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Endocrinology Vol. 145, No. 11 4997-5005
Copyright © 2004 by The Endocrine Society

Ghrelin Degradation by Serum and Tissue Homogenates: Identification of the Cleavage Sites

Carine De Vriese, Francoise Gregoire, Roger Lema-Kisoka, Magali Waelbroeck, Patrick Robberecht and Christine Delporte

Department of Biochemistry and Nutrition (C.D.V., F.G., M.W., P.R., C.D.) and L. Deloyers Laboratory for Experimental Surgery (R.L.-K.), Faculty of Medicine, Université Libre de Bruxelles, Brussels B-1070, Belgium

Address all correspondence and requests for reprints to: Christine Delporte, Department of Biochemistry and Nutrition, Faculty of Medicine, Université Libre de Bruxelles, Bat G/E, CP 611, 808 route de Lennik, B-1070 Brussels, Belgium. E-mail: cdelport{at}ulb.ac.be.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The endogenous ligand for the GH secretagogue receptor is ghrelin, a peptide recently purified from the stomach. Ghrelin is n-octanoylated on the Ser3 residue, and this modification is essential for its interaction with the receptor. The degradation of ghrelin by rat and human serum, purified commercial enzymes, and tissues homogenates was analyzed by combining HPLC and mass spectrometry. In serum, ghrelin was desoctanoylated, without proteolysis. The desoctanoylation was significantly reduced by phenylmethylsulfonyl fluoride, a serine proteases and esterases inhibitor. In rat serum, the carboxylesterase inhibitor bis-p-nitrophenyl-phosphate totally inhibited ghrelin desoctanoylation, and a correlation was found between ghrelin desoctanoylation and carboxylesterase activity. Moreover, purified carboxylesterase degraded ghrelin. Thus, carboxylesterase could be responsible for ghrelin desoctanoylation in that species. In human serum, ghrelin desoctanoylation was partially inhibited by eserine salicylate and sodium fluoride, two butyrylcholinesterase inhibitors, but not by bis-p-nitrophenyl-phosphate and EDTA. Purified butyrylcholinesterase was able to degrade ghrelin, and there was a correlation between the butyrylcholinesterase and ghrelin desoctanoylation activities in human sera. This suggested that several esterases, including butyrylcholinesterase, contributed to ghrelin desoctanoylation in human serum. In contact with tissues homogenates, ghrelin was degraded by both desoctanoylation and N-terminal proteolysis. We identified five cleavage sites in ghrelin between residues -Ser2-(acyl)Ser3- (stomach and liver), -(acyl?)Ser3-Phe4- (stomach, liver, and kidney), -Phe4-Leu5- (stomach and kidney), -Leu5-Ser6- and -Pro7-Glu8- (kidney). In all cases, the resulting fragments were biologically inactive.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GHRELIN, A 28-AMINO ACID peptide containing a n-octanoyl modification on Ser3, is the main endogenous ligand for the GH secretagogue receptor (GHS-R) (1) that was recently purified and identified from rat stomach (2). Octanoylation is essential for biological activity of ghrelin such as the stimulation of GH release from the pituitary (3). Endocrine cells of the stomach are the main source of ghrelin, but other tissues such as the intestine, kidney, pituitary, pancreas, placenta, lung, testis, and ovary (4) may also contribute to blood ghrelin. Apart from GH release, ghrelin seems to exert various important physiological effects such as induction of adiposity and body weight gain (5) due to appetite-stimulating effects and increased food intake (6), and stimulation of gastric acid secretion and motility (7).

Many studies have evaluated the plasma or serum ghrelin levels in human and animal models and attempted to correlate the results with several parameters like diet-induced weight loss or fasting (8, 9). It must be noted that the major circulating form of ghrelin is the des-acyl ghrelin (10), the biologically inactive form of ghrelin (at least on the GHS-R), and that very little is known about circulating ghrelin and des-acyl ghrelin catabolism. Only acylated forms of ghrelin bind to the GHS-R and exert endocrine actions. Recently, des-acyl ghrelin, devoid of any endocrine activity through the GHS-R, was shown to stimulate cell proliferation in prostate carcinoma cell lines (11) and adipogenesis (12), induce cardiovascular effects (13), and inhibit apoptosis in cardiomyocytes and endothelial cells (14). These effects could be mediated by an unidentified ghrelin receptor, distinct from the GHS-R. Tissues have been shown to contain both ghrelin and des-acyl ghrelin (10). One can hypothesize that both forms of ghrelin are secreted via two differently regulated pathways and, as a consequence, induce distinct physiological effects. Therefore, it would be appropriate to determine whether ghrelin and des-acyl ghrelin could be interconverted and/or degraded in tissues and sera.

