From the Pacific Northwest Research Institute,
Seattle, Washington 98122 and the ¶ Departments of Medicine
and Physiology, New England Medical Center and Tufts University School
of Medicine, Boston, Massachusetts 02111
Received for publication, December 13, 2000, and in revised form, March 27, 2001
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ABSTRACT |
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Glucose regulates proinsulin biosynthesis
via stimulation of the translation of the preproinsulin mRNA in
pancreatic Pancreatic islet Glucose stimulates general protein synthesis in the Specific translational regulation of individual mRNAs can involve
elements within their untranslated regions (UTRs). For example, the
translation of the ferritin mRNA is regulated by intracellular iron
levels acting through an element in the 5'-UTR (13), known as an iron
response element, that includes a cis-acting stem-loop secondary structure (dG = Virus Construction--
Rat preproinsulin cDNA was cloned
from isolated rat islets by RT-PCR, and the sequence was verified by
DNA sequencing. A 6-histidine tag was inserted at the SmaI
site that lies within the coding region, 213 bp downstream from the
preproinsulin initiation codon, by the insertion of a hybridized
oligonucleotide pair that give the sense sequence
GCACCACCACCACCACCACGC. This histidine-tagged preproinsulin (named
HisPI) coding region was used to make four gene constructs (Fig.
2). In the first of these (5HisPI3) the His-tagged preproinsulin coding region was flanked by the UTRs of the rat II preproinsulin mRNA. In the second construct (5HisPI) the 3'-UTR of 5HisPI3 was replaced with that of SV40, while retaining the preproinsulin mRNA 5'-UTR. In contrast, in the third construct (HisPI3) the preproinsulin 3'-UTR was retained, but the 5'-UTR was
replaced with an artificial sequence
(UGAAUACAAGCUCACGACCCACUACACAAGCTACCAGATACAACAACAAGCATCCACC) that was
based upon a sequence that is predicted to have little secondary
structure and that should act as a strong, unregulated 5'-UTR
(17). Finally, both the 5'- and 3'-UTRs of the 5HisPI3 were
replaced with the artificial 5'-UTR and the SV40 3'-UTR to create
HisPI. These gene constructs were placed under the control of the
cytomegalovirus (CMV) promoter and used to construct attenuated, recombinant adenoviruses. A luciferase-expressing adenovirus was generated using the firefly luciferase coding region from the plasmid
pSP-luc+NF (Promega). These constructs were subcloned into pAC-CMV
(27), between the SacI site that lies in the proximal part
of the CMV promoter, 12 bp from the transcription initiation site, and
restriction endonuclease sites within the pAC-CMV multiple cloning
site. Use of this SacI site within the CMV promoter sequence allowed detailed positioning of the transcription initiation site to
match that used in the endogenous preproinsulin mRNA.
Mapping the 5' and 3' Termini of mRNAs--
The 5'-ends of
the virally expressed RNAs were mapped by primer extension using
poly(A)+ RNA purified from primary hepatocytes or the Islet Isolation and Culturing--
Pancreatic islets were
isolated from 200-250-g male Sprague-Dawley rats as described
previously (9). Islets were cultured overnight in RPMI 1640 with 5.6 mM glucose, 10% fetal bovine serum, 100 units/ml
penicillin, 100 µg/ml streptomycin, either in the absence or the
presence of adenovirus at ~1010 pfu/ml. Islets were then
cultured in Krebs Ringer HEPES-buffered saline with 2.8 mM
glucose for 60 min to bring biosynthesis to a basal level, and then at
the desired glucose concentration for 40 min, before being labeled with
250 µCi/ml [35S]methionine for 20 min in the same
medium. Islets were collected by centrifugation at 500 × g for 2 min and lysed.
Hepatocyte Isolation and Culturing--
Primary hepatocytes were
isolated from 200-250-g male Sprague-Dawley rats according to
previously published methods (29). Briefly, the liver was digested with
101 units/ml colagenase type 2 (Worthington Biochemical Corporation)
for ~10 min. Digestion was halted with Dulbecco's modified Eagle's
medium (Life Technologies, Inc.) supplemented with 10% fetal bovine
serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. The
hepatocytes were plated for 4 h in this medium after which
adenovirus infection was carried out by including ~1010
pfu/ml adenovirus in the overnight culture medium (Dulbecco's modified
Eagle's medium supplemented with 100 nM dexamethasome, 100 units/ml penicillin, and 100 µg/ml streptomycin). Subsequent experiments were conducted as described for islet isolation with insulin added where indicated at 100 nM. Lactate levels
were assayed using a lactate kit (Sigma) used according to the
manufacturers instructions.
