Structure, measurement, and secretion of human glucagon-like peptide-2

Peptides 21 (2000) 73– 80

Structure, measurement, and secretion of human glucagon-like peptide-2 Bolette Hartmanna, Anders H. Johnsenb, Cathrine Ørskovc, Kim Adelhorstd,1, Lars Thimd, Jens J. Holsta,* a

Department of Medical Physiology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark b Department of Medical Anatomy, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark c Department of Clinical Biochemistry, Rigshospitalet, DK-2100, Copenhagen Ø, Denmark d Novo Nordisk A/S, DK-2880 Bagsvaerd, Denmark Received 15 July 1999; accepted 27 September 1999

Abstract By using radioimmunoassays toward the cDNA-predicted amino acid sequence of human glucagon-like peptide-2, a peptide was isolated from extracts of human ileum. By mass spectrometry and Edman sequencing, this peptide was identified as human proglucagon 126-158. High-performance liquid chromatography analyses indicated that a similar immunoreactive peptide (iGLP-2) was present in human plasma. Human plasma concentrations of iGLP-2 were elevated 3- to 4-fold at 1 to 2 h after ingestion of 800 to 1200 kcal meals. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Proglucagon; Radioimmunoassay; GLP-2; GLP-1; DPP-IV

1. Introduction In mammals, the glucagon gene is expressed, not only in the pancreas, but also in the mucosa of the gastrointestinal tract [21,24]. The single glucagon gene [1] encodes a common precursor, proglucagon (PG), a peptide of 160 amino acids that undergoes tissue-specific post-translational processing [10] (Fig. 1). Thus, in the pancreas, PG is predominantly cleaved to produce glicentin-related pancreatic polypeptide (GRPP), corresponding to PG 1-30, glucagon itself (PG 33-61) [22,26], a hexapeptide corresponding to PG 64-69 (intervening peptide 1), and the so-called major PG fragment (MPGF; PG 72-158) [11,28]. The processing pattern in the small intestine differs markedly. Here, the major secreted products are the 69 amino acid glucagoncontaining peptide, glicentin, (PG 1-69) [29], the two glucagon-like peptides GLP-1 (PG 78-107NH2) [12,29] and GLP-2 (PG 126-158) [5,30], and the so-called intervening peptide 2 (PG 111-123) [5]. So far, very little is known about GLP-2 [13]. However, * Corresponding author. Tel.: ⫹45-3532-7518; fax: ⫹45-3532-7537. E-mail address: [email protected] (J.J. Holst) 1 Present address: Dako A/S, Glostrup, DK-2600, Denmark.

Drucker et al. have recently shown that GLP-2 induces intestinal proliferation in mice [8,27]. To investigate human GLP-2 in greater detail, we determined its exact chemical structure, developed a specific radioimmunoassay for fully processed GLP-2, and studied its secretion over a 24-h period in which healthy subjects received three mixed meals.

2. Materials and methods 2.1. Peptides Synthetic GLP-2 1-11 (PG 126-136), bovine GLP-2 with Thr123 Tyr12 substitution, and recombinant human GLP-2 (PG 126-158) made in yeast, were all produced at Novo Nordisk (Bagsvaerd, Denmark). The structure and purity of these peptides were confirmed by high-performance liquid chromotography (HPLC), mass and sequence analysis. 2.2. Tissue extraction Fresh pieces of human ileum and pancreas were obtained from transplantation donors (approved by the Local Ethical Committee) and immediately frozen.

0196-9781/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 6 - 9 7 8 1 ( 0 0 ) 0 0 1 7 6 - X

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Fig. 1. Simplified diagram of the intestinal and pancreatic processing of human proglucagon (PG). The numbers indicate the positions of the amino acid residues in PG. The vertical lines indicate positions of basic amino acids, typical cleavage sites (see Ref. [16] for further details).