The aim of this paper was to study ghrelin degradation in contact with serum and tissue homogenates to identify the cleavage sites and the enzymes involved.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Peptide synthesis
All of the peptides used were synthesized by solid phase methodology using the Fmoc (9-fluorenyl-methoxy-carbonyl) strategy (15) with a Symphony Multiplex apparatus and purified by reverse phase-HPLC (RP-HPLC) on Amberchrome-type resin 78 CG-162sd (15.9 x 2.5 cm), Vydac 259HP510A (25 x 1 cm; Alltech, Laarne, Belgium) and Vydac 259VHP54 (25 x 0.46 cm; Alltech). Selective protection of Ser3 was obtained using Fmoc Ser-Trt, instead Fmoc Ser-Tbu for Ser2, to allow its selective deprotection and by a solution of 1% trifluoroacetic acid (TFA) in dichloromethane before subsequent octanoylation. The Ser3 hydroxyl group of ghrelin was acylated with n-octanoic acid using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of 4-(dimethylamino)pyridine according to Bednarek et al. (16). All ghrelin analogs were based on the human sequence. Peptide purity was assessed (>95%) by capillary electrophoresis (P/ACE MDQ, Beckman, Fullerton, CA), and molecular weight was verified by electrospray mass spectrometry using a VG Platform ns 8230E (Waters, Milford, MA).

Preparation of sera and tissue homogenates
Sera were prepared from blood obtained either from male Wistar rats (Iffa Credo, Brussels, Belgium) anesthetized using sevoflurane (Abbott N.V., Ottignies, Belgium), or from healthy human volunteers after informed consent. After achievement of coagulation during 20 min, blood samples were centrifuged for 15 min at 2000 x g at room temperature to allow sera collection. The sera were immediately used or frozen without less of activity. The protocols were approved by the local Ethics Committees for animal and for human studies. Tissues were removed from killed animals, weighed, and homogenized in 50 mM Tris-HCl (pH 7.4). Sera or rat tissue homogenates were then diluted either in the absence or presence of enzyme inhibitor. Protein concentrations were determined using the method of Bradford (17), using BSA as standard.

Ghrelin degradation by sera and tissue homogenates
Ten micrograms of ghrelin or analog were incubated at 37 C for various periods with 200 µl of human or rat serum, or rat stomach-, liver-, or kidney homogenate, in a final volume of 300 µl. The incubation was stopped by the addition of TFA (2% final concentration). An internal standard (2 nmol of rat atrial natriuretic peptide [1–28] for the sera, or 20 nmol of Arg-Tyr for the homogenates) was subsequently added. Samples were centrifuged at 4 C for 30 min at 20,000 x g 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. For the tissue homogenates, peptides were directly eluted with 2 ml of 80% CH3CN/0.1% TFA, whereas for the sera, 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 metabolites
The Sep-Pak eluates were subjected to RP-HPLC on a C18 column Vydac 218TP54 (25 x 0.46 cm; Alltech) equilibrated with 3% CH3CN/0.1% TFA. For the tissue homogenates, RP-HPLC was performed using a linear gradient of CH3CN from 3–80% in 0.1% TFA for 50 min. For the sera, RP-HPLC was performed using a gradient of CH3CN from 3–20% for 5 min, then from 20–60% for 20 min and finally from 60–80% for 5 min in 0.1% TFA. The remaining substrate and the resulting products were monitored at 226 nm using a Shimadzu CR6A integrator. RP-HPLC fractions were collected and lyophilized. Products were identified by electrospray mass spectrometry using a VG Platform ns 8230E (Waters, Milford, MA) and by their elution position. Each substrate, internal standard, and fragment was quantified in nanomoles using calibration curves obtained with the corresponding synthetic peptide.

