Departments of 1 Physiology and 2 Medicine, University of Toronto, Toronto, Ontario M5S 1A8; and 3 Banting and Best Diabetes Centre, Toronto Hospital, Toronto, Ontario, Canada M5G 2C4
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ABSTRACT |
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The intestinotropic hormone glucagon-like peptide
(GLP)-2-(133) is cleaved in vitro to GLP-2-(3
33) by dipeptidyl
peptidase IV (DP IV). To determine the importance of DP IV versus renal clearance in the regulation of circulating GLP-2-(1
33) levels in
vivo, GLP-2-(1
33) or the DP IV-resistant analog
[Gly2]GLP-2 was
injected in normal or DP IV-negative rats and assayed by HPLC and RIA.
Normal rats showed a steady degradation of GLP-2-(1
33) to
GLP-2-(3
33) over time, whereas little or no conversion was detected
for GLP-2-(1
33) in DP IV-negative rats and for
[Gly2]GLP-2 in normal
rats. To determine the role of the kidney in clearance of GLP-2-(1
33)
from the circulation, normal rats were bilaterally nephrectomized, and
plasma immunoreactive GLP-2 levels were measured. The slope of the
disappearance curves for both GLP-2-(1
33) and
[Gly2]GLP-2 were
significantly reduced in nephrectomized compared with nonnephrectomized
rats (P < 0.01). In contrast to both
GLP-2-(1
33) and
[Gly2]GLP-2,
GLP-2-(3
33) did not stimulate intestinal growth in a murine assay in
vivo. Thus the intestinotropic actions of GLP-2-(1
33) are determined
both by the actions of DP IV and by the kidney in vivo in the rat.
dipeptidyl peptidase IV; kidney; clearance; degradation
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INTRODUCTION |
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PEPTIDES WITH
NH2-terminal
Xxx1-Ala2
sequences, such as glucagon-like peptide (GLP)-1, glucose-dependent
insulinotropic polypeptide (GIP), and growth hormone-releasing hormone
(GHRH), are degraded and inactivated by the enzyme dipeptidyl peptidase
(DP) IV (6, 7, 12, 14, 19). DP IV, also known as CD26, is an
ectopeptidase on several tissues and is also present as a circulating
enzyme in serum (4, 5, 16, 27, 29). DP IV-mediated cleavage of some
peptide hormones is extremely rapid, with DP IV substrates such as
GLP-1 and GIP exhibiting in vivo half-lives of 0.9 and 2 min,
respectively, compared with 6-10 min for GHRH (Table
1; see Refs. 7, 12, and 14). These studies have also
implicated DP IV as a significant factor in terminating the bioactivity
of these peptides.
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We have recently identified GLP-2-(133) as an intestinal growth
factor that increases intestinal wet weight and villus height, due to
both increased crypt cell proliferation and inhibition of apoptosis at
the villus tips (3, 9, 10, 25). After GLP-2 administration, the murine
intestine is fully functional and exhibits a significant increase in
the activities of brush-border digestive enzymes such as sucrase,
lactase, and maltase (3). Recent studies have also demonstrated that
exogenous administration of GLP-2-(1
33) reduces the severity of
intestinal inflammation in a murine model of colitis (11) and enhances
the adaptive response of the small intestine to massive resection in
the rat (24).
The NH2-terminal sequence of
GLP-2-(133) is identical to that of GLP-1
(His1-Ala2)
and similar to that of GIP and GHRH
(Tyr1-Ala2;
Table 1), suggesting that DP IV may be an important determinant of
GLP-2 bioactivity in vivo. Consistent with this hypothesis, we have
recently shown that GLP-2 is degraded by DP IV in vitro, yielding
GLP-2-(3
33) (10). Furthermore, we have detected the presence of
circulating GLP-2-(3
33) in the plasma of both rats and humans (2),
suggesting that DP IV degradation of GLP-2-(1
33) also occurs in vivo.
In contrast, modification of the native peptide by substitution of
Ala2 with glycine,
[Gly2]GLP-2, was shown
to confer DP IV resistance in vitro, and
[Gly2]GLP-2 was more
potent than wild-type
[Ala2]GLP-2 in the
induction of rat small bowel growth in vivo (10). These findings
suggest that the Gly2 substitution
renders the
[Gly2]GLP-2 analog
more potent by reducing DP IV degradation in vivo.
