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Laboratory of Molecular Medicine, Department of Internal Medicine, University of Rome Tor Vergata (P.B., O.P., D.P., S.C., R.M., M.F., G.S., D.L., L.N.J.L.M.), 00133 Rome, Italy; the Institute of Bioimaging and Pathophysiology of the Central Nervous System, National Research Council (V.D., S.C.), Catania 95100, Italy; and the Institute of Experimental Medicine, National Research Council (L.N.J.L.M.), Rome 00133, Italy
Address all correspondence and requests for reprints to: Patrizia Borboni, M.D., Department of Internal Medicine, University of Rome Tor Vergata, Via di Tor Vergata 135, 00133 Rome, Italy. E-mail: borboni{at}uniroma2.it
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Introduction |
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In the rat pancreas, PACAP-like immunoreactivity has been localized in the nerve fibers, around blood vessels, and in the central portion of islets (5). Previous studies demonstrated that PACAP is a potent stimulator of glucose-induced insulin release (6). PACAP exerts its biological action by binding to specific membrane receptors, which can be divided into at least three subtypes: PAC1 (7, 8, 9, 10, 11), is PACAP selective, whereas VPAC1 (12, 13, 14, 15) and VPAC2 (16, 17, 18, 19, 20) exhibit similar affinitiesfor PACAP and VIP. Several PAC1 variants, generated by an alternative splicing mechanism, have been identified. The splicing of a 21-amino acid domain in the N-terminal extracellular region modulates the binding and relative potencies of PACAP-27 and PACAP-38 (10), whereas the presence or absence of either one or two cassettes, designated hip and hop, in the third intracellular loop regulates the coupling to adenylate cyclase (AC) and phospholipase C (PLC) (11). VPAC1 are coupled to AC (12, 13, 15, 21), whereas VPAC2 can additionally activate PLC (16, 17, 18, 19, 20). PACAP/VIP receptors with distinct transducing systems may subserve different functional assignments. Previous studies demonstrated the presence of PAC1 and VPAC2 in HIT-T15 and RINm5F insulinoma cells (17, 22, 23).
In the present study we determined the molecular and functional characterization of PACAP receptors isoforms in a rat insulinoma cell line (RIN 1046–38 cells) and analyzed the signal transduction pathways activated by PACAP-38; furthermore we investigated whether the insulinotropic action of PACAP-38 involves modification of the expression of components of the ß-cell glucose-sensing system.
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Materials and Methods |
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Pancreatic islets were isolated from neonatal rats (5 days old) as previously described (25). Briefly, pancreas were excised, digested with collagenase type V (Sigma, St. Louis, MO), and seeded in 35-mm petri dishes in RPMI 1640 medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% FCS, 2 mM glutamine, 50 IU penicillin, and 50 µg/ml streptomycin at 37 C in an atmosphere of 95% humidified air-5% CO2. After 6 h, most of fibroblasts, present as contaminating cells in the cultures, were attached to the bottom of the dishes; the supernatant, containing islets and exocrine tissue, was replated in a new dishes. After 24 h, the supernatant containing undigested material and debris was discarded, and fresh medium was added for an additional 24 h. Thereafter, islets were processed for RNA extraction or insulin release experiments.
Analysis of PACAP/VIP-receptor messenger RNAs (mRNAs) by RT-PCR
Primers used to identify PAC1 receptor mRNAs by
RT-PCR were based on the reported sequence for rat PAC1
hip-hop1 isoform (11) (GenBank accession no. Z23272). Oligonucleotides
were synthesized by Pharmacia Biotech (Milan, Italy).
