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Beta Cell Biology, Novo Nordisk A/S (A.B.D., H.K.), Novo Allé, DK-2880 Bagsvaerd, Denmark; the Department Physiology, University of British Columbia (A.M.J.B.), Vancouver, British Columbia, Canada V6T 1Z3; and the Department of Medical Physiology, The Panum Institute, University of Copenhagen (J.J.H.), DK-2200 Copenhagen N, Denmark
Address all correspondence and requests for reprints to: Dr. Hans Kofod, Beta Cell Biology, Novo Nordisk A/S, Novo Allé, 6B3.99, DK-2880 Bagsvaerd, Denmark. E-mail: hko{at}novo.dk
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Abstract |
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However, some of the cleavage sites in Pg have not proven to be substrates exclusively for either PC2 or PC1/3, and the cleavage profile of Pg in a primary cultured L cell has not yet been correlated with the actual presence of PC2 and PC1/3 in the L cell.
We demonstrate here the presence of PC1/3, PC2, and the PC2 chaperone 7b2, in L cells using light immunohistochemistry on sections from canine ileum and on a canine intestinal cell culture enriched for L cells. Analysis of the cultured L cells, using gel chromatography and RIA, confirms the classical intestinal cleavage profile of Pg, resulting in mainly glicentin, oxyntomodulin, GLP-1-(7–37), and GLP-2. Despite the presence of 7b2 and mature PC2, as demonstrated by Western blot, absolute minimal amounts of glucagon were detected.
These data show that the presence of intracellular PC2 and 7b2 in a primary cell possessing Pg does not have to lead to the formation of glucagon. This formation must then require an additional element to occur, or alternatively, the results could be explained by a canine specific organization of PC2 and Pg into separate compartments, which would prevent interaction.
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Introduction |
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) (1). These conversions from Pg are
thought to be catalyzed by intracellular proteolytic enzymes belonging
to a subgroup of the family of subtilisin-like convertases termed
prohormone convertases (PC) (2). Two of these enzymes, PC1/3 (also
called PC-1 or PC-3) and PC2, have been detected in Pg-expressing
cells. In the human intestinal L cells, PC1/3 has been detected,
whereas PC2 was not (3), and in the pancreatic A cell, PC2 has been
found, but PC1/3 was not (4). The classical cleavage sites for these
PCs are at dibasic sequences, exemplified by proinsulin in the
pancreatic ß-cell, where the Lys-Arg site is primarily cleaved by
PC2, and Arg-Arg is primarily cleaved by PC1/3, resulting in insulin
and C peptide (5).
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We used a model based on cultured L cells (13, 14), obtained from canine small intestine (ileum), in which the production and release of Pg-derived peptides can be investigated. We investigated the presence of PC1/3 and PC2 in this primary culture of L cells and the resulting effect on Pg processing. The presence of PC1/3, PC2, and 7b2, a modulator of PC2 (15, 16), in the canine L cell was examined by means of immunocytochemistry and Western blot. Size exclusion chromatography and specific RIAs were used to determine the profile of Pg products released from the culture of canine L cells.
We report here that the canine intestinal L cell of ileal origin expresses both PC1/3 and PC2 in mature form together with 7b2. Nevertheless, the cleavage of Pg in these cells does not lead to the formation of glucagon, whereas the classical products of the L cell, glicentin, oxyntomodulin, GLP-1, and GLP-2, are all produced.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tissue processing
The ileal segments and pancreatic samples were fixed in Bouin’s
solution (71% saturated aqueous picric acid; Sigma Chemical Co., St. Louis, MO), 24% formalin (Merck & Co., Inc., Rahway, NJ; 40% formaldehyde), and 4% glacial acetic
acid (Merck & Co., Inc.) for 2 h at room temperature,
washed 3 times, and stored in 70% ethanol. A 1-cm portion was cut from
the center of each segment to ensure correct orientation during the
embedding process. The piece of intestine was dehydrated through graded
alcohol and embedded in paraffin wax at 60 C. Care was taken to ensure
that the intestine was embedded perpendicularly to the resultant block
face. Serial 20-µm sections were cut and mounted on glass slides.
