(Received for publication, February 19, 1997)
From the Section of Gastroenterology, Boston University School of Medicine and Boston Medical Center, Boston, Massachusetts 02118
Glucose-dependent insulinotropic
polypeptide (GIP) is a 42-amino acid gastrointestinal regulatory
peptide that, in the presence of glucose, stimulates insulin secretion.
GIP is expressed in K cells of the small intestine and in cells of the
submandibular salivary gland. Using a rat GIP cDNA as a specific
probe, we screened a number of established cell lines for the
expression of GIP mRNA. STC-1 cells, a cell line derived from a
mouse neuroendocrine tumor, were found to express high levels of GIP
mRNA. GIP-specific transcripts were not detected in other cell
lines tested, which included cells of intestinal, salivary, and
endocrine origin. Analysis of GIP-luciferase fusions identified two
promoters, a distal and a proximal promoter, upstream of the
translation initiation codon for GIP. The distal promoter, located
upstream of position +1, corresponds to the principal promoter of the
GIP gene and can promote cell-specific transcription. Sequential
deletion and site-directed mutational analysis of the distal promoter
demonstrated that the sequence between 193 and
182 determines
cell-specific expression of GIP. Contained in this region is a
consensus GATA motif, suggesting that a member of the GATA family of
DNA-binding proteins is involved in the cell-specific regulation of the
GIP gene.
Glucose-dependent insulinotropic polypeptide
(GIP),1 first isolated in 1969 from porcine
small intestine, was originally named gastric inhibitory polypeptide,
on the basis of its ability to inhibit gastric acid secretion in dogs
(1). The primary structure of GIP was described in 1971 (2), and its
amino acid sequence placed it in the secretin family of
gastrointestinal regulatory peptides. GIP immunoreactivity has been
demonstrated in the cytoplasmic granules of K cells in the mucosa
distributed throughout the length of the small intestine (3). After
intestinal glucose perfusion, the primary site of endogenous GIP
release is the duodenum and proximal jejunum (4). Although several
studies have supported the role of GIP as a physiological inhibitor of
acid secretion (5-8), some investigators have challenged this notion
(9, 10). Moreover, subsequent studies of the physiological properties of GIP demonstrated that, in addition to its inhibitory effects in the
stomach, when GIP is administered in physiological doses, the peptide
is a potent stimulator of insulin release by pancreatic -cells.
(11-13). Thus, it has been suggested that GIP may function as an
incretin, the proposed substance that mediates the enteroinsular axis
and plays a physiological role in maintaining glucose homeostasis (14).
Recently, the cDNAs encoding human and rat GIP have been isolated
from libraries prepared from duodenal RNA (15-17). Using the rat
cDNA as a specific probe, Northern hybridization analysis has
demonstrated GIP expression in both the rat small intestine and
salivary gland (17). In the small intestine, GIP mRNA levels were
highest in the duodenum and jejunum, with lower concentrations detected
in the ileum. In addition, recent studies have demonstrated that
glucose and fat induce GIP gene expression (18, 19). Despite these
findings, little is known about the regulatory factors that control the
cell-specific and the nutrient-regulated expression of the GIP gene.
One reason for this lack of information is the inability to adequately
purify populations of GIP-producing K cells, which are scattered
throughout the small intestinal mucosa. Therefore, the characterization
of the factors that regulate GIP expression, including
cis-acting elements in the promoter/enhancer regions,
requires the identification of surrogate cell lines that can be used as
a model for in vitro studies. STC-1 cells are an intestinal
cell line derived by Rindi et al. (20) from transgenic mice
expressing viral oncogenes under the control of the insulin promoter.
Immunochemical analysis has demonstrated GIP staining in these cells
(21). In this report, we use Northern analysis to identify a
GIP-specific transcript that has the same electrophoretic mobility as
that observed in rat intestinal tissue. In addition, we describe the
cloning of the 5-flanking region of the rat GIP gene and the
characterization of the transcriptional activity of these sequences in
the neuroendocrine cell line STC-1.
