Endocrinology Vol. 142, No. 1 37-42
Copyright © 2001 by The Endocrine Society
Coregulation of Glucagon-Like Peptide-1 Synthesis with Proglucagon and Prohormone Convertase 1 Gene Expression in Enteroendocrine GLUTag Cells1
Savita Dhanvantari2,
Angelo Izzo,
Erik Jansen and
Patricia L. Brubaker
Section on Cellular Neurobiology Laboratory of Developmental
Neurobiology (S.D.), National Institute of Child Health and Human
Development, National Institutes of Health, Bethesda, Maryland;
Departments of Physiology (A.I., P.L.B.) and Medicine (P.L.B.),
University of Toronto, Toronto, Ontario, M5S 1A8, Canada; and
Laboratory for Molecular Oncology (E.J.), Center for Human Genetics,
University of Leuven and Flanders Interuniversity Institute for
Biotechnology, Leuven, Belgium
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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Abstract
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The insulinotropic hormone glucagon-like peptide-1 (GLP-1)
is synthesized in the intestinal L cell by prohormone convertase 1
(PC1)-mediated posttranslational processing of proglucagon. Previous
studies have demonstrated that proglucagon gene transcription in the L
cell is stimulated by the protein kinase A (PKA) pathway through a cAMP
response element (CRE). Because the PC1 gene contains two functional
CREs, the present studies were conducted to investigate whether the PC1
and proglucagon genes are coregulated by PKA, and to elucidate the
temporal relationship(s) of PC1 and proglucagon gene expression with
production of GLP-1, in the intestinal cell. The GLUTag
enteroendocrine cell line, which is known to express the proglucagon
gene and to synthesize and secrete GLP-1, was used as a
model. Proglucagon and PC1 messenger RNA transcript levels were
both increased after 12 h (but not 24 h) of treatment of
GLUTag cells with forskolin/isobutylmethylxanthine (IBMX), by 2.7
± 0.3- and 2.4 ± 0.3-fold, respectively, compared with controls
(P < 0.01–0.001). Activation of PKA
resulted in a 2.1 ± 0.1-fold increase in PC1 reporter construct
expression (P < 0.001) at 12 h, which was
dependent on the presence of the CRE, and a 13- to 24-fold increment in
PC1 protein levels (P < 0.01) at 12 and 24 h.
Similarly, forskolin/IBMX increased secretion of GLP-1, by
1.8 ± 0.2- and 2.2 ± 0.6-fold at 12 and 24 h,
respectively (P < 0.05–0.01). Although the cell
content of GLP-1 was diminished after 12 h of
treatment (P < 0.001), GLP-1 levels
increased back to control values after 24 h of forskolin/IBMX
treatment (P < 0.01 vs. 12-h
levels). Thus, PKA-induced secretion of GLP-1 from the L
cell is followed by restoration of the cellular peptide levels through
a PKA-mediated, CRE-dependent up-regulation of proglucagon and PC1 gene
expression.
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Introduction
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THE INTESTINAL HORMONE glucagon-like
peptide-17–36NH2 (GLP-1) has
generated considerable interest, over the past decade, as a potential
treatment for type II diabetes. The actions of GLP-1 on
glucose homeostasis are pleiotropic, and they include stimulation of
glucose-dependent insulin secretion, inhibition of glucagon release and
gastric emptying, and possibly, enhancement of peripheral insulin
sensitivity and regulation of food and water intake
(1, 2, 3). Consistent with these actions, administration of
GLP-1 to patients with type II diabetes is associated with
reductions in glycemia (1, 2, 3, 4). Furthermore, the essential
role of GLP-1 in the metabolic response to glucose
administration has been established by studies demonstrating that acute
administration of a GLP-1 antagonist leads to an increase
in blood glucose levels in humans (5, 6), whereas
disruption of the GLP-1 receptor in mice through
homologous recombination results in glucose intolerance and mild
diabetes (7). Current studies are therefore directed
toward the therapeutic potential of GLP-1 in the treatment
of type II diabetes.
