A Growth Hormone-Releasing Peptide that Binds Scavenger Receptor CD36 and Ghrelin Receptor Up-Regulates Sterol Transporters and Cholesterol Efflux in Macrophages through a Peroxisome Proliferator-Activated Receptor γ-Dependent Pathway

Abstract

Macrophages play a central role in the pathogenesis of atherosclerosis by accumulating cholesterol through increased uptake of oxidized low-density lipoproteins by scavenger receptor CD36, leading to foam cell formation. Here we demonstrate the ability of hexarelin, a GH-releasing peptide, to enhance the expression of ATP-binding cassette A1 and G1 transporters and cholesterol efflux in macrophages. These effects were associated with a transcriptional activation of nuclear receptor peroxisome proliferator-activated receptor (PPAR)γ in response to binding of hexarelin to CD36 and GH secretagogue-receptor 1a, the receptor for ghrelin. The hormone binding domain was not required to mediate PPARγ activation by hexarelin, and phosphorylation of PPARγ was increased in THP-1 macrophages treated with hexarelin, suggesting that the response to hexarelin may involve PPARγ activation function-1 activity. However, the activation of PPARγ by hexarelin did not lead to an increase in CD36 expression, as opposed to liver X receptor (LXR)α, suggesting a differential regulation of PPARγ-targeted genes in response to hexarelin. Chromatin immunoprecipitation assays showed that, in contrast to a PPARγ agonist, the occupancy of the CD36 promoter by PPARγ was not increased in THP-1 macrophages treated with hexarelin, whereas the LXRα promoter was strongly occupied by PPARγ in the same conditions. Treatment of apolipoprotein E-null mice maintained on a lipid-rich diet with hexarelin resulted in a significant reduction in atherosclerotic lesions, concomitant with an enhanced expression of PPARγ and LXRα target genes in peritoneal macrophages. The response was strongly impaired in PPARγ^+/− macrophages, indicating that PPARγ was required to mediate the effect of hexarelin. These findings provide a novel mechanism by which the beneficial regulation of PPARγ and cholesterol metabolism in macrophages could be regulated by CD36 and ghrelin receptor downstream effects.

MACROPHAGES PLAY A key role in the early events of atherogenesis by accumulating cholesteryl ester and oxidized derivatives through increased uptake of oxidized low-density lipoprotein (oxLDL) by scavenger receptors such as CD36, thereby contributing to excessive lipid loading and atherogenic formation of foam cells. In addition to initiating a proinflammatory response in monocytes/macrophages, such internalization of oxLDL by CD36 provides a source of oxidized fatty acids and oxysterols that serve as endogenous ligands for the respective activation of the nuclear receptors peroxisome proliferator-activated receptor (PPAR) and liver X receptor (LXR) (1, 2).

The identification of several natural ligands, such as prostanoid derivatives and polyunsaturated and oxidized forms of fatty acids, have depicted all three isoforms of PPARs as important metabolic fatty acid sensors (3). Of these, PPARγ is highly expressed in macrophages, and studies using synthetic PPARγ ligands of the thiazolidinedione family have provided a role for PPARγ in exerting antiatherogenic effects in animal models of atherosclerosis (4–6). PPARγ was shown to increase in a positive feedback fashion the expression of scavenger receptor CD36, an effect strongly associated with excessive lipid accumulation and macrophage foam cell formation (7, 8). However, PPARγ activation also produces opposite effects by promoting the expression of LXRα, which, upon activation by endogenous ligands such as oxysterols, induces the expression of genes involved in peripheral cholesterol efflux and transport from macrophages, such as apolipoprotein E (apoE) and ATP-binding cassette transporters ABCA1 and ABCG1 (1, 2, 9, 10). The metabolic cascade involving PPARγ and LXRα is therefore proposed as an attempt by the macrophage to enhance its ability to remove oxLDL from the vessel wall and shunt free cholesterol into the reverse cholesterol transport pathway, thus providing a protective effect against plaque formation in vivo. Hence, interfering with the balance between macrophage lipid uptake and efflux would be predicted to influence atherosclerotic lesion formation.

The GH-releasing peptides (GHRPs) belong to a class of small synthetic peptides known to stimulate GH release through binding to the GH secretagogue-receptor 1a (GHS-R1a), a G protein-coupled receptor originally identified in hypothalamus and pituitary (11). The endogenous ligand of GHS-R1a was later recognized as ghrelin, a 28-amino-acid peptide primarily expressed in the mucosal layer of the stomach but also in several other tissues (12). The peripheral distribution of GHS-R1a in tissues such as heart, adrenals, fat, prostate, and bone has supported physiological roles of ghrelin and GHRPs not exclusively linked to GH release. For example, GH-independent effects on orexigenic properties, fat metabolism, bone cell differentiation, and hemodynamic control have been reported for ghrelin and GHRPs (13, 14). Also, in conditions in which GH release was not promoted or in GH-deficient animals, the GHRP hexarelin was shown to feature cardioprotective effects by preventing ventricular dysfunction (15, 16) and by protecting the heart from damages induced by postischemic reperfusion (17). Our recent findings demonstrating that hexarelin serves as a ligand for CD36 receptor in myocardium (18), and that it interferes with the binding of oxLDL by sharing the same interaction site on CD36 (19), have prompted us to evaluate the ability of hexarelin to modulate macrophage cholesterol metabolism.

Here, we demonstrate that hexarelin promotes cholesterol efflux from macrophages through the enhanced expression of LXRα and ABC transporters, an effect severely impaired in treated peritoneal macrophages isolated from PPARγ heterozygote mice, implying an essential role for PPARγ in mediating the response to hexarelin. The interaction of hexarelin with GHS-R1a, and to a lesser extent with CD36, resulted in transcriptional activation of PPARγ, an effect also observed with ghrelin, the endogenous ligand of GHS-R1a. Using apoE-null mice, we show that the beneficial effect of hexarelin on macrophage cholesterol metabolism also occurred in vivo with a significant reduction in atherosclerotic lesions in treated mice. These findings therefore support a regulatory pathway by which CD36- and GHS-R1a-mediated effects may translate into antiatherosclerotic properties.