Sequences and location of the primers with respect to the initiation of
translation were as follows: A1, 5'-CATCCTTGTACAGAAGCTGC-3' (forward
primer, matching the beginning of the third intracellular loop and
corresponding to bases 1074–1093); A2, 5'-ACAAATTTAAGACTGAGAGT-3'
(forward primer, matching the beginning of the hip cassette and
corresponding to bases 1135–1154); A3, 5'-TCCACCATTACTCTACGGCT-3'
(forward primer, matching the end of the hop cassette and corresponding
to bases 1288–1307); and A4, 5'-GGTGCTTGAAGTCCATAGTG-3' (reverse
primer, matching a region of the cytoplasmic tail and corresponding to
bases 1527–1546). The following are the sizes of the expected PCR
products using primer pair A1/A4: 305 bp for PAC1, 386 bp
for PAC1-hop2, 389 bp for PAC1-hip and
PAC1-hop1, and 473 bp for PAC1-hip-hop1. The
predicted PCR products using primer pair A2/A4 are 328 bp for
PAC1-hip and 412 for PAC1-hip-hop1, whereas
those obtained with primer pair A3/A4 are 256 bp for
PAC1-hop2 and 259 bp for PAC1-hop1 and
PAC1-hip-hop1. To investigate the existence of the 21-amino
acid domain in the N-terminal extracellular region of PAC1
we used two primers external to the exonic cassette encoding for this
domain. Oligonucleotide sequences and location of the primers with
respect to the initiation of translation were as follows: A5,
5'-CTGCATCTTCAAGAAGGAGC-3' (forward primer corresponding to bases
159–178); and A6, 5'-CACAAGCATCGAAGTAGTGG-3' (reverse primer
corresponding to bases 471–490). The sizes of the expected PCR
products are 332 and 269 bp in the presence or absence of the cassette,
respectively. Primers used to identify VPAC1 mRNA by RT-PCR
were based on the reported sequence (13) (GenBank accession no.
M86835). Oligonucleotide sequences and locations of the primers with
respect to the initiation of translation were as follows: B1,
5'-CACGAGTGTGAGTACCTGCA-3' (forward primer, corresponding to bases
161–180); and B2, 5'-CGGTCTTCACGGTATTGTAG-3' (reverse primer,
corresponding to bases 475–494). Primers used to identify
VPAC2 mRNA by RT-PCR were based on the reported sequence
(18) (GenBank accession no. Z25885). Oligonucleotide sequences and
locations of the primers with respect to the initiation of translation
were as follows: C1, 5'-GTGCTGGTCAAGGACAGTGT-3' (forward primer,
corresponding to bases 591–610); and C2, 5'-AGGCGAGTTGCTATCCATG-3'
(reverse primer, corresponding to bases 838–856). The following are
the sizes of the expected PCR products: 334 bp for VPAC1
(primer pair B1/B2) and 584 bp for VPAC2 (primer pair
C1/C2). To control for the integrity of RNA and for differences
attributable to errors in experimental manipulation from tube to tube,
primers for rat phosphoglycerate kinase 1 (26) were included in each
PCR reaction and generated a 183-PCR product. Oligonucleotide sequences
and locations of the primers were as follows:
5'-AGGTGCTCAACAACATGGAG-3' (forward primer, residues 777–796) and
5'-TACCAGAGGCCACAGTAGCT-3' (reverse primer, residues 940–959). Total
RNA from RIN 1046–38 or pancreatic islets was treated with
ribonuclease-free deoxyribonuclease (Roche Molecular Biochemicals, Milan, Italy) to remove any residual genomic DNA.
Single stranded complementary DNAs (cDNAs) were synthesized using
total RNA (1 µg), Moloney murine leukemia virus reverse transcriptase
(200 U; Roche Molecular Biochemicals, Milan, Italy), and
oligo-(deoxythymidine)18 primer (100 nM) in
presence of deoxy-NTPs (1 mM) and ribonuclease inhibitor
(40 U; Roche Molecular Biochemicals) in a final volume of
20 µl. The reaction was terminated by incubation at 70 C for 10 min,
and cDNA samples were diluted by adding 80 µl sterile
H2O. For each amplification, 40 µl of a PCR master mix
were added to 10 µl diluted cDNA sample to yield the following final
concentrations: 1 µM specific primers, 200
µM deoxy-NTPs, 1.25 U Taq DNA polymerase
(Roche Molecular Biochemicals), and Taq buffer
containing 1.5 mM MgCl2. PCR was performed on a
Perkin-Elmer Corp./Cetus thermal cycler (35 cycles of 95 C
for 30 sec, 57 C for 30 sec, and 72 C for 45 sec). At the end of PCR,
samples were kept at 72 C for 10 min for final extension and stored at
4 C. Amplification products arising from RT-PCR were separated by
electrophoresis (2.0% agarose gel in 0.045 M Tris-borate
and 1 mM EDTA buffer) and visualized by ethidium bromide
staining. In a preliminary series of experiments, the use of RT-PCR for
the identification of PACAP/VIP receptors was validated on the bases of
specificity of the primers, and sizes and sequences of the
amplification products.
