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Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University (S.Y., I.K.), Maebashi, 371-8512, Japan; and Department of Aging Medicine and Geriatrics, School of Medicine (M.K., Y.S., K.Y., K.H.), and Center for Health Services (T.A.), Shinshu University, Matsumoto 390-8621, Japan
Address all correspondence and requests for reprints to: Dr. Toru Aizawa, Center for Health Services, Shinshu University, 3-1-1 Asahi, Matsumoto 390-8621, Japan. E-mail: traizawa{at}hsp.md.shinshu-u.ac.jp.
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Abstract |
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5.5 mM) of glucose caused TDP, and 6 µM forskolin (an activator of adenylyl cyclase) significantly enhanced it (3.1-fold at most). Forskolin produced an 8.0-fold increase in islet cell cAMP; however, it returned to the baseline after washout by the time of stimulation of exocytosis. Two millimoles of dibutyryl cAMP (a cAMP analog), 0.1 mM isobutylmethylxanthine (a phosphodiesterase inhibitor), and 100 nM glucagon-like peptide-1 (an incretin hormone) also enhanced glucoseinduced TDP. The time-dependent effect of cAMP was not attenuated by protein kinase A inhibitors (200 µM adenosine 3',5'-cyclic monophosphothioate, Rp isomer, and 10 µM H89). Although glucose-induced TDP was attenuated by NaN3 (a mitochondrial poison) and cerulenin (an inhibitor of protein acylation), cAMP enhancement of it was unaffected by these agents. In conclusion, cAMP time-dependently stimulates insulin exocytosis, provided the extracellular glucose concentration is equivalent to or higher than ambient plasma levels. Protein kinase A, mitochondrial metabolism, and protein acylation are not involved in this cAMP action. Incretin stimulation of insulin exocytosis may occur in part via this mechanism. |
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Materials and Methods |
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Measurement of cAMP
The cAMP content of the islet cells was determined as previously reported (15) to demonstrate the level of cAMP during the different stages of the experiment, i.e. immediately before removal of forskolin, after 20 min on ice, and after 15-min incubation at 37 C. To this end, five size-matched islets were first incubated for 60 min in KRB buffer containing 11.1 mM glucose with or without 6 µM forskolin (preincubation). To determine the cAMP content at the end of exposure to forskolin, preincubation buffer was aspirated, and 300 µl 0.2 M HCl were immediately added to the tube for extraction of cAMP. The cAMP content at the time of stimulation of insulin release was determined under two different conditions. In one group of islets, after preincubation with forskolin, the buffer was replaced with 1 ml ice-cold basal KRB buffer, and the tubes were placed in an ice-cold water bath for 20 min (washout incubation). The other group of islets was incubated at 37 C for 15 min with 1 ml basal KRB buffer (washout incubation) immediately after removal of forskolin-containing preincubation buffer. At the end of washout incubation, buffer was replaced with 300 µl 0.2 M HCl. Finally, all tubes were placed in boiling water for 5 min with occasional vortexing, and the contents of the tubes were evaporated using a Speed-Vac system (Savant Instruments, Farmingdale, NY). After reconstitution with 50 µl distilled water, cAMP was determined by RIA using commercially available kits (Yamasa, Chiba, Japan).
Measurement of insulin release
Insulin release was measured in static incubation experiments as previously reported (12). In brief, five size-matched islets were first incubated for 60 min in KRB buffer containing the indicated concentrations of test substances at 37 C (preincubation). At the end of preincubation, the buffer was aspirated, and the islets were washed once with 1 ml ice-cold KRB buffer containing 2.5 mM CaCl2, 2.8 mM glucose, and 250 µM diazoxide. Then, 1 ml fresh KRB buffer containing 2.5 mM CaCl2, 2.8 mM glucose, 250 µM diazoxide, and 50 mM KCl was introduced, and incubation was carried out for an additional 30 min at 37 C (test incubation). It took a minimum of 20 min for buffer change (collection of the preincubation buffer, addition and removal of washout buffer, and introduction of buffer for the test incubation, for 80–120 tubes). The rack (with all the tubes) was placed into an ice-cold water bath during this period. Therefore, carry-over of a high rate of nutrient metabolism from the preincubation period to the test incubation period could be prevented.
