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Citrulline increases cholesterol efflux from macrophages in vitro and ex vivo via ATP-binding cassette transporters

Reverse cholesterol transport (RCT) is a mechanism critical to the anti-atherogenic property of HDL. Although citrulline contributes to the amelioration of atherosclerosis via endothelial nitric oxide production, it remains unclear whether it affects RCT. This study was undertaken to clarify the effects of citrulline on expressions of specific transporters such as ATP binding cassette transporters (ABC)A1 and ABCG1, and the cholesterol efflux from macrophages to apolipoprotein (apo) A-I or HDL in vitro and ex vivo. Citrulline increased ABCA1 and ABCG1 mRNA and protein levels in THP-1 macrophages, translating into enhanced apoA-I- and HDL-mediated cholesterol efflux. In the human crossover study, 8 healthy male volunteers (age 30–49 years) consumed either 3.2 g/day citrulline or placebo for 1 week. Citrulline consumption brought about significant increases in plasma levels of citrulline and arginine. Supporting the in vitro data, monocyte-derived macrophages (MDM) differentiated under autologous post-citrulline sera demonstrated enhancement of both apoA-I- and HDL-mediated cholesterol efflux through increased ABCA1 and ABCG1 expressions, compared to MDM differentiated under pre-citrulline sera. However, the placebo did not modulate these parameters. Therefore, in addition to improving endothelium function, citrulline might have an anti-atherogenic property by increasing RCT of HDL.

Introduction H DL has been shown to be inversely associated with the risk of atherosclerotic cardiovascular disease (CVD) and is thus considered to be an anti-atherogenic lipoprotein. (1,2) However, failure in recent clinical trials with HDL-C raising agents such as nicotinic acid and the cholesteryl ester transfer protein inhibitor torcetrapib to provide cardiovascular benefits despite raising HDL-C levels, indicate that the focus should be on quality (functionality), and not necessarily quantity (HDL-C levels) regarding therapeutic targets. (3) Anti-atherogenic functions of HDL include anti-inflammatory, (4) anti-oxidative, (5) anti-coagulative, (6) and endothelial-improving functions, (7) as well as enhancement of macrophage reverse cholesterol transport (RCT), a process by which cholesterol is transported from macrophages to the liver for ultimate fecal excretion. Although these functions may comprehensively be involved in the prevention of atherosclerosis progression, (8,9) RCT itself has been postulated to play a major role in HDL-mediated atheroprotection. (10) Indeed, quantitative measures of macrophage RCT have been shown to be more strongly associated with atherosclerosis than plasma HDL-C concentrations in mice and humans. (11,12) The first critical step of macrophage-derived foam cell RCT involves efflux of cellular cholesterol to circulating HDL particles.
Research in recent years has documented a specific role for the macrophage transporters ATP-binding cassette subfamilies (ABC) A1 and ABCG1 in cholesterol efflux. ABCA1 has been shown to play an important role in apolipoprotein (apo) A-Imediated cholesterol efflux from peripheral cells and macrophages while ABCG1 promotes cholesterol efflux from macrophages to HDL particles. (13,14) These findings have stimulated efforts to target the macrophage at the cellular level as a means of enhancing overall RCT.
Endothelial dysfunction is a key feature of early atherosclerotic lesions in both humans and animal models and elevated plasma concentrations of risk factors for CVD. (15)(16)(17)(18) Citrulline is unusually abundant in watermelon (Citrullus vulgaris) and arginine is synthesized from it in endothelial cells. (19) The conversion of citrulline to nitric oxide (NO) via arginine forms part of a recycling pathway. (20)(21)(22) NO is synthesized by NO synthase (NOS), which produces citrulline, a precursor of arginine. Chronic consumption of citrulline-rich food has been known to be effective in increasing plasma concentrations of arginine in healthy humans, (23) and Hayashi et al. (24) reported that citrulline decreased the progression of atherosclerosis in rabbits fed a high cholesterol diet, via endothelial NOS (eNOS). Although citrulline contributes to antiatherosclerosis via endothelial nitric oxide, it remains unclear whether it affects the anti-atherogenic properties of HDLmediated RCT functions, or how it might do this. Therefore, the present study was undertaken to investigate the effects of citrulline on ABCA1 and ABCG1 expressions and HDL-mediated cholesterol efflux from macrophages, both in vitro and ex vivo situations.