RESULTS

Hexarelin Reduces Lipid Accumulation and Increases Cholesterol Efflux in Human THP-1 Macrophages

Human monocytic THP-1 cells were differentiated into adherent macrophage-like cells by the addition of phorbol myristate acetate (PMA) and were tested for CD36 and GHS-R1a mRNA and protein expression (Fig. 1A). To determine whether hexarelin could modulate the scavenging function of CD36 to internalize oxLDL into macrophages, THP-1 cells were incubated with oxLDL to promote lipid accumulation and were then treated with hexarelin. As determined by staining neutral lipids with Oil red O, the increase in size and number of lipid vesicles upon oxLDL loading was markedly diminished by treating cells with hexarelin (Fig. 1B). Hexarelin reduced the lipid loading in a significant and dose-dependent manner with a respective 23% and 40% decrease at 10⁻⁷ and 10⁻⁵m hexarelin (Fig. 1C), suggesting that hexarelin prevents lipid accumulation from oxLDL uptake in THP-1 cells. Loading macrophages with oxLDL is known to provide the necessary ligands that promote PPARγ activation and cholesterol removal (8, 9). To test whether hexarelin by itself could promote cholesterol removal from macrophages, THP-1 cells were labeled with tritiated cholesterol and efflux of cholesterol was monitored in absence of oxLDL. As shown in Fig. 1D, treatment of THP-1 cells with 10⁻⁷m hexarelin resulted in a significant 30% increase in cholesterol efflux to extracellular high-density lipoprotein (HDL) acceptors, compared with untreated cells.

Fig. 1.

Effects of Hexarelin on Lipid Accumulation and Cholesterol Efflux in Human THP-1 Macrophages A, RNA and protein levels of CD36 and GHS-R1a receptors were measured in predifferentiated and PMA-differentiated THP-1 cells. B, Representative images of differentiated THP-1 cells incubated with 50 μg/ml oxLDL for 24 h in the presence or absence of 10⁻⁷m hexarelin. Lipids were stained with Oil red O. C, THP-1 cells were incubated for 24 h with hexarelin and/or 50 μg/ml oxLDL as indicated. Lipids were stained with Oil red O, extracted, and quantified by photometry. Data are presented as mean ± sem of at least three separate experiments. *, P < 0.02; and **, P < 0.01 vs. controls. D, Hexarelin promotes cholesterol efflux from THP-1 cells. THP-1 cells were loaded with ³H-cholesterol and incubated with vehicle or 10⁻⁷m hexarelin. Efflux was measured 16 h after addition of 50 μg/ml HDL acceptors. Data are presented as the percentage (±sem) of efflux relative to the total radioactivity in the cells and medium calculated from three independent experiments. *, P < 0.02.

Hexarelin Increases the Metabolic PPARγ-LXRα-ABC Pathway in THP-1 Macrophages

To evaluate the cellular mechanism by which cholesterol is removed from macrophages treated with hexarelin, we measured the expression of components of the PPAR-LXR-ABC pathway described to be involved in cholesterol efflux (9). Although differentiating THP-1 into macrophage-like cells with PMA contributed to increase the expression of PPARγ and LXRα, higher mRNA levels were consistently measured for PPARγ and LXRα after treatment of THP-1 cells with hexarelin, reaching, respectively, a 5.4- and 3.3-fold increases at the 48-h treatment period, which correspond to 2.5- and 1.9-fold increases when normalized to untreated differentiated cells (Fig. 2, A and B). Consistent with these changes, we also measured a 1.9- and a 3.2-fold increase in expression levels of, respectively, ABCA1 and ABCG1 in differentiated cells. These results indicate that hexarelin can induce the expression of components of the PPARγ-LXRα-ABC pathway in THP-1 macrophages. Similarly, the expression of apoE, also described as an LXR target (10) with ABCA1 and ABCG1, was significantly increased under the same conditions. To ascertain whether these effects correlated with increases in protein levels, we observed by Western analysis a marked increase in PPARγ, LXRα, and transporters ABCA1 and ABCG1 protein levels upon treatment of cells for 24 and 48 h with hexarelin (Fig. 2C). Consistent with our data on cholesterol efflux, these results indicate that THP-1 macrophages respond to hexarelin by increasing the expression of key markers involved in cholesterol removal.

Fig. 2.

Hexarelin Stimulates the PPARγ-LXRα-ABC Pathway in THP-1 Macrophages A, RT-PCR analysis from THP-1 cells treated or not with 10⁻⁷m hexarelin for 48 h. Representative images of mRNA expression are shown for the indicated genes. B, Time study of THP-1 cells treated with 10⁻⁷m hexarelin. Relative amounts of mRNA for the indicated genes were quantified by densitometry. Data are presented as relative fold changes (±sem) compared with undifferentiated cells, obtained from three to four separate experiments. C, Representative Western analysis of THP-1 cells treated with 10⁻⁷m hexarelin.