Although DP IV appears to be a critical determinant limiting GLP
actions, the kidney has also been identified as a major organ for
clearance of GLP-1 and GIP from the circulation (15,
21-23). Because GLP-2 shares ~40% sequence homology with GLP-1,
these findings raise the possibility that GLP-2 may also be removed from the circulation by the kidney. In the present study, we have analyzed the relative contributions of DP IV and the kidney to the
regulation of circulating levels of GLP-2-(133) in the rat in vivo.
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EXPERIMENTAL PROCEDURES |
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Peptides. Rat GLP-2-(133), rat
GLP-2-(3
33), and human
[Gly2]GLP-2 were kind
gifts from Allelix Biopharmaceuticals (Mississauga, ON, Canada).
Animals. Fed control male (Wistar and
Fischer) rats (Charles River, St. Constant, QC, Canada) and
Fischer-derived (28) DP IV-negative rats (a kind gift from Dr. R. Pederson, University of British Columbia, Vancouver, BC, Canada),
350-375 g, were anesthetized by intraperitoneal injection of 65 mg/kg pentobarbital sodium. In some studies, the kidneys of the rats
were exposed and decapsulated, and the rats were functionally
nephrectomized by ligation of the ureter, renal artery, and renal vein.
The right jugular vein and the left carotid artery of all rats were
cannulated with PE-50 tubing (Becton-Dickinson, Sparks, MD) filled with
0.5% BSA-heparinized saline, and at time
(t) = 0 min, 1 µg of GLP-2-(133)
or [Gly2]GLP-2 was
injected into the jugular vein. At t = 0.5, 2, 5, 10, 30, and 60 min, 1-ml blood samples were collected from
the carotid artery into 100 µl Trasylol-EDTA-Diprotin A
[5,000 kallikrein-inactivating units Trasylol (Bayer, ON,
Canada)-12 mg/ml EDTA-0.1 M Diprotin A (Sigma Chemical, St. Louis,
MO)] to inhibit further proteolytic degradation. During sampling,
some Wistar rats were reinfused with red blood cells that had been
reconstituted in 0.5% BSA-heparinized saline at
t = 5, 10, and 30 min. Because no
differences were detectable in GLP-2 levels between these rats and rats
that were not reinfused with red blood cells, all data were combined
for analysis and presentation. At t = 60 min, 2 ml of 0.1% fast green (Sigma Chemical), a dye known to be
rapidly cleared by the kidney (1), was injected in the jugular vein of
nephrectomized rats. The bladders were then exposed and monitored for
10 min to ensure that no green dye appeared in the urine. To assess
recovery of peptides, 500 µl of plasma obtained from control Wistar
rats that had not been injected with peptides were "spiked" with
10 ng of GLP-2-(1
33) or GLP-2-(3
33). Peptides contained in 500 µl
of plasma were extracted by reversed-phase adsorption to
C18 Silica
(C18 Sep-Pak; Waters Associates,
Milford, MA), as described previously (2, 10), and briefly stored at
20°C before analysis by HPLC and/or RIA. Recovery of GLP-2
using this method has previously been reported to be >80% (2).
Peptide recovery did not differ between GLP-2-(1
33) and GLP-2-(3
33)
(unpublished data).
For assessment of GLP-2 bioactivity in vivo, 6-wk-old female CD1 mice
(Charles River), 22-25 g, were injected subcutaneously two times
daily with 2.5 µg of GLP-2-(133), GLP-2-(3
33), or [Gly2]GLP-2 in 0.5 ml
PBS, or with vehicle alone (PBS), for 10 days. Mice were then fasted
overnight and killed, and the small intestine was removed, rinsed with
saline, blotted to remove excess liquid, and weighed.
HPLC. GLP-2-(133) was separated from
GLP-2-(3
33) by HPLC utilizing a
C18 µ-Bondapak HPLC column
(Waters Associates) with a gradient of 30-60%
solvent B
[solvent A: 0.1%
trifluoroacetic acid (TFA) in water; solvent
B: 0.1% TFA in acetonitrile] over 45 min
followed by a 10-min purge at 99% solvent
B (2). The flow rate was 1.5 ml/min, and fractions were
collected every 0.3 min. Trace amounts of iodinated GLP-2 (<200
counts/min) were added to all samples to serve as an internal standard;
this did not interfere with the RIA. The elution positions of
GLP-2-(1
33) and GLP-2-(3
33) were determined by similar analyses of
the elution of the synthetic peptides. In some HPLC analyses, the
presence of an earlier eluting immunoreactive (IR) peptide was
observed; this was determined, in a separate experiment, to represent
oxidized GLP-2 (unpublished data).