The sequence for GLP-1 is contained within the proglucagon
molecule. Although the proglucagon gene is expressed in both the
pancreatic A and intestinal L cells, tissue-specific posttranslational
processing of proglucagon leads to production of GLP-1
only within the L cell (8, 9, 10). A variety of different
approaches have now been used to demonstrate that the specificity of
this processing is determined by the actions of prohormone convertase 1
(PC1, also known as PC3), which plays a key role in the synthesis of
GLP-1 in the L cell (11, 12, 13, 14). The physiologic
factors that regulate GLP-1 biosynthesis are not well
understood. However, the recent development of the GLUTag L cell line
from intestinal tumors in proglucagon-SV40 large T antigen transgenic
mice has facilitated studies on the factors that regulate proglucagon
gene expression in the intestine (15, 16). Studies to date
have demonstrated that GLUTag cells express the proglucagon gene, and
synthesize and secrete bioactive GLP-1 in a manner that is
consistent with that found in the normal L cell (15, 16).
However, it has not yet been established whether GLUTag cells express
PC1.
Previous studies have demonstrated that activation of the protein
kinase A (PKA) pathway in the GLUTag cell line, as well as in primary
fetal rat intestinal L cells in culture, is associated with increased
proglucagon messenger RNA (mRNA) transcript levels and stimulation of
GLP-1 synthesis and secretion (15, 16, 17, 18). The
increase in proglucagon mRNA transcript levels in the GLUTag cells
occurs consequent to increased proglucagon gene transcription, mediated
through a cAMP-response element (CRE) in the 5'-flanking sequence of
the gene. PC1 is also known to contain two CREs in its 5'-flanking
sequence, a complete CRE at -283 bp and a partial CRE at -263 bp
(19, 20), with some (20, 21, 22), but not all
(23, 24), studies showing induction of PC1 gene expression
through the PKA pathway. Thus, the aim of the present study was to
determine whether PC1 and proglucagon are regulated in parallel by PKA
in the intestinal L cell, and to establish the temporal relationship(s)
between expression of these genes and production of
GLP-1.
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Materials and Methods
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Cell culture
GLUTag cells were grown in DMEM containing 10% FBS, as
described previously (15, 16). Several days before each
experiment, the cells were split into 24-well plates (for peptide
experiments), 6-cm dishes (for PC1 transfection experiments), or 10-cm
dishes (for Northern and Western analyses) and were allowed to reach
80–90% confluence. In all experiments, cells were incubated for
12–24 h with either media alone (DMEM with 0.5% FBS; control) or
media supplemented with 10 µM forskolin plus 10
µM isobutylmethylxanthine (IBMX; Sigma, St.
Louis, MO).
Northern blot analysis
Total cellular RNA was extracted by the guanidium isothiocyanate
method (25) and size-fractionated on a
formaldehyde-agarose gel. The integrity of the RNA was determined by
ethidium bromide staining, and the RNA was then transferred to a nylon
membrane and fixed with UV light. Hybridization to full-length probes
for rat proglucagon (a kind gift from Dr. D. J. Drucker, Toronto,
ON, Canada), mouse PC1 (kindly donated by Dr. N. G. Seidah,
Montreal, Québec, Canada), and 18S ribosomal RNA (from Dr.
D. J. Drucker) and washing were carried out as previously
described (10, 15).
PC1 analyses
PC1 reporter plasmid constructs [-971-, -288-, and -244-LUC
(luciferase) in pGL2-basic (20)] or control
constructs (cytomegalovirus-LUC: positive control; and the
promoterless (SacI 
KPNI-Bluescript) LUC: negative control; a kind
gift from Dr. D. J. Drucker) were transfected into GLUTag
cells using FuGENE6 (Roche Molecular Biochemicals,
Laval, Québec, Canada). Control cells were then treated
with media alone, while cells transfected with the PC1-constructs were
treated with media alone or medium supplemented with forskolin/IBMX,
for 12 h. Cells were extracted in 50 mM
Tris/2-[N-morpholino]ethanesulfonic acid containing
1 mM dithiothreitol and 0.1% Triton X-100, and cell
lysates were mixed with equal volumes of 750 mM
Tris/2-[N-morpholino]ethanesulfonic acid (containing 150
mM MgAcetate and 40 mM ATP) and 500
µM luciferin (ICN Pharmaceuticals, Inc.,
Costa Mesa, CA). Luminescence was determined using a microplate
luminometer (courtesy of Dr. Attisano, Toronto, Ontario, Canada).