Membrane preparation and displacement studies
RIN 1046–38 cells membranes were prepared following the
technique previously described by Andersson et al. (22) with
minor modifications. Cells were grown to confluence, washed in 50
mM Tris (pH 7.4), and scraped off with a rubber policeman
in a buffer containing 50 mM Tris, 1.5 mM
MgCl2, and 150 µg/ml bacitracin, pH 7.4. After
centrifugation (1200 x g at 4 C for 15 min), the
pellet was resuspended in Tris buffer and homogenized with a Polytron
homogenizer (Brinkmann Instruments, Inc., Westbury, NY) at
4 C. The homogenate was than centrifuged at 100,000 x
g at 4 C for 30 min, the pellet containing the membranes was
resuspended in Tris buffer, the protein content was evaluated by the
Bradford method, and aliquots were stored at -80 C until use. For
displacement studies RIN 1046–38 membranes were resuspended in a
buffer containing 50 mM Tris, 1.5 mM
MgCl2, 300 µg/ml bacitracin, and 1% BSA at a protein
concentration of 600 µg/ml. Membranes were incubated at 15 C in a
final volume of 250 µl with [125I]PACAP-27 (50
pM) in the absence or presence of increasing concentrations
of native PACAP-38 (ranging from 0.1 nM to 1
µM). After 1 h, aliquots of the incubation mix were
transferred to microtubes and centrifuged at 10,000 x
g in a microfuge, the pellets were washed twice, and the
microtube tips containing the pellets with the bound radioactivity were
cut and counted in a 
-counter. Nonspecific binding, considered to be
binding in the presence of 1 µM native
PACAP-38, was less than 15% of total binding and was subtracted from
each point to obtain the specific binding. Experiments were performed
in triplicate.
Cellular cAMP content
Cells were seeded in 24-well plates and washed twice with
glucose-free HEPES buffer containing 114 mM NaCl, 25.5
mM NaHCO3, 10 mM HEPES, 2.5
mM CaCl2, 4.7 mM KCl, 1.21
mM KH2PO4, 1.16 mM
MgSO4, and 0.1% BSA (pH 7.2). Thereafter, cells were
incubated for 10 min in a final volume of 250 µl with PACAP-38 (0.1
pM) in the absence or presence of 2.8 mM
glucose. The incubation was stopped by adding ice-cold ethanol to a
final concentration of 65%; thereafter, cells were scraped off with a
rubber policeman and centrifuged at 1200 x g for 15
min. Aliquots of the supernatants were dried with a Speed-Vac (Savant
Instruments, Hicksville, NY) and stored at -20 C until analysis of
protein content (27) and cAMP RIA.
Measurement of inositol phospholipid (IP) hydrolysis
The rate of IP hydrolysis was estimated by measuring the
accumulation of [3H]IP in the presence of
Li+, which blocks the conversion of IP to free
inositol. Cells were seeded in six-well plates in medium
containing 20 nM myo-[2-3H]inositol (SA, 20
Ci/mmol) to label membrane inositol phospholipids. Cells were washed
twice at 37 C with Krebs Henseleit buffer containing 119 mM
NaCl, 4.74 mM KCl, 2.54 mM CaCl2,
1.19 mM MgSO4, 1.19 mM
KH2PO4, 25 mM NaHCO3,
10 mM HEPES, and 0.1% BSA, pH 7.4, with 10 mM
glucose and 10 mM LiCl and incubated in the same buffer for
1 h in the presence of PACAP-38 (0.1 pM). The
supernatant was removed, the reaction was stopped by adding 1.5 ml
ice-cold methanol-H2O (1:1, vol/vol), and the cells were
scraped off with a rubber policeman. After the addition of 0.75 ml
chloroform, the cell suspension was centrifuged at 500 x
g for 2 min. Aliquots (800 ml) of the aqueous phase were
applied to columns containing 1 ml Dowex 1x8 resin (100–200 mesh,
formate form, Bio-Rad Laboratories, Inc., Hercules, CA),
and the phosphate esters were eluted by stepwise addition of formate
solution of increasing strength. Inositol monophosphate was eluted with
0.2 M ammonium formate and 0.1 M formic acid.
Fractions were collected, and the radioactivity was measured by liquid
scintillation counting. Protein was measured by the Bradford method
(27).
