Augmentation of basal insulin release from rat islets by preexposure to a high concentration of glucose

Shimpei Fujimoto1, Yoshiyuki Tsuura1, Hitoshi Ishida2, Kazuo Tsuji3, Eri Mukai1, Mariko Kajikawa1, Yoshiyuki Hamamoto1, Tomomi Takeda1, Yuichiro Yamada1, and Yutaka Seino1

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

GLUCOSE IS THE MOST IMPORTANT physiological regulator of insulin secretion from pancreatic beta -cells. Insulin release from pancreatic beta -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 beta -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 beta -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 beta -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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Mycophenolic acid (MPA), oligomycin, clonidine, potassium aspartate, and 12-O-tetradecanoyl-phorbol-13-acetate (TPA), guanosine 5'-O-(2-thiodiphosphate) (GDPbeta S), 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).


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 1.   Schematic presentation of protocols in static incubation of intact islets (A) and of permeabilized islets (B). Protocols were performed basically as indicated by bold characters, although some experiments were performed as indicated in parentheses. Detailed protocols are indicated in RESULTS (see Figs. 1-5 and Tables 1-9). Buffer A contains 5 mM K+ and 2.7 mM Ca2+, unless otherwise specified. Nos. are mM concentration of glucose, K+ and Ca2+. G, glucose; Fors, forskolin; Mh, mannoheptulose; Dz, diazoxide; Bis, bisindolylmaleimide I; 2-DG, 2-deoxyglucose; GDPbeta S, guanosine 5'-O-(2-thiodiphosphate).



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 2.   A and B: time course of basal insulin release from 16.7 mM glucose-primed intact islets. Values in A and in B are means ± SE of 5 determinations in same experiment. Two groups of cultured islets were preincubated with 2.8 mM glucose for 30 min. In 1 group of islets, medium containing 2.8 mM glucose was changed to that containing 16.7 mM glucose at -30 min (priming period). In primed group, medium containing 16.7 mM glucose was then changed to that containing 2.8 mM glucose at 0 min to observe basal insulin release. Another group of islets continued to be incubated with 2.8 mM glucose during priming period and final incubation. Values from -35 to 15 min are indicated in A, and those from 9 to 90 min are indicated in (B). G, glucose. To convert insulin release in ng · 10 islets-1 · min-1 to ng/ml, multiply by 3.6. (A) All values measured except at -35, -33, -32, -30, -29, and -28 min were significantly greater in 16.7 mM glucose-primed islets than corresponding values in control islets (P < 0.01, except 6-15 min, P < 0.05). A ( inset): time course of washout of insulin and glucose in perifusion system. In absence of islets, medium containing 16.7 mM glucose and 3.8 ng/ml insulin was changed to that containing 2.8 mM glucose without insulin at 0 min. x, not detected (<0.1 ng/ml). B: values from 9 to 30 min were significantly greater with 16.7 mM glucose-primed islets than corresponding values in control islets (dagger P < 0.05). C: time course of basal insulin release from 8.3 mM glucose-primed intact islets. Experimental protocol was same as in A and B except 8.3 mM glucose was used instead of 16.7 mM glucose during priming period. (C, inset) Values from 15 to 30 min from same experiment as C are indicated. Values are means ± SE of 6 determinations in same experiment. D: time course of basal insulin release from intact islets after exposure to 5 µM forskolin and 30 mM K+. Two groups of cultured islets were preincubated with 2.8 mM glucose and 5 mM K+ for 30 min. In 1 group of islets, medium was changed to that containing 2.8 mM glucose, 5 µM forskolin and 30 mM K+ at -30 min and was then returned to that containing 2.8 mM glucose at 0 min to observe basal insulin release. Another group of control islets continued to be incubated with 2.8 mM glucose and 5 mM K+during observation. Values are means ± SE of 6 determinations in same experiment.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of 16.7 mM glucose-induced priming on basal insulin release from intact islets cultured in medium containing 150 µM adenine (control), 150 µM adenine plus 25 µg/ml mycophenolic acid (MPA), or 100 µM guanine + 25 µg/ml MPA for 18 h. Values are means ± SE of 8 determinations in same experiment. They were preincubated with buffer A containing 2.8 mM glucose for 30 min. Each group of islets was then subdivided into 2 groups, 1 of which was exposed to 16.7 mM glucose and another that continued to be incubated with 2.8 mM glucose for another 30 min (priming period). After all groups of islets were incubated with 2.8 mM glucose again for the next 15 min (interval period), they were incubated with 2.8 mM glucose for another 30 min (final incubation period), and basal insulin release was measured. * Significant at P < 0.01 vs. corresponding nonprimed controls.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 4.   Time course of basal insulin release from 16.7 mM glucose-preexposed intact islets cultured in medium containing 150 µM adenine + 25 µg/ml MPA. Values are means ± SE of 6 determinations in same experiment. After being cultured in medium containing 150 µM adenine + 25 µg/ml MPA for 18 h, 2 groups of cultured islets were preincubated with 2.8 mM glucose and for 30 min. In 1 group of islets, medium was changed to that containing 16.7 mM glucose at -30 min and was then returned to that containing 2.8 mM glucose at 0 min to observe basal insulin release. Another group of control islets continued to be incubated with 2.8 mM glucose during observation.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of GDPbeta S on enhanced insulin release by 16.7 mM glucose-induced priming from electrically permeabilized islets at 30 nM Ca2+ in the absence of ATP. Values are means ± SE of 10 determinations in same experiment. Cultured islets were preincubated with buffer A containing 2.8 mM glucose for 30 min. They were then subdivided into 2 groups, 1 of which was exposed to 16.7 mM glucose and another which continued to be incubated with 2.8 mM glucose for another 30 min (priming period). After all groups of islets were incubated with 2.8 mM glucose again for the next 15 min (interval period), they were permeabilized and incubated with buffer B without ATP at 30 nM Ca2+ with or without 0.5 mM GDPbeta S for 30 min (final incubation), and insulin release was measured. GDPbeta S was present only during final incubation. * P < 0.01 vs. nonprimed, incubated without GDPbeta S; dagger  P < 0.01 vs. 16.7 mM glucose-primed, incubated without GDPbeta S.