KRB buffer containing 8.3 mM glucose and a basal (4.8 mM) concentration of KCl without diazoxide was used during test incubation in one experiment. In this particular experiment diazoxide was omitted from the washout solution. In another experiment the islets were washed with basal KRB buffer without diazoxide and a 15-min washout incubation at 37 C in basal KRB buffer was performed between the preincubation and the test incubation. Insulin release during the test incubation was employed as a quantitative index of TDP. Insulin release during the preincubation was determined in some experiments as needed. Insulin was measured by RIA using commercially available kits (Eiken, Tokyo, Japan) in which rat insulin was used as a standard. The conversion factor of picograms to nanomoles is 0.1739 in all figures.
Materials
Forskolin; 
-ketoisocaproic acid (-KIC); NaN3; cerulenin; N6,2'-O- dibutyryl cAMP; 1,9-dideoxy-forskolin; adenosine 3',5'-cyclic monophosphothioate, Rp isomer (Rp-cAMPS); H-89; GLP-1; 3-isobutyl-1-methylxanthine (IBMX); and diazoxide were obtained from Sigma (St. Louis, MO).
Statistical analysis
Statistical analysis was performed by one-way ANOVA, with pairwise comparison by Fisher’s protected least significance difference test, unless otherwise indicated, and comparison of two mean values was performed by Mann-Whitney U test where indicated (StatView, SAS Institute, Inc., Cary, NC). P < 0.05 was considered significant. Data are expressed as the mean ± SE.
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Results |
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, column 1 vs. 2). However, the cAMP level 20 min after removal of forskolin was not significantly different from the baseline (Fig. 1, column 3 vs. 1). After a 15-min of washout incubation at 37 C without forskolin, the cAMP level was slightly, but not significantly, lower than the baseline (Fig. 1, column 4 vs. 1). The cAMP level in the islets kept at low temperature and that in islets incubated at 37 C after removal of forskolin, respectively, were not significantly different (Fig. 1, column 3 vs. 4). The findings are compatible with a high turnover rate of cAMP in the islet cells (16). The data imply that the islet cell cAMP level is not significantly higher than baseline at the beginning of the test incubation.
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, left panel) (9, 10, 11, 12). Glucose at concentrations higher than 5.5 mM caused TDP in a concentration-dependent manner as previously described (11, 12, 17), which was significantly augmented by forskolin (Fig. 2A, left panel). The maximal effect of forskolin (a 2-fold enhancement) was observed at 11.1 mM glucose. We therefore used 11.1 mM glucose in most of the following experiments. The time-dependent nature of the forskolin effect was confirmed in the experiment with a placement of a 15-min washout incubation between preincubation and test incubation. In this experiment insulin release during the test incubation was 174.7 ± 12.6, 211.9 ± 18.2, 1621.2 ± 118.4, and 2608.1 ± 240.3 with preincubation with 2.8 mM glucose alone, 2.8 mM glucose plus 6 µM forskolin, 11.1 mM glucose, and 11.1 mM plus 6 µM forskolin, respectively. The difference between the latter two was highly significant (P < 0.0001).
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-KIC (a mitochondrial fuel) was used in place of glucose, it caused TDP as previously reported (18, 19), and inclusion of forskolin during preincubation clearly enhanced -KIC-induced TDP (Fig. 2A
, middle panel).
Effects of NaN3 and cerulenin on forskolin enhancement of TDP
NaN3 (a disrupter of mitochondrial respiratory chain) was tested at a concentration of 2 mM. This concentration of azide only partially impedes mitochondrial metabolism in the ß-cell, and it does not significantly suppress Ca2+-triggered insulin release (20, 21). NaN3 (2 mM) greatly attenuated nutrient-induced TDP itself as expected (Fig. 2B). However, it did not attenuate the forskolin enhancement of glucose- and -KIC-induced TDP (Fig. 2B).
