the four different genotypes (data not shown).
      Table IPlasma cholesterol profiles in mice with different genotypes
      GenotypeCholesterol
      CMVLDLLDLHDL
      mg/dl
      ApoE+/+;LRP5+/+0.10 ± 0.122.26 ± 0.284.59 ± 1.0539.9 ± 2.8
      ApoE+/+;LRP5−/−0.03 ± 0.023.58 ± 0.406.11 ± 1.0141.2 ± 1.5
      ApoE;LRP5+/+0.16 ± 0.08180 ± 35
      p < 0.01 versus ApoE+/+; LRP5+/+ and ApoE+/+; LRP5−/−.
      145 ± 7
      p < 0.01 versus ApoE+/+; LRP5+/+ and ApoE+/+; LRP5−/−.
      21.7 ± 3.8
      p < 0.01 versus ApoE+/+; LRP5+/+ and ApoE+/+; LRP5−/−.
      ApoE;LRP5−/−0.12 ± 0.05244 ± 24
      p < 0.01 versus ApoE+/+; LRP5+/+ and ApoE+/+; LRP5−/−.
      ,
      p < 0.01 versus ApoE−/−; LRP5+/+.
      171 ± 21
      p < 0.01 versus ApoE+/+; LRP5+/+ and ApoE+/+; LRP5−/−.
      ,
      p < 0.01 versus ApoE−/−; LRP5+/+.
      22.9 ± 1.6
      p < 0.01 versus ApoE+/+; LRP5+/+ and ApoE+/+; LRP5−/−.
      Plasma samples from mice of each genotype at 4 months of age were separated by HPLC, and cholesterol contents were determined as described under “Experimental Procedures.” Values are mean ± S.D. of six mice.
      a p < 0.01 versus ApoE+/+; LRP5+/+ and ApoE+/+; LRP5−/−.
      b p < 0.01 versus ApoE+/+; LRP5+/+ and ApoE+/+; LRP5−/−.
      c p < 0.01 versus ApoE−/−; LRP5+/+.
      Fig. 2 shows the SDS-polyacrylamide gel electrophoresis of apoproteins in pooled lipoprotein fraction from mice of four different genotypes. Consistent with the previous work by Ishibashi et al. (
      • Ishibashi S.
      • Herz J.
      • Maeda N.
      • Goldstein J.L.
      • Brown M.S.
      ), the amounts of apoB48 were markedly increased in the apoE knockout mice as well as in the apoE;LRP5 double knockout mice. Despite the severe hypercholesterolemia in the apoE;LRP5 double knockout mice, there were no apparent differences in the pattern of apoproteins between the apoE- and apoE;LRP5 double knockout mice.
      Figure thumbnail gr2
      Figure 2SDS-polyacrylamide gel electrophoresis of total lipoprotein fractions. Equal volumes (1 ml) of plasma were pooled from four mice of different genotypes fed a normal diet and total lipoprotein fractions (d < 1.215 g/ml) were isolated by ultracentrifugation, and the delipidated apoproteins were subjected to electrophoresis on an SDS/5–15% polyacrylamide gradient gel. Proteins were stained with Coomassie Blue. Positions of migration of apoB100, apoB48, apoA-VI, apoE, and apoA1 are denoted. Representative data from four independent experiments is shown.

