Angiopoietin-like Protein 3 Mediates Hypertriglyceridemia Induced by the Liver X Receptor*,

Toshimori Inaba {ddagger} §, Morihiro Matsuda § ¶ ||, Mitsuru Shimamura ¶, Norihide Takei ¶, Naoki Terasaka {ddagger}, Yosuke Ando **, Hiroaki Yasumo {ddagger}, Ryuta Koishi {ddagger}{ddagger}, Makoto Makishima ¶ §§ and Iichiro Shimomura ¶ ¶¶ ||||

From the Department of Medicine and Pathophysiology, Graduate School of Frontier Bioscience and Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan, {ddagger}Pharmacology and Molecular Biology Research Laboratories and {ddagger}{ddagger}Biomedical Research Laboratories, Sankyo Co., Ltd., 2-58 Hiromachi 1-tyome, Shinagawaku, Tokyo 140-8710, Japan, **Medical Safety Research Laboratories, Sankyo Co., Ltd., 717 Horikoshi, Fukuroi, Shizuoka 437-0065, Japan, he ||21st Century COE Program, the Japan Society for the Promotion of Science, Tokyo 102-8471, Japan, and ¶¶PRESTO, Japan Science and Technology Corporation (JST), Kawaguchi, Saitama 332-0012, Japan

Received for publication, December 26, 2002 , and in revised form, March 11, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The KK/San obese and diabetic mouse, a mutant strain from KK obese mice, exhibits significantly low plasma triglyceride levels. In KK/San mice, genetic analysis identified a mutation in the gene encoding angiopoietinlike protein 3 (Angptl3), a liver-specific secretory protein, which had suppressive effect on lipoprotein lipase activity. In the current study, LXR ligands augmented Angptl3 mRNA expression and protein production in hepatoma cells. LXR ligands and LXR·retinoid X receptor (RXR) complex increased the promoter activity of Angptl3 gene. Serial deletion and point mutation of Angptl3 promoter identified an LXR response element (LXRE). Gel mobility shift assay showed the direct binding of LXR·RXR complex to the LXRE of the Angptl3 promoter. Furthermore, treatment of mice with synthetic LXR ligand caused triglyceride accumulation in the liver and plasma, which was accompanied by induction of hepatic mRNAs of several LXR target genes, including sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS), and Angptl3. In Angptl3-deficient C57BL/6J mice, LXR ligand did not cause hypertriglyceridemia but accumulation of triglyceride in the liver. Our results demonstrate that Angptl3 is a direct target of LXR and that induction of hepatic Angptl3 accounts for hypertriglyceridemia associated with the treatment of LXR ligand.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
There is a growing realization that hypertriglyceridemia plays a central role in the pathophysiology of insulin resistance syndrome. Hypertriglyceridemia induces triglyceride accumulation in various tissues including adipose tissue, skeletal muscles, liver, and pancreatic islets, which results in disturbed insulin signaling in each tissue (1). Hypertriglyceridemia is also directly involved in the development of atherosclerosis (2). Therefore, the factors that regulate the amount of triglycerides in plasma could be viewed as potentially attractive pharmaceutical targets (3).

In the colony of KK obese mice, which exhibit diabetes and hypertriglyceridemia, we found a subgroup of KK mice (KK/San) with significantly low plasma triglyceride levels, although the phenotype of obesity and diabetes was still maintained (4). Genetic analysis and positional cloning in the KK/San mice identified a 4-bp nucleotide insertion in exon 6 of the gene encoding angiopoietin-like protein 3 (Angptl3),1 which caused a premature termination after frameshift (4). Angptl3 is a ~70-kDa secretory protein, and its mRNA is expressed exclusively in the livers of humans and mice (5). The protein contains a coiled-coil region and fibrinogen-like motif and shows similarity in amino acid sequence and structure with angiopoietin (5). Angptl3 can also induce angioneogenesis at a lesser degree than angiopoietin (6). Consistent with the hypotriglyceridemia in KK/San mice exhibiting a genetic defect of Angptl3, Angptl3 protein has a physiological capability to increase the plasma triglyceride levels. Recent studies reported that either the injection of recombinant ANGPTL3 protein or adenovirus-mediated production of ANGPTL3 acutely increased plasma triglyceride levels in both KK/San and wild-type lean mice (4). We have recently demonstrated that the effect of Angptl3 on plasma triglyceride was mediated by suppression of lipoprotein lipase activity with subsequent inhibition of hydrolysis of plasma triglyceride (7).

Liver X receptor (LXR) is a nuclear receptor that forms a heterodimer with RXR and activates the transcription of several genes involved in lipid metabolism by targeting the DR4 element in the promoters (8). There are two isoforms of LXR known to date. LXR{alpha} (NR1H3) is expressed selectively in the liver, fat, and macrophages, whereas LXR{beta} (NR1H2) is expressed ubiquitously (912). Several cholesterol derivatives such as (22R)-hydroxycholesterol and (24S),25-epoxycholesterol are known endogenous ligands of LXR (13, 14). T09013 [GenBank] 17 is a synthetic LXR ligand broadly used for the research in LXR biology (1518). Since LXR activation increases the transcription of ABCA1 through its LXRE on the promoter, T09013 [GenBank] 17 seems to enhance the ABCA1-mediated efflux of cellular cholesterol in various cells, especially macrophages (16). This effect of LXR ligand leads to a reduction of atherosclerotic plaque in vivo (19). However, treatment of rodents with T09013 [GenBank] 17 caused triglyceride accumulation in the liver and plasma. Accumulation of triglycerides in the liver was explained by increased expression of the sterol regulatory element-binding protein-1c (SREBP1c) and fatty acid synthase (FAS), both of which are direct targets of LXR. However, the mechanism underlying LXR-induced hypertriglyceridemia is not known at present (20).

