The Caudal Homeobox Protein cdx-2/3 Activates Endogenous Proglucagon Gene Expression in InR1-G9 Islet Cells
Tianru Jin1,
D. K. Y. Trinh,
Feng Wang and
Daniel J. Drucker
Department of Medicine Banting and Best Diabetes Centre The
Toronto Hospital University of Toronto Toronto, Ontario, M5G
2C4, Canada
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ABSTRACT
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The proglucagon gene is expressed in a highly
cell-specific manner in islet and enteroendocrine cells. DNA sequences
within the proximal proglucagon G1 promoter region bind the homeobox
protein cdx-2/3, and cdx-2/3 activates the proglucagon promoter in
fibroblasts. We show here that cdx-2/3 activates the proglucagon
promoter in both islet (InR1-G9) and enteroendocrine (STC-1 and GLUTag)
cell lines. Furthermore, transfected cdx-2/3 increased the levels of
endogenous proglucagon mRNA transcripts in both transient and stable
transfections of InR1-G9 islet cells. The cdx-2/3-dependent induction
of endogenous proglucagon mRNA transcripts in stable islet lines was
associated with a corresponding increase in the transcriptional
activity of proglucagon promoter-luciferase plasmids. An
amino-terminally truncated cdx-2/3 derivative containing the
homeodomain and carboxy-terminal region of the molecule inhibited both
the cdx-2/3 activation of the proglucagon promoter and the induction of
endogenous proglucagon mRNA transcripts. These observations demonstrate
that cdx-2/3, acting through the proximal G1 element, is a major
transcriptional determinant of cell-specific proglucagon gene
expression in pancreatic islet cells.
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INTRODUCTION
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The proglucagon gene encodes a number of biologically active
peptide hormones important for the control of plasma glucose in
vivo. Glucagon, released from the pancreatic A cell, regulates
glycogenolysis and gluconeogenesis, whereas a truncated form of GLP-1,
liberated from the intestinal L cell, stimulates glucose-dependent
insulin secretion from the islet ß-cell (1). Accordingly, the
molecular factors important for the control of proglucagon gene
transcription in the endocrine pancreas and enteroendocrine cells of
the intestine are of considerable interest and importance.
Experiments using transgenic mice and gene transfer in vitro
have identified several regions within the first few kilobases of the
rat proglucagon promoter that appear to be important for control of
proglucagon gene transcription (2, 3). Expression of proglucagon
gene-SV40 T antigen transgenes in mice led to the delineation of an
upstream element, designated the proglucagon gene upstream enhancer,
that is necessary for proglucagon gene transcription in enteroendocrine
cells in vivo (4). A number of control elements in the more
proximal proglucagon promoter have been identified that function as
islet cell-specific enhancers (5). DNA sequences within the first 100
bp of the promoter are functionally important for restricting
proglucagon gene transcription to islet and intestinal endocrine cell
types (6). This proximal promoter element, termed G1, contains a number
of AT-rich sequences characteristic of the binding sites for homeobox
genes.
A combination of transfection and electrophoretic mobility shift assay
experiments have suggested that the homeobox transcription factor isl-1
binds to the proglucagon G1 element and activates the proglucagon
promoter (7). Reduction of isl-1 levels in InR1-G9 islet cells was
associated with decreased levels of proglucagon mRNA transcripts,
providing further support for the importance of isl-1 in the control of
proglucagon gene expression (7). The caudal homeobox protein
cdx-2/3, present in both islet and intestinal nuclear extracts, also
binds to the AT-rich sequences in the proglucagon G1 element (8), and
transfected cdx-2/3 activates the proglucagon promoter in BHK
fibroblasts. To delineate a role for cdx-2/3 in the control of
proglucagon gene transcription in endocrine cells, we have now examined
the cdx-2/3-dependent regulation of proglucagon gene transcription in
islet and enteroendocrine cell lines.