Materials and Methods
Materials. Actinomysin D (ActD) and human apoA-I were purchased from Sigma (St. Louis, MO). N G -nitro-L-arginine methyl ester (L-NAME) and phorbol 12-myristate 13-acetate (PMA) were purchased from Wako Pure Chemical (Tokyo, Japan). HDL was isolated by sequential ultracentrifugation and acetylated LDL (acLDL) was prepared according to the previously reported methods. (25,26) L-Citrulline for the in vitro and in vivo study was purchased from Kanto Chemical CO., INC. (Tokyo, Japan). Placebo and citrulline for the ex vivo study were kindly H donated by Kyowa Hakko Bio Co., Ltd. (Tokyo, Japan).
Quantitative real time PCR. At the indicated hours after treatment with the compounds, total RNA was extracted from the cells, and first-strand cDNA was synthesized from the total RNA (250 ng) by placing in a Reverse Transcription Reagent (Applied Biosystems, Foster City, CA). Quantitative real-time PCR was performed using an ABI 7900 PCR machine, TaqMan PCR master mix and FAM-labeled TaqMan probes (Assays-on-Demand, Applied Biosystems) for human ABCA1, ABCG1, scavenger receptor class B type I (SR-BI) and 18S ribosomal RNA. The expression data were normalized for 18S levels.
Western blot analyses. The cells were harvested and protein extracts prepared as previously described. (25)(26)(27) They were then subjected to Western blot analyses (10% SDS-PAGE; 30 μg protein per lane) using rabbit anti-ABCG1 (Novus Biologicals, Littleton, CO) and anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies. Rat anti-ABCA1 was kindly donated by Dr. S, Yokoyama. The proteins were visualized and quantified using a chemiluminescence method (ECL Pus Western Blotting Detection System; GE Healthcare UK Ltd., Buckinghamshire, UK) and the NIH image analysis software program.
Determination of cholesterol efflux. Cholesterol efflux experiments were performed as previously described. (25)(26)(27) The cells were labeled with [ 3 H] cholesterol (1.0 μCi/ml) in media containing 0.1% bovine serum albumin (BSA) for 24 h. The cells were washed with phosphate buffered saline (PBS) and incubated in RPMI 1640 containing 0.1% BSA in the presence and absence of apoA-I (10 μg/ml) or HDL (50 μg/ml) for 24 h. The percentage cholesterol efflux was calculated by dividing the media-derived radioactivity by the sum of the radioactivity in the media and the cells.
Human study. Eight healthy male volunteers (age 30-49 years) were recruited for a crossover study. After an overnight (12 h) fast, the study subjects consumed placebo or 1.6 g citrulline twice daily for 1 week. (28) Blood samples were taken at 0 and 1 week after consumption of placebo or citrulline. All subjects provided written informed consent. The study was approved by the Ethical Committee of the National Defense Medical College.
Human monocyte isolation and macrophage differentia tion. Human monocytes were isolated as previously described. (25)(26)(27)29) The remaining blood was drawn into heparinized blood collection tubes. Human peripheral blood monocytes were isolated using the method of Fogelman et al. (30) with Ficoll/ Hypaque gradient centrifugation. The mononuclear cells were re-suspended in RPMI 1640 (Sigma) supplemented with 20% autologous serum, plated on to serum-treated 10-cm dishes and incubated for 2 h. Non-adherent cells were removed by washing three times with PBS, and adherent cells were then detached by incubation in PBS containing 10% autologous serum and 0.02% EDTA at 4°C for 30 min. The adherent cells were then washed extensively and re-suspended in RPMI 1640 supplemented with 10% autologous serum. They were then plated on 12-well plates and incubated for 10 days so that they would differentiate into monocyte derived macrophages (MDM). The culture media was changed every 3-4 days during this 10-days period.
Analysis of citrulline and arginine in plasma. Blood samples were rapidly centrifuged and deproteinized with a 5% (w/v) sulfosalicylic acid solution. The supernatant fractions were stored at -80°C for analysis of amino acids. Amino acids were separated and quantified by ion exchange chromatography using an amino acid autoanalyser (Amino Tac, JLC-300; Jeol Ltd, Tokyo, Japan) and the detection sensitivity was 125-500 nM. Amino acid analysis was performed using an LC-R-6 column (6.0 × 90 mm I,D., Mitsubishi Chemical Co., Tokyo, Japan). The mobile phase was performed using lithium citrate buffer solution. (28) The autoanalyzer system consisted of the reaction coil (0.25 × 10 m I.D.) and a 570 and 440 nm at 132°C.
Serum TC, TG, LDL C, HDL C, glucose and NO3. Serum total cholesterol (TC), triglycerides (TG), LDL-cholesterol (LDL-C) and HDL-C levels were determined by standard enzymatic methods (Measure L, Kyowa Mediceed, Tokyo, Japan). The serum concentration of glucose was determined using an Olympus AU 600 analyser. Blood nitrate (NO3) was quantified by the HPLC-UV method. (31,32) Statistical analysis. Statistical analyses were performed using the Stat View ver. 5.0 software package (SAS Institute Inc., NC). For the cell culture study, two-way factorial ANOVA ( Fig. 1A and B) or one-way ANOVA, followed by post-hoc analysis using Bonferroni/Dunn test, with a value of p<0.05 considered to be significant. All results were expressed as the mean ± SEM.

