Abstract
Excessive formation of advanced glycation end products (AGE) and lipid accumulation in macrophages play a pivotal role in the progression of atherosclerosis in diabetes mellitus. This study aimed to determine the molecular link between AGE-induced fatty acid binding protein 4 (FABP4) expression and macrophage lipid accumulation. AGE–BSA markedly increased macrophage FABP4 expression via engagement of RAGE, a 35-kDa transmembrane receptor that is able to bind extracellular AGE and responsible for the corresponding signal transduction, whereas knockdown of RAGE significantly reversed the FABP4 up-regulation. This effect was further paralleled with elevated intracellular total cholesterol and triacylglycerol levels. Finally, administration of FABP4 inhibitor totally abolished the increased lipid contents in response to AGE–BSA. These results indicate that FABP4 up-regulation is responsible for the enhanced macrophage lipid accumulation by AGE, which may underlie the accelerated formation of foam cells and development of atherosclerotic cardiovascular diseases in diabetic patients.
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Introduction
Accelerated atherosclerosis accounts for about 80% of morbidity and mortality in patients with diabetes [1, 2]. Increased formation of advanced glycation end products (AGE) is recognized as an important player in diabetic vascular complications [3, 4]. After longstanding exposure to hyperglycemic milieu, proteins and lipids in blood and tissues undergo non-enzymatic Maillard reaction, and finally give rise to the irreversible formation of AGE [4]. AGE exert adverse effects largely through engagement of receptor for AGE (RAGE) on the cell membrane of vascular wall [5–7]. Postmortem studies have consistently shown that RAGE expression was mainly localized in macrophages within or around necrotic cores of atherosclerotic plaques, and was substantially increased in diabetic patients [8].
Macrophages actively participate in atherogenic processes, including formation of lipid-laden foam cells and atheromatous lipid cores [9]. FABP4, also known as adipocyte protein 2 (aP2), is highly expressed in macrophages [10]. It functions in cellular lipid metabolism, cholesterol trafficking and other biologic responses [11, 12]. Expression of FABP4 in THP-1 monocytes/macrophages was greatly increased upon treatment with phorbol 12-myristate 13-acetate (PMA) and oxidized low-density lipoprotein (oxLDL), while transformation of THP-1 monocytes into foam cells was significantly suppressed by FABP4 inhibitor, a small synthetic molecule designed to competitively prevent FABP4 from binding fatty acids [13, 14]. Likewise, inhibition of FABP4 expression either by macrophage-specific gene deficiency or administration of FABP4 inhibitor in vivo led to a protection from the development of atherosclerosis [15]. Iwashima et al. [6] observed that macrophage lipoprotein receptors, including CD36, macrophage scavenger receptor (MSR) class A and lectin-like oxLDL receptor 1 (LOX-1) were up-regulated in response to AGE stimulation, whereas key proteins localized on the cell membrane that mediate macrophage cholesterol efflux, including ABCA1 and ABCG1, were down-regulated or destabilized [16, 17].
It is still unclear, however, whether macrophage FABP4 remains, equally or to a greater extent, an active pro-atherogenic participator in the context of diabetes where AGE are greatly enriched. In this study, we tested the hypothesis that AGE-induced FABP4 expression in THP-1 macrophages was potentially associated with an increase in intracellular lipid levels. The assessment of lipid contents after FABP4 inhibitor administration was also performed.
Materials and Methods
Cell Culture
Human monocytic leukemia THP-1 cells were obtained from ATCC and cultured in Gibco RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 50 U/ml penicillin and 50U/ml streptomycin (Gibco, Auckland, New Zealand) at 37 °C in 5% CO2. THP-1 monocytes were differentiated into macrophages by induction of 100nM phorbol 12-myristate 13-acetate (Sigma-Aldrich, MO) for 48 h. Cells were then incubated in the presence or absence of bovine serum albumin (BSA; fatty acid free, catalog no. 126575; Calbiochem, CA) or AGE–BSA (catalog no. 121800; Calbiochem, CA) for the indicated time intervals. FABP4 inhibitor (Calbiochem, CA) dissolved in dimethyl sulfoxide (DMSO) at the indicated concentrations was administrated for study purpose.