Ghrelin degradation by purified butyrylcholinesterase and carboxylesterase
Two nanomoles of peptide were incubated at 37 C for various periods in 50 mM Tris-HCl (pH 7.4) with 2.5 U of purified human serum butyrylcholinesterase (Sigma Aldrich, St. Louis, MO) or 0.1 U of porcine liver carboxylesterase (Sigma), in a final volume of 200 µl. The reaction was stopped by the addition of 400 µl of 6% CH3CN/0.2% TFA. The samples were immediately subjected to HPLC analysis.

Assay for butyrylcholinesterase activity
Butyrylcholinesterase activity was measured by the method of Ellman et al. (18) using a LKB Ultrospec Plus 4054 UV/visible spectrophotometer (Pharmacia, Roosendaal, The Netherlands). Purified human butyrylcholinesterase (0.01 U) (Sigma) or 1 µl of human serum was added to 100 mM butyrylthiocholine iodide and 0.25 mM 5',5'-dithiobis-2-nitrobenzoic acid in 50 mM Tris-HCl (pH 7.4). The absorbance was read at 405 nm every 30 sec for up to 5 min. The enzyme activity was calculated as micromoles of the product·min–1·ml–1 (after correction for nonenzymatic hydrolysis of the substrate) using the extinction coefficient (13,300 M–1·cm–1) of the product.

Assay for carboxylesterase activity
Carboxylesterase activity was determined by measuring the hydrolysis of {alpha}-naphtylacetate (19, 20). Human and rat sera were preincubated during 20 min with 10 µM eserine to inhibit acetyl- and butyrylcholinesterases and 10 mM EDTA to inhibit paraoxonase. Ten microliters of 0.02 M {alpha}-naphtylacetate were added to 0.01 U purified porcine liver carboxylesterase, 50 µl of human serum, or 5 µl of rat serum in a 100 mM phosphate buffer (pH 7.0). The absorbance was measured at 321 nm every 10 min for up to 30 min for human serum, and every 30 sec for up to 12 min for rat serum. The enzyme activity was calculated as micromoles of the product·min–1·ml–1 using the extinction coefficient (2200 M–1·cm–1) of the product.

Assay for paraoxonase/arylesterase activity
Paraoxonase activity was determined in human serum by measuring the hydrolysis of phenylacetate (21). Five microliters of human serum were added to 1 mM phenylacetate in 20 mM Tris-HCl (pH 8.0). The absorbance was recorded at 270 nm and calculated as micromoles of the product·min–1·ml–1 using the extinction coefficient (1310 M–1·cm–1) of the product.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ghrelin degradation by human serum
The incubation of Ghr [1–28] with serum from healthy volunteers led to the sole production of des-acyl Ghr [1–28]. After 240 min incubation, 47.1 ± 5.8% and 52.5 ± 5.7% (n = 3) of Ghr [1–28] and des-acyl Ghr [1–28] were detected, respectively. Identical results were obtained when Ghr [1–23] was added to serum: after 240 min incubation, Ghr [1–23] and des-acyl Ghr [1–23] represented 48.2 ± 3.9% and 52.0 ± 4.0% (n = 11) of Ghr [1–23] at time zero, respectively (Fig. 1Go, A and B). Ghrelin hydrolysis activity was 0.039 ± 0.003 10–3 µmol·min–1·ml–1 (n = 11; Table 1Go).



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  		FIG. 1. Degradation of Ghr [1–23] by human and rat serum. Ghr [1–23] was incubated with either human (A and B) or rat (C and D) serum. After Sep-Pak extraction, peptides were submitted to HPLC analysis and Ghr [1–23] (A and C) and des-acyl Ghr [1–23] (B and D) were detected. The results are expressed as the percentage of the quantity of Ghr [1–23] at zero time (3.64 nmol). The mean is indicated by a line.

 

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  		TABLE 1. Carboxylesterase and ghrelin hydrolysis activities in human and rat sera

 
We verified that the observed fragments, des-acyl Ghr [1–28] and des-acyl Ghr [1–23], were not degraded by human serum after 240 min incubation (data not shown).