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RESULTS
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Because cdx-2/3 activated the proglucagon promoter in BHK
fibroblasts (that do not express cdx-2/3) (8), we wished to determine
whether cdx-2/3 introduced into islet or intestinal cell lines also
increased the activity of the proglucagon promoter. Cotransfection of
cdx-2/3 and a series of 5'-deleted proglucagon-luciferase plasmids into
GLUTag and STC-1 intestinal cells and InR1-G9 islet cells demonstrated
a cell-specific activation pattern of proglucagon promoter activity
(Fig. 1
). The cdx-2/3-dependent activation of the
proglucagon promoter plasmids containing the largest amounts of
5'-flanking sequences, such as (-2292)GLU-Luc, was somewhat greater in
InR1-G9 cells compared with GLUTag cells. In contrast, cdx-2/3 did not
function as an activator of the longer proglucagon-luciferase plasmids
in STC-1 cells until proglucagon gene 5'-flanking sequences upstream of
-155 were deleted. Further deletion of promoter sequences to -60
eliminated the cdx-2/3-dependent activation (data not shown),
consistent with the known location of the cdx-2/3-binding site in the
rat proglucagon promoter (8). Taken together, the differential
cdx-2/3-dependent activation of the proglucagon promoter in STC-1
vs. InR1-G9 and GLUTag cells suggests that the cell-specific
expression of DNA-binding proteins, as previously documented for the
upstream proglucagon enhancer sequences (4), may modify the
transcriptional activity of the proglucagon promoter, and its response
to cdx-2/3 in different cell types. Furthermore, the transfection data
shown here clearly establish that cdx-2/3 activates the proglucagon
promoter not only in fibroblasts (8), but also in islet and intestinal
cell lines.

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Figure 1. Cdx-2/3-Dependent Transcriptional Activation of the
Proglucagon Promoter in Different Cell Types
GLUTag (a), STC-1 (b), and InR1-G9 (c) proglucagon-producing cell lines
were transfected with 5 µg (InR1-G9) or 7.5 µg (STC-1 and GLUTag)
of luciferase reporter genes driven by different proglucagon promoter
fragments in the presence (+) or absence (-) of 5 µg hamster cdx-3
under the control of a CMV promoter (8). The data are expressed as mean
relative luciferase activity ± SEM normalized to the
activity obtained after transfection of the promoterless luciferase
plasmid in the same experiment.
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The finding that exogenous cdx-2/3 activated proglucagon promoter
plasmids in endocrine cells prompted us to determine whether cdx-2/3
transfected into InR1-G9 cells was also capable of activating the
endogenous proglucagon gene. The results of this experiment are shown
in Figs. 2
and 3
. Increasing amounts of
cdx-2/3 transiently transfected into InR1-G9 cells resulted in a
corresponding increase in the levels of proglucagon mRNA transcripts.
In contrast, no increase in the levels of tubulin mRNA transcripts was
detected in the same experiment. The transcription factor isl-1 also
increased proglucagon gene expression in transfected InR1-G9 cells,
consistent with previous findings that depletion of isl-1 in InR1-G9
cells caused a reduction in the basal levels of proglucagon mRNA
(7).

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Figure 2. Cdx-2/3 Activates Endogenous Proglucagon Gene
Expression in InR1-G9 Cells
a, InR1-G9 cells were either mock-transfected (WT) or transfected with
the CMV expression vector alone (pBAT7), with cdx-3 in the antisense
orientation (AS), or with various amounts (in micrograms) of isl-1 or
cdx-3. RNA was extracted for Northern analysis with cDNA probes for
glucagon (G) or tubulin (T) 16 h after the transfection. The
ethidium bromide stain of the RNA gel showing the migration positions
of 28 and 18S ribosomal RNA is shown. b, GLUTag cells were transfected
with cdx-3 or pBAT7 in the amounts indicated (micrograms DNA in
parentheses). RNA was extracted 18 h after the
transfection, and Northern blots were hybridized with the cDNA probes
for proglucagon (G) and tubulin (T). The relative densitometric values
for the RNA signals are shown below.
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Figure 3. Northern Analysis
Northern analysis of InR1-G9 G418-resistant stable islet cell lines
(wt, wild type), transfected with the expression vector alone (pSR1neo-
V1 and V2), a pool of 50100 clones (M1), or individual clones
(designated C). The blots were probed with radiolabeled cDNAs for
glucagon (G), cdx-2/3, or rat chromogranin (C). The ethidium
bromide-stained gel before transfer, along with the integrity of 28S
and 18S ribosomal RNA, is shown. Various exposures of the blots were
scanned with a laser densitometer, and the relative densitometric
units, normalized to the values obtained for the signals in the InR1-G9
wild type lane, are shown below.