                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Augmentation of basal insulin release from intact islets by prior exposure to a high concentration of glucose


                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Insulin release during priming period, insulin content at end of priming period and basal insulin release during final incubation


                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Inhibition of glucose-induced priming effect on basal insulin release by exposure to 20 mM MH and by decreasing Ca2+ influx during priming period


                              
View this table:
[in this window]
[in a new window]
 
Table 4.   Fluorescence measurement of [Ca2+]i in presence of 16.7 mM and 200 µM diazoxide in dispersed islet cells


                              
View this table:
[in this window]
[in a new window]
 
Table 5.   Insulin release during priming period, and basal insulin release after priming, calculated from perifusion experiments


                              
View this table:
[in this window]
[in a new window]
 
Table 6.   Enhanced insulin release from electrically permeabilized islets incubated in the absence of ATP by glucose-induced priming effect


                              
View this table:
[in this window]
[in a new window]
 
Table 7.   Effect of oligomycin, Ca2+ deprivation, and clonidine on basal insulin release from intact islets primed by glucose


                              
View this table:
[in this window]
[in a new window]
 
Table 8.   Augmentation of basal insulin release from TPA-cultured islets by preexposure to high-concentration glucose and from non-TPA-cultured islets by preexposure to high-concentration glucose with bisindolylmaleimide


                              
View this table:
[in this window]
[in a new window]
 
Table 9.   Effect of incubation and glucose on ATP content and ATP-to-ADP ratio in MPA-cultured islets and control islets