       Fat Tolerance Test

      In a pervious study, we showed that LRP5 plays a role in the hepatic uptake of dietary cholesterol. The LRP5 knockout mice displayed dietary derived hypercholesterolemia due to decreased plasma clearance of chylomicron remnants (
      • Fujino T.
      • Asaba H.
      • Kang M.J.
      • Ikeda Y.
      • Sone H.
      • Takada S.
      • Kim D.H.
      • Ioka R.X.
      • Ono M.
      • Tomoyori H.
      • Okubo M.
      • Murase T.
      • Kamataki A.
      • Yamamoto J.
      • Magoori K.
      • Takahashi S.
      • Miyamoto Y.
      • Oishi H.
      • Nose M.
      • Okazaki M.
      • Usui S.
      • Imaizumi K.
      • Yanagisawa M.
      • Sakai J.
      • Yamamoto T.T.
      ). To further define the role of LRP5, fat tolerance test was carried out using mice of four different genotypes. Mice were fasted for 16 h, and olive oil (1 ml/30 g body weight) was administered intragastrically. As shown in Fig. 3, plasma levels of total triglyceride increased and peaked at about 2 h and then declined toward base line 6 h after loading in both apoE-knockout mice and normal controls. In contrast, the increased levels of plasma triglyceride were sustained for several h after loading in both LRP5 knockout and apoE;LRP5 double knockout mice, indicating that the plasma clearance of intragastrically loaded triglyceride was markedly impaired by LRP5 deficiency. HPLC analysis of plasma lipoproteins revealed that the majority of particles at 6 h after fat loading were in the VLDL fraction.
      Figure thumbnail gr3
      Figure 3Effects of intragastric fat loading on plasma triglyceride levels in mice with different genotypes. Six males (6 months old) of each genotype received an intragastrically administration of olive oil (1 ml/30 g body weight). At the indicated times, 50 μl of blood was taken from the tail vein and subjected to HPLC analysis. Data are mean ± S.E. of six mice. *,p < 0.01; Student's t test.
      In addition, we noticed that 16 h of fasting increased the levels of VLDL-triglyceride in the apoE knockout, LRP5 knockout, and apoE;LRP5 double knockout mice. This result may indicate that both apoE and LRP5 mediate the plasma clearance of VLDL-triglyceride induced by fasting.

       Atherosclerosis

      Aortic atherosclerotic lesions of the apoE knockout and apoE;LRP5 double knockout mice were first analyzed byen face lipid staining (Fig.4 A). At 4 months of age, the area of the thoracic and abdominal aortas stained by Oil Red O of the apoE;LRP5 double knockout mice was approximately the same as that in the apoE knockout mice. In contrast, at 6 months of age, the lesions in the apoE;LRP5 double knockout mice were ∼3-fold larger than those in the apoE knockout mice (Fig. 4 B).
      Figure thumbnail gr4
      Figure 4Atherosclerotic lesions in apoE and apoE ;LRP5 double knockout mice. A, en face lipid staining of aortas. Thoracic and abdominal aorta from the indicated genotype was cut open with the luminal surface facing up, and the inner aortic surface was stained with Oil Red O. Representative data of each genotype are shown. Bar, 5 mm. B, quantitative analysis of en face lipid staining. The inner aortic surface area stained with Oil Red O was quantified by NIH Image 1.62f software analysis of the digitized microscopic images. Results are expressed as percentage of lipid-accumulating lesion area of the total aortic area analyzed. Data are mean ± S.D. of six mice. *,p < 0.01; Student's t test.C–F, representative histopathological features of the aorta. Bars, 100 μm. C, an apoE-knockout mouse (aged 6 months) shows a slightly atheromatous lesion characteristic of the accumulation of foam cells, which is not associated with the destruction of internal elastic lamina (dark brown-colored) or the degenerative change of muscle layer of the aorta (elastica-Masson staining). D, one of the multiple atheromatous lesions developed in an apoE;LRP5 double knockout mouse (aged 6 months) manifests a hump structure associated with cholesterin deposits, fibrosis (light green-colored), and elastosis (dark brown-colored). Destruction of the internal elastic lamina adjacent with a degenerative lesion of muscle layer of the aorta is remarkable (elastica-Masson staining). E, an atheromatous lesion in an apoE;LRP5 double knockout mouse (aged 6 months) reveals a remarkable accumulation of foam cells, especially marked in the superficial region of atheroma, and a crystal structure of cholesterin deposits (hematoxylin and eosin staining). F, an atheromatous lesion in an apoE;LRP5 double knockout mouse (aged 6 months) reveals severe deposition of neutral lipid in the aortic wall, resulting in the destruction of lamellar structure of the elastic fibers (Oil Red O staining).
      In histopathology under light microscopic examination, the lesions in the apoE knockout mice at 6 months of age were relatively modest, showing slightly atheromatous lesions with a fatty streak-like structure, which were localized on the surface of aortic intima but were not associated with the destruction of internal elastic lamina or the medial muscle layer (Fig. 4 C). In contrast, the apoE;LRP5 double knockout mice developed multiple atheromatous lesions manifesting a hump structure, which were associated with cholesterin deposits, fibrosis, and elastosis (Fig. 4 D). Some of them showed the destruction of internal elastic lamina and the degenerative change of medial muscle layers of the aorta (Fig. 4 E). In these lesions, severe deposition of neutral lipid was observed (Fig.4 F).