Immunoprecipitation, Nickel Affinity Purification, and Protein
Gel Electrophoresis--
Cultured islets and hepatocytes were either
lysed in immunoprecipitation lysis buffer (50 mM HEPES,
0.1% Triton X-100, 1 µM phenylmethylsulfonyl fluoride, 1 µM E64, 1 µM pepstatin A, 1 µM 1-chloro-3-tosylamido-7-amino-2-heptanone, 1.0%
sodium azide, pH 8.0) for immunoprecipitation by guinea pig anti-bovine
insulin (Sigma) and rabbit anti-firefly luciferase (Cortex) antibodies as described previously (9) or were lysed in a denaturing nickel affinity lysis buffer containing 6 M guanidine
hydrochloride, as recommended by Qiagen. Samples were briefly
sonicated, and aliquots were taken for trichloroacetic acid
precipitation. The 6-histidine-tagged proinsulin was purified from the
lysate by addition of nickel-nitrilotriacetic acid-agarose (Qiagen) to
the sample and incubation at room temperature for 2 h. The
nickel-nitrilotriacetic acid-agarose was washed twice in native wash
buffer and eluted in gel loading buffer (24% glycerol, 8% SDS, 10%
RNA Analysis--
The RNA levels in islets and hepatocytes
cultured as for protein analysis were analyzed by the RNase protection
assay, using the Direct lysis kit (Ambion). RNA was protected using
[32P]uridine-labeled antisense RNA fragments
corresponding to the coding region of the histidine-tagged
preproinsulin or to part of the glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) coding region (Ambion). This yielded protected
RNA fragments of 336 bp of the His-tagged preproinsulin-expressing
mRNA, two fragments of 200 bp and 110 bp for the endogenous
preproinsulin mRNA, and 318 bp of the GAPDH mRNA. Samples were
resolved on denaturing 5% acrylamide/Tris-borate EDTA-buffered
gels and analyzed by autoradiography and phosphorimaging.
Virus Construction and Evaluation--
To examine the role of the
untranslated regions of the rat preproinsulin II mRNA, a series of
gene constructs were designed that would express mRNA molecules
that mirror the endogenous rat preproinsulin II mRNA as closely as
possible, except for defined alterations (Fig. 2). These mRNAs
encode a His-tagged proinsulin flanked by the preproinsulin 5'- and
3'-UTRs (5HisPI3), the preproinsulin 5'-UTR and SV40 3'-UTR (5HisPI),
the preproinsulin 3'-UTR and a synthetic 5'-UTR that is based upon a
previously characterized sequence that is A/T-rich and predicted to
have little secondary structure (HisPI3; see Ref. 17), or the synthetic
5'-UTR and the SV40 3'-UTR (HisPI). These gene constructs, under the
control of the CMV promoter, were used to construct attenuated
recombinant adenoviruses. In addition, a recombinant adenovirus
expressing firefly luciferase was used as a control.
The termini of the RNAs expressed by these adenoviruses were mapped and
compared with those of the endogenous preproinsulin mRNA. The
5'-end was mapped by primer extension using poly(A) RNA isolated from
hepatocytes and the The Levels of the Endogenous and HisPI-expressing RNAs Do Not
Respond to Glucose--
Total RNA levels were analyzed by RNase
protection assay, allowing assessment of both the endogenous and
His-tagged preproinsulin mRNAs. RNA levels were measured relative
to the endogenous GAPDH level, in uninfected islets or in islets
infected with each of the four His-tagged proinsulin expressing viruses
or a luciferase-expressing control virus (Fig.
4). The His-tagged
preproinsulin-expressing mRNAs were present at levels below that of
the endogenous preproinsulin mRNA but, nonetheless, were at
equivalent levels relative to each other. Incubation of isolated islets
at basal (2.8 mM) or stimulatory (11.1 mM)
glucose for 1 h showed that neither endogenous nor the His-tagged
preproinsulin mRNA levels responded to glucose, relative to the
GAPDH mRNA, in accordance with previous observations (4, 18).
The 5'-UTR Is Necessary and Acts Cooperatively with the 3'-UTR to
Specifically Regulate Preproinsulin mRNA Translation--
The
level of translation from the endogenous and His-tagged
proinsulin-expressing mRNAs was examined in uninfected islets or
islets infected with the luciferase or HisPI adenoviruses. Islets were
incubated for 1 h over a range of glucose concentrations (2.8-16.7 mM) to determine the biosynthetic response of
the endogenous and His-tagged proinsulins to a glucose stimulus (Fig.