Hexarelin Signals to Promote PPARγ Transcriptional Activity

To determine whether the up-regulation of PPARγ and LXRα downstream targets in response to hexarelin was dependent upon PPARγ activity in macrophages, we tested the effect of the highly selective PPARγ inhibitor GW9662. We found that the increases in protein levels of PPARγ, LXRα, and ABCG1 in THP-1 cells treated with 10⁻⁷m hexarelin were completely abrogated in the presence of increasing doses of GW9662 (Fig. 3A). ABCA1 protein levels were also diminished but not to basal levels, suggesting a residual regulation by possibly other PPAR isoforms, as opposed to ABCG1, which was found to be more dependent on PPARγ (20). Our data therefore suggest that the transcriptional activation of PPARγ is necessary to mediate the effects of hexarelin. To further elucidate whether hexarelin could signal to activate PPARγ in macrophages, we used a cell reporter luciferase assay to directly assess PPARγ activity. Murine RAW264.7 macrophages, which express both CD36 and GHS-R1a receptors (data not shown), were transfected with a Gal4-PPARγ expression plasmid in the presence of a UAStkLuc reporter containing the luciferase gene under the control of a Gal4 response element. As a control, treatment of transfected cells with the PPARγ-selective ligand troglitazone resulted in a 2.1-fold increase in PPARγ activity, whereas similar activation levels were also observed upon treatment with 10⁻⁷ and 10⁻⁵m hexarelin, suggesting that hexarelin could promote PPARγ activity in macrophages (Fig. 3B). Because hexarelin has the potential to act through CD36 and/or GHS-R1a, we evaluated the ability of hexarelin to activate PPARγ through each receptor using human 293 cells, which do not express either receptor. We found that, although transfection of 293 cells with either CD36 or GHS-R1a expression plasmid did not result in activation of PPARγ, addition of 10⁻⁷m hexarelin contributed to increase PPARγ activity (Fig. 3C), suggesting that both receptors needed to be activated to signal to PPARγ. In addition, these effects were shown to be additive because expression of CD36 and GHS-R1a together conferred a stronger activation of PPARγ. Similarly, the potential of CD36 and GHS-R1a to translate the effects of hexarelin on PPARγ activity was also monitored using a PPREtkLuc reporter in cells transiently expressing PPARγ and RXRα. As shown in Fig. 3D, treating transfected 293 cells with increasing doses of hexarelin enhanced PPARγ activity in the presence of either CD36 or GHS-R1a, suggesting that expression of either receptor was necessary to mediate the activation of PPARγ by hexarelin. Given such agonist-independent activation of PPARγ, we also tested the response of the other PPAR isoforms to hexarelin signaling. Similar to what we observed for PPARγ, both PPARα and PPARβ/δ were activated in cells expressing GHS-R1a and treated with hexarelin (Fig. 3E). However, using the same conditions, no significant activation of LXRα or LXRβ was observed in response to hexarelin. These data suggest that activation of GHS-R1a by hexarelin results in the selective activation of PPAR isoforms, without directly modulating LXR activity.

Fig. 3.

Hexarelin Signals to Activate PPARγ A, Inhibition of PPARγ activity impairs the increase in LXRα, ABCA1, and ABCG1 protein levels by hexarelin. PMA-differentiated THP-1 cells were treated with 10⁻⁷m hexarelin and with 1 and 5 μm of the PPARγ inhibitor GW9662 for 48 h and analyzed by Western blot. B, Murine RAW264.7 macrophage cells were transfected with the expression plasmid coding for Gal4-PPARγ in the presence of the reporter gene UAStkLuc. Cells were then treated with 1 μm troglitazone (Tro) or hexarelin for 16 h before harvest. Results are expressed as fold changes in luciferase activity compared with untreated cells, determined from duplicates from at least three independent experiments. C, Activation of PPARγ by hexarelin through CD36 and GHS-R1a receptors. Human 293 cells were transfected with Gal4-PPARγ plasmid and UAStkLuc reporter, and treated with 10⁻⁷ or 10⁻⁵m hexarelin for 16 h before harvest. Cells were cotransfected with CD36 and/or GHS-R1a expression plasmid as indicated. Normalized reporter activity was determined and presented as fold change ± sem compared with untreated cells set at 1.0. D, Activation of PPARγ by hexarelin is dose dependent. 293 cells were transfected with a PPREtkLuc reporter and expression vectors for PPARγ and RXRα in the absence (open circle) or presence of a plasmid coding for GHS-R1a (filled circles) or CD36 (open squares). After transfection, cells were treated with increasing doses of hexarelin and harvested for luciferase activity. Each point represents the percentage changes (mean ± sem) compared with untreated cells set at 100%. E, Hexarelin can promote the activity of PPARs but not LXRs. 293 cells were transfected with Gal4 fusions of PPAR or LXR isoforms as indicated, in the presence of the UAStkLuc reporter and GHS-R1a plasmid. Cells were treated with receptor ligands or hexarelin for 16 h before harvest for luciferase activity. Ligands used are WY14643 (1 μm) for PPARα, carbacyclin (1 μm) for PPARδ, and 22R-hydroxycholesterol (5 μm) for LXRs. Results are expressed as in B.

The Activation of PPARγ through GHS-R1a Activation Is Activation Function (AF)-1 Dependent

The transcriptional ability of most nuclear receptors to regulate gene expression is dependent upon their AF-1 and AF-2 domain activity. Although AF-2 activity is triggered by ligand binding to promote coregulator recruitment, it is also recognized that receptor AF-1 activity can mediate transcriptional activation in response to intracellular signaling transduction pathways. Our results on the activation of PPARγ by hexarelin through the GHS-R1a receptor suggest that PPARγ activity might be under the control of such intracellular signaling by GHS-R1a. To determine whether AF-1 activity is involved in the PPARγ response to GHS-R1a activation, we generated a Gal4-ABCDγ [amino acids (aa) 1–254] truncated version of hPPARγ, in which the entire ligand binding domain was deleted. As shown in Fig. 4A, using 293 cells transfected with GHS-R1a, hexarelin contributed to increase the Gal4-ABCDγ activity to a similar extent as the full-length PPARγ. The activation levels of the ABCDγ construct in response to hexarelin were dose dependent in GHS-R1a-expressing cells (Fig. 4B). These data therefore suggest a role of AF-1 in the regulation of PPARγ activity through GHS-R1a activation by hexarelin.

Fig. 4.

GHS-R1a-Dependent Transactivation of PPARγ by Hexarelin Is Ligand-Independent A, Hexarelin promotes PPARγ AF-1 activity. Human 293 cells were transfected with Gal4 fusions of full-length PPARγ or a ligand binding domain-truncated ABCDγ (aa 1–254) and the UAStkLuc reporter in the presence or absence of GHS-R1a. After transfection, cells were treated with 10⁻⁷m hexarelin or left untreated for 16 h before harvest. Results are expressed as fold changes compared with untreated cells, which were arbitrarily set at 1.0 for each PPARγ construct. B, Dose response of cells transfected with Gal4-ABCDγ and treated with increasing concentrations of hexarelin as indicated. Cells were either mock transfected (open circles) or transfected (filled circles) with a GHS-R1a plasmid. Each point represents the percentage changes (mean ± sem) compared with untreated cells set at 100%. C, Hexarelin promotes PPARγ phosphorylation in THP-1 macrophages. THP-1 cells were differentiated, serum-deprived, and treated with hexarelin for the indicated time. Cell extracts were immunoprecipitated with an antibody against PPARγ and analyzed by Western blot using an antiphosphoserine antibody. PPARγ content was also monitored by Western analysis.