Transfection of RAGE-Specific siRNA
Transient transfection of THP-1 macrophages with RAGE-specific and negative control siRNA (Ambion CA) was scheduled as follows: Silencing RAGE expression was performed using human RAGE-specific siRNA and siRNA transfection reagent (Lipofectamine 2000, Invitrogen, CA) to macrophages. Macrophages grown in 6-well plates were transfected with various concentrations of siRNA according to the manufacturer’s protocol. The RAGE-specific siRNA (100 pM) for reducing RAGE expression was added 48 h before AGE stimulation; macrophages were then exposed to BSA or AGE–BSA (Calbiochem CA) for 24 or 48 h for RNA or protein analysis and ORO-staining, respectively.
Assessment of Intracellular Lipid Contents
Cultured and transfected macrophages were washed twice in phosphate-buffered saline (PBS, Gibco, Auckland, New Zealand) and then fixed in 4% paraformaldehyde for 30 min. After rinsing with ddH2O, macrophages were incubated with filtered Oil Red O solution (0.6 g/L ORO in 60% isopropanol) (ORO, Sigma-Aldrich, MO) for 10 min and with 1 mL of Mayer’s hemalum (Merck, Darmstadt) for 1 min. After rinsing with PBS four times, intracellular lipid droplets were evaluated under Olympus DP-71 microscope. Spectrophotometric quantification of the staining was performed as described previously [18]. Briefly, stained oil droplets were dissolved in 100% isopropanol for 10 min. Then optical density was measured at 500 nm and equalized with the cell numbers estimated by microscope. Quantifications of intracellular total cholesterol and triacylglycerol levels were performed following the protocols from the manufacture (Biovision, CA) and analyzed with a micro-titer plate reader (Ex/Em = 538/587 nm). Total cholesterol and triacylglycerol concentrations were expressed as micrograms per milligram protein and nanomoles per milligram protein sample, respectively.
Quantitative Real-Time PCR Analysis
Total RNA was isolated using Trizol reagent (Invitrogen, CA). For reverse transcription, 1 μg of the total RNA was converted to first strand complementary DNA in 20-μl reactions using a reverse transcription kit (Promega, WI). Quantitative real-time PCR analysis was performed (StepOne, Applied Biosystems) using SYBR Green (Takara, Dalian, China). The thermal cycling program was 10 s at 95 °C for enzyme activation and 40 cycles of denaturation for 5 s at 95 °C, 31 s 60 °C for annealing and extension. The comparative cycle threshold (CT) method was used to determine relative mRNA expression of genes as normalized by β-actin housekeeping gene. Primers used are as follows: 5′-TGGGATGGAAAATCAACC-3′ for FABP4-F, 5′-TCTCTCATAAACTCTCGTGG-3′ for FABP4-R, 5′-GGCAGAAAAACTCAGAC-3′ for FABP5-F, 5′-GACACACTCCACCACTAA-3′ for FABP5-R, 5′-GCTGGTGTTCCCAATAA-3′ for AGER-F, 5′-AGTGTGAAGAGCCCTGT-3′ for AGER-R, 5′-CGTGGACATCCGCAAAG-3′ for β-actin-F, and 5′-TGGAAGGTGGACAGCGA-3′ for β-actin-R.
Western Blot
Total proteins were prepared by standard procedures and assessed by microplate protein assay (BSA; Pierce, IL). Thirty micrograms of protein per sample and known molecular weight markers were loaded onto a 12% SDS-polyacrylamide gel. After SDS-polyacrylamide gel electrophoresis, proteins were transferred to PVDF membranes (Millipore, MA). The blocked membranes (5% nonfat milk in TBS buffer containing 0.1% Tween 20) were incubated with anti-FABP4 (1:1000; Santa Cruz Biotechnology, CA), anti-RAGE (1:1000; Abcam, MA) and anti-β-actin antibody (1:1000; Cell Signaling Technology, MA) for overnight at 4 °C. The membranes were then treated with horseradish peroxidase-conjugated rabbit anti-goat, goat anti-rabbit or goat anti-mouse antibodies (1:5000; Bio-Rad, Hercules, CA). After washing, immunodetection analysis was accomplished using the Immobilon Western Chemiluminescent HRP Substrate (Millipore, MA).
Statistical Analysis
Data are expressed as means ± SEM. The mean values for biochemical measurements from each group were compared using Student’s t test. A P < 0.05 was considered statistically significant.
Results
AGE–BSA-Induced Up-Regulation of FABP4 Expression
After incubation with AGE–BSA for 24 h at various concentrations (from 25 to 200 μg/ml), FABP4 expression was substantially up-regulated, and reached the maximum level at 100 μg/ml (3.59 ± 0.53-fold vs. con; P = 0.001), whereas BSA did not affect FABP4 expression significantly (0.95 ± 0.14-fold vs. con, P = 0.577; Fig. 1a). A 3.9-fold increase in FABP4 expression was observed by 48 h of incubation (P = 0.0003; Fig. 1b). FABP5, another fatty acid binding protein expressed in macrophages, was not affected by AGE (Fig. 1c).
RAGE Mediates AGE–BSA-Induced FABP4 Expression
AGE–BSA moderately increased RAGE expression by 1.2-fold in cells transfected with non-specific negative control siRNA (P = 0.010, Fig. 2a). After gene silencing of RAGE, AGE exposure no longer affected FAPB4 expression both at mRNA (1.38 ± 0.24-fold vs. BSA, P = 0.069; Fig. 2b) and protein (Fig. 2c) levels when compared with negative control-treated groups (2.78 ± 0.36-fold vs. BSA, P = 0.001).
AGE–BSA-Induced Macrophage Lipid Accumulation
Oil red O (ORO) staining of lipid droplets was most evident at 100 and 200 μg/ml of AGE–BSA (Fig. 3a, b). A similar trend was also obtained for intracellular TC (100 μg/ml AGE–BSA: 37.20 ± 2.52 μg/mg, P = 0.0002; 200 μg/ml AGE–BSA: 29.89 ± 4.67 μg/mg, P = 0.0066 vs. con: 15.10 ± 1.61 μg/mg; Fig. 3c) and TAG levels (100 μg/ml AGE–BSA: 4.35 ± 1.35 nmol/mg, P = 0.0252; 200 μg/ml AGE–BSA: 2.89 ± 0.75 nmol/mg, P = 0.0463 vs. con: 1.64 ± 0.08 nmol/mg; Fig. 3d). RAGE knockdown markedly reduced lipid contents in response to AGE exposure (TC, 21.92 ± 3.09 μg/mg vs. BSA: 17.23 ± 2.20 μg/mg, P = 0.0980; TAG, 5.64 ± 1.43 nmol/mg vs. BSA: 1.85 ± 1.10 nmol/mg, P = 0.0220) when compared with negative control-treated cells, as demonstrated by ORO staining (Fig. 3e, f), TC (Fig. 3g) and TAG (Fig. 3h) quantification.
Changes After Incubation with FABP4 Inhibitor
After incubation with FABP4 inhibitor for 48 h, AGE-induced lipid accumulation in macrophages was substantially suppressed and reached the maximum effect at 40 μM as demonstrated by ORO staining (Fig. 4a, b), TC (14.92 ± 1.54 μg/mg vs. BSA: 14.56 ± 0.81ug/mg, P = 0.7356; Fig. 4c) and TAG (0.79 ± 0.35 nmol/mg vs. BSA: 0.52 ± 0.24 nmol/mg, P = 0.2656; Fig. 4d) quantification when compared with macrophages without FABP4 treatment. AGE–BSA up-regulated FABP4 protein levels irrespective of FABP4 inhibitor at each concentration (Fig. 4e). FABP5 expression was not affected by FABP4 inhibitor (Fig. 4f).
Discussion
In THP-1 macrophages, AGE-induced FABP4 expression via a RAGE-dependent signaling pathway resulted in increased intracellular lipid levels, which could be attenuated by FABP4 inhibitor.
AGE are increasingly regarded as an indispensable factor for severity and progression of atherosclerosis in patients with diabetes [8]. AGE-albumin, a further glycation modified protein derived from Amadori adduct exerts more severe pathogenic influence. Elevated serum AGE-albumin and decreased serum endogenous secretary RAGE (esRAGE) levels highly correlated with the extent of angiographic severity in patients with type 2 diabetes [19, 20]. In contrast, in vivo studies showed that administration of soluble RAGE (sRAGE), another truncated form of RAGE acting as a decoy for AGE, completely suppressed diabetic atherosclerosis in glycemia- and lipid-independent manners [21]. Other approaches utilizing inhibitors for AGE formation, including aminoguanidine and an AGE cross-link breaker ALT-711, also led to a striking reduction in atherosclerotic lesions in diabetic apoE-deficient mice [22].
Despite the important contribution of AGE to the accelerated atherosclerosis in diabetes, the specific molecular mechanisms in response to AGE within a macrophage (a central player in atherogenesis) remain unclear. In this study, we discovered that exposure of THP-1 macrophages to AGE was time-dependently associated with a significant elevation of FABP4 expression, reaching its maximum level when 100 μg/ml AGE–BSA was used. At the same time, the enhanced expression of FABP4 was paralleled with increases in intracellular TC and TAG levels. RAGE-specific gene silencing led to reversions both in FABP4 expression and lipid contents, suggesting that AGE-elicited atherogenic effects in macrophages were, at least partly, RAGE-dependent. Finally, this increase in lipid levels was totally abolished by simultaneous administration of FABP4 inhibitor.
Our study is the first to show that macrophage FABP4 expression, a critical participant in atherogenesis, is further enhanced in the context of diabetes. In an animal experiment, Gerrity et al. [2] observed that FABP4 deficiency protected against the development of insulin resistance, diabetes, and atherosclerotic cardiovascular disease. Recent population studies revealed that genetic variations at the FABP4 locus in humans led to lowered serum triglyceride levels, and a markedly reduced risk of coronary heart disease in type 2 diabetes [23]. These anti-atherosclerotic effects were mainly attributed to the improvement of glucose control, insulin resistance and dyslipidemia [24, 25]. In this study, we demonstrated that exposure of macrophages to AGE was capable of increasing intracellular FAPB4 levels and lipid contents, similar to that when transforming macrophages to foam cells in the presence of oxLDL stimulation [14]. These observations suggest that excessive formation of AGE could render diabetic patients under high risk of developing atherosclerosis like those with dyslipidemia. [26–28].
Furthermore, our results imply that FABP4 might be a potential therapeutic target for the treatment of diabetic patients with atherosclerosis. In this study, mild doses of FABP4 inhibitor (10–20 μM) greatly attenuated AGE–BSA elicited macrophage lipid accumulation, whereas lipid levels in the absence of AGE were not affected. These findings indicate that FABP4 inhibitor may not disturb physiological lipid metabolism in macrophages, but, on the contrary, suppresses exclusively pathological excessive lipid accumulation.
This study does not specify the mechanisms by which lipid contents and trafficking are affected by AGE besides the elevated FAPB4 level. Deficiency of FABP4 enhanced CD36-mediated lipoprotein entry and, at the same time, activated ABCA1-dependent lipid efflux to a greater extent, thereby lowering intracellular lipid contents [29]. Likewise, AGE stimulation on macrophages could increase SRA-1 and CD36 and decrease ABCG1 protein levels [6, 16, 17]. Thus, enhanced expression of FABP4 by AGE might extensively affect lipid influx and efflux, directly or indirectly, at the same time to increase lipid accumulation. Further studies including analysis of lipid trafficking in macrophages from FABP4-deficient mice and blocking lipoprotein transporters by their specific siRNA or antibodies, are needed to elucidate the mechanisms.
In conclusion, this study demonstrates a causal molecular link between AGE and FABP4 in macrophage lipid accumulation. FABP4 inhibitor may be useful for suppressing the atherosclerotic process in patients with diabetes.
Abbreviations
- AGE:
-
Advanced glycation end products
- aP2:
-
Adipocyte protein 2
- BSA:
-
Bovine serum albumin
- DMSO:
-
Dimethyl sulfoxide
- esRAGE:
-
Endogenous secretary RAGE
- FABP4:
-
Fatty acid binding protein 4
- FABP5:
-
Fatty acid binding protein 5
- FBS:
-
Fetal bovine serum
- LOX-1:
-
Lectin-like oxidized low-density lipoprotein receptor 1
- MSR:
-
Macrophage scavenger receptor
- OD:
-
Optical density
- ORO:
-
Oil red O
- oxLDL:
-
Oxidized low-density lipoprotein
- PBS:
-
Phosphate-buffered saline
- PMA:
-
Phorbol 12-myristate 13-acetate
- RAGE:
-
Receptor for advanced glycation end products
- sRAGE:
-
Secretary RAGE
- TAG:
-
Triacylglycerol
- TC:
-
Total cholesterol
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Acknowledgments
This work was supported by grant from Chinese National Nature Science Foundation (No. 30871084).
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Wang, X.Q., Yang, K., He, Y.S. et al. Receptor Mediated Elevation in FABP4 Levels by Advanced Glycation End Products Induces Cholesterol and Triacylglycerol Accumulation in THP-1 Macrophages. Lipids 46, 479–486 (2011). https://doi.org/10.1007/s11745-011-3542-4
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DOI: https://doi.org/10.1007/s11745-011-3542-4