Results
Upregulation of ABCA1 and ABCG1 expressions by citrul line. The effect of citrulline on ABCA1 and ABCG1 mRNA levels in THP-1 macrophages was first investigated. As shown in Fig. 1A and B, 1 mM of citrulline increased ABCA1 and ABCG1 mRNA levels by 74.5 ± 21.5% (p<0.05) and 58.3 ± 22.0% (p<0.05), respectively. In contrast, citrulline did not affect SR-BI expression in THP-1 macrophages (data not shown). To further investigate an association between ABCA1 and ABCG1 expressions and NOS, we performed experiments using the NOS inhibitor L-NAME. To our surprise, citrulline-induced ABCA1 and ABCG1 mRNA expression with or without 5 mM of L-NAME in THP-1 macrophages ( Fig. 1C and D), indicating that citrulline-induced ABCA1 and ABCG1 expressions were NOindependent. Mirroring the increased mRNA levels, citrulline dose-dependently increased the protein levels of ABCA1 and ABCG1 (Fig. 1E).
Effect of ABCA1 and ABCG1 mRNA stability by citrulline.
To further explore the mechanisms by which the citrulline increased ABCA1 and ABCG1 mRNA levels in the macrophages, we investigated whether they transcriptionally induced these genes using Act D. As shown in Fig. 2A and B, treatment with 10 μg/ml of ActD did not abolish the inducible effects of citrulline on ABCA1 and ABCG1 expression in THP-1 macrophages. Further, ABCA1 and ABCG1 mRNA decay induced by ActD were inhibited in the presence of the citrulline ( Fig. 2C and D), thus indicating they enhanced mRNA stability of ABCA1 by 44.5 ± 7.0% (p<0.05) and ABCG1 by 39.1 ± 5.5% (p<0.05). These data indicated that citrulline enhanced mRNA stability of ABCA1 and ABCG1.
Citrulline increases serum citrulline and arginine, but has no effects on serum TC, TG, LDL C, HDL C, glucose or NO3.
Our next objective was to clarify the matter of whether the above in vitro observations translate into human physiology. As shown in Fig. 4, 1 week of citrulline consumption markedly increased its concentration, by 595% (p<0.05). Further, arginine and ornithine, which are metabolically linked with citrulline through the Krebs-Henseleit cycle, (33) were also increased, by 44.7% (p<0.05) and 12.4%, respectively. As expected, placebo consumption did not produce any changes. We also found that 1 week of citrulline consumption did not change other parameters, including plasma lipids, glucose, and NO3 (a metabolite of NO) (data not shown).
Increased ABCA1 and ABCG1 expression in MDM in pres ence of post citrulline sera. Individual changes in ABCA1 and ABCG1 mRNA levels in the MDM differentiated using autologous sera obtained before and after citrulline or placebo consumption are shown in Fig. 5. Although there was some individual variation, ABCA1 and ABCG1 mRNA levels were increased in the MDM treated with post-citrulline sera as compared to pre-citrulline cells and sera combinations by 95.9% (p<0.05) and 162% (p<0.05), respectively, in most study subjects. As expected, placebo did not affect the expression of the MDM genes. As shown in Fig. 6, parallel increases in ABCA1 and ABCG1 protein levels were observed. ABCA1 and ABCG1 protein levels in MDM cultured under post-citrulline sera were significantly increased, by 85.7 ± 24.1% (p<0.05) and 34.7 ± 10.5% (p<0.05), respectively ( Fig. 6C and D). Again, there was no difference between pre-and post-placebo cells and sera ( Fig. 6A and B).

Enhanced ApoA I and HDL mediated cholesterol efflux in
MDM treated with post citrulline sera. Finally, we performed an HDL-mediated cholesterol efflux assay using human MDM cultured in media containing sera obtained before and after citrulline consumption. Consistent with the increased ABCA1/G1 expressions observed above, ApoA-I and HDL-mediated cholesterol effluxes were also significantly increased, by 78.0% (p<0.05) and 11.0% (p<0.05), respectively, in MDM cultured under post-citrulline sera, as compared to pre-citrulline cells and sera ( Fig. 7A and B). Again, no changes were observed for placebo.