Insulin release experiments and intracellular insulin content
evaluation
For short term experiments, cells were seeded in 24-well plates;
thereafter, medium was removed, and cells were washed twice at 37 C for
30 min each time with glucose-free HEPES buffer. Thereafter, cells were
incubated for 1 h in the same buffer with PACAP-38 (0.01, 0.1, and
1 pM) in the absence or presence of glucose (1 and 2.8
mM). At the end of the incubation period, aliquots of the
supernatant were collected and stored at -20 C for subsequent insulin
RIA; cells were extracted overnight at 4 C with a solution of acidified
ethanol for intracellular insulin content assay. For long term
experiments, PACAP-38 was added to the culture medium for 12 h
(renewed after 6 h) in the absence or presence of glucose (6, 12,
or 18 mM); 6 mM glucose was the lowest glucose
concentration allowing the cells to remain attached during the entire
incubation period. Aliquots of the medium were collected after 3, 6,
and 12 h for subsequent insulin RIA. Cells were extracted
overnight at 4 C with a solution of acidified ethanol for intracellular
insulin content assay.
Insulin and cAMP RIA
Insulin was determined using a dextran-charcoal method as
previously described (28), with an antiinsulin antibody raised in
guinea pig, porcine insulin standard (Sigma), and
[125I]insulin from New England Nuclear (Boston, MA).
For cAMP determination, sample were previously acetylated and successively processed for RIA using a commercial kit (Amersham Pharmacia Biotech, Arlington Heights, IL).
Analysis of insulin mRNA expression by Northern blot
Insulin mRNA expression was analyzed after 12 h of
treatment with 0.1 pM PACAP-38 in the absence or presence
of 6, 12, or 18 mM glucose. Total RNA (20 µg for each
condition) was denatured at 65 C and applied to a 1% agarose gel
containing 5% (vol/vol) formaldehyde. RNAs were then blotted onto
nylon membrane (Schleicher & Schuell, Inc., Keene, NH)
using a Posiblot apparatus (Stratagene, La Jolla, CA) and
cross-linked. The blots were hybridized with a 450-kb mouse insulin
cDNA (provided by Dr. A. L. Notkins, NIH, NIDR, Bethesda, MD)
labeled with [
-32P]deoxy-CTP (Amersham Pharmacia Biotech, Arlington Heights, IL) using a random priming procedure
with 2 U Klenow polymerase (Boehringer Mannheim, Milan, Italy). The
hybridization were carried out overnight at 42 C in 50% formamide,
6 x SSC, 0.5% SDS, and 100 µg/ml single stranded DNA. The
membrane were then washed with 1 x SSC (standard saline citrate)
and 0.5% SDS at 60 C. Blots were subsequently exposed overnight to
Kodak films (Eastman Kodak Co., Rochester,
NY) at -80 C with intensifying screens. Densitometric analysis was
performed using an image analyzer (Fluor-S, Bio-Rad Laboratories, Inc.).
Analysis of hexokinase (HK) and GLUT1 mRNA by competitive
RT-PCR
HK and GLUT1 mRNA expressions were analyzed after 12 h of
treatment with 0.1 pM PACAP-38 in the absence or presence
of 6, 12, or 18 mM glucose.
Competitive RT-PCR assay was performed by coamplification of increasing
amounts of HK or GLUT1 complementary RNA internal standard (i.s.) with
a constant amount of wild-type (w.t.) RNA isolated from RIN 1046–38
cells, as previously described, with minor modifications regarding w.t.
synthesis (29). Briefly, a 100-bp fragment was introduced into the HK
i.s. sequence or the GLUT1 i.s. sequence by PCR-based, site-directed
mutagenesis, allowing us to distinguish the amplification products
arising from the i.s. RNA from those arising from the w.t. RNA. Known
amounts of HK or GLUT1 i.s. complementary RNA were incubated in the
presence of a constant amount of w.t. RNA (0.5 µg) isolated from RIN
1046–38 cells. The i.s./w.t. RNA mixtures were reverse transcribed and
amplified for 31 cycles (94 C/45 sec, 62 C/45 sec, and 72 C/1 min, with
a final extension at 72 C/5 min) by 2.5 U Taq DNA polymerase
(Promega Corp., Madison, WI) in a mixture containing 2.1
mM MgCl2 and 100 pmol upstream and downstream
amplification primers in the presence of 0.5 x 106
cpm [-32P]ATP-labeled upstream primer (Amersham Pharmacia Biotech, Arlington Heights, IL). The upstream primer
was labeled with 10 U T4 kinase (Roche Molecular Biochemicals) and purified on Stratagene
purification columns (Stratagene).