Male Sprague-Dawley rats were
purchased from Charles River Laboratories (Kingston, MA). STC-1 cells
(mouse endocrine) were kindly provided by Dr. D. Hanahan (University of
California at San Francisco, CA), Rin5AH cells (rat insulinoma) were a
gift from Dr. Å. Lernmark (University of Washington, Seattle, WA), and L cells (mouse connective tissue) were kindly donated by Dr. T. B. Usdin (National Institute of Mental Health, Bethesda, MD). NIH 3T3 (NIH
Swiss mouse embryo), SCA-9 (mouse submandibular gland), HS124 (human
sublingual gland), GH4 (rat pituitary), HIT T15 (Syrian hamster cell), and 407 (human embryonic intestine) cells were purchased from the American Type Tissue Collection (Bethesda, MD).
STC-1, SCA-9, HS124, GH4, and Rin5AH cells were grown in Dulbecco's
minimal essential medium containing 10% fetal bovine serum. NIH 3T3
cells were grown in Dulbecco's minimal essential medium containing
10% calf serum, and 407 cells were grown in Dulbecco's minimal
essential medium containing 15% fetal bovine serum. HIT cells were
grown in modified Ham's F12K media with 12.5% calf serum and 2.5%
fetal bovine serum. All cells were grown at 37 °C in an atmosphere
of 5% CO2 in media containing 100 units/ml penicillin G,
100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B.
Cells were grown to 80% confluence, after which medium was aspirated, and the cells were washed twice with ice-cold phosphate-buffered saline prior to RNA isolation. Intestinal mucosa was prepared for RNA extraction by surgically removing an 8-10-cm segment of duodenum immediately distal to the pylorus and scraping the mucosa with a glass slide. Total RNA from duodenal mucosa or STC-1 cells was extracted using the acid/phenol method of Chomczynski and Sacchi (22).
Northern hybridization analysis was done as described previously, using
the EcoRI fragment of the rat GIP cDNA (17) radiolabeled with [-32P]deoxycytidine triphosphate.
A rat
genomic DASH® library, purchased from Strategene, was
screened by plaque hybridization with the rat GIP cDNA clone, as
described previously (17). The hybridization was carried out using
standard methods (23). The DNA from two positive clones was digested
with a variety of enzymes and subjected to Southern analysis using the
oligonucleotide 5
-GCCAACAGCTCTTCTTAGCT-3
, which is complementary to
the 5
-untranslated region immediately upstream from the ATG codon, as
a GIP-specific probe. Two overlapping restriction fragments, a
2.8-kilobase pair (kbp) BamHI-HindIII fragment
and a 5.9-kbp SacI fragment, were identified and subcloned into pBSKII+ (Strategene) to form the plasmids pBSBH and
pBSSac3, respectively (Fig. 1A). The 5
-flanking region was
then sequenced using double-stranded template and synthetic
oligonucleotides.
GIP-Luciferase Plasmids
The promoterless plasmid pGL2-basic
(Promega, Madison, WI), containing the firefly luciferase reporter
gene, was used to examine the transcriptional regulation of the GIP
gene. Two series of chimeric clones were constructed (Fig.
1, B and C). In the first series,
which included pGL-943, pGL-425, pGL-397, pGL-363, pGL-259, pGL-203,
pGL193, pGL-182, pGL-173, and pGL-143, all constructs contained
GIP-specific inserts with the same 3-end, a BstYI site at
base pair +8 in exon 1, ligated to the BglII site in the
pGL2-basic polycloning region. In the second series, which included
pGL-943i, pGL-778i, pGL-425i, pGL-397i, pGL-363i, pGL-182i, and
pGL+111i, the GIP-specific inserts extended 3
to include sequences for intron 1. Each of these clones ends with the AluI site at
base pair +781, ligated to an EcoRV site that was placed
immediately in front of the HindIII site in the polycloning
region of pGL2-basic.
GIP promoter-specific primers
containing various base substitutions were synthesized by Life
Technologies, Inc. Mutations were introduced by polymerase chain
reaction using forward primers with the following sequences:
5-GGGGTACCCAGATGACACTGCAGATACCCAAA-3
(mut1),
5
-GGGGTACCCAAAAAACACTGCAGATACCCAAA-3
(mut2),
5
-GGGGTACCCAGATGACACTGCAGAAAAACCCAAA-3
(mut3), and
5
-GGGGTACCCAAAAAACACTGCAAAAACCCAAA-3
(mut4).
The luciferase-specific primer GLprimer2 (Promega, Madison, WI) was used as the reverse primer with the wild type pGL-193 DNA as the template for each reaction. The polymerase chain reaction products were digested with KpnI and HindIII before ligation into pGL2-basic.