We previously reported that cerulenin, which inhibits protein acylation at a concentration of 30–100 µg/ml, specifically inhibits nutrient-induced insulin release and glucose-induced TDP (12, 22). We therefore examined whether protein acylation was involved in the enhancement of nutrient-induced TDP by forskolin. The islets were first incubated in KRB buffer containing 2.5 mM CaCl2, 2.8 mM glucose, and 30 µg/ml cerulenin for 30 min. Subsequently, the preincubation was carried out in the presence of cerulenin. During the test incubation, cerulenin was not included. As reported, treatment with 30 µg/ml cerulenin significantly attenuated glucose-induced TDP itself (Fig. 2C). However, enhancement of the TDP by forskolin was resistant to this treatment (Fig. 2C), indicating that the phenomenon occurs independently of protein acylation.
Membrane depolarization and Ca2+ entry are not required for forskolin enhancement of TDP
Knowing that glucose-induced TDP occurs independently of extracellular Ca2+ (12), it was anticipated that forskolin enhancement of the TDP does not require the ionic events either. Nonetheless, we verified this. To this end, the effects of addition of diazoxide, an activator of the ATP-sensitive K+ channel (K+ATP channel), and removal of extracellular Ca2+, respectively, were examined. The islets were preincubated with 11.1 mM glucose in KRB buffer containing 2.5 mM Ca2+, 2.5 mM Ca2+ and 250 µM diazoxide, or 1 mM EGTA without added Ca2+, each of these with or without 6 µM forskolin. Insulin release subsequently triggered by a depolarizing concentration of K+ during the test incubation was employed as an index of TDP. The enhancement of glucose-induced TDP by forskolin under each condition was not significantly different (2.4 ± 0.1-, 2.9 ± 0.3-, and 2.7 ± 0.2-fold; n = 16–18 for each condition), implying that membrane depolarization and Ca2+ entry were not required for this forskolin action.
Involvement of cAMP but not protein kinase A (PKA) in the action of forskolin
To verify that cAMP is the molecule responsible for forskolin enhancement of nutrient-induced TDP, we tested the effect of dibutyryl cAMP, a cell-permeable cAMP analog, on glucose-induced TDP. Dibutyryl cAMP (2 mM), like forskolin, did not cause TDP when applied with 2.8 mM glucose (data not shown), but it augmented TDP induced by 11.1 mM glucose (Fig. 2A, right panel). The effect of 2 mM dibutyryl cAMP was not significantly different from that of 6 µM forskolin by a direct comparison using the same batch of islets for both agents (Fig. 2A, right panel; P = 0.204). However, the effect of forskolin was greater in other experiments in which dibutyryl cAMP was not included than in the experiment shown in Fig. 2A, right panel. Overall, 2 mM dibutyryl cAMP may not be as effective as 6 µM forskolin in raising cAMP. An inactive analog of forskolin, 1,9-dideoxyforskolin, did not mimic the effect of forskolin (data not shown).
We then investigated the role of PKA by using Rp-cAMPS (200 µM), an inhibitor of PKA (23, 24, 25). When the effect of Rp-cAMPS was examined, islets were first incubated in KRB buffer containing 2.8 mM glucose and 200 µM Rp-cAMPS for 30 min before preincubation. Rp-cAMPS was included also during preincubation, but not during the final test incubation. During the preincubation, 11.1 mM glucose directly stimulated insulin release (Fig. 3A, column 2), and this glucose effect was significantly potentiated by forskolin (Fig. 3A, column 4) as previously described (15, 26). Such a forskolin effect was partially suppressed by Rp-cAMPS (Fig. 3A, column 6). The finding is in accord with the idea that there exists PKA-dependent and -independent components for insulinotropic effect of cAMP (24, 27, 28). In marked contrast, forskolin enhancement of glucose-induced TDP was not attenuated by Rp-cAMPS (Fig. 3B, column 4 vs. 6). H-89 (10 µM), a different class of PKA inhibitor (25), showed qualitatively the same effects as Rp-cAMPS, i.e. suppression of the immediate, but not the time-dependent, effect of forskolin (Fig. 4, A and B).
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, 100 nM GLP-1 significantly enhanced TDP induced by 11.1 mM glucose, and addition of 0.1 mM IBMX, an inhibitor of phosphodiesterase, further augmented the effect of GLP-1. The peptide clearly augmented glucose-induced TDP when the glucose concentration was 8.3 mM or higher (Fig. 6B).
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Discussion |
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In the past it has been regarded that an increase in intracellular cAMP does not participate in nutrient-induced TDP (9, 10, 11, 12). However, a synergistic effect of