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Departments of Physiology (S.D., P.L.B.) and Medicine (P.L.B.), University of Toronto, Toronto, Ontario M5S 1A8, Canada
Address all correspondence and requests for reprints to: Dr. P. L. Brubaker, Room 3366, Medical Sciences Building, University of Toronto, Toronto, Ontario M5S 1A8, Canada. E-mail: p.brubaker{at}utoronto.ca
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) renders GLP-1 biologically active, as
GLP-17–37/36NH2. The insulinotropic effects of truncated
GLP-1 have been well documented (1, 2), and the results of these
studies have led to clinical trials to determine if hyperglycemia in
type II diabetes may be controlled through administration of GLP-1.
Although the function of glicentin is not known, oxyntomodulin is an
inhibitor of pentagastrin-stimulated gastric acid secretion (3, 4).
Finally, the biological function of GLP-2 as an intestinal growth
factor has recently been elucidated (5, 6).
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To examine the role of PC1 in the processing of proG in vivo, and to clarify the role of PC2, we used a well known pancreatic cell line, InR1-G9, to provide a stable model of proG processing. InR1-G9 cells are derived from a BK virus-induced hamster insulinoma (18), and express large amounts of proG messenger RNA (mRNA) relative to other pancreatic hormone mRNA transcripts (19). The pattern of proG processing is consistent with the pancreatic origin of the cells. InR1-G9 cells produce mainly glucagon, along with much smaller amounts of glicentin, oxyntomodulin, and GLP- 11–36NH2 (20, 21). We have also shown that these cells express PC2, but not PC1, mRNA (21). We therefore hypothesized that: 1) overexpression of PC1 in InR1-G9 cells would result in the increased production of the intestinal proG-derived peptides; and 2) antisense-mediated deletion of PC2 would decrease glicentin, and perhaps glucagon, production. Our findings demonstrate a role for PC1 in the production of the intestinal proG-derived peptides, and describe a role for PC2 in the production of glicentin, but not glucagon.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stable transfection of InR1-G9 cells with PC1 or ASPC2
Wild-type (WT) InR1-G9 cells (a gift from Dr. D. J.
Drucker, Toronto, Ontario, Canada) were maintained in DMEM (Life
Technologies Inc., Grand Island, NY) containing 10% (vol/vol) FBS and
4.5 g/liter glucose. Cells were transfected with vector controls,
pRcCMV/PC1, or pZeo/ASPC2 using the calcium phosphate method (22).
Briefly, cells were plated at approximately 30% confluency in 10-cm
dishes 16 h before transfection. Cells were incubated with 10 µg
DNA in 1 ml 0.1 x Tris-EDTA containing 2 x HEPES-buffered
saline and 2 M CaCl2 for 4 h at 37 C.
Cells were then subjected to a 3-min glycerol shock [15% (vol/vol)
glycerol in HEPES-buffered saline], washed in PBS, and cultured for
60 h to allow for expression of transfected DNA. On day 3 after
transfection, cells were trypsinized and plated at a dilution of 1:2 in
culture medium containing 800 µg/ml G418 (Sigma Chemical Co.,
Oakville, Ontario, Canada) or 250 µg/ml Zeocin (Invitrogen), as
appropriate. The cells were maintained in the selection medium for 3–4
weeks before isolation of individual clones. After transfection of
InR1-G9 cells with PC1, 24 individual clones were screened for
secretion of GLP-1. Eight clones were chosen for assessment of PC1 mRNA
expression, four of which secreted high amounts of GLP-1 (31–40
ng/dish) relative to WT cells (10 ng/dish). The two clones that most
highly expressed PC1 mRNA transcripts were selected for full
characterization of proG processing. As the processing profiles were
similar for both clones, results for only one clone are shown.
InR1-G9/ASPC2 cells were used as a mixed population, as single cell
clones proved to be unstable.
RNA analysis
RNA extraction and analysis was carried out as described
previously (21). Twenty micrograms of total RNA were used for all
analyses. cDNA probes for mouse PC1 and mouse PC2 were generated by
excision of the full-length fragments from the Bluescribe vector (gifts
from Dr. N. G. Seidah), and the full-length rat proglucagon and
actin probes were gifts from Dr. D. J. Drucker.