Given that the metabolism of the [1–23] ghrelin fragment (with full biological activity) was not different from that of the longer form [1–28], all the subsequent studies were performed on that easier to synthesize peptide. Besides, because the amino acid sequences of human and rat only differ in positions 11 and 12 (Arg11 and Val12 in human, vs. Lys11 and Ala12 in rat), human ghrelin was used throughout in this study.

To identify the enzyme responsible for the desoctanoylation of ghrelin in serum, several enzyme inhibitors were tested after 240 min incubation. Phenylmethylsulfonyl fluoride (PMSF, 1 mM), a reported serine proteases and esterases inhibitor, inhibited Ghr [1–23] desoctanoylation by 59.7 ± 4.4% (n = 6) (Fig. 2AGo). Eserine salicylate (1 mM) and NaF (59.5 mM), two cholinesterase inhibitors, inhibited Ghr [1–23] desoctanoylation by 31.5 ± 9.5% (n = 6) and 22.4 ± 6.2% (n = 6), respectively (Fig. 2AGo). In contrast, the desoctanoylation of Ghr [1–23] was not inhibited by 4-(amidino-phenyl)methanesulfonyl fluoride (50 µM), a serine protease inhibitor, EDTA (5 mM), a metallopeptidase and paraoxonase inhibitor or bis-p-nitrophenyl-phosphate (BNPP, 1 mM), a carboxylesterase inhibitor (data not shown).



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  		FIG. 2. Effect of inhibitors on the degradation of Ghr [1–23] by human and rat serum. Ghr [1–23] was incubated with human (A) or rat (B) serum in the absence or presence of 1 mM eserine, 59.5 mM NaF, 1 mM PMSF, or 1 mM BNPP. 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 = 6 for human sera and n = 3 for rat sera. *, P < 0.05.

 
Ghrelin degradation by rat serum
Ghr [1–23] was rapidly desoctanoylated into des-acyl Ghr [1–23] by rat serum. After 30 min incubation, Ghr [1–23] and des-acyl Ghr [1–23] represented 42.2 ± 3.8% and 57.0 ± 3.8% (n = 10) of Ghr [1–23] added at time zero, respectively (Fig. 1Go, C and D). A representative RP-HPLC profile of Ghr [1–23] desoctanoylation by rat serum is shown in Fig. 3AGo. Ghrelin hydrolysis activity was 0.347 ± 0.023 10–3 µmol·min–1·ml–1 (n = 10; Table 1Go). PMSF (1 mM) and BNPP (1 mM) inhibited Ghr [1–23] desoctanoylation by 88.5 ± 3.5% (n = 3) and 91.5 ± 3.3% (n = 3), respectively (Fig. 2BGo).



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  		FIG. 3. RP-HPLC profiles of ghrelin degradation by rat serum, and rat stomach, liver, and kidney homogenates. Ghr [1–23] was incubated with rat serum (A) for 15 min; or rat stomach homogenate (B); rat liver homogenate (C); or rat kidney homogenate (D) for 120 min. After Sep-Pak extraction, peptides were submitted to RP-HPLC analysis as described in Materials and Methods. The absorbance was monitored at 226 nm. The CH3CN gradient was shown by the line. The degradation fragments identified were: 1: des-acyl Ghr [1–23]; 2: Ghr [1–23]; 3: Ghr [5–23]; 4: Ghr [4–23]; 5: acyl Ghr [3–23]; 6: Ghr [8–23]; 7: Ghr [6–23].

 
Ghrelin degradation by commercially purified butyrylcholinesterase and carboxylesterase
We examined the ability of purified butyrylcholinesterase (EC 3.1.1.8) and carboxylesterase (EC 3.1.1.1) to convert ghrelin into des-acyl ghrelin.

After 240 min incubation of Ghr [1–23] in the presence of 2.5 U of human serum butyrylcholinesterase, 20.0 ± 0.6% of Ghr [1–23] and 80.3 ± 4.2% of des-acyl Ghr [1–23] were detected (n = 3). Butyrylcholinesterase activity of the purified enzyme was verified using the method of Ellman and butyrylthiocholine as substrate (see Materials and Methods; data not shown). The purified enzyme was totally inhibited by PMSF (1 mM), eserine salicylate (1 mM), or NaF (59.5 mM), but not by EDTA (5 mM) or BNPP (1 mM) (data not shown).

