Endocrinology Vol. 139, No. 2 491-495
Copyright © 1998 by The Endocrine Society
Differential Expression of Rat Insulin I and II Messenger Ribonucleic Acid after Prolonged Exposure of Islet ß-Cells to Elevated Glucose Levels1
Zhidong Ling,
Harry Heimberg,
André Foriers,
Frans Schuit and
Daniel Pipeleers
Diabetes Research Center, Vrije Universiteit Brussel, Brussels,
Belgium
Address all correspondence and requests for reprints to: Dr. D. Pipeleers, Diabetes Research Center, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium.
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Abstract
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Prolonged exposure of rat islet ß-cells to 10 mmol/liter glucose has
been previously shown to activate more cells into a glucose-responsive
state (>90%) than has exposure to 6 mmol/liter glucose (50%). The
present study demonstrates that this recruitment of more activated
cells results in 4- to 6-fold higher levels of proinsulin I and
proinsulin II messenger RNA (mRNA). However, only the rate of
proinsulin I synthesis is increased. Failure to increase the rate of
proinsulin II synthesis in the glucose-activated cells results in
cellular depletion of the insulin II isoform, which can be responsible
for degranulation of ß-cells cultured at 10 mmol/liter glucose.
Higher glucose levels (20 mmol/liter) during culture did not correct
this dissociation between the stimulated insulin I formation and the
nonstimulated insulin II formation. On the contrary, the rise from 10
to 20 mmol/liter glucose resulted in a 2-fold reduction in the levels
of proinsulin II mRNA, but not of proinsulin I mRNA; this process
further increased the ratio of insulin I over insulin II to 5-fold
higher values than those in freshly isolated ß-cells. The present
data suggest that an elevated insulin I over insulin II ratio in
pancreatic tissue is a marker for a prolonged exposure to elevated
glucose levels. The increased ratio in this condition results from a
transcriptional and/or a posttranscriptional failure in elevating
insulin II formation while insulin I production is stimulated in the
glucose-activated ß-cells.
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Introduction
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BOTH ACUTE and chronic elevations in the
glucose concentration stimulate the rate of insulin biosynthesis. Acute
stimulation is achieved at the translational level (1), whereas the
chronic effect enhances the formation and/or stability of preproinsulin
messenger RNA (mRNA) (2, 3, 4). Rodent ß-cells synthesize two different
insulins that are encoded by two nonallelic genes with more than 90%
homology (5, 6, 7, 8). The primary translation products, preproinsulin I and
II, differ by three amino acids in the preregion, two in the C
peptides, and two in the B chain (6, 8). The conversion products
insulin I and II are usually stored in unequal amounts. The ratio of
the cellular contents in insulin I over insulin II fluctuates between 1
and 2 in a basal fed or fasting state, but increases 2- to 4-fold
during pregnancy, excessive GH secretion, or chronic hyperglycemia (5, 9, 10, 11, 12, 13, 14). This variation was taken as evidence for an independent
regulation of the two genes, whereby the insulin I gene is
preferentially expressed under stimulatory conditions (10). The use of
purified rat ß-cells allowed us to assess their direct acute and
chronic regulation by glucose without interference of other cell types
that can influence the glucose response of islet ß-cells in
vitro (15). It was thus shown that the glucose concentration in
the culture medium dose-dependently increases the percentage of islet
ß-cells that are glucose responsive (16); when the cells were
chronically exposed to excessively high glucose levels (20 mmol/liter),
they reached a sustained state of activation, during which they
exhibited a glucose hypersensitivity rather than a glucose toxicity or
glucose desensitization (16). In the present study, we have examined
whether these in vitro conditions of stimulation also lead
to an increased ratio of the cellular insulin I over insulin II content
and whether this results from differences at the transcriptional or
translational level; the results may indicate cellular markers for the
state of activation in the ß-cell population.
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Materials and Methods
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Preparation and culture of purified B cells
Pancreatic islets were isolated from adult Wistar rats and
dissociated into single cells using calcium-free medium supplemented
with trypsin and deoxyribonuclease (17). Purified ß-cells were
collected after autofluorescence-activated sorting of dissociated cells
(17). Single ß-cells were reaggregated for 3 h in a rotatory
shaking incubator (37 C; Braun, Melsugen, Germany) (15) and then
statically cultured for 9 days (37 C; 95% air/5% CO2) in
serum-free Ham’s F-10 medium at 6, 10, or 20 mmol/liter glucose (16).