Western blots for PC1 were carried out after treatment of cells with
control media alone or medium supplemented with forskolin/IBMX for 12
or 24 h. Media were lyophilized and reconstituted in 1 ml of 10
mM HCl, followed by precipitation of protein in 20%
trichloroacetic acid for 20 min on ice, with 25 µg/ml ribonuclease A
added as carrier protein. Equal aliquots of protein were separated on a
12% Tris-glycine precast gel (Novex, San Diego, CA) and
transferred to a nitrocellulose membrane. PC1 was detected using an
N-terminal antiserum (a kind gift from Dr. I. Lindberg, New Orleans,
LA) at a final dilution of 1:1000 in 1% BSA (in PBS). Detection of
immunoreactive PC1 was by the SuperSignal Chemiluminescence System
(Pierce Chemical Co., Rockford, IL) according to the
supplier’s protocol. Bands were quantified using ImageQuant version
1.2 software, and the detection range was linear.
GLP-1 analysis
Peptides were extracted from media by addition of 10% (vol/vol)
trifluoroacetic acid (TFA), followed by passage twice through a
cartridge of C18 silica (C18 Sep-Pak, Waters Corp.,
Milford, MA) and elution with 80% isopropanol containing 0.1% TFA.
Peptides contained in the cells were extracted by homogenization in 1
N HCl containing 5% HCOOH, 1% TFA, and 1% NaCl, followed
by passage through a C18 Sep-Pak and elution as above. This methodology
has previously been reported to permit greater than 80% recovery of
the proglucagon-derived peptides from cells and tissues (26, 27). RIA for immunoreactive GLP-1 was carried out
using an antiserum directed toward the C-terminal end of
GLP-136NH2 (Affinity Research Products
Ltd., Mamhead, UK), which has previously been demonstrated to detect
predominantly GLP-17–36NH2 in
GLUTag cells (15). Total content of GLP-1 was
determined as the sum of the media and cell content of
GLP-1.
Data analysis
All data are expressed as mean ± SEM.
Statistical differences between groups were determined by ANOVA using
n-1 custom hypotheses tests or by unpaired Student’s t
test, as appropriate, using the SAS system (Statistical Analysis
Systems, Cary NC). Some data were
log10-transformed before analysis to normalize
variances.
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Results
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To establish whether GLUTag cells express the PC1 gene, total
cellular RNA was probed for the presence of PC1 mRNA (Fig. 1
). Two transcripts were detected
(approximately 3 and 5 kb in size), which is in agreement with reports
on PC1 mRNA transcripts in other endocrine cell types
(18). Two proglucagon mRNA transcripts were also detected
in the GLUTag cells, consistent with previous findings
(15). Treatment of GLUTag cells with forskolin/IBMX for
12 h significantly increased proglucagon mRNA transcript levels,
by 2.7 ± 0.3-fold, compared with control cells (P
< 0.001), and induced a parallel increment in PC1 mRNA transcripts
(2.4 ± 0.3-fold; P < 0.01). In contrast, no
changes in either proglucagon or PC1 transcript levels were seen in
GLUTag cells treated with forskolin/IBMX for 24 h.
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Figure 1. GLUTag cells were treated with control media alone
or media with 10 µM forskolin plus 10 µM
IBMX for 12 or 24 h, after which total cellular RNA was extracted
for Northern blot analysis using probes for proglucagon, PC1, and 18S
RNA. A, Representative Northern blot of proglucagon, PC1, and 18S RNA
in 6 dishes of GLUTag cells treated with either control media alone (C)
or with forskolin plus IBMX (F) for 12 h. B, Proglucagon and PC1
mRNA transcript levels (normalized for 18S RNA) after treatment of
GLUTag cells with control media alone (open bars) or
with forskolin/IBMX (closed bars) for 12 h (two to
three dishes were used for each treatment group in each of three
independent experiments for a total of n = 8). C, Proglucagon and
PC1 mRNA transcript levels (normalized for 18S RNA) after treatment of
GLUTag cells with control media alone (open bars) or
forskolin/IBMX (closed bars) for 24 h (n = 3
dishes were used for each treatment group). **, P
< 0.01; and ***, P < 0.001 vs.
controls.
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Although proglucagon gene transcription is enhanced by forskolin/IBMX
in GLUTag cells (15), no information is available about
the regulation of PC1 gene expression in these cells. Therefore, to
determine whether the PKA-mediated increase in PC1 mRNA transcripts was
attributable to enhanced PC1 gene expression, GLUTag cells were
transfected with PC1 promoter-luciferase reporter constructs that
contained both (-971- and -288-LUC) or neither (-224-LUC) of the
CREs, and were treated for 12 h with forskolin/IBMX (Fig. 2). Activation of PKA increased reporter
activity by 2.1 ± 0.1-fold (P < 0.01–0.001) in
cells expressing the -971 and -288 constructs but not in those cells
expressing the truncated -224 construct. To establish whether this
increment in PC1 gene expression was associated with increased PC1
protein levels, Western blot analysis was carried out after treatment
of GLUTag cultures with forskolin/IBMX for 12 or 24 h. As
previously reported (28), two different bands of PC1
protein were detected, an 87-kDa long form and 64-kDa C-terminally
truncated form, both of which are biologically active (Fig. 3).