For static incubation experiments (Fig. 1A), unless otherwise specified, preincubation, incubation during priming period and interval period, and final incubation were performed in buffer A at 37°C. The cultured islets were preincubated with 2.8 mM glucose for 30 min. After groups of islets were exposed to 16.7 mM glucose or continued to be exposed to 2.8 mM glucose for 30 min (priming period) with subsequent incubation with 2.8 mM glucose for 15 min (interval period), groups of five islets were then incubated in 0.4 ml of medium for 30 min to observe basal insulin release in the presence of 2.8 mM glucose at 5 mM K+ (final incubation period). At the end of the preincubation and interval period, the spent medium was discarded, and the islets were washed once with cold buffer A containing 2.8 mM glucose, and at the end of priming period, they were washed twice. Each washing procedure was completed within 1 min. In the experiments summarized in Table 1, the concentrations of glucose during the priming period and during the final incubation and duration of preincubation, priming, and interval were changed. Detailed protocols are indicated in RESULTS (see Figs. 1-5 and Tables 1-9). At the end of the final incubation period, islets were pelleted by centrifugation (10,000 g, 180 s), and aliquots of the buffer were sampled.

For perifusion experiments, groups of 25 islets were placed in each of the parallel chambers of a perifusion apparatus and perifused with buffer A supplemented with 2.8 mM glucose and 10 mM HEPES adjusted to pH 7.4, at a rate of 0.7 ml/min at 37°C. The medium was continuously gassed with 95% O2-5% CO2. Islets were usually perifused for 30 min to establish a stable insulin secretory rate at the basal level of glucose. The priming process and the incubation to observe basal insulin release then were performed. The time intervals at which perifusate samples were collected and the detailed protocols are indicated in Figs. 2 and 4.

The amount of immunoreactive insulin (IRI) was determined by RIA using rat insulin as standard (20). Experiments using the same protocol were repeated at least three times to ascertain reproducibility.

In the experiment summarized in Fig. 2A (inset), after the medium containing 16.7 mM glucose and 3.8 ng/ml rat insulin was allowed to flow in the absence of islets at a rate of 0.7 ml/min for 30 min, the medium containing 2.8 mM glucose without insulin was then added at the same rate. The insulin concentration and glucose concentration in the collected samples were then determined by RIA and by the glucose oxidase method as described previously (20), respectively.

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 islets-1 · 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.

To determine, in a perifusion apparatus, the time of the washout of released insulin from stimulated islets during a priming period with a high concentration of glucose in the priming medium, the medium containing 16.7 mM glucose and 3.8 ng/ml insulin was changed to a medium containing 2.8 mM glucose without insulin, in the absence of islets. In this condition, the glucose concentration decreased to a subthreshold level (<4 mM) within 3 min, and no insulin was detected within 6 min (Fig. 2A, inset). According to this result, in the presence of islets, infusion of medium containing 2.8 mM glucose for 9 min after cessation of infusion with medium containing 16.7 mM glucose is sufficient to determine the precise basal insulin release.

Basal insulin release from primed islets at 9 min was significantly greater than control (0.22 ± 0.03, 16.7 mM glucose-primed vs. 0.10 ± 0.02 ng · 10 islets-1 · min-1 control, n = 5, P < 0.05). Moreover, the basal insulin release from primed islets decreased gradually after 9 min (~2.5 × 10-3 ng · 10 islets-1 · min-2). Basal insulin release at 30 min was still significantly greater than that of the control group (0.17 ± 0.02, 16.7 mM glucose-primed vs. 0.09 ± 0.02 ng · 10 islets-1 · min-1, control, at 30 min, n = 5, P < 0.05). No significant augmentation of basal insulin release was observed after 40 min (Fig. 2B).

Basal insulin release after 8.3 mM glucose exposure was also slightly augmented compared with control (Fig. 2C). Total basal release after a 15-min interval for 30 min (from 15 min to 45 min in Fig. 2C) was significantly augmented in the 8.3 mM glucose-primed group compared with control (P < 0.01; Table 5; experiment 2).

Basal insulin release after 5 µM forskolin and 30 mM K+ exposure in the presence of 2.8 mM glucose was not enhanced at all (Fig. 2D). Total basal release after 15-min interval for 30 min (from 15 min to 45 min in Fig. 2D) was not significantly augmented (Table 5, experiment 3).

Afterward, the amount of insulin release was determined under static incubation, both because of the small discrepancy observed between static and dynamic methods (Tables 1, 2, and 5) and because experiments under more various conditions can be done simultaneously.