Approximately 25 sections/1 cm segment of tissue were used for this
study from each of the 3 segments of small intestine. Tissue samples
from rat pancreatic islets were processed similarly. Before staining,
the sections were dewaxed in xylene for 5 min and cleared in petroleum
ether (Fluka, Buchs, Switzerland) for 5 min and then washed in
PBS [80 mM Na2HPO4 (Merck & Co., Inc.), 1.47 mM KH2PO4
(Merck & Co., Inc.), 2.86 KCl (Merck & Co., Inc.), and 137 mM NaCl (Merck & Co., Inc.), pH 7.2) including 0.1% Triton X-100 (PBS/Triton X-100;
Sigma Chemical Co.).
Cell isolation and culture
Cell cultures were obtained as previously described (14).
Briefly, a 40-cm piece of small intestine (including ileum), proximal
from the cecum, was removed by surgery and rinsed in ice-cold HBSS
(Life Technologies, Inc., Grand Island, NY) with 0.65
mM dithiothreitol (DTT; Merck & Co., Inc.),
1% (wt/vol) BSA (Sigma Chemical Co.), 10 ml/liter
penicillin/streptomycin (1000 IU/ml; Life Technologies, Inc.), 9.05 mM NaHCO3 (Merck & Co., Inc.), and 20 mM HEPES (Sigma Chemical Co.; HBSS/DTT), and the mucosa was stripped of the submucosa and
minced. The tissue was digested sequentially with 75 U/ml collagenase
(type 1 co130, Sigma Chemical Co.) in basal Eagle’s
medium (Life Technologies, Inc.) including 26.4
mM NaHCO3, 0.1% BSA, and 10 mM
HEPES at pH 6.9 for 1 h at 37 C in a shaking water bath. In the
last 15 min of the digest, EDTA (Merck & Co., Inc.; 3
mM final concentration) was added. The resulting cell
suspension was washed three times in ice-cold HBSS/DTT, filtered
through an 80-µm pore size nylon filter (Nytal P80, SST Thal, Thal,
Switzerland), and diluted to a single cell solution at 6 x
106 cells/ml in HBSS/DTT. Enrichment of L cells and
purification from bacteria were achieved by centrifugal elutriation
using a Curamé 3000 Elutriator (Heraeus, Osterode,
Germany). One hundred milliliters of cell suspension were loaded
at 2600 rpm at a constant flow rate of 15 ml/liter at room temperature
and washed for 4 min, then washed for another 6 min at 2000 rpm. The
cells used for release experiments were obtained by collecting 50 ml
cell suspension of the fraction elutriated between 1300–2000 rpm. The
cell suspension was centrifuged at 800 x g and
resuspended to 2 x 106 cells/ml in 47.5% DMEM
(Life Technologies, Inc.) and 47.5% Ham’s F-12 medium
(Life Technologies, Inc.) including 5% dog serum and 10
ml/liter penicillin/streptomycin supplemented with 1 µg/ml
hydrocortisone (Sigma Chemical Co.), 4 ng/ml insulin
(Novo Nordisk A/S), 5 ng/ml nerve growth factor
(Sigma Chemical Co.), and 11 mM glucose (Pro
Labo, Paris, France). The suspension was plated onto 24-well
plates (0.5 ml/well) coated with rat tail collagen type 1 (Sigma Chemical Co.) and incubated for 36–48 h at 37 C in 5%
CO2. The resulting culture formed a monolayer that
consisted of approximated 25% L cells and 30% N cells; the remaining
cells were mucin producing (14).
For immunocytochemistry, cultured cells were fixed in Bouin’s fixative for 10 min and washed three times in PBS/Triton X-100.
Double staining
For immunocytochemistry, wax-embedded segments and cell cultures
were washed in PBS/Triton X-100 and incubated in 10% horse
serum/PBS/Triton X-100 (Life Technologies, Inc.-BRL) for
15 min and then incubated with two primary antibodies, applied together
in the same mixture, overnight at 4 C. All tissue and cell samples were
incubated with an appropriate set of secondary antibodies, applied
together in the same mixture, for 1 h at room temperature.