Due to the lack of commercially available human liver carboxylesterase, ghrelin degradation was performed in the presence of the corresponding porcine enzyme. In the presence of 0.1 U porcine liver carboxylesterase, the sole degradation fragment of Ghr [1–23] was des-acyl Ghr [1–23]. After 120 min incubation, 23.6 ± 1.9% of Ghr [1–23] and 77.9 ± 1.1% of des-acyl Ghr [1–23] were observed (n = 3). Carboxylesterase activity of the purified enzyme was verified using {alpha}-naphtylacetate as substrate (see Materials and Methods; data not shown). The purified enzyme was totally inhibited by PMSF (1 mM), BNPP (1 mM), and NaF (59.5 mM), inhibited by about 27% by eserine salicylate (1 mM) but not affected by EDTA (5 mM) (data not shown).

Butyrylcholinesterase activity in human sera
A correlation was found between the butyrylcholinesterase and ghrelin hydrolysis activities in human sera from 11 healthy volunteers (r = 0.7187, P < 0.05; Fig. 4AGo).



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  		FIG. 4. Correlation between ghrelin hydrolysis and butyrylcholinesterase, or paraoxonase activities in human sera. Ghrelin hydrolysis, butyrylcholinesterase (A) and paraoxonase (B) activities in sera were determined as described in Materials and Methods. Ghrelin hydrolysis was expressed as 10–3 µmol·min–1·ml–1, and butyrylcholinesterase (BchE) and paraoxonase (PON) activities in sera were expressed as µmol·min–1·ml–1.

 
Carboxylesterase activity in human and rat sera
The carboxylesterase activity of rat serum (3.52 ± 0.15 µmol·min–1·ml–1, n = 10) was larger than that of human serum (0.019 ± 0.001 µmol·min–1·ml–1, n = 11) (Table 1Go).

In contrast to human sera from 11 healthy volunteers (r = 0.2570, P > 0.05; Fig. 5AGo), a correlation was found between the carboxylesterase and ghrelin hydrolysis activities in rat sera from 10 animals (r = 0.7936, P < 0.01; Fig. 5BGo).



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  		FIG. 5. Correlation between ghrelin hydrolysis and carboxylesterase activities in human and rat sera. Ghrelin hydrolysis and carboxylesterase activities in human (A) and rat (B) sera were determined as described in Materials and Methods. Ghrelin hydrolysis and carboxylesterase (CE) activities in sera were expressed as 10–3 µmol·min–1·ml–1 and µmol·min–1·ml–1, respectively.

 
Paraoxonase/arylesterase activity in human sera
A negative correlation was found between the paraoxonase and ghrelin hydrolysis activities in human sera from 11 healthy volunteers (r = –0.6028, P < 0.05; Fig. 4BGo).

Ghrelin degradation in presence of rat stomach homogenate
Several degradation products were identified after incubating Ghr [1–23] with a rat stomach homogenate diluted to 0.05% (wt/vol): des-acyl Ghr [1–23], Ghr [5–23], Ghr [4–23] and acyl Ghr [3–23]. Almost all the substrate was degraded after 120 min incubation and the fragment concentrations increased with time (Fig. 6AGo). After 120 min incubation, Ghr [1–23], des-acyl Ghr [1–23], Ghr [5–23], Ghr [4–23] and acyl Ghr [3–23] represented 6.6 ± 3.2, 20.9 ± 7.9, 28.4 ± 4.1, 4.0 ± 1.1 and 3.1 ± 1.5% of the amount of Ghr [1–23] present at time zero, respectively (n = 3). All the fragments identified represented 63 ± 2% (n = 3) of Ghr [1–23] present at time zero. A representative RP-HPLC profile of Ghr [1–23] degradation by rat stomach homogenate is shown in Fig. 3BGo. The degradation of Ghr [1–23] after 120 min incubation was inhibited by about 76%, by PMSF (1 mM) (data not shown).