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-alpha activation in the induction of priming, islets were cultured in medium containing 1 µM TPA for 18 h to deplete PKC-alpha 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-alpha ) 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 · islet-1 · 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 GDPbeta S 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 GDPbeta S. GDPbeta 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 GDPbeta S, although that from 16.7 mM glucose-primed islets was significantly suppressed by 0.5 mM GDPbeta 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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha -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 alpha -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 alpha -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-alpha 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-alpha downregulated islets, which were cultured with a high concentration of TPA, a known PKC activator (18, 32, 52), to evaluate the involvement of PKC-alpha 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-alpha 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 GTPgamma S, a poorly hydrolyzable analog of GTP, enhances insulin release from electrically permeabilized beta -cells in the absence of Ca2+ (49, 50). With the use of capacitance measurement of a single beta -cell, it was demonstrated that intracellular GTP or GTPgamma 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 gamma -subunits of trimeric G proteins undergo glucose-induced posttranslational modifications and may play an important role in glucose-induced insulin release in pancreatic beta -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 GDPbeta S, a GDP stable analog. The fact that GDPbeta 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 GDPbeta 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 beta -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 alpha -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.


    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).


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aizawa, T, Sato Y, Ishihara F, Taguchi N, Komatsu M, Suzuki N, Hashizume K, and Yamada T. ATP-sensitive K+ channel-independent glucose action in rat pancreatic beta -cell. Am J Physiol Cell Physiol 266: C622-C627, 1994[Abstract/Free Full Text].

2.   Ashby, JP, and Shirling D. Evidence for priming and inhibitory effects of glucose on insulin secretion from isolated islets of Langerhans. Diabetologia 18: 417-421, 1980[ISI][Medline].

3.   Band, AM, Jones PM, and Howell SL. The mechanism of arachidonic acid-induced insulin secretion from rat islets of Langerhans. Biochim Biophys Acta 1176: 64-68, 1993[ISI][Medline].

4.   Chalmers, JA, and Sharp GWG The importance of Ca2+ for glucose-induced priming in pancreatic islets. Biochim Biophys Acta 1011: 46-51, 1989[ISI][Medline].

5.   Detimary, P, Jonas JC, and Henquin JC. Stable and diffusible pools of nucleotides in pancreatic islet cells. Endocrinology 137: 4671-4676, 1996[Abstract].

6.   Detimary, P, Van den Berghe G, and Henquin JC. Concentration dependence and time course of the effects of glucose on adenine and guanine nucleotides in mouse pancreatic islets. J Biol Chem 271: 20559-20565, 1996[Abstract/Free Full Text].

7.   Detimary, P, Xiao C, and Henquin JC. Tight links between adenine and guanine nucleotide pools in mouse pancreatic islets: a study with mycophenolic acid. Biochem J 324: 467-471, 1997[ISI][Medline].

8.   Flamez, D, Van-Breusegem A, Scrocchi LA, Quartier E, Pipeleers D, Drucker DJ, and Schuit F. Mouse pancreatic beta -cells exhibit preserved glucose competence after disruption of the glucagon-like peptide-1 receptor gene. Diabetes 47: 646-652, 1998[Abstract].

9.   Fujimoto, S, Ishida H, Kato S, Okamoto Y, Tsuji K, Mizuno N, Ueda S, Mukai E, and Seino Y. The novel insulinotropic mechanism of pimobendan: direct enhancement of the exocytotic process of insulin secretory granules by increased Ca2+ sensitivity in beta -cells. Endocrinology 139: 1133-1140, 1998[Abstract/Free Full Text].

10.   Gembal, M, Detimary P, Gilon P, Gao ZY, and Henquin JC. Mechanisms by which glucose can control insulin release independently from its action on adenosine triphosphate-sensitive K+ channels in mouse B cells. J Clin Invest 91: 871-880, 1993[ISI][Medline].

11.   Gembal, M, Gilon P, and Henquin JC. Evidence that glucose can control insulin release independently from its action on ATP-sensitive K+ channels in mouse B cells. J Clin Invest 89: 1288-1295, 1992[ISI][Medline].

