1 Department of Metabolism and Clinical Nutrition, Graduate School of Medicine, Kyoto University, Kyoto 606 - 8507; 2 Third Department of Internal Medicine, Kyorin University Faculty of Medicine, Mitaka, Tokyo 181 - 8611; and 3 Department of Internal Medicine, Kitano Hospital, Osaka 530-0026, Japan
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ABSTRACT |
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We have found that preexposure
to an elevated concentration of glucose reversibly induces an
enhancement of basal insulin release from rat pancreatic islets
dependent on glucose metabolism. This basal insulin release augmented
by priming was not suppressed by reduction of the intracellular ATP or
Ca2+ concentration, because even in the absence of ATP at
low Ca2+, the augmentation was not abolished from primed
electrically permeabilized islets. Moreover, it was not inhibited by an
-adrenergic antagonist, clonidine. A threshold level of GTP is
required to induce these effects, because together with adenine,
mycophenolic acid, a cytosolic GTP synthesis inhibitor, completely
abolished the enhancement of basal insulin release due to the
glucose-induced priming without affecting the glucose-induced increment
in ATP content and ATP-to-ADP ratio. In addition, a GDP analog
significantly suppressed the enhanced insulin release due to priming
from permeabilized islets in the absence of ATP at low
Ca2+, suggesting that the GTP-sensitive site may play a
role in the augmentation of basal insulin release due to the
glucose-induced priming effect.
glucose-induced priming effect; basal insulin release; guanosine 5'-triphosphate; rat pancreatic islets
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INTRODUCTION |
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GLUCOSE IS THE
MOST IMPORTANT physiological regulator of insulin secretion from
pancreatic -cells. Insulin release from pancreatic
-cells is
augmented by a stimulatory level (>6 mM) of glucose, while a basal
level of insulin release is also observed at a subthreshold level of glucose.
The mechanism of the enhancement of insulin release at a stimulatory
concentration of glucose has been examined extensively. The
entry of glucose into the -cells is followed by an acceleration of
glycolysis and glucose oxidation that results in an increased ATP
content and ATP-to-ADP ratio that close the ATP-sensitive K+ channels (KATP channel). This produces a
decrease in K+ conductance that leads to membrane
depolarization with subsequent opening of the voltage-dependent
Ca2+ channels (VDCC). Ca2+ influx through the
VDCC then increases and leads to the rise in intracellular
Ca2+ concentration ([Ca2+]i )
that eventually triggers exocytosis of the insulin granules (17). This KATP channel-dependent pathway of
glucose action may contribute in determining the
[Ca2+]i in pancreatic
-cells according to
the extracellular glucose concentration. It has been shown that glucose
can also promote insulin release when the KATP channel is
kept in the open state using diazoxide, if
[Ca2+]i is elevated by a depolarizing
concentration of K+. This KATP
channel-independent action of glucose is also dependent on accelerated
glucose metabolism that correlates with increments in the ATP-to-ADP
ratio and may play an important role in increasing Ca2+
efficacy in stimulation-secretion coupling (1, 10, 11). Moreover, the stimulatory effect of glucose is observed under stringent
Ca2+-deprived conditions when protein kinase A (PKA) and
protein kinase C (PKC) are strongly activated simultaneously. This
Ca2+-independent action of glucose is also dependent on
glucose metabolism (24). From these observations, basal
insulin release occurs not only at a resting level of
[Ca2+]i but also at a low level of glucose
metabolic signals.
Glucose potentiates the insulin release triggered by glucose
itself and other fuel and nonfuel secretagogues. It has been demonstrated that a previous exposure within 60 min to an elevated concentration of glucose augments the insulin secretory response to a
second stimulation with glucose or other insulinotropic agent (2,
4, 13-15). This enhancing effect on insulin release from -cells is termed the priming effect, a time-dependent potentiation or "memory effect" of glucose. This priming effect of glucose has
been examined in the case of stimulated insulin release. However, there
are few reports regarding the priming effect of glucose on basal
insulin release.
Littman et al. (29) has previously reported that preexposure to a high concentration of glucose for 20 min augments basal insulin release in mouse. Because this phenomenon can be reversed by the addition of glutathione, it was regarded as a functional defect by Littman et al. However, there are some concerns about these results. In their experiments using islets without treatment of glutathione, the augmentation of insulin release in response to elevated glucose was poor compared with that observed in other examinations (8, 36, 41), possibly a result of islets that were oxidatively stressed or injured during the isolation procedure. Moreover, the priming effect on basal insulin release was determined only by comparison of the secretion after exposure to the preexposure secretion. No comparison of primed basal release with nonprimed basal release in a parallel simultaneous experiment was performed.