Initially, all secondary antibodies were controlled for nonspecific binding by incubating the antibodies separately and together without primary antibodies on all types of tissue used in the study. Background staining was found to be minimal (data not shown). Similarly, each of the secondary antibodies was controlled for nonspecific binding to the primary antibodies by incubating secondary antibodies separately and together with each of the primary antibodies on all types of tissue used in the study (data not shown).
For detection of prohormone convertases in the pancreatic A cell, sections from wax-embedded islets of Langerhans from rat were doubled stained with the monoclonal anti-Pg mouse IgG 23.8B6 (50 µg/ml) (17) in combination with the two rabbit antisera RS20 (1:25), toward PC1/3 and PC2-P4 (1:25) toward PC2 (3). Similarly, rat islets were exposed to anti-7b2-(23–39) rabbit serum (1:3) (15) in combination with anti-Pg mouse IgG in a costaining procedure.
PC1/3 and PC2 were detected in intestinal L cells, both in sections obtained from wax-embedded canine ileum and in cultured L cells by the two rabbit antisera RS20, toward PC1/3 and PC2-P4 toward PC2 (3). For a control, sections were also stained for PC1/3 and PC2 by another set of rabbit sera, either anti-PC1/3 or anti-PC2 rabbit serum (1:200/1:100) (18), which showed the same results as with RS20 and PC2-P4. Each of these four antibodies was used in a double staining procedure together with anti-Pg IgG 23.8.B6. Similarly, the anti-Pg was used together with anti-7b2-(23–39) rabbit serum in both sections and cultured cells.
The primary antibodies were visualized using combinations of antimouse and antirabbit fluorophore-conjugated (fluorescein dichlorotriazine, fluorescein isothiocyanate, Cy3) antibodies (10 µg/ml; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).
Release experiments
After incubation, medium and unattached cells were removed. The
cells attached to the wells were washed in DMEM including 0.1% BSA
(DMEM/BSA) and preincubated for 2 h at 37 C in 5% CO2
in 0.5 ml DMEM/BSA and 5.5 mM glucose. After preincubation,
the medium was replaced with 450–475 µl DMEM/BSA and 5.5
mM glucose. Stock solutions of secretagogues were added in
volumes of 25 µl in a 20-fold concentration and incubated for 2
h in the absence (basal condition) or presence of GLP-1
secretagogues [400 nM 4ß-phorbol 12ß-myristate
13
-acetate (PMA; Molecular Probes Europa BV, Leiden, The
Netherlands), 1 µM forskolin (Sigma Chemical Co.), or 100 nM glucose-dependent insulinotropic
peptide (GIP; Peninsula Laboratories, Inc., Belmont,
CA)]. After incubation, the release medium was recovered. Cells that
were incubated under basal conditions were lysed by 0.3% formic acid
diluted 1/10 in ELISA buffer, and the pH was adjusted to 7. All samples
were then centrifuged at 4000 x g, and supernatant was
kept at -20 C for GLP-1 measurement.
GLP-1 measurement
We have used a sensitive sandwich enzyme-linked immunoabsorbent
assay for determination of GLP-1 (19). Monoclonal mouse-Ab
[F5A3, Novo Nordisk A/S; raised against
GLP-1-(26–36)] was used as catching antibody.
Biotinylated polyclonal rabbit-Ab [91022–2, Dr. J. J. Holst,
Copenhagen, Denmark; raised against GLP-1-(7–17)] was
used as detecting antibody. GLP-1-(7–37) (Novo Nordisk A/S) was diluted in DMEM/BSA and used as standard.
Eighty microliters of sample were added to 20 µl detecting antibody
solution, corresponding to 2.4 µg antibody/well. For
GLP-1 measurement, some samples were diluted in
DMEM/BSA.
Western blot
SDS-PAGE was performed using 10% SDS and 1-mm 15-well precast
gels (Novex, San Diego, CA) using the appropriate
protocol described by Novex. The proteins from the gels
were blotted onto a nitro-cellulose filter and stained with either
anti-PC1/3 rabbit serum RS20 (dilution 1:500) (3) or anti-PC2 rabbit
serum PC2-P4 (dilution 1:500) (3). The nitro-cellulose filter was
sequentially incubated with horseradish peroxidase-conjugated goat
antirabbit IgG (Bio-Rad Laboratories, Inc., Hercules, CA)
and visualized by enhanced chemiluminescence Western blotting detection
reagent RPN 2106 (Amersham Pharmacia Biotech, Arlington
Heights, IL).