12.   Ghosh, A, Ronner P, Cheong E, Khalid P, and Matschinsky FM. The role of ATP and free ADP in metabolic coupling during fuel-stimulated insulin release from islet beta -cells in the isolated perfused rat pancreas. J Biol Chem 266: 22887-22892, 1991[Abstract/Free Full Text].

13.   Grill, V. Time and dose dependencies for priming effect of glucose on insulin secretion. Am J Physiol Endocrinol Metab 240: E24-E31, 1981[Abstract/Free Full Text].

14.   Grill, V, Adamson U, and Cerasi E. Immediate and time-dependent effects of glucose on insulin release from rat pancreatic tissue. Evidence for different mechanisms of action. J Clin Invest 61: 1034-1043, 1978[ISI][Medline].

15.   Grill, V, and Rundfeldt M. Effects of priming with D-glucose on insulin secretion from rat pancreatic islets: increased responsiveness to other secretagogues. Endocrinology 105: 980-987, 1979[ISI][Medline].

16.   Hampp, R. Luminometric method. In: Methods of Enzymatic Analysis, edited by Bergmeyer HU.. Weinheim: Verlagsgeselschaft, 1986, p. 370-379.

17.   Henquin, JC. Cell biology of insulin secretion. In: Joslin's Diabetes Mellitus, 13th ed, edited by Kahn CR, and Weir GC.. Malvern, PA: Lea and Febiger, 1994, p. 56-80.

18.   Hii, CST, Jones PM, Persaud SJ, and Howell SL. A re-assessment of the role of protein kinase C in glucose-stimulated insulin secretion. Biochem J 246: 489-493, 1987[ISI][Medline].

19.   Hopcroft, DW, Mason DR, and Scott RS. Standardization of insulin secretion from pancreatic islets: validation of a DNA assay. Horm Metab Res 17: 559-561, 1985[ISI][Medline].

20.   Kato, S, Ishida H, Tsuura Y, Tsuji K, Nishimura M, Horie M, Taminato T, Ikehara S, Okada H, Ikeda H, Okada Y, and Seino Y. Alterations in basal and glucose-stimulated voltage-dependent Ca2+ channel activities in pancreatic beta  cells of noninsulin-dependent diabetes mellitus GK rats. J Clin Invest 97: 2417-2425, 1996[Abstract/Free Full Text].

21.   Kennedy, ED, Rizzuto R, Theler JM, Pralong WF, Bastianutto C, Pozzan T, and Wollheim CB. Glucose-stimulated insulin secretion correlates with changes in mitochondrial and cytosolic Ca2+ in aequorin-expressing INS-1 cells. J Clin Invest 98: 2524-2538, 1996[Abstract/Free Full Text].

22.   Knight, DE, and Scrutton MC. Gaining access to the cytosol: the technique and some applications of electropermeabilization. Biochem J 234: 497-506, 1986[ISI][Medline].

23.   Komatsu, M, Noda M, and Sharp GWG Nutrient augmentation of Ca2+-dependent and Ca2+-independent pathways in stimulus-coupling to insulin secretion can be distinguished by their guanosine triphosphate requirements: studies on rat pancreatic islets. Endocrinology 139: 1172-1183, 1998[Abstract/Free Full Text].

24.   Komatsu, M, Schermerhorn T, Aizawa T, and Sharp GWG Glucose stimulation of insulin release in the absence of extracellular Ca2+ and in the absence of any increase in intracellular Ca2+ in rat pancreatic islets. Proc Natl Acad Sci USA 92: 10728-10732, 1995[Abstract].

25.   Kowluru, A, Li G, and Metz SA. Glucose activates the carboxyl methylation of gamma  subunits of trimeric GTP-binding proteins in pancreatic beta  cells. Modulation in vivo by calcium, GTP, and pertussis toxin. J Clin Invest 100: 1596-1610, 1997[Abstract/Free Full Text].

26.   Kowluru, A, Li G, Rabaglia ME, Segu VB, Hofmann F, Aktories K, and Metz SA. Evidence for differential roles of the Rho subfamily of GTP-binding proteins in glucose- and calcium-induced insulin secretion from pancreatic beta  cells. Biochem Pharmacol 54: 1097-1108, 1997[ISI][Medline].