      DISCUSSION

      In the present study, we show extreme hypercholesterolemia in mice lacking both apoE and LRP5. It has been well established that both LDLR and apoE are critical in the plasma clearance of cholesterol-carrying lipoproteins, including LDL and apoE-containing intermediate density lipoprotein and chylomicron remnants (
      • Brown M.S.
      • Goldstein J.L.
      ,
      • Mahley R.W.
      • Weisgraber K.H.
      • Innerarity T.L.
      • Rall S.J.
      ). In contrast to the mice lacking apoE (
      • Piedrahita J.A.
      • Zhang S.H.
      • Hagaman J.R.
      • Oliver P.M.
      • Maeda N.
      ,
      • Zhang S.H.
      • Reddick R.L.
      • Piedrahita J.A.
      • Maeda N.
      ,
      • Kashyap V.S.
      • Santamarina F.S.
      • Brown D.R.
      • Parrott C.L.
      • Applebaum B.D.
      • Meyn S.
      • Talley G.
      • Paigen B.
      • Maeda N.
      • Brewer H.J.
      ) or LDLR (
      • Ishibashi S.
      • Brown M.S.
      • Goldstein J.L.
      • Gerard R.D.
      • Hammer R.E.
      • Herz J.
      ), the lack of LRP5 alone did not increase the plasma levels of cholesterol on a normal diet, whereas high fat feeding results in hypercholesterolemia in the LRP5 knockout mice (
      • Fujino T.
      • Asaba H.
      • Kang M.J.
      • Ikeda Y.
      • Sone H.
      • Takada S.
      • Kim D.H.
      • Ioka R.X.
      • Ono M.
      • Tomoyori H.
      • Okubo M.
      • Murase T.
      • Kamataki A.
      • Yamamoto J.
      • Magoori K.
      • Takahashi S.
      • Miyamoto Y.
      • Oishi H.
      • Nose M.
      • Okazaki M.
      • Usui S.
      • Imaizumi K.
      • Yanagisawa M.
      • Sakai J.
      • Yamamoto T.T.
      ). Ishibashi et al. (
      • Ishibashi S.
      • Herz J.
      • Maeda N.
      • Goldstein J.L.
      • Brown M.S.
      ) showed that the plasma cholesterol levels in the double knockout mice lacking both apoE and LDLR were not significantly different from the levels in the apoE knockout mice. The severe hypercholesterolemia developed in the double knockout mice lacking both apoE and LRP5 suggests the presence of an alternative pathway for cholesterol catabolism mediated by LRP5, which appears to be independent of the LDLR pathway.
      Consistent with the previous work (
      • Fujino T.
      • Asaba H.
      • Kang M.J.
      • Ikeda Y.
      • Sone H.
      • Takada S.
      • Kim D.H.
      • Ioka R.X.
      • Ono M.
      • Tomoyori H.
      • Okubo M.
      • Murase T.
      • Kamataki A.
      • Yamamoto J.
      • Magoori K.
      • Takahashi S.
      • Miyamoto Y.
      • Oishi H.
      • Nose M.
      • Okazaki M.
      • Usui S.
      • Imaizumi K.
      • Yanagisawa M.
      • Sakai J.
      • Yamamoto T.T.
      ), the LRP5 knockout mice and the apoE;LRP5 double knockout mice displayed markedly impaired fat tolerance. In contrast, the plasma clearance of intragastrically loaded triglyceride was not significantly impaired in the apoE-knockout mice. These observations suggest that LRP5 modulates the plasma clearance of diet-derived triglycerides in the absence of apoE by stimulating the hydrolysis of triglycerides. In this context, it is important to note that LRP5 and LRP6 can bind Dickkopf (Dkk), an antagonist of Wnt proteins (