The tissues were extracted at neutral pH as previously described (extraction of small samples and acidic peptides in [14]). In short, frozen tissue was submerged in boiling water (10 ml/g tissue), boiled for 15 min, homogenized in a Potter homogenizer and centrifuged, and the supernatant was retained. 2.3. HPLC purification of GLP-2 We used LKB equipment with an MN Nucleosil cartridge system (Macherey-Nagel, Duren, Germany) at room temperature with on-line UV detection at 226 nm. Forty milliliters of ileal extract was applied to a 4 ⫻ 100 mm Nucleosil 300-5 ␮m C18 column in 10-ml portions and eluted at a flow rate of 1 ml/min with linear gradients of acetonitrile (AcN, HPLC grade; Rathburn, Walkerburn, Scotland) in water containing, in addition, 0.1% trifluoroacetic acid (TFA; Rathburn, Walkerburn, Scotland) (0 to 35% AcN over 5 min, followed by 35 to 45% AcN over 20 min, and 45 to 75% AcN over 5 min). One-minute fractions were collected and subjected to analysis for both N- and C-terminal sequences of the assumed GLP-2 molecule corresponding to PG 126-158 (see below). All immunoreactive material from the four preliminary HPLC runs was collected, diluted ⫻ 2 with 0.1% TFA, and rechromatographed using the same column and gradient. Again, 1- min fractions were collected, subjected to analysis for both N-terminal and C-terminal PG 126-158 immunoreactivity (IR), and all immunoreactive material was collected and rechromatographed with a 4 ⫻ 100 mm Nucleosil 300-7 ␮m C6H5 column; gradient, 0 to 30% AcN over 5 min, followed by 30 to 40% AcN over 20 min, and 40 to75% AcN over 5 min. The final purification step was performed at 50°C on a 2.1 ⫻ 150 mm 5 ␮m C8 column (Vydac, Hesperia, CA, USA) at a flow rate of 200 ␮l/min by using a HewlettPackard HP 1090 system (Hewlett–Packard, Waldbronn, Germany) equipped with diode array detector. The absor-

bance was monitored at 214 nm and peak fractions were collected manually. The gradient was 10 to 30% AcN over 5 min, followed by 30 to 45% AcN over 30 min. Four milliliters of the extract used for the HPLC purification was subjected to gel permeation chromatography on Sephadex G50 Fine grade column (Pharmacia, Uppsala, Sweden) equilibrated and eluted with 40 mmol/l sodium phosphate buffer pH 7.5 containing, in addition, 0.1% w/v human serum albumin, 0.1 mol/l NaCl, 10 mmol/l EDTA and 0.6 mmol/l Thimerosal (16 ⫻ 1000 mm at a flow rate of 0.3 ml/min at 4°C). Trace amounts of 125I-labeled albumin and 22NaCl were added for internal calibration. All fractions were collected and analyzed for PG 126-158-IR by using both a “side-viewing” and an N-terminal specific assay (see below). 2.4. Mass spectrometry and protein sequence analysis The HPLC fraction containing the purified peptide was lyophilized, reconstituted in 5 ␮l of 0.1% TFA in 30% AcN. For mass spectrometry, 0.5 ␮l was mixed with 0.5 ␮l of 33 mmol/l ␣-cyano-4-hydroxycinnamic acid in AcN/methanol (Hewlett–Packard). A 0.5-␮l aliquot of this mixture was analyzed by matrix-assisted laser desorption mass spectrometry using a Biflex Instrument (Bruker–Franzen, Bremen, Germany) in the reflector mode with positive acceleration at 19.5 kV and the reflector set to 20 kV. The method has an accuracy of 0.02%. The remainder of the sample was utilized for determination of the amino acid sequence of the peptide in an automated protein sequencer (Procise 494A, ADB, Perkin Elmer, Foster City, CA, USA). All reagents and solvents were from Perkin Elmer. 2.5. Radioimmunoassays In anticipation of a structure of GLP-2 corresponding to PG 126-158, a specific N-terminal radioimmunoassay for