In the present study, our purpose was to find if a prior short exposure (within 30 min) to a high concentration of glucose augments basal insulin release, and we have demonstrated that basal insulin release is enhanced after such exposure. In addition, the mechanism of induction of this augmentation of basal insulin release by priming was investigated.
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METHODS |
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Materials.
Mycophenolic acid (MPA), oligomycin, clonidine, potassium
aspartate, and 12-O-tetradecanoyl-phorbol-13-acetate
(TPA), guanosine 5'-O-(2-thiodiphosphate) (GDPS),
mannoheptulose, ADP, phosphoenolpyruvate, RPMI 1640 medium,
and diazoxide were obtained from Sigma (St. Louis, MO). The oligomycin
is a mixture of several oligomycins (A, B, and C). ATP was purchased
from Kohjin (Tokyo, Japan). Rat insulin was obtained from Novo Nordisk
(Bagsvaerd, Denmark). Bisindolylmaleimide I was obtained from
Calbiochem-Novabiochem (La Jolla, CA). Cell-Tak was obtained from
Collaborative Biomedical Products (Bedford, MA). Fura 2-acetoxymethyl
ester (AM) was obtained from Molecular Probes (Eugene, OR). All other
reagents were of analytical grade and were obtained from Nacalai Tesque
(Kyoto, Japan). Clonidine, forskolin, TPA, diazoxide,
bisindolylmaleimide I, fura 2-AM, and oligomycin were first prepared in
DMSO and then were diluted to 1:1,000 with buffer. The final
concentration of DMSO did not exceed 0.2%, and the same concentration
of DMSO was added to control. MPA was first prepared in ethanol and
then diluted to 1:400 with RPMI medium. The same concentration of
ethanol was added to control.
Animals. Male Wistar rats were obtained from Shimizu (Kyoto, Japan). The animals were fed on standard lab chow ad libitum and allowed free access to water in an air-conditioned room with a 12:12-h light-dark cycle until used in the experiments. All experiments were carried out with rats aged 8-12 wk.
Solutions. The medium used for islet isolation and incubation of intact islets was Krebs-Ringer bicarbonate buffer, containing 119.4 mM NaCl, 3.7 mM KCl, 2.7 mM CaCl2, 1.3 mM KH2PO4, 1.3 mM MgSO4, 24.8 mM NaHCO3 (equilibrated with 5% CO2-95% O2, pH 7.4), 0.2% bovine serum albumin (BSA), and various concentrations of glucose, hereafter referred to as buffer A. The medium used for islet permeabilization and incubation of permeabilized islets was potassium aspartate buffer, containing 140 mM potassium aspartate, 7 mM MgSO4, 2.5 mM EGTA, 30 mM HEPES, and 0.5% BSA (pH 7.0), with CaCl2 added to give a Ca2+ concentration of 30 nM (and without ATP and glucose), hereafter referred to as buffer B. The Ca2+ concentration in buffer B was determined as previously described (9).
Measurement of insulin release from intact islets.
Islets of Langerhans were isolated from Wistar rats by collagenase
digestion as described previously (20). Islets were
cultured for 18 h in RPMI 1640 medium (containing 10% fetal calf
serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 5.5 mM
glucose) with or without test materials as indicated in RESULTS
(see Figs. 1-5 and Tables 1-9), at 37 °C in
humidified air containing 5% CO2. Insulin release
from intact islets was monitored using either static incubation or
perifusion conditions as described previously (9).
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Measurement of insulin and DNA content.
In the experiment in Table 2, after an
aliquot of incubation medium was taken, total insulin contents were
extracted by boiling the islets with 1 M acetic acid as described
previously (37) and were measured by RIA. In the
experiment with MPA-cultured islets to determine the insulin content
and the DNA content simultaneously, after an aliquot of final
incubation medium for insulin assay was taken (final volume 40 µl,
containing 5 islets), 360 µl of hypoosmotic 5 mM HEPES solution was
added to each tube. They were frozen at 20°C, after which they were
thawed and sonicated to lyse the islet cells. This procedure was
repeated three times. Determination of insulin content was performed by
RIA as described previously (20). The amount of
insulin content extracted by this method was not significantly
different from that extracted by acetic acid (data not shown). DNA
content was determined according to fluorometric assay as described by
Hopcroft et al. (19) using bisbenzimidazole (compound
Hoechst 33258, Nacalai Tesque) as fluorochrome and calf thymus DNA
(Type I; Sigma) as standard. Experiments using the same protocol were
repeated at least three times to ascertain reproducibility.
