Ghrelin Interacts with Human Plasma Lipoproteins

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Endocrinology 148(5):2355–2362 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2006-1281

Ghrelin Interacts with Human Plasma Lipoproteins Carine De Vriese, Mirjam Hacquebard, Franc¸oise Gregoire, Yvon Carpentier, and Christine Delporte Department of Biochemistry and Nutrition (C.D.V., F.G., C.D.) and L. Deloyers Laboratory for Experimental Surgery (M.H., Y.C.), Faculty of Medicine, Universite´ Libre de Bruxelles, B-1070 Brussels, Belgium Ghrelin, a peptide hormone produced predominantly by the stomach, stimulates food intake and GH secretion. The Ser3 residue of ghrelin is mainly modified by a n-octanoic acid. In the human bloodstream, ghrelin circulates in two forms: octanoylated and desacylated. We previously demonstrated that ghrelin is desoctanoylated in human serum by butyrylcholinesterase (EC 3.1.1.8) and other esterase(s), whereas in rat serum, only carboxylesterase (EC 3.1.1.1) is involved. The aims of this study were to determine the role of lipoprotein-associated enzymes in ghrelin desoctanoylation and the role of lipoproteins in the transport of circulating ghrelin. Our results show that ghrelin desoctanoylation mostly occurred in contact with low-density lipoproteins (LDLs) and lipoproteinpoor plasma subfractions. Butyrylcholinesterase and plate-

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let-activating factor acetylhydrolase (EC 3.1.1.47) were responsible for the ghrelin hydrolytic activity of the lipoproteinpoor plasma and LDL subfractions, respectively. Moreover, we observed that ghrelin is associated with triglyceride-rich lipoproteins (TRLs), high-density lipoproteins (HDLs), very high-density lipoproteins (VHDLs), and to some extent LDLs. In conclusion, we report that the presence of the acyl group is necessary for ghrelin interaction with TRLs and LDLs but not HDLs and VHDLs. Ghrelin interacts via its N- and C-terminal parts with HDLs and VHDLs. This suggests that, whereas TRLs mostly transport acylated ghrelin, HDLs and VHDLs transport both ghrelin and des-acyl ghrelin. (Endocrinology 148: 2355–2362, 2007)

HRELIN, A 28 AMINO acid peptide produced predominantly by the X/A-like cells of the stomach submucosa, is an endogenous ligand for the GH secretagogue receptor (GHS-R) 1a (1). Ser3-octanoylation of ghrelin is required for binding the GHS-R 1a and for its biological actions including stimulation of GH release from the pituitary, stimulation of food intake, induction of adiposity, control of gastric acid secretion and motility, cardiovascular actions, and modulation of cell proliferation (2). Since its discovery, other forms of ghrelin were purified from the stomach: desGln14-ghrelin resulting from an alternative splicing of rat ghrelin gene (3), decanoyl ghrelin (1–28), decenoyl ghrelin (1–28), octanoyl ghrelin (1–27), decanoyl ghrelin (1–27), and des-acyl ghrelin (1–27) produced by posttranslational processing of the ghrelin precursor in human stomach (4). Ghrelin circulates in the bloodstream mainly as a desacylated form, the des-acyl ghrelin, originally described as biologically inactive and unable to bind the GHS-R 1a. However, des-acyl ghrelin has been shown to exert several effects such as modulation of cell proliferation in prostate carcinoma cell lines (5), stimulation of adipogenesis (6), cardiovascular effects (7), inhibition of apoptosis in cardiomyocytes and endothelial cells (8), decrease of food intake, and gastric

emptying (9). These effects could be mediated by a stillunidentified ghrelin receptor, distinct from the GHS-R. Desacyl ghrelin could result from desoctanoylation of ghrelin or an incomplete octanoylation of the peptide, or both forms could be secreted via two differently regulated pathways. We previously showed that, in contact with human serum, ghrelin was desoctanoylated by butyrylcholinesterase and other esterase(s), whereas only carboxylesterase was involved in rat serum (10). Few studies suggested an interaction between high-density lipoproteins (HDLs) and circulating ghrelin (11, 12). Lipoprotein particles can bind hydrophobic drugs (cyclosporine, amphotericin B, halofantrine) as well as vitamins (A, E) (13) and also bind to enzymes, such as platelet-activating factor acetylhydrolase (PAF-AH) (14) and paraoxonase (PON) (15). A previous study suggested an interaction between ghrelin and HDLs (11), and we showed that ghrelin was desoctanoylated by butyrylcholinesterase and other unidentified esterase(s) in the serum (10). Therefore, the aims of this study were to determine the implication of lipoprotein-associated enzymes in ghrelin desoctanoylation, test the effects of esterase inhibitors on ghrelin stability in human plasma samples, and determine the role of lipoproteins in the transport of circulating ghrelin.