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PG 126-158 was developed, using an antiserum raised in Danish white rabbits against synthetic GLP-2 1-11 (PG 126-136) coupled to bovine serum albumin (BSA) with carbodiimide as previously described [14]. For standards, we used recombinant human GLP-2(1–33) and the tracer was bovine GLP-2 with Thr123 Tyr12 substitution, 125Ilabeled using the standard stoichiometric chloramine T method as described elsewhere [14] (the first eleven Nterminal residues are identical in human and bovine GLP-2). The assay buffer was 80 mmol/l sodium phosphate buffer, pH 7.5, containing in addition 0.1% wt/vol human serum albumin (ORHA 20/21, Behring, Marburg, Germany), 10 mmol/l EDTA and 0.6 mmol/l Thimerosal (no. T-5125, Sigma Chemical Co., St. Louis, MO, USA). Free and bound moieties were separated with plasma-coated charcoal (E. Merck, Darmstadt, Germany) [14]. For assays of the assumed C-terminus of GLP-2, antiserum code no. 8772 raised against PG 149-158 was used, with PG 149-158 or PG 126-158 as standard [30] and 125 I-labeled bovine Thr123 Tyr12 GLP-2. This assay reacts fully with PG 126-158 but with neither PG 126-159 nor PG 151-160 nor any of the other peptides tested. A side-viewing GLP-2 assay (reacting with a mid-sequence of GLP-2) was developed using the same protocol described above for the N-terminal assay except we used antiserum cat.No. RAS 7167, Peninsula Laboratories, and rat GLP-2 with Asp333 Tyr33 substitution for iodination. In agreement with its specificity for a mid-region of GLP-2, the antiserum does not bind the Tyr12-GLP-2 tracer. To study whether the N-terminal GLP-2 assay crossreacts with MPGF, we subjected 4 ml of extract of human pancreas (which contains high concentrations of MPGF) to gel filtration as described above. All fractions were analyzed for immunoreactive MPGF, glucagon and N-terminal GLP-2. To measure MPGF we used antiserum 91022 raised against synthetic PG 78-87 with PG 78-107amide as standard and 125I-labeled PG 78-107amide as tracer as described in [11]. This assay cross-reacts fully with MPGF. For glucagon determination we used the side-viewing antiserum 4304 (measuring the 6 –15 sequence of glucagon) and labeled and unlabeled glucagon as described in [15]. 2.6. Plasma studies Six normal healthy nonsmoking volunteers (four female, two male), median age 27 (range, 24 –34) years (mean body mass index ⫾ SEM: 20.9 ⫾ 1.4 kg/m2) were studied after an overnight fast. The study was approved by the local ethical committee. The subjects received breakfast at 0900 h (two rolls with butter and jam, one piece of Danish pastry, a glass of orange juice, and a glass of milk; 825 kcal; carbohydrate (C), 58%; fat (F), 33%; protein (P), 9%), lunch at 1300 h (lasagne, a glass of grape juice, and a piece of cake; 1079 kcal; C, 52%; F, 35%; P, 12%), and dinner at 1900 h (chili con carne, a piece of cake, and a beer; 1188

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kcal; C, 62%; F, 25%; P, 13%). The volunteers were allowed to drink water between the meals. Blood samples were drawn at regular intervals before and after the meals and collected into chilled tubes containing, in final concentrations: diprotin-A (a specific DPP-IV inhibitor; 0.1 mmol/l; Bachem Feinchemikalen, Bubendorf, Switzerland), ethylenediaminetetraacetic acid (EDTA) (3.7 mmol/l) and aprotinin (500 Kallikrein Inhibitory Unit (KIU)/ml, Trasylol®; Bayer, Marburg, Germany), kept on ice, and centrifuged within 0.5 h. Plasma samples were kept at -20°C until assay. Other aliquots of the samples were previously analyzed for GLP-1 [31]. All plasma samples were extracted in a final concentration of 75% ethanol before GLP-2 measurements (using the N-terminal specific assay) to remove unspecific cross-reacting substances. Briefly, 2.5 ml of 96% ethanol were added to 700 ␮l plasma/sample, thoroughly mixed, centrifuged at 3300 G for 30 min at 4°C, whereafter the supernatant was dried down overnight and redissolved in assay buffer. All samples were assayed in duplicate. The recovery of synthetic GLP-2 added to plasma before extraction and assay was 68%. 2.7. HPLC analysis of endogenous GLP-2 In order to investigate the nature of the circulating GLP-2, we compared HPLC elution profiles of plasma samples with a high concentration of endogenous GLP-2 (n ⫽ 6) with those of fasting plasma spiked with synthetic GLP-2 (n ⫽ 3). Forty-milliliter blood samples were collected 1 h after the lunch as described above. Plasma was separated by centrifugation at 4°C and concentrated on Sep-Pak C18 cartridges (Waters–Millipore, Milford, MA, USA) eluted with 70% AcN, 0.1% TFA. Eluates were lyophilized, reconstituted in 0.1% TFA (500 ␮l), and subjected to analytical reverse phase HPLC using LKB equipment with an MN Nucleosil cartridge system (Macherey-Nagel) fitted with a Nucleosil 120-5 ␮m C8 column. The column was eluted at a flow rate of 2 ml/min, with stepwise linear gradients of AcN in 0.1% TFA (0% AcN for 3 min, followed by 0 to 35% over 3 min, 35 to 42% over 21 min, and 42 to 75% over 3 min). Fractions were collected every 0.25 min and subjected to analysis for N-terminal GLP-2-immunoreactivity (IR).