Transient Transfection AssaysOne day prior to
transfection, cells were plated at a density of ~3-6 × 105 cells per 60-mm dish in the appropriate growth medium.
A mixture containing 2.5 µg of pGL2 reporter plasmid, 14 µl of
lipofectamine (Life Technologies, Inc.), 0.5 µg of pCMV-gal DNA (a
control for transfection efficiency), and 600 µl of serum-free medium was incubated at room temperature. After 15 min, 2.4 ml of media was
added, and the DNA mixture was added to cells previously washed twice
with serum-free medium. After 5 h, 3 ml of medium containing twice
the normal concentration of serum was added, and the incubation was
continued for 48 h, after which the cells were harvested. For
luciferase assays, the cells were first washed twice with phosphate-buffered saline and then were lysed in 500 µl of lysis buffer following the manufacturer's instructions (Analytical
Luminescence, San Diego, CA).
To assay
luciferase activity, 100 µl of the cell lysate was mixed with 100 µl of luciferase substrate solution A (Analytical Luminescence).
Using a luminometer with automatic injection, 100 µl of substrate
solution B (Analytical Luminescence) was then added, and luciferase
activity was measured as the light emission over a 30-s period.
-Galactosidase activity in 40 µl of the cell lysate was determined
after a 5-30-min incubation at 37 °C with 2 mM
chlorophenol red
-galactopyranoside (Boehringer Mannheim) in 2 mM MgCl2, 0.1 mM MnCl2,
45 mM 2-mercaptoethanol, and 100 mM
NaHPO4, pH 8. The reactions were stopped by adding 500 µl
of 0.5 M EDTA, pH 8.0, and the absorbance at 570 nm was
measured using a spectrophotometer. Within each experiment, luciferase activity was determined in duplicate and normalized to
-galactosidase activity for each dish. Each plasmid was tested at
least six times in two separate experiments.
Initially, a number of established cell lines were screened for
the expression of immunoreactive GIP. Because GIP expression was
initially detected in both intestinal and salivary tissues, cells of
intestinal origin, including STC-1 and intestinal 407, and cells
derived from the salivary gland, including SCA-9 and Hs124, were
screened. In addition, three endocrine cell lines, HIT T15, Rin5AH, and
GH4, were also screened for expression of immunoreactive
GIP. Expression of GIP was demonstrated only in the mouse
neuroendocrine tumor cell line STC-1 and the human embryonic intestinal
407 cell line (data not shown). The level of immunoreactive GIP
expression was significantly greater in STC-1 cells than in 407 cells.
Consistent with this observation, Northern hybridization analysis using
a GIP cDNA probe demonstrated a specific band in RNA isolated from
STC-1 cells with the same mobility as rat duodenal GIP mRNA, while
no hybridization was detectable in RNA isolated from any of the other
cell lines screened, including 407 cells; comparable levels of actin
transcripts were detected in the same samples (Fig.
2).
To characterize the tissue-specific transcriptional activity of the GIP
promoter, the GIP gene was isolated from a rat genomic library after
screening 106 plaques with a rat intestinal GIP cDNA.
Two overlapping clones, each having inserts of approximately 20 kbp,
were identified. Restriction fragments that contained upstream
sequences were identified by restriction endonuclease digestion and
Southern hybridization analysis using a synthetic oligonucleotide
homologous to the 5-end of the GIP cDNA sequence. Two fragments, a
5.9-kbp SacI-SacI fragment and a 2.8-kbp
BamHI-HindIII fragment, were identified,
subcloned into pBSKII, and partially sequenced (Fig. 1A).
The sequence of the GIP gene was found to be identical to a previously
published sequence (24) with the exception of a one-base pair
substitution (G for T) at position
800. This substitution introduces
an AluI site.
To define DNA regions upstream to the GIP start site responsible for basal transcriptional activity (24), a series of restriction fragments of varying sizes were placed upstream to the promoterless luciferase gene of the reporter plasmid pGL2-basic. Two series of GIP-Luc chimeric plasmids were constructed. One series contained sequences upstream of position +8 with respect to the putative transcription start site (Fig. 1B), and the second series of clones contained sequences upstream of position +781 and included intron 1 (Fig. 1C).