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To determine whether cdx-2/3 was capable of activating endogenous
proglucagon gene expression in various cell types, we transfected
increasing amounts of cdx-2/3 into mouse intestinal GLUTag cells. No
increase in the levels of proglucagon mRNA transcripts was observed in
GLUTag cells (Fig. 2b
), suggesting that the activation of endogenous
proglucagon gene expression by cdx-2/3 is highly cell-specific.
Furthermore, we did not observe activation of proglucagon gene
transcription (as assessed by Northern analysis) after transfection of
cdx-2/3 into STC-1 enteroendocrine cells, or BHK fibroblasts (data not
shown).
To provide complementary evidence correlating increased expression of
cdx-2/3 with activation of endogenous proglucagon gene expression, we
transfected either isl-1 or cdx-2/3 [ligated in the plasmid pSR1neo
(7)] into InR1-G9 cells and selected transfectants with the antibiotic
G418. Analysis of a few randomly selected stably transfected
G418-resistant InR1-G9 clones that expressed cdx-2/3 displayed
increased basal levels of proglucagon mRNA transcripts in three of four
lines examined (data not shown). Accordingly, to correlate more
precisely the levels of cdx-2/3 expression with potential changes in
expression of the endogenous proglucagon gene, a larger number of
clones were isolated and examined (Fig. 3
). Increased cdx-2/3
expression was generally associated with induction of proglucagon gene
expression in InR1-G9 cells. Of 21 clones examined, 18 had increased
levels of both cdx-2/3 and proglucagon mRNA, whereas three clones had
normal levels of cdx-2/3 and proglucagon (Fig. 3
and data not shown).
Nevertheless, we did not observe a perfect quantitative correlation
between cdx-2/3 and proglucagon gene expression, as some clones,
e.g. C4, had very high levels of cdx-2/3 mRNA, but
comparatively modest induction of proglucagon gene expression. In
contrast to the activation of the proglucagon gene detected in
association with increased cdx-2/3 expression, the levels of mRNA
transcripts for tubulin (Fig. 3
) or the neuroendocrine gene
chromogranin (Fig. 3
) were not induced by cdx-2/3 in the G418-resistant
cell lines. The results of these experiments strongly suggest that
cdx-2/3 activates the endogenous proglucagon gene in InR1-G9 islet
cells.
The increased levels of proglucagon mRNA transcripts were likely
attributable in part to an increase in proglucagon promoter activity.
To examine this directly, we transfected proglucagon-luciferase
plasmids into wild type InR1-G9 cells, and InR1-G9 clones C2 and C4,
that exhibited normal and increased levels of endogenous proglucagon
mRNA transcripts, respectively. Proglucagon promoter activity was
clearly increased in the C4 InR1-G9 clone with all three proglucagon
promoter plasmids that contained the cdx-2/3-binding site, consistent
with the increased levels of both cdx-2/3 and proglucagon mRNA
transcripts detected in the C4 cell line (Fig. 4
). In
contrast, no increase in luciferase activity was observed after
transfection of the same plasmids in the C2 clone, which exhibited wild
type levels of proglucagon mRNA transcripts. Furthermore, the increased
activity of proglucagon promoter plasmids was not seen after deletion
of 5'-flanking sequences to -60, consistent with the elimination of
the cdx-2/3-binding site. Taken together, these experiments clearly
demonstrate a correlation between expression of the cdx-2/3 gene and
activation of both the endogenous proglucagon gene and the transfected
proglucagon promoter in InR1-G9 islet cells.

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Figure 4. Proglucagon Promoter Activity in InR1-G9 Cells
Expressing cdx-3
Five micrograms of the promoterless control plasmid (pBLUC) or the
different proglucagon-luciferase reporter plasmids were transfected
into wild type InR1-G9 cells, (WT), clone C2 (a G418-resistant
cdx-3-transfected InR1-G9 cell clone showing no increase in either
proglucagon or cdx-3 expression), and clone C4 (a G418-resistant
cdx-3-transfected InR1-G9 cell clone that exhibits increased expression
of both proglucagon and cdx-3). The cells were harvested 16 h
after transfection, and reporter gene activity was measured as
described in Fig. 2 .