RIA. HPLC fractions or extracted
peptides were dried in vacuo and assayed for IR GLP-2 using antiserum
UTTH-7. This antiserum recognizes the mid-sequence of GLP-2 (amino
acids 25-30) and cross-reacts equally with GLP-2-(133),
GLP-2-(3
33), and
[Gly2]GLP-2 (Ref. 2
and unpublished data). The working range of the assay was 10-2,000
pg/tube.
DP IV assay. Blood was removed via
cardiac puncture from anesthetized Wistar, Fischer, and DP IV-negative
rats (350-375 g). Serum was collected and stored at
20°C. At the time of assay, 450 µl of 1.11 mM
Gly-Pro-p-nitroanilide (Sigma
Chemical) and 450 µl of 0.1 mM Tris buffer (pH 7.4) were incubated at
37°C for 15 min, after which 100 µl of the test serum were added.
Absorbance at 410 nm was recorded immediately upon addition of the
serum, and then at 5-min intervals for 30 min, to monitor the
appearance of the product
p-nitroaniline (14). A standard curve
was prepared using p-nitroaniline
(Sigma Chemical), and the slope of the curve was used to determine
serum DP IV activity in nanomoles per minute per milliliter.
Data analysis. All data are expressed
as means ± SE. Areas under the curve for HPLC peaks were determined
as the sum of the peak fraction plus three immediately neighboring
fractions, as appropriate, for a total of four fractions per peak.
Statistical analyses were performed by ANOVA using
n 1 "post
hoc" custom hypotheses tests or by paired or unpaired Student's
t-test, as appropriate, using the
Statistical Analysis System (SAS, Cary, NC).
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RESULTS |
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DP IV activity in the serum of control rats was 105 ± 18 nmol · min1 · ml
1
(n = 7-9). In contrast, DP
IV-like activity was significantly reduced but clearly detectable in
serum from DP IV-negative rats (43 ± 7 nmol · min
1 · ml
1,
P < 0.01, n = 7-9). HPLC analysis of plasma
collected from control rats injected with 1 µg of GLP-2-(1
33)
demonstrated only small amounts of GLP-2-(3
33) at
t = 5 min; however, increasing levels of this NH2-terminally cleaved
peptide were detected over the subsequent 1-h sampling period (Fig.
1). Although the areas under the curve were
determined for both GLP-2-(1
33) and GLP-2-(3
33), the peaks are
presumed to represent both exogenously administered and endogenous
peptide. Therefore, the half-life for conversion of GLP-2-(1
33) to
GLP-2-(3
33) in control rats could only be estimated at ~6 min (Fig.
2). Only limited degradation of
GLP-2-(1
33) to GLP-2-(3
33) was detected in DP IV-negative rats
compared with control animals (Fig. 2). Indeed, the half-life for
conversion of GLP-2-(1
33) to GLP-2-(3
33) could not be calculated in
these experiments, since only small amounts of GLP-2-(3
33) could be detected over the 60-min sampling period. Consistent with the results
of previous in vitro studies (10), degradation of
[Gly2]GLP-2 to
GLP-2-(3
33) in control rats in vivo was also markedly reduced
compared with that of GLP-2-(1
33), and the half-life for conversion
to GLP-2-(3
33) could not be determined (Fig. 2).
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Further analysis of the sequential HPLC profiles for the rats
administered GLP-2-(133) or
[Gly2]GLP-2
demonstrated that both peptides, as well as GLP-2-(3
33), disappeared
from the circulation over time (Figs. 1 and 2). To test the hypothesis
that these peptides were being cleared from the circulation by the
kidneys, the disappearance curves for both GLP-2-(1
33) and
[Gly2]GLP-2 were
compared in normal and bilaterally nephrectomized rats (Fig.
