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Departments of Physiology (P.L.B.) and Medicine (P.L.B., P.E., C.-H.T., D.J.D.) and Banting and Best Diabetes Center (D.J.D.), University of Toronto, Toronto, Canada; and Allelix Biopharmaceuticals, Inc. (A.C.), Mississauga, Ontario, Canada
Address all correspondence and requests for reprints to: Dr. P. L. Brubaker, Room 3366, Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada M5S 1A8. E-mail: p.brubaker{at}utoronto.ca
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have recently identified GLP-2 as a stimulator of intestinal epithelial proliferation (15, 16, 17). Subcutaneous administration of GLP-2 to mice twice daily for 10 days induces a 1.5- to 2-fold increment in small intestine weight. These changes occur consequent to stimulation of crypt cell proliferation and result in increased villous height (15, 16, 17). The establishment of a biological activity for GLP-2 has therefore necessitated the development of specific RIA and HPLC methods to analyze the synthesis, secretion, and molecular forms of this peptide so that its regulation might be studied in detail.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peptides contained in 5 cm rat ileum or half of a rat pancreas (n = 3) were extracted by homogenization in 1 N HCl containing 5% (vol/vol) formic acid, 1% (vol/vol) TFA, and 1% (wt/vol) NaCl, followed by reverse phase extraction using C18 silica (4, 5, 6). The tissue protein contents of ileal and pancreatic extracts were 2411 ± 207 and 3027 ± 269 µg, respectively, as determined by the Lowry protein assay (20).
In feeding experiments, rats (n = 4–5) were fasted for 22–24 h
(fasted) or were allowed free access to chow without fasting (fed).
Normal human volunteers (males and females, aged 23–32 yr; n = 6)
were fasted overnight, and a blood sample was collected before (fasted)
and after (fed) a small breakfast, consisting of coffee and a donut
(
220 cal). Plasma was also collected from two groups of patients
using a protocol approved by the Toronto Hospital ethics committee. In
brief, one group of patients (n = 4) was examined before and after
abdominal surgery, and a second group of patients with chronic renal
failure (n = 6) was studied pre- and postdialysis. All plasma
samples were collected into a 10% volume of Trasylol-EDTA-diprotin A
[5000 kallikrein inhibitory units/ml (Miles Canada, Etobicoke,
Canada):1.2 mg/ml:0.1 mM; a dipeptidylpeptidase IV (DP IV;
CD26; EC 3.4.14.5) inhibitor (21); Sigma Chemical Co., St. Louis, MO]
and were subjected to reverse phase extraction using C18
silica after addition of 2 vol 1% TFA (pH adjusted to 2.5 with
diethylamine) (4).
Proglucagon was obtained by reverse phase extraction of Baby Hamster Kidney (BHK) proglucagon cells, which express the proglucagon gene under the control of a metallothionein promoter, but do not process proglucagon to any of the PGDPs (6, 22).
HPLC separation of GLP-2-related peptides was carried out using one of three protocols. Separation of proglucagon, glicentin, oxyntomodulin, glucagon, and GLP-2 was accomplished using a C18 µBondapak column (Waters Associates) with a linear gradient of acetonitrile and TFA [25–62.5% (vol/vol) B over 45 min, followed by a 10-min purge; A = 1.0% (vol/vol) TFA (pH adjusted to 2.5 with diethylamine); B = 80% (vol/vol) acetonitrile] (4, 5, 6). The flow rate was 1.5 ml/min, and fractions were collected every 18 sec. Trace amounts of [125I]glucagon were added to all samples to serve as an internal control. Mol wt analysis of pancreatic immunoreactive GLP-2 was carried out on two Protein-Pak 125 columns (Waters Associates) in series with isocratic elution using 40% (vol/vol) acetonitrile and 0.1% (vol/vol) TFA. The flow rate was 1 ml/min, and 0.5-min fractions were collected. Cytochrome c (12.5 kDa), Trasylol (6.5 kDa), and [125I]glucagon (3.5 kDa) were used as mol wt markers (6). Finally, GLP-2-related peptides in plasma were separated using a gradient of 30–60% (vol/vol) B over 45 min [A = 0.1% (vol/vol) TFA in water; B = 0.1% (vol/vol) TFA in acetonitrile], followed by a purge. The flow rate was 1.5 ml/min, and 18-sec fractions were collected. Trace amounts of [125I]GLP-2-(1–33)-Tyr34 were added to all samples as an internal marker.
Incubation of hGLP-2-(1–33) and [Gly2]hGLP-2-(1–33) (from California Peptide Research) was carried out in 40 mM PBS, pH 6.5. Ten micrograms of peptide in 50 µl PBS were incubated with 2.5 µl (0.125 mU) human DP IV (SA, 5000 mU/mg protein; Calbiochem, San Diego, CA) at 37 C for 24 h. The reaction was quenched by addition of 200 µg diprotin A. The samples were then analyzed by HPLC as described above for plasma samples, using a Rainin Dynamax C18 column (Mandel, Guelph, Ontario, Canada). Elution was monitored by absorption at 214 nm.