5). At a stimulatory (11.1 mM) glucose concentration endogenous proinsulin biosynthesis was strongly stimulated relative to that at a basal (2.8 mM) glucose concentration (Fig. 5a),
specifically increasing above total islet protein synthesis, which was
increased ~2-fold. Biosynthesis of luciferase in islets expressing
the control luciferase adenovirus was not regulated by glucose, despite
a marked glucose-induced stimulation of endogenous proinsulin synthesis
in the same islet The Signal Peptide Is Not Involved in the Specific Regulation of
Preproinsulin Translation--
A modest response to glucose of the
translation of the HisPI mRNA suggested that sequences within the
His-tagged preproinsulin coding region might influence translation.
Therefore the potential role of the signal peptide-encoding region of
the preproinsulin mRNA in glucose-induced translational regulation
was examined. An additional recombinant adenovirus was generated in
which the signal peptide of 5HisPI3 was removed (5HisPI3 Translational Regulation of the Proinsulin Biosynthesis Is Specific
to Pancreatic Preproinsulin mRNA was one of the first mRNAs found to be
specifically regulated at the translational level (4). However, over
the subsequent 20 years the mechanism of this regulation has remained
unknown. There are generic effects of glucose on up-regulating total
protein synthesis in pancreatic islet In this study, using a recombinant adenovirus-mediated expression of a
His-tagged proinsulin reporter in isolated rat islets, we have revealed
that the specific translational regulation of proinsulin biosynthesis
by glucose was largely dependent upon elements that lie in the
untranslated regions of preproinsulin mRNA. The 5'-UTR of
preproinsulin mRNA encoded an element, most likely residing in a
conserved stem-loop secondary structure (14, 15), that was necessary
for the specific stimulation of proinsulin biosynthesis translation in
response to glucose. The 3'-UTR of preproinsulin mRNA contained an
element, most likely the conserved UUGAA sequence (Fig. 1), that had a
tendency to suppress glucose-induced proinsulin biosynthesis but also
to stabilize the preproinsulin mRNA in a pancreatic islet
-cells. However, the mechanism by which this occurs has
remained unclear. Using recombinant adenoviruses that express the
preproinsulin mRNA with defined alterations, the untranslated
regions (UTRs) of the preproinsulin mRNA were examined for elements
that specifically control translation of the mRNA in rat pancreatic
islets. These studies revealed that the preproinsulin 5'-UTR was
necessary for glucose stimulation of preproinsulin mRNA
translation, whereas the 3'-UTR appeared to suppress translation.
However, together the 5'- and 3'-UTRs acted cooperatively to markedly
increase glucose-induced proinsulin biosynthesis. In primary
hepatocytes the presence of the preproinsulin 3'-UTR led to reduced
mRNA levels compared with the presence of the SV40 3'-UTR,
consistent with the presence of mRNA stability determinants in the
3'-UTR that stabilize the preproinsulin mRNA in a pancreatic
-cell-specific manner. Translation of these mRNAs in primary
hepatocytes was not stimulated by glucose, indicating that regulated
translation of the preproinsulin mRNA occurs in a pancreatic
-cell-specific manner. Thus, the untranslated regions of the
preproinsulin mRNA play crucial roles in regulating insulin
production and therefore glucose homeostasis by regulating the
translation and the stability of the preproinsulin mRNA.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cells secrete insulin in response to
increases in circulating nutrients, the most physiologically relevant of which is glucose (1). To replenish secreted insulin and maintain
optimal intracellular insulin stores there is a corresponding rapid and
specific stimulation of proinsulin biosynthesis (1-3). Under normal
circumstances this occurs by increasing translation of the existing
preproinsulin mRNA and is mostly controlled at the initiation phase
of the translational mechanism. Preproinsulin mRNA is mobilized
from an inert intracellular storage pool (in which most of the mRNA
is free) to membrane-bound polyribosomes, marking the entry of newly
synthesized proinsulin into the
-cell secretory pathway (4, 5).
Translation of the preproinsulin mRNA is targeted to the
endoplasmic reticulum through an interaction between its signal
peptide and the signal recognition particle (SRP).1 It has been reported
that SRP-mediated translocation of secretory pathway mRNAs to the
endoplasmic reticulum is glucose-regulated in pancreatic
-cells (6,
7).
-cell
approximately 2-fold (7-9). This occurs through an increase in the
activity of the general translation machinery, largely through protein
phosphorylation regulation of eukaryotic initiation factor (eIF)
activity (10). In
-cells, glucose stimulation of general translation
has been shown to occur through the regulation of the activity of two
basal translation factors, eIF-2B and PHAS-1/eIF-4E-BP (11, 12).
However, glucose-stimulated proinsulin biosynthesis can increase as
much as 30-fold in 1 h, implying a specific control mechanism
above the general effect of glucose on total protein synthesis. We
reveal in this study that such specific translational regulation of
preproinsulin mRNA lies within the molecule itself.