3). Total IR GLP-2 was observed to
disappear from the circulation of rats injected with either peptide in
both normal and nephrectomized animals. When the data from Fig. 3
were linearized by a
log10 (minute) transformation and
the slope of each line was calculated as the change in percent IR GLP-2
per unit time, no significant differences between the clearance of
GLP-2-(1
33) and
[Gly2]GLP-2 were
observed in either normal (
51.5 ± 2.4 and
52.5 ± 0.7) or nephrectomized (
40.0 ± 2.9 and
44.5 ± 5.0) rats. When taken together, the clearance of total IR GLP-2 from
nephrectomized rats was found to be significantly reduced compared with
nonnephrectomized animals (n = 6-9, P < 0.01). HPLC analysis
of plasma from nephrectomized rats injected with GLP-2 revealed that
the ratio of GLP-2-(3
33) to GLP-2-(1
33) was 3:2 at 0.5 min and 1:1
at 30 min (n = 3, data not shown).
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To ascertain the putative intestinotropic activity of GLP-2-(333),
the primary product of DP IV-mediated degradation of GLP-2-(1
33), mice were injected two times per day for 10 days with PBS,
GLP-2-(1
33), [Gly2]GLP-2, or
GLP-2-(3
33), and the small intestinal weights were determined (Fig.
4). GLP-2-(1
33) and
[Gly2]GLP-2 induced
significant 30-70% increases in intestinal wet weight compared
with controls (n = 6, P < 0.01-0.001),
whereas the intestinal weight of GLP-2-(3
33)-treated mice was not
different from that of PBS-treated animals.
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DISCUSSION |
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GLP-2 has recently been demonstrated to be a potent intestinotropic
peptide (3, 9-11, 24, 25). We previously demonstrated that
GLP-2-(133) is degraded by DP IV in vitro to GLP-2-(3
33) (10) and
that this degradation product is present in the circulation of rats and
humans (2). Because DP IV-mediated cleavage leads to inactivation of
several structurally related, biologically active peptides, including
GLP-1, GHRH, and GIP (12, 14, 19), it was therefore important to
determine the role of DP IV in the degradation of GLP-2-(1
33) in vivo
and the effects of such a cleavage on the biological activity of this
peptide. The results of the present study have demonstrated that
GLP-2-(1
33) is rapidly degraded by DP IV in vivo to produce
GLP-2-(3
33), a peptide that does not stimulate intestinal growth.
These findings implicate the NH2
terminus of GLP-2 as an essential structural determinant of GLP-2
biological activity, as is also the case for GLP-1, GIP, and GHRH (6,
7, 12, 14, 19, 27). Furthermore, our in vivo data demonstrating that
GLP-2-(3
33) does not stimulate intestinal growth extend the recent
finding that
His1-Ala2
is important for GLP-2 receptor binding and activation (20).
Interestingly, although the extreme
NH2-terminal sequences
(His1-Ala2) of GLP-1 and GLP-2 are identical
(Table 1), the half-life for DP IV cleavage of GLP-2-(133) in vivo in
the rat (~6 min) was found to be substantially longer than that
reported for GLP-1 (0.9 min; see Refs. 7 and 14). Structural
differences between these peptides likely account for such differential
sensitivity to DP IV cleavage, as even a small change to the
midsequence of GHRH (Gly15Ala)
reduces the rate of DP IV-mediated
NH2-terminal degradation by 45%
(17). Nevertheless, despite the differences in rates of cleavage by DP
IV, the importance of DP IV for inactivation of peptides is illustrated
by the development of DP IV-resistant analogs of GHRH, GLP-1, and GLP-2
for pharmaceutical treatment of specific human diseases (7, 10, 13,
17).
Consistent with a role for DP IV in the degradation of GLP-2-(133),
cleavage of this peptide was markedly reduced in DP IV-negative rats.
The DP IV-deficient rats are a Fischer-344-derivative strain (28) in
which a mutation of Gly633 to Arg
in the active site
(Gly-Xxx-Ser-Xxx-Gly633)
results in rapid intracellular degradation of the protein (26). It
would appear from the results of the present study, however, that one
or more functional enzyme(s) with DP IV-like activity persist in the
circulation of the DP IV-negative rat, as detectable levels of DP IV
activity were consistently observed in the DP IV-negative rats studied.
A previous report has also demonstrated very low but detectable levels
of DP IV in animals from the same colony (14). These findings suggest
the presence of a DP IV-like enzyme in the plasma of DP IV-deficient
rats that is capable of cleaving both the substrate
(Gly-Pro-p-nitroanilide) used in our in vitro assay and, to a lesser extent, GLP-2. The exopeptidase DP I is
one possible enzyme, as it exhibits a general dipeptidase activity,
cleaving NH2-terminal dipeptides
from most peptides and proteins, including those that are also
substrates for the more limited actions of DP IV (18).