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  		FIG. 6. Degradation of Ghr [1–23] by rat tissue homogenates. Ghr [1–23] was incubated with rat stomach (A), liver (B), kidney (C) homogenate. After Sep-Pak extraction, peptides were submitted to HPLC analysis. 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 three experiments.

 
Ghrelin degradation by rat liver homogenate
The incubation of Ghr [1–23] in presence of a 0.025% (wt/vol) rat liver homogenate led to three major degradation products: des-acyl Ghr [1–23], Ghr [4–23], and acyl Ghr [3–23]. All the fragments increased with time, and the major fragment was des-acyl Ghr [1–23] (Fig. 6BGo). After 120 min, 11.4 ± 2.4, 65.2 ± 2.8, 11.0 ± 1.5, and 7.6 ± 1.0% of Ghr [1–23], des-acyl Ghr [1–23], Ghr [4–23] and acyl Ghr [3–23], were detected, respectively (n = 3). All the fragments identified represented 105 ± 4% (n = 3) of Ghr [1–23] present at time zero. A representative RP-HPLC profile of Ghr [1–23] degradation by rat liver homogenate is shown in Fig. 3CGo. The degradation of Ghr [1–23] after 120 min incubation was inhibited, by about 65.8% by PMSF (1 mM) (data not shown).

Ghrelin degradation by rat kidney homogenate
Incubation of Ghr [1–23] with a 0.006% (wt/vol) rat kidney homogenate produced five major degradation fragments: the des-acyl Ghr [1–23], Ghr [8–23], Ghr [6–23], Ghr [5–23] and Ghr [4–23]. All the fragments increased with time (Fig. 6CGo). After 120 min, 15.7 ± 2.4, 8.7 ± 1.7, 27.7 ± 1.5, 3.8 ± 1.3, 5.9 ± 1.6 and 6.2 ± 0.8% of Ghr [1–23], des-acyl Ghr [1–23], Ghr [8–23], Ghr [6–23], Ghr [5–23] and Ghr [4–23] were measured, respectively. All the fragments identified represented 68 ± 6% (n = 3) of Ghr [1–23] present at time zero. A representative RP-HPLC profile of Ghr [1–23] degradation by rat kidney homogenate is shown in Fig. 3DGo. The degradation of Ghr [1–23] after 120 min incubation was inhibited by about 71% by PMSF (1 mM) (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Because Ghr [1–23], a C-terminal shortened ghrelin analog, was equipotent to Ghr [1–28] (16), and similarly degraded, most of our experiments were performed using that peptide as substrate. After incubation of ghrelin with serum, the only metabolite was des-acyl ghrelin. Ghrelin desoctanoylation was strongly inhibited by PMSF, a serine proteases and esterases inhibitor (22, 23, 24), but not by 4-(amidino-phenyl) methanesulfonyl fluoride, a specific irreversible inhibitor of serine proteases (25).

Serum esterases have a broad substrate specificity with interspecies and individual differences (26). They are classified into three groups, A-, B-, and C-esterases, based on their interaction with organophosphates (27, 28). A-esterases, including arylesterase (EC 3.1.1.2)/paraoxonase (EC 3.1.8.1), rapidly hydrolyze organophosphates. B-esterases, including acetylcholinesterase (EC 3.1.1.7), butyrylcholinesterase (EC 3.1.1.8) and nonspecific carboxylesterase (EC 3.1.1.1), are inhibited by organophosphates. C-esterases, such as acetylesterase (EC 3.1.1.6), do not interact with organophosphates.

The human serum paraoxonase/arylesterase (EC 3.1.8.1; formerly EC 3.1.1.2) catalyzes the hydrolysis of organophosphorous esters like paraoxon, as well as phenylacetate, aromatic carboxylic acid esters and carbamates (29). Calcium is required for enzyme stability and activity (30, 31) and the activity is therefore reduced by EDTA. A recent study, showing an interaction between ghrelin and high-density lipoproteins that contain the plasma paraoxonase activity, concluded that this enzyme might be involved in the conversion of ghrelin to des-acyl ghrelin (32). The present study did not support this hypothesis: ghrelin desoctanoylation was not inhibited by EDTA and was negatively not correlated with the paraoxonase activity. Furthermore, the inhibition of ghrelin desoctanoylation by PMSF suggested that B-esterases, rather than A- and C-esterases, contributed to that reaction.