Processing of Pg
For determination of the processing of Pg with respect to
glucagon, 10 24-well plates with cultured L cells were incubated for
2 h in the presence of 1 µM forskolin in a volume of
0.5 ml/well. Release medium was recovered as described above and
analyzed with or without concentration [42 ml were applied to a
Sep-Pak column (Waters Corp., Milford, MA) and eluted with
4 ml 60% acetonitrile according to the manufacturer’s instructions]
and as unconcentrated. Cell extract was obtained using formic acid as
previously described.
For size exclusion chromatography, 4-ml samples were applied to 16
x 1000-mm columns packed with Sephadex G-50 (fine grade,
Pharmacia Biotech, Uppsala, Sweden), equilibrated, and
eluted with 0.05 M sodium phosphate (pH 7.4), 0.1
M NaCl, 0.1% human serum albumin, 0.6 mM
thimerosal, and 10 mM EDTA at a flow rate at 0.3 ml/min at
4 C. Trace amounts of 125I-labeled albumin and
22NaCl were added to all samples for internal calibration.
The samples were analyzed by RIA as previously described (20, 21, 22),
using the following rabbit polyclonal antibodies: 1) code 4830,
reacting with Pg-(33–42), but side-viewing; 2) code 4304, reacting
with Pg-(38–48), but side-viewing; 3) code 4305, reacting with
Pg-(52–61), requires free C-terminal glucagon; 4) code 2135, reacting
with GLP-1 midsequence side-viewing; 5) code 89390,
reacting with Pg-(99–106)amide, amidated GLP-1; and 6)
code 92160, reacting with Pg-(126–135), requiring a free N-terminal
GLP-2. The term side-viewing means that the antibody is reacting with a
certain sequence regardless of its position in larger peptides or of
modification of the termini of the larger peptide (see Fig. 1
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Results |
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Double staining of the L cells
In sections from canine small intestine (ileum), Pg-immunoreactive
cells L cells were equally abundant in villi and crypts.
Pg-immunoreactive cells costained for PC1/3, PC2, or 7b2
immunoreactivity (
Figs. 2–4),
using RS20, PC2-P4, or anti-7b2-(23–39) in combination with anti-Pg
23.8B6. We obtained similar results applying a second set of antisera
(18) for control of sera (data not shown).
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The same results were obtained in L cell-enriched canine intestinal
cell culture. L cells, defined as Pg-immunoreactive cells, costained
for PC1/3, PC2, and 7b2 antisera (
Figs. 5–7)
using RS20, PC2-P4, and anti-7b2-(23–39). This indicates that the
canine L cell expresses all of these proteins, and that the primary
culture preparation reflects the normal L cells in situ.
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Western blot analysis demonstrated that both cell extract and release
medium contained PC1/3 and PC2. Bands occurred in both cases close to
the 69-kDa marker protein corresponding to approximately 66 kDa (Fig. 8). These bands for PC1/3 and PC2 can
equally well arise from the L cells as from the neurotensin-containing
N cells, as human N cells have been shown to contain the two PCs (3).
No pro-PC1/3 or pro-PC2 could be detected. The smear occurring at the
blot above 69 kDa (Fig. 8, lanes A1 and B1) resulted from medium
alone.
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and 10
show 12 representative chromatograms of the analysis.
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shows the cleavage profile of the N-terminal part of Pg in
cell extract and release medium. The side-viewing assays identifying
the N-terminus and a midregion of glucagon (4830 and 4304); both
identify immunoreactive moieties eluting at Kd 0.30 and
0.75, the elution positions of glicentin, Pg-(1–69), and
oxyntomodulin, Pg-(33–69), whereas very little fully processed
glucagon could be identified at the elution position of glucagon
(Kd, 0.81) using the specific C-terminal assay (4305; Fig. 9). The difference between the amounts of glicentin identified by the
two side-viewing assays is due to the fact that the cross-reactivity of
the N-terminally extended form is lower in the N-terminal assay. The
elution profile for the release medium was very similar; however, the
concentrations were, as expected, considerably lower.