Activation of PKA increased both forms of PC1 at both time points, by
13.1 ± 8.2- and 24.3 ± 6.5-fold, respectively
(P < 0.01).
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Figure 2. GLUTag cells were transfected with SK- (negative
control), CMV- (positive control), or PC1 promoter-LUC constructs
(-224, -288, and -971) and were then treated with control media
alone (C) or media with 10 µM forskolin plus 10
µM IBMX (F) for 12 h, followed by determination of
cell lysate luminescence (n = 4 dishes were used for each
treatment group). Schematics of the 3 PC1 promoter constructs used
(-971, -288, and -224 bp) indicate the approximate position of the
two CREs, at -283 and -263 bp ( ), followed by the luciferase (LUC)
coding sequence. **, P < 0.01; and ***,
P < 0.001 vs. controls.
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Figure 3. GLUTag cells were treated with control media alone
or with 10 µM forskolin plus 10 µM IBMX for 12 or
24 h, after which media was analyzed by Western blot analysis for
PC1 protein. A, Western blots of PC1 from GLUTag cells treated with
control media alone (C) or media with forskolin plus IBMX (F) for 12 or
24 h. Molecular mass markers are indicated on the left
side (in kDa). Equal amounts of protein were loaded in each
lane. B, Densitometric analysis of blots shown in A. Open
bars, Control media alone; closed bars, cells
treated with forskolin/IBMX (n = 4 dishes were used for each
treatment group); **, P < 0.01 vs.
control.
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Finally, to determine whether GLP-1 synthesis and
secretion were increased in parallel with proglucagon and PC1 mRNA
transcript levels, the media and cell content of GLP-1 was
quantitated in GLUTag cells after 12 and 24 h of treatment with
forskolin/IBMX (Fig. 4).
GLP-1 secretion into the media was increased by 2-fold at
both time points, both in absolute terms (to 182 ± 22% and
215 ± 58% of controls at 12 and 24 h, respectively,
P < 0.05 - 0.01) and as a percent of the total culture
content of GLP-1 [from 29.9 ± 2.2% to 57.9 ±
3.7% of total content at 12 h (P < 0.001), and
from 40.2% ± 3.8 to 60.4 ± 5.1% of total content at 24 h
(P < 0.01)]. Consistent with the release of
GLP-1 into the media, the cell content of
GLP-1 was diminished after 12 h of treatment with
forskolin/IBMX, to 59 ± 4% of control values (P
< 0.001). In contrast, the cell content of GLP-1 was not
different from that of controls after 24 h of treatment (84
± 5% of controls, P = not significant), despite
continued secretion of GLP-1 into the media. Thus, after
24 h of forskolin/IBMX treatment, compared with the 12-h time
point, there was a significant increase in both the media
(P < 0.05) and the cell content (P <
0.01) of GLP-1.
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Figure 4. GLUTag cells were treated with control media alone
(open bars) or media with 10 µM forskolin
plus 10 µM IBMX (closed bars) for 12 or 24 h,
after which cell and media peptides were extracted and analyzed by RIA
for immunoreactive GLP-1 levels (n = 6 culture wells
were used for each treatment group). *, P < 0.05;
**, P < 0.01; and ***, P <
0.001 vs. controls. #, P < 0.05;
and ##, P < 0.01 vs. the same
treatment group at 12 h.
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Discussion
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PC1 is an essential enzyme for the posttranslational processing of
many neuroendocrine prohormones, including POMC, proinsulin,
proneurotensin, prodynorphin, and proglucagon (29).