27.   Kowluru, A, Seavey SE, Li G, Sorenson RL, Weinhaus AJ, Nesher R, Rabaglia ME, Vadakekalam J, and Metz SA. Glucose- and GTP-dependent stimulation of the carboxyl methylation of Cdc 42 in rodent and human pancreatic islets and pure beta  cells. Evidence for an essential role of GTP-binding proteins in nutrient-induced insulin secretion. J Clin Invest 98: 540-555, 1996[Abstract/Free Full Text].

28.   Lambillotte, C, Gilon P, and Henquin JC. Direct glucocorticoid inhibition of insulin secretion. An in vitro study of dexamethasone effects in mouse islets. J Clin Invest 99: 414-423, 1997[Abstract/Free Full Text].

29.   Littman, ED, Opara EC, and Akwari OE. Glutathione-mediated preservation and enhancement of isolated perifused islet function. J Surg Res 59: 694-698, 1995[ISI][Medline].

30.   Maechler, P, Kennedy ED, Pozzan T, and Wollheim CB. Mitochondrial activation directly triggers the exocytosis of insulin in permeabilized pancreatic beta -cells. EMBO J 16: 3833-3841, 1997[Abstract/Free Full Text].

31.   Meredith, M, Rabaglia ME, and Metz SA. Evidence of a role for GTP in the potentiation of Ca2+-induced insulin secretion by glucose in intact rat islets. J Clin Invest 96: 811-821, 1995[ISI][Medline].

32.   Metz, SA. Is protein kinase C required for physiologic insulin release? Diabetes 37: 3-7, 1988[Abstract].

33.   Metz, SA, Meredith M, Rabaglia ME, and Kowluru A. Small elevations of glucose concentration redirect and amplify the synthesis of guanosine 5'-triphosphate in rat islets. J Clin Invest 92: 872-882, 1993[ISI][Medline].

34.   Metz, SA, Meredith M, Vadakekalam J, Rabaglia ME, and Kowluru A. A defect late in stimulus-secretion coupling impairs insulin secretion in Goto-Kakizaki diabetic rats. Diabetes 48: 1754-1762, 1999[Abstract].

35.   Metz, SA, Rabaglia ME, and Pintar TJ. Selective inhibitors of GTP synthesis impede exocytotic insulin release from intact rat islets. J Biol Chem 267: 12517-12527, 1992[Abstract/Free Full Text].

36.   Miyawaki, K, Yamada Y, Yano H, Niwa H, Ban N, Ihara Y, Kubota A, Fujimoto S, Kajikawa M, Kuroe A, Tsuda K, Hashimoto H, Yamashita T, Jomori T, Tashiro F, Miyazaki J, and Seino Y. Glucose intolerance caused by a defect in the entero-insular axis: a study in gastric inhibitory polypeptide receptor knockout mice. Proc Natl Acad Sci USA 96: 14843-14847, 1999[Abstract/Free Full Text].

37.   Okamoto, Y, Ishida H, Taminato T, Tsuji K, Kurose T, Tsuura Y, Kato S, Imura H, and Seino Y. Role of cytosolic Ca2+ in impaired sensitivity to glucose of rat pancreatic islets exposed to high glucose in vitro. Diabetes 41: 1555-1561, 1992[Abstract].

38.   Papas, KK, and Jarema MAC Glucose-stimulated insulin secretion is not obligatorily linked to an increase in O2 consumption in beta HC9 cells. Am J Physiol Endocrinol Metab 275: E1100-E1106, 1998[Abstract/Free Full Text].

39.   Papas, KK, Long RC, Jr, Constantinidis I, and Sambanis A. Role of ATP and Pi in the mechanism of insulin secretion in the mouse insulinoma beta TC3 cell line. Biochem J 326: 807-814, 1997[ISI][Medline].

40.   Persaud, SJ, Jones PM, and Howell SL. Effects of Bordetella pertussis toxin on catecholamine inhibition of insulin release from intact and electrically permeabilized rat islets. Biochem J 258: 669-675, 1989[ISI][Medline].