Confirmation of the importance of DP IV in the regulation of GLP-2
bioactivity derives from analysis of the biological activity and
degradation of
[Gly2]GLP-2, a GLP-2
analog that is not cleaved by DP IV in vitro (10). [Gly2]GLP-2 was
significantly more potent compared with native GLP-2-(133) in the
induction of intestinal growth in rats in vivo (10). Furthermore,
[Gly2]GLP-2 exhibited
very little DP IV-mediated cleavage over time in vivo, consistent with
the known specificity of DP IV for proteins or peptides bearing
NH2-terminal penultimate Ala or
Pro residues (27). Reduced DP IV degradation has also been observed for
several long-acting analogs of GLP-1 and GHRH that have
Ala2 substitutions, including
D-Ala2,
Gly2,
Ile2,
Ser2,
Thr2, and
Val2 (7, 13, 17). When taken
together, therefore, the results of these studies provide strong
evidence that DP IV is a critical determinant limiting the bioactivity
of GLP-2 in vivo.
The findings of the present study extend previous concepts of
GLP-2-(133) inactivation by presenting evidence for both DP IV-dependent and -independent mechanisms. Clearance of both
GLP-2-(1
33) and
[Gly2]GLP-2 was
significantly decreased in nephrectomized rats compared with
nonnephrectomized animals, demonstrating that the kidney plays a key
role in the clearance of GLP-2 from the circulation. It is recognized
that the blood sampling protocol used in the present study may have
altered renal hemodynamics and/or regional blood flow. However, the 1:1
ratio of GLP-2-(1
33) to GLP-2-(3
33) in nephrectomized Fischer rats
30 min after injection with GLP-2-(1
33) indicated that both the
active and inactive forms of GLP-2 are present in the circulation of
nephrectomized animals and that both forms contribute to the elevated
levels of IR GLP-2 observed in the clearance curves. A previous study
also identified the kidney as an important organ in the clearance of
[125I]GLP-2 in rats,
through a mechanism involving both glomerular filtration and tubular
catabolism (23). However, because some clearance of both native GLP-2
and [Gly2]GLP-2 was
still observed in nephrectomized animals, this suggests that other
organs and mechanisms may also play a role in GLP-2 clearance. A study
involving exogenous administration of GLP-1 to pigs has also identified
the liver and the lung as clearance organs for this peptide (8); hence,
it is possible that these organs may also play a role in the removal of
GLP-2 from the circulation. Further studies involving the measurement
of differences in arteriovenous concentrations of GLP-2 across the lung
and liver will be required to determine if these organs are indeed
involved in GLP-2 clearance.
In summary, the present study has identified DP IV as a key enzyme
involved in the degradation of the intestinotropic hormone GLP-2-(133) in the circulation of rats in vivo. The major DP IV
cleavage product, GLP-2-(3
33), is biologically inactive in a murine
intestinal growth assay in vivo. These findings provide a rationale for
the design of potent GLP-2 analogs, such as
[Gly2]GLP-2, that are
DP IV-resistant in vivo. The kidney was identified as a major organ for
the clearance of both GLP-2-(1
33) and
[Gly2]GLP-2 from the
circulation. Given the structural similarity of rat and human GLP-2,
and the recent detection of both GLP-2-(1
33) and GLP-2-(3
33) in
human plasma (2), it seems likely that the findings demonstrated here
in the rat may be extended to studies of human GLP-2 metabolism and
clearance in future experiments.
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ACKNOWLEDGEMENTS |
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We are grateful to Allelix Biopharmaceuticals (Mississauga, ON, Canada) for providing peptides and to Dr. R. Pederson (University of British Columbia, Vancouver, BC, Canada) for supplying the dipeptidyl peptidase IV-deficient rats.
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FOOTNOTES |
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This work was supported in part by Allelix Biopharmaceuticals and by Medical Research Council of Canada operating grants to P. L. Brubaker and D. J. Drucker. W. Tavares was the recipient of a University of Toronto Open Fellowship, and D. J. Drucker is a Senior Scientist of the Medical Research Council, and a consultant with Allelix Biopharmaceuticals.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. L. Brubaker, Rm. 3366, Medical Sciences Bldg., Univ. of Toronto, Toronto, ON Canada M5S 1A8 (E-mail: p.brubaker{at}utoronto.ca).
Received 4 January 1999; accepted in final form 30 August 1999.
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