After transient transfection of the GIP-Luc constructs into STC-1
cells, transcriptional activity was determined by measuring luciferase
activity in cell extracts. All GIP-Luc constructs produced luciferase
activities higher than the control plasmid, pGL2-basic (Fig.
3). Transfection of STC-1 cells with the plasmids
pGL-943, pGL-425, pGL-397, pGL-363, pGL-259, pGL-203, and pGL-193
resulted in activities approximately 200-fold over control (Fig.
3A). In contrast, transfection of pGL-182 induced only an
approximately 30-fold increase in luciferase activity when compared
with pGL2-basic (Fig. 3A). When the construct pGL-173 was
compared with pGL2-basic, only an 11-fold enhancement was observed. The
GIP-specific insert in the plasmid pGL-173 contains a TATA box sequence
(27 to
24), a consensus sequence of an enhancer core (
133 to
126), and two CAAT box sequences (
158 to
154 and
171 to
167)
and may represent the minimal promoter of the GIP gene. This minimal
promoter activity was almost completely abolished when sequences from
173 to
144 were deleted from pGL-173, producing the plasmid pGL-143
(Fig. 3A).
The differences in the transcriptional activities observed between
constructs pGL-182 and pGL-193 indicated the possible presence of a
specific element(s) that can enhance basal GIP gene expression is
present in the region of base pairs 193 to
182. Analysis of the DNA
sequence in this region revealed a potential GATA binding motif
starting at position
190, with the sequence AGATAA. This sequences
conforms to the consensus sequence, (A/T)GATA(A/G), for a GATA binding
motif (25). A mutational analysis was performed to determine the
importance of this site, as well as another site located 6 base pairs
downstream that shares five of the six bases with the consensus
sequence for a GATA motif having the sequence AGATAC. The sequences of
the wild type (WT) and four mutants (Mut1-4) in the region
of interest are shown in Fig. 4B. An A to G
substitution at position
186 (Mut1) yielded a 40% reduction in
promoter activity (Fig. 4C). The enhancer effect was
abolished by mutating two sites within the upstream GATA motif, a G to
A and a T to A substitution at positions
189 and
186 (Mut2),
respectively (Fig. 4C). In contrast, a similar mutation in
the downstream GATA motif (Mut3) produced only a 35% reduction in
promoter activity from the wild type sequence (Fig. 4C). A
construct with two mutated GATA motifs (Mut4) behaved the same as the
construct with only the upstream motif (Mut2) altered (Fig.
4C), suggesting that the upstream site is more important in
determining promoter activity.
As described previously, consensus sequences for cis-acting
elements, including a TATA box and a CAAT box, are present within the
first intron of the rat GIP gene (24). To examine the properties of
these sequences, a second series of GIP-Luc chimeric constructs containing intron 1 sequences (Fig. 1C) was transfected into
STC-1 cells. Transfection of STC-1 cells with the plasmid pGL+111i, which does not contain sequences upstream of the putative
transcriptional start site, did increase luciferase activity over
control (Fig. 3B), suggesting that intron one sequences may
possess promoter activity. The transcriptional activity of pGL+111i is
equivalent to the activity of pGL-182i that, in addition to the intron
one sequences, includes exon 1 and upstream sequences that are
responsible for the low level of promoter activity observed with
pGL-182. Similar to the first series of GIP-Luc constructs, enhancement of transcriptional activity could be seen when the sequences from 190
to
182 were included (Fig. 3B). Interestingly,
transcription rates did not differ significantly between GIP-Luc
fusions that contained intron 1 from those GIP-Luc fusions that lacked
these sequences (Figs. 3, A and B), suggesting
that the intron 1 sequences do not contribute to the activity of the
upstream promoter. To determine whether the putative enhancer located
between
190 and
182 might influence transcription initiated within
intron 1, the plasmids pGL-
363i and pGL-
425i were constructed by
deletion of sequences between
177 and +111 from the plasmids pGL-363i and pGL-425i, respectively. When pGL-
363i and pGL-
425i were transfected into STC-1 cells, no further increase in luciferase activity, over that for pGL+111, was detected (Fig. 3B).
To determine whether changes in transcriptional activity represent
cell-specific expression, luciferase activity was measured in a number
of additional cell lines transfected with GIP promoter constructs (Fig.