In the present study, we show that the Angptl3 is a direct target gene of LXR. Both T09013 [GenBank] 17 and endogenous LXR ligands increased the hepatic mRNA expression and the secretion of Angptl3. In Angptl3-deficient mice, T09013 [GenBank] 17 treatment did not increase plasma triglyceride levels. Our results indicated that hypertriglyceridemia associated with synthetic LXR ligand treatment was due to overproduction of hepatic ANGPTL3 protein into plasma, which inhibited the hydrolysis of plasma triglyceride in the body.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—T0901317 was purchased from Cayman Chemical. 9-cis-Retinoic acid (9-cis-RA) and (22R)-hydroxycholesterol were obtained from Sigma. (24S),25-Epoxycholesterol was purchased from Steraloids Inc. (Newport, RI). Plasmids expressing cDNA for human RXR{alpha} (pCMX-hRXR{alpha}) and human LXR{alpha} (pCMX-hLXR{alpha}) were kindly provided by Dr. David Mangelsdorf (Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX).

Cell Cultures—HepG2 cells were obtained from the American Type Culture Collection (Manassas, VA). All cultures were performed under standard conditions (37 °C and 5% CO2 in air). The cells were seeded into six-well plates (Corning, NY) and allowed to grow in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. At 80% confluence, the cells were replaced with fresh 10% fetal calf serum DMEM, containing T09013 [GenBank] 17, 9-cis-RA, (22R)-hydroxycholesterol, or (24S),25-epoxycholesterol.

Enzyme-linked Immunosorbent Assay Construction and Assay—Recombinant human ANGPTL3 protein was prepared as described previously (4). Two ANGPTL3 mouse monoclonal antibodies were produced using the recombinant human ANGPTL3 as the antigen and were introduced in a double-antibody sandwich enzyme immunoassay system to detect human ANGPTL3. Culture media containing ANGPTL3 protein were condensed with Microcon YM-10 (Millipore Corp., Bedford, MA) and subjected to the immunoassay system using a horseradish peroxidase enzyme method. Two ANGPTL3 rabbit polyclonal antibodies against two peptides (CLSQQHMQIKEIEKQLR and CSFDSAPSEPKSRFAML) of mouse ANGPTL3 were produced and were introduced in a double antibody sandwich enzyme immunoassay system to detect mouse ANGPTL3.

Plasmid Construction—The 5'-flanking region of human Angptl3 gene (–781/+79, relative to the transcription start site) was prepared by PCR using human genomic DNA (Roche Applied Science) as a template and a forward primer tailed with a KpnI restriction site (5'-AAGGTACCAATTCTAGTTTGGTCCTAGA-3') and a reverse primer tailed with BamHI (5'-TTGGATCCTCTTGATCAATTCTGCAGGA-3'). The PCR product was digested with KpnI and BamHI and subcloned into the KpnI/BglII-digested luciferase reporter plasmid pGL3-basic (Promega, Madison, WI), generating pGL3/–781+79hAngptl3. For the construction of pGL3/–282+79hAngptl3 or pGL3/–126+79hAngptl3, pGL3/–781+79hAngptl3 was digested with DraI/HindIII or HpaI/HindIII, respectively, and subcloned into the SmaI/HindIII-digested pGL3 basic vector. For the construction of pGL3/–151+79hAngptl3, a DNA fragment was generated by PCR using pGL3/–781+79hAngptl3 as a template and a KpnI site-tailed forward primer (5'-CGGGGTACCGAAGGTTACATTCGTGCAAGTT-3') and a BamHI-tailed reverse primer (5'-TTGGATCCTCTTGATCAATTCTGCAGGA-3'). After digestion with KpnI and BamHI, it was subcloned into the KpnI/BglII-digested pGL3 basic vector. Site-directed mutagenesis of the construct pGL3/–781+79hAngptl3 was accomplished using the QuikChangeTM site-directed mutagenesis kit (Stratagene, La Jolla, CA) and two pairs of 32-mer oligonucleotides containing mutations corresponding, respectively, to nucleotide –146(T -> A)/–147(G -> A) and nucleotide –136(G -> A)/–137(T -> A) of the DR4 element in human Angptl3 promoter. The integrity of each plasmid was verified by DNA sequencing.