Despite its importance as a determinant of mature peptide hormone
biosynthesis, relatively little is known about the factors involved in
the regulation of PC1 gene expression. Much of the data available to
date is supportive of the concept of prohormone and PC1 gene
coregulation. For example, POMC and PC1 mRNA transcript levels change
in parallel in the rat neurointermediate pituitary in response to
administration of dopamine receptor-2 agonists and antagonists
(30, 31, 32). Similarly, these transcripts are coordinately
regulated by dexamethasone and CRH in the AtT-20 anterior pituitary
cell line (31, 32). Coregulation of proinsulin and PC1 by
glucose has also been demonstrated in rodent islets and islet cell
lines, with modulation at both the transcriptional and translational
levels (23, 28, 33, 34). Because the PC1 gene is known to
contain two functional CREs (19, 20, 21, 22), a number of studies
have also addressed possible PKA-mediated coregulation of PC1 with
relevant prohormone genes. Recently, PKA stimulation of PC1 mRNA
transcript and neurotensin levels has been reported for the pancreatic
BON carcinoid cell line (22). However, although
prodynorphin mRNA transcripts are increased by activation of PKA in 223
TC-3 cells, neither proinsulin nor PC1 transcript levels are enhanced.
Thus, the role of PKA in the coordinated regulation of PC1 and
prohormone gene expression remains controversial, and may require
additional, cell-type specific factors.
The results of the present study demonstrate that PC1 is expressed in
the GLUTag cell line. GLUTag cells are a model of the intestinal L cell
that expresses the proglucagon gene and synthesizes and secretes
bioactive GLP-1 in a normal, regulated fashion (15, 16). The presence of PC1 in GLUTag cells is therefore consistent
with their ability to process proglucagon to its constituent intestinal
peptides, including GLP-1. These results also confirm the
findings of previous studies using heterologous cell lines or
recombinant proteins in vitro, demonstrating that PC1 is
essential for the liberation of GLP-1 from proglucagon
(11, 12, 13, 14).
Although PC1 is known to be present in the L cell (35, 36), these studies are the first to examine possible
coregulation of PC1 with its substrate, proglucagon. The results
indicate that proglucagon and PC1 mRNA transcript levels are increased
in parallel by activation of the PKA pathway in GLUTag cells.
Interestingly, these effects were observed only at the 12-h time point,
with a return to basal levels by 24 h. The proglucagon gene
contains a CRE in its 5'-flanking sequence, and previous studies have
demonstrated that the PKA-induced increment in proglucagon mRNA
transcripts in the GLUTag cells is mediated at the transcriptional
level (15). Peak increments in proglucagon mRNA transcript
levels in this study were also seen after 12 h of treatment with
forskolin/IBMX (15). Consistent with these findings,
PC1-LUC constructs that contained the two CREs, but not a
construct lacking these consensus sites, were also found to be
responsive to PKA, and PC1 protein was increased coordinately by
treatment of the GLUTag cells with forskolin/IBMX. Thus, activation of
PKA in this L cell line is associated with increments in both
proglucagon and PC1 gene expression, leading to a peak in mRNA
transcript levels at 12 h and increased PC1 protein at both 12 and
24 h.
GLP-1 secretion by the GLUTag cells was also increased by
treatment with forskolin/IBMX for 12–24 h. However, the
GLP-1 content of the cells did not increase until the 24-h
time point. Previous studies have similarly shown that
GLP-1 secretion by both GLUTag and primary fetal rat
intestinal L cells is increased at 2 and 24 h after activation of
PKA but that the cell GLP-1 content is only increased at
24 h (15, 18). The present studies further these
observations by the demonstration of a temporal relationship between
the increments in proglucagon and PC1 mRNA transcripts, PC1 protein,
and the changes in cell GLP-1 content. These findings are
strongly suggestive of increased proglucagon and PC1 gene
transcription, followed by translation of the proglucagon and PC1 mRNA
transcripts, and then processing of proglucagon by PC1 to liberate
GLP-1 in secretory granules. A similar study in pancreatic
BON cells showed PKA-induced elevations in PC1 mRNA transcript levels
over 4–24 h, with replenishment of cellular neurotensin content only
after 16 h (22).
In summary, the results of the present study indicate that biosynthesis
of the antidiabetic peptide GLP-1 in the intestinal L cell
is regulated by the PKA pathway through coordinated regulation of the
processing enzyme PC1 with its substrate, proglucagon. Such information
about the factors regulating GLP-1 biosynthesis will be
important for future studies in which cells are genetically engineered
to produce GLP-1 for encapsulation and implantation into
patients with type II diabetes (37).
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Acknowledgments
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The authors are grateful to Dr. I. Lindberg for the gift of PC1
antiserum and to Drs. Drucker and Seidah for the gifts of cDNA
probes.
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Footnotes
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1 This work was supported by operating grants (to P.L.B.) from the
Canadian Diabetes Association and the Medical Research Council of
Canada. 
2 Supported by a postdoctoral fellowship from the Canadian Diabetes
Association.
Received July 18, 2000.
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Abstract
Introduction