      • Mao B.
      • Wu W.
      • Li Y.
      • Hoppe D.
      • Stannek P.
      • Glinka A.
      • Niehrs C.
      ,
      • Boyden L.M.
      • Mao J.
      • Belsky J.
      • Mitzner L.
      • Farhi A.
      • Mitnick M.A.
      • Wu D.
      • Insogna K.
      • Lifton R.P.
      ). Dkk is involved in Xenopus head formation and the impaired action of Dkk at LRP5 increases bone density in humans (
      • Boyden L.M.
      • Mao J.
      • Belsky J.
      • Mitzner L.
      • Farhi A.
      • Mitnick M.A.
      • Wu D.
      • Insogna K.
      • Lifton R.P.
      ). The Dkk sequence consists of two cysteine-rich domains. The C-terminal domain has the typical cysteine pattern of colipase, which is required by pancreatic lipase for the efficient lipid hydrolysis (reviewed in Ref.
      • van Tilbeurgh H.
      • Bezzine S.
      • Cambillau C.
      • Verger R.
      • Carrière F.
      ). The C-terminal domain of colipase binds to the C-terminal noncatalytic domain of pancreatic lipase, which is thought to stabilize an active conformation of the lipase, and is also conserved among various lipases, including hepatic and lipoprotein lipases. Detailed sequence analysis and molecular modeling of the Dkk sequence onto the colipase structure suggest that Dkk and colipase have the same disulfide pattern and very similar three-dimensional structures (
      • van Tilbeurgh H.
      • Bezzine S.
      • Cambillau C.
      • Verger R.
      • Carrière F.
      ). This structural analogy implies a common function (lipid interaction) and raises the possibility that Dkk bound to LRP5 stimulates lipid hydrolysis by interacting with hepatic lipase and/or lipoprotein lipase. Furthermore, the impaired fat tolerance caused by the deficiency of LRP5 may lead to severe hypercholesterolemia in the absence of apoE.
      Another explanation for the impaired lipoprotein metabolism in the apoE;LRP5 double knockout mice is that LRP5 may recognize other lipoproteins in addition to apoE-containing lipoproteins. The candidate apoproteins that may be recognized by LRP5 remain unidentified, since the LRP5 deficiency did not significantly alter the pattern of apoproteins in the plasma lipoproteins of the apoE knockout mice or that of normal mice.
      In addition to the role of LRP5 in embryonic development and bone development, our current data provide further evidence that LRP5 also plays a role in the metabolism of plasma lipoproteins. Furthermore, consistent with the marked elevation of plasma cholesterol, severe arthrosclerosis developed in the apoE;LRP5 double knockout mice. The remarkable destruction of the internal elastic lamina seen in the lesion of the double knockout mice is characteristic of highly advanced atherosclerosis. The apoE;LRP5 double knockout mice manifesting extreme hypercholesterolemia and highly advanced arthrosclerosis will provide a useful animal model for the research and development of therapeutic agents against hypercholesterolemia and atherosclerosis.

      Acknowledgement

      We thank N. Suzuki for preparing the manuscript.

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