Hexarelin Promotes Phosphorylation of PPARγ in THP-1 Macrophages

Posttranslational modification such as phosphorylation is well known to regulate the AF-1 activity of many nuclear receptors, including PPARγ (21). Given our results on the effects of hexarelin to regulate the AF-1 activity of PPARγ, we next determined whether hexarelin could modulate the phosphorylation of PPARγ. Using differentiated THP-1 macrophages, we found an increase in serine phosphorylation of PPARγ in response to hexarelin treatment, as determined by immunoprecipitation of endogenous PPARγ and Western analysis using an antiphosphoserine antibody (Fig. 4C). Such enhanced PPARγ phosphorylation was readily observed after 5 min of treatment with hexarelin and in a dose-dependent manner, as compared with untreated cells, indicating that hexarelin may induce kinase activation within that period.

Hexarelin Differently Affects the Expression and Promoter Occupancy of CD36 and LXRα Genes by PPARγ in THP-1 Cells

Given the potential role of GHS-R1a and CD36 receptors to transduce the effect of hexarelin in macrophages, we wished to determine whether their expression was modulated under these conditions. The mRNA and protein levels of GHS-R1a were not significantly modified in differentiated THP-1 cells treated with hexarelin (Fig. 5, A and B), indicating that GHS-R1a expression was apparently not under the regulation of PPARγ. In the same conditions, CD36 expression was also not affected by hexarelin, with a slight decrease in mRNA levels and no change in its protein steady-state levels. The expression of SR-A, another scavenger receptor that mediates internalization of modified LDL, also remained unaffected, suggesting that macrophages treated with hexarelin may not result in enhanced lipid uptake through CD36 and SR-A scavenger receptors. The apparent down-regulation in CD36 mRNA levels was rather surprising because CD36 has been described to be up-regulated by ligands of PPARγ in macrophages (7, 8). To further investigate the mechanism by which this unexpected regulation of CD36 by hexarelin may result, we performed chromatin immunoprecipitation (ChIP) assay to assess the relative occupancy by PPARγ onto the promoter region of the CD36 gene, in comparison with LXRα, which, in contrast to CD36, was up-regulated in THP-1 cells treated with hexarelin (Fig. 2, A and B). The CD36 and LXRα promoters were described to contain each a functional PPAR response element (PPRE) that mediates their enhanced expression by PPARγ ligands (7, 22). After treatment of THP-1 cells, chromatin was immunoprecipitated using an anti-PPARγ antibody, and the relative amount of the CD36 and LXRα promoter region that contains the PPRE was assessed by PCR using specific primers, as depicted in Fig. 5C. As predicted, both CD36 and LXRα promoters were found to be occupied by PPARγ in a greater extent in response to the PPARγ agonist troglitazone. We also obtained a similar rise in the occupancy of LXRα promoter in cells treated with hexarelin, indicating that LXRα up-regulation by hexarelin may result from a preferred recruitment of PPARγ to the LXRα promoter. However, the occupancy of the promoter region of CD36 by PPARγ was slightly diminished in response to hexarelin (Fig. 5C), consistent with the changes in CD36 mRNA expression. These findings suggest a selective mechanism used to differently regulate PPARγ-targeted genes in response to hexarelin.

Fig. 5.

The Expression of CD36 and GHS-R1a Is Not Increased by Hexarelin in THP-1 Macrophages A, THP-1 cells were treated with 10⁻⁷m hexarelin for 48 h and analyzed for GHS-R1a, CD36, and SR-A expression by RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase expression was used as a loading control. B, Representative Western analysis of GHS-R1a and CD36 receptors in THP-1 cells treated as in A. β-Actin was used as a loading control. C, ChIP analysis of PPARγ recruitment to CD36 and LXRα promoters in response to hexarelin. THP-1 cells treated with troglitazone (Tro) or hexarelin for 3 h were subjected to ChIP analysis using an antibody against PPARγ. PCR was performed on immunoprecipitated DNA from samples and on total DNA extracted (input), using pairs of primers that cover the PPRE-containing regions of CD36 and LXRα gene promoters as shown. The fold changes compared with untreated THP-1 cells represent the mean of two independent experiments.

In Vivo Activation of the PPARγ-LXRα-ABC Pathway in Peritoneal Macrophages from Mice Treated with Hexarelin

Concurrent with our findings that hexarelin can promote activation of the PPAR-LXR-ABC cascade in cultured THP-1 macrophages, we next addressed the question as to whether hexarelin might promote similar effects in vivo using apoE-null mice, which have been widely studied for the development of atherosclerosis, because they develop spontaneous atherosclerotic lesions and fibrous plaques at the aortic root and branch points, similar to what occurs in human atherosclerosis (23, 24). We therefore maintained apoE^−/− mice on a high-fat, high-cholesterol diet for 12 wk, a condition known to promote atherosclerotic plaque formation, and then we treated them daily with either saline or hexarelin. The concentration of 100 μg/kg·d hexarelin used in our study was reported to not elicit GH release and therefore prevented any undesired effects of GH (Ref. 16 and data not shown). Chronic treatment of mice with hexarelin did not significantly change their weight and food intake, and no adverse health problems were noticed throughout the study. We measured a 10% decrease in total cholesterol and a 33% increase in HDL-associated cholesterol in hexarelin-treated mice (data not shown), which suggests that although these changes did not reach statistical significance, hexarelin is likely to promote circulating cholesterol into the HDL fraction. Plasma triglycerides were not affected, indicating that hexarelin did not induce hypertriglyceridemia, as is often observed with the use of LXR ligands (2). To further assess the effect of hexarelin in vivo, we observed that macrophages collected from the peritoneal cavity of apoE^−/− mice fed a cholesterol-rich diet and treated with hexarelin had a reduced ability to accumulate lipids in response to oxLDL loading compared with controls (Fig. 6A). More noticeably, we found that these changes were accompanied by an increase in the expression of PPARγ (7.5-fold), LXRα (2.9-fold), ABCA1 (1.9-fold), and ABCG1 (1.5-fold) in oxLDL-loaded macrophages from mice treated with hexarelin compared with the saline group (Fig. 6B). In the same conditions, a slight decrease in CD36 expression was observed, whereas expression of SR-A and GHS-R1a remained unchanged. To preclude any undesired effects of the apoE-null phenotype and also to avoid possible activation of PPAR and LXR by exogenous ligands provided with loading of oxLDL, we performed RT-PCR analysis on macrophages collected from wild-type C57Bl/6 mice (i.e. the same genetic background as the apoE-null), without prior activation with oxLDL. In these conditions, maximal increases for PPARγ (2.8-fold), LXRα (1.8-fold), ABCA1 (2.4-fold), ABCG1 (2.1-fold), and apoE (2.4-fold) were obtained in macrophages treated with hexarelin compared with control, whereas CD36 expression remained unaffected (Fig. 6C). These changes also correlated with protein levels, notably for LXRα and ABCG1 (Fig. 6D). These findings are consistent with the expression profile seen in THP-1 cells (Figs. 2A and 5A) and also indicate that the macrophage PPARγ-LXRα-ABC pathway could be activated in vivo by hexarelin. Using isolated macrophages from apoE-null mice, we found that the cholesterol efflux was significantly increased in cells treated with hexarelin compared with untreated cells (Fig. 6E). Although the use of peritoneal macrophages may not truly reflect the actions of artery wall macrophages, these results suggest that hexarelin may be sufficient by itself to favor protective mechanisms within macrophages through activation of the PPAR-LXR-ABC pathway and cholesterol removal.