15.4 kcal/mole). This element
up-regulates ferritin mRNA translation in response to increased
cellular iron through an interaction with a trans-acting
iron-binding protein. Iron response elements are evolutionarily
conserved in ferritin H chain mRNAs from several mammalian species.
Intriguingly, the preproinsulin mRNA also has evolutionarily
conserved features. RNA structural analysis of the rat preproinsulin II
mRNA predicts the presence of a stem-loop structure in the 5'-UTR
(dG =
10.8 kcal/mole) that closely resembles structures
predicted to form in the 5'-UTR of other mammalian preproinsulin
mRNAs (14, 15). By comparative analysis of mammalian preproinsulin
mRNA sequences we have also found the presence of a highly
conserved 12-bp element within the 3'-UTR (Fig.
1) that lies between the polyadenylation signal (AAUAAA) and the polyadenylation site. Elements within the
3'-UTR of other mRNAs have been shown to be necessary for the
regulation of mRNA localization, translation, polyadenylation, and
stability (16). These observations are consistent with a role for the
untranslated regions of the preproinsulin mRNA in regulating its
specific translation in response to glucose, which has been directly
tested in this study.
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Fig. 1.
Conserved features exist within the
untranslated regions of the preproinsulin mRNA. Analysis of
the sequence of the untranslated regions of mammalian preproinsulin
mRNAs indicates that certain features are conserved, including a
primary sequence that was identified in the sequences of published
mammalian preproinsulin mRNAs that lies between the polyadenylation
signal and the site of cleavage and polyadenylation.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Gene constructs were designed to express a
His-tagged proinsulin. Four gene constructs were made in which the
preproinsulin coding region (boxes are representative as
follows: SP, signal peptide; B, B peptide of
insulin; C, C peptide; A, A peptide) was tagged
with 6 histidine residues (hatched), leading to production
of HisPI. The untranslated regions of 5HisPI3 were replaced with a
synthetic (Syn) 5'-UTR (HisPI3 and HisPI) and/or the SV40
3'-UTR (5HisPI and HisPI).
-cell line,
INS-1, that had been infected overnight with the virus of
interest. Primer extension was carried out as previously described (28)
using an oligonucleotide (AGGAAGCGGATCCACAGG) that hybridizes between
nucleotides 6 and 23 of the preproinsulin coding region. The 3'-ends of
the mRNAs were mapped by performing RT-PCR (Ready To Go kit;
Amersham Pharmacia Biotech) upon total RNA isolated from islets
infected with the HisPI3 adenovirus using the RNeasy kit (Qiagen).
Fragments were digested by BamHI and NotI,
subcloned into pBluescript (Stratagene), and sequenced with the
Sequenase kit (Amersham Pharmacia Biotech) using the universal primer.
-mercaptoethanol, 0.4 M Tris, pH 6.8) containing 400 mM imidazole. Nickel affinity-purified histidine-tagged
proinsulin and immunoprecipitated endogenous proinsulin were resolved
by Tricine-SDS-polyacrylamide gel electrophoresis. Immunoprecipitated
luciferase was resolved by glycine-SDS-polyacrylamide gel
electrophoresis. Gels were fixed in 50% methanol/10% acetic acid,
dried, and analyzed by phosphorimaging.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cell line, INS-1, infected with the
adenoviruses expressing the His-tagged proinsulin (Fig. 3a). These results showed that
the initiation of transcription of the endogenous preproinsulin
mRNA in INS-1 cells occurred 55 bp from the initiation codon.
Transcription of the preproinsulin 5'-UTR from the CMV promoter of the
5HisPI adenovirus was shown to occur 1 bp before this (56 bp from the
initiation codon) in INS-1 cells and within 2 bp of this in primary
hepatocytes. The synthetic 5'-UTR of HisPI3 was shown to be ~58 bp in
length. The preproinsulin 5'-UTR of 5HisPI3 and the synthetic 5'-UTR of
HisPI matched those of 5HisPI and HisPI3, respectively (data not
shown). The 3'-end of the endogenous and virally expressed mRNAs
were mapped by RT-PCR, subcloning, and sequencing of 3'-ends generated from RNA prepared from rat islets infected with the HisPI3 adenovirus (Fig. 3b). In the endogenous preproinsulin 3'-UTR five sites
of polyadenylation were identified over a window of 7 bp, downstream of
the conserved UUGAA sequence. Polyadenylation of the HisPI3 mRNA
occurred at one of these sites and leaves intact the conserved primary
sequence element (Fig. 1) that lies between the polyadenylation signal
(AAUAAA) and the polyadenylation site. Thus, the preproinsulin 5' and
3' termini in the HisPI constructs mimic those found in the endogenous
preproinsulin mRNA.