First Published Online February 8, 2007 Abbreviations: Apo, Apolipoprotein; BMI, body mass index; d, density; EC, Enzyme Commission; Ghr, ghrelin; GHS-R, GH secretagogue receptor; HDL, high-density lipoprotein; IR, immunoreactive; LDL, lowdensity lipoprotein; LPP, lipoprotein-poor plasma; PAF-AH, plateletactivating factor acetylhydrolase; PMSF, phenylmethylsulfonyl fluoride; PON, paraoxonase; RP-HPLC, reverse phase-HPLC; TFA, trifluoroacetic acid; TRL, triglyceride-rich lipoprotein; VHDL, very high-density lipoprotein. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

Materials and Methods Plasma pool Blood samples, obtained from healthy human volunteers [n ⫽ 11; seven females and four males; 21– 43 yr old, mean 32 ⫾ 2; body mass index (BMI) 19.5 to 29.7, mean 22.8 ⫾ 0.9] after informed consent, were collected in tubes containing EDTA (1 mg/ml) and centrifuged at 4 C in a Beckman J2.21 centrifuge (Beckman Instruments Inc., Fullerton, CA) at 2000 ⫻ g for 10 min. Plasma samples were pooled, and the various lipoprotein subfractions were immediately separated as described thereafter. The protocols were approved by the local ethics committees for human studies.

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Endocrinology, May 2007, 148(5):2355–2362

De Vriese et al. • Ghrelin and Lipoproteins

Preparation of plasma lipoprotein subfractions

Effects of esterase inhibitors on ghrelin stability

Different plasma subfractions were separated by sequential ultracentrifugation of plasma in a Beckman L8 –55 ultracentrifuge using an angular Beckman 50.2Ti rotor (16). Each fraction was isolated sequentially by a successive increase of plasma density with potassium bromide. The triglyceride-rich lipoprotein (TRL) subfraction was obtained at density (d) ⬍ 1.019 g/ml (20 h; 5 C; 227,000 g). Low-density lipoprotein (LDL) subfraction was separated at 1.019 ⬍ d ⬍ 1.063 g/ml (20 h; 5 C; 227,000 g). HDL (1.063 ⬍ d ⬍ 1.210 g/ml) and the lipoprotein-poor plasma (LPP) (d ⬎ 1.210 g/ml) subfraction were also separated (48 h; 5 C; 227,000 g). After isolation, each subfraction, excepted LPP, was dialyzed in the dark against a saline solution [0.19 m with 0.1 mg/ml EDTA, 0.1 g/ml glycerol (pH 7.4)] with five changes of dialysis solution over a 12-h period. The plasma subfractions were frozen at ⫺80 C.

Plasma from 10 healthy volunteers (n ⫽ 10; six females and four males; 26 –59 yr old, mean 42 ⫾ 4; BMI 19.0 –29.3, mean 24.2 ⫾ 1.0) was prepared using disodium EDTA (1 g/liter) within 30 min after blood collection. Then 2 ml of plasma was immediately incubated without (control) or with phenylmethylsulfonyl fluoride (PMSF; 1 mm), NaF (59.5 mm), eserine salicylate (1 mm) for 1 h at room temperature. The incubation was stopped by the addition of 2 ml of 10% CH3CN/0.1% TFA. The samples were subsequently loaded onto a Sep-Pak C18 cartridge equilibrated with 5 ml of 3% CH3CN/0.1% TFA. The Sep-Pak was washed with 5 ml of 10% CH3CN/0.1% TFA, and the peptides were eluted with 2 ml of 50% CH3CN/0.1% TFA. The eluates were lyophilized in a Speed-Vac concentrator and submitted to ghrelin RIA (see below), therefore allowing measurement of octanoylated and total ghrelin levels.

Ghrelin degradation by plasma lipoprotein subfractions To remove NaCl, EDTA, and glycerol, the plasma lipoprotein subfractions were desalted on a PD-10 desalting column (GE Healthcare, Buckinghamshire, UK) and eluted in 50 mm Tris-HCl (pH 7.4). Ten micrograms of ghrelin were incubated with the plasma lipoprotein subfractions at 37 C for 240 min [time during which about 50% of ghrelin was degraded (10)] in the absence or presence of enzyme inhibitors, in a final volume of 700 ␮l. TRL, LDL, HDL, and LPP subfractions represented, in terms of protein concentration, 1.3, 18.4, 31.1, and 49.2% of the whole plasma, respectively. Therefore, to represent 200 ␮l of whole plasma as reported elsewhere (10), we incubated ghrelin with 442, 458, 456, and 156 ␮l of TRL, LDL, HDL, and LPP subfractions, respectively. The incubation was stopped by the addition of trifluoroacetic acid (TFA; 2% final concentration). An internal standard [2 nmol of rat atrial natriuretic peptide (1–28)] was subsequently added. Samples were centrifuged at 4 C for 30 min at 20,000 ⫻ g in a JA-21 Beckman centrifuge. Supernatants were collected and loaded onto a Sep-Pak C18 cartridge (Waters, Milford, MA) equilibrated with 3% CH3CN/0.1% TFA. The Sep-Pak was washed with 5 ml of 10% CH3CN/0.1% TFA, and the peptides were eluted with 2 ml of 50% CH3CN/0.1% TFA. The eluates were lyophilized in a Speed-Vac concentrator and subjected to HPLC analysis.