Peptide extraction
Extraction of PDGPs was carried out as described previously (10, 19, 21). Extraction of PC1 and PC2 protein was carried out as described
by Rothenberg et al. (12). Briefly, cells were scraped in
0.5 M sodium phosphate buffer with 9 mM EDTA,
pH 10, at 4 C, and subsequently sonicated in five 10-sec bursts.
Extracts were then freeze-thawed once in a dry ice-acetone bath and a
65 C water bath, centrifuged at 12,000 x g for 10 min,
and stored at -70 C.
HPLC
Glicentin, oxyntomodulin, glucagon, and GLP-2 were separated on
the basis of hydrophobicity as described previously (10, 19, 21, 23).
All samples included [125I]glucagon as an internal
control for normalization of data. The GL1 and GLP-2 content of
fractions 1–60/111–160 and 1–110/141–160, respectively, were below
the limits of detection and are therefore not reported. Full-length and
truncated forms of GLP-1 were separated as described previously (10, 19, 21). Synthetic forms of GLP-1 were used as controls for elution
positions. Equal amounts of immunoreactive peptide were loaded onto
each column to normalize the relative amounts of peptides measured.
RIAs
Two antisera were used to distinguish proglucagon and its N-
terminally derived peptides (glicentin, oxyntomodulin, and
glucagon) as previously described (10, 19, 21). 1) Antiserum K4023
(Biospacific, Emeryville, CA) recognizes the mid-sequence of glucagon,
thus detecting the glucagon-like immunoreactive (GLI) peptides,
proglucagon, glicentin, oxyntomodulin, and glucagon (Fig. 1
). 2)
Antiserum 04A (Dr. R. H. Unger, Dallas, TX) recognizes the free
carboxy terminal end of glucagon, thereby measuring immunoreactive
glucagon (IRG). The detection range of both assays was 4–400
pg/tube.
Processed forms of GLP-1 (GLP-11–37,
GLP-17–37, GLP-11–36NH2, and
GLP-17–36NH2) were detected using two different antisera,
as previously reported (10, 19, 21) (Fig. 1). 1) The b5 antiserum (a
gift from Dr. S. Mojsov, New York, NY) recognizes the C-terminally
glycine-extended forms of GLP-1, GLP-11–37 and
GLP-17–37. 2) C-terminally amidated forms of GLP-1
(GLP-11–36NH2 and GLP-17–36NH2) were detected
using an anti-GLP-17–36NH2 antiserum (Affinity Research,
Nottingham, UK). The detection ranges for the GLP-1x-37 and
GLP-1x-36NH2 assays were 1–160 and 3–800 pg/tube,
respectively.
Processed forms of GLP-2 were detected using antiserum no. 92160 (a
gift from Dr. J. J. Holst, Copenhagen, Denmark), which recognizes
the free N-terminus of GLP-2 (Fig. 1) (23). The detection range of the
GLP-2 assay was 10–2000 pg/tube.
PC1 and PC2 protein levels were kindly assessed by Dr. B. D. Noe (Emory University, Atlanta, GA) as previously described (11, 12), using RIAs that recognize the N-terminus of the catalytic domain of each PC (11). The detection ranges were 10–1280 pg for the PC1 assay and 5–1500 pg for the PC2 assay. Total cellular protein levels were determined using the Lowry protein assay (24).
Statistics
Optical density readings for PC1 and PC2 mRNA transcripts and
total cellular immunoreactivity (IR) of PC1 and PC2 were assessed for
differences between means by ANOVA with n-1 custom hypotheses tests.
Peak areas generated by HPLC/RIA for each of the PGDPs (glicentin,
oxyntomodulin, glucagon, GLP-11–36NH2,
GLP-17–36NH2 and GLP-2) were quantified for three
independent experiments. Peak areas of GLP-11/7–37 were
not quantified because little or no GLP-11/7–37 was
detected by HPLC. Differences between means were assessed by ANOVA with
Tukey multiple comparisons tests. All data were
log10-transformed before analysis to normalize variances.
All statistical manipulations were performed using SAS software for IBM
computers (Statistical Analysis Systems, Cary, NC).