Discussion
There is a strong inverse correlation between plasma concentrations of HDL-C and risk of atherosclerotic cardiovascular disease. (34) Although the mechanism by which HDL may exert a direct protective effect against the development of atherosclerosis is not yet well understood, it has been postulated that HDL facilitates the efflux of cholesterol from peripheral tissues and transports it back to the liver in a process called RCT. (35) Here we showed, for the first time, that citrulline consumption lead to increased ApoA-I-and HDL-mediated cholesterol efflux from macrophages by inducing ABCA1 and ABCG1 expressions, both in an in vitro and ex vivo study.
With regard to mechanisms by which citrulline-mediated induction of ABCA1 and ABCG1 gene expression is transcriptionally regulated, preliminary results showed that citrulline did not increase these promoter activities of well-defined human ABCA1  (−940 to +110 bp relative to transcription start site) and ABCG1 (−1104 to +38 bp relative to transcription start site in exon 1) or (−1180 to +144 bp in exon 5) promoter-luciferase constructs (data not shown). (27,36,37) We then investigated effects of citrulline on mRNA stability of ABCA1 and ABCG1 by using ActD to find that citrulline enhanced ABCA1 and ABCG1 mRNA stability. Indeed, we showed that citrulline enhanced ABCA1 and ABCG1 mRNA stability (Fig. 2C and D), thus resulting in increased steady-state expression of these genes in the macrophage.
Consistent with the in vitro findings, the ex vivo human study showed that MDM differentiated under autologous post-citrulline sera enhanced apoA-I-and HDL-mediated cholesterol efflux by increasing ABCA1 and ABCG1 expression without changing plasma HDL-C levels. In theory, since ex vivo experiments can  provide a situation that is closer to the in vivo situation in humans than the in vitro setting, comparable results from in vitro and in vivo studies would provide a reasonable basis for hypothesizing that citrulline promotes RCT by enhancing cholesterol efflux via ABCA1/G1 pathways in humans in vivo. This has been highlighted as an important concept in the recent study by Rader et al. (11) which found that quantitative measures of macrophage RCT are more strongly associated with atherosclerotic disease morbidity than plasma HDL-C concentrations. Enthusiasm concerning cholesterol efflux and its modulation began about 10 years ago when ABCA1 was discovered as the mutation causing human HDL deficiency in Tangier disease. However, although research efforts have been intensely focused on enhancing ABCA1 function, drugs that achieve this are still not available. In the meantime, natural products that enhance cholesterol efflux might be realistic alternatives.
Several studies have reported an association between citrulline and anti-atherosclerosis. The activation of adhesion molecules elicited by oxidized LDL and the uptake of oxidized LDL by macrophages are key steps in the progression of atherothrombotic cardiovascular diseases. (38) L-citrulline itself is an efficient radical scavenger and is a strong antioxidant. (39) In addition, NO has potent antioxidant properties that exert anti-inflammatory effects and can inhibit LDL oxidation. (40) Recently, Morita et al. (41) reported that 800 mg/day of L-citrulline intake for 8 weeks reduced serum oxidized LDL and lectin-like oxidized LDL receptor 1 (LOX-1) ligand containing ApoB, an indicator of the biological activity of oxidized lipoprotein binding to LOX-1 in humans with vasospastic angina. NO is a widespread signaling molecule in the cardiovascular system, which functions in multiple ways to protect against the initiation and progression of atherosclerosis. (42)(43)(44) Interestingly, in our study, citrulline selectively induced ABCA1 and ABCG1 independently of L-NAME. Also, citrulline consumption did not induce any changes in serum NO3 levels (data not shown), indicating that citrulline increases HDL-mediated cholesterol efflux from macrophages via an NOSindependent pathway. Thus, overall, our results do not completely exclude the possibility that enhanced cholesterol efflux due to citrulline is NO dependent. We will therefore need to perform an in vivo RCT assay using NOS knockout mice as a further study.
There are several limitations in the present study. First, as mentioned above, the subjects in the human study were healthy volunteers. Therefore, it is difficult to generalize the findings to dyslipidemic patients who are at increased risk for atherosclerosis. Second, 1 week of oral citrulline represents an acute effect, which may differ from the effect of taking it habitually. Third, the present study did not test for the presence of other factors potentially affecting cholesterol efflux nor factors along the pathway by which citrulline consumption could affect NO. Likewise, arginine might increase cholesterol efflux by citrulline administration because citrulline administration increased serum arginine concentration, although arginine concentration is very lower than citrullin concentration. Finally, we did not directly examine whether citrulline actually removed oxidized cholesterol, oxidized fatty acids or oxidized phospholipids from peripheral tissues. Such interesting a posteriori considerations deserve future studies.
In conclusion, the comparable results from the in vitro and ex vivo studies indicate that potential anti-atherogenic properties of citrulline could be explained, at least in part, as being due to upregulated cholesterol efflux from macrophages mediated through ABCA1 and ABCG1 pathways. Combined with its previouslyproven endothelial NO-related properties, this favorable effect of citrulline on HDL suggests great synergistic potential with regard to cardioprotective properties.

Acknowledgments
We thank Fumiko Watanabe of KYOWA HAKKO Bio Co., Ltd., for her helpful assistance. Supported, in part, by a research grant from KYOWA HAKKO Bio Co., Ltd. Placebo and Citrulline for the ex vivo study were kindly donated by KYOWA HAKKO Bio Co., Ltd.