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The results of recent experiments using RT-PCR for the analysis of
cdx-2/3 gene expression suggested that an amino-terminally-truncated
cdx-2/3 protein (that contains the homeodomain and carboxy-terminal
cdx-2/3 sequences), binds the G1 promoter element but fails to activate
transcription from a G1-linked promoter in InR1-G9 cells (9). To
determine the putative importance of the N-terminally-deleted cdx-2/3
protein for proglucagon promoter activity, we cotransfected increasing
amounts of cdx-2/3 alone or cdx-2/3 in the presence of
NT-cdx-2/3,
an internally deleted cdx-2/3 cDNA (that we generated by PCR), which
lacks amino acids 8180 (Fig. 5
). Activation of the
proglucagon promoter by transfected cdx-2/3 in InR1-G9 cells was found
to be maximal at concentrations of transfected cdx-2/3 approximating
100 ng, and cotransfection with the
NT-cdx-2/3 plasmid completely
abrogated the cdx-2/3 induction of proglucagon promoter activity,
despite increasing amounts of cdx-2/3 in the transfection experiments
(Fig. 5a
). The
NT-cdx-2/3 plasmid also attenuated the basal activity
of the proglucagon promoter in InR1-G9 cells (Fig. 5b
), consistent with
the recently described competition of this protein with wild type
cdx-2/3 for binding to the G1 promoter site (9). To ascertain whether
the N-terminal cdx-2/3 deletion might also influence the levels of
endogenous proglucagon mRNA transcripts, the
NT-cdx-2/3 plasmid was
transiently transfected into InR1-G9 cells alone or in the presence of
cdx-2/3, after which the levels of proglucagon mRNA transcripts were
examined (Fig. 5c
). The results of this experiment demonstrate that the
NT-cdx-2/3 plasmid abrogates the cdx-2/3-dependent induction of
proglucagon mRNA transcripts in InR1-G9 islet cells.

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Figure 5. Effect of [8180] cdx-2/3 on the
Transcriptional Activity of the Proglucagon Promoter in InR-1-G9 Cells
a, Proglucagon promoter plasmids (5 µg) containing 2,292, 329, 217,
or 82 bp of rat proglucagon gene 5'-flanking sequences fused to the
luciferase cDNA were transfected into InR1-G9 cells along with 10 µg
of pBAT8 (the promoterless expression vector) or 10 µg of
N[8180] cdx-2/3. The values are depicted as the mean ±
SEM of three different transfections. *,
P < 0.05;**, P < .01. b,
Inhibition of the cdx-2/3 induction of proglucagon promoter activity by
N[8180] cdx-2/3. All transfections contained 5 µg of -82
proglucagon-luciferase as the reporter and varying amounts of cdx-2/3
(100400 ng) in the presence of 15 µg N[8180] cdx-2/3, or 15
µg pBAT7. The values are depicted as the mean ± SEM
of three different transfections. *, P < 0.05. c,
Effect of cdx-2/3 plasmids on proglucagon gene expression in InR1-G9
islet cells. Ten micrograms of each of the various plasmids shown were
transfected into InR1-G9 cells, and RNA was harvested 24 h after
transfection for Northern analysis. G, Glucagon; T, tubulin. The
relative densitometric values for the RNA signals are shown below.
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DISCUSSION
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Cdx-2/3-binding sites have been identified in the
sucrase-isomaltase, carbonic anhydrase 1, apolipoprotein B, and
proglucagon gene promoters (8, 10, 11, 12). Although cdx-2/3 activates the
sucrase-isomaltase and proglucagon promoters in transfection of
heterologous cell types (8, 10), cdx-2/3-binding sites in the third
intron of the human apoB gene mediate repression of gene transcription,
possibly through interaction with members of the CCAAT/enhancer binding
protein transcription factor family (11). Despite increasing evidence
supporting a role for cdx-2/3 as a regulator of intestinal gene
transcription, the only genetic target identified for cdx-2/3, in
extraintestinal tissues such as the endocrine pancreas, is the
proglucagon gene. Furthermore, no experiments to date have demonstrated
that cdx-2/3 activates the expression of the mRNAs for
sucrase-isomaltase, carbonic anhydrase 1, or apolipoprotein B.