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Fig. 3.
Mapping of the termini of the His-tagged
proinsulin-expressing mRNAs revealed that they mimic those of the
endogenous preproinsulin mRNA. To determine that the
translation of the His-tagged proinsulin-expressing mRNAs mirrored
the endogenous preproinsulin mRNA as closely as possible, the
termini were mapped. A, primer extension performed upon
poly(A)+ from the -cell line, INS-1 (I), or
primary hepatocytes (H), either uninfected
(UNINF) or infected with adenoviruses expressing the 5HisPI
or HisPI3 mRNAs. Polyacrylamide gels showing the primer extension
productions are presented alongside sequencing reactions of 5HisPI and
HisPI3 that used the same oligonucleotide primer. CMV
indicates CMV promoter sequences, PI 5'-UTR indicates the
preproinsulin mRNA 5'-UTR, and Syn 5'-UTR indicates the
synthetic 5'-UTR. B, the 3' termini of the endogenous
preproinsulin (ppI) mRNA and the HisPI3 mRNA were
mapped by RT-PCR, subcloning, and DNA sequencing. Italics
indicate the polyadenylation signal (AAUAAA). Bold indicates
the evolutionarily conserved sequence element. Bases found to
immediately precede the poly(A)+ tail are
underlined. Five sites of polyadenylation were found in the
endogenous message, one of which was used in the preproinsulin 3'-UTR
of HisPI3.
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Fig. 4.
Measurement of RNA levels by RNase protection
assay. Arrows indicate the positions of HisPI-,
endogenous proinsulin (ppI)-, and GAPDH-encoding mRNAs
in pancreatic islets infected with the indicated adenovirus and
cultured for 1 h at 2.8 or 11.1 mM glucose. The
His-tagged proinsulin probe fragment protected the preproinsulin coding
region and 3'-UTR, thereby protecting a larger fragment in mRNAs
carrying the preproinsulin 3'-UTR (HisPI3 and 5HisPI3) compared with
those carrying the SV40 3'-UTR (HisPI and 5HisPI).
-cells (Fig. 5a). As such, regulation
of proinsulin biosynthesis was unaffected by the recombinant adenovirus
infection. Glucose dose-response analysis of the biosynthesis of the
endogenous proinsulin outlined a threshold response to glucose at about
3 mM glucose, a marked increase between 3 and 8 mM glucose, and maximum stimulation was reached at 11 mM glucose, with proinsulin levels 14.7-fold (S.E. ± 1.4;
n = 5) above those at 2.8 mM glucose, when
corrected for effects of glucose upon general protein synthesis. These
results are in accordance with previous reports (9, 19). The
biosynthetic response to glucose for the His-tagged proinsulin was
dependent upon the UTRs present on the adenovirally expressed mRNA
(Fig. 5a). Translation of the HisPI mRNA, which lacks
preproinsulin mRNA UTR sequences, was stimulated in response to
11.1 mM glucose 2.30-fold (S.E. ± 0.14; n = 4) above the level observed at 2.8 mM glucose (Fig. 5,
a and c). In the presence of the 3'-UTR of the
preproinsulin mRNA, a 1.76-fold (S.E. ± 0.23; n = 3) stimulation of the translation of the HisPI3 mRNA at 11.1 mM glucose (above that at basal, 2.8 mM,
glucose) was modestly, yet significantly (p < 0.05),
below the response observed with the HisPI mRNA. In contrast, the
5'-UTR of the preproinsulin mRNA was stimulatory, in that the
translation of 5HisPI at 11.1 mM glucose was 3.22-fold (S.E. ± 0.28; n = 3) above the levels at 2.8 mM glucose and significantly above the response observed
with HisPI and HisPI3 (p < 0.05; see Fig.
5c). However, the strongest translational response to
glucose was seen with the 5HisPI3 mRNA, which was 5.73-fold (S.E. ± 0.32; n = 3) stimulated at 11.1 mM
glucose above the level at 2.8 mM glucose and above the
responses observed for the HisPI, HisPI3, and 5HisPI translation at
11.1 mM glucose (p < 0.05; Fig.
5c). This indicated that a cooperativity existed between
sequences in the 5'- and 3'-UTRs for glucose-regulated preproinsulin
mRNA translation. The glucose dose-response for 5HisPI3 mRNA
translation (Fig. 5c) followed the same qualitative pattern
as that for endogenous proinsulin biosynthesis (Fig. 5b),
although the magnitude of the response was lower. However, it should be
considered that adenovirus-mediated HisPI expression, as driven by the
CMV-promoter, would result in constitutive His-tagged proinsulin
expression in the non-
-cells of the islet. This, in turn, would
obscure the glucose stimulation of the specific control of 5HisPI3
mRNA translation in glucose-sensitive islet
-cells. Indeed there
is precedence for this, in that high constitutive expression of
prohormone convertase (PC) 2 in the non-
-cells of the pancreatic
islets masked the specific glucose-induced translational control of PC2
biosynthesis in the
-cells (20, 21).