Media were recovered on days 5 and 9 and analyzed by reverse phase
HPLC.
Pulse-chase labeling
After 9 days of culture, ß-cell aggregates were collected from
the petri dishes and washed in Earle’s HEPES medium (EH; composition
given in Ref.16). In the first series of experiments, 5 x
104 cells were labeled for 2 h with
L-[3,5-3H]tyrosine (TRK 200, Amersham,
Aylesbury, UK; 50 Ci/mmol/liter) in 200 µl EH supplemented with 10
mmol/liter glucose and 1% (wt/vol) BSA (fraction V, RIA grade, Sigma
Chemical Co., St. Louis, MO) (18). Under this condition, insulin
synthesis is maximally stimulated by glucose (16). At the end of this
incubation, the cells were washed in EH with 1 mmol/liter tyrosine and
then extracted in 1 ml acetic acid (2 mol containing 0.25% BSA) (18).
In the second series of experiments, 105 cells were labeled
for 20 min at 5 or 20 mmol/liter glucose using 50 µCi
L-[3,5-3H]tyrosine (TRK 200, Amersham; 50
Ci/mmol/liter) alone or in combination with 50 µCi
[35S]methionine (Amersham; >1000 Ci/mmol/liter). At the
end of this 20-min incubation, the cells were washed in medium with 1
mmol/liter tyrosine and 1 mmol/liter methionine. One sample (5 x
104 cells) was taken for extraction in 2 M
acetic acid-0.25% BSA, and the rest (5 x 104 cells)
was further incubated for 150 min in 200 µl medium with 1 mmol/liter
tyrosine and 2.5 mmol/liter glucose (chase). At the end of this chase
period, cells and medium were collected and analyzed by reverse phase
HPLC.
Reverse phase HPLC
Cell extracts and media were injected into a Millipore Waters
HPLC system (Millipore Waters, Milford, MA) consisting of a
µBondapack C18 column (30 x 0.46 cm; 10 µm). The
mobile phase was buffer-acetonitrile (70.6:29.4, vol/vol) with 0.1
M phosphoric acid and 0.05 M ethanolamine as
buffer, pH 2.3. Twenty minutes after injection, the acetonitrile
concentration was linearly increased from 29.4% to 32.4% over 15 min
and then kept constant at 32.4% for the last 15 min of the run.
Fractions of 0.5 ml were collected at a flow rate of 1 ml/min. Samples
of 200 µl were counted in a liquid scintillation counter (Beckman,
Palo Alto, CA); another 200 µl of sample were assayed for insulin
(17).
Figure 1
shows a representative HPLC
elution profile of a ß-cell extract after 2-h labeling with
[3H]tyrosine. The three major radioactive peaks were
immunoprecipitable with antiinsulin serum. The first two peaks
contained the majority of the cellular hormone as detected by RIA (Fig. 1). When the cells were pulse labeled for 20 min with
[3H]tyrosine and [35S]methionine, which is
not incorporated in insulin I, all radioactivity eluted in the third
peak (data not shown), suggesting that the latter contains both
proinsulin I and proinsulin II. After a 150-min chase period, the
3H radioactivity was recovered in both the first and the
second peaks, whereas 35S radioactivity was only measured
in the second peak (data not shown), indicating that insulin I elutes
in the first peak, and insulin II elutes in the second. An insulin
standard was recovered for more than 90% in the eluate.
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Figure 1. RP-HPLC elution profile of newly synthesized
proteins (top) and insulin immunoreactivity
(bottom) in extracts of rat ß-cells after 2-h
incubation at 10 mmol/liter glucose with [3H]tyrosine. No
insulin immunoreactivity was detectable in the RIA of the proinsulin
peak, as this fraction contains only 2% of the total cellular insulin
content, and only 20% of proinsulin is picked by the present insulin
assay.
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Analysis of mRNA in ß-cells
Insulin II complementary DNA (cDNA) was provided in a pGEM
vector (19), whereas insulin I cDNA was subcloned in this vector.