HPLC analysis of ghrelin degradation products The Sep-Pak eluates were subjected to reverse phase-HPLC (RPHPLC) on a C18 column Vydac 218TP54 (25 ⫻ 0.46 cm; Alltech, Wallisellen, Switzerland) equilibrated with 3% CH3CN/0.1% TFA. RPHPLC was performed using a gradient of CH3CN from 3 to 20% for 5 min, then from 20 to 60% for 20 min, and finally from 60 to 80% for 5 min in 0.1% TFA. The elution profile was monitored at 226 nm using a Shimadzu CR6A integrator. Fractions were collected and lyophilized. Degradation products were identified by electrospray mass spectrometry using a VG Platform ns 8230E (Micromass, Zellik, Belgium) and by their elution position. Each substrate, internal standard, and fragment was quantified in nanomoles using calibration curves obtained with the corresponding synthetic peptide.

Assay for PAF-AH activity and ghrelin hydrolytic activity Blood samples, obtained from healthy human volunteers (n ⫽ 11; seven females and four males; 25–59 yr old, mean 41 ⫾ 4; BMI 19.3–29.7, mean 24.0 ⫾ 1.0) after informed consent, were collected and centrifuged for 20 min at 2000⫻ g at room temperature. PAF-AH activity was measured by the method of Ellman et al. (17) using an Ultrospec Plus 4054 UV/visible spectrophotometer (LKB, Mt. Waverley, Australia). Ten microliters of human serum were added to 200 ␮m 2-thio PAF and 0.25 mm 5⬘,5⬘-dithiobis-2-nitrobenzoic acid in 100 mm Tris-HCl (pH 7.2) containing 1 mm EGTA. The absorbance was read at 414 nm every 30 sec for up to 10 min. The enzyme activity was calculated as micromoles of the product min⫺1䡠ml⫺1 (after correction for nonenzymatic hydrolysis of the substrate) using the extinction coefficient (13,600 m⫺1䡠cm⫺1) of the product. Ghrelin hydrolytic activity in human sera was determined as previously described (10).

Peptide extraction from plasma lipoprotein subfractions Plasma lipoprotein subfractions were heated at 100 C for 5 min in the presence of CH3COOH (1 m final concentration). Samples were centrifuged at 20,000 ⫻ g for 30 min at 4 C in a JA-21 Beckman centrifuge. Supernatants were collected and loaded onto a Sep-Pak C18 cartridge (Waters) equilibrated with 3% CH3CN/0.1% TFA. The Sep-Pak was washed with 5 ml of 10% CH3CN/0.1% TFA, and the peptides were eluted with 2 ml of 50% CH3CN/0.1% TFA. The eluates were lyophilized in a Speed-Vac concentrator and subjected to HPLC analysis (see above). RP-HPLC fractions were collected, lyophilized, and submitted to RIA (see below), therefore allowing measurement of octanoylated and desacyl ghrelin levels.

Effects of esterase inhibitors on ghrelin stability Plasma from 10 healthy volunteers was prepared using disodium EDTA (1 g/liter) within 30 min after blood collection. Then 2 ml of plasma were immediately incubated without (control) or with PMSF (1 mm), NaF (59.5 mm), and eserine salicylate (1 mm) for 1 h at room temperature. The incubation was stopped by the addition of 2 ml of 10% CH3CN/0.1% TFA. The samples were subsequently loaded onto a SepPak C18 cartridge equilibrated with 5 ml of 3% CH3CN/0.1% TFA. The Sep-Pak was washed with 5 ml of 10% CH3CN/0.1% TFA and the peptides were eluted with 2 ml of 50% CH3CN/0.1% TFA. The eluates were lyophilized in a Speed-Vac concentrator and subjected to ghrelin RIA.

Peptide synthesis and radioiodination All the peptides used were synthesized by solid-phase methodology using the Fmoc (9-fluorenyl-methoxy-carbonyl) strategy (18) and purified as previously described (19). All ghrelin analogs were based on the human sequence, unless specified otherwise, and those possessing a C-terminal acidic function instead of an amide were noted by -OH. [Tyr24]Ghr (1–23) and [Tyr0]Ghr (13–28)-OH were radioiodinated on the tyrosine by the iodogen method (20) and purified on a Sep-Pak C18 cartridge (Waters).