Measurement of [Ca2+]i. After cultured islets were washed in phosphate-buffered saline (PBS) and incubated with 0.25% trypsin and 1 mM EDTA solution (Life Technologies, Grand Island, NY) for 2 min at 37°C, digestion was terminated by rinsing the cells in cold PBS (9). They were washed in PBS again, placed on small glass coverslips (15 × 4 mm), coated with Cell-Tak to accelerate cell adhesion, and incubated in buffer A containing 2.8 mM glucose in humidified air containing 5% CO2 at 37°C until used for experiments. One micromole of fura 2-AM was then loaded with freshly dispersed islet cells for 30 min at 37°C. A heat-controlled chamber on the stage of an inverted microscope kept at 36 ± 1°C was superfused with buffer A containing 2.8 mM glucose and 10 mM HEPES adjusted to pH 7.4. The cells were exited successively at 340 and 380 nm, and the fluorescence emitted at 510 nm was captured by a charge-coupled device camera (Micro Max 5 MHz System, Roper Industries, Trenton, NJ). The images were analyzed with the Meta Fluor image analyzing system (Universal Imaging, West Chester, PA). The 340 nm (F340) and 380 nm (F380) fluorescence signals were detected every 10 s, and the ratios (F340/F380) were calculated. In vitro calibration was performed using a fura 2 calcium imaging calibration kit (Molecular Probes), and F340/F380 was converted into calibrated values of [Ca2+]i.
Measurement of insulin release from permeabilized islets. Islets were cultured in RPMI 1640 medium containing 5.5 mM glucose, for 18 h. They were preincubated with buffer A containing 2.8 mM glucose for 30 min. After the islets were washed, they were then divided into two groups; one group was exposed to buffer A containing 16.7 mM glucose while the other group of islets continued to be incubated with 2.8 mM glucose for another 30 min (priming period). After all the groups of islets were washed and incubated with buffer A containing 2.8 mM glucose again for the following 15 min (interval period), they were washed twice in cold buffer B. The islets were then permeabilized by high-voltage discharge (4 exposures each of 450-µs duration to an electrical field of 4.0 kV/cm) in cold buffer B and washed once with the same buffer (9). These procedures were completed within 5 min. Groups of five electrically permeabilized islets were then batch-incubated for 30 min at 37°C in 0.4 ml buffer B with or without test materials. Detailed protocols are indicated in RESULTS (see Figs. 1B and 5, and Table 6). At the end of the incubation period, permeabilized islets were pelleted by centrifugation (15,000 g, 180 s) and aliquots of the buffer were sampled for IRI determination. Experiments using the same protocol were repeated three times to ascertain reproducibility.
Measurement of nucleotide content.
After groups of cultured intact islets were preincubated at 2.8 mM
glucose for 30 min, they were batch-incubated in 1 ml buffer A containing 2.8 mM or 16.7 mM glucose at 37°C. The incubation was stopped by the addition of 0.2 ml of trichloroacetic acid (TCA) to
a final concentration of 5%. The tubes were immediately mixed with
vortex and then sonicated in the ice-cold water for 3 min. They were
then centrifuged (2,000 g, 180 s), and a fraction (0.9 ml) of the supernatant was mixed with 1 ml of water-saturated diethylether. The ether phase containing TCA was discarded. The step
was repeated four times. After the extracts (0.4 ml) were diluted with
0.1 ml 40 mM HEPES solution (final pH 7.4), they were frozen at
80°C until assays. ATP and ADP were assayed by a luminometric
method (6, 16). For measurement of the sum of ATP + ADP, ADP was first converted into ATP by adding 210 µl of solution
containing 20 mM HEPES (pH 7.75), 3 mM MgCl2, 1.5 mM
phosphoenolpyruvate, and 2.2 U/ml pyruvate kinase to 70 µl of the thawed extracts, with incubation at 37°C for 15 min. The ATP
concentration in the solutions was measured by adding 200 µl of
luciferin-luciferase solution (Turner Designs, Sunnyvale, CA) to a
fraction of sample (100 µl) in a bioluminometer (Luminometer Model
20e, Turner Designs, Sunnyvale, CA). The ATP concentration was shown as
a signal that was the integrated luminescence strength for 10 s
from 5 s after the reaction started. For the measurement of ATP,
the same procedure was performed, except that the incubation step was
done without pyruvate kinase. The ADP concentration was calculated as
the difference between the value of ATP + ADP and that of ATP from
the same sample. To draw a standard curve and ascertain that the
conversion of ADP into ATP was complete, blank, ADP, and ATP standards
were run through the entire procedure, including the extraction steps.
Experiments using the same protocol were repeated three times to
ascertain reproducibility.
Statistical analysis. Results are means ± SE. Statistical significance was evaluated by unpaired Student's t-test. P < 0.05 was considered significant.
