A lincRNA-DYNLRB2-2/GPR119/GLP-1R/ABCA1dependent signal transduction pathway is essential for the regulation of cholesterol homeostasis Yan-Wei Hu,1,* Jun-Yao Yang,1,* Xin Ma,1,† Zhi-Ping Chen,* Ya-Rong Hu,* Jia-Yi Zhao,* Shu-Fen Li,* Yu-Rong Qiu,* Jing-Bo Lu,§ Yan-Chao Wang,* Ji-Juan Gao,* Yan-Hua Sha,* Lei Zheng,2,* and Qian Wang2,* Laboratory Medicine Center,* and Departments of Anesthesiology† and Vascular Surgery,§ Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China
This study was supported by the National Natural Sciences Foundation of China (81271905 and 81301489), Guangdong Provincial Natural Sciences Foundation of China (S2012020010920), and the President Foundation of Nanfang Hospital, Southern Medical University (2013B004). The authors declare no conflicts of interest. Manuscript received 6 October 2013 and in revised form 30 January 2014. Published, JLR Papers in Press, January 31, 2014 DOI 10.1194/jlr.M044669
Supplementary key words long intervening noncoding ribonucleic acid-DYNLRB2-2 • G protein-coupled receptor 119 • glucagon-like peptide 1 receptor • ATP binding cassette transporter A1 • atherosclerosis
Atherosclerosis is a complex disease, involving many cell types and circulating mediators and resulting in an inflammatory state. Atherosclerotic lesions form de novo from focal accumulation of lipoproteins, monocyte-derived macrophages, and lymphocytes within the arterial wall (1, 2). However, cholesterol and other lipids can be mobilized and excreted to prevent atherosclerosis in a process termed reverse cholesterol transport (RCT), in which ABCA1 plays a key role. ABCA1 is a member of a family of highly conserved transmembrane transport proteins. It plays a crucial role in the efflux of cellular cholesterol to HDL and its apolipoproteins (3). In humans, ABCA1 mutations can cause a severe HDL-deficiency syndrome, leading to the development of sterol deposits in tissue macrophages and premature atherosclerosis (4). Because of its role in macrophage lipid transport, ABCA1 is an important target for the prevention and treatment of atherosclerosis. G protein-coupled receptor 119 (GPR119), which has been described as a deorphanized G protein-coupled receptor, plays a significant role in mediating systemic metabolic homeostasis. It has recently attracted attention because evidence from in vitro systems and animal models suggests that its modulation has a favorable effect on glucose homoeostasis (5, 6). Because GPR119 promotes glucose-stimulated insulin
Abbreviations: Ac-LDL, acetylated LDL; CaMK, Ca2+/calmodulindependent protein kinase; CE, cholesteryl ester; FC, free cholesterol; GLP-1R, glucagon-like peptide 1 receptor; GPR119, G protein-coupled receptor 119; HFD, high-fat diet; IL, interleukin; lincRNA, long intervening noncoding RNA; lncRNA, long noncoding RNA; LPS, lipopolysaccharide; LV, lentivirus; Ox-LDL, oxidized LDL; PI-3K, phosphoinositide 3-kinase; PK, protein kinase; RCT, reverse cholesterol transport; SAA, serum amyloid A; TC, total cholesterol. 1 Y-W. Hu, J-Y. Yang, and X. Ma contributed equally to this work. 2 To whom correspondence should be addressed. e-mail: [email protected] (Q.W.); [email protected] (L.Z.)
Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at http://www.jlr.org
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Abstract Accumulated evidence shows that G protein-coupled receptor 119 (GPR119) plays a key role in glucose and lipid metabolism. Here, we explored the effect of GPR119 on cholesterol metabolism and inflammation in THP-1 macⴚ/ⴚ rophages and atherosclerotic plaque progression in apoE mice. We found that oxidized LDL (Ox-LDL) significantly induced long intervening noncoding RNA (lincRNA)-DYNLRB2-2 expression, resulting in the upregulation of GPR119 and ABCA1 expression through the glucagon-like peptide 1 receptor signaling pathway. GPR119 significantly decreased cellular cholesterol content and increased apoA-I-mediated cholesterol efflux in THP-1 macrophage-derived foam cells. ⴚ/ⴚ mice were randomly divided into two In vivo, apoE groups and infected with lentivirus (LV)-Mock or LVGPR119 for 8 weeks. GPR119-treated mice showed decreased liver lipid content and plasma TG, interleukin (IL)-1, IL-6, and TNF-␣ levels, whereas plasma levels of apoA-I were significantly increased. Consistent with this, atherosclerotic lesion development was significantly inhibⴚ/ⴚ mice with LV-GPR119. Our ited by infection of apoE findings clearly indicate that, Ox-LDL significantly induced lincRNA-DYNLRB2-2 expression, which promoted ABCA1-mediated cholesterol efflux and inhibited inflammation through GPR119 in THP-1 macrophage-derived foam cells. Moreover, GPR119 decreased lipid and serum inflammatory cytokine levels, decreasing atheroscleⴚ/ⴚ mice. These suggest that GPR119 may rosis in apoE be a promising candidate as a therapeutic agent.—Hu, Y-W., J-Y. Yang, X. Ma, Z-P. Chen, Y-R. Hu, J-Y. Zhao, S-F. Li, Y-R. Qiu, J-B. Lu, Y-C. Wang, J-J. Gao, Y-H. Sha, L. Zheng, and Q. Wang. A lincRNA-DYNLRB2-2/GPR119/ GLP-1R/ABCA1-dependent signal transduction pathway is essential for the regulation of cholesterol homeostasis. J. Lipid Res. 2014. 55: 681–697.
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GPR119. In addition, lincRNA-DYNLRB2-2 and GPR119 promoted ABCA1-mediated cholesterol efflux from THP-1 macrophage-derived foam cells, and inhibited inflammatory responses in these cells. Overexpression of GPR119 markedly decreased lipid levels and serum inflammatory cytokine levels to prevent atherosclerosis in apoE⫺/⫺ mice.
MATERIALS AND METHODS Materials Human lipoproteins [acetylated (Ac)-LDL, Ox-LDL, and HDL] were obtained from Biomedical Technologies Inc. (Stoughton, MA). The PrimeScript RT reagent kit (Perfect Real Time) (DRR037A; TaKaRa Bio, Inc., Shiga, Japan) and SYBR® Premix Ex TaqTM II (Tli RNaseH Plus) (DRR820A; TaKaRa Bio, Inc.) were obtained as indicated. Chemicals were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise noted.
Animals and diets The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication number 85-23, revised 1996) and was approved by the Animal Experimental Committee at Nanfang Hospital. Male apoE⫺/⫺ mice with a C57BL/6 background were purchased from the Laboratory Animal Center of Peking University (Beijing, China) and housed five per cage at 25°C on a 12 h light/dark cycle. To detect the effect of GPR119 on lipid metabolism, 6-week-old apoE⫺/⫺ mice were randomized into two groups and injected via the tail vein with control lentivirus [lentivirus (LV)-Mock, n = 15] or LV encoding mouse GPR119 (LV-GPR119, n = 15), respectively. The mice were fed a high-fat diet (HFD) for a period of 8 weeks. The diet was a commercially prepared mouse food supplemented with 21% (w/w) butterfat, 0.15% (w/w) cholesterol, and 19.5% (w/w) casein (Beijing Keao Xieli Feed Co., LTD, Beijing, China). At week 8, mice were anesthetized and 1 ml of blood was collected by cardiac puncture before the mice were euthanized by cervical dislocation and tissues were collected for further analysis.
Cell culture Human monocytic THP-1 cells were obtained from ATCC (Manassas, VA). THP-1 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% FCS and differentiated for 72 h with 100 nM PMA. Macrophages were transformed into foam cells by incubation in the presence or absence of 50 g/ml of Ox-LDL in serum-free RPMI 1640 medium containing 0.3% BSA for 48 h. Cells were incubated at 37°C in an atmosphere of 5% CO2. Cells were seeded in 6- or 12-well plates or 60 mm dishes and grown to 60–80% confluence before use.
Fig. 1. Dose-dependent and time-dependent effects of Ox-LDL/Ac-LDL on lincRNA-DYNLRB2-2 and GPR119 expression in THP-1 macrophages. A, B: THP-1 macrophages were treated with Ox-LDL/Ac-LDL at 0, 25, 50, and 100 g/ml for 24 h, and THP-1 macrophages were treated with 50 g/ml Ox-LDL/Ac-LDL for 0, 12, 24, and 48 h. lincRNA-DYNLRB2-2 levels were detected by real-time quantitative PCR. C–E: THP-1 macrophages were infected with LV-Mock or LV-DYNLRB2-2. lincRNA-DYNLRB2-2 levels and GPR119 gene levels were measured by real-time quantitative PCR and GPR119 protein expression was measured by Western blotting. F–I: THP-1 macrophages were treated with Ox-LDL/Ac-LDL at 0, 25, 50, and 100 g/ml for 24 h, and THP-1 macrophages were treated with 50 g/ml Ox-LDL/Ac-LDL for 0, 12, 24, and 48 h. F, G: GPR119 gene levels were measured by real-time quantitative PCR. H, I: GPR119 protein levels were measured by Western blotting. Similar results were obtained in three independent experiments. All the results are expressed as mean ± SD of three independent experiments, each performed in triplicate. *P < 0.05 versus vehicle.
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secretion, pancreatic -cell function, and glucagon-like peptide 1 (GLP-1) release, its therapeutic potential for the treatment of type 2 diabetes mellitus is getting more attention (7, 8). Moreover, GPR119 coupling to G␣ stimulatory proteins induces adenylate cyclase activity, resulting in an increase in intracellular cAMP, which regulates the GPR119-mediated release of GLP-1 (9). In addition, the GPR119-mediated release of GLP-1 from intestinal L-cells is protein kinase (PK)A dependent (10). Furthermore, cAMP, as a common second messenger that can activate MAPKs via PKA (11), can stimulate ABCA1 expression with species specific (12), alleviating the accumulation of cholesterol in macrophages by promoting ABCA1-mediated cellular cholesterol efflux (13, 14). Moreover, GPR119 can be activated by oleoylethanolamide and several other endogenous lipids containing oleic acid (15). These data suggest that GPR119 not only improves systemic glucose homeostasis but also affects lipid metabolism homeostasis. Long noncoding RNAs (lncRNAs) are nonprotein coding transcribed RNA molecules consisting of more than 200 nucleotides and fall into one of three categories including: 1) long intervening noncoding RNAs (lincRNAs) are transcribed from regions far away from protein-coding genes; 2) natural antisense transcripts are transcribed from the opposite strand of a protein-coding gene; and 3) intronic lncRNAs are transcribed from within introns of protein-coding genes (16). lncRNAs have emerged as key components of the address code, allowing protein complexes, genes, and chromosomes to be trafficked to appropriate locations, and subjected to proper activation and deactivation (17). lncRNA-based mechanisms control cell fates during development, and their dysregulation underlies some human disorders caused by chromosomal deletions (18). An increasing number of studies have revealed that lncRNAs have a variety of important functions, including their role in transcription, splicing, translation, nuclear factor trafficking, imprinting, genome rearrangement, and chromatin modification (19–21). Aberrant lncRNA expression is associated with human diseases such as cancer, Alzheimer’s disease, and cardiovascular disease (22–24). Thus, a better understanding of the roles of lncRNA will advance our understanding of cell regulatory and disease mechanisms. In the present study, we demonstrated that oxidized LDL (Ox-LDL) significantly induced lincRNA-DYNLRB2-2 expression, which upregulated ABCA1 expression through
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Fig. 2. Effect of lincRNA-DYNLRB2-2 and GPR119 on lipid absorption, lipid content and lipid efflux. A–D: THP-1 macrophages were treated as indicated. A: GPR119 protein levels were measured by Western blotting. B: Cells were incubated with 5 g/ml of DiI-labeled Ox3 LDL for 1 h and uptake of DiI-labeled Ox-LDL was analyzed by flow cytometry. C, D: The cells were labeled with 0.2 Ci/ml [ H]cholesterol 3 or 2 Ci/ml of [ H]choline chloride for 72 h and then apoA-I-mediated cellular cholesterol was analyzed using liquid scintillation counting assays. apoA-I-mediated phospholipid efflux was calculated by subtracting the efflux to the medium and expressed as the percentage of total cellular and medium phospholipid amounts. All the results are expressed as mean ± SD from three independent experiments, each performed in triplicate. *P < 0.05 versus control group.