Butyrylcholinesterase is capable of hydrolyzing low doses of organophosphorous and carbamates pesticides, and several drugs (for example, cocaine, succinylcholine, and aspirin) (33). Acetyl and butyrylcholinesterases are inhibited by eserine (34, 35), and by NaF (36, 37, 38). Purified human butyrylcholinesterase desoctanoylated ghrelin. However, eserine and NaF inhibited only partially the ghrelin desoctanoylation by human serum, and a correlation was found between the butyrylcholinesterase and ghrelin desoctanoylation activities. Because butyrylcholinesterase represents a significant proportion of the total esterase activity in human serum, its participation in the ghrelin desoctanoylation process is likely, but it did not seem to be the sole enzyme responsible for the reaction. Compared with human serum, rat serum has a reduced butyrylcholinesterase activity (39, 40) but a higher ghrelin hydrolyzing activity, suggesting that another esterase was responsible for this reaction in rat serum.

B-esterases also include nonspecific carboxylesterases found in serum and in tissues (41). These enzymes catalyze the hydrolysis of a variety of drugs or prodrugs containing ester and amide bonds, as well as endogenous compounds such as short- and long-chain acyl-glycerols and long-chain acyl-coenzyme A esters (42, 43, 44), suggesting their participation in the metabolism of drugs and endogenous lipids. In humans and rats, several carboxylesterases isoenzymes are encoded by multiple genes (45, 46). Purified porcine liver carboxylesterase (EC 3.1.1.1) was able to degrade ghrelin to des-acyl ghrelin, suggesting that carboxylesterases may indeed participate to ghrelin desoctanoylation. Ghrelin desoctanoylation was faster in rat serum (half-life of 27 ± 2 min, n = 10) than in human serum (236 ± 18 min, n = 11). This discrepancy is consistent with the higher carboxylesterase activity in rat serum, compared with that in human serum, observed by us (a 9-fold difference; Table 1Go) and others (47). Moreover, the correlation between the carboxylesterase and ghrelin desoctanoylation activities in rat serum, the ability of the purified porcine liver carboxylesterase to hydrolyze ghrelin, and the inhibition of ghrelin desoctanoylation by the carboxylesterase inhibitor BNPP supported the hypothesis that carboxylesterase contributed to the desoctanoylation of ghrelin in rat serum.

In human serum, in contrast, we did not observe any inhibition of ghrelin desoctanoylation by BNPP, ghrelin desoctanoylation, and carboxylesterase activities were not correlated, and the carboxylesterase activity was 185 times lower than in rat serum, whereas ghrelin hydrolysis activity was only nine times lower (Table 1Go). Those observations did not support the hypothesis that carboxylesterase participated in ghrelin desoctanoylation by human serum and suggested the involvement of an additional esterase in that process.

So far, determinations of plasma concentrations of ghrelin revealed that the concentration of the desoctanoylated form of ghrelin was much higher than that of the octanoylated one (10). This might result from ghrelin desoctanoylation in the blood circulation and/or during blood collection and handling of the samples despite the half-life of 240 min. Nevertheless, it seems appropriate to add PMSF into the blood collecting tubes.

Degradation of ghrelin by rat stomach, liver, and kidney homogenates, at variance with the serum, led to the generation of several C-terminal fragments in addition to desoctanoylated ghrelin. This indicated the participation of both esterases and aminopeptidases. In view of these results, it would be useful to reevaluate ghrelin and des-acyl ghrelin levels in tissues in the presence of esterases and proteases inhibitors because ghrelin degradation activity was very high in tissue homogenates. Carboxylesterases are widely distributed throughout the tissues and present in several subcellular organelles (41). They are particularly abundant in rat liver microsomes, loosely bound to the luminal surface of the endoplasmic reticulum (48, 49). The use of synthetic ghrelin derivatives, containing more stable or thioether bonds capable of replacing the octanoyl ester bond of ghrelin without modifying much the activity on the ghrelin receptors (50), could be very advantageous to extend the half-life of ghrelin in vivo.