Recovery of exogenously added GLP-2 from intestinal, pancreatic, and plasma samples after reverse phase extraction was 84 ± 17%. Statistical analyses were carried out using paired Student’s t test or ANOVA with n-1 custom hypotheses tests, as appropriate, using an SAS system for IBM computers (Statistical Analysis Systems, Cary, NC).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Binding of
[125I]hGLP-2-(1–33)-Tyr34 to antiserum
UTTH-7 was displaced by synthetic human GLP-2-(1–34) as well as by
N-terminally extended [acetylated GLP-2-(1–33)] or truncated
[GLP-2-(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33) and GLP-2-(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33)] analogs of GLP-2 (Fig. 1b).
Similarly, binding was not affected by addition of a C-terminal
extension to GLP-2 [GLP-2-(1–33)NH2], or removal of the
three C-terminal amino acids [GLP-2-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)]. However, antiserum
binding of the trace was not displaced by GLP-2-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25), which is
lacking eight C-terminal amino acids. Thus, the antigenic site
recognized by UTTH-7 was localized to amino acids 25 and 30 of GLP-2.
UTTH-7 did not bind any other peptides that are structurally related to
GLP-2, including other PGDPs (Fig. 1c).
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). One
major peak of immunoreactive GLP-2 was detected that eluted with the
same retention time as synthetic rat GLP-2-(1–33). Unexpectedly,
variable amounts of a minor peak of GLP-2 were also detected in all
samples (16 ± 7% of total GLP-2), which eluted with the same
retention time as synthetic rat GLP-2-(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33). In contrast with the
intestinal samples, rat pancreatic extracts contained glucagon, but no
other glucagon-containing peptide (e.g. glicentin or
oxyntomodulin). No peak of GLP-2 could be detected after HPLC
separation of pancreatic peptides despite the detection of
immunoreactive peptide in the whole extract before HPLC.
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). Antiserum UTTH-7 also cross-reacted
with proglucagon, as determined by HPLC and RIA analysis of BHK
proglucagon cells (data not shown).
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). Analysis of the immunoreactive GLP-2
by HPLC demonstrated the existence of three main peaks in plasma from
fasted rats, two of which eluted with the same retention time as
synthetic GLP-2-(1–33) and GLP-2-(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33) (Fig. 4b). The levels of
GLP-2-(1–33) and GLP-2-(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33) were both markedly increased in plasma
from fed rats. The minor peak of immunoreactive GLP-2 (fraction 63) has
not been identified, but did not change with feeding.
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). To determine the contribution of
intact GLP-2 to these values, a second GLP-2 RIA was established using
antiserum 92160, which cross-reacts with the free N-terminal end of rat
and human GLP-2, but not with N-terminally truncated forms of GLP-2
(e.g. GLP-2-(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33); Fig. 5a, inset). Analysis of
several of the human plasma samples using antiserum 92160 indicated
that GLP-2 levels rose from 116 ± 22 to 197 ± 98 pg/ml
2 h postfeeding. Thus, intact GLP-2 accounts for 15 ± 4% of
the total GLP-2 immunoreactivity in human plasma. Total immunoreactive
GLP-2 levels were also determined using antiserum UTTH-7 in plasma from
patients undergoing abdominal surgery as well as dialysis. Pre- and
postsurgery concentrations of GLP-2 were not different from those found
in normal fasted humans. By contrast, patients with chronic renal
failure exhibited markedly increased levels of circulating GLP-2
(4.1-fold vs. normal humans; P < 0.01), and
these levels were not significantly decreased after dialysis. HPLC
analysis of plasma from fed humans indicated the presence of two main
peaks of GLP-2 that eluted with the same retention times as
GLP-2-(1–33) and GLP-2-(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33) (Fig. 5b).
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). Greater than 50% of the
hGLP-2-(1–33) was cleaved to GLP-2-(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33) by DP IV, as confirmed by
mass spectrometry and amino acid analysis of the products. To confirm
that Ala2, a known consensus sequence for DP IV cleavage
(24), is important for the action of DP IV on GLP-2, an analog of GLP-2
was synthesized in which Ala2 was replaced by
Gly2 ([Gly2]hGLP-2-(1–33)]; this peptide
was completely resistant to cleavage by DP IV (Fig. 6b).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Consistent with the results of previous studies, the present analyses
have demonstrated that GLP-2-(1–33) is liberated from proglucagon in
the intestinal L cell, whereas pancreatic GLP-2 is contained within the
sequence of MPGF, a large protein that contains the sequences of both
GLP-1 and GLP-2 (1, 2, 10, 11). The results of the present study extend
these earlier findings by demonstrating significant amounts of
GLP-2-(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33) in rat ileum. This peptide has not previously been
detected by mol wt sieving of rat, pig, or human intestinal tissues (2, 10, 11). However, separation of GLP-2-(1–33) and GLP-2-(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33) is
difficult, requiring reverse phase HPLC with a relatively shallow
gradient (0.67% acetonitrile/min) and a very rapid fraction
collection rate (18 sec/fraction).