3. Results 3.1. N-terminal radioimmunoassay The selected antiserum (code no. 92160) could be used in a final dilution of 1:35.000 and had a binding affinity for PG 126-159 of approximately 1010 l/mol (Scatchard analysis). It showed no cross-reaction with GLP-1 1-36NH2, GLP-1 7-36NH2, glicentin, oxyntomodulin, glucagon, pituitary adenylyl cyclase activating polypeptide (PACAP), vasoactive

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Fig. 3. Final purification step of human GLP-2 by HPLC on a 2.1 ⫻ 150 mm Vydac 5 ␮m C8 column eluted with a 30 to 45% gradient of acetonitrile in 0.1% trifluoroacetic acid as indicated (broken line). Optical density is plotted against retention time (solid line). The material eluting at 26.1 min (corresponding to 38% acetonitrile) was collected for mass spectrometry and sequence analysis.

Fig. 2. Gel filtration of an extract of human pancreas analyzed with assays for Major Proglucagon Fragment (MPGF; 91022, Œ); Glucagon-Like Peptide-2 (GLP-2: 92160, F) and Glucagon (4304, 䡺). Note the 50-fold expanded scale at the right y-axis showing immunoreactive GLP-2 concentrations.

intestinal peptide (VIP), growth hormone releasing factor (GHRF), gastric inhibitory peptide (GIP), secretin, and peptide histidine isoleucine amide (PHI) in concentrations up to 5 nmol/l. With synthetic human GLP-2(3–33), we found a minor cross-reaction of maximum 5.6 ⫾ 1.8%. The experimental detection limit was 5 pmol/l, and the intra-assay coefficient of variation was 5% at a concentration of 40 pmol/l. In an extract of human pancreas subjected to gel filtration (Fig. 2), a single peak corresponding to MPGF was identified with the 91022 assay at coefficient of distribution, Kd, 0.25 and the 4304 assay identified a single glucagon peak at Kd 0.8. The 92160 assay revealed a single peak at Kd 0.5 corresponding to GLP-2, confirming the lack of cross-reaction of the GLP-2 assay with MPGF. The total amount of GLP-2 constituted 0.7% of the amount of MPGF. 3.2. Purification and structure of human GLP-2 Extracts of human intestine were subjected to HPLC purification, and the final HPLC run showed one predominant peak with GLP-2 immunoreactivity eluting at 38% AcN (Fig. 3). Sequence analysis of this purified peptide established that its amino acid sequence corresponded to human PG 126-158. Amino acid no. 1, 2, and 4 were ambiguous due to high background levels. Laser desorption mass spectrometry showed the molecular mass to be 3765.8 compared to the calculated mol wt 3766.1 for human PG 126-158 (Fig. 4).

Gel filtration of the ileal extract showed identical homogeneous elution profiles of GLP-2-IR at Kd 0.50 for sideviewing as well as N-terminal specific assays (not shown). The tissue concentration of GLP-2 as calculated by dividing the total amount of eluted GLP-2 by the weight of the extracted tissue was 107.2 pmol/g tissue, wet weight. 3.3. Secretion The mean plasma concentration of GLP-2 in fasting subjects was 15 ⫾ 2 pmol/l (n ⫽ 6). After all three meals there were significant increases in plasma levels, with maximum GLP-2-IR (61 ⫾ 9 pmol/l) being seen 60 min after lunch (Fig. 5). The plasma GLP-1 concentrations, obtained in a previous study [31], showed a similar pattern, with a correlation between plasma GLP-1 and GLP-2 of r ⫽ 0.66 ⫾ 0.06, P ⬍ 0.0001. 3.4. HPLC analysis of endogenous GLP-2 Reverse phase HPLC showed identical elution positions of endogenous human GLP-2 and synthetic GLP-2 1-33 added to fasting plasma (Fig. 6). All N-terminal immunoreactive material eluted at 35.9% AcN in the six samples with high endogenous GLP-2 concentration as well as in the three samples spiked with synthetic GLP-2.