Fig. 6.

Activation of the PPARγ-LXRα-ABCA1/G1 Pathway in Peritoneal Macrophages and Reduction of Atherosclerotic Lesions in apoE-Null Mice Treated with Hexarelin A, Representative images of Oil red O-stained peritoneal macrophages from apoE^−/− mice kept on a high-fat, high-cholesterol diet for 6 wk and treated with daily sc injections of 0.9% NaCl or 100 μg/kg·d hexarelin. Thioglycolate-elicited macrophages were collected from treated mice and incubated in vitro with oxLDL before lipid staining. B, Enhanced PPARγ-LXRα-ABC pathway in peritoneal macrophages from apoE^−/− mice treated with hexarelin. apoE-null mice were treated as in A, and peritoneal macrophages were collected after injection of oxLDL or saline and subjected to RT-PCR analysis for the indicated genes. C, Hexarelin promotes the PPARγ-LXRα-ABC pathway in C57Bl/6 mouse peritoneal macrophages. Macrophages were collected from C57Bl/6 mice maintained on a standard diet and treated in culture with increasing amounts of hexarelin for 24 h. Cells were harvested and RT-PCR analysis was performed as in B. D, Macrophages were isolated and treated as in C and harvested for Western analysis. E, Hexarelin increases cholesterol removal from mouse macrophages. Cholesterol efflux to HDL was determined in peritoneal macrophages collected from apoE-null mice and incubated for 16 and 24 h with 10⁻⁷m hexarelin or vehicle. Also shown are the basal rates without HDL acceptors. Data are presented as the percentage (±sem) of efflux relative to the total radioactivity in the cells and medium calculated from four independent experiments. *, P < 0.05 vs. respective controls. F, Reduction of atherosclerotic lesions in apoE-null mice fed a high-fat, high-cholesterol diet for 12 wk and treated with hexarelin as compared with mice treated with saline. The entire aortic root was dissected and stained for lipids using Oil red O for en face analysis. G, Quantification of aortic tree lesion area in mice treated with hexarelin compared with controls. Aortic surface covered by Oil red O-stained lesions was quantified and expressed as a percent of the total aortic area. *, P < 0.01 vs. controls.

Hexarelin Protects apoE-Null Mice from Developing Atherosclerotic Lesions

We next addressed the question of whether the response of macrophages to hexarelin could correlate with a beneficial effect on plaque formation induced in apoE-null mice maintained on a lipid-rich diet for 12 wk. En face analysis of aortic tree sections stained with Oil red O showed a significant reduction of 28% in atherosclerotic lesions in hexarelin-treated mice compared with controls (Fig. 6, F and G). These results demonstrate that hexarelin could reduce plaque formation in an animal model of atherosclerosis.

PPARγ Is Fully Required to Mediate the Response to Hexarelin in Peritoneal Macrophages

The complete disruption of the PPARγ gene causes embryonic lethality in mice and therefore precludes obtaining mature macrophages that lack PPARγ (25, 26). To partly circumvent this, we isolated macrophages from genetically altered PPARγ^+/− mice, which are viable and show an impaired PPARγ function (27), and performed expression analysis in response to hexarelin. We observed that mRNA levels of LXRα, ABCA1, ABCG1, and apoE, all shown to be potently up-regulated in wild-type macrophages, remained mostly unaffected (fold increases ranged from 0.8–1.4) in PPARγ^+/− macrophages treated with hexarelin (Fig. 7A). The protein levels of LXRα and ABCG1 were also unaffected in treated PPARγ^+/− macrophages (Fig. 7B). This impaired response in PPARγ^+/− macrophages strongly supports a functional role for PPARγ in mediating the effect of hexarelin in macrophages.

Fig. 7.

Activation of the LXRα-ABCA1/G1 Pathway by Hexarelin in Peritoneal Macrophages Is Dependent on PPARγ A, The activation of the LXR-ABCA1/G1 pathway by hexarelin is severely impaired in PPARγ^+/− macrophages. Peritoneal macrophages were collected from PPARγ^+/− mice maintained on a standard diet and treated in culture with hexarelin for 24 h. Cells were harvested and RT-PCR analysis was performed from two separate experiments. B, LXRα and ABCG1 proteins were not increased by hexarelin in PPARγ^+/− macrophages. PPARγ^+/− peritoneal macrophages were collected and treated as in A before being harvested for Western analysis. Equal amounts of proteins were loaded in each well.