Transient Transfection and Reporter Assay—HepG2 cells were seeded at a density of 3.2 x 104 cells/well of 96-well plates in DMEM containing 10% fetal bovine serum 24 h prior to transfection. Cells were transfected with plasmids in serum-free Opti-MEM I medium using LipofectAMINETM 2000 transfection reagent (Invitrogen) according to the instructions provided by the supplier. Typically, each well contained 1 µl of LipofectAMINETM 2000, 15 ng of pCMX-hRXR{alpha} and/or pCMX-hLXR{alpha} expression vector, 60 ng of pGL3 basic luciferase reporter plasmids containing the 5'-flanking region of the human Angptl3 gene, and 60 ng of pCMX-{beta}-galatactosidase as an internal control for transfection efficiency. After adding the reagents, cells were transfected for 4 h at 37 °C in an atmosphere of 5% CO2. The cells were then incubated for ~40–48 h in fresh DMEM containing 5% fetal bovine serum with T09013 [GenBank] 17, 9-cis-RA, (22R)-hydroxycholesterol, (24S),25-epoxycholesterol, or vehicle. Cell lysates were produced using passive lysis buffer (Promega) using the protocol recommended by the manufacturer. Luciferase activity in cell extracts was determined using luciferase assay buffer (Promega) in a Lmax luminometer (Molecular Devices). {beta}-Galactosidase activity was determined using {beta}-D-galactopyranoside (Calbiochem) as described previously (21). Luciferase activity was normalized to {beta}-galactosidase activity individually for each well.

In Vitro Transcription/Translation and Electrophoretic Mobility Shift Analysis—Human RXR{alpha} and human LXR{alpha} proteins were synthesized in vitro from pCMX-hRXR{alpha} and pCMX-hLXR{alpha} expression vectors by using the TNT® Quik Coupled transcription/translation system (Promega) using the protocol recommended by the manufacturer. Double-stranded oligonucleotides corresponding to the LXR response element of the human Angptl3 gene promoter (LXREwt; 5'-GGAAGAAGGTTACATTCGTGCAAGTTAA-3') were 32P-radiolabeled with polynucleotide kinase (Promega). Protein-DNA binding assays were performed as described previously (22). Salmon sperm DNA (0.5 µg/µl) was added in binding assays to reduce nonspecific binding of labeled oligonucleotides. A 10- or 50-fold molar excess of unlabeled double-stranded LXREwt, the mutant LXR response element of the human Angptl3 gene promoter (LXREmut; 5'-GGAAGAAGAATACATTCGAACAAGTTAA-3') or nonspecific oligonucleotides (5'-ACTATTCTGTATGCAGCTGCGAGCCCCAGCCCCAGG-3') were used for the competition experiments. The samples were electrophoresed on a 4% polyacrylamide gel in 0.5x TBE buffer (4.5 mM Tris, 45 mM boric acid, 10 mM EDTA). The gels were dried and autoradiographed at –80 °C.

Animal Studies—C57BL/6J mice were obtained from Charles River, and C57BL/ANGPTL3hypl mice were from the Medical Safety Research Laboratories (Sankyo Co., Ltd., Shizuoka, Japan) and housed in a room under controlled temperature (23 ± 1 °C) and humidity (45–65%) and had free access to water and chow (Oriental Yeast). Experiments were conducted when the mice (males) were between 8 and 9 weeks of age. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Osaka University and Sankyo Co., Ltd.

On the day before the start of treatment (day 0), blood was collected to determine the initial plasma total cholesterol (TC) and triglyceride (TG) levels. Then the mice were divided into two groups, arranged so that the mean values of TC and TG in each group were almost equal. Mice were treated orally with T09013 [GenBank] 17 at a dose of 10 mg/kg daily in a propylene glycol/Tween 80 (4:1) formulation. Blood was collected under nonfasting conditions.

Lipid and Lipoprotein Analysis—Plasma lipid (TC and TG) levels were determined using commercial kits (Cholesterol CII-test Wako and Triglycerides E-test Wako, respectively; Wako Pure Chemical Industries, Osaka, Japan). Hepatic lipids were extracted by the method of Folch et al. (23). The extract was dissolved in 10% Triton X-100 (Sigma) in 2-propanol. Then both hepatic TC and TG contents were determined by using commercial kits as described above.

The plasma (20 µl) was further diluted with 80 µl of 10 mmol/liter sodium phosphate buffer (pH 7.4) containing 0.15 mol/liter NaCl (PBS) and was used for the analysis of lipoproteins by a high performance liquid chromatography (HPLC) system with two tandem gel permeation columns (TSK gel Lipopropak XL, 7.5 x 300 mm; Tosoh Co., Tokyo, Japan) and an online enzymatic detection system for total cholesterol.

RNA Analysis—Total RNAs from HepG2 cells were prepared with an RNA STAT-60 kit (Tel-Test, Friendswood, TX). The cDNA was produced using TaqMan reverse transcription kits (PerkinElmer Life Sciences). Real time PCR was performed on a LightCycler using the FastStart DNA Master SYBR Green I (Roche Applied Science) using the instructions provided by the manufacturer. Primers were designed as 5'-CCAGAACACCCAGAAGTAACT-3' and 5'-TCTGTGGGTTCTTGAATACTAGTC-3' for human Angptl3. Primers of 18 S ribosomal RNA were obtained from PerkinElmer Life Sciences. The amount of Angptl3 mRNA was expressed relative to the level of 18 S RNA mRNA.