4. Discussion The present study provides information about the exact chemical structure of human GLP-2 and its secretion as revealed by the peripheral plasma concentrations in healthy human subjects. Earlier studies on proglucagon processing in human intestine and pancreas revealed a differential processing of the precursor [32]. Thus, the pancreas was shown

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Fig. 4. Mass spectrum of human GLP-2. Approximately 0.5 pmol was analyzed by matrix-assisted laser desorption mass spectrometry using a Biflex Instrument (Bruker–Franzen) in the reflector mode. The spectrum was averaged from 110 laser beam shots. The value given above the peak indicates the molecular mass of the molecular ion (MH⫹).

to contain and secrete mainly the so-called MPGF [28,33], corresponding to residues no. 72 to 158 of proglucagon [11], in which the GLP-2 sequence is contained [1], whereas the intestinal mucosa was found to contain and secrete each of the two glucagon-like peptides [28,32]. Immunochemical and chromatographic studies of human GLP-2 and comparisons with porcine GLP-2, the structure of which was elucidated by sequence analysis, led to the conclusion that human GLP-2 would correspond to the proglucagon residues 126 to 158 [5,30]. However, from the sequence data of porcine GLP-2, it could not be excluded that GLP-2 molecules terminating with Arg 159 or Lys 160 of proglucagon could also exist as well as N-terminally extended forms of GLP-2 that were actually demonstrated in pigs [5]. Finally, O-glycosylation, reported to occur in the MPGF of the rat [25], might have passed unnoticed. The present study unequivocally establishes the structure of human GLP-2 to correspond to PG 126-158, and its molecular mass excludes the presence of glycosylations. Because GLP-2 was isolated using C-terminal as well as N-terminal assays, and because no immunoreactive material was discarded during the purification, it is unlikely that significant amounts of N- or C-terminally extended molecules are produced in the human intestine. Furthermore, size exclusion chromatography of the ileal extract used for isolation showed identical elution profiles for the N-terminal and the side-viewing GLP-2 assays, proving absence of extended or truncated molecular forms in the extract. Based on these data, it was now possible to synthesize

GLP-2 of the correct structure, allowing the development of a specific radioimmunoassay for human GLP-2. Earlier processing studies, confirmed here, indicated that the GLP-2 sequence resulting from pancreatic expression of the glucagon gene was contained almost exclusively in the MPGF, with only trace amounts of proglucagon being processed to release GLP-2 [11]. As a strategy for the development of a specific assay for fully processed intestinal GLP-2 we, therefore, raised antibodies against the free N-terminus of GLP-2. Antisera against other epitopes would be predicted to crossreact with MPGF and other molecules carrying an N-terminal extension of the GLP-2 sequence. Using a short, synthetic N-terminal fragment of GLP-2 coupled to albumin as antigen, one of the resulting antisera turned out to exhibit the desired specificity. The specificity was tested by investigating the performance of the resulting radioimmunoassay toward fractionated pancreatic extracts containing high concentrations of MPGF which contains the entire GLP-2 sequence at the C-terminus. Indeed, in such extracts there was no cross-reaction with MPGF at all, and in intestinal extracts, GLP-2 was found in amounts corresponding to GLP-1 as expected, if the two are processed with equal efficiency in the intestinal L-cells [28]. In addition the tissue concentration of GLP-2 determined here (107.2 pmol/g tissue) is equal to or higher than GLP-1 or glicentin levels found in human intestinal whole wall biopsies earlier [32]. We, therefore, believe that our assay reliably and specifically measures intact GLP-2. In a previous radioimmunoassay for GLP-2 [34] we used as label, GLP-2 iodinated at

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Fig. 5. Diurnal mean plasma concentrations of human glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2) in six healthy subjects. The plasma concentrations in pmol/l (⫾SEM) are plotted against time. The arrows indicate the time points of the meals.