Ghrelin RIA Assays were performed as previously described using radioiodinated [Tyr24]Ghr (1–23) and [Tyr0]Ghr (13–28)-OH and rabbit polyclonal SB801 and SB969 antibodies, directed, respectively, toward the synthetic [Cys12]Ghr (1–11) and [Cys0]Ghr (13–28)-OH peptides (19). SB801 and SB969 allowed to measure respectively octanoylated ghrelin and total ghrelin (octanoylated and des-acyl ghrelin). When samples were previously analyzed by RP-HPLC, RIAs allowed measurement of octanoylated and des-acyl ghrelin (instead of octanoylated and total ghrelin without RP-HPLC analysis).

Affinity chromatography of plasma lipoprotein subfractions To investigate whether plasma lipoprotein subfractions could interact with ghrelin, five analogs of ghrelin were synthesized and covalently coupled to a SulfoLink coupling gel using the SulfoLink kit (Pierce, Rockford, IL): octanoyl Ghr (1–11) [Cys12], decanoyl Ghr (1–11) [Cys12], des-acyl Ghr (1–11) [Cys12], [Cys12] Ghr (13–28)-OH, and [Cys12] Ghr


(13–28). The SulfoLink coupling gel is an iodoacetyl-activated agarose support used for covalent immobilization of cysteine-containing proteins and peptides. The sample (2 ml of plasma lipoprotein subfraction) was applied to each affinity column. The columns were successively washed with 8 ml PBS, 6 ml PBS-1 m NaCl, 6 ml PBS-2 m NaCl and 6 ml PBS. The remaining bound proteins were eluted with 8 ml of a 100 mm glycine buffer (pH 2.8) and collected by 1-ml fractions. The elution was monitored by measuring the absorbance at 280 nm. The fractions were then lyophilized in a Speed-Vac concentrator and resolubilized in 500 ␮l of a Tris buffer [10 mm Tris-HCl, 1 mm EDTA (pH 7.4)] before proceeding to apolipoprotein assay.

Apolipoprotein assay Apolipoproteins (Apo) AI and Apo B were determined by sandwich ELISA in plasma subfractions and elution fractions from the affinity column chromatography (21). Apo AI was measured in HDL and LPP subfractions, whereas Apo B was measured in TRL and LDL subfractions.

Protein assay Protein concentration was determined using Bradford’s method (22).

Data analysis Data are summarized as mean values ⫾ sem. Results were statistically analyzed using the ANOVA and Dunnett t tests. All the statistical values reported were obtained using GraphPad InStat version 3.02 for Windows (GraphPad Software, San Diego, CA). P ⬍ 0.05 was considered significant.

Results Ghrelin degradation by plasma lipoprotein subfractions

We have previously shown that the metabolism of ghrelin (Ghr) (1–23) (with full biological activity) was not different from that of Ghr (1–28) (10). Therefore, ghrelin degradation by plasma lipoprotein subfractions was performed using Ghr (1–23). The incubation of Ghr (1–23) with plasma lipoprotein subfractions led to the sole production of des-acyl Ghr (1–23) (Fig. 1). After 240 min incubation with lipoprotein subfractions, the entire amount of des-acyl Ghr (1–23) offered was retrieved, showing that des-acyl Ghr (1–23) was not further degraded (data not shown). To identify the enzyme responsible for the desoctanoylation of ghrelin in each plasma lipoprotein subfraction, we tested the effects of several enzyme inhibitors after 240 min incubation. Ghrelin desoctanoylation induced by LDL was signifi-

FIG. 1. Degradation of Ghr (1–23) by human plasma lipoprotein subfractions. Ghr (1–23) was incubated with TRL, LDL, HDL, and LPP for 240 min. After Sep-Pak extraction, peptides were submitted to HPLC analysis: Ghr (1–23) and des-acyl Ghr (1–23) were detected. The results are expressed as the percentage of the quantity of Ghr (1–23) at zero time (3.64 nmol) and are the mean ⫾ SEM of n ⫽ 3 experiments.