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Results |
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). In
contrast, PC1 mRNA was highly expressed in InR1-G9/PC1 cells relative
to WT cells (P < 0.001). The PC1 mRNA transcripts
detected in InR1-G9/ASPC2 cells were not significantly different from
background levels, as assessed by densitometry (Fig. 2B). PC2 mRNA
transcripts were detected in all cells. Densitometric analysis revealed
that PC2 mRNA levels were decreased by 62 ± 9%
(P < 0.001) in InR1-G9/ASPC2 cells compared with
control cells (Fig. 2B).
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).
Consistent with mRNA levels, the PC1 protein levels in WT and control
cells were below the limits of detection of the PC1 RIA (11), whereas
PC1 protein levels were significantly increased in InR1-G9/PC1 cells
(P < 0.01). Immunoreactive PC1 was present only at
background levels in InR1-G9/ASPC2 cells, in agreement with PC1 mRNA
levels. Immunoreactive PC2 was detected in all cells, consistent with
mRNA levels, whereas PC2 levels, were decreased by 63 ± 17%
(P < 0.05) in InR1-G9/ASPC2 cells compared with
transfected controls. Unexpectedly, PC2 levels were increased
(P < 0.05) in InR1-G9/PC1 cells.
Production of glicentin, oxyntomodulin, and glucagon
Identification of GLI and IRG peptides by HPLC revealed that WT
InR1-G9 cells produced glicentin and oxyntomodulin in a ratio of 1:3,
and that most of the GLI eluted as glucagon (Fig. 3). This profile mirrors that which is
found in normal pancreatic A cells (21) and is in agreement with
previous results (20, 21). Overexpression of PC1 caused an increase in
the production of glicentin (P < 0.01) and
oxyntomodulin (P < 0.05) relative to WT cells, and
increased the ratio of glicentin:oxyntomodulin to 1:1. Glucagon
production was not significantly altered in InR1-G9/PC1 cells.
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Identification of the first GLI peak as oxyntomodulin was made on the
basis of its known elution position at fraction 82 relative to elution
of the internal control (10, 19, 21, 23). However, in each experiment,
significant immunoreactivity was observed in a single peak that spanned
from fractions 79–84; the early eluting shoulder on the oxyntomodulin
peak (fraction 82) has not been identified, but may represent oxidized
oxyntomodulin. Glucagon production in InR1-G9/ASPC2 cells decreased
significantly when compared with WT cells (from 39 ± 5 to 19
± 2 ng/peak, P < 0.05), but not compared with
vector-transfected controls.
Production of GLP-1
The total levels of IR GLP-1 in WT InR1-G9 cells were very low
(approximately 1/20th that of GLI/IRG) but were detectable. HPLC
analysis showed that, in WT cells, the major form of GLP-1 produced was
GLP-17–36NH2; little or no GLP-11–37,
GLP-11–36NH2 or GLP-17–37 was detected (Fig. 4). These results are consistent with
previous descriptions of GLP-1 production in InR1-G9 cells (20, 21).
The apparent increase in unamidated GLP-1x-37 levels in
control cells was not consistently observed and did not reach
statistical significance. Overexpression of PC1 appeared to increase
processing to GLP-11–36NH2, although the difference
between peak areas was not significant. Processing to
GLP-17–36NH2 was not altered in InR1-G9/PC1 cells,
compared with WT cells.
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).
Production of GLP-2
No GLP-2 peaks were observed in WT or control InR1-G9 cells after
HPLC (Fig. 5). In contrast, one large
peak of GLP-2 was seen after transfection of PC1 into InR1-G9 cells.
HPLC analysis showed that little or no GLP-2 was present in cells
expressing ASPC2 (Fig. 5).
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shows a summary of the
changes observed in proG processing upon overexpression of PC1 or
transfection of ASPC2. In InR1-G9/PC1 cells, production of glicentin,
oxyntomodulin, and GLP-2 increased as compared with WT cells, as well
as compared with control cells (data not shown). Expression of ASPC2
decreased glicentin, but not glucagon, production. Interestingly,
generation of GLP-17–36NH2 increased in InR1-G9/ASPC2
cells, but not in InR1-G9/PC1 cells, compared with controls.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In agreement with previous work from our lab (21), but in contrast to the results of others (25), we were unable to detect PC1 mRNA transcripts in WT InR1-G9 cells. Consistent with our mRNA data, we also showed that protein levels of PC1 were not detectable in WT InR1-G9 cells using a highly sensitive and specific RIA. We therefore conclude that PC1 is not expressed in the InR1-G9 cells employed in this study. That PC1 is not expressed in InR1-G9 cells is also consistent with its pancreatic glucagon-producing phenotype. PC1 cannot be colocalized with glucagon by immunocytochemistry in adult mouse islets (26) and cannot be detected by Western blot of non-B cells from islets (27). Our results therefore indicate that the InR1-G9 cell line is a faithful reproduction of the pancreatic A cell in terms of proG processing.