Analysis of the PCR-amplified products
PCR products were analyzed on 1% agarose-0.5 x
Tris-borate EDTA gel, showing a 574-bp band for HK w.t. or a
674-bp band for HK i.s. and a 475-bp band for GLUT1 w.t. or a 575-bp
for GLUT1 i.s. To quantitate the amount of product arising from the
amplified i.s. or w.t. RNA, the corresponding ethidium bromide-stained
bands were excised from the gel, and the amount of incorporated
radioactivity was determined in a ß-counter (LKB,
Rockville, MD). Gel slices corresponding to lanes containing a control
sample (water) were also excised at the sizes of i.s. and w.t. bands to
determine the amount of background radioactivity. Data were plotted as
the ratio of counts per min incorporated into the w.t. amplification
product and the counts per min incorporated into the i.s. amplification
product as a function of the known amount of i.s. The plot was analyzed
by logarithmic regression, and the extrapolated point of equivalence
corresponds to the amount of HK or GLUT1 mRNA present in the unknown
sample.
Statistical analysis
Data are presented as the average ± SEM.
Statistical analysis was carried out by one-way ANOVA followed by
Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and in rat pancreatic islets (Fig. 1B), we analyzed their mRNAs by the use of four primer pairs.
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, A and B, lane 1). Primer pair A2/A4
produced no amplification product (Fig. 1, A and B, lane 2), whereas
primer pair A3/A4 produced an amplification product of about 250 bp
(Fig. 1, A and B, lane 3), indicating the existence of
PAC1-R and -hop receptor variants. In addition, to
investigate whether these variants contained the exon encoding the
21-amino acid domain in the N-terminal extracellular region, we used
primers A5/A6, which are external to the site of insertion of this
cassette. Amplification with these primers produced a single
amplification product of about 330 bp (Fig. 1, A and B, lane 4), which
indicates the presence of the 21-amino acid domain in all of the
PAC1 receptors expressed in both rat insulinoma cells and
pancreatic islets.
To determine the mRNA expression of VPAC1 and
VPAC2 we used primer pairs B1/B2 and C1/C2, respectively;
amplification products obtained with these primers were indicative of
the VPAC1 and VPAC2 mRNAs (Fig. 1, A and B,
lanes 5 and 6).
Displacement studies
PACAP-38 displaced [125I]PACAP-27 (50
pM) in RIN 1046–38 cells in a concentration-dependent
manner within the range of 0.1 nM to 1 µM.
IC50 was obtained with 0.2 nM PACAP-38 (Fig. 2).
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Cellular cAMP content
PACAP-38 induced a rapid increase in intracellular cAMP, as
demonstrated in Table 1, in both the
absence and presence of glucose. The basal intracellular cAMP contents
were 198 ± 40 and 350 ± 72 fmol/µg protein, respectively,
in the absence and presence of 2.8 mM glucose. When 0.1
pM PACAP was added for 10 min, the intracellular cAMP
contents were 602 ± 93 and 1106± 181 fmol/µg protein,
respectively, in the absence and presence of 2.8 mM
glucose.
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Insulin release experiments and intracellular insulin content
evaluation
Insulin release from RIN 1046 38 cells was 2.16 ± 0.36% of
the total insulin content in the absence of glucose; increasing glucose
concentrations (1 and 2.8 mM) stimulated insulin release
(4.59 ± 0.52% and 4.30 ± 0.72% of the total insulin
content, respectively). Short term treatment (1 h) with PACAP-38
increased basal insulin release in a dose-dependent manner (2.94
± 0.57%, 4.00 ± 0.16%, and 3.38 ± 0.49% of the total
insulin content, respectively, at 0.1, 1, and 10 pM);
insulin release induced by glucose was also increased by PACAP-38 in a
dose-dependent manner (5.28 ± 0.79%, 7.09 ± 0.54%, and
6.46 ± 0.54%, respectively, with 0.01, 0.1, and 1 pM
PACAP-38 in the presence of 1 mM glucose; 4.83 ±
0.69%, 7.41 ± 0.49%, and 6.59 ± 0.25%, respectively,
with 0.01, 0.1, and 1 pM PACAP-38 in the presence of 2.8
mM glucose; Fig. 3).