Establishment of stable intestinal epithelial IEC-6 cell lines
expressing high levels of mouse cdx-2 was followed by proliferation
arrest and differentiation in association with expression of cdx-2. The
proliferation arrest was released after several days, despite the
continuing expression of cdx-2. Although no induction of the
cdx-2-responsive SI gene was observed after 40 days of culture, IEC-6
cells expressing cdx-2 for more than 50 days did express SI gene
transcripts (13). These observations suggest an indirect link between
prolonged expression of cdx-2 and the activation of endogenous target
gene expression.
The cdx-2/3-dependent activation of endogenous proglucagon gene
expression reported here was observed in InR1-G9 islet cells but not in
enteroendocrine or fibroblast cell lines. In contrast, cdx-2/3
activated the proglucagon promoter in transfection experiments in
fibroblasts and intestinal endocrine cells. The mechanisms determining
the cell-specific cdx-2/3 responsiveness of the endogenous proglucagon
gene promoter remain unknown. We have demonstrated by RT-PCR that
InR1-G9 and GLUTag cells express a different profile of homeobox genes
(data not shown), and it is possible that homeobox proteins with
different affinities for the G1-binding site may modify the effect of
transfected cdx-2/3 on activation of the endogenous proglucagon
promoter in GLUTag cells. Similar cell-specific differences in target
gene responsiveness have recently been reported for the IPF-1 induction
of insulin mRNA transcripts (14). The insulin gene homeobox
transcription factor IPF-1 induced the expression of the insulin and
amylin genes in stably transfected, glucagon-producing AN 697 islet
cells, but not in rat embryo fibroblasts. Furthermore, transient
transfection of IPF-1 into either
TC1.9 or AN697 glucagon-producing
islet cell lines was not associated with induction of endogenous
insulin gene expression (14). These observations differ from our
demonstration that cdx-2/3 activates the proglucagon promoter and
endogenous proglucagon gene in both transient transfections and in
stable cell lines expressing increased levels of cdx-2/3. The
mechanisms for the inability of IPF-1 to induce insulin gene expression
in transient transfections remain unknown, but it has been suggested
that IPF-1 action may require the expression of other factors necessary
for activation of the insulin gene promoter, and induction of these
factors may require more than the 4872 h duration of a transient
transfection (14).
The data reported here demonstrate that increased expression of cdx-2/3
correlates with the increased expression of a specific endogenous mRNA
and not simply a transfected fusion gene. The LIM domain homeobox gene
isl-1 was previously shown to bind to an adjacent site in the
proglucagon gene G1 promoter region, and reduced levels of isl-1 were
associated with a decrease in the levels of proglucagon mRNA
transcripts (7). The results of our experiments demonstrate that
transfected isl-1 is also associated with an increase in endogenous
proglucagon mRNA transcripts, consistent with the postulated role for
isl-1 as a positive activator of proglucagon gene transcription.
Although cdx-2/3 activated the expression of proglucagon-luciferase
reporter genes in three different endocrine cell lines, considerable
heterogeneity was observed with respect to the relative degree and
pattern of reporter gene activation in the different cell types. We
have previously documented that the relative profile of basal
proglucagon promoter transcriptional activation differs after
transfection of STC-1, InR1-G9, and GLUTag cell lines (4). Whereas the
latter two cell types appear to more accurately represent the A and L
cell phenotype, respectively, STC-1 cells are plurihormonal and much
less representative of a pure population of differentiated
glucagon-producing cells (15). Furthermore, we have recently shown that
the pattern of DNA-protein interaction using STC-1 extracts and
proglucagon promoter sequences clearly differs from the profile of
binding events detected with extracts from InR1-G9 and GLUTag cells
(4). Taken together, these observations suggest that cell heterogeneity
and differential expression of transcription factors may account for
the observed differences in proglucagon gene promoter activity in
various cell types.