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cantly inhibited by 50.5 ⫾ 9.7% (n ⫽ 3; P ⫽ 0.017) and 49.4 ⫾ 4.4% (n ⫽ 3; P ⫽ 0.007) in the presence of, respectively, 1 mm PMSF, a reported serine protease and esterase inhibitor, and 59.5 mm sodium fluoride (NaF), an esterase inhibitor (Fig. 2). Ghrelin desoctanoylation induced by LDL was not inhibited by 1 mm bis-p-nitrophenyl-phosphate, a carboxylesterase inhibitor, 1 mm eserine salicylate, a butyrylcholinesterase inhibitor, and 5 mm EDTA, a paraoxonase inhibitor (data not shown). Ghrelin desoctanoylation induced by HDL was not significantly affected by the presence of any of the above-mentioned inhibitors. Ghrelin desoctanoylation induced by LPP was significantly inhibited by 41.7 ⫾ 5.8% (n ⫽ 3; P ⫽ 0.021) and 42.5 ⫾ 8.3% (n ⫽ 3; P ⫽ 0.030) in the presence of, respectively, 1 mm PMSF and 1 mm eserine salicylate, whereas 1 mm bis-pnitrophenyl-phosphate, 59.5 mm NaF, and 5 mm EDTA had no significant effect (data not shown). Due to the low ghrelin degradation observed in the presence of TRL after 240 min incubation, the effects of the inhibitors were not investigated. PAF-AH activity in human sera

A correlation was found between PAF-AH and ghrelin hydrolytic activities in human sera from 11 healthy volunteers (r ⫽ 0.7919, P ⫽ 0.004; Fig. 3). Effects of esterase inhibitors on ghrelin stability

We determined whether the addition of PMSF, NaF, and eserine salicylate to blood sample could improve ghrelin

FIG. 2. Effect of inhibitors on the degradation of Ghr (1–23) by plasma LDL and LPP subfractions. Ghr (1–23) was incubated with plasma LDL or LPP subfractions for 240 min in the absence or presence of 1 mM PMSF, 59.5 mM NaF, or 1 mM eserine. After Sep-Pak extraction, peptides were submitted to HPLC analysis. The results are expressed as the percentage of inhibition of Ghr (1–23) desoctanoylation and are the mean ⫾ SEM of n ⫽ 3 experiments. 多, P ⬍ 0.05; 多多, P ⬍ 0.01.

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FIG. 3. Correlation between ghrelin hydrolytic and PAF-AH activities in human sera. Ghrelin hydrolytic and PAF-AH activities in sera were determined as described in Materials and Methods. Ghrelin hydrolytic activity is expressed as 10⫺3 ␮mol 䡠 min⫺1.ml⫺1, and PAF-AH activity is expressed as ␮mol䡠min⫺1䡠ml⫺1. r ⫽ 0.7919; P ⫽ 0.004.

stability. For this purpose, plasma from 10 healthy volunteers was incubated in the absence or presence of PMSF (1 mm), NaF (59.5 mm), and eserine salicylate (1 mm) for 1 h at room temperature and then submitted to ghrelin RIA. Octanoylated ghrelin level was significantly increased when using PMSF (P ⫽ 0.005) and eserine salicylate (P ⫽ 0.045) but not NaF, compared with the absence of esterase inhibitor (Table 1). Total ghrelin level was not significantly modified by esterase inhibitors (Table 1). Ghrelin identification in plasma lipoprotein subfractions

To determine whether ghrelin was transported by lipoproteins in the bloodstream, we measured the amount of ghrelin present in each plasma lipoprotein subfraction. In TRL, LDL, HDL, and LPP subfractions, immunoreactive (IR) ghrelin eluting at the octanoylated ghrelin position was 159 ⫾ 17, 32 ⫾ 9, 15 ⫾ 8, and 56 ⫾ 19 fmol/mg Apo (n ⫽ 5), respectively, and IR ghrelin eluting at des-acylated ghrelin position was 130 ⫾ 42, 13 ⫾ 4, 45 ⫾ 18, and 190 ⫾ 48 fmol/mg Apo (n ⫽ 5), respectively (Fig. 4). Affinity chromatography of plasma lipoprotein subfractions

To investigate the ability of the lipoproteins to bind ghrelin and determine the ghrelin domain involved in this binding, plasma lipoprotein subfractions were applied to affinity columns in which octanoyl Ghr (1–11) [Cys12], decanoyl Ghr (1–11) [Cys12], des-acyl Ghr (1–11) [Cys12], [Cys12] Ghr (13– 28)-OH, and [Cys12] Ghr (13–28) were coupled. To identify

FIG. 4. IR-ghrelin determination in human plasma lipoprotein subfractions. After Sep-Pak extraction, peptides were submitted to HPLC analysis followed by RIA. IR-octanoylated ghrelin (A) and IR-des-acyl ghrelin (B) are expressed as femtomoles per milligram Apo. Results were normalized to Apo AI for HDL and LPP and to Apo B for TRL and LDL. The results are the mean ⫾ SEM of n ⫽ 5 experiments.