We identified five cleavage sites in ghrelin between residues -Ser2-(acyl)Ser3- in stomach and liver, -(acyl?)Ser3-Phe4- in stomach, liver and kidney, -Phe4-Leu5- in stomach and kidney, -Leu5-Ser 6- and -Pro7-Glu8- in kidney (Fig. 7Go). All those relatively long C-terminal fragments are probably inactive because the minimal structural requirement for ghrelin’s activity resides in the N-terminal tetrapeptide with an octanoyl group on Ser3 (16, 50). After 120 min incubation of ghrelin with stomach or kidney homogenate, 70% (compared with 100% with liver homogenate) of the original ghrelin at time zero can be accounted for by the observed fragments, suggesting the presence of additional proteolysis for which fragments could not be detected. The cleavage between residues -Ser2-(acyl)Ser3- might be due to a dipeptidyl-peptidase, perhaps the dipeptidyl-peptidase II (EC 3.4.14.2), present in the stomach and the liver, or the dipeptidyl-peptidases I and III (EC 3.4.14.1 and EC 3.4.14.4), present in the liver. Neprilysin (EC 3.4.24.11), which is able to cleave between hydrophobic residues, could be responsible for the formation of Ghr [4–23] and Ghr [5–23] in the kidney. Aminopeptidase N (EC 3.4.11.2), cytosol alanyl aminopeptidase (EC 3.4.11.14) and cathepsin H (EC 3.4.22.16), releasing N-terminal amino acid from a wide range of peptides, might be responsible for the cleavage between Ser3-Phe4, Phe4-Leu5, and Leu5-Ser6 in the liver and the kidney if sequential cleavage occurs. Dipeptidyl-peptidase IV (EC 3.4.14.5), which is able to release the N-terminal dipeptide Xaa-Xbb from a peptide containing Xaa-Xbb-Xcc, preferentially when Xbb is Pro and provided that Xcc is neither Pro nor hydroxyproline, might be responsible for the formation of Ghr [8–23] from Ghr [6–23] in the kidney.



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  		FIG. 7. Ghr [1–23] degradation by rat tissue homogenates: cleavage sites. The cleavage sites of Ghr [1–23] after incubation with rat stomach (St), liver (L), or kidney (K) homogenate, or serum (Se) are indicated by the arrows.

 
Recent studies suggest that the kidneys and liver play a role in the clearance of ghrelin (51, 52). Even though ghrelin degradation was studied using tissue homogenates, thereby exposing ghrelin to some enzymes that might never reach it in vivo, part of the ghrelin clearance could be due to the action of enzymes at the observed cleavage sites. Further experiments using perfused organs or whole animals are required to assess the relative importance of ghrelin desoctanoylation and proteolysis. Due to ghrelin degradation by serum and tissues, it is difficult to accurately determine the bioactive serum, plasma, or tissue levels of this hormone and consequently assess its physiological and pathophysiological roles. Inhibitors of esterases and proteases will represent valuable tools to limit ghrelin degradation and allow accurate determinations of ghrelin and des-acyl ghrelin levels.

In summary, the degradation of ghrelin by serum led to the sole generation of desoctanoylated ghrelin. In human serum, butyrylcholinesterase and other esterase(s) participated to ghrelin desoctanoylation, whereas in rat serum carboxylesterase was involved. At variance with serum, ghrelin degradation by stomach, liver, or kidney homogenates resulted from both N-terminal proteolysis and desoctanoylation.


   Acknowledgments
 
The authors thank Prof. Y. Carpentier (L. Deloyers Laboratory for Experimental Surgery, ULB, Belgium) for his suggestions, M. Stiévenart for his secretarial assistance, and P. De Neef and M.-D. Martin-Martinez for their technical assistance (all from the Department of Biochemistry and Nutrition, ULB, Belgium).


   Footnotes
 
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).

Abbreviations: BNPP, Bis-p-nitrophenyl-phosphate; GHS-R, GH secretagogue receptor; PMSF, phenylmethylsulfonyl fluoride; RP-HPLC, reverse phase-HPLC; TFA, trifluoroacetic acid.

Received May 5, 2004.

Accepted for publication July 8, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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