Insulin I and insulin II sense strand RNAs were in vitro
transcribed starting from the T7 promoter after linearization of the
construct with HindIII. Run-off transcripts were blotted and
hybridized with 5'-radiolabeled oligonucleotides. The sequence-specific
oligonucleotide probes were complementary to the sequence of codon
17–23, numbered according to the start codon: insulin I,
5'-GGGCAGGCTTGGGCTCCC; and insulin II,
5'-GGGCAGGGCG-GGGCTCCC (6). Specific hybridization was
achieved by overnight incubation at 50 C in the presence of 4.5 x
106 µCi/µg probe (0.13 x 10-12 µg
probe/ml) and stringent washing in 3 M
tetramethylammoniumchloride for 30 min at 57.5 C (Fig. 2). RNA was extracted and blotted as
previously described (20), and membranes were probed for insulin I and
insulin II. During the insulin I hybridization, a membrane strip
containing islet RNA was reprobed with insulin II to directly compare
signal intensities from the subsequent hybridizations. Signal
intensities on autoradiographs were measured on an Ultroscan XL
densitometer (Pharmacia, Uppsala, Sweden). Data were derived from the
integrated area under the curve and related to the signals of
ß-actin.
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Figure 2. Dot blot of insulin I and II cDNA and RNA. Plasmid
DNA of pGEM1-insulin I and pGEM1-insulin II as well as run-on
transcripts from these plasmids were dot-blotted on a nylon membrane
and hybridized with 32P-radiolabeled sequence-specific
oligonucleotide probes as described in Materials and
Methods.
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Data expression
Results were expressed as the mean ± SEM, and
statistical significance of differences was calculated by ANOVA and
Student’s t test.
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Results
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Insulin I and insulin II contents in rat ß-cells
In freshly isolated rat ß-cells, 57% of insulin was identified
as insulin I, and 43% was identified as insulin II, leading to a ratio
of 1.4 (Table 1). Culture increased this
ratio with 5-fold higher values when the medium contained 20 mmol/liter
glucose (Table 1). For each of the tested glucose concentrations, the
insulin content of cultured cells was lower than that of freshly
isolated cells (Table 1). The decrease occurred to a greater extent for
insulin II than for insulin I. After culture with 20 mmol/liter
glucose, the cellular insulin I and insulin II contents were,
respectively, 3- and 16-fold lower than those in freshly isolated cells
(Table 1). The medium that was collected during the culture period
contained both insulin I and II in a ratio (1.9) not significantly
different from that in freshly isolated cells, indicating that the
higher ratio in 20 mmol/liter cultured cells is not the result of a
preferential release of insulin II.
Biosynthesis of insulin I and insulin II by cultured ß-cells
At the end of the 9-day culture period, ß-cells cultured at 10
or 20 mmol/liter glucose exhibited 2- to 3-fold higher maximal rates of
proinsulin synthesis than 6 mmol/liter cultured cells, but their rates
of proinsulin conversion were comparable (Table 2). This higher biosynthetic activity at
maximal glucose stimulation was restricted to the production of insulin
I, leading to a higher ratio of newly formed insulin I over insulin II
(Table 2). After culture at 20 mmol/liter glucose, the capacity for
insulin II production was comparable to that after culture at 6
mmol/liter glucose (Table 2).
Pulse labeling with [35S]methionine, which is only
incorporated in proinsulin II, indicated that the cells cultured in 20
mmol/liter glucose did not exhibit a higher rate of proinsulin II
synthesis than those cultured in 6 mmol/liter glucose at either
intermediate (5 mmol/liter) or maximal (20 mmol/liter) glucose
stimulation (Fig. 3). On the other hand,
their total proinsulin synthesis, as expressed by
[3H]tyrosine labeling, was markedly higher at both
glucose concentrations (Fig. 3); these higher rates are thus entirely
attributable to a higher production of proinsulin I.
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Figure 3. Reverse phase HPLC elution profiles of ß-cell
extracts prepared after 9-day culture with 6 or 20 mmol/liter glucose
and after a 20-min incubation with [3H]tyrosine and
[35S]methionine at 5 mmol/liter (solid
line) or 20 mmol/liter glucose (dotted line).
The respective 3H and 35S radioactivities are
plotted on the top (PROINS. I+II) and
bottom (PROINS. II).