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RESULTS |
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Glucose-induced priming effects on basal insulin release from intact islets. When the islets were incubated with 16.7 mM glucose for 30 min (priming time) and then incubated with 2.8 mM glucose for 15 min (interval time), basal insulin release (in the presence of 2.8 mM glucose and 5 mM K+) was significantly enhanced compared with controls. When the interval time was extended to 30 min and the priming time and glucose concentration during the priming time remained the same, basal insulin release was still significantly enhanced. However, when the interval period was extended to 60 min, the augmentation of basal insulin release was abolished (Table 1, group A). When the interval time was 15 min and the glucose concentration during the priming period was 16.7 mM, a 10-min exposure to glucose during the priming period produced a significant enhancement of basal insulin release, whereas a 5-min exposure did not bring about a significant enhancement (Table 1, group B). When the interval period was 15 min and the priming time 30 min, exposure to 8.3 mM glucose during the priming time enhanced basal insulin release significantly but exposure to 5.5 mM glucose did not (Table 1, group C). When the interval period was 15 min and the priming time 30 min, exposure to 16.7 mM glucose during the priming time enhanced basal insulin release significantly in the condition when the glucose concentration during final incubation was reduced to 0 mM or 1.4 mM (Table 1, group D). These augmentations of basal insulin release are not due to the increase in insulin content of the islets induced by priming, because the insulin content in the islets was not different in nonprimed and primed islets. Moreover, it does not correlate with the amount of insulin release during the priming period. Incubation with 16.7 mM glucose and 5 mM K+ brought about approximately the same amount of insulin release during the priming period as that produced by incubation with 2.8 mM glucose, 30 mM K+, and 5 µM forskolin, although basal insulin release was not enhanced in the latter case (Table 2). Prior exposure to 20 mM mannoheptulose, a glucokinase inhibitor, in addition to 2.8 mM glucose did not affect basal insulin release from islets, but exposure to 16.7 mM glucose with 20 mM mannoheptulose during the priming period significantly reduced enhancement of basal insulin release due to the glucose-induced priming effect (Table 3, group A). Augmentation of basal insulin release from diazoxide-treated hyperpolarized islets was not observed despite the presence of 16.7 mM glucose during the priming period, but once depolarization was introduced to the condition described above during the priming period, it was observed. Moreover, a reduction of Ca2+ concentration in the incubation medium during the priming period significantly decreased the augmentation of basal insulin release from diazoxide-treated islets induced by exposure to 16.7 mM glucose and a depolarizing concentration of K+ during the period (Table 3, group B). Fluorescence measurement of [Ca2+]i in dispersed islet cells in the presence of 16.7 mM glucose and 200 µM diazoxide revealed that depolarization induced by 30 mM K+ significantly increased [Ca2+]i and that extracellular Ca2+ omission significantly reduced [Ca2+]i in the 30 mM K+-depolarized condition (Table 4).
Time course of basal insulin release from high concentration
glucose-primed intact islets.
Before the priming period, insulin release in the presence of 2.8 mM
glucose was not significantly different in the two groups. One group of
islets then was exposed to 16.7 mM glucose for 30 min, while the other
continued to be exposed to 2.8 mM glucose (priming period). In the 16.7 mM glucose-primed islets, the typical biphasic glucose-induced insulin
release was observed during the priming period. At the end of priming
period, insulin release reached ~1 ng · 10 islets1 · min
1 (insulin
concentration in the sample, 3.6 ng/ml) (Fig. 2A). Both groups of islets were then exposed to 2.8 mM glucose for 90 min to
observe basal insulin release.
Effect of low intracellular Ca2+ and ATP concentration on enhanced basal insulin release induced by priming. In electrically permeabilized islets, the intracellular Ca2+ and ATP concentration can be manipulated according to the extracellular concentration without gross distortion of the architecture of the exocytotic apparatus (22). The fact that raising the Ca2+ concentration from 30 to 1,000 nM elicited approximately a six fold insulin release in the presence of 5 mM ATP and that ATP dose dependently increased insulin release in the presence of 1,000 nM Ca2+ from these islets (data were not shown) indicates that the exocytotic system is intact and that the [Ca2+]i and ATP can be manipulated effectively according to the extracellular medium in our preparation. To determine whether the augmentation of basal insulin release was brought about by the intracellular increase in the Ca2+ concentration or the ATP concentration, insulin release from ATP-deprived and very low (30 nM) Ca2+-clamped islets was examined by use of permeabilized islets incubated with 30 nM Ca2+ in the absence of ATP. Even in the absence of ATP, at 30 nM Ca2+, an enhancement of insulin release by the glucose-induced priming effect was still observed. To exclude minute intracellular metabolism as the cause of the enhancement of insulin release from the 16.7 mM glucose-primed islets at 30 nM Ca2+ without ATP, the value of insulin release in the presence of 4 µg/ml oligomycin (an inhibitor of mitochondrial respiratory chain; Ref. 43) and 20 mM 2-deoxyglucose (an inhibitor of glycolysis due to phosphoglucose isomerase inhibition; Refs. 47, 55) was measured. These metabolic inhibitors did not alter the insulin release significantly, and the values were still significantly larger than those of control islets (Table 6). Moreover, augmented basal insulin release from intact islets by the 16.7 mM glucose-induced priming effect was not suppressed by Ca2+ deprivation and 2 µg/ml oligomycin, at which concentration complete suppression of depolarization-induced insulin release was observed (Table 7, group A).