Figure 10 illustrates the cleavage profile of the C-terminal part of Pg
in cell extracts and release medium. The side-viewing
GLP-1 assay reacting with a midregion of
GLP-1 (2135) identified two immunoreactive peaks eluting
at the position of GLP-1 (7–37) (Kd, 0.55)
and somewhat later at Kd 0.65–0.70. The latter position
does not correspond to a previously identified Pg-derived product and
may represent a degradation product occurring during incubation
in vitro. The assay for the amidated C-terminus of
GLP-1 (89390) also identified material at these positions,
but in much smaller amounts, indicating that the amidation capacity of
the L cells is limited in vitro. Similar profiles were
observed for the release medium, although the ratio of metabolite
vs. intact GLP-1 seemed to be larger, perhaps
indicating a more extensive degradation in the release medium. The
assay against the N-terminus of GLP-2 (92160) identified large amounts
of immunoreactivity, mainly eluting at the position of GLP-2.
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Discussion |
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The role of PCs in the conversion of prohormones such as proinsulin and Pg into mature products has been debated over the last decade (23). Studies using cell lines that secrete Pg-derived peptides or immunopurified proteins showed that Pg is a physiologically relevant substrate for PC activity (6, 7, 8, 9, 10, 11, 12, 24). Some of these studies provided evidence that PC2 is responsible for the cleavage of glicentin into glucagon and that PC1/3 is involved in the cleavage of MPgF into GLP-1 (6, 7, 8, 24). These separate actions of PC1/3 and PC2 are supported by immunohistochemistry (3), which shows that human L cells only express PC1/3 and not PC2, and it has been demonstrated that pancreatic A cells only express PC2 and not PC1/3 (25, 26, 27, 28). Other studies, also using Pg-producing cell systems or purified proteins, have, on the other hand, cast doubt on the function of PC2 alone to produce glucagon from Pg when present together (9, 10, 11, 12). Taken together, there is consensus about the role of PC1/3 in the conversion of Pg into GLP-1, whereas the role of PC2 in the conversion of Pg into glucagon still seems open for debate.
In the present study we investigate by immunocytochemistry the possible colocalization of Pg with the two prohormone convertases, PC1/3 and PC2, or the PC2 chaperone 7b2 in the in vivo and in vitro canine L cells. We furthermore analyze the composition of Pg-derived peptides in cell extracts and release medium from cultured canine L cells (14) to correlate peptide data with those from immunocytochemistry.
Our results show that the canine L cell expressed both PC1/3 and PC2 as well as 7b2. The presence of PC1/3, PC2, and 7b2 was observed both in vivo, as seen in sections obtained from the canine ileum, and in vitro, as seen in the cultured L cell. As light microscopy is not a direct quantitative measurement of protein concentrations, one can argue that the amount of PC2 or 7b2 present would be insufficient to produce glucagon, but as no general differences in intensities were observed in PC1/3, PC2, and 7b2 immunostaining on the L cells, one could expect that PC2 should process Pg, as does PC1/3. Similarly, no differences in intensities were observed between the PC-positive cells that stained for Pg and those that did not.
Western blot analysis showed that the two PC were found in both the cell extract and the release medium. In both cases PC1/3- and PC2-immunoreactive bands were observed at approximately 66 kDa, indicating that both enzymes were present in the L cell and secreted into the medium as mature PCs (29, 30). The latter suggests that both enzymes were secreted along with the peptides secreted to the medium, although one can argue that the prohormone convertases may arise not only from the L cells, but also from other endocrine cells present in the culture (14).
As the canine L cells apparently do express PC2, it is important to know whether PC2 is present in its mature form. However, in both extracts and release medium, no band was observed at 75 kDa, corresponding to immature PC2 (29, 30), suggesting that it is mature PC2 that is present in the L cell. The reason for absence of the pro-PCs is not clear, but in a study using the same antisera for PC2 (PC2-P4), the pro-PC2 could not be seen in the Pg-processing cell line STC-1 (6), although PC2 was present in the mature form.