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). Insulin accumulation in the medium
was not influenced by higher glucose concentrations (12 and 18
mM) in either the absence or presence of PACAP-38 (data not
shown).
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shows that intracellular insulin
content was significantly increased by glucose (6, 12, and 18
mM); the addition of 0.1 pM PACAP-38
significantly potentiated the effect of glucose.
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, upper panel). Densitometric analysis indicated that
PACAP-38 increased insulin mRNA expression by 19.31 ± 5.70%,
35.39 ± 4.85%, and 61.35 ± 4.94%, respectively, in the
presence of 6, 12, or 18 mM glucose (Fig. 5, lower panel).
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shows that glucose
increased the expression of GLUT1 mRNA in a dose-dependent manner; the
basal GLUT1 mRNA level in the presence of 6 mM glucose were
7.61 ± 0.08 pg i.s./µg total RNA, whereas in the presence of 12
or 18 mM glucose they were, respectively, 14.16 ±
1.26 and 21.29 ± 1.06 pg i.s./µg total RNA. The addition of 0.1
pM PACAP-38 to 6 or 12 mM glucose produced an
increase in GLUT1 mRNA expression compared with that using glucose
alone (14.26 ± 1.21 and 17.61 ± 0.83 pg i.s./µg total
RNA, respectively); on the contrary, in the presence of 18
mM glucose, PACAP-38 slightly decreased GLUT1 mRNA
expression induced by glucose (15.94 ± 0.76 pg i.s./µg total
RNA).
Figure 6B shows that glucose increased the expression of HK1 mRNA in a
dose-dependent manner. The value of HK mRNA in the presence of 6
mM glucose was 28.82 ± 0.98 pg i.s./µg total RNA,
whereas in the presence of 12 or 18 mM glucose they were,
respectively, 36.43 ± 1.55 and 51.19 ± 1.73 pg i.s./µg
total RNA. The addition of 0.1 pM PACAP-38 produced an
increase in HK1 mRNA expression compared with that using glucose alone,
which was more pronounced in the presence of 6 and 12 mM
glucose but very weak in the presence of 18 mM glucose
(47.69 ± 0.65, 53.91 ± 0.90, and 59.36 ± 1.05 pg
i.s./µg total RNA, respectively).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Glucose-induced insulin release is proportional to the rate of glucose metabolism, which, therefore, represents a rate-limiting step. The range of glucose concentrations to which ß-cells respond is regulated by the plasma membrane glucose transporters and the glucose-phosphorylating enzyme HKs (31, 32), representing the two major components of the glucose-sensing system (31, 33). Normal, fully differentiated, mature ß-cells express mainly GLUT2 and glucokinase (HK4), whereas insulinoma cell lines express preferentially GLUT1 and HK1 (33, 34). Glucose transporter isoforms and hexokinase isoforms are coexpressed in a specific manner, based on the reciprocal biochemical and functional characteristics of each isoform; for example, the high Km GLUT2 is coexpressed with the high Km glucokinase, whereas the low Km GLUT1 is coexpressed with the low Km HK1, and GLUT4 is coexpressed with HK2 (35). In RIN 1046–38 cells, GLUT1 and HK1 are quantitatively more expressed than GLUT2 and glucokinase, as this cell line looses the high Km components with time in culture; therefore, RIN 1046–38 cells metabolize glucose using preferentially the low Km components GLUT1 and HK1 and release insulin at subphysiological glucose concentrations (36).