the possible lipoproteins eluted, the Apo AI and Apo B were measured in each elution fraction. Because TRL and LDL contain mostly Apo B, Apo B was measured in elution fractions obtained when TRL and LDL subfractions were applied to the columns. For the TRL subfraction, Apo B was detected in two elution fractions from the octanoyl Ghr (1–11) [Cys12] and the decanoyl Ghr (1–11) [Cys12] columns but not in the elution fractions from the other columns (Fig. 5). For LDL subfraction, Apo B was detected in elution fractions from the octanoyl Ghr (1–11) [Cys12] and the decanoyl Ghr (1–11) [Cys12] columns and in very low amount in the des-acyl Ghr (1–11) [Cys12] and [Cys12] Ghr (13–28)-OH columns but not the [Cys12] Ghr (13–28) column (Fig. 5). Because Apo AI is the major apolipoprotein of the HDL and very high-density lipoprotein (VHDL), Apo AI was measured in elution fractions obtained when plasma HDL and LPP subfractions were applied to the column. For HDL and LPP subfractions, Apo AI was detected

TABLE 1. Effects of esterase inhibitors on ghrelin stability Esterase inhibitor

Octanoylated ghrelin (fmol/ml of plasma)

Total ghrelin (fmol/ml of plasma)

Control PMSF NaF Eserine salicylate

13 ⫾ 2 32 ⫾ 5a 18 ⫾ 3 27 ⫾ 4b

87 ⫾ 13 86 ⫾ 13 88 ⫾ 19 84 ⫾ 15

Effect of the esterase inhibitors on ghrelin stability was determined by measuring octanoylated and total ghrelin using RIA (see Materials and Methods). Plasma from 10 healthy volunteers was incubated without (control) or with PMSF (1 mM), NaF (59.5 mM), and eserine salicylate (1 mM) for 1 h at room temperature (as described in Materials and Methods). The results are expressed as femtomoles per milliliter of plasma and are the mean ⫾ SEM of n ⫽ 10. a P ⬍ 0.01 for octanoylated ghrelin. b P ⬍ 0.05 for octanoylated ghrelin.



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FIG. 5. Affinity chromatography profiles of ghrelin interaction with human plasma lipoprotein subfractions. Two milliliters of plasma lipoprotein subfractions were applied to the five columns described in Materials and Methods. The columns were successively washed with 8 ml PBS, 6 ml PBS-NaCl 1 M, 6 ml PBS-NaCl 2 M, and 6 ml PBS. The remaining bound proteins were eluted with 8 ml of a glycine buffer [glycine 100 mM (pH 2.8)]. Apo B was measured in the elution fractions from TRLs and LDLs, whereas Apo AI was measured in the elution fractions from HDLs and LPP.

in elution fractions from the octanoyl Ghr (1–11) [Cys12], decanoyl Ghr (1–11) [Cys12], des-acyl Ghr (1–11) [Cys12], and [Cys12] Ghr (13–28)-OH columns but not the [Cys12] Ghr (13–28) column (Fig. 5). Discussion

In contact with human serum, we showed that ghrelin is desoctanoylated, without proteolysis. The partial inhibition of this desoctanoylation by eserine salicylate, a butyrylcholinesterase inhibitor, and the existence of a correlation between the butyrylcholinesterase and ghrelin desoctanoylation activities suggest a major contribution of butyrylcholinesterase to this phenomenon (10). However, butyrylcholinesterase does not seem to be the sole esterase responsible for ghrelin desoctanoylation because eserine salicylate inhibits only partially the reaction. To locate and identify other relevant esterases involved in ghrelin desoctanoylation in human serum, we studied ghrelin degradation in contact with different plasma lipoprotein subfractions. Plasma lipoprotein subfractions were separated, based on their density, by sequential ultracentrifugation into four main categories: 1) TRL subfraction (containing chylomicrons, VLDLs, and intermediate-density lipoproteins); 2) LDL subfraction; 3) HDL subfraction; and 4) LPP subfraction (containing VHDLs and plasma proteins). After incubation of ghrelin with LDL, HDL, and LPP subfractions, the only degradation product detected was desacyl ghrelin. Ghrelin desoctanoylation was insignificant in contact with TRL, low in contact with HDL, and occurred mostly in contact with LDL and LPP. This suggests that one