DISCUSSION

Alterations in the rates of macrophage cholesterol uptake or efflux each have the potential to influence foam cell formation and the development of atherosclerotic lesions. Therefore, an understanding of the regulatory pathways that control cellular lipid flux may identify new opportunities for intervention in the process of lipid disorders. In this study, we demonstrate the ability of scavenger receptor CD36 and ghrelin receptor to enhance PPARγ transcriptional activity in response to hexarelin, a shared peptide ligand. These effects were associated with an enhanced expression of downstream mediators, such as LXRα, apoE, and ABCA1 and ABCG1 transporters, that coordinate cholesterol removal from treated mouse macrophages and a significant reduction in plaque formation in apoE-null mice maintained on a high-fat, high-cholesterol diet.

Although activation of PPARγ by selective agonists was shown to promote beneficial effects in cholesterol removal from macrophages and subsequent plaque reduction in atherosclerotic mice (2), our findings suggest that CD36 and/or GHS-R1a receptors may signal to enhance PPARγ activity resulting in similar effects on macrophage cholesterol metabolism. As such, given the ability of hexarelin to interfere with oxLDL binding to CD36 (19), thereby lowering the supply in oxidative fatty acids and other potential ligands of PPARγ, it seems unlikely that activation of PPARγ by hexarelin would depend on a greater intake of exogenous oxidized lipids by macrophages. In agreement with this, treatment of peritoneal macrophages with hexarelin in absence of oxLDL resulted in an increase of the PPARγ-LXRα-ABC pathway, suggesting that hexarelin signaling may be sufficient by itself to activate PPARγ. In addition, scavenger receptor SR-A, which also mediates modified LDL uptake by macrophages, was not up-regulated by hexarelin. Our observation that CD36 and/or GHS-R1a ligands enhanced PPARγ activity may therefore rely on more ligand-independent effects. Consistent with this, we observed that activation of GHS-R1a receptor with hexarelin, or its natural ligand ghrelin (unpublished observations), led to activation of a PPARγ construct missing the ligand binding domain, suggesting that such an effect may translate through activation of PPARγ AF-1 function. Ligand-independent activation of PPARγ has been reported to involve receptor posttranslational modifications such as phosphorylation, and our results that hexarelin can promote phosphorylation of PPARγ in THP-1 cells may therefore provide a basis on how PPARγ can respond to hexarelin signaling. However, the effects of phosphorylation on PPARγ activity have been reported to vary, often in opposite directions, given the stimulus, the kinase involved, the modified residue, and the cellular and promoter context (21). For example, PPARγ activity was enhanced in cells treated with insulin through a MAPK-dependent phosphorylation of its N-terminal region (28–30). Both ligand-dependent and -independent activation of PPAR isoforms was shown to require PPAR phosphorylation in response to protein kinase A activators (31). Although the signaling events triggered by the interaction of hexarelin with CD36 remain to be defined, CD36 has been shown to interact with the src kinases Lyn and Fyn to initiate downstream activation of MAPKs in response to ligands such as thrombospondin-1 and β-amyloid (32, 33). Similarly, activation of GHS-R1a by ghrelin was shown to initiate a signaling cascade leading to MAPK activation (34–36), and the neuroprotective effects of hexarelin in a rat model of neonatal hypoxia-ischemia were correlated with activation of Akt (37). On the other hand, mechanisms related to receptor phosphorylation have also been described to inhibit PPARγ activity. Phosphorylation of a specific site within its AF-1 domain resulted in an inhibition of ligand-activated PPARγ and reduced adipocyte differentiation (38–40). Treatment of THP-1 macrophages with TGFβ also resulted in PPARγ phosphorylation and inhibition with a decreased expression of CD36 (41). Therefore, the exact mechanism(s) by which PPARγ activity is modulated in response to CD36 and/or GHS-R1a ligands remain to be clearly defined. However, given the ability by which posttranslational modifications such as phosphorylation could regulate PPARγ transcriptional activity and that ligand-independent recruitment of transcriptional coregulators is favored by nuclear receptor phosphorylation (21, 42), our results suggest that such mechanism may contribute in the cellular response to hexarelin.

It is recognized that CD36 is considered proatherogenic. Studies using CD36-null mice have provided evidence that CD36 might favor atherogenesis (43, 44), and elevated levels of CD36 have been associated with foam cell formation and atherosclerosis in humans (45). Internalization of oxLDL through CD36 is known to provide a source of fatty acid derivatives that promote PPARγ activation and further increase in CD36 that results in massive entry of lipids in macrophages (1, 7, 8). Unexpectedly, we found that although PPARγ was activated by hexarelin, the expression of CD36 was not increased in cultured and in peritoneal macrophage cells, as opposed to LXRα, also described as a direct target for PPARγ (7, 9). The exact mechanism by which hexarelin exerts such apparent opposite regulation of CD36 and LXRα expression is not yet clear, but our results suggest that hexarelin signaling might disrupt CD36 up-regulation normally seen upon activation of PPARγ with ligands. Consistent with these findings, we observed in a ChIP assay a preferred occupancy of the LXRα promoter by PPARγ, as opposed to the CD36 promoter, in THP-1 cells treated with hexarelin. Activation of PPARγ by selective agonists has been shown to differently regulate downstream effectors in macrophage lipid metabolism. For example, troglitazone, in contrast to other PPARγ ligands, repressed the expression of ABCA1 while increasing CD36 in macrophages, suggesting paradoxical effects on gene expression upon PPARγ activation (46). More recently, although both PPARα- and PPARγ-selective ligands markedly reduced plaque formation in atherosclerotic mice, they induced an opposite pattern in CD36 expression in isolated peritoneal macrophages of treated mice (20). Clearly, the regulation of CD36 expression may not solely depend on PPARγ activity, as exemplified in this study and others using PPARγ-deficient macrophages (46). The potential of hexarelin to lower CD36 expression, as observed in mouse peritoneal macrophages either from treated animals or maintained in culture, was not as significant as in THP-1 cells, suggesting that in vivo other mechanisms might affect CD36 expression. Inflammatory cytokines and growth factors were described to variably regulate CD36 expression in vascular and peritoneal macrophages (47). These effects were reported to involve both transcriptional and posttranscriptional mechanisms, resulting in changes in the intracellular pool and surface expression of CD36 (47, 48). Therefore, we can expect that intracellular signaling triggered by hexarelin and possibly through GHS-R1a may likely be involved in limiting CD36 expression as opposed to PPARγ ligands. Consistent with this, disruption of insulin receptor signaling was shown to result in a posttranscriptional increase of CD36 in macrophages (49). Our observations that protein levels of CD36 were not significantly changed in macrophages treated with hexarelin or with another GHRP selective for CD36 (44) suggest that the cellular mechanism by which steady-state levels of CD36 are maintained but not increased is likely shared among the GHRP class of peptides. Altogether, the apparent different regulation of PPARγ targets may provide an interesting potential for hexarelin to up-regulate PPARγ-dependent activation pathway toward LXRα, ABC transporters, and cholesterol efflux in macrophages, without increasing CD36 receptor, and therefore limiting its ability to mediate excessive oxLDL uptake, to protect macrophages from becoming foam cells.