Effect of clonidine on enhanced basal insulin release induced by priming. Depolarization-induced insulin release from islets was almost completely suppressed by 10 µM clonidine: however, enhanced basal insulin release from 16.7 mM glucose-primed islets was not suppressed by 10 µM clonidine at all (Table 7, group B).
The high-concentration glucose-induced priming effect on basal
insulin release from TPA-cultured islets.
To determine the involvement PKC isozyme- activation in the
induction of priming, islets were cultured in medium containing 1 µM
TPA for 18 h to deplete PKC-
activity (18, 32,
52). Basal insulin release in the presence of 2.8 mM glucose
with 5 mM K+ from non-TPA-cultured islets was significantly
augmented by exposure to 16.7 mM glucose during the priming period
(Table 8A). Moreover, 100 nM
TPA significantly augmented the insulin release in the presence of a
depolarizing concentration (30 mM) of K+ and 2.8 mM glucose
from non-TPA-cultured, nonprimed islets (0.59 ± 0.06, control,
vs. 3.26 ± 0.21 ng · islet
1 · 30 min
1, 100 nM TPA-incubated, P < 0.01, n
= 5). On the contrary, 100 nM TPA had no insulinotropic effect on
insulin release from TPA-cultured, nonprimed islets in the presence of
30 mM K+ and 2.8 mM glucose, although an insulinotropic
effect of 2 mM dibutyryl cAMP (DBcAMP) was detected (1.64 ± 0.11, control, vs. 1.58 ± 0.12 ng · islet
1 · 30 min
1, 100 nM TPA-incubated, not significant, n = 5; control, vs.
3.11 ± 0.20 ng · islet
1 · 30 min
1, 2 mM DBcAMP-incubated, P < 0.01, n = 5). However, it was found that basal insulin release in
the presence of 2.8 mM glucose and 5 mM K+ from
TPA-cultured islets was significantly enhanced by priming with 16.7 mM
glucose (Table 8, group A). Moreover, addition of 1 µM
bisindolylmaleimide I, a known PKC (including PKC-
) inhibitor during
16.7 mM glucose preexposure did not suppress 16.7 mM
glucose-preexposure-induced augmentation of basal insulin release from
non-TPA-cultured islets (Table 8, group B).
Evaluation of islets cultured with MPA. To deplete cellular GTP content and maintain the ATP content and ATP-to-ADP ratio, islets were cultured with 25 µg/ml MPA and 150 µM adenine for 18 h (23, 31, 33, 35). Control islets were cultured with 150 µM adenine without MPA (23). No significant difference between the values of DNA content and insulin content of MPA-cultured islets and those of control islets was observed (DNA content, 30.0 ± 0.9, MPA-treated vs. 28.3 ± 0.8 ng/islet, control, n = 50, not significant; insulin content, 27.3 ± 1.4, MPA-treated vs. 24.8 ± 1.0 ng/islet, control, n = 50, not significant). During stimulation with 16.7 mM glucose for 30 min, the ATP content and ATP-to-ADP ratio of MPA-cultured and control islets were significantly increased ~1.4-fold and 1.6-fold those of basal 2.8 mM glucose-incubated islets, respectively. The basal and stimulated ATP content and ATP-to-DP ratio were not significantly different between MPA-cultured and control islets (Table 9).
The high-concentration glucose-induced priming effect on basal
insulin release from MPA-cultured islets.
In islets cultured without MPA, a significant enhancement of basal
insulin release in the presence of 2.8 mM glucose with 5 mM
K+ was observed from 16.7 mM glucose-preexposed islets
compared with that from 2.8 mM glucose-preexposed islets (0.39 ± 0.02, 16.7 mM glucose preexposed vs. 0.22 ± 0.03 ng/islet · 30 min, 2.8 mM glucose preexposed, n = 8, P < 0.01). On the other hand, in MPA-cultured islets,
a significant enhancement of basal insulin release was not observed
from 16.7 mM glucose preexposed islets (0.26 ± 0.02, 16.7 mM
glucose preexposed vs. 0.27 ± 0.03 ng · islet1 · 30 min
1, 2.8 mM glucose preexposed, n = 8, not significant).