of the enzymes responsible for ghrelin desoctanoylation is associated with lipoproteins and mainly with LDL. The inhibition of LDL-induced ghrelin desoctanoylation by PMSF and NaF suggested the involvement of a serine esterase. In human plasma, a serine esterase, PAF-AH (EC 3.1.1.47), circulates mainly bound to LDLs and, to some extent, HDLs (14, 23). PAF-AH is a phospholipase A2 that hydrolyzes PAF, inactivating it to lysoPAF, and has a marked preference for phospholipids with short-chain fatty acid moiety at the sn-2 position (24). In contrast to most phospholipase A2 enzymes, PAF-AH does not require Ca2⫹ for enzymatic activity and is therefore insensitive to EDTA but is sensitive to serine esterase inhibitors such as PMSF and NaF (14). A recent study showed that a lysophospholipase was able to desoctanoylate ghrelin in rat stomach (25). In human sera, we observed a correlation between PAF-AH activity and ghrelin desoctanoylation activity, suggesting that PAF-AH contributed to the desoctanoylation of ghrelin in human sera. This hypothesis is also supported by the inhibition of LDL-induced ghrelin desoctanoylation by PMSF and NaF but not EDTA. Two esterases are associated with HDLs, the PON and PAF-AH (26, 27). A study showing an interaction between ghrelin and HDL suggested that PON, a Ca2⫹-dependent esterase, might be involved in the desoctanoylation of ghrelin (11). We previously showed that, in human serum, ghrelin desoctanoylation was not inhibited by EDTA and was negatively correlated with the paraoxonase activity, excluding PON as enzyme responsible of ghrelin desoctanoylation (10). The low HDL-induced ghrelin desoctanoylation

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observed by us could be due to PAF-AH because this enzyme is also associated with HDL. This could not be verified due to the low ghrelin desoctanoylation activity and the nonsignificant effects of the inhibitors on that activity. LPP-induced ghrelin desoctanoylation was partially inhibited by PMSF and eserine salicylate, suggesting the involvement of butyrylcholinesterase (EC 3.1.1.8) as well as an additional esterase. Butyrylcholinesterase is likely located in LPP subfraction, containing VHDL and plasma proteins, because it does not circulate bound to lipoproteins. Albumin is the most abundant plasma protein contained in the LPP subfraction. Whereas albumin does not possess an enzyme commission number, it has been described to possess an esterase-like activity (28). Albumin has been found to catalyze the hydrolysis of various compounds such as pnitrophenyl acetate (29), ketoprofen glucuronide (30), cyclophosphamide (31), organophosphate insecticides (32), carbaryl (33), nicotinate esters (34), and long- and short-chain fatty acid esters (35). Albumin esterase activity is resistant to inhibition by diisopropylfluorophosphate, ecothiophate, paraoxon, and EDTA (36). Studies have also suggested that the esterase-like activity of serum albumin could result from a contamination by butyrylcholinesterase or other esterases (37, 38). At present, it is not clear whether the esterase activity of albumin is intrinsic to the protein or due to the presence of an esterase impurity. Therefore, it is presently not possible to determine whether albumin could account for the LPP-induced ghrelin desoctanoylation activity that was not inhibited by PMSF and eserine. The present study confirms that butyrylcholinesterase participates in ghrelin desoctanoylation in human plasma (10) and suggests the additional involvement of PAF-AH, a lipoprotein-associated phospholipase. In obese subjects, the plasma concentrations of total ghrelin (ghrelin and des-acyl ghrelin) were lower, compared with that of normal-weight control subjects (39, 40). Lower ghrelin levels were also associated with a higher prevalence of the metabolic syndrome; this was largely explained by the greater BMI of individuals with lower levels of ghrelin (41). Recent studies demonstrated that human obesity was associated with lower levels of both octanoylated and des-acyl ghrelins (42, 43), but the mechanism by which octanoylated ghrelin decreases in systemic circulation of obese subjects is still unknown. Higher levels of butyrylcholinesterase activity were found in the serum of patients with hyperlipidemias, diabetes, and obesity as compared with healthy and lower body weight individuals, and butyrylcholinesterase activity has been suggested as a marker for metabolic syndrome (44, 45). In obese subjects it is tempting to suggest that higher serum butyrylcholinesterase activity could partially account for the decrease of octanoylated ghrelin concentration. Ghrelin desoctanoylation can occur in blood circulation as well as during blood collection and subsequent blood handling at room temperature. Generally, ghrelin levels are measured in plasma. Therefore, we tested the effects of esterase inhibitors on ghrelin stability in human plasma samples. Compared with the control in the absence of esterase inhibitor, PMSF and eserine salicylate significantly increased the amount of octanoylated ghrelin by about 146 and 108%,