The recent report that an amino-terminally truncated cdx-2/3 RNA
transcript was detected in InR1-G9 cells by RT-PCR (9) prompted us to
examine the potential significance of such a molecule for the control
of proglucagon promoter activity. Although the relative degree of
inhibition of promoter activity varied somewhat with the various
proglucagon-promoter plasmids analyzed, we consistently observed an
inhibition of the cdx-2/3-dependent induction of proglucagon promoter
activity in the presence of the
NT-cdx-2/3 plasmid. Furthermore, the
NT-cdx-2/3 plasmid diminished the ability of the wild type cdx-2/3
plasmid to activate endogenous proglucagon mRNA transcripts in InR1-G9
islets, but the
NT-cdx-2/3 plasmid alone did not reduce the basal
levels of proglucagon mRNA. This may be due to competition for binding
to the proglucagon G1 promoter site (by other homeobox proteins), or to
other mechanisms that remain to be elucidated.
The results of our experiments with the
NT-cdx-2/3 plasmid suggest
that such an N-terminally-truncated protein, if expressed in islet
cells, might possibly modulate the activity of cdx-2/3 on the
proglucagon promoter, and therefore the ratio of wild type and
truncated cdx-2/3 proteins could be an important determinant of cdx-2/3
activity. Nevertheless, we have not observed a smaller cdx-2/3 RNA
transcript by Northern blotting, and Western blot analysis of InR1-G9
cells using antisera detected against the carboxy-terminal region of
cdx-2/3 did not detect the presence of any smaller immunoreactive
cdx-2/3 proteins that might correspond in size to the predicted
N-terminal cdx-2/3 deletion (8). Accordingly, the biological
significance of the N-terminal cdx-2/3 deletion in the control of
proglucagon gene expression awaits the definitive detection of this
protein in islet and enteroendocrine cells and remains uncertain.
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MATERIALS AND METHODS
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Plasmids
The plasmid pBAT7.cdx-3 was kindly provided by M. S. German (San
Francisco, CA) (16). The expression of hamster cdx-3 (313 amino acids)
in this plasmid is under the control of a cytomegalovirus (CMV)
promoter. The series of 5'-deleted proglucagon-luciferase plasmids used
here has been previously described (4, 8). The rat isl-1 expression
plasmid was reported previously (7). The plasmid
NT-cdx-2/3, which
contains a deletion from amino acids 8180 in hamster cdx-3, was
constructed by PCR, and the sequence of the N-terminal-deleted cdx-3
cDNA was verified by DNA sequencing. For generation of stable
G418-resistant cell lines, hamster cdx-3 was ligated into the psR1neo
expression vector, which contains the neo resistance cassette (7).
Cell Lines, Northern Analysis, and Transfections
The InR1-G9, STC-1, and GLUTag cell lines were propagated and
transfected as previously described (15, 17, 18, 19). RNA isolation used
the acid phenol extraction method (20), and Northern blotting was
carried out using nylon membranes as described (21, 22). The luciferase
activity was analyzed by normalizing each transfection relative to the
protein concentration in each transfected extract, and luciferase
activity was measured as described (23). The reporter gene activity was
expressed relative to the activity obtained using the promoterless
luciferase plasmid pBluc in the same experiment. All transfections were
carried out in triplicate on at least three separate occasions, and
statistical significance was assessed by Students t
test.
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FOOTNOTES
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Address requests for reprints to: Dr. D. Drucker, The Toronto Hospital, 200 Elizabeth Street CCRW3-838, Toronto M5G 2C4, Canada.
This work was supported by an operating grant from the Medical Research
Council of Canada. T.J. was supported by a fellowship award from the
Ontario Ministry of Health. D.T. was supported by a fellowship award
from the Banting and Best Diabetes Centre. D.J.D. is a Scientist of the
Medical Research Council of Canada.
1 Present address: Oncology Research Laboratories, The Toronto Hospital,
University of Toronto, 67 College Street, Toronto, Ontario M5G 2M1,
Canada. 
Received for publication October 4, 1996.
Revision received October 29, 1996.
Accepted for publication November 4, 1996.
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