The role of the GHS-R1a receptor in atherosclerosis has not been well characterized. GHS-R1a is expressed in a variety of tissues and cell types including macrophages (50). Its natural ligand ghrelin and synthetic GHS were shown to mediate endocrine and nonendocrine activities such as GH release, orexigenic action, cardiovascular and antiproliferative effects, and influence gastroenteropancreatic functions and adipocyte differentiation (13). For example, treatment of rat adipocytes with ghrelin contributed to significantly increase the expression of PPARγ and adipogenesis (51). More recently, studies have emerged associating GHS-R1a ligands with antiinflammatory function in endothelial cells and macrophages. Both GHRP-2 and ghrelin were shown to prevent the endotoxin-induced release of IL-6 from peritoneal macrophages isolated from adjuvant-induced arthritic rats (52). In addition, ghrelin was reported to inhibit cytokine release and nuclear factor κB activity in human endothelial cells (53). With studies using PPAR isoform-null macrophages that have outlined the role of PPARs in mediating antiinflammatory processes (54, 55), and given the ability of PPAR ligands to inhibit the expression of inflammatory genes in macrophages and other vascular cells (2), it is tempting to speculate that activation of CD36 and/or GHS-R1a by hexarelin might impact the inflammatory response in macrophages through PPARγ or other PPAR isoforms. In support of such an expanded role of hexarelin toward all PPAR isoforms, we found that PPARα and PPARδ, but not LXR isoforms, were up-regulated, suggesting a selective response of PPARs to hexarelin. In addition, although it is likely that alterations in plasma cholesterol may contribute in part to the beneficial effects of hexarelin, the changes we measured did not obviously reflect the extent of lesion reduction, which may therefore support a role for antiinflammatory pathways in cooperating to the beneficial effects of hexarelin. Clearly, more studies will be needed to address this possibility.

In summary, we describe a novel regulatory pathway to promote the PPARγ-LXRα-ABC cascade in macrophages involving interaction with CD36 and ghrelin receptor. Such modulation requires PPARγ and is associated with enhanced cholesterol efflux by macrophages and reduction of lesions in atherosclerotic mice. Consequently, a detailed knowledge of the concerted modulation of CD36 and ghrelin receptor signaling pathways may help to provide additional strategies in pathologic conditions such as atherosclerosis.

MATERIALS AND METHODS

Cell Culture and Treatments

THP-1 cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum (FBS). Macrophage differentiation was initiated with the addition of 5 ng/ml PMA in culture medium for 48 h. Human embryonic kidney 293 cells and macrophage RAW264.7 cells were cultured in DMEM containing 5% and 10% FBS, respectively. Treatments with troglitazone (1 μm), hexarelin, and GW9662 (Sigma, St. Louis, MO) were replaced with fresh medium every 24 h. To prepare oxLDL, LDLs (1.5 μg/ml) isolated from human plasma (Intracel, Frederick, MD) were oxidized with 5 μm CuSO₄ for 2 h at 37 C. The extent of LDL oxidation ranged between 6 and 10 nmol malondialdehyde/mg of proteins as determined by the amount of thiobarbituric acid-reactive substances.

Lipid Staining

THP-1 cells were fixed with 3.7% formaldehyde/PBS and stained with Oil red O (Sigma). Quantification of lipid accumulation was achieved by extracting Oil red O from stained cells with isopropyl alcohol and measuring the OD of the extracts at 510 nm.

Cholesterol Efflux from Macrophages

THP-1 cells were differentiated as described above and labeled with 1 μCi/ml ³H-cholesterol for 48 h to allow for equilibration with cellular cholesterol. Cholesterol-loaded cells were washed and incubated in serum-free media containing 0.2% fatty acid free BSA, and efflux was initiated by adding 50 μg/ml HDL in the presence of 10⁻⁷m hexarelin or vehicle. The amount of ³H-cholesterol was measured by liquid scintillation spectrometry in the medium and in the cell lysate (intracellular content) after 16 h of treatment. Cholesterol efflux is presented as specific percentage efflux calculated from total counts of the medium and intracellular fractions. Cholesterol efflux was also determined in peritoneal macrophage cells isolated from apoE-deficient mice (see below). Cells were incubated for 2 h at a density of 10⁶ cells/well in DMEM containing 10% FBS and were washed to remove nonadherent cells before cholesterol efflux determination.

RNA Isolation and RT-PCR Analysis

Total RNA was isolated from THP-1 cells using TRIzol reagent (Invitrogen, Burlington, Ontario, Canada), and from mouse peritoneal macrophages using the RNeasy kit (QIAGEN Inc., Mississauga, Ontario, Canada), each according to the manufacturer’s protocol. RT-PCR analysis was performed as described (44), and relative signal intensities were determined with an image analyzer (Alpha Innotech, San Leandro, CA).

Antibodies and Immunoblotting Analysis

Antibodies to ABCA1 and ABCG1 were obtained from Novus Biologicals (Littleton, CO), PPARγ and LXRα were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and β-actin was obtained from Abcam (Cambridge, MA). The antibody against CD36 has been described (19). Antibodies against GHS-R1a were generated by immunizing rabbits with a peptide corresponding to positions 250–262 of human GHS-R1a using a protocol described previously (18). The antiphosphoserine antibody was from Chemicon (Temecula, CA). Immunoprecipitation and immunoblotting procedures were performed as described (44, 56).