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A pulse-chase experiment makes it unlikely that the dissociation
between the higher insulin I production and the unaffected insulin II
production (Table 2
) was caused by a delay in proinsulin II conversion.
After 20-min pulse labeling at 20 mmol/liter glucose, ß-cells were
chased for 150 min at 2.5 mmol/liter glucose. In both 6 and 20
mmol/liter cultured cells, all newly formed proinsulin was converted at
the end of the chase period. The ratio of newly formed insulin I over
insulin II was again more than 2-fold higher in the cells cultured at
20 mmol/liter glucose (data not shown).
Relative amounts of insulin I and insulin II mRNA in cultured
ß-cells
After culture at 10 or 20 mmol/liter glucose, 2-fold higher
amounts of RNA were extracted from the cells than at 6 mmol/liter
glucose (data not shown). From similar amounts of RNA, the three
preparations were then compared for their relative abundance in insulin
I and insulin II mRNA, using ß-actin mRNA as an internal standard. In
cells cultured in 10 and 20 mmol/liter glucose, the signal intensity of
proinsulin I mRNA was 2- to 3-fold higher than that in cells cultured
in 6 mmol/liter glucose. As these preparations were analyzed at a
2-fold higher dilution than the 6 mmol/liter cultured cells, it is
concluded that they contain 4- or 6-fold more proinsulin I mRNA than
the 6 mmol/liter cultured cells. The signal for proinsulin II mRNA was
also 2-fold higher in 10 mmol/liter glucose cultured cells than in 20
mmol/liter cultured cells (Fig. 4). These
data result in a ratio of proinsulin I mRNA over proinsulin II mRNA
that is comparable in 6 and 10 mmol/liter cultured ß-cells (
1),
but 3-fold elevated in 20 mmol/liter cultured cells.
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Figure 4. Analysis of mRNA from ß-cells cultured for 9
days in 6, 10, or 20 mmol/liter glucose. Blots were hybridized with
oligonucleotide probes specific for insulin I or insulin II as well as
with a cDNA probe specific for ß-actin. Blots shown on
top are representative for three independent
experiments. The intensity of the autoradiographic signals was scanned
on a laser densitometer and normalized for ß-actin mRNA. Data
represent the mean ± SEM of three independent
measurements and are expressed as a function of the values measured in
cells cultured with 6 mmol/liter glucose.
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Discussion
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In vitro studies of purified ß-cells have indicated
that the glucose levels to which ß-cells are chronically exposed
determine the percentage of cells that can rapidly respond to acute
variations in glucose concentration (16). Culture at 10 mmol/liter
glucose recruits virtually all rat ß-cells to such a responsive state
(16). Total glucose-induced insulin synthesis thus becomes 2- to 3-fold
higher than that in ß-cell preparations that are cultured at 6
mmol/liter glucose, a condition that activates only 50% of the cells
(15). Activation of more ß-cells also leads to a 4- to 6-fold higher
content in proinsulin mRNA, which reflects the response of a greater
number of cells to the well known stimulatory effects of glucose on
proinsulin mRNA formation (2, 3, 4, 21, 22, 23, 24). The degree of stimulation was
comparable for both proinsulin isoforms. However, despite this rise in
the level of mRNA for both isoforms, only insulin I was synthesized at
higher rates; the formation of insulin II occurred at similar rates in
cells cultured in 10 and 6 mmol/liter glucose. Failure to increase the
rate of insulin II synthesis to the same extent as that of insulin I
explains the difference in isoform content between cultured and freshly
isolated ß-cells. We have previously observed that culture at 10
mmol/liter glucose reduces the cellular insulin content by 40–50%
compared with that of freshly isolated cells (16). It now appears that
this reduction is almost entirely due to a decrease in the cellular
insulin II content, probably as a result of a failure of the
glucose-activated cells to increase insulin II formation during
culture. Analysis of the insulin isoforms in the culture medium
excluded that preferential release of insulin II was responsible for
the disproportionally low cellular insulin II content. We have not
studied whether higher degradation of insulin II accounts in part for
its lower cellular content.