5). All cell lines examined demonstrated a high level of
luciferase activity following transfection with pRSVLUC (data not
shown). As demonstrated in Fig. 5, only minimal GIP promoter activity
was detected in these cell lines. Moreover, the enhancer activity
observed in STC-1 cells was greatly diminished in all the other cell
lines (Fig. 5). Interestingly, GIP promoter activity was not enhanced
at all in the human embryonic intestinal 407 cell line, which expressed
detectable levels of immunoreactive GIP. These results are consistent
with Northern hybridization analysis that demonstrated GIP-specific
transcripts only in STC-1 cells (Fig. 2). In addition, with the
exception of the NIH 3T3 cells, minimal promoter activity elicited from
constructs containing intron 1 sequences was similar to activity
resulting from constructs containing sequences +8 to 182 to (Fig. 5).
In the NIH 3T3 cell line, reduced promoter activity was detected in
cells transfected with constructs containing intron 1 sequences
only.
Because of their presumed importance in the pathogenesis of
noninsulin-dependent diabetes mellitus (NIDDM), renewed
interest in incretins, such as GIP and its closely related peptide
glucagon-like peptide 1 (GLP-1), has been evident in recent years.
Elevated circulating levels of GIP have been found in some patients
with NIDDM (26, 27), and recent clinical trials have demonstrated the
therapeutic potential of GLP-1 in the treatment of some forms of NIDDM
(28). Both GIP and GLP-1 are synthesized in the small intestine and
augment insulin secretion after binding to specific G-coupled protein
receptors present on pancreatic -cell membranes (29).
Although substantial information has been accrued concerning
tissue-specific expression of GLP-1, relatively little information is
available regarding the biosynthesis of GIP. The gene encoding GLP-1 is
transcribed in pancreatic -cells, in L cells of the small intestine,
and in the brain (30, 31). The tissue-specific expression of GLP-1 is
regulated at both the transcriptional and posttranslational levels.
GLP-1 results from limited proteolysis of proglucagon, a process
restricted to the L cells of the intestine and possibly the brain (32).
Like GLP-1, the GIP gene is expressed in more than one tissue. We have
previously demonstrated GIP immunoreactivity in K cells of the small
intestine and in ductal cells of the rat submandibular salivary gland
(17, 33). GIP-(1-42) is the main functional proteolytic product of
proGIP, the primary product of the GIP gene in the upper small
intestine. Recently, GIP-(7-42) was purified from the upper part of
the porcine intestine and was shown to have antibacterial activity
(34). It is unknown whether the immunoreactivity detected in the
salivary gland represents either GIP or GIP-(7-42) or whether it is a
closely related peptide derived by alternate posttranslational
processing of proGIP.
In the present study, we have used a combination of Northern hybridization analysis and cell-mediated transcription assays to identify an established cell line that can be used to examine tissue-specific regulation of GIP. The identification of a surrogate cell line was necessary to enable investigation of the regulation of GIP gene expression because of difficulties encountered when trying to isolate and culture intestinal K cells. Canine K cells, which represent less than 0.1% of duodenal mucosal cells, have been enriched as much as 100-fold; however, the viability of these cells in culture has been limited to less than 2 days (35).
Only one cell line we tested, STC-1, expressed detectable levels of GIP-specific transcripts. The mRNA had an electrophoretic mobility identical to GIP mRNA previously identified in rat duodenal mucosa (17). STC-1 cells were derived from an intestinal endocrine tumor isolated from a transgenic mouse carrying two oncogenes, SV40 large T antigen and polyoma small T antigen, both of which were linked to the rat insulin promoter. Like the tumors that developed in these same transgenic mice, STC-1 cells are plurihormonal and, in addition to GIP, express GLP-I, GLP-II, glicentin, secretin, somatostatin, gastrin-CCK, neurotensin, and pancreatic polypeptide. Rindi et al. (20) suggested that the plurihormonal nature of these tumors arises from the ability of their proliferating cells to switch to multiple differentiated states, a quality that has apparently prevailed in the establishment of the STC-1 cell line (20). Therefore, like many tumor-derived cell lines, the STC-1 cell line must be viewed as a mixture of different cell types (36, 37), and interactions among the various cell types must be taken into consideration when studying the regulation of hormone expression. Interestingly, while attempting to clone a pure GIP-expressing cell from parental STC-1 cells, Kieffer et al. (21) found that only 30% of the expanded clone still expressed GIP immunoreactivity, suggesting that dedifferentiation of STC-1 cells was possible.