A test of function was performed on the same batch of L cells as that used for immunocytochemistry. The L cells responded to forskolin, PMA, and GIP by increasing the release of GLP-1 to levels similar to those previously reported (14), indicating that the secretory and transport systems were intact in terms of GLP-1 release.
The presence of PC2 and 7b2 in the canine L cell suggests the
possibility that cleavage of Pg should include the formation of
glucagon. To further examine this, we analyzed the composition of
Pg-derived peptides in cell extracts and in release medium from the L
cell culture by means of size exclusion chromatography and specific
RIAs. The overall cleavage profile of Pg in the cultured canine L cells
was generally in accordance with that previously reported (31). Our
analysis can be summarized as follows. Pg was exclusively cleaved (Fig. 1) at Pg-(70–71), Pg-(109–110), and Pg-(124–125), resulting in the
formation of glicentin, GLP-1, and GLP-2. In addition,
Pg-(31–32) was cleaved to a minor extent, resulting in oxyntomodulin.
The cleavage of Pg-(62–63) was limited, as very little glucagon (at
the level of detection in the RIA) was found in cell extracts, and none
was found in the release medium when the cells were stimulated with
PMA. The predominant form of GLP-1 appeared to be
GLP-1-(7–37), although a small amount of
GLP-1-(1–37) was detected. Consequently, most of the Pg
is cleaved at Pg (77). In vitro, GLP-1 appears
to degraded into a smaller form at Kd 0.60–0.70 in both
cell extracts and release medium.
It has been shown that PC2 null-mutated mice had marked decreases in fully processed glucagon and insulin, although the precursors of these hormones were present. This is in accordance with the hypothesis that PC2 plays a major role in the maturation of Pg into glucagon (32, 33). It was therefore surprising that a primary culture of endocrine cells, containing Pg together with PC1/3, PC2, and 7b2 was not capable of producing glucagon.
The peptide 7b2 is preferentially distributed in neural and endocrine tissues and acts as a modulator of PC2 (15, 34, 35, 36, 37), in that the N-terminal part of the 7b2 protein takes part in the maturation of PC2 (13). However, the C-terminal part, termed CT, can act as an inhibitor of PC2 (38, 39), an effect that, in turn, can be reversed by the cleavage of CT, a process that can be accomplished by PC2 (36). We detected the 7b2 protein in the primary canine L cells, and as shown by Western blot, the maturation of PC2 appeared to be complete. However, it could be argued that although mature PC2 is present in the L cell, the inhibitory effect of CT on PC2 action prevented the cleavage of Pg into glucagon. Thus, the interplay among PC2, 7b2, and CT in the canine L cell requires further elucidation.
To date, the majority of studies concerning the conversion of Pg into its products have been conducted in artificial systems, all of which assumed the absence of PC2 and 7b2 in intestinal L cells. The data presented here clearly indicate that both PC2 and 7b2 as well as PC1/3 are expressed in both canine L cells in vivo and short term culture. Furthermore, in the culture, the conditions for the processing of Pg are similar to those in vivo, as the intracellular arrangement of the Golgi apparatus and secretory granules and the functions of the secretory pathway must be expected to be intact.
The presence of PC2 in the L cell may be species specific, as PC2 was not demonstrated in human L cells (3). However, the present studies indicate that the presence of PC2 along with 7b2 in a primary culture of Pg-processing cells does not have to lead to the production of glucagon. Our data are in agreement with those from a recently published study in which PC2 was found to generate glicentin [cleavage of K(70)-R(71)], but failed to generate glucagon (12).
In conclusion, this study confirms the action of PC1/3 in the processing of Pg to GLP-1, but indicates that PC2 in combination with its chaperone 7b2 is not sufficient to process Pg to glucagon, a process that seems to require an additional convertase to reach glucagon as end product. It is possible that PC2 only cleaves Pg at K(70)-R(71), producing glicentin and MPgF, and this may be the role of PC2 in the processing of Pg. It can be speculated that Pg is sequentially cleaved first by PC2 and then by tissue-specific PC, giving rise to GLP-1 from MPgF by PC1/3 in the L cell and glucagon in the pancreatic A cell by an as yet unidentified PC.
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Acknowledgments |
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Received February 4, 1999.
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