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Fig. 5.
Translation of the endogenous and His-tagged
preproinsulin mRNAs. The translation of the endogenous
preproinsulin mRNA and the virally encoded HisPI, HisPI3, 5HisPI,
5HisPI3, and luciferase mRNAs were measured. A,
representative gels of [35S]methionine-labeled endogenous
and His-tagged proinsulin and luciferase from isolated rat islets
cultured at 2.8 or 11.1 mM glucose. Graphs show
the dose response to glucose of the translation of endogenous
proinsulin (B) and His-tagged proinsulin
(C) measured from islets cultured at a range of
glucose concentrations (2.8-16.7 mM). Values are expressed
as mean ± S.E. Endogenous proinsulin, n = 5;
HisPI, n = 4; HisPI3, 5HisPI, and 5HisPI3,
n = 3.
SP; see Fig.
6a). In isolated pancreatic
islets infected with recombinant adenovirus to express the 5HisPI3 or
5HisPI3
SP mRNAs, incubation for 1 h at basal (2.8 mM) or stimulatory (11.1 mM) glucose resulted
in no change in 5HisPI3 or 5HisPI3
SP mRNA levels, in parallel
with endogenous preproinsulin and GAPDH mRNAs (Fig. 6b).
This was despite a marked glucose-induced increase in endogenous
proinsulin biosynthesis in the same islets (Fig. 6b).
Although removal of the signal peptide reduced the response to
stimulatory (11.1 mM) glucose 1.44-fold (S.E. ± 0.19;
n = 4), the translational response to glucose was nonetheless still evident (Fig. 6b). Thus, it appeared that
specific translational regulation of proinsulin biosynthesis by glucose does not require the preproinsulin signal peptide region. However, 5HisPI3
SP mRNA translation was reduced at 11.1 mM
glucose compared with that of 5HisPI3 mRNA, implicating a general
effect imparted by the signal peptide on the synthesis of proteins
destined to the
-cell secretory pathway. The presence of the signal
peptide probably also accounted for a modest glucose effect on HisPI
mRNA translation (Fig. 5, a and c).
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Fig. 6.
The role of the signal peptide in the
translational regulation of preproinsulin. A, an
adenovirus with a new gene construct, 5HisPI3 SP, removed the signal
peptide from the coding region of 5HisPI3. SP, signal
peptide; B, B peptide of insulin; A, A peptide.
B, protein synthesis (endogenous and His-tagged proinsulin)
and RNA levels (endogenous and His-tagged proinsulin and GAPDH)
examined in isolated pancreatic islets infected with the 5HisPI3 and
5HisPI3
SP adenoviruses were cultured at the indicated glucose
concentrations.
-cells--
To examine whether the regulation of
proinsulin biosynthesis was because of a
-cell-specific mechanism,
primary hepatocytes were infected with the His-tagged
proinsulin-expressing adenoviruses. Primary hepatocytes were chosen as
the liver is also a recognized glucose-sensing tissue. Analysis of the
expression of the His-tagged preproinsulin mRNAs in primary
hepatocytes showed no change in mRNA levels in response to glucose.
However, an unexpected, yet intriguing observation of HisPI mRNA
expression in primary hepatocytes was that the levels of the mRNAs
carrying the rat preproinsulin 3'-UTR (HisPI3 and 5HisPI3) were much
lower than those with the SV40 3'-UTR (HisPI and 5HisPI), despite
equivalent GAPDH mRNA levels (Fig.