Luciferase Assay

For transient transfection, 293 cells were seeded in DMEM supplemented with 5% charcoal-dextran-treated FBS, and plasmid constructs were introduced into cells using the calcium phosphate precipitation method essentially as described (42). Typically, a mixture containing 500 ng of a PPREtkLuc reporter plasmid, 50–100 ng each of pCMX-hPPARγ and -hRXRα, and 100 ng pcDNA vector encoding human GHS-R1a or CD36 were added in a total of 2 μg per well. Cells were also transfected with pCMX encoding a Gal4 DBD fusion with PPARγ or with a LBD-truncated ABCDγ (aa 1–254), and UAStkLuc reporter. Gal4 fusions with PPARα, PPARδ, LXRα, and LXRβ were also used in transfection. RAW264.7 cells were transfected using LipofectAMINE reagent (Life Technologies Inc.) according to the manufacturer’s instructions. After transfection, cells were refed with medium containing receptor ligands for 16 h and were harvested for luciferase activity. Luciferase values were normalized for transfection efficiency to β-galactosidase activity and expressed as relative fold response compared with controls. Luciferase assays were performed in duplicate in at least three independent experiments.

ChIP Assay

ChIP assays were performed as previously described (56). Differentiated THP-1 cells were grown at a density of 10⁷/100-mm dish and were treated with troglitazone or hexarelin for 3 h. Cells were cross-linked with 1% formaldehyde, washed, and resuspended in sodium dodecyl sulfate lysis buffer. Lysates were then sonicated, clarified, and subjected to immunoprecipitation using an anti-PPARγ antibody (Santa Cruz Biotechnology). Specific genomic DNA fragments from immunoprecipitated samples and inputs were quantitated by PCR using 2–5 μl of sample DNA solution and primer pairs encompassing the PPRE-containing regions of CD36 (7) and LXRα (22) promoters.

Animals

Both apoE-deficient mice and wild-type C57Bl/6 littermates were previously described (43) and were given a standard pelleted diet with water ad libitum. At 6 wk of age, male mice were housed individually and maintained on a high-fat, high-cholesterol diet (D12108, cholate-free AIN-76A semi-purified diet containing 40% wt/wt fat and 1.25% wt/wt cholesterol; Research Diets Inc., New Brunswick, NJ) for 12 wk with water ad libitum. During that period, mice were treated with sc injections of 100 μg/kg·d hexarelin, a dose known to not promote GH release (16), or 0.9% NaCl (vehicle). Age-matched PPARγ^+/− mice were maintained on standard diet with water ad libitum as described (27). All experimental procedures were carried out in accordance with the Institutional Animal Ethics Committee of the Université de Montréal, the Canadian Council on Animal Care guidelines, and the Cantonal Veterinary Service of the Canton of Vaud for use of experimental animals.

Macrophage Isolation and Foam Cell Assay

apoE-deficient mice were fed a high-cholesterol, high-fat diet for a period of 6 wk. Thioglycolate-elicited peritoneal macrophages were collected from both hexarelin-treated and control mice in saline containing 10 U heparin/ml. Peritoneal cells were cultured on sterile glass coverslip in DMEM containing 10% FBS. Adherent cells were incubated with 50 μg/ml oxLDL for 24 h, fixed in paraformaldehyde, and stained for neutral lipids with Oil red O. For RT-PCR analysis, apoE-null mice were maintained on a lipid-rich diet for a period of 12 wk and were treated daily with either saline or hexarelin. Before collection of macrophages, mice received an ip injection of oxLDL (250 μg/cavity) or 0.9% NaCl. RT-PCR was performed on macrophage RNA as described above. Expression studies were also performed on cultured peritoneal macrophages from age-matched wild-type C57Bl/6 and PPARγ^+/− mice without prior thioglycolate stimulation and injection of oxLDL. Peritoneal cells were collected and cultured in DMEM containing 10% FBS for 2 h. Cells were then treated with hexarelin or saline for 24 h and harvested for RT-PCR and Western analyses.

Histology and Morphometric Analysis of Lesions

For en face analysis, the entire aortic section from hexarelin-treated and control mice was dissected out using a stereo-microscope (NI-150; Nikon, Melville, NY), opened longitudinally from the heart to the iliac arteries, and the lesions were stained with Oil red O, as described previously (43). Morphometric evaluation of the aortic lesion areas was performed using a video image analysis software (Scion Corp., Frederick, MD). Data are expressed as the percentage of the total aortic surface area covered by lesions for each treatment.

Acknowledgments

The authors thank Vincent Giguere and Maria Febbraio for providing plasmids. We also acknowledge the expert technical assistance of Eve Marie Charbonneau and Elisabeth Joye. Hexarelin was a generous gift from Europeptides (Argenteuil, France).

A.D. holds a doctoral award from the Canadian Institutes of Health Research (CIHR) and A.R.-W. is supported by a doctoral award from the Natural Sciences and Engineering Research Council of Canada. A.T. is a New Investigator of the CIHR. This work was supported by the CIHR, the Canadian Foundation for Innovation, the Swiss National Science Foundation, and Ardanna Bioscience (Edinburgh, Scotland, UK).

Present address for R.A.: Ricerca Farmacologica, Sanofi Midy Research Center, Sanofi-Synthelabo S.p.A., via Piranesi 38, 20137 Milano, Italy.

Disclosure statement: The authors have nothing to disclose.

¹

R.A. and A.D. contributed equally to this work.

Abbreviations

 
• aa

  Amino acids;

 
• ABC

  ATP-binding cassette;

 
• AF

  activation function;

 
• apoE

  apolipoprotein E;

 
• ChIP

  chromatin immunoprecipitation;

 
• FBS

  fetal bovine serum;

 
• GHRP

  GH-releasing peptide;

 
• GHS-R1a

  GH secretagogue-receptor 1a;

 
• HDL

  high-density lipoprotein;

 
• LDL

  low-density lipoprotein;

 
• LXR

  liver X receptor;

 
• oxLDL

  oxidized LDL;

 
• PMA

  phorbol myristate acetate;

 
• PPAR

  peroxisome proliferator-activated receptor;

 
• PPRE

  PPAR response element.