Overexpression of PC1 in InR1-G9 cells resulted in significant
increases in glicentin, oxyntomodulin, and GLP-2 production, consistent
with our previous results using vv- infected heterologous cell lines
(10). It must be noted that the observed increases in glicentin and
oxyntomodulin did not appear to be accompanied by a concomitant
decrease in glucagon production. However, because the amounts of
glicentin and oxyntomodulin were very low in WT cells, an increase of
about 5–6 ng/peak would translate into a significant increase (up to
100%; Fig. 6), whereas a similar change in the large amount of
glucagon produced would not translate into a change of comparable
percentage. In contrast to glicentin and oxyntomodulin, no
statistically significant increase in peak areas of
GLP-11–36NH2 or GLP-17–36NH2 could be found
in InR1-G9/PC1 cells. Nonetheless, the increased prominence of the
GLP-11–36NH2 peak in the InR1-G9/PC1 cells, and the
complete lack of change in the GLP-17–36NH2 peak, suggests
that GLP-11–36NH2 was not being processed by PC1 to the
truncated peptide. These results are in contrast to those of our
previous study, in which we have shown that coinfection of vv:PC1 and
vv:proG in GH3 cells results in the production of both
full-length and truncated forms of GLP-1 (10). The fact that very
little C-terminal GLP-1 IR could be detected in GH3 cells
infected with vv:proG alone indicated that cleavage was not occurring
at Arg109-Arg110 (Fig. 1) in the absence of
PC1. Taken together with the results of the present study, these data
suggest that PC1 may mediate processing of proG at
Lys70-Arg71 and
Arg109-Arg110 to yield
GLP-11–36NH2, and that another enzyme may cleave GLP-1 at
Arg77 to yield GLP-17–36NH2.
This is the first study to demonstrate the production of fully processed GLP-2 by PC1 in vivo. Through HPLC analysis of GLP-2 IR, a single GLP-2 peptide was observed in InR1-G9/PC1 cells, which eluted in the same position as synthetic rat GLP-21–33. This extends the observations from our previous study (10), in which an incompletely processed form of GLP-2 was detected in GH3 cells coinfected with vv:proG and vv:PC1. We had previously speculated that this GLP-2 peak represented GLP-2-Lys159-Lys160, as the overexpression of proG may have overwhelmed the capacity of endogenous carboxypeptidase H. Because InR1-G9 cells express proG endogenously, overexpression of proG relative to carboxypeptidase H did not occur in the present study, thus ensuring appropriate levels of both substrate and enzyme to permit complete processing of GLP-2. Of some considerable interest was the observation that overexpression of PC1 in InR1-G9 cells resulted in an increase in IR PC2, albeit not of PC2 mRNA transcript levels. Although we (10) and others (11, 12) have previously demonstrated that PC2 can cleave proglucagon to glicentin, no effects of PC2 on production of GLP-1 or GLP-2 were found. Thus, it is highly unlikely that the increased production of PC2 in the InR1-G9/PC1 cells could account for the observed change in processing phenotype in these cells. However, it is important to note that the GLP-2 antiserum used in this study is specific for the free N-terminal end of GLP-2 (23). Thus, our ability to detect IR-GLP-2 in all of these studies was dependent upon cleavage at Arg124-Arg125, which appears to require the presence of PC1. In contrast, the detection of small amounts of free C-terminal GLP-1 IR in WT and control InR1-G9 cells suggests that PC2 may process proglucagon slightly, albeit inefficiently, at Arg109-Arg110.