7a). This was in contrast to
their expression in pancreatic islets, where the same titers of the
purified recombinant adenoviruses gave similar levels for all four
His-tagged proinsulin-expressing mRNAs (Fig. 4). Because the
transcription of these mRNAs was regulated by the same CMV promoter
sequences, it appeared that a pancreatic islet-specific mechanism
exists for specific stabilization of the preproinsulin mRNA via an
element within the 3'-UTR. The biosynthesis of the His-tagged
proinsulin in hepatocytes tended to parallel HisPI mRNA expression
levels. Therefore, although HisPI3 and 5HisPI3 biosynthesis was lower
than that of HisPI and 5HisPI (Fig. 7b), no response was
observed in His-tagged proinsulin biosynthesis to 11.1 mM
glucose above that seen at 2.8 mM glucose from any of the
virally expressed mRNAs (Fig. 7b). These hepatocytes
were confirmed as glucose responsive in that, after a 6-h incubation at
either 2.8 or 11.1 mM glucose, a significant
(p < 0.01) increase in lactate output at 11.1 mM glucose (1.53 ± 0.09 mg/dl in the absence of
insulin, 1.46 ± 0.53 in the presence of insulin) was observed
compared with hepatocytes cultured at 2.8 mM glucose (0.45 ± 0.02 mg/dl in the absence of insulin, 0.42 ± 0.03 in the presence of insulin). Measurement of total protein synthesis in these primary hepatocytes showed that the incorporation of
[35S]methionine into trichloroacetic acid-precipitable
material was not stimulated in response to glucose, unlike isolated
islets of Langerhans, where a stimulation of 2.23-fold (S.E. ± 0.14; n = 26) was observed. To stimulate total protein
synthesis, primary hepatocytes were cultured in the presence or absence
of 100 nM insulin, a known stimulator of general protein
synthesis in hepatocytes (22). Indeed, total protein synthesis was
stimulated by insulin (1.51-fold; S.E. ± 0.19; n = 3), but no specific stimulation of His-tagged proinsulin biosynthesis
by insulin at either 2.8 or 11.1 mM glucose was observed
(Fig. 7c). These results show that proinsulin translation
was regulated by a
-cell-specific mechanism and reaffirmed that this
was not because of a general glucose-sensing mechanism or an
up-regulation of the general translation machinery.
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Fig. 7.
His-tagged proinsulin expression in primary
hepatocytes. Primary hepatocytes infected with the indicated
adenovirus were cultured at 2.8 or 11.1 mM glucose.
A, RNA levels of His-tagged proinsulin (ppI) were
measured by RNase protection assay. B,
[35S]methionine-labeled His-tagged proinsulin levels from
islets infected with the HisPI, HisPI3, 5HisPI, or 5HisPI3 adenoviruses
and cultured at 2.8 or 11.1 mM glucose. C,
[35S]methionine-labeled His-tagged proinsulin levels from
islets infected with the 5HisPI3 adenovirus cultured at 2.8 or 11.1 mM glucose in the absence or presence of 100 nM
insulin.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cells, which in turn have a
modest effect of increasing proinsulin synthesis. Glucose stimulates
increases in general protein synthesis in pancreatic islet
-cells,
most likely by the phosphorylation of certain general translation
initiation factors (11, 12). Moreover, there is an additional general
control mechanism applied to newly synthesized proteins destined for
the
-cell secretory pathway, mediated via the nascent signal
peptide, most likely through the signal peptide/SRP interaction and
alleviation of SRP-mediated arrest of translation (5, 23). Both of
these general mechanisms contribute to the up-regulation of proinsulin
synthesis but are relatively minor and do not account for the specific
nature of glucose-induced preproinsulin mRNA translation.
-cell-specific manner. When expressed together, the 5'- and 3'-UTRs
of preproinsulin mRNA acted synergistically to markedly stimulate
the His-tagged proinsulin biosynthesis translation in a manner
reminiscent of the endogenous proinsulin (Fig. 5). The finding of a
cooperativity between the 5'- and 3'-UTRs of preproinsulin mRNA for
translational control of proinsulin biosynthesis complements recent
findings with the basal translational machinery that show an
interaction between the cap binding complex subunit, eIF-4G, and the
poly(A)-binding protein, PABP (24). Based on the findings in this
study, we propose a model for the translation of the preproinsulin
mRNA, illustrated in Fig. 8. Firstly,
there are two generic effects; one is an effect of glucose on the
-cell translational machinery, mostly at the initiation phase (11, 12), resulting in an ~2-fold increase in total protein synthesis. The
other is directed at a nascent signal peptide/SRP interaction (5-7),
which is probably adaptive to an up-regulation in biosynthesis of
proteins destined for the pancreatic
-cell secretory pathway, of
which proinsulin is included. However, the major specific translational control of proinsulin biosynthesis by glucose is via the UTRs of
preproinsulin mRNA. As the glucose concentration rises above 3 mM glucose, the 5'-UTR promotes a marked increase in
translation of preproinsulin mRNA, and simultaneously there is an
alleviation of suppressing preproinsulin mRNA translation mediated
at the 3'-UTR, probably via an interaction with the 5'-UTR. Sequences in the 3'-UTR also specifically increase preproinsulin mRNA
stability in
-cells, which will also contribute proinsulin
biosynthesis at the translational level by preserving the amount of
available preproinsulin mRNA template. Thus, there are multiple
points for specific regulation of preproinsulin mRNA translation
that, when operating coordinately, account for the fine control of
proinsulin biosynthesis by glucose in pancreatic islet
-cells. It is
likely that elements within the 5'- and 3'-UTRs of preproinsulin
mRNA will interact with trans-acting factors, probably
proteins, to confer their effects in glucose-regulated proinsulin
biosynthesis translation and/or preproinsulin mRNA stability. Such
trans-acting factors, and/or their regulation, are likely to
be specific to the pancreatic
-cell, because the preproinsulin
mRNA UTRs could not confer specific glucose-regulated translation
of the preproinsulin mRNA in hepatocytes. Because glucose
metabolism is required for glucose-induced proinsulin biosynthesis (1),
one would predict that trans-acting factors should associate
with the elements in the 5'- and 3'-UTRs of the preproinsulin mRNA
in a manner that responds to secondary signals that arise from glucose
metabolism and that these interactions should up-regulate preproinsulin
mRNA translation. Future experimentation will be directed to
identify such
-cell-specific preproinsulin
mRNA-regulating trans-acting factors.