However, the addition of 100 µM guanine in place of 150 µM adenine
in the culture completely reversed the inhibitory effect of MPA on the enhancement of basal insulin release induced by 16.7 mM glucose-priming (0.40 ± 0.01, 16.7 mM glucose preexposed vs. 0.24 ± 0.02 ng · islet
1 · 30 min
1, 2.8 mM glucose preexposed, n = 8, P < 0.01) (Fig. 3). In perifusion experiments
using MPA-cultured islets, biphasic insulin release was observed during
16.7 mM glucose exposure, although augmentation of basal insulin
release was not observed at all from 16.7 mM glucose-preexposed islets
after 6-min interval period (Fig. 4).
Effects of GDPS on enhanced basal insulin release induced by
priming from permeabilized islets.
To deduce the role of GTP-binding proteins (G proteins) in the
augmentation of basal insulin release by the glucose-induced priming
effect, we examined, using electrically permeabilized islets, to find
if, once insulin release is augmented by priming, which is not
suppressed by a reduction of intracellular ATP and [Ca2+]i, it can be suppressed by GDP
S.
GDP
S is expected to lock activated G proteins in an inactive state
(42). In the absence of ATP, at 30 nM Ca2+,
insulin release from 16.7 mM glucose-primed permeabilized islets was
significantly greater than that from control islets (0.41 ± 0.03, 16.7 mM glucose-primed vs. 0.24 ± 0.01 ng · islet
1 · 30 min
1,
control, n = 10, P < 0.01). Insulin release
without ATP, at 30 nM Ca2+ from control islets, was not
affected by 0.5 mM GDP
S, although that from 16.7 mM glucose-primed
islets was significantly suppressed by 0.5 mM GDP
S (0.30 ± 0.02 ng · islet
1 · 30 min
1,
n =1 0, P < 0.01) compared with that from 16.7 mM glucose-primed, control islets (Fig.
5).
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DISCUSSION |
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In this study, we observed an enhancement of basal insulin release that was induced by the exposure to 16.7 mM glucose within 10 min that can be reversed completely by ~60-min exposure to 2.8 mM glucose. The glucose-induced priming effect on stimulated insulin release has been explored by many investigators using dissected pancreases or isolated islets, but enhancement of basal release has not been discussed and, in many cases, was not observed (2, 4, 13-15). Many investigators used freshly dissected pancreases or isolated islets of ad libitum-fed animals, in which the primed condition is more directly affected by the postprandial milieu dependent on the different feeding states of the individual animals before death. On the contrary, the threshold level of glucose-cultured islets was used in our experiments to exclude these variables. It is also possible that, in the case of experiments using islets, the sensitivity of the RIA of insulin or the number of islets per chamber in the perifusion system was not sufficient to detect small changes in basal insulin release.
This augmentation does not represent irreversible leakage of insulin, because it is a reversible process. Moreover, it does not merely reflect the increase in insulin content of the islets and does not simply correlate to the amount of insulin release during the priming period. In addition, as previously reported in the case of the priming effect on stimulated insulin secretion (15), the induction of the priming effect on basal insulin release is also dependent on glucose metabolism, because the inclusion of mannoheptulose, a glucokinase inhibitor with a high concentration of glucose during the priming period, reduced the priming effect. Moreover, the induction of the priming effect on basal insulin release requires sufficient Ca2+ influx during the priming period: reduction of Ca2+ influx during the 16.7 mM glucose-treated priming process by diazoxide, a KATP channel opener, which inhibits glucose metabolism-induced membrane depolarization but does not inhibit glucose metabolism itself (1, 10, 11) abolished augmentation of basal insulin release by priming; increasing Ca2+ influx by introduction of 30 mM K+-induced depolarization in the presence of diazoxide and 16.7 mM glucose during the priming period restored it; and reduction of Ca2+ influx by decreasing the extracellular Ca2+ concentration in diazoxide, 16.7 mM glucose, and 30 mM K+- treated depolarized islets during the priming period attenuated it.
With the use of electrically permeabilized islets incubated at low
Ca2+ in the absence of ATP, in which intracellular
Ca2+ and ATP are clamped to low concentrations, it was
revealed that augmentation of basal insulin release by the
glucose-induced priming effect from already primed islets does not
necessarily require elevation of the [Ca2+]i
or ATP. This result using electrically permeabilized islets is
compatible with the results using intact islets, in which enhanced basal insulin release by priming was not suppressed even in the presence of oligomycin, which decreases the intracellular ATP concentration, and at a very low concentration of extracellular Ca2+, which decreases the
[Ca2+]i. Furthermore, this augmentation was
not inhibited by clonidine, a known -adrenergic blocker, which acts
at several sites in the stimulus-secretion coupling pathway, including
a late stage in the secretion process (40, 48), although
depolarization-induced insulin release was almost completely suppressed
by clonidine. This result is consistent with the previous result that
arachidonic acid-induced insulin release is also independent of changes
in intracellular Ca2+ or ATP and that this kind of
secretion also was not inhibited by
-adrenergic blocker, which
clearly shows an augmentation of insulin release independent of
intracellular Ca2+ or ATP, acting at a site distal to the
effect of
-adrenergic blocker (3). Our data also show
glucose to exert its effect at this stage in stimulus-secretion coupling.