In the present study we demonstrate that PACAP-38 regulates the expression of GLUT1 and HK1 in RIN 1046–38 cells. GLUT1 and HK1 mRNA expressions are increased by glucose in a dose-dependent manner, and the addition of PACAP-38 at very low concentration significantly enhances this effect. The effect on GLUT1 mRNA may be due to an increased transcription by cAMP. In fact, a cAMP-responsive element (CRE) has been described in the GLUT1 gene (37) and cAMP is known to increase the GLUT1 transcription rate (38, 39). HK1 mRNA can be regulated by cAMP as previously demonstrated in other cell lines (40). Therefore, it is possible to hypothesize that PACAP-38 regulates GLUT1 and HK1 mRNA expression via the same cAMP-mediated mechanism. Likewise, we have observed that insulin mRNA expression is increased by glucose in a dose-dependent manner and that PACAP-38 potentiates this effect. Insulin gene transcription is increased by cAMP as previously demonstrated in ßTC-1 cells (41), suggesting that the same mechanism can be activated by PACAP-38 in RIN 1046–38. Actually, mutational analysis showed that forskolin increases the activity of the proinsulin promotor through a specific CRE present in this gene (42). Furthermore, there is evidence that at least two different nuclear proteins that bind to CRE and the recently cloned CRE-binding protein (CREB) 327/341 are expressed in ß-cells (43, 44). Therefore, it is possible to suggest that PACAP-38, after binding to its receptor, activates the cAMP-dependent PKA pathway, leading to phosphorylation of CREB, which, in turn, acts as a third messenger, triggering gene transcription.
It has been demonstrated that transfection of RINr cells with GLUT1 cDNA induces higher insulin mRNA level and intracellular insulin content (45); moreover, islets from mice overexpressing the high affinity yeast HK show increased glucose utilization, insulin synthesis, and insulin secretory capacity (46). Therefore, it is possible to hypothesize that up-regulation of the glucose-sensing components induced by PACAP-38 may secondarily increase the expression of insulin mRNA and insulin secretion.
In conclusion, our results indicate that RIN 1046–38 cells express a specific pattern of PACAP/VIP receptors, and that PACAP-38 binds to these receptors and preferentially activates the cAMP-dependent PKA pathway, triggering insulin secretion through short and long term mechanisms of action. It has been demonstrated that the short term effects of PACAP on insulin secretion are mediated by increased cytoplasmic Ca2+ concentration by both voltage-dependent and -independent mechanisms, the latter involving elevation of intracellular cAMP, which, in turn, may stimulate Ca2+ release from intracellular stores (47). We suggest that the long term effects of PACAP on insulin secretion are mediated by transcriptional mechanisms involving the cAMP-CRE pathway, leading to transcription of insulin gene and genes of the glucose-sensing system, which, in turn, can further increase insulin biosynthesis and secretion.
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Footnotes |
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Received May 4, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Y. Li, T. Hansotia, B. Yusta, F. Ris, P. A. Halban, and D. J. Drucker Glucagon-like Peptide-1 Receptor Signaling Modulates beta Cell Apoptosis J. Biol. Chem., January 3, 2003; 278(1): 471 - 478. [Abstract] [Full Text] [PDF]
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 M. Tsutsumi, T. H. Claus, Y. Liang, Y. Li, L. Yang, J. Zhu, F. Dela Cruz, X. Peng, H. Chen, S. L. Yung, et al. A Potent and Highly Selective VPAC2 Agonist Enhances Glucose-Induced Insulin Release and Glucose Disposal: A Potential Therapy for Type 2 Diabetes Diabetes, May 1, 2002; 51(5): 1453 - 1460. [Abstract] [Full Text] [PDF]
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F. Jamen, R. Puech, J. Bockaert, P. Brabet, and G. Bertrand Pituitary Adenylate Cyclase-Activating Polypeptide Receptors Mediating Insulin Secretion in Rodent Pancreatic Islets Are Coupled to Adenylate Cyclase But Not to PLC Endocrinology, April 1, 2002; 143(4): 1253 - 1259. [Abstract] [Full Text] [PDF]
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 S. L. Gray, K. J. Cummings, F. R. Jirik, and N. M. Sherwood Targeted Disruption of the Pituitary Adenylate Cyclase-Activating Polypeptide Gene Results in Early Postnatal Death Associated with Dysfunction of Lipid and Carbohydrate Metabolism Mol. Endocrinol., October 1, 2001; 15(10): 1739 - 1747. [Abstract] [Full Text] [PDF]
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K. Filipsson, M. Kvist-Reimer, and B. Ahren The Neuropeptide Pituitary Adenylate Cyclase-Activating Polypeptide and Islet Function Diabetes, September 1, 2001; 50(9): 1959 - 1969. [Abstract] [Full Text] [PDF]
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D. Vaudry, B. J. Gonzalez, M. Basille, L. Yon, A. Fournier, and H. Vaudry Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors: From Structure to Functions Pharmacol. Rev., June 1, 2000; 52(2): 269 - 324. [Abstract] [Full Text] [PDF]
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