View larger version (19K):
[in a new window]
Fig. 8.
A model for the translational regulation of
the preproinsulin mRNA in response to glucose. Translation of
the preproinsulin mRNA is regulated by glucose acting through
general mechanisms (general translational factors and secretory
pathway) and by specific mechanisms that require sequences in both the
5'- and 3'-UTRs. 4F refers to the cap binding
complex, eIF-4F. sig indicates the signal peptide encoded by
the preproinsulin mRNA.
The untranslated regions of mammalian preproinsulin mRNAs contain
conserved features that may be necessary for the regulation of
proinsulin translation. Previous studies have shown that the translation of ~50 proteins in -cells is stimulated greater than 10-fold in response to glucose (7). Intriguingly, a predicted stem-loop
secondary structure in the 5'-UTR of the mRNA encoding two of these
proteins, the prohormone convertases, PC2 and PC3, revealed a similar
structure to that predicted to form in the 5'-UTR of the preproinsulin
mRNA. It is likely that this secondary structure is involved in
glucose-induced translational regulation of proPC2 and proPC3
biosynthesis in
-cells parallel to that of proinsulin (9, 20, 21).
However, preproPC2 and preproPC3 mRNAs lack the conserved UUGAA
element in the 3'-UTR that may explain their less robust translational
response to glucose (15), as well as a shorter mRNA half-life
relative to preproinsulin mRNA (25). Notwithstanding, it is quite
likely that certain aspects of the mechanism for glucose-induced
translation control of proinsulin biosynthesis unveiled in this study
also apply to controlling the biosynthesis of other proteins destined
for the insulin secretory granule compartment, particularly those that catalyze proinsulin sorting, processing, and regulated secretion (7).
Moreover, the mechanisms that underlie nutrient-induced translational
regulation of proinsulin biosynthesis are likely to be crucial in
understanding wider aspects of
-cell physiology and metabolic
homeostasis, because this is the major control of insulin production in
mammals under normal physiological conditions (1-3, 18). Indeed, there
is disregulation of proinsulin biosynthesis in an animal model of type
II diabetes, which contributes to
-cell dysfunction and decreased
availability of insulin (26). Finally, it is quite likely that the
translational control mechanism for glucose-stimulated proinsulin
biosynthesis in pancreatic
-cells may serve as a model for tightly
regulating production of the major polypeptide product in other
neuroendocrine cells. Neuroendocrine cells that produce a primary
polypeptide product and store it intracellularly in secretory vesicles
will have that store depleted by secretion of the polypeptide in
response to an extracellular stimulus. Translational regulation of
pre-existing mRNA of a polypeptide hormone/neurotransmitter by the
same stimulus provides a means to rapidly and economically replenish
intracellular stores lost by exocytosis and maintain them at optimal
levels, as such preserving efficient secretory function of the
neuroendocrine cell.
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ACKNOWLEDGEMENTS |
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We thank Professor Kevin Docherty for useful discussions.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant DK50610 and by a Juvenile Diabetes Foundation International fellowship (to B. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Dept. of Anatomy and Physiology, MSI/Wellcome Trust Bldg., University of Dundee, Dow St., Dundee DD1 5EH, Scotland.
To whom correspondence should be addressed: Pacific Northwest
Research Inst., 720 Broadway, Seattle, WA 98122. Tel.: 206-860-6777; Fax: 206-726-1202; E-mail: cjr@pnri.org.
Published, JBC Papers in Press, April 10, 2001, DOI 10.1074/jbc.M011214200
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ABBREVIATIONS |
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The abbreviations used are: SRP, signal recognition particle; eIF, eukaryotic (translation) initiation factor; UTR(s), untranslated regions; HisPI, 6 Histidine-tagged proinsulin; CMV, cytomegalovirus; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PC, prohormone convertase; bp, base pair(s); PCR, polymerase chain reaction; Tricine, N- [2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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