Our previous study (10) showed that coexpression of vv:proG and vv:PC2
in a number of heterologous cell lines did not result in the production
of glucagon. Although we had shown that vv:PC2 was bioactive in our
system by demonstrating that vv:PC2 could cleave POMC to 
MSH in
AtT-20 cells (10), the liberation of glucagon from proG is a very late
processing event (17), and it is known that vv infection may interfere
with some late events. Production of 
Lys-MSH from POMC is seen after
3 h of chase in AtT-20 cells stably expressing PC2 (7) but is not
observed in a vv system (28). As well, pro-nerve growth factor is not
processed by vv:PC2 but is processed in PC12 cells stably expressing
PC2 (29). Therefore, whereas the vv system provided a useful model of
proG processing, we predicted that the InR1-G9 stable cell model used
in the present study might provide a more appropriate intracellular
environment for the production of glucagon.
Transfection of InR1-G9 cells with ASPC2 resulted in a 62% decrease in PC2 mRNA levels and a 63% decrease in PC2 immunoreactivity, consistent with other reports that used antisense-mediated inhibition of PC expression (12, 30, 31, 32, 33). Of particular importance, studies that used cells stably transfected with ASPC2 show concomitant alterations in PC2 expression and propeptide processing. GH3 cells cotransfected with POMC and ASPC2 express almost no PC2 protein and demonstrate very little production of ACTH-related peptides (34). Expression of both ASPC2 and proenkephalin in RIN5f cells resulted in a decrease, but not abolition, of PC2 expression, and proenkephalin processing was altered in a small but significant fashion (32). These two studies used a similar ASPC2 construct, which consisted of a 480-bp fragment spanning the translation start site, and we therefore designed a similar construct from mouse PC2 cDNA.
Expression of ASPC2 in InR1-G9 cells resulted in the virtual
disappearance of glicentin, but glucagon production did not change
compared with vector-transfected controls. Although glucagon production
decreased relative to WT InR1-G9 cells, it must be noted that glucagon
production also decreased in vector-transfected controls, despite the
fact that neither PC2 mRNA nor protein levels changed. Therefore, a
reduction in PC2 expression in InR1-G9/ASPC2 cells correlated with a
reduction in glicentin, but not glucagon, production. These results are
in agreement with those of Rothenberg et al. (12), who
showed that a 90% decrease in PC2 protein levels decreased glicentin,
but not glucagon, production in the pancreatic cell line, TC1–6.
Furthermore, stable expression of PC2 in PC12 cells (29) does not
significantly increase the production of glucagon from proG in
transient transfection experiments (Dhanvantari, S., N. G. Seidah,
and P. L. Brubaker, unpublished observations). However, a very
recent study of PC2 null mice has clearly demonstrated the absence of
mature glucagon in the A cell, and a concomitant increase in the levels
of unprocessed proglucagon (35). These findings are not incompatable
with the hypothesis that PC2 cleaves proglucagon to glicentin, and that
glicentin is converted through the actions of another, yet to be
identified enzyme, to glucagon.
Interestingly, an increase in GLP-17–36NH2 production was seen in InR1-G9/ASPC2 cells. Because we (10) and others (11, 12, 13, 17) have not shown a role for PC2 in the generation of GLP-1, and because PC2 immunoreactivity is not found in the L cells of the ileum (14), PC2 is not a candidate enzyme for GLP-17–36NH2 production. Furthermore, as the PC1 immunoreactivity measured in InR1-G9/ASPC2 cells was not significantly different from background levels, the increase in GLP-17–36NH2 production cannot be attributed to an increase in PC1. We have not analyzed WT InR1-G9 cells for the presence of other PCs, such as PC5a, which has recently been shown to colocalize with glucagon and PC2 in pancreatic A cells (36). The reduction in PC2 expression in InR1-G9/ASPC2 cells may lead to increased expression of another endogenous enzyme that is responsible for the production of GLP-17–36NH2. Finally, the absence of GLP-2 in InR1-G9/ASPC2 cells correlates well with the relative absence of PC1, again demonstrating that PC1 is required for the production of GLP-2.
In conclusion, when taken together with the results of our previous study (10), these results confirm a role for PC1 in the processing of proG to the intestinal PGDPs, glicentin, oxyntomodulin, GLP-11–36NH2, and GLP-2. A role for PC2 in the production of glicentin was also demonstrated; however, we were again unable to show a role for PC2 in the liberation of glucagon from proG. We therefore conclude that PC2 generates glicentin, and another, unidentified enzyme may be responsible for the production of glucagon.
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Acknowledgments |
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Footnotes |
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2 Recipient of a University of Toronto Open Fellowship and the Edward
Christie Stevens Fellowship in Medicine.
Received September 18, 1997.
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