When the rat ß-cells were chronically exposed to excessively high
glucose levels (20 mmol/liter), their proinsulin mRNA levels were also
markedly higher than after culture in 6 mmol/liter glucose, but this
effect was less pronounced for the proinsulin II isoform. The content
of proinsulin I mRNA was comparable to that in cells cultured in 10
mmol/liter glucose, suggesting that it reflects the number of activated
cells, which in both culture conditions was nearly 100% (16). On the
other hand, the cellular content of proinsulin II mRNA was 2- to 3-fold
lower than that after 10 mmol/liter glucose culture, indicating that
culture with 20 mmol/liter glucose was associated with an inhibitory
effect at the level of proinsulin II mRNA formation or with decreased
mRNA stability. The ratio of newly synthesized insulin I over insulin
II was more than 2-fold higher than that after 6 mmol/liter glucose
culture. This explains the higher ratio of insulin I over insulin II in
the cellular hormone store. This store is markedly decreased after
culture at 20 mmol/liter glucose, during which the rates of hormone
synthesis are inadequate to compensate for its increased release rates
(18); furthermore, as shown by the present data, the 20 mmol/liter
glucose-cultured cells failed to raise the rates of insulin II
synthesis as they did for insulin I synthesis, leading to near
depletion of the cellular insulin II store (>15-fold lower than that
in freshly isolated ß-cells).
A previous study had shown that chronically elevated glucose
levels impaired insulin gene transcription in the HIT-T15 ß cell line
(25). The present work demonstrates that this culture condition
inhibits the expression of insulin II in normal rat ß-cells at both
the transcriptional and posttranscriptional levels. It is not yet known
whether it also influences the rates of intracellular degradation of
one or both insulin isoforms (12). Although a chronic excess of
intracellular glucose might be responsible for processes that suppress
the expression of insulin II, it is conceivable that the elevated
extracellular insulin levels that occur in this condition have caused
this inhibitory effect, as they also suppress other ß-cell functions
(26, 27).
The present findings indicate that shifts in the functional
heterogeneity of the rat ß-cell population will also influence the
absolute amounts of proinsulin mRNA. A prolonged glucose stimulation
keeps an increased percentage of ß-cells in an activated state, even
during subsequent culture at reduced glucose levels (6 mmol/liter for
up to 24 h; our unpublished observations). This phenomenon is
responsible for a higher total transcriptional activity, in particular
for glucose-inducible genes such as preproinsulin I and II. This higher
mRNA content for both hormone isoforms is associated with an increased
production of insulin I, but not of insulin II. Activation of more
cells is thus responsible for more hormone synthesis, but only of the
insulin I isoform. Exposure to excessively high glucose levels does not
eliminate this dissociation; in fact, it results in statistically
significant increases in the ratio of insulin I over insulin II in
terms of both their respective mRNA content as well as their peptide
content. It can be concluded that rat ß-cells exhibit a differential
regulation of the biosynthesis of two insulin isoforms at the level of
both transcription and translation. This differential regulation became
apparent during prolonged in vitro stimulation by glucose,
leading to an increased ratio of insulin I over insulin II content at
excessively high glucose levels. The degree to which this duality is
expressed may vary with other regulatory factors. It is, therefore,
conceivable that prolonged exposure to high glucose leads to different
ratios of insulin I over insulin II content in purified ß-cells
compared with those in isolated islets and/or intact pancreatic tissue.
However, similar variations in the pancreatic content of both isoforms
were observed after prolonged in vivo activation of the
ß-cell population (9, 10); the present in vitro
observations may thus be representative of conditions of sustained
ß-cell stimulation rather than of a glucose-specific effect.
According to the present data, the dissociation between insulin I and
insulin II is the result of an impaired stimulation of insulin II
production rather than a preferential stimulation of insulin I
synthesis. It has yet to be examined whether chronically activated
ß-cells become resistant to the stimulatory effects of glucose on
proinsulin II gene transcription and/or translation.
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Acknowledgments
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The authors thank Erik Quartier for expert technical assistance,
and Nadine Van Slycke for secretarial support.
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Footnotes
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1 This work was supported by grants from the Belgian Fund for
Scientific Research (3.0057.94 and G3.3132.91), the Flemish Community
(Concert Action, GOA 92/97–1807), and the Juvenile Diabetes Foundation
International. 
Received August 18, 1997.
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Top
Abstract
Introduction