This augmentation of basal insulin release by preexposure to a high concentration of glucose is not a delay in the shutting off of glucose-stimulated release. It has been reported that the intracellular ATP-to-ADP ratio and the intracellular Ca2+ return to basal level after glucose is shut off within 5 min (6, 21). Therefore, the stimulated concentration of glucose-preexposed insulin release after a 15-min interval is the insulin release under the condition where both intracellular ATP-to-ADP ratio and Ca2+ are at the basal level.
We tried to observe the progression of the basal insulin release in the presence of 16.7 mM glucose directly by abolishing Ca2+-stimulated insulin release, which obscures Ca2+-independent insulin release, using diazoxide, which can abolish Ca2+ influx by hyperpolarization in the presence of high glucose. But this failed, because to induce augmentation of basal insulin release requires not only metabolic signals in the presence of high glucose but also sufficient Ca2+ influx, and treatment with diazoxide inhibited this induction. However, augmentation was gradually induced and increased during high-concentration glucose exposure, indicated by the dependence on the duration of high-concentration glucose exposure.
Our results also indicate that the augmentation of basal insulin
release is different from the Ca2+-independent
insulinotropic action of glucose previously reported by Komatsu et
al. (23, 24), for the following reasons.
Ca2+-independent insulinotropic action of glucose is
induced under stringent Ca2+-free conditions, but the
augmentation of basal insulin release needs a sufficient level of
[Ca2+]i to be induced. The Ca2+-
independent action of glucose is completely suppressed by
-adrenergic blocker, but the augmentation of basal insulin release
is resistant to it. The Ca2+-independent action of glucose
is induced when PKA and PKC are strongly activated simultaneously, but
the augmentation of basal insulin release does not require strong activations.
We investigated the factors linked to glucose metabolism that are
involved in the induction of augmentation of basal insulin release by
glucose priming. Zawalich et al. (53, 56) reported that
prior exposure to cholinergic agonists or cholecystokinin, which
activate phosphoinositide hydrolysis, augmented subsequent insulin
secretory responses to glucose and that events that are associated with
persistent stimulant-induced increases in phosphoinositide hydrolysis
are involved in the induction and maintenance of glucose-induced priming effects (54). They proposed that PKC activation
may participate in the priming process induced by glucose
(51) and that PKC- activation may play an important
role in glucose-induced insulin secretion (52).
Accordingly, we examined the priming effect on basal insulin release by
glucose in PKC-
downregulated islets, which were cultured with a
high concentration of TPA, a known PKC activator (18, 32,
52), to evaluate the involvement of PKC-
activation in the
glucose-induced enhancement of basal insulin release. A depolarizing
concentration of K+-induced insulin secretion was enhanced
from these islets by DBcAMP, a PKA activator, but it was not by 100 nM
TPA, which did augment that from control islets. However, the
enhancement of basal insulin release due to glucose-induced priming was
nevertheless confirmed, indicating that PKC-
activation is not a
critical factor in the induction of the augmentation of basal insulin
release by the priming effect of glucose. However, the involvement of
other PKC isoforms that are not downregulated by TPA exposure
(46) in glucose-induced augmentation of basal insulin
release remains unknown.
It was previously reported that GTPS, a poorly hydrolyzable analog
of GTP, enhances insulin release from electrically permeabilized
-cells in the absence of Ca2+ (49, 50).
With the use of capacitance measurement of a single
-cell, it was
demonstrated that intracellular GTP or GTP
S stimulated exocytosis in
the absence of Ca2+ (42). These results
suggest that GTP augments insulin secretion in the exocytotic process
directly, Ca2+ independently. Moreover, Meredith et
al. (31) demonstrated that KATP
channel-independent augmentation of Ca2+-induced insulin
secretion by glucose (1, 10, 11), which may correlate with
the priming effect of glucose (45), was inhibited by MPA,
an inhibitor of cytosolic GTP synthesis. Therefore, we examined the
priming effect of glucose on basal insulin release in MPA-cultured
islets in which the intracellular GTP level was decreased (23,
31, 33, 35).
Augmentation of basal insulin release by the glucose-induced priming effect was completely abolished from MPA-cultured intact islets. In addition, the provision of guanine, which restores the GTP content of islets (23, 31, 33, 35), reversed the inhibitory effect of MPA on the enhanced basal insulin release due to glucose priming.