respectively. This confirmed the implication of butyrylcholinesterase in ghrelin desoctanoylation in serum and LPP subfraction. Therefore, it seems appropriate to add PMSF and eserine salicylate into the blood-collecting tubes to preserve ghrelin. Whereas NaF significantly inhibited ghrelin desoctanoylation induced by LDLs, no significant effect could be observed on ghrelin desoctanoylation in human plasma samples by NaF. This could be explained by the low PAF-AH activity in human plasma, compared with the butyrylcholinesterase activity. NaF could not inhibit ghrelin desoctanoylation if the PAF-AH activity is too low to induce a significant desoctanoylation of ghrelin in 1 h incubation at room temperature. To determine whether lipoproteins could behave as ghrelin transporters in human bloodstream, we studied ghrelin interaction with four lipoprotein subfractions (TRL, LDL, HDL, and LPP). Exogenous compounds like nystatin, cyclosporine, amphotericin B, and halofantrine but also endogenous compounds like vitamin E, vitamin A, and PAF are known to be associated with lipoproteins (13). Like albumin and ␣1-glycoproteins, lipoproteins have generally a low affinity and low specificity, but their plasma levels are high enough to bind significant amounts of ligand (46). By reverse-phase chromatography followed by RIAs, we found that total ghrelin (IR-octanoylated ghrelin and IR-des-acyl ghrelin) was present in each plasma subfraction but were more abundant in TRLs and LPP. TRLs and LDLs contained 61 and 71% of octanoylated ghrelin, respectively, whereas HDLs and LPP contained only 25 and 23% of octanoylated ghrelin, respectively. Octanoylated ghrelin interacts mainly with Apo B-containing lipoproteins, whereas des-acyl ghrelin interacts mainly with Apo AI-containing lipoproteins and plasma proteins or circulates freely in the LPP subfraction. Using affinity chromatography, we showed that, in plasma lipoprotein subfractions, Apo B-containing lipoproteins interact only with the N-terminal portion of the ghrelin containing the octanoylated or decanoylated Ser3, whereas Apo AI-containing lipoproteins interact with the N-terminal portion of the ghrelin, containing or not the octanoylated or decanoylated Ser3, but also with the C-terminal portion of the ghrelin possessing a C-terminal acidic function. The presence of an esterase activity could explain the detection of IR-desacyl ghrelin in LDLs but not TRLs. Because IR-octanoylated ghrelin represented the major proportion of total IR-ghrelin in TRL and LDL subfractions, the amount of lipoprotein eluted from the des-acyl Ghr (1–11) [Cys12] and [Cys12] Ghr (13–28)-OH columns could be too weak to be detected in our Apo B assay. Indeed, with LDL subfraction, Apo B was detected in very weak amount in elution fractions from these columns, suggesting a minor interaction with des-acyl ghrelin. Lipoproteins of the HDL and LPP subfractions interact with the N-terminal portion of the ghrelin, independently of the octanoylated or decanoylated Ser3, and with the C-terminal portion of the ghrelin possessing a C-terminal acidic function. These results could account for the presence of both IR-octanoylated ghrelin and IR-des-acyl ghrelin in those subfractions. In LPP, IR-octanoylated ghrelin and IR-des-acyl ghrelin could also be associated with plasma proteins, but this should be verified in a further study. In all lipoprotein subfractions, no interaction with the C-terminal portion of the


ghrelin possessing a C-terminal amide function was observed, suggesting the importance of the natural C-terminal acid for the interaction of ghrelin with lipoproteins. The present study supports the possible role of HDL particles as ghrelin transporters in the bloodstream as previously observed by Beaumont et al. (11). Furthermore, our study also supports the role of TRL, VHDL, to some extent LDL, and probably plasma proteins in the transport of ghrelin in the bloodstream. Although IR-des-acyl ghrelin was detected in TRLs, TRL interaction with ghrelin necessitated the presence of the acyl group on Ser3. LDL interaction with ghrelin mostly necessitated the N-terminal portion of ghrelin containing acyl Ser3. HDL and VHDL interaction with ghrelin involved both the N-terminal portion containing either acyl or des-acyl Ser3 and the C-terminal portion of ghrelin. Taken together, our data suggested that both ghrelin and des-acyl ghrelin are carried by TRLs, LDLs, HDLs, and VHDLs in the human bloodstream and that both butyrylcholinesterase and PAF-AH are involved in ghrelin desoctanoylation in human plasma.


10. 11.

12.

13. 14.

15. 16. 17. 18.

Acknowledgments The authors thank Dr. L. Portois and R. Lema-Kisoka for helpful discussion, M. Stie´venart for his secretarial assistance, and V. Delforge for her technical assistance. Received September 18, 2006. Accepted January 29, 2007. Address all correspondence and requests for reprints to: Christine Delporte, Department of Biochemistry and Nutrition, Faculty of Medicine, Universite´ Libre de Bruxelles, Bat G/E, CP 611, 808 Route de Lennik, B-1070 Brussels, Belgium. E-mail: [email protected]. This work was supported by Grant 3.4510.03 from the Fund for Medical Scientific Research (Belgium) and by an “Interuniversity Poles of Attraction Program-Belgian State, Prime Minister’s Office, Federal Office for Scientific, Technical, and Cultural Affairs.” C.D.V. is a recipient of a doctoral fellowship from FRIA (Belgium). Disclosure Statement: The authors have nothing to disclose.

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