Total RNAs from mouse liver were isolated using TRIzol Reagent (Invitrogen) based on the protocol provided by the manufacturer. Total RNA (1 µg) was used to generate cDNA using oligo(dT) oligonucleotide primer (T12–18) following the protocol for the First Strand cDNA Synthesis Kit (Amersham Biosciences). Real time quantitative PCR analysis was performed using ABI Prism 7700 (Applied Biosystems). Equal amounts of cDNA were used and amplified with the TaqMan Universal PCR Master Mix (Applied Biosystems). Levels of various mRNAs were normalized to those of cyclophilin as described previously (24). The sequences of primers and TaqMan probes were designed as follows: forward primer, 5'-ACATGTGGCTGAGATTGCTGG-3'; reverse primer, 5'-CCTTTGCTCTGTGATTCCATGTAG-3'; Taqman probe, 5'-CCTCCCAGAGCACACAGACCTGATGTTT-3' for mouse Angptl3; forward primer, 5'-AAGCTGTCGGGGTAGCGTCT-3'; reverse primer, 5'-GGAGCATGTCTTCAAATGTGC-3'; Taqman probe, 5'-CTAGGGGATCGGCGCGGACCACGGAGC-3' for mouse SREBP1c; forward primer, 5'-GAGGCCTGTACGGGATCATA-3'; reverse primer, 5'-CCGAGCCTTGTAAGTTCTGTG-3'; Taqman probe, 5'-TACATGACCAGCGCTCTGGGCATCACAGCC-3' for mouse stearoyl-CoA desaturase 1; forward primer, 5'-CGATGACGAGCCCTTGG-3'; reverse primer, 5'-TCTGCTCTTTGGAACTTTGTC-3'; Taqman probe, 5'-CGCGTCTCCTTTGAGCTGTTTGCA-3' for cyclophilin.

Statistical Analysis—All data were expressed as mean ± S.E. Differences between groups were examined for statistical significance using Student's t test or Dunnett's multiple comparison test. A p value less than 0.05 denoted the presence of a statistically significant difference.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Angptl3 Gene Expression and Protein Production Are Induced by LXR Agonists in HepG2 Cells—Human hepatoma HepG2 cells were used to analyze the regulation of Angptl3 mRNA expression and protein secretion. Angptl3 mRNA expression increased in a dose-dependent manner by the treatment with T09013 [GenBank] 17, with 6-fold peak increase following the addition of 1 µM T09013 [GenBank] 17 (Fig. 1A, upper panel). To investigate whether the Angptl3 gene is regulated by the LXR·RXR heterodimer, we added RXR agonist 9-cis-RA (1 µM) with 1 µM T09013 [GenBank] 17. Angptl3 mRNA expression increased further up to 7-fold by the addition of 9-cis-RA (Fig. 1A, upper panel). ANGPTL3 protein secreted in the media also increased in a dose-dependent manner with T09013 [GenBank] 17 treatment, and the addition of 9-cis-RA further increased ANGPTL3 secretion in the media (Fig. 1A, lower panel). The mRNA and protein secretion of Angptl3 were increased by 1 µM T09013 [GenBank] 17 in a time-dependent manner (Fig. 1B). Thus, the increased expression of Angptl3 mRNA was accompanied by increased production of ANGPTL3 protein after T09013 [GenBank] 17 treatment. Angptl3 mRNA levels were increased by the addition of 30 µM (24S),25-epoxycholesterol, (22R)-hydroxycholesterol, both of which are endogenous LXR ligands, or 1 µM T09013 [GenBank] 17 (Fig. 1C). Similar results of the effect of synthetic and natural LXR ligands on the Angptl3 mRNA and protein were seen in rat hepatoma H4IIEC3 cells (data not shown). Thus, LXR agonists induced the expression and secretion of Angptl3 in hepatocytes.



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FIG. 1.
Angptl3 gene expression and protein secretion is induced by synthetic and endogenous LXR ligands in HepG2 cells. A, dose-dependent induction of Angptl3 mRNA expression and protein secretion into media by T09013 [GenBank] 17. HepG2 cells were incubated with DMEM containing 0, 0.01, 0.1, or 1 µM T09013 [GenBank] 17 or 1 µM T09013 [GenBank] 17 plus 1 µM 9-cis-RA for 24 h. B, time-dependent induction of Angptl3 mRNA expression and protein secretion into media by T09013 [GenBank] 17. HepG2 cells were incubated with DMEM containing 1 µM T09013 [GenBank] 17 (black circles) or vehicle (white circles). Cells were harvested, and medium was collected at the indicated time. C, induction of Angptl3 mRNA by natural and synthetic LXR ligands. HepG2 cells were incubated with DMEM containing 10% fetal calf serum with 30 µM (24S),25-epoxycholesterol (E.C.; thick-lined hatched bar), 30 µM (22R)-hydroxycholesterol (H.C.; thin-lined hatched bar), 1 µM T09013 [GenBank] 17 (gray bar), or vehicle alone (open bar) for 12 h. A–C, total mRNA was extracted and subjected to mRNA analysis as described under "Experimental Procedures." Angptl3 mRNA expression levels were normalized by 18 S ribosomal RNA (18S), and the results were expressed as -fold increase compared with control treatment with vehicle alone. A and B, secreted ANGPTL3 protein was quantified with an enzyme-linked immunosorbent assay system as described under "Experimental Procedures." *, p < 0.01, compared with the control group using Student's t test. All results are shown as mean ± S.E. of more than three experiments.

 

T0901317 and Endogenous LXR Ligands Enhance LXR·RXR-mediated Increase of Angptl3 Promoter Activity through the DR4 Element on Its Promoter—Fig. 2 shows the sequence of the 5'-flanking region of human Angptl3 gene. Computer analysis revealed the putative binding sites of transcriptional factors, including CCAAT/enhancer-binding protein, glucocorticoid receptor, hepatic nuclear factor, and LXR. To investigate the effect of T09013 [GenBank] 17 on the transcription of the Angptl3 gene, we cloned its promoter region from human genomic DNA and constructed a luciferase reporter plasmid containing the 5'-flanking region (–781/+79) of the human Angptl3 gene. In transfection assays with the luciferase reporter constructs, T09013 [GenBank] 17 enhanced the luciferase activity driven by the 5'-flanking region of the Angptl3 gene in HepG2 cells, and the addition of 9-cis-RA augmented this increase (Fig. 3A). Co-expression of LXR{alpha} and RXR{alpha} further augmented the T09013 [GenBank] 17-mediated increase of luciferase activity compared with the expression of LXR{alpha} or RXR{alpha} alone (Fig. 3A). Fig. 3B shows the effects of endogenous LXR ligands, (24S),25-epoxycholesterol and (22R)-hydroxycholesterol, on luciferase activity driven by the human Angptl3 promoter in HepG2 cells. The endogenous ligands also augmented luciferase activity, in addition to T09013 [GenBank] 17, similar to the mRNA regulation depicted in Fig. 1C, and a further increase was observed when LXR{alpha} and RXR{alpha} were co-expressed, compared with the expression of either construct (Fig. 3B).



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FIG. 2.
Sequence of the 5'-flanking region of the human Angptl3 gene. Putative transcription factor-binding sites are predicted by the sequence motif search program, TFSEARCH, version 1.3. A DR4 element is bundled within the box. The transcription start site is designated as +1. The exon region is shown in boldface type. The transcription site for exon 1 is indicated by the arrow. C/EBP, CCAAT/enhancer-binding protein; GR, glucocorticoid receptor; AP-1, activator protein-1; HFH, forkhead domain factor; HNF, hepatic nuclear factor.

 


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FIG. 3.
T0901317 augments LXR·RXR-mediated increase of human Angptl3 gene promoter activity through the DR4 element. A and B, promoter activity of Angptl3 induced by LXR ligands. HepG2 cells (3.2 x 104 cells/well of 96-well plate) were transfected with 15 ng of expression plasmids encoding human RXR{alpha}, human LXR{alpha}, or the empty pCMX vector as control; 60 ng of reporter plasmids containing the firefly luciferase gene driven by 5'-flanking region of the human Angptl3 gene; and 60 ng of pCMX-lacZ, using the LipofectAMINETM 2000 transfection reagent. A, after 4-h transfection, cells were treated with ethanol (open bars), 100 nM T09013 [GenBank] 17 (hatched bars), or 100 nM T09013 [GenBank] 17 plus 1 µM of 9-cis-retinoic acid (solid bars) for 40 h, before harvest. B, cells were treated with ethanol (open bars), 30 µM (22R)-hydroxycholesterol (22(R)H.C.; gray bars), 30 µM (24S),25-epoxycholesterol (24,25E.C.; hatched bars), or 100 nM T09013 [GenBank] 17 (solid bars) for 40 h before harvest. The cell lysates were used for determination of luciferase and {beta}-galactosidase activities as described under "Experimental Procedures." Reporter plasmid containing the –781/+79 region of the human Angptl3 gene was used for transfection assays. C, HepG2 cells were transfected with the reporter plasmids containing the serially deleted promoter of the human Angptl3 gene and expression vectors of human LXR{alpha} and human RXR{alpha} or control vector (pCMX). After transfection, cells were treated without (gray bars) or with 100 nM T09013 [GenBank] 17 (solid bars) before harvest, and luciferase assays were conducted similarly to A and B. A–C, luciferase activities were normalized to {beta}-galactosidase activity for each well. *, p < 0.01, compared with the control group using Student's t test. Each value represents the mean ± S.E. of six determinations. D, sequence comparison of the DR4 in the promoter of the Angptl3 gene with the LXRE in LXR target genes reported previously.

 

In the next step, transient reporter assays were performed using various Angptl3 gene promoter constructs. Serial deletions showed that the T09013 [GenBank] 17-mediated induction of luciferase activities was highly maintained when the constructs contained the region of –151/–126 (Fig. 3C). This region contained the putative LXRE suggested by computer search. Fig. 3D shows comparison of the sequences of the DR4 element in the promoters of human and mouse Angptl3 genes with the DR4 previously described in other LXR target genes (18, 2527).

To further determine the significance of LXRE in the promoter of the Angptl3 gene, we generated a construct containing the point mutation in LXRE and compared the response to T09013 [GenBank] 17 between the wild-type and mutant constructs (Fig. 4A). The point mutation of LXRE in the Angptl3 gene promoter totally abolished the response to T09013 [GenBank] 17 treatment (Fig. 4B).



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FIG. 4.
Point mutation in the LXRE abolished T09013 [GenBank] 17-induced transactivation of the human Angptl3 gene. A, schematic illustration of the human Angptl3 promoter and luciferase reporter construct. The putative LXRE sequence at position from –150 to –135 is indicated. Point mutations from GT to AA and from TG to AA were introduced at –148/–147 and –132/–133, respectively. B, comparison of the transcriptional activities of the wild-type and mutant reporter described for A. HepG2 cells were transfected with wild-type or mutant plasmids and expression vectors of human LXR{alpha} and RXR{alpha} or control vector. After transfection, cells were treated with ethanol (open bars), 30 µM (22R)-hydroxycholesterol (22(R)H.C.; gray bars), 30 µM (24S),25-epoxycholesterol (24,25E.C.; hatched bars) or 100 nM T09013 [GenBank] 17 (solid bars) for 40 h. The cell lysates were used for determination of luciferase and {beta}-galactosidase activities as described under "Experimental Procedures." Luciferase activities were normalized to {beta}-galactosidase activity for each well. *, p < 0.01, compared with the control group using Student's t test. Each value represents the mean ± S.E. of three determinations.

 

LXR{alpha}·RXR{alpha} Complex Binds to the Angptl3 LXRE—To determine whether LXR{alpha} binds to the LXRE as complexes with RXR{alpha}, gel mobility shift assays were performed with double-stranded oligonucleotides corresponding to the Angptl3 LXRE (Fig. 5). The 32P-radiolabeled double-stranded LXREwt oligonucleotides were incubated with in vitro translated LXR{alpha} and/or RXR{alpha} protein. Neither LXR{alpha} nor RXR{alpha} alone bound to Angptl3 LXRE significantly (lanes 3 and 4). When LXR{alpha} and RXR{alpha} were produced together, the mobility of 32P-radiolabeled LXRE oligonucleotides were shifted to a higher range (lane 5 versus lane 2), indicating the binding of LXR{alpha}·RXR{alpha} complex to the Angptl3 LXRE. The addition of excessive unlabeled LXREwt oligonucleotides reduced the signal of 32P-radiolabeled LXREwt oligonucleotide binding to LXR{alpha}·RXR{alpha} complex (lanes 6 and 7 versus lane 5). On the other hand, the addition of either LXREmut oligonucleotides (28) (Fig. 4A) or nonspecific oligonucleotides did not reduce the specific signal (lanes 8–11 versus lane 5). These results demonstrated the specific binding of LXR{alpha}·RXR{alpha} complex to the Angptl3 LXRE.



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FIG. 5.
LXR{alpha}·RXR{alpha} complex specifically binds to the DR4 element in the human Angptl3 gene promoter. Electrophoretic mobility shift assays were performed as described under "Experimental Procedures." The 32P-radiolabeled double-stranded oligonucleotide corresponding to the LXR response element of human Angptl3 promoter (LXREwt) was incubated with in vitro synthesized LXR and RXR proteins. Competitive experiments were performed using unlabeled oligonucleotides corresponding to wild type (LXREwt), mutant LXR response element (LXREmut) (25) (Fig. 4A) of the human Angptl3 promoter, or nonspecific oligonucleotides as competitors by 10- and 50-fold molar excess. The arrow indicates shifted bands of 32P-radiolabeled wild type LXRE oligonucleotide binding to LXR{alpha}·RXR{alpha} protein complex. The asterisk indicates nonspecific bands.

 

T0901317 Increases Hepatic mRNA Expression and Plasma Concentration of ANGPTL3 and Plasma Triglycerides in C57BL/6J Mice—To determine whether T09013 [GenBank] 17 increases the hepatic mRNA expression and plasma concentration of ANGPTL3 in vivo, T09013 [GenBank] 17 was administered orally at a dose of 10 mg/kg/day for 2 days. Hepatic mRNA expression of Angptl3 increased by 1.6-fold at day 2 of the treatment (Fig. 6A). Plasma concentration of ANGPTL3 increased by 3.3-fold on the same day (Fig. 6B). This was associated with an increase in plasma triglyceride level, reaching a 4-fold increase at day 2 (Fig. 6C).



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FIG. 6.
Treatment with T09013 [GenBank] 17 increased hepatic Angptl3 mRNA, plasma ANGPTL3 protein, and plasma triglyceride levels in C57BL/6J. Mice were orally administered either vehicle (propylene glycol/Tween 80 = 4:1, open bar) or 10 mg/kg/day of T09013 [GenBank] 17 (black bar) for 2 days (n = 5 per group). At day 2, mice were sacrificed. A, total RNAs were isolated from the livers. Angptl3 mRNA expression levels were determined by real time quantitative reverse transcriptase-PCR and normalized to cyclophilin. Data are represented as mRNA expression relative to the vehicle control. B, plasma ANGPTL3 concentrations were measured with an enzyme-linked immunosorbent assay as described under "Experimental Procedures." C, plasma TG levels were measured at day 0 and 2. **, p < 0.01, compared with the control group using Dunnett's multiple comparison test (plasma TG) and Student's t test (Angptl3 mRNA and plasma ANGPTL3).

 

T0901317 Increases Hepatic Triglyceride Contents but Not Plasma Triglyceride Level in C57BL/6J-Angptl3hypl Mice—Angptl3-defective KK/San mice were backcrossed to C57BL/6J mice by five generations to establish the genuine C57BL/6J-Angptl3hypl mice, which did not produce Angptl3 mRNA or protein (data not shown). To determine whether T09013 [GenBank] 17-induced hypertriglyceridemia (Fig. 6) was mediated by Angptl3, we compared the response of C57BL/6J wild-type and C57BL/6J-Angptl3hypl mice to a 2-day course of oral T09013 [GenBank] 17. Fig. 7A shows triglyceride and cholesterol contents in livers of T09013 [GenBank] 17-treated mice. T09013 [GenBank] 17 treatment increased hepatic triglyceride contents but not cholesterol contents in both Angptl3-deficient and wild-type mice (Fig. 7A). This triglyceride accumulation in liver by the treatment was accompanied by the increased mRNA expressions of hepatic lipogenic genes, SREBP1c, FAS, and stearoyl-CoA desaturase-1, all of which are direct targets of LXR, in both Angptl3-deficient and wild-type mice (Fig. 8). Fig. 7B shows plasma triglyceride and cholesterol concentration in T09013 [GenBank] 17-treated mice. Plasma triglyceride and cholesterol concentrations were lower at base line in Angptl3-deficient mice, as described previously (4). T09013 [GenBank] 17 treatment significantly increased plasma triglyceride levels in the wild-type mice but failed to increase these levels in the Angptl3-deficient mice (Fig. 7B). Plasma cholesterol slightly increased in both Angptl3-deficient and wild-type mice (Fig. 7B). The results demonstrated that T09013 [GenBank] 17-induced hypertriglyceridemia was accounted for by LXR-mediated induction of Angptl3.



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FIG. 7.
Mutation of Angptl3 abolished T09013 [GenBank] 17-induced plasma TG elevation but increased hepatic triglyceride content. A and B, C57BL/6J (WT) or C57BL/6J-Angptl3hypl mice (Angptl3hypl) were orally administered either vehicle (white bar) or 10 mg/kg/day of T09013 [GenBank] 17 (black bar) for 2 days. A, hepatic lipid contents. TG and TC contents in the livers were determined at day 2 as described under "Experimental Procedures." B, the levels of plasma TG and TC were determined at day 2 as described under "Experimental Procedures." *, p < 0.05; **, p < 0.01, compared with the control group using Student's t test.

 


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FIG. 8.
T0901317 increased mRNA expressions of the genes involved in lipogenesis in C57BL/6J-Angptl3hypl. C57BL/6J (wild type) or C57BL/6J-Angptl3hypl mice (Angptl3hypl) were orally administered either vehicle (white bar) or 10 mg/kg/day of T09013 [GenBank] 17 (black bar) for 2 days. Total RNAs were isolated from the livers, and mRNA levels of the genes involved in lipogenesis, SREBP1c, FAS, and stearoyl-CoA desaturase 1 (SCD-1), were measured by real time reverse transcriptase-PCR. **, p < 0.01, compared with the control group using Student's t test.

 

Fig. 9 shows plasma lipoprotein profiles of wild-type and Angptl3-deficient mice treated with or without T09013 [GenBank] 17 for 2 days. T09013 [GenBank] 17 treatment increased VLDL fraction in wild-type mice. On the other hand, Angptl3-deficient mice failed to increase the plasma VLDL fraction after treatment with T09013 [GenBank] 17.



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FIG. 9.
Mutation of Angptl3 abolished T09013 [GenBank] 17-induced VLDL increase. C57BL/6J (WT; A and B) or C57BL/6J-Angptl3hypl mice (Angptl3hypl; C and D) were orally administered either vehicle (A and C) or 10 mg/kg/day of T09013 [GenBank] 17 (B and D) for 2 days. Lipoprotein profiles were analyzed with HPLC at day 2 as described under "Experimental Procedures."

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study demonstrated that LXR agonists increased the mRNA expression and secretion of Angptl3 in human hepatoma cells. Serial deletion-promoter analysis identified an LXRE site at –150/–135 in the 5'-flanking region of the Angptl3 gene. Furthermore, point mutation analysis revealed that this element was essential for transactivation of the Angptl3 promoter by LXR ligand. Electrophoretic mobility shift assay demonstrated the direct binding of LXR·RXR heterodimer to this LXRE site. In addition, Angptl3 mRNA and plasma concentration of ANGPTL3 protein were induced in wild-type mice treated with T09013 [GenBank] 17, a synthetic ligand of LXR. These findings indicate that Angptl3 is a direct target of LXR. During the preparation of this manuscript, Kaplan et al. (29) reported the identification of the putative LXRE in human and mouse Angptl3 promoters and showed the LXR-dependent regulation of Angptl3 mRNA expression, although they did not show specific binding of LXR·RXR complex to this element or ANGPTL3 protein regulation by LXR ligand. Our data were consistent with their results. We further elucidated the molecular mechanism underlying LXR-induced hypertriglyceridemia in vivo. In wild-type mice, T09013 [GenBank] 17 increased the triglyceride content in liver and plasma. On the other hand, T09013 [GenBank] 17 failed to increase plasma triglyceride concentration in Angptl3-deficient mice with the accumulation of hepatic triglyceride similarly to the wild-type mice. These results indicate that the effect of LXR ligand on plasma triglyceride was solely attributed to the LXR-mediated induction of Angptl3 gene expression.

Angptl3 is a secretory protein, and its mRNA is expressed exclusively in the liver, and the name "angiopoietin-like protein 3" was coined for this protein because of the similarity of its amino acid sequences and protein structure to those of angiopoietin (30, 31). We previously found a subgroup of KK obese mice (KK/San) that had significantly low plasma triglyceride concentrations compared with wild-type KK obese mice and identified a causative frameshift mutation in the Angptl3 gene in KK/San mice (4). Low plasma triglyceride in KK/San mice was explained by the genetic defect of Angptl3, which acts to increase plasma triglyceride by suppressing peripheral LPL activities (7).

Several target genes of LXR have been reported in lipid metabolism, including Cyp7A1 (15), a limiting enzyme for converting cholesterol to bile acid, and ABCA1, a transporter that facilitates cholesterol efflux from the cells (16). LXR-mediated induction of ABCA1 enhances cholesterol efflux from macrophages, accompanying the supply of cholesterol to ApoA1 and the increase of plasma high density lipoprotein (16). Furthermore, LXR-mediated induction of ABCG5/G8 in liver and small intestine increases excretion of cholesterol into bile and feces, resulting in reduction in cholesterol absorption (32). Based on these effects on macrophages and intestinal mucosa, LXR agonists were expected to act as antiatherogenic and antihypercholesterolemic agents (19, 33). However, in vivo, T09013 [GenBank] 17 treatment resulted in triglyceride accumulation in the liver and in plasma. Increased hepatic triglyceride synthesis by LXR agonists could be explained by increased mRNA expressions of SREBP1c and FAS, both of which are direct targets of LXR (20). SREBP1c is a transcriptional factor that induces the transcription of the genes encoding enzymes involved in fatty acid and triglyceride synthesis (17). FAS is a limiting enzyme for fatty acid synthesis. Therefore, transactivation of SREBP1c and FAS by LXR agonists leads to accumulation of fatty acid and triglyceride in the liver.

Increased plasma triglyceride levels by T09013 [GenBank] 17 are probably not due to the induced expression of SREBP1c, because plasma triglyceride levels did not increase in the transgenic mice that overexpressed the active form of SREBP1c in the liver despite increased hepatic synthesis of triglyceride (34). In the present study, treatment of wild-type mice with T09013 [GenBank] 17 resulted in a rise in plasma triglyceride in parallel with the induction of Angptl3 mRNA in the liver and plasma concentration of ANGPTL3 protein. On the other hand, in Angptl3-deficient mice, such treatment did not alter plasma triglyceride levels, although it increased the mRNA expression levels of SREBP1c and FAS and triglyceride contents in the liver. The HPLC analysis of the plasma in the wild-type mice showed a significant increase of the VLDL fraction after treatment with T09013 [GenBank] 17 (Fig. 9, B versus A). On the other hand, Angptl3-deficient mice failed to increase the plasma VLDL fraction after treatment with T09013 [GenBank] 17 (Fig. 9, D versus C). Other lipoprotein fractions were not affected significantly. We tried to correlate the in vivo effect of LXR agonist or Angptl3 on LPL activities but failed because significant amounts of triglyceride in plasma of T09013 [GenBank] 17-treated mice interfered with the assay of LPL activity after the injection of heparin. Instead, we measured the LPL protein amount in the postheparinized plasma of hamsters before and after T09013 [GenBank] 17 treatment. The protein amount of LPL did not change between before and after T09013 [GenBank] 17 treatment (supplemental data). From our previous study, ANGPTL3 inhibited the enzymatic activity of LPL, but not that of hepatic lipase, in vitro (7). Taken together with the data of plasma ANGPTL3 and triglyceride in mice (Figs. 6, 7, 8, 9), we concluded that the rise of the plasma VLDL fraction after T09013 [GenBank] 17 treatment should be due to the inhibitory effect of Angptl3 on LPL activities by increased ANGPTL3 protein in plasma.

LXR agonists were expected to be used for the treatment of atherosclerosis, but their hypertriglyceridemic effect prevented clinical application. Our results identified the liver-specific LXR target gene, Angtpl3, as the cause of hypertriglyceridemia. Pharmaceutical regulation of the function or expression of Angptl3 should be useful for treatment of hypertriglyceridemia, which is an associated risk factor of atherosclerosis. Furthermore, LXR-targeting therapy that selectively targets macrophages and intestinal mucosa but not hepatocytes or accompanying Angptl3 management is a potentially useful strategy for the treatment of atherosclerosis and hypercholesterolemia.


    FOOTNOTES
 
* This work was supported by grants from the Daiwa Securities Health Foundation, Suzuken Memorial Foundation, Tokyo Biochemical Research Foundation, Takeda Medical Research Foundation, Uehara Memorial Foundation, Takeda Science Foundation, Novartis Foundation (Japan) for the Promotion of Science, Cell Science Research Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research, a Grant-in-Aid of the Japan Medical Association, the Naito Foundation, the Kanae Foundation for Life and Socio-Medical Science, and a Japan Heart Foundation Research Grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains an additional figure. Back

§ These two authors contributed equally to this work. Back

§§ To whom correspondence may be addressed: Dept. of Medicine and Pathology, Graduate School of Frontier Bioscience and Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-3272; Fax: 81-6-6879-3279; E-mail: maxima{at}fbs.osaka-u.ac.jp. |||| To whom correspondence may be addressed: Dept. of Medicine and Pathology, Graduate School of Frontier Bioscience and Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-3272; Fax: 81-6-6879-3279; E-mail: ichi{at}fbs.osaka-u.ac.jp.

1 The abbreviations used are: Angptl3, angiopoietin-like protein 3; LPL, lipoprotein lipase; LXR, liver X receptor; SREBP1c, sterol regulatory element-binding protein-1c; FAS, fatty acid synthase; RXR, retinoid X receptor; 9-cis-RA, 9-cis-retinoic acid; DMEM, Dulbecco's modified Eagle's medium; LXRE, LXR-responsive element; TC, total cholesterol; TG, triglyceride; HPLC, high performance liquid chromatography; VLDL, very low density lipoprotein. Back


    ACKNOWLEDGMENTS
 
We thank Tadashi Koieyama for animal experiments, Ayano Hiroshima for genetic analysis, and Dr. Hidehiko Furukawa for helpful suggestions.



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