We tried to rule out the possibility that MPA also decreases ATP and the ATP-to-ADP ratio despite the provision of adenine, because the guanine and adenine pools have been reported to be tightly linked and so might not be affected specifically by MPA in mouse islets (7), although this was not observed in rat islets in other reports (23, 31). According to our measurements, the ATP-to-ADP ratio at 2.8 mM glucose and at 16.7 mM glucose were not significantly different between MPA-cultured and control islets together with adenine and, in addition, the elevation of the glucose concentration from 2.8 mM to 16.7 mM significantly increased them both ~1.6-fold. Because the DNA content and insulin content per islet were not significantly different between control and MPA-cultured islets in our study, the sizes of the stable pool of nucleotides in an islet, which occurs mainly in the insulin granules, and the diffusible pool, which occurs mainly in the cytosol (5), were not very different, the comparison of total nucleotide content is valid. Accordingly, induction of augmentation of basal insulin release by the glucose-induced priming effect cannot be fully explained only by the elevation of ATP-to-ADP ratio during the priming period and appears to require a threshold concentration of GTP, although the possibility that a threshold level of the ATP-to-ADP ratio also is required for the induction of priming and the participation of other metabolic signals, including unknown mitochondrial factors (30), cannot be ruled out.
Recently, it has been found that low molecular weight G proteins and
-subunits of trimeric G proteins undergo glucose-induced posttranslational modifications and may play an important role in
glucose-induced insulin release in pancreatic
-cells
(25-27). To examine the role of the GTP-sensitive
site in the glucose-induced augmentation of basal insulin release, we
used permeabilized islets to determine if, once insulin release is
augmented by the glucose-induced priming effect, which was observed
even in the absence of ATP at low Ca2+, it could be
reversibly suppressed by GDP
S, a GDP stable analog. The fact that
GDP
S significantly suppressed the enhanced insulin release by
priming in the absence of ATP at low Ca2+ but did not
affect that from nonprimed islets suggests that the GTP-sensitive site
may play a role in the augmentation of basal insulin release due to
glucose-induced priming. In addition, the finding that, once augmented,
basal release can be suppressed in the presence of GDP
S, indicates
clearly that this augmentation is not derived from slow washout of
earlier released insulin.
It is well established that glucose elicits a biphasic insulin release
from islets. The second phase (after ~10 min exposure) of insulin
release is characterized by a gradually rising response. This rising
response cannot be explained simply by
[Ca2+]i elevation and/or by the elevation of
metabolic signals, including the ATP-to-ADP ratio, because elevated
[Ca2+]i and the ATP-to-ADP ratio do not
elicit a rising response (6, 20, 28, 34). Therefore,
factors other than Ca2+ and the ATP-to-ADP ratio are
required to explain this phenomenon. The augmentation of basal insulin
release induced by a stimulatory concentration of glucose is
Ca2+ and ATP independent and may increase mainly during
second phase of insulin release, according to the results of the
experiments on the time dependency of the induction. Therefore, this
augmentation may be a small part of the rising response in the second
phase of insulin release and may play a more important role if the
augmentation has a synergistic effect on Ca2+-induced
insulin release. But the physiological relevance of this phenomenon in
vivo is uncertain, because our examination was not performed using
nutrient-rich media. It was revealed, recently, that an increase in
oxygen consumption and thus the increase in the intracellular level of
ATP on glucose stimulation are not observed when pancreatic -cells
are incubated in nutrient-rich media, although glucose-induced insulin
release is normally increased (12, 38, 39). Because these
phenomena were not reported in the experiments using isolated islets,
further studies dealing with insulin release from isolated islets using
nutrient-rich media must be performed.
In conclusion, prior exposure to glucose reversibly enhances basal
insulin release from rat pancreatic islets. This priming process is
induced dependent on glucose metabolism and requires sufficient
Ca2+ influx. Basal insulin release once augmented by
priming was not abolished by reducing the intracellular ATP and
[Ca2+]i and was not suppressed by an
-adrenergic antagonist. In addition, the GTP-sensitive site may play
a role in this augmentation of basal insulin release, because such
augmentation was not observed in GTP-depleted islets, and GDP analog
significantly suppressed the augmentation in ATP- and
Ca2+-depleted permeabilized islets.
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ACKNOWLEDGEMENTS |
---|
This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan; Grants-in-Aid for Creative Basic Research (10NP0201) from the Ministry of Education, Science, Sports and Culture of Japan; and by a grant from "Research for the Future" Program of the Japan Society for the Promotion of Science (JSPS-RFTF97I00201).
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FOOTNOTES |
---|
Address for reprint requests and other correspondence: S Fujimoto, Dept. of Metabolism and Clinical Nutrition, Graduate School of Medicine, Kyoto Univ., 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan (E-mail: fujimoto{at}metab.kuhp.kyoto-u.ac.jp).
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.
Received 29 November 1999; accepted in final form 19 May 2000.
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