Immunoreactive GLP-2 levels were elevated 1.5- to 3.6-fold in fed compared with fasted rats and humans, consistent with previous reports of a 50% rise in immunoreactive GLP-2 levels after nutrient ingestion in humans (12). Similar studies have demonstrated that GLP-1 levels in humans also rise after nutrient ingestion (12, 25) in parallel with the increments in circulating levels of immunoreactive GLP-2 (12). Such findings are anticipated based upon the known cosynthesis of GLP-1 and GLP-2 from proglucagon within the L cell (1, 2, 3, 4, 5, 6). However, the results of the present study further demonstrate that in addition to full-length GLP-2, amino-terminally truncated GLP-2 [e.g. GLP-2-(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33)] is a major constituent of circulating GLP-2 (50 ± 6% of the total). It must be noted, however, that GLP-2-(1–33) appears to account for only a small percentage of total immunoreactive GLP-2 in the circulation. Thus, the higher levels of total GLP-2 immunoreactivity detected using antiserum UTTH-7 probably reflect contributions from GLP-2-(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33) as well as pancreatic MPGF.
DP IV-mediated cleavage of rat GLP-2-(1–33) results in the production of GLP-2-(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33) and appears to be an important determinant of GLP-2 bioactivity (23). DP IV removes the first two amino acids from proteins containing N-terminal penultimate alanine or proline residues (24). The results of our experiments establish that human GLP-2 is also subject to DP IV-mediated cleavage. Furthermore, a GLP-2 analog containing glycine at position 2 is resistant to DP IV cleavage, consistent with the importance of Ala2 for DP IV action. Similar studies on the structurally related hormones GLP-1, glucose-dependent insulinotropic peptide, GH-releasing hormone, and peptide-histidine-methionine, have demonstrated that all of these structurally related peptides that contain alanine at position 2 are also subject to degradation by DP IV (26, 27, 28, 29, 30, 31).
DP IV is distributed in a number of tissues, with the highest levels found in kidney, followed by lung, skin, and intestine (32). Interestingly, within the small intestine, DP IV concentrations increase in the aboral direction (33) and along the crypt-villous axis (34). The expression of DP IV within the intestinal epithelium may account for the detection of GLP-2-(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33) in ileal extracts. However, circulating and/or tissue DP IV may contribute to the presence of larger amounts of GLP-2-(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33) in plasma. Further studies are clearly required to elucidate the site of degradation of circulating GLP-2. It must be noted, however, that GLP-2-(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33) was detected in plasma even in the presence of diprotin A, a DP IV-specific inhibitor (21), thereby suggesting that GLP-2-(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33) is a normally circulating peptide. These findings emphasize the importance of collection of plasma for GLP-2 analyses in diprotin A to prevent further degradation of the peptide during storage and demonstrate the importance of analytical methods that distinguish between these two forms of the peptide.
Analysis of plasma from a limited number of patients undergoing abdominal surgery did not demonstrate any changes in the circulating levels of GLP-2. It must be noted, however, that this study did not include any patients undergoing resection of the small intestine, which might be expected to alter circulating levels of the intestinal PGDPs, including GLP-2. In contrast, GLP-2 levels were markedly elevated in patients with chronic renal failure, indicating a role for the kidney in the clearance of circulating immunoreactive GLP-2. Renal clearance accounts for removal of a number of the intestinal PGDPs, including glicentin, oxyntomodulin, GLP-1, and GLP-2 (35, 36, 37). However, most of the circulating immunoreactive GLP-2 in human plasma appears to be consequent to accumulation of immunoreactive peptides other than intact GLP-2, such as truncated GLP-2 and/or MPGF. Thus, the results of these studies clearly demonstrate the need for detailed studies on the clearance of GLP-2 and related peptides from the circulation.
In summary, the results of the present study demonstrate that GLP-2-(1–33) is a major form of intestinal GLP-2 that is released into the circulation in both rats and humans. However, GLP-2-(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33), a degradation product produced by the actions of DP IV, is present in both the intestine and circulation, implicating this enzyme as a regulator of the molecular forms of GLP-2 in vivo.
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Acknowledgments |
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Footnotes |
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2 Scientist of the Medical Research Council of Canada and a consultant
to Allelix Biopharmaceuticals.
Received April 8, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S. Dhanvantari and P. L. Brubaker Proglucagon Processing in an Islet Cell Line: Effects of PC1 Overexpression and PC2 Depletion Endocrinology, April 1, 1998; 139(4): 1630 - 1637. [Abstract] [Full Text] [PDF]
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). b, HPLC analysis of
immunoreactive GLP-2 in 1 ml plasma collected from fed human subjects.
Synthetic hGLP-2-(1–33) and rat GLP-2-(3–33) eluted in fractions 67
and 72, respectively, using this HPLC method. Data are representative
of two experiments.