the N-terminal His. This tracer would be expected to react only with antisera that were not directed against the Nterminal, and, in agreement with this, the assay cross-reacted strongly with MPGF [28,32]. Our choice of substituting Thr 12 by Tyr was inspired by the reported presence of Tyr at this position in bovine GLP-2 [19]. If this residue is not conserved between species, such a tracer might also be suitable for receptor studies, as opposed to the N-terminally labeled tracer, since all of the peptides in this family seem to depend on a free and unmodified N-terminus for biologic activity [20]. Subsequent inquiries revealed, however, that

the indicated Tyr was erroneous and that the residue present at position 12 in bovine GLP-2 is indeed Thr as also indicated from the nucleotide sequence [19]. By the usual criteria for radioimmunoassays of peptide hormones in plasma, our analysis seems accurate [2]. Thus, sensitivity, precision, and specificity seemed adequate. In addition, the recovery of GLP-2 added to plasma was satisfactory, when corrected for the losses inherent in the ethanol extraction step required. The accuracy was also evaluated by reverse-phase HPLC analysis of samples with a high concentration of endogenous GLP-2. The endogenous immunoreactive material eluted at the position of exogenous GLP-2 subjected to chromatography under identical conditions. The diurnal profile of GLP-2 was highly similar to that of GLP-1, in agreement with the assumption that the two are secreted in parallel and in equimolar amounts [16]. The fact that rather similar concentrations were obtained for the two peptides and that the decreases after the meals were almost superimposable, suggest that the two peptides are also metabolized at about similar rates. However, recent research has established that GLP-1 is initially metabolized by the ubiquitous enzyme, DPP-IV [6], whereby the peptide loses its two N-terminal amino acid residues as well as its biologic activity. Thereby the elimination half-life of the intact peptide becomes as low as 1 to 1.5 min, while its metabolic clearance rate exceeds cardiac output by a factor of two [7]. The studies by Mentlein et al. [20] suggested that GLP-2 would also be a substrate for DPP-IV and studies by Drucker et al. [9] indicated that a significant DPP-IV mediated degradation of GLP-2 takes place in rats. In our N-terminal assay, the metabolite shows only minimal cross reaction, and the values we obtained here therefore apply to the intact peptide. It can, therefore, be concluded that GLP-2 is metabolized more slowly than GLP-1. Brubaker et al. recently [4] reported an assay for GLP-2, based on an antibody reacting with amino acids no. 25 to 30 of GLP-2, similar to the assay described by us in 1987 [34]. As discussed above, a ‘side-viewing’ assay cannot discriminate between intact GLP-2 and other GLP-2-containing proglucagon-derived peptides including the metabolite, as also indicated by the very high GLP-2 levels reported for rat and human plasma in that study. Using our antibody 92160, the same authors reported fasting and postfeeding levels of intact GLP-2 of 30 ⫾ 6 and 52 ⫾ 26 pmol/l, i.e. similar to the levels found here. Using the side-viewing assay these authors also found significant amounts of the metabolite GLP-2(3–33) in human and rat plasma. In a comparison of the effect on intestinal proliferation in mice of intact GLP-2 and the metabolite, the latter was found to be ineffective (Hartmann, unpublished data). Accurate determination of intact bioactive GLP-2, therefore, requires absence of interference by this metabolite in the assay. Evidence is accumulating that GLP-2 plays an important role as a growth factor for intestinal cells [8,27]. A role for proglucagon-derived peptides in the adaptation and growth

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Fig. 6. Human plasma from a representative healthy subject collected 1 h after lunch was analyzed by reverse phase HPLC on a Nucleosil C8 column and measured with the N-terminally specific radioimmunoassay (92160). The arrow indicates the elution position of synthetic human GLP-2.

of the intestinal mucosa has long been suspected [3] and, whereas the two other main products of intestinal proglucagon processing, glicentin and GLP-1, seem to have little effect (see [17] for review), it now seems that GLP-2 may be responsible for the endocrine regulation of intestinal growth and adaptation under a variety of experimental and pathophysiological situations that are associated with a marked release of proglucagon derived peptides [3,17]. Our findings show that under normal physiological circumstances, its release, and hence its effects, would be related to meal ingestion. This seems to be in agreement with the fact that feeding by the oral route stimulates intestinal growth whereas parenteral nutrition, bypassing the intestinal lumen and thereby devoid of a stimulatory effect on GLP-2 secretion, is associated with marked atrophy of the intestinal mucosa [3]. However, GLP-2 may also have other, yet unmasked, effects. A receptor for GLP-2 was recently cloned [23]. An earlier report that GLP-2 may activate adenylate cyclase in brain sections [18] is consistent with the reported expression of the receptor in the hypothalamus [23].

Acknowledgments We gratefully acknowledge the technical assistance of Allan Kastrup, Lene Albæk, and Rigmor Holck. This study was supported by The Danish Medical Research Council.

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