Despite the heterogeneous nature of the STC-1 cells, they have been
used as a convenient model to study both gastrointestinal peptide
release and transcriptional regulation (21, 38, 39). We have used
variable length restriction fragments, representing the 5-flanking
region of the rat GIP gene, fused to the luciferase reporter gene in
transient transfection assays to investigate GIP gene transcription in
STC-1 cells. Two separate regions of the GIP gene were able to promote
gene transcription in STC-1 cells. The major promoter located upstream
had been previously identified by primer extension analysis and RNase
protection analysis of GIP mRNA expressed in rat duodenal tissue
(24). The first 173 base pairs of this promoter encode minimal promoter
activity that is lost if base pairs
144 to
173 are deleted.
Interestingly, two putative CCAAT boxes are located in this sequence.
In addition, the sequence from
154 to
161, TCACCCAT, is similar to
the sequence, TCACCAA, that is located from
152 to
159 in the human
GIP promoter and that confers cAMP responsiveness in a hamster
insulinoma cell line (40).
In the present study, the addition of base pairs 183 to
193
resulted in a significant increase in promoter activity, suggesting the
presence of an enhancer sequence in this region. The further addition
of sequences 5
to position
193 up to
963 did not significantly change the level of promoter activity. We have also found similar rates
of transcription with a sequence that extends to approximately position
2500 of the rat GIP gene. This observation does not preclude the
presence of additional tissue-specific enhancers as well as repressors
upstream of position
193. Earlier studies of the proglucagon promoter
demonstrated that sequences that were capable of promoting gene
expression in STC-1 cells could not promote intestine-specific gene
expression in a transgenic mouse model. A 5
-flanking segment extending
only to position
238 promoted enhanced transcriptional activity in
STC-1 cells, while a segment extending past position
1400 was
required for intestine-specific promoter activity in the transgenic
model (39).
The sequence between base pairs 173 and
193 contains two consensus
GATA binding motifs. In the present study, a mutational analysis of
this region demonstrated the upstream motif to be more important than
the downstream sequence in enhancing expression of the GIP gene.
Disrupting this motif resulted in a loss of 90% of the activity.
Interestingly, of the two GATA motifs, only the sequence of the
upstream motif is conserved between rats and humans (41). DNA
regulatory elements conforming to the sequence (A/T)GATA(A/G) were
first identified to play a role in the transcriptional regulation of
erythroid genes (42). Since then, GATA binding motifs have been
implicated in the tissue-specific expression of numerous other genes.
(43-45). Intestinal K cells that express GIP are thought to be derived
from the endoderm during development like other neuroendocrine tissue.
Since GATA-binding proteins, including GATA4/5 and 6 (25, 46, 47), have
been found in a number of different endodermally derived tissues, it is
possible that the upstream GATA motif in the GIP gene could represent a
target for one of these transcription factors.
The second GIP promoter, located within the first intron, conferred
only minimal promoter activity in STC-1 cells. This more proximal
promoter does contain a TATAA box, as well as a putative CCAAT box, but
does not include an enhancer core consensus sequence (24). The rate of
transcription of this promoter was not affected by putative enhancers
located between base pairs 173 and
193. This was demonstrated by
the removal of sequences required for the initiation of transcription
of the primary (or distal) promoter (
143 to +8), while retaining
sequences in the proximal promoter (+111 to +781) that conferred
minimal gene transcription. The resulting GIP-Luc chimera produced the
same transcriptional activity as the GIP-Luc construct that included
only intron 1 sequences. Primer extension analysis and RNase protection
analysis have indicated that GIP transcription initiates from the
distal promoter in the duodenum of adult rats (24). Little is presently
known about either the regulation of GIP gene expression during the
ontogeny of rats or GIP expression in the salivary gland, and it is
conceivable that coordinate regulation of the two GIP promoters could
be involved in either of these two processes.
In conclusion, the results of this present study indicate that STC-1
cells represent a suitable model for the examination of cell-specific
expression of the GIP gene. Future studies using these cells will
enable investigators to identify the transcription factors that
interact with and regulate the GIP gene. More advanced approaches,
possibly involving transgenic mouse models, may be necessary to
investigate the contributions of the sequences upstream of position
193, as well as the proximal promoter located within intron 1.