41.   Pontoglio, M, Sreenan S, Roe M, Pugh W, Ostrega D, Doyen A, Pick AJ, Baldwin A, Velho G, Froguel P, Levisetti M, Bonner-Weir S, Bell GI, Yaniv M, and Polonsky KS. Defective insulin secretion in hepatocyte nuclear factor 1alpha -deficient mice. J Clin Invest 101: 2215-2222, 1998[Abstract/Free Full Text].

42.   Proks, P, Eliasson L, Ämmälä C, Rorsman P, and Ashcroft FM. Ca2+- and GTP-dependent exocytosis in mouse pancreatic beta -cells involves both common and distinct steps. J Physiol (Lond) 496: 255-264, 1996[Abstract].

43.   Rutenbeck, I, Herrmann C, and Grimmsmann T. Energetic requirement of insulin secretion distal to calcium influx. Diabetes 46: 1305-1311, 1997[Abstract].

44.   Sener, A, and Malaisse WJ. The metabolism of glucose in pancreatic islets. Diabetes Metab 4: 127-133, 1978[ISI].

45.   Taguchi, N, Aizawa T, Sato Y, Ishihara F, and Hashizume K. Mechanism of glucose-induced biphasic insulin release: Physiological role of adenosine triphosphate-sensitive K+ channel-independent glucose action. Endocrinology 136: 3942-3948, 1995[Abstract].

46.   Tang, SH, and Sharp GWG Atypical protein kinase C isozyme mediates carbachol-stimulated insulin secretion in RINm5F cells. Diabetes 47: 905-912, 1998[Abstract].

47.   Tsuura, Y, Ishida H, Hayashi S, Sakamoto K, Horie M, and Seino Y. Nitric oxide opens ATP-sensitive K+ channels through suppression of phosphofructokinase activity and inhibits glucose-induced insulin release in pancreatic beta  cells. J Gen Physiol 104: 1079-1099, 1994[Abstract].

48.   Ullrich, S, and Wollheim CB. GTP-dependent inhibition of insulin secretion by epinephrine in permeabilized RINm5F cells. Lack of correlation between insulin secretion and cyclic AMP levels. J Biol Chem 263: 8615-8620, 1988[Abstract/Free Full Text].

49.   Vallar, L, Biden TJ, and Wollheim CB. Guanine nucleotides induce Ca2+-independent insulin secretion from permeabilized RINm5F cells. J Biol Chem 262: 5049-5056, 1987[Abstract/Free Full Text].

50.   Wollheim, CB, Ullrich S, Meda P, and Vallar L. Regulation of exocytosis in electrically permeabilized insulin-secreting cells. Evidence for Ca2+ dependent and independent secretion. Biosci Rep 7: 443-454, 1987[ISI][Medline].

51.   Zawalich, WS. Regulation of insulin secretion by phosphoinositide-specific phospholipase C and protein kinase C activation. Diabetes Rev 4: 160-176, 1996.

52.   Zawalich, WS, Bonnet-Eymard M, Zawalich KC, and Yaney GC. Chronic exposure to TPA depletes PKCalpha and augments Ca-dependent insulin secretion from cultured rat islets. Am J Physiol Cell Physiol 274: C1388-C1396, 1998[Abstract/Free Full Text].

53.   Zawalich, WS, and Diaz VA. Prior cholecystokinin exposure sensitizes islets of Langerhans to glucose stimulation. Diabetes 36: 118-122, 1987[Abstract].

54.   Zawalich, WS, Diaz VA, and Zawalich KC. Role of phosphoinositide metabolism in induction of memory in isolated perifused rat islets. Am J Physiol Endocrinol Metab 254: E609-E616, 1988[Abstract/Free Full Text].

55.   Zawalich, WS, Dye ES, Rognstad R, and Matschinsky FM. On the biochemical nature of triose- and hexose- stimulated insulin secretion. Endocrinology 103: 2027-2034, 1978[Abstract].

56.   Zawalich, WS, Zawalich KC, and Rasmussen H. Cholinergic agonists prime the beta -cell to glucose stimulation. Endocrinology 125: 2400-2406, 1989[Abstract].


Am J Physiol Endocrinol Metab 279(4):E927-E940
0193-1849/00 $5.00 Copyright © 2000 the American Physiological Society