lncRNA microarray analysis Briefly, THP-1 macrophages (three samples) and THP-1 macrophage-derived foam cells (three samples) were used to isolate total RNA. Total RNA from each sample was quantified using the
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NanoDrop ND-1000 spectrophotometer and RNA integrity was assessed using standard denaturing agarose gel electrophoresis. For microarray analysis, the Agilent Array platform was employed. The sample preparation and microarray hybridization
TABLE 1.
Effect of lincRNA-DYNLRB2-2 and GPR119 on cholesterol content in THP-1 macrophage
Control LV-Mock LV-DYNLRB2-2 pcDNA3.1-Mock pcDNA3.1-GPR119
TC (g/mg cell protein)
FC (g/mg cell protein)
CE (g/mg cell protein)
CE/TC (%)
115 ± 11 120 ± 9 95 ± 6a 116 ± 10 87 ± 6b
109 ± 9 113 ± 8 89 ± 5a 110 ± 7 82 ± 5b
6±3 7±3 6±2 6±3 5±2
5.2 5.8 6.3 5.2 5.7
THP-1 cells were differentiated for 72 h with 100 nM PMA and then THP-1 macrophage cells were divided into five groups as indicated. Cellular TC, FC, and CE were determined by HPLC. The results are expressed as mean ± SD from three independent experiments, each performed in triplicate. a P < 0.05 versus LV-Mock group. b P < 0.05 versus pcDNA3.1-Mock group.
RNA isolation and real-time quantitative PCR analysis Total RNA from cultured cells or mouse tissues was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA) in accordance with the manufacturer’s instructions. lincRNA-DYNLRB2-2 was detected with a commercial reagent (Promega, Madison, Wisconsin) according to the instruction manual in 20 l reaction volumes. Real-time PCR was performed on a real-time PCR ABI 7500 Fast system (Applied Biosystems, Foster City, CA). The expression of U6 RNA was used as an endogenous control. Primers used for lincRNA-DYNLRB2-2 were as follows: lincRNA-DYNLRB2-2-F, 5′-TACAGGCATGAGGCATCGT-3′; lincRNA-DYNLRB2-2-R, 5′AGAGGTTGGGATTCATGCTAGA-3′; and lincRNA-DYNLRB2-2-RT, 5′-GCAAGACTTGGAGTTGGTGAT-3′. Primers used for U6 were as follows: U6-F, 5′-CTCGCTTCG GCAGCACA-3′; U6-R, 5′-AACGCTTCACGAATTTGCGT-3′; and U6-RT, 5′-AACGCTTCACGAATTTGCGT-3′. The mRNA levels were evaluated by real-time quantitative PCR using an ABI 7500 Fast real-time PCR system with SYBR Green detection chemistry (TaKaRa Bio, Inc.). Glyceraldehyde 3-phosphate dehydrogenase expression was used as the internal control. Quantitative measurements were determined using the ⌬⌬Ct method. All samples were measured in triplicate and the mean value was considered for comparative analysis.
Western blot analyses Cytokine assays and measurement of serum biochemical parameters The levels of human TNF-␣, interleukin (IL)-1, and IL-6 present in the culture media (R&D Systems, Minneapolis, MN); the serum concentrations of IL-1, IL-6, and TNF-␣ (R&D Systems); the serum serum amyloid A (SAA) amount (IBL, Hamburg, Germany); and the levels of serum apoA-I and apoB100 (Cusabio Biotech Co., Ltd., Wuhan, China) were measured by ELISA according to the manufacturer’s instructions. The TG and total cholesterol (TC) concentrations were determined enzymatically using an automated analyzer.
TABLE 2.
Cells and tissues were harvested and protein extracts prepared according to established methods. Extracts were then separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and subjected to Western blot analysis using rabbit polyclonal anti-LXR␣, -SRA1, -CD36, -SREBP1c, -SREBP2, and -GPR119 antibodies (Proteintech Group, Inc., Chicago, IL); rabbit polyclonal anti-NF-B, -PPAR␥, and -ABCA1 antibodies (Abcam Inc., Cambridge, MA); rabbit polyclonal anti-ABCG1, -SR-B1, -LDLR, -HMGCR, -HMGS, and -Niemann-Pick C1-Like 1 antibodies (Epitomics, Burlingame, CA); and rabbit polyclonal anti-ABCG5, -ABCG8, -MTP-PXR, and --actin antibodies (Santa Cruz Biotechnology, Inc.,
Effect of lincRNA-DYNLRB2-2 and GPR119 on cholesterol content in THP-1 macrophage-derived foam cells
Control LV-Mock LV-DYNLRB2-2 pcDNA3.1-Mock pcDNA3.1-GPR119
TC (g/mg cell protein)
FC (g/mg cell protein)
CE (g/mg cell protein)
CE/TC (%)
489 ± 15 495 ± 16 300 ± 13a 482 ± 13 306 ± 8b
190 ± 12 195 ± 13 128 ± 11a 186 ± 11 131 ± 10b
299 ± 15 300 ± 13 172 ± 14a 296 ± 14 175 ± 16b
61.1 60.7 57.5 61.5 57.1
THP-1 cells were differentiated for 72 h with 100 nM PMA and then macrophages were transformed into foam cells by incubation in the presence of 50 g/ml of Ox-LDL for 48 h. THP-1 macrophage-derived foam cells were divided into five groups as indicated. Cellular TC, FC, and CE were determined by HPLC. The results are expressed as mean ± SD from three independent experiments, each performed in triplicate. a P < 0.05 versus LV-Mock group. b P < 0.05 versus pcDNA3.1-Mock group.
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were performed using the manufacturer’s standard protocols with minor modifications. Briefly, mRNA was purified from 1 g of total RNA after removal of rRNA (mRNA-ONLY™ eukaryotic mRNA isolation kit; Epicentre Biotechnologies, Madison, WI). Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3′ bias utilizing a random priming method. The labeled cRNAs were hybridized onto the Human LncRNA Array v2.0 (8 × 60K; Arraystar, Inc., Rockville, MD). After the slides were washed, the arrays were scanned using the Agilent scanner G2505B. Agilent Feature Extraction software (version 10.7.3.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v11.5.1 software package (Agilent Technologies, Santa Clara, CA). After quantile normalization of the raw data, lncRNAs and mRNAs, in which at least three out of six samples had flags in present or marginal (“All Targets Value”), were chosen for further data analysis. Differentially expressed lncRNAs and mRNAs with statistical significance were identified through Volcano Plot filtering. All of the microarray data were uploaded to the NCBI Gene Expression Omnibus (GEO) with a GEO accession number (GSE54039).
TABLE 3.
Effect of lincRNA-DYNLRB2-2 and GPR119 on LPS-induced inflammatory cytokines in THP-1 macrophage cells TNF-␣ (pg/ml)
IL-1 (pg/ml)
IL-6 (pg/ml)
136 ± 29 861 ± 45a 121 ± 37 879 ± 56 392 ± 55b 115 ± 32 839 ± 46 352 ± 46c
42 ± 11 521 ± 35a 53 ± 15 551 ± 41 262 ± 28b 48 ± 9 542 ± 47 281 ± 33c
186 ± 31 506 ± 43a 178 ± 39 525 ± 39 312 ± 43b 173 ± 31 531 ± 42 295 ± 35c
Control LPS LV-DYNLRB2-2 LV-Mock + LPS LV-DYNLRB2-2 + LPS pcDNA3.1-GPR119 pcDNA3.1-Mock + LPS pcDNA3.1-GPR119 + LPS
THP-1 macrophage cells were divided into eight groups as indicated and cultured in medium at 37°C for 24 h. Secreted inflammatory cytokines were quantified by ELISA. LPS (lipopolysaccharide) indicates treatment with 10 ng/ml LPS. The results are expressed as the mean ± SD of three independent experiments, each performed in triplicate. a P < 0.05 versus control group. b P < 0.05 versus LV-Mock + LPS group. c P < 0.05 versus pcDNA3.1-Mock + LPS group.
Analysis was performed on a fluorescent activated cell sorting (FACScalibur) flow cytometer (Becton Dickinson, Franklin Lakes, NJ) with Cell Quest Pro software (BD Biosciences, San Jose, CA).
LV production and infection Human monocytic THP-1 cells were cultured. Packed empty LVs with green fluorescent protein (LV-Mock) and LV-mediated lincRNA-DYNLRB2-2 overexpression vector (LV-DYNLRB2-2) were generated. The cells were infected with the LV stock at a multiplicity of infection of 100 (THP-1 cells) transducing units per cell in the presence of 8 g/ml of polybrene. The cells were washed with fresh complete media after 24 h of incubation.
Plasmid construction and transfection The PCR-XL-TOPO vector containing GPR119, vector PIRES2EGFP, Platinum HIFI Taq polymerase, and Accuprime Pfx DNA polymerase were purchased from Invitrogen Biotechnology (Shanghai, China). The pCDNA3.1(+) vector, competent DH5␣ cells, XhoI and EcoRI restriction enzymes, and T4 DNA ligase were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). The fragment of EcoRI-GPR119-IRES-EGFP-XhoI was achieved from the PCR-XL-TOPO vector and PIRES2-EGFP vector by overlap extension PCR and was linked to pcDNA3.1 to create the recombinant plasmid pcDNA3.1-GPR119-IRES-EGFP. The inserted gene was identified by electrophoresis and sequencing. The recombinant plasmid was then transfected into the cultured cells by Lipofectamine 2000 (Invitrogen), and the over-expression of GPR119 was confirmed by RT-PCR and Western blotting.
Transfection with siRNA mimics THP-1 macrophages were transfected with 50 nM siRNAs against GPR119, ABCA1, and an irrelevant 21-nucleotide control siRNA (negative control) purchased from Ribo Biotechnology. Cells (2 × 106 cells/well) were transfected using Lipofectamine 2000 transfection reagent for 48 h according to the manufacturer’s instructions. All experimental control samples were treated with an equal concentration of a nontargeting control mimic sequence (negative controls). After 48 h of transfection, real-time RT-PCR and Western blotting were performed.
Cellular cholesterol efflux experiments THP-1 macrophages were cultured and labeled with 0.2 Ci/ml [3H]cholesterol. After 72 h, cells were subsequently washed with PBS and incubated overnight in RPMI 1640 medium containing 0.1% (w/v) BSA to allow equilibration of [3H]cholesterol in all cellular pools. Equilibrated [3H]cholesterol-labeled cells were washed with PBS and incubated in 2 ml of efflux medium containing RPMI 1640 medium and 0.1% BSA with 25 g/ml human plasma apoA-I for 12 h. A 150 l sample of efflux medium was obtained at the times designated and passed through a 0.45 m filter to remove any floating cells. Monolayers were washed twice in PBS, and cellular lipids were extracted with isopropanol. Medium and cell-associated [3H]cholesterol was then measured by liquid scintillation counting. Percent efflux was calculated by the following equation: [total media counts/(total cellular counts + total media counts)] × 100%.
Phospholipid efflux experiments Cells were cultured as indicated above, and then incubated with 2 Ci/ml of [3H]choline chloride to label the phospholipids. After 72 h, cells were subsequently washed with PBS and incubated overnight in RPMI 1640 medium containing 0.1% (w/v) BSA. After 6 h of incubation in medium containing 10 mg/ml apoA-I, efflux medium was collected, centrifuged to remove cell debris as above, and aliquots were taken for extraction and separated by thin-layer chromatography with the use of silica G plates developed in chloroform/methanol/ammonia (25%, w/v)/water (50:65:5:4, v/v/v/v). Phospholipid spots were visualized by I2 vapors and identified by comigration with standards. Relative radioactivity was measured by Phosphoscreen and quantified by PhosphorImager (Molecular Dynamics Inc.). Phospholipid efflux was expressed as percent counts in the supernatant versus the total counts for each individual lipid.
En face plaque area Dil-Ox-LDL uptake assays PMA-differentiated THP-1 cells were treated with LV lincRNADYNLRB2-2 (LV-DYNLRB2-2) or plasmid pcDNA3.1-GPR119 and its own negative control as indicated, then fluorescent-tagged Dil-Ox-LDL was added, and the cells were incubated for 1 h. Adherent cells were harvested, and washed three times with PBS.
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Immediately after the mice were euthanized, the aortas were excised and fixed in 10% buffered formalin for quantification of the en face plaque areas. Briefly, after the adventitial tissue was carefully removed, the aorta was opened longitudinally, stained with Oil Red O (Sigma), and pinned on a blue wax surface. En face images were obtained by a stereomicroscope
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Santa Cruz, CA). The proteins were visualized using a chemiluminescence method (ECL Plus Western blot detection system; Amersham Biosciences, Foster City, CA).
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Fig. 3. GPR119 is involved in lincRNA-DYNLRB2-2-induced upregulation of ABCA1 expression and function. A–I: THP-1 macrophages were treated as indicated. A, C: The expression of ABCA1 was detected by real-time quantitative PCR. B, D–F, I: Protein expression was detected by Western blotting. E, F: THP-1 macrophages were treated as indicated and protein expression was detected by Western blotting. G, H: The cells 3 3 were labeled with 0.2 Ci/ml [ H]cholesterol or 2 Ci/ml of [ H]choline chloride for 72 h and then apoAI-mediated cellular cholesterol efflux and phospholipid efflux were analyzed using liquid scintillation counting assays. All the results are expressed as mean ± SD from three independent experiments, each performed in triplicate. *P < 0.05 versus control group.
(SZX12; Olympus, Tokyo, Japan) equipped with a digital camera (Dxm1200, Nikon, Tokyo, Japan) and analyzed using Adobe Photoshop version 7.0 and Scion Image software. The percentage of the luminal surface area stained by Oil Red O was determined.
beginning at the aortic root, were collected for a distance of 400 m. Sections were stained with Oil Red O. The area of positive staining for Oil Red O was calculated as a percentage of the total section area and an average lipid droplet size was calculated utilizing ImagePro Plus software (Media Cybernetics) from five views per animal.
Quantification of atherosclerosis in the aortic sinus The upper portion of the heart and proximal aorta were obtained, embedded in OCT compound (Fisher, Tustin, CA), and stored at ⫺70°C. Serial 10 m thick cryosections of the aorta,
Liver histology and Oil Red O staining To examine hepatic lipid deposition, lipid was assessed in samples collected in OCT compound by Oil Red O staining. Briefly,
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liver cryosections were fixed for 10 min in 60% isopropanol and stained with 0.3% Oil Red O in 60% isopropanol for 30 min and subsequently washed with 60% isopropanol. Sections were counterstained with Gill’s hematoxylin, washed with acetic acid solution (4%), and mounted with aqueous solution. The area of positive staining for Oil Red O was calculated as a percentage of the total section area and an average lipid droplet size was calculated TABLE 4.
utilizing ImagePro Plus software (Media Cybernetics) from five views per animal.
Immunohistochemistry For this procedure, each frozen liver tissue or aortic root sample was sectioned to 5 M thickness and fixed in microscope slides. Sections of frozen tissue specimens were mounted
ABCA1 is involved in the regulation of LPS-induced inflammatory cytokines by GPR119 in THP-1 macrophage cells
Control pcDNA3.1-GPR119 + NC pcDNA3.1-GPR119 + ABCA1-siRNA pcDNA3.1-Mock + NC + LPS pcDNA3.1-GPR119 + NC + LPS pcDNA3.1-GPR119 + ABCA1-siRNA + LPS
TNF-␣ (pg/ml)
IL-1 (pg/ml)
IL-6 (pg/ml)
131 ± 27 121 ± 22 145 ± 39 796 ± 53 a 387 ± 46 731 ± 55
39 ± 12 30 ± 13 46 ± 15 529 ± 42 296 ± 42a 563 ± 57
197 ± 35 181 ± 43 191 ± 47 506 ± 35 268 ± 52a 491 ± 42
THP-1 macrophage cells were divided into six groups as indicated and cultured in medium at 37°C for 24 h. Secreted inflammatory cytokines were quantified by ELISA. LPS indicates treatment with 10 ng/ml LPS. The results are expressed as the mean ± SD of three independent experiments, each performed in triplicate. a P < 0.05 versus pcDNA3.1-Mock + NC + LPS group.
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Continued.
Fig. 3.
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Fig. 4. GLP-1R is involved in GPR119-induced upregulation of ABCA1. A–C: THP-1 macrophages were treated as indicated and Western blot assays were performed to detect protein expression. D, E: THP-1 macrophages were treated with or without inhibitors of CaMK (KN93, 5 M), ERK1/2 (PD98059, 50 M), PKA (H89, 10 M), PKB (LY294002, 10 M), and inhibitor of PKC- (rottlerin, 10 M) for 30 min, then transfected with pc-DNA3.1-GPR119 or pc-DNA3.1-Mock for 48 h. ABCA1 mRNA and protein levels were measured by real-time quantitative PCR and Western blotting, respectively. All results are expressed as mean ± SD of three independent experiments, each performed in triplicate. *P < 0.05 versus control group.
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TABLE 5.
Effect of GPR119 on serum lipids and lipoprotein values in apoE⫺/⫺ mice (n = 10) TG (mmol/L)
TC (mmol/l)
apoA-I (g/l)
apoB (g/l)
1.35 ± 0.17 a 0.97 ± 0.15
32.04 ± 2.65 32.32 ± 3.13
0.05 ± 0.01 0.07 ± 0.02a
0.16 ± 0.02 0.15 ± 0.03
LV-Mock LV-GPR119
Data are expressed as mean ± SD. a P < 0.05 versus LV-Mock group.
on Poly-Lysine (Sigma)-coated slides, air dried, and fixed with acetone. Immunohistochemical staining was performed for CD68 using rabbit polyclonal antibody to CD68 at a dilution of 1:100 (Abcam, Cambridge, MA). Images were acquired and quantitated on an Olympus BX50 microscope using Optimis software (version 6.2) and digitized using a color video camera (threecharge coupled device; JVC, Wayne, NJ).
Statistical analysis
RESULTS Ox-LDL induces GPR119 expression by upregulating lincRNA-DYNLRB2-2 in THP-1 macrophages Monocyte recruitment to the vessel wall is a rate-limiting step in atherogenesis. Monocytes are recruited from the circulation into the subendothelial space, where they differentiate into mature macrophages and internalize modified lipoproteins to differentiate into lipid-laden foam cells (25). Studies have shown that lncRNAs play an essential role in cardiovascular diseases (24). To explore possible changes in RNA expression during macrophage formation, we performed microarray analysis of THP-1 macrophages and THP-1 macrophage-derived foam cells in the presence of Ox-LDL using the Arraystar probe dataset, which included 24,748 lncRNAs and 24,420 coding transcripts. lncRNA and mRNA expression profiles from three pairs of THP-1 macrophages and THP-1 macrophagederived foam cells were produced using the Arraystar Human LncRNA Array v2.0 platform. Expression values were normalized based on the mean expression value for each probe set. Differently expressed probe sets were identified based on Student’s t-test for paired samples’ normalized expression values using the following cutoff: absolute fold change value larger than 3 and P value less than 0.01. The results showed that lincRNA-DYNLRB2-2 (BM973870; 16q23.3, chr16:79,831,946-79,861,047) expression was upregulated in THP-1 macrophage-derived foam cells (4.688-fold, P < 0.001), suggesting that Ox-LDL and TABLE 6.
LV-Mock LV-GPR119
⫺/⫺
Effect of GPR119 on serum cytokine levels in apoE
mice (n = 10)
IL-1 (pg/ml)
IL-6 (pg/ml)
TNF-␣ (pg/ml)
SAA (g/ml)
12.37 ± 1.18 a 8.41 ± 1.25
78.67 ± 5.82 56.65 ± 4.27a
11.35 ± 1.56 7.15 ± 1.28a
30.35 ± 3.23 18.26 ± 2.50a
Data are expressed as mean ± SD. P < 0.05 versus LV-Mock group.
a
690
The effect of lincRNA-DYNLRB2-2 and GPR119 on cholesterol metabolism and inflammation Atherosclerosis can be considered as both a lipid metabolism disorder and a chronic inflammatory disease. Innate immunity pathways have long been suspected to contribute to the initiation and progression of atherosclerosis. This suggests that crosstalk between lipid metabolism and innate immunity pathways play an important role in the development and/or prevention of atherosclerosis (26). To determine whether lincRNA-DYNLRB2-2 and GPR119 affect cholesterol metabolism and inflammation, we first examined the effect of lincRNA-DYNLRB2-2 and GPR119 on cholesterol metabolism in THP-1 macrophagederived foam cells. LV-DYNLRB2-2 and plasmid vector pcDNA3.1-GPR119 were created and transfected into THP-1 macrophages (Fig. 1C, Fig. 2A). As shown in Fig. 2B, Dil-Ox-LDL uptake significantly decreased in GPR119 overexpressing or lincRNA-DYNLRB2-2 overexpressing THP-1 macrophages. Moreover, LV-DYNLRB2-2 and GPR119 significantly decreased TG, free cholesterol (FC), and cholesteryl ester (CE) levels (Tables 1, 2). The defect in
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Data are expressed as mean ± SD. Results were analyzed by one-way ANOVA followed by the Student-Newman-Keuls test and the Student’s t-test using SPSS v13.0 statistical software (SPSS, Inc., Chicago, IL). A two-tailed P value 0.05).
aortic valve section and en face analyses. In mice infected with LV-GPR119, the average lesion area was decreased compared with that in controls by both en face and aortic valve section analyses (Fig. 6A). Quantification of Oil Red O-stained lesions in en face preparations of aortas revealed that treatment with LV-GPR119 resulted in a significant 30% decrease in lesion area in apoE⫺/⫺ mice compared with the controls. To confirm the positive effects of GPR119 on atherosclerosis, Oil Red O-stained aortic valve sections were quantified, which showed a significant 27% reduction in lesion area in apoE⫺/⫺ mice treated with LV-GPR119 compared with controls. Immunohistochemical staining was performed to visualize macrophages in atherosclerotic lesions (using an antibody against CD68). As shown in Fig. 6B, LV-GPR119-treated mice showed a considerably lower number of CD68+ cells than control mice, indicating that macrophage infiltration was significantly decreased in lesions of apoE⫺/⫺ mice (P < 0.05). These observations indicated that the positive effect of GPR119 is ongoing in atherosclerotic lesions of apoE⫺/⫺ mice. Western blot assessment of the levels of proteins related to cholesterol metabolism and inflammation in aortic tissue of apoE⫺/⫺ mice showed that the levels of SR-A1, CD36, and NF-B were decreased, whereas protein levels of ABCA1 and SR-B1 were increased in the GPR119 group (Fig. 6C). However, no effect was observed on ABCG1 protein expression in the GPR119 group compared with the control group (P > 0.05). In addition, assessment of the levels of nuclear receptor proteins related to cholesterol metabolism and inflammation, including LXR␣, PPAR␣, PPAR␥, PXR, HNF-1␣, and LRH1, showed no significant differences between the GPR119 and control groups (P > 0.05, data not shown).
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Fig. 6. Effect of GPR119 on atherosclerosis initiation and development in apoE⫺/⫺ mice. A–C: apoE⫺/⫺ mice were randomized into a control group and a GPR119 group and fed a HFD. A: Representative staining of en face aorta (a) with Oil Red O and aortic valves (b) with ⫺/⫺ mice. B: Cryosections of aortic valves from Oil Red O. En face lesions (c) and total lesions in the aortic valves (d) were analyzed in apoE ⫺/⫺ mice were immunohistochemically stained for the macrophage marker CD68 (a). The integral optical density of CD68 in the apoE
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the mobilized cholesterol (34). The control of macrophage cholesterol homeostasis is of critical importance in the pathogenesis of atherosclerosis, as alterations in the balance between cholesterol influx, intracellular transport and efflux can lead to excessive accumulation of cholesterol in macrophages and their transformation into foam cells (35). In the present study, GPR119 exerted an antiatherogenic effect by decreasing cellular cholesterol content and upregulating the expression of ABCA1, which plays a crucial role in mediating the transport of cholesterol across cellular membranes (36–38). Its regulation is cAMP- and sterol-dependent, and cAMP is responsible for the functional activation of ABCA1 or the upregulation of ABCA1 expression (39, 40). In the present study, we showed that GPR119 activates GLP-1R and its downstream pathways (CaMK and cAMP/PKA) and PI-3K/AKT/PKB pathways, and it inhibits the MAPK-ERK1/2 and PKC- pathways, finally resulting in the upregulation of ABCA1 expression and apoA-I mediated cholesterol efflux. These results provide strong evidence supporting the notion that GPR119 exerts its anti-atherogenic effects by increasing the rate of RCT. Furthermore, GPR119 enhanced the overall rate of RCT by activating pathways that mediate the delivery of HDL-cholesterol to the liver. Mature HDL can transfer its cholesterol to the liver directly via SR-B1 or indirectly via CETP-mediated transfer to apoB-containing lipoproteins, with subsequent uptake by the liver via the
LDLR (41). We showed that GPR119 upregulated the ⫺/⫺ mice and induced the exexpression of SR-B1 in apoE pression of LDL receptors in the liver, increasing the catabolism of plasma LDL and decreasing the plasma concentration of cholesterol. In addition, apolipoproteins are important constituents of plasma lipoproteins and essential for RCT. We performed in vivo experiments and showed that GPR119 can significantly increase apoA-I levels in apoE⫺/⫺ mice. apoA-I is secreted predominantly by the liver and is present in the majority of HDL particles, and its concentration is closely correlated with plasma HDL (42)· Atherosclerosis is a chronic disease that can remain asymptomatic for decades. Inflammatory processes take part in all stages of the atherosclerotic process, from lesion initiation to plaque rupture (43). Macrophages, whether engorged with lipids or not, play a key role in the mediation and modulation of inflammation, and much atherosclerosis research has targeted the role of macrophages in the inflammatory pathways that underlie atherogenesis (44, 45). Evidence from several recent studies indicate that inflammation, along with other atherogenic-related mediators, plays a distinct regulating role in ABCA1 expression. Proatherogenic cytokines such as IFN-␥ and IL-1 inhibit the expression of ABCA1, while anti-atherogenic cytokines, including IL-10 and transforming growth factor TGF1, promote the expression of ABCA1. Moreover, some
aortic valve cryosections from apoE⫺/⫺ mice was analyzed (b). C: Protein expression levels in tissues of apoE⫺/⫺ mice were analyzed by Western blotting (n = 5 mice/group). All data are presented as mean ± SD (n = 10). All experiments were performed in triplicate, except as indicated. *P < 0.05 versus control group.
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Fig. 7. ABCA1 expression and function can be induced by GPR119 to protect from atherosclerosis. OxLDL upregulates lincRNA-DYNLRB2-2 expression, resulting in increased ABCA1 expression through GPR119. GLP-1R-mediated signaling pathways including CaMK, cAMP/PKA, PI-3K/AKT/PKB, MAPKERK1/2, and PKC- are involved in GPR119-induced ABCA1 expression. Moreover, GPR119 inhibits inflammation and promotes apoA-I-mediated cellular cholesterol efflux through inducing ABCA1 expression. These beneficial effects are accompanied by a reduction of macrophage-derived foam cell formation, inhibition of inflammatory gene expression, adhesion molecule expression in the aorta, and increase of cholesterol efflux from peripheral tissues, thus preventing of atherosclerotic plaque formation.
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and inflammatory cytokine levels in serum to further pre⫺/⫺ mice, indicating that vent atherosclerosis in apoE GPR119 may represent a promising candidate as a therapeutic agent.
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cytokines such as TNF-␣ regulate ABCA1 expression in a species-specific and dose-dependent manner (46). Chronic activation of inflammatory pathways mediates the pathogenesis of insulin resistance, and the macrophage/adipocyte nexus provides a key mechanism underlying decreased insulin sensitivity. Recently, a family of G protein-coupled receptors has been identified that exhibits high affinity for inflammation, glucose metabolism, and insulin sensitivity (47). In the present study, we showed that GPR119 significantly downregulated IL-1, IL-6, and TNF-␣ levels through the regulation of ABCA1 expression in LPSinduced THP-1 macrophage-derived foam cells. To further investigate the mechanisms whereby GPR119 treatment inhibited plaque progression and stabilization, changes in gene expression of inflammatory molecules ⫺/⫺ mice fed a high-fat/high-chowere explored in apoE lesterol diet. We found that IL-1, IL-6, TNF-␣, and SAA were markedly repressed in GPR119-overexpressing ⫺/⫺ mice. Our results suggest that GPR119-induced apoE suppression of inflammatory molecule expression could block or retard the development of atherosclerotic lesions and thus have a positive influence on disease outcomes. The diverse roles of regulatory RNAs extend well beyond the epigenome, as they regulate transcription in different ways (48). lncRNAs are nonprotein coding transcribed RNAs that target the minor dihydrofolate reductase gene promoter, which binds to the general transcription factor IIB and to the DNA of the dihydrofolate reductase major promoter forming a specific and stable tripartite complex, resulting in the dissociation of the preinitiation complex and transcriptional repression (49). The lncRNAs also function as transcriptional coactivators, modulating protein localization and function. Ox-LDL contributes to atherosclerotic plaque formation and progression by several mechanisms, including the induction of endothelial cell activation and dysfunction, macrophage foam cell formation, and smooth muscle cell migration and proliferation. Ox-LDL upregulates ABCA1 expression at both protein and mRNA levels (31). In the present study, we showed that Ox-LDL induces GPR119 expression through lincRNA-DYNLRB2-2 in THP-1 macrophages. In response to extracellular signals, lincRNA-DYNLRB2-2 upregulated GPR119 expression, which increased ABCA1 expression resulting in promotion of ABCA1-mediated cholesterol efflux leading to the protection from atherosclerosis. However, the detailed mechanism of lincRNADYNLRB2-2-induced upregulation of GPR119 expression needs further clarification. Moreover, in the present study, we showed that Ox-LDL and Ac-LDL had similar effects on lincRNA-DYNLRB2-2 levels. Remarkably, little is known about the direct downstream target lncRNAs of Ox-LDL and Ac-LDL. Thus, whether lincRNA-DYNLRB2-2 can be directly regulated by Ox-LDL or Ac-LDL and whether another mechanism is involved needs to be further explored. In conclusion, we have demonstrated that Ox-LDL significantly induced lincRNA-DYNLRB2-2 expression (Fig. 7), which promoted ABCA1-mediated cholesterol efflux and inhibited inflammation through GPR119 in THP-1 macrophagederived foam cells. In addition, GPR119 decreased lipid
34. Tall AR. An overview of reverse cholesterol transport. Eur Heart J. 1998; 19 Suppl A:A31–5. 35. Tabas, I. 2000. Cholesterol and phospholipid metabolism in macrophages. Biochim. Biophys. Acta. 1529: 164–174. 36. Rader, D. J., E. T. Alexander, G. L. Weibel, J. Billheimer, and G. H. Rothblat. 2009. The role of reverse cholesterol transport in animals and humans and relationship to atherosclerosis. J. Lipid Res. 50(Suppl): S189–S194. 37. Ohashi, R., H. Mu, X. Wang, Q. Yao, and C. Chen. 2005. Reverse cholesterol transport and cholesterol efflux in atherosclerosis. QJM. 98: 845–856. 38. Hu, Y. W., L. Zheng, and Q. Wang. 2010. Regulation of cholesterol homeostasis by liver X receptors. Clin. Chim. Acta. 411: 617–625. 39. Haidar, B., M. Denis, L. Krimbou, M. Marcil, and J. Genest, Jr. 2002. cAMP induces ABCA1 phosphorylation activity and promotes cholesterol efflux from fibroblasts. J. Lipid Res. 43: 2087–2094. 40. Lin, G., and K. E. Bornfeldt. 2002. Cyclic AMP-specific phosphodiesterase 4 inhibitors promote ABCA1 expression and cholesterol efflux. Biochem. Biophys. Res. Commun. 290: 663–669. 41. Rigotti, A., H. E. Miettinen, and M. Krieger. 2003. The role of the high-density lipoprotein receptor SR-BI in the lipid metabolism of endocrine and other tissues. Endocr. Rev. 24: 357–387. 42. Rubin, E. M., R. M. Krauss, E. A. Spangler, J. G. Verstuyft, and S. M. Clift. 1991. Inhibition of early atherogenesis in transgenic mice by human apolipoprotein AI. Nature. 353: 265–267. 43. Navab, K. D., O. Elboudwarej, M. Gharif, J. Yu, S. Y. Hama, S. Safarpour, G. P. Hough, L. Vakili, S. T. Reddy, M. Navab, et al. 2011. Chronic inflammatory disorders and accelerated atherosclerosis: chronic kidney disease. Curr. Pharm. Des. 17: 17–20. 44. Libby, P. 2012. Inflammation in atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 32: 2045–2051. 45. Mantovani, A., C. Garlanda, and M. Locati. 2009. Macrophage diversity and polarization in atherosclerosis: a question of balance. Arterioscler. Thromb. Vasc. Biol. 29: 1419–1423. 46. Hu, Y. W., X. Ma, J. L. Huang, X. R. Mao, J. Y. Yang, J. Y. Zhao, S. F. Li, Y. R. Qiu, J. Yang, L. Zheng, et al. 2013. Dihydrocapsaicin attenuates plaque formation through a PPAR␥/LXR␣ pathway in apoE-/- mice fed a high-fat/high-cholesterol diet. PLoS ONE. 8: e66876. 47. Oh, D. Y., and W. S. Lagakos. 2011. The role of G-protein-coupled receptors in mediating the effect of fatty acids on inflammation and insulin sensitivity. Curr. Opin. Clin. Nutr. Metab. Care. 14: 322–327. 48. Goodrich, J. A., and J. F. Kugel. 2006. Non-coding-RNA regulators of RNA polymerase II transcription. Nat. Rev. Mol. Cell Biol. 7: 612–616. 49. Schonrock, N., R. P. Harvey, and J. S. Mattick. 2012. Long noncoding RNAs in cardiac development and pathophysiology. Circ. Res. 111: 1349–1362.
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This study was supported by the National Natural Sciences Foundation of China (81271905 and 81301489), Guangdong Provincial Natural Sciences Foundation of China (S2012020010920), and the President Foundation of Nanfang Hospital, Southern Medical University (2013B004). The authors declare no conflicts of interest. Manuscript received 6 October 2013 and in revised form 30 January 2014. Published, JLR Papers in Press, January 31, 2014 DOI 10.1194/jlr.M044669
Supplementary key words long intervening noncoding ribonucleic acid-DYNLRB2-2 • G protein-coupled receptor 119 • glucagon-like peptide 1 receptor • ATP binding cassette transporter A1 • atherosclerosis
Atherosclerosis is a complex disease, involving many cell types and circulating mediators and resulting in an inflammatory state. Atherosclerotic lesions form de novo from focal accumulation of lipoproteins, monocyte-derived macrophages, and lymphocytes within the arterial wall (1, 2). However, cholesterol and other lipids can be mobilized and excreted to prevent atherosclerosis in a process termed reverse cholesterol transport (RCT), in which ABCA1 plays a key role. ABCA1 is a member of a family of highly conserved transmembrane transport proteins. It plays a crucial role in the efflux of cellular cholesterol to HDL and its apolipoproteins (3). In humans, ABCA1 mutations can cause a severe HDL-deficiency syndrome, leading to the development of sterol deposits in tissue macrophages and premature atherosclerosis (4). Because of its role in macrophage lipid transport, ABCA1 is an important target for the prevention and treatment of atherosclerosis. G protein-coupled receptor 119 (GPR119), which has been described as a deorphanized G protein-coupled receptor, plays a significant role in mediating systemic metabolic homeostasis. It has recently attracted attention because evidence from in vitro systems and animal models suggests that its modulation has a favorable effect on glucose homoeostasis (5, 6). Because GPR119 promotes glucose-stimulated insulin
Abbreviations: Ac-LDL, acetylated LDL; CaMK, Ca2+/calmodulindependent protein kinase; CE, cholesteryl ester; FC, free cholesterol; GLP-1R, glucagon-like peptide 1 receptor; GPR119, G protein-coupled receptor 119; HFD, high-fat diet; IL, interleukin; lincRNA, long intervening noncoding RNA; lncRNA, long noncoding RNA; LPS, lipopolysaccharide; LV, lentivirus; Ox-LDL, oxidized LDL; PI-3K, phosphoinositide 3-kinase; PK, protein kinase; RCT, reverse cholesterol transport; SAA, serum amyloid A; TC, total cholesterol. 1 Y-W. Hu, J-Y. Yang, and X. Ma contributed equally to this work. 2 To whom correspondence should be addressed. e-mail: [email protected] (Q.W.); [email protected] (L.Z.)
Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at http://www.jlr.org
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Abstract Accumulated evidence shows that G protein-coupled receptor 119 (GPR119) plays a key role in glucose and lipid metabolism. Here, we explored the effect of GPR119 on cholesterol metabolism and inflammation in THP-1 macⴚ/ⴚ rophages and atherosclerotic plaque progression in apoE mice. We found that oxidized LDL (Ox-LDL) significantly induced long intervening noncoding RNA (lincRNA)-DYNLRB2-2 expression, resulting in the upregulation of GPR119 and ABCA1 expression through the glucagon-like peptide 1 receptor signaling pathway. GPR119 significantly decreased cellular cholesterol content and increased apoA-I-mediated cholesterol efflux in THP-1 macrophage-derived foam cells. ⴚ/ⴚ mice were randomly divided into two In vivo, apoE groups and infected with lentivirus (LV)-Mock or LVGPR119 for 8 weeks. GPR119-treated mice showed decreased liver lipid content and plasma TG, interleukin (IL)-1, IL-6, and TNF-␣ levels, whereas plasma levels of apoA-I were significantly increased. Consistent with this, atherosclerotic lesion development was significantly inhibⴚ/ⴚ mice with LV-GPR119. Our ited by infection of apoE findings clearly indicate that, Ox-LDL significantly induced lincRNA-DYNLRB2-2 expression, which promoted ABCA1-mediated cholesterol efflux and inhibited inflammation through GPR119 in THP-1 macrophage-derived foam cells. Moreover, GPR119 decreased lipid and serum inflammatory cytokine levels, decreasing atheroscleⴚ/ⴚ mice. These suggest that GPR119 may rosis in apoE be a promising candidate as a therapeutic agent.—Hu, Y-W., J-Y. Yang, X. Ma, Z-P. Chen, Y-R. Hu, J-Y. Zhao, S-F. Li, Y-R. Qiu, J-B. Lu, Y-C. Wang, J-J. Gao, Y-H. Sha, L. Zheng, and Q. Wang. A lincRNA-DYNLRB2-2/GPR119/ GLP-1R/ABCA1-dependent signal transduction pathway is essential for the regulation of cholesterol homeostasis. J. Lipid Res. 2014. 55: 681–697.
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GPR119. In addition, lincRNA-DYNLRB2-2 and GPR119 promoted ABCA1-mediated cholesterol efflux from THP-1 macrophage-derived foam cells, and inhibited inflammatory responses in these cells. Overexpression of GPR119 markedly decreased lipid levels and serum inflammatory cytokine levels to prevent atherosclerosis in apoE⫺/⫺ mice.
MATERIALS AND METHODS Materials Human lipoproteins [acetylated (Ac)-LDL, Ox-LDL, and HDL] were obtained from Biomedical Technologies Inc. (Stoughton, MA). The PrimeScript RT reagent kit (Perfect Real Time) (DRR037A; TaKaRa Bio, Inc., Shiga, Japan) and SYBR® Premix Ex TaqTM II (Tli RNaseH Plus) (DRR820A; TaKaRa Bio, Inc.) were obtained as indicated. Chemicals were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise noted.
Animals and diets The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication number 85-23, revised 1996) and was approved by the Animal Experimental Committee at Nanfang Hospital. Male apoE⫺/⫺ mice with a C57BL/6 background were purchased from the Laboratory Animal Center of Peking University (Beijing, China) and housed five per cage at 25°C on a 12 h light/dark cycle. To detect the effect of GPR119 on lipid metabolism, 6-week-old apoE⫺/⫺ mice were randomized into two groups and injected via the tail vein with control lentivirus [lentivirus (LV)-Mock, n = 15] or LV encoding mouse GPR119 (LV-GPR119, n = 15), respectively. The mice were fed a high-fat diet (HFD) for a period of 8 weeks. The diet was a commercially prepared mouse food supplemented with 21% (w/w) butterfat, 0.15% (w/w) cholesterol, and 19.5% (w/w) casein (Beijing Keao Xieli Feed Co., LTD, Beijing, China). At week 8, mice were anesthetized and 1 ml of blood was collected by cardiac puncture before the mice were euthanized by cervical dislocation and tissues were collected for further analysis.
Cell culture Human monocytic THP-1 cells were obtained from ATCC (Manassas, VA). THP-1 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% FCS and differentiated for 72 h with 100 nM PMA. Macrophages were transformed into foam cells by incubation in the presence or absence of 50 g/ml of Ox-LDL in serum-free RPMI 1640 medium containing 0.3% BSA for 48 h. Cells were incubated at 37°C in an atmosphere of 5% CO2. Cells were seeded in 6- or 12-well plates or 60 mm dishes and grown to 60–80% confluence before use.
Fig. 1. Dose-dependent and time-dependent effects of Ox-LDL/Ac-LDL on lincRNA-DYNLRB2-2 and GPR119 expression in THP-1 macrophages. A, B: THP-1 macrophages were treated with Ox-LDL/Ac-LDL at 0, 25, 50, and 100 g/ml for 24 h, and THP-1 macrophages were treated with 50 g/ml Ox-LDL/Ac-LDL for 0, 12, 24, and 48 h. lincRNA-DYNLRB2-2 levels were detected by real-time quantitative PCR. C–E: THP-1 macrophages were infected with LV-Mock or LV-DYNLRB2-2. lincRNA-DYNLRB2-2 levels and GPR119 gene levels were measured by real-time quantitative PCR and GPR119 protein expression was measured by Western blotting. F–I: THP-1 macrophages were treated with Ox-LDL/Ac-LDL at 0, 25, 50, and 100 g/ml for 24 h, and THP-1 macrophages were treated with 50 g/ml Ox-LDL/Ac-LDL for 0, 12, 24, and 48 h. F, G: GPR119 gene levels were measured by real-time quantitative PCR. H, I: GPR119 protein levels were measured by Western blotting. Similar results were obtained in three independent experiments. All the results are expressed as mean ± SD of three independent experiments, each performed in triplicate. *P < 0.05 versus vehicle.
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secretion, pancreatic -cell function, and glucagon-like peptide 1 (GLP-1) release, its therapeutic potential for the treatment of type 2 diabetes mellitus is getting more attention (7, 8). Moreover, GPR119 coupling to G␣ stimulatory proteins induces adenylate cyclase activity, resulting in an increase in intracellular cAMP, which regulates the GPR119-mediated release of GLP-1 (9). In addition, the GPR119-mediated release of GLP-1 from intestinal L-cells is protein kinase (PK)A dependent (10). Furthermore, cAMP, as a common second messenger that can activate MAPKs via PKA (11), can stimulate ABCA1 expression with species specific (12), alleviating the accumulation of cholesterol in macrophages by promoting ABCA1-mediated cellular cholesterol efflux (13, 14). Moreover, GPR119 can be activated by oleoylethanolamide and several other endogenous lipids containing oleic acid (15). These data suggest that GPR119 not only improves systemic glucose homeostasis but also affects lipid metabolism homeostasis. Long noncoding RNAs (lncRNAs) are nonprotein coding transcribed RNA molecules consisting of more than 200 nucleotides and fall into one of three categories including: 1) long intervening noncoding RNAs (lincRNAs) are transcribed from regions far away from protein-coding genes; 2) natural antisense transcripts are transcribed from the opposite strand of a protein-coding gene; and 3) intronic lncRNAs are transcribed from within introns of protein-coding genes (16). lncRNAs have emerged as key components of the address code, allowing protein complexes, genes, and chromosomes to be trafficked to appropriate locations, and subjected to proper activation and deactivation (17). lncRNA-based mechanisms control cell fates during development, and their dysregulation underlies some human disorders caused by chromosomal deletions (18). An increasing number of studies have revealed that lncRNAs have a variety of important functions, including their role in transcription, splicing, translation, nuclear factor trafficking, imprinting, genome rearrangement, and chromatin modification (19–21). Aberrant lncRNA expression is associated with human diseases such as cancer, Alzheimer’s disease, and cardiovascular disease (22–24). Thus, a better understanding of the roles of lncRNA will advance our understanding of cell regulatory and disease mechanisms. In the present study, we demonstrated that oxidized LDL (Ox-LDL) significantly induced lincRNA-DYNLRB2-2 expression, which upregulated ABCA1 expression through
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Fig. 2. Effect of lincRNA-DYNLRB2-2 and GPR119 on lipid absorption, lipid content and lipid efflux. A–D: THP-1 macrophages were treated as indicated. A: GPR119 protein levels were measured by Western blotting. B: Cells were incubated with 5 g/ml of DiI-labeled Ox3 LDL for 1 h and uptake of DiI-labeled Ox-LDL was analyzed by flow cytometry. C, D: The cells were labeled with 0.2 Ci/ml [ H]cholesterol 3 or 2 Ci/ml of [ H]choline chloride for 72 h and then apoA-I-mediated cellular cholesterol was analyzed using liquid scintillation counting assays. apoA-I-mediated phospholipid efflux was calculated by subtracting the efflux to the medium and expressed as the percentage of total cellular and medium phospholipid amounts. All the results are expressed as mean ± SD from three independent experiments, each performed in triplicate. *P < 0.05 versus control group.
lncRNA microarray analysis Briefly, THP-1 macrophages (three samples) and THP-1 macrophage-derived foam cells (three samples) were used to isolate total RNA. Total RNA from each sample was quantified using the
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NanoDrop ND-1000 spectrophotometer and RNA integrity was assessed using standard denaturing agarose gel electrophoresis. For microarray analysis, the Agilent Array platform was employed. The sample preparation and microarray hybridization
TABLE 1.
Effect of lincRNA-DYNLRB2-2 and GPR119 on cholesterol content in THP-1 macrophage
Control LV-Mock LV-DYNLRB2-2 pcDNA3.1-Mock pcDNA3.1-GPR119
TC (g/mg cell protein)
FC (g/mg cell protein)
CE (g/mg cell protein)
CE/TC (%)
115 ± 11 120 ± 9 95 ± 6a 116 ± 10 87 ± 6b
109 ± 9 113 ± 8 89 ± 5a 110 ± 7 82 ± 5b
6±3 7±3 6±2 6±3 5±2
5.2 5.8 6.3 5.2 5.7
THP-1 cells were differentiated for 72 h with 100 nM PMA and then THP-1 macrophage cells were divided into five groups as indicated. Cellular TC, FC, and CE were determined by HPLC. The results are expressed as mean ± SD from three independent experiments, each performed in triplicate. a P < 0.05 versus LV-Mock group. b P < 0.05 versus pcDNA3.1-Mock group.
RNA isolation and real-time quantitative PCR analysis Total RNA from cultured cells or mouse tissues was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA) in accordance with the manufacturer’s instructions. lincRNA-DYNLRB2-2 was detected with a commercial reagent (Promega, Madison, Wisconsin) according to the instruction manual in 20 l reaction volumes. Real-time PCR was performed on a real-time PCR ABI 7500 Fast system (Applied Biosystems, Foster City, CA). The expression of U6 RNA was used as an endogenous control. Primers used for lincRNA-DYNLRB2-2 were as follows: lincRNA-DYNLRB2-2-F, 5′-TACAGGCATGAGGCATCGT-3′; lincRNA-DYNLRB2-2-R, 5′AGAGGTTGGGATTCATGCTAGA-3′; and lincRNA-DYNLRB2-2-RT, 5′-GCAAGACTTGGAGTTGGTGAT-3′. Primers used for U6 were as follows: U6-F, 5′-CTCGCTTCG GCAGCACA-3′; U6-R, 5′-AACGCTTCACGAATTTGCGT-3′; and U6-RT, 5′-AACGCTTCACGAATTTGCGT-3′. The mRNA levels were evaluated by real-time quantitative PCR using an ABI 7500 Fast real-time PCR system with SYBR Green detection chemistry (TaKaRa Bio, Inc.). Glyceraldehyde 3-phosphate dehydrogenase expression was used as the internal control. Quantitative measurements were determined using the ⌬⌬Ct method. All samples were measured in triplicate and the mean value was considered for comparative analysis.
Western blot analyses Cytokine assays and measurement of serum biochemical parameters The levels of human TNF-␣, interleukin (IL)-1, and IL-6 present in the culture media (R&D Systems, Minneapolis, MN); the serum concentrations of IL-1, IL-6, and TNF-␣ (R&D Systems); the serum serum amyloid A (SAA) amount (IBL, Hamburg, Germany); and the levels of serum apoA-I and apoB100 (Cusabio Biotech Co., Ltd., Wuhan, China) were measured by ELISA according to the manufacturer’s instructions. The TG and total cholesterol (TC) concentrations were determined enzymatically using an automated analyzer.
TABLE 2.
Cells and tissues were harvested and protein extracts prepared according to established methods. Extracts were then separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and subjected to Western blot analysis using rabbit polyclonal anti-LXR␣, -SRA1, -CD36, -SREBP1c, -SREBP2, and -GPR119 antibodies (Proteintech Group, Inc., Chicago, IL); rabbit polyclonal anti-NF-B, -PPAR␥, and -ABCA1 antibodies (Abcam Inc., Cambridge, MA); rabbit polyclonal anti-ABCG1, -SR-B1, -LDLR, -HMGCR, -HMGS, and -Niemann-Pick C1-Like 1 antibodies (Epitomics, Burlingame, CA); and rabbit polyclonal anti-ABCG5, -ABCG8, -MTP-PXR, and --actin antibodies (Santa Cruz Biotechnology, Inc.,
Effect of lincRNA-DYNLRB2-2 and GPR119 on cholesterol content in THP-1 macrophage-derived foam cells
Control LV-Mock LV-DYNLRB2-2 pcDNA3.1-Mock pcDNA3.1-GPR119
TC (g/mg cell protein)
FC (g/mg cell protein)
CE (g/mg cell protein)
CE/TC (%)
489 ± 15 495 ± 16 300 ± 13a 482 ± 13 306 ± 8b
190 ± 12 195 ± 13 128 ± 11a 186 ± 11 131 ± 10b
299 ± 15 300 ± 13 172 ± 14a 296 ± 14 175 ± 16b
61.1 60.7 57.5 61.5 57.1
THP-1 cells were differentiated for 72 h with 100 nM PMA and then macrophages were transformed into foam cells by incubation in the presence of 50 g/ml of Ox-LDL for 48 h. THP-1 macrophage-derived foam cells were divided into five groups as indicated. Cellular TC, FC, and CE were determined by HPLC. The results are expressed as mean ± SD from three independent experiments, each performed in triplicate. a P < 0.05 versus LV-Mock group. b P < 0.05 versus pcDNA3.1-Mock group.
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were performed using the manufacturer’s standard protocols with minor modifications. Briefly, mRNA was purified from 1 g of total RNA after removal of rRNA (mRNA-ONLY™ eukaryotic mRNA isolation kit; Epicentre Biotechnologies, Madison, WI). Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3′ bias utilizing a random priming method. The labeled cRNAs were hybridized onto the Human LncRNA Array v2.0 (8 × 60K; Arraystar, Inc., Rockville, MD). After the slides were washed, the arrays were scanned using the Agilent scanner G2505B. Agilent Feature Extraction software (version 10.7.3.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v11.5.1 software package (Agilent Technologies, Santa Clara, CA). After quantile normalization of the raw data, lncRNAs and mRNAs, in which at least three out of six samples had flags in present or marginal (“All Targets Value”), were chosen for further data analysis. Differentially expressed lncRNAs and mRNAs with statistical significance were identified through Volcano Plot filtering. All of the microarray data were uploaded to the NCBI Gene Expression Omnibus (GEO) with a GEO accession number (GSE54039).
TABLE 3.
Effect of lincRNA-DYNLRB2-2 and GPR119 on LPS-induced inflammatory cytokines in THP-1 macrophage cells TNF-␣ (pg/ml)
IL-1 (pg/ml)
IL-6 (pg/ml)
136 ± 29 861 ± 45a 121 ± 37 879 ± 56 392 ± 55b 115 ± 32 839 ± 46 352 ± 46c
42 ± 11 521 ± 35a 53 ± 15 551 ± 41 262 ± 28b 48 ± 9 542 ± 47 281 ± 33c
186 ± 31 506 ± 43a 178 ± 39 525 ± 39 312 ± 43b 173 ± 31 531 ± 42 295 ± 35c
Control LPS LV-DYNLRB2-2 LV-Mock + LPS LV-DYNLRB2-2 + LPS pcDNA3.1-GPR119 pcDNA3.1-Mock + LPS pcDNA3.1-GPR119 + LPS
THP-1 macrophage cells were divided into eight groups as indicated and cultured in medium at 37°C for 24 h. Secreted inflammatory cytokines were quantified by ELISA. LPS (lipopolysaccharide) indicates treatment with 10 ng/ml LPS. The results are expressed as the mean ± SD of three independent experiments, each performed in triplicate. a P < 0.05 versus control group. b P < 0.05 versus LV-Mock + LPS group. c P < 0.05 versus pcDNA3.1-Mock + LPS group.
Analysis was performed on a fluorescent activated cell sorting (FACScalibur) flow cytometer (Becton Dickinson, Franklin Lakes, NJ) with Cell Quest Pro software (BD Biosciences, San Jose, CA).
LV production and infection Human monocytic THP-1 cells were cultured. Packed empty LVs with green fluorescent protein (LV-Mock) and LV-mediated lincRNA-DYNLRB2-2 overexpression vector (LV-DYNLRB2-2) were generated. The cells were infected with the LV stock at a multiplicity of infection of 100 (THP-1 cells) transducing units per cell in the presence of 8 g/ml of polybrene. The cells were washed with fresh complete media after 24 h of incubation.
Plasmid construction and transfection The PCR-XL-TOPO vector containing GPR119, vector PIRES2EGFP, Platinum HIFI Taq polymerase, and Accuprime Pfx DNA polymerase were purchased from Invitrogen Biotechnology (Shanghai, China). The pCDNA3.1(+) vector, competent DH5␣ cells, XhoI and EcoRI restriction enzymes, and T4 DNA ligase were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). The fragment of EcoRI-GPR119-IRES-EGFP-XhoI was achieved from the PCR-XL-TOPO vector and PIRES2-EGFP vector by overlap extension PCR and was linked to pcDNA3.1 to create the recombinant plasmid pcDNA3.1-GPR119-IRES-EGFP. The inserted gene was identified by electrophoresis and sequencing. The recombinant plasmid was then transfected into the cultured cells by Lipofectamine 2000 (Invitrogen), and the over-expression of GPR119 was confirmed by RT-PCR and Western blotting.
Transfection with siRNA mimics THP-1 macrophages were transfected with 50 nM siRNAs against GPR119, ABCA1, and an irrelevant 21-nucleotide control siRNA (negative control) purchased from Ribo Biotechnology. Cells (2 × 106 cells/well) were transfected using Lipofectamine 2000 transfection reagent for 48 h according to the manufacturer’s instructions. All experimental control samples were treated with an equal concentration of a nontargeting control mimic sequence (negative controls). After 48 h of transfection, real-time RT-PCR and Western blotting were performed.
Cellular cholesterol efflux experiments THP-1 macrophages were cultured and labeled with 0.2 Ci/ml [3H]cholesterol. After 72 h, cells were subsequently washed with PBS and incubated overnight in RPMI 1640 medium containing 0.1% (w/v) BSA to allow equilibration of [3H]cholesterol in all cellular pools. Equilibrated [3H]cholesterol-labeled cells were washed with PBS and incubated in 2 ml of efflux medium containing RPMI 1640 medium and 0.1% BSA with 25 g/ml human plasma apoA-I for 12 h. A 150 l sample of efflux medium was obtained at the times designated and passed through a 0.45 m filter to remove any floating cells. Monolayers were washed twice in PBS, and cellular lipids were extracted with isopropanol. Medium and cell-associated [3H]cholesterol was then measured by liquid scintillation counting. Percent efflux was calculated by the following equation: [total media counts/(total cellular counts + total media counts)] × 100%.
Phospholipid efflux experiments Cells were cultured as indicated above, and then incubated with 2 Ci/ml of [3H]choline chloride to label the phospholipids. After 72 h, cells were subsequently washed with PBS and incubated overnight in RPMI 1640 medium containing 0.1% (w/v) BSA. After 6 h of incubation in medium containing 10 mg/ml apoA-I, efflux medium was collected, centrifuged to remove cell debris as above, and aliquots were taken for extraction and separated by thin-layer chromatography with the use of silica G plates developed in chloroform/methanol/ammonia (25%, w/v)/water (50:65:5:4, v/v/v/v). Phospholipid spots were visualized by I2 vapors and identified by comigration with standards. Relative radioactivity was measured by Phosphoscreen and quantified by PhosphorImager (Molecular Dynamics Inc.). Phospholipid efflux was expressed as percent counts in the supernatant versus the total counts for each individual lipid.
En face plaque area Dil-Ox-LDL uptake assays PMA-differentiated THP-1 cells were treated with LV lincRNADYNLRB2-2 (LV-DYNLRB2-2) or plasmid pcDNA3.1-GPR119 and its own negative control as indicated, then fluorescent-tagged Dil-Ox-LDL was added, and the cells were incubated for 1 h. Adherent cells were harvested, and washed three times with PBS.
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Immediately after the mice were euthanized, the aortas were excised and fixed in 10% buffered formalin for quantification of the en face plaque areas. Briefly, after the adventitial tissue was carefully removed, the aorta was opened longitudinally, stained with Oil Red O (Sigma), and pinned on a blue wax surface. En face images were obtained by a stereomicroscope
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Santa Cruz, CA). The proteins were visualized using a chemiluminescence method (ECL Plus Western blot detection system; Amersham Biosciences, Foster City, CA).
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Fig. 3. GPR119 is involved in lincRNA-DYNLRB2-2-induced upregulation of ABCA1 expression and function. A–I: THP-1 macrophages were treated as indicated. A, C: The expression of ABCA1 was detected by real-time quantitative PCR. B, D–F, I: Protein expression was detected by Western blotting. E, F: THP-1 macrophages were treated as indicated and protein expression was detected by Western blotting. G, H: The cells 3 3 were labeled with 0.2 Ci/ml [ H]cholesterol or 2 Ci/ml of [ H]choline chloride for 72 h and then apoAI-mediated cellular cholesterol efflux and phospholipid efflux were analyzed using liquid scintillation counting assays. All the results are expressed as mean ± SD from three independent experiments, each performed in triplicate. *P < 0.05 versus control group.
(SZX12; Olympus, Tokyo, Japan) equipped with a digital camera (Dxm1200, Nikon, Tokyo, Japan) and analyzed using Adobe Photoshop version 7.0 and Scion Image software. The percentage of the luminal surface area stained by Oil Red O was determined.
beginning at the aortic root, were collected for a distance of 400 m. Sections were stained with Oil Red O. The area of positive staining for Oil Red O was calculated as a percentage of the total section area and an average lipid droplet size was calculated utilizing ImagePro Plus software (Media Cybernetics) from five views per animal.
Quantification of atherosclerosis in the aortic sinus The upper portion of the heart and proximal aorta were obtained, embedded in OCT compound (Fisher, Tustin, CA), and stored at ⫺70°C. Serial 10 m thick cryosections of the aorta,
Liver histology and Oil Red O staining To examine hepatic lipid deposition, lipid was assessed in samples collected in OCT compound by Oil Red O staining. Briefly,
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liver cryosections were fixed for 10 min in 60% isopropanol and stained with 0.3% Oil Red O in 60% isopropanol for 30 min and subsequently washed with 60% isopropanol. Sections were counterstained with Gill’s hematoxylin, washed with acetic acid solution (4%), and mounted with aqueous solution. The area of positive staining for Oil Red O was calculated as a percentage of the total section area and an average lipid droplet size was calculated TABLE 4.
utilizing ImagePro Plus software (Media Cybernetics) from five views per animal.
Immunohistochemistry For this procedure, each frozen liver tissue or aortic root sample was sectioned to 5 M thickness and fixed in microscope slides. Sections of frozen tissue specimens were mounted
ABCA1 is involved in the regulation of LPS-induced inflammatory cytokines by GPR119 in THP-1 macrophage cells
Control pcDNA3.1-GPR119 + NC pcDNA3.1-GPR119 + ABCA1-siRNA pcDNA3.1-Mock + NC + LPS pcDNA3.1-GPR119 + NC + LPS pcDNA3.1-GPR119 + ABCA1-siRNA + LPS
TNF-␣ (pg/ml)
IL-1 (pg/ml)
IL-6 (pg/ml)
131 ± 27 121 ± 22 145 ± 39 796 ± 53 a 387 ± 46 731 ± 55
39 ± 12 30 ± 13 46 ± 15 529 ± 42 296 ± 42a 563 ± 57
197 ± 35 181 ± 43 191 ± 47 506 ± 35 268 ± 52a 491 ± 42
THP-1 macrophage cells were divided into six groups as indicated and cultured in medium at 37°C for 24 h. Secreted inflammatory cytokines were quantified by ELISA. LPS indicates treatment with 10 ng/ml LPS. The results are expressed as the mean ± SD of three independent experiments, each performed in triplicate. a P < 0.05 versus pcDNA3.1-Mock + NC + LPS group.
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Continued.
Fig. 3.
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Fig. 4. GLP-1R is involved in GPR119-induced upregulation of ABCA1. A–C: THP-1 macrophages were treated as indicated and Western blot assays were performed to detect protein expression. D, E: THP-1 macrophages were treated with or without inhibitors of CaMK (KN93, 5 M), ERK1/2 (PD98059, 50 M), PKA (H89, 10 M), PKB (LY294002, 10 M), and inhibitor of PKC- (rottlerin, 10 M) for 30 min, then transfected with pc-DNA3.1-GPR119 or pc-DNA3.1-Mock for 48 h. ABCA1 mRNA and protein levels were measured by real-time quantitative PCR and Western blotting, respectively. All results are expressed as mean ± SD of three independent experiments, each performed in triplicate. *P < 0.05 versus control group.
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TABLE 5.
Effect of GPR119 on serum lipids and lipoprotein values in apoE⫺/⫺ mice (n = 10) TG (mmol/L)
TC (mmol/l)
apoA-I (g/l)
apoB (g/l)
1.35 ± 0.17 a 0.97 ± 0.15
32.04 ± 2.65 32.32 ± 3.13
0.05 ± 0.01 0.07 ± 0.02a
0.16 ± 0.02 0.15 ± 0.03
LV-Mock LV-GPR119
Data are expressed as mean ± SD. a P < 0.05 versus LV-Mock group.
on Poly-Lysine (Sigma)-coated slides, air dried, and fixed with acetone. Immunohistochemical staining was performed for CD68 using rabbit polyclonal antibody to CD68 at a dilution of 1:100 (Abcam, Cambridge, MA). Images were acquired and quantitated on an Olympus BX50 microscope using Optimis software (version 6.2) and digitized using a color video camera (threecharge coupled device; JVC, Wayne, NJ).
Statistical analysis
RESULTS Ox-LDL induces GPR119 expression by upregulating lincRNA-DYNLRB2-2 in THP-1 macrophages Monocyte recruitment to the vessel wall is a rate-limiting step in atherogenesis. Monocytes are recruited from the circulation into the subendothelial space, where they differentiate into mature macrophages and internalize modified lipoproteins to differentiate into lipid-laden foam cells (25). Studies have shown that lncRNAs play an essential role in cardiovascular diseases (24). To explore possible changes in RNA expression during macrophage formation, we performed microarray analysis of THP-1 macrophages and THP-1 macrophage-derived foam cells in the presence of Ox-LDL using the Arraystar probe dataset, which included 24,748 lncRNAs and 24,420 coding transcripts. lncRNA and mRNA expression profiles from three pairs of THP-1 macrophages and THP-1 macrophagederived foam cells were produced using the Arraystar Human LncRNA Array v2.0 platform. Expression values were normalized based on the mean expression value for each probe set. Differently expressed probe sets were identified based on Student’s t-test for paired samples’ normalized expression values using the following cutoff: absolute fold change value larger than 3 and P value less than 0.01. The results showed that lincRNA-DYNLRB2-2 (BM973870; 16q23.3, chr16:79,831,946-79,861,047) expression was upregulated in THP-1 macrophage-derived foam cells (4.688-fold, P < 0.001), suggesting that Ox-LDL and TABLE 6.
LV-Mock LV-GPR119
⫺/⫺
Effect of GPR119 on serum cytokine levels in apoE
mice (n = 10)
IL-1 (pg/ml)
IL-6 (pg/ml)
TNF-␣ (pg/ml)
SAA (g/ml)
12.37 ± 1.18 a 8.41 ± 1.25
78.67 ± 5.82 56.65 ± 4.27a
11.35 ± 1.56 7.15 ± 1.28a
30.35 ± 3.23 18.26 ± 2.50a
Data are expressed as mean ± SD. P < 0.05 versus LV-Mock group.
a
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The effect of lincRNA-DYNLRB2-2 and GPR119 on cholesterol metabolism and inflammation Atherosclerosis can be considered as both a lipid metabolism disorder and a chronic inflammatory disease. Innate immunity pathways have long been suspected to contribute to the initiation and progression of atherosclerosis. This suggests that crosstalk between lipid metabolism and innate immunity pathways play an important role in the development and/or prevention of atherosclerosis (26). To determine whether lincRNA-DYNLRB2-2 and GPR119 affect cholesterol metabolism and inflammation, we first examined the effect of lincRNA-DYNLRB2-2 and GPR119 on cholesterol metabolism in THP-1 macrophagederived foam cells. LV-DYNLRB2-2 and plasmid vector pcDNA3.1-GPR119 were created and transfected into THP-1 macrophages (Fig. 1C, Fig. 2A). As shown in Fig. 2B, Dil-Ox-LDL uptake significantly decreased in GPR119 overexpressing or lincRNA-DYNLRB2-2 overexpressing THP-1 macrophages. Moreover, LV-DYNLRB2-2 and GPR119 significantly decreased TG, free cholesterol (FC), and cholesteryl ester (CE) levels (Tables 1, 2). The defect in
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Data are expressed as mean ± SD. Results were analyzed by one-way ANOVA followed by the Student-Newman-Keuls test and the Student’s t-test using SPSS v13.0 statistical software (SPSS, Inc., Chicago, IL). A two-tailed P value 0.05).
aortic valve section and en face analyses. In mice infected with LV-GPR119, the average lesion area was decreased compared with that in controls by both en face and aortic valve section analyses (Fig. 6A). Quantification of Oil Red O-stained lesions in en face preparations of aortas revealed that treatment with LV-GPR119 resulted in a significant 30% decrease in lesion area in apoE⫺/⫺ mice compared with the controls. To confirm the positive effects of GPR119 on atherosclerosis, Oil Red O-stained aortic valve sections were quantified, which showed a significant 27% reduction in lesion area in apoE⫺/⫺ mice treated with LV-GPR119 compared with controls. Immunohistochemical staining was performed to visualize macrophages in atherosclerotic lesions (using an antibody against CD68). As shown in Fig. 6B, LV-GPR119-treated mice showed a considerably lower number of CD68+ cells than control mice, indicating that macrophage infiltration was significantly decreased in lesions of apoE⫺/⫺ mice (P < 0.05). These observations indicated that the positive effect of GPR119 is ongoing in atherosclerotic lesions of apoE⫺/⫺ mice. Western blot assessment of the levels of proteins related to cholesterol metabolism and inflammation in aortic tissue of apoE⫺/⫺ mice showed that the levels of SR-A1, CD36, and NF-B were decreased, whereas protein levels of ABCA1 and SR-B1 were increased in the GPR119 group (Fig. 6C). However, no effect was observed on ABCG1 protein expression in the GPR119 group compared with the control group (P > 0.05). In addition, assessment of the levels of nuclear receptor proteins related to cholesterol metabolism and inflammation, including LXR␣, PPAR␣, PPAR␥, PXR, HNF-1␣, and LRH1, showed no significant differences between the GPR119 and control groups (P > 0.05, data not shown).
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Fig. 6. Effect of GPR119 on atherosclerosis initiation and development in apoE⫺/⫺ mice. A–C: apoE⫺/⫺ mice were randomized into a control group and a GPR119 group and fed a HFD. A: Representative staining of en face aorta (a) with Oil Red O and aortic valves (b) with ⫺/⫺ mice. B: Cryosections of aortic valves from Oil Red O. En face lesions (c) and total lesions in the aortic valves (d) were analyzed in apoE ⫺/⫺ mice were immunohistochemically stained for the macrophage marker CD68 (a). The integral optical density of CD68 in the apoE
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the mobilized cholesterol (34). The control of macrophage cholesterol homeostasis is of critical importance in the pathogenesis of atherosclerosis, as alterations in the balance between cholesterol influx, intracellular transport and efflux can lead to excessive accumulation of cholesterol in macrophages and their transformation into foam cells (35). In the present study, GPR119 exerted an antiatherogenic effect by decreasing cellular cholesterol content and upregulating the expression of ABCA1, which plays a crucial role in mediating the transport of cholesterol across cellular membranes (36–38). Its regulation is cAMP- and sterol-dependent, and cAMP is responsible for the functional activation of ABCA1 or the upregulation of ABCA1 expression (39, 40). In the present study, we showed that GPR119 activates GLP-1R and its downstream pathways (CaMK and cAMP/PKA) and PI-3K/AKT/PKB pathways, and it inhibits the MAPK-ERK1/2 and PKC- pathways, finally resulting in the upregulation of ABCA1 expression and apoA-I mediated cholesterol efflux. These results provide strong evidence supporting the notion that GPR119 exerts its anti-atherogenic effects by increasing the rate of RCT. Furthermore, GPR119 enhanced the overall rate of RCT by activating pathways that mediate the delivery of HDL-cholesterol to the liver. Mature HDL can transfer its cholesterol to the liver directly via SR-B1 or indirectly via CETP-mediated transfer to apoB-containing lipoproteins, with subsequent uptake by the liver via the
LDLR (41). We showed that GPR119 upregulated the ⫺/⫺ mice and induced the exexpression of SR-B1 in apoE pression of LDL receptors in the liver, increasing the catabolism of plasma LDL and decreasing the plasma concentration of cholesterol. In addition, apolipoproteins are important constituents of plasma lipoproteins and essential for RCT. We performed in vivo experiments and showed that GPR119 can significantly increase apoA-I levels in apoE⫺/⫺ mice. apoA-I is secreted predominantly by the liver and is present in the majority of HDL particles, and its concentration is closely correlated with plasma HDL (42)· Atherosclerosis is a chronic disease that can remain asymptomatic for decades. Inflammatory processes take part in all stages of the atherosclerotic process, from lesion initiation to plaque rupture (43). Macrophages, whether engorged with lipids or not, play a key role in the mediation and modulation of inflammation, and much atherosclerosis research has targeted the role of macrophages in the inflammatory pathways that underlie atherogenesis (44, 45). Evidence from several recent studies indicate that inflammation, along with other atherogenic-related mediators, plays a distinct regulating role in ABCA1 expression. Proatherogenic cytokines such as IFN-␥ and IL-1 inhibit the expression of ABCA1, while anti-atherogenic cytokines, including IL-10 and transforming growth factor TGF1, promote the expression of ABCA1. Moreover, some
aortic valve cryosections from apoE⫺/⫺ mice was analyzed (b). C: Protein expression levels in tissues of apoE⫺/⫺ mice were analyzed by Western blotting (n = 5 mice/group). All data are presented as mean ± SD (n = 10). All experiments were performed in triplicate, except as indicated. *P < 0.05 versus control group.
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Fig. 7. ABCA1 expression and function can be induced by GPR119 to protect from atherosclerosis. OxLDL upregulates lincRNA-DYNLRB2-2 expression, resulting in increased ABCA1 expression through GPR119. GLP-1R-mediated signaling pathways including CaMK, cAMP/PKA, PI-3K/AKT/PKB, MAPKERK1/2, and PKC- are involved in GPR119-induced ABCA1 expression. Moreover, GPR119 inhibits inflammation and promotes apoA-I-mediated cellular cholesterol efflux through inducing ABCA1 expression. These beneficial effects are accompanied by a reduction of macrophage-derived foam cell formation, inhibition of inflammatory gene expression, adhesion molecule expression in the aorta, and increase of cholesterol efflux from peripheral tissues, thus preventing of atherosclerotic plaque formation.
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and inflammatory cytokine levels in serum to further pre⫺/⫺ mice, indicating that vent atherosclerosis in apoE GPR119 may represent a promising candidate as a therapeutic agent.
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cytokines such as TNF-␣ regulate ABCA1 expression in a species-specific and dose-dependent manner (46). Chronic activation of inflammatory pathways mediates the pathogenesis of insulin resistance, and the macrophage/adipocyte nexus provides a key mechanism underlying decreased insulin sensitivity. Recently, a family of G protein-coupled receptors has been identified that exhibits high affinity for inflammation, glucose metabolism, and insulin sensitivity (47). In the present study, we showed that GPR119 significantly downregulated IL-1, IL-6, and TNF-␣ levels through the regulation of ABCA1 expression in LPSinduced THP-1 macrophage-derived foam cells. To further investigate the mechanisms whereby GPR119 treatment inhibited plaque progression and stabilization, changes in gene expression of inflammatory molecules ⫺/⫺ mice fed a high-fat/high-chowere explored in apoE lesterol diet. We found that IL-1, IL-6, TNF-␣, and SAA were markedly repressed in GPR119-overexpressing ⫺/⫺ mice. Our results suggest that GPR119-induced apoE suppression of inflammatory molecule expression could block or retard the development of atherosclerotic lesions and thus have a positive influence on disease outcomes. The diverse roles of regulatory RNAs extend well beyond the epigenome, as they regulate transcription in different ways (48). lncRNAs are nonprotein coding transcribed RNAs that target the minor dihydrofolate reductase gene promoter, which binds to the general transcription factor IIB and to the DNA of the dihydrofolate reductase major promoter forming a specific and stable tripartite complex, resulting in the dissociation of the preinitiation complex and transcriptional repression (49). The lncRNAs also function as transcriptional coactivators, modulating protein localization and function. Ox-LDL contributes to atherosclerotic plaque formation and progression by several mechanisms, including the induction of endothelial cell activation and dysfunction, macrophage foam cell formation, and smooth muscle cell migration and proliferation. Ox-LDL upregulates ABCA1 expression at both protein and mRNA levels (31). In the present study, we showed that Ox-LDL induces GPR119 expression through lincRNA-DYNLRB2-2 in THP-1 macrophages. In response to extracellular signals, lincRNA-DYNLRB2-2 upregulated GPR119 expression, which increased ABCA1 expression resulting in promotion of ABCA1-mediated cholesterol efflux leading to the protection from atherosclerosis. However, the detailed mechanism of lincRNADYNLRB2-2-induced upregulation of GPR119 expression needs further clarification. Moreover, in the present study, we showed that Ox-LDL and Ac-LDL had similar effects on lincRNA-DYNLRB2-2 levels. Remarkably, little is known about the direct downstream target lncRNAs of Ox-LDL and Ac-LDL. Thus, whether lincRNA-DYNLRB2-2 can be directly regulated by Ox-LDL or Ac-LDL and whether another mechanism is involved needs to be further explored. In conclusion, we have demonstrated that Ox-LDL significantly induced lincRNA-DYNLRB2-2 expression (Fig. 7), which promoted ABCA1-mediated cholesterol efflux and inhibited inflammation through GPR119 in THP-1 macrophagederived foam cells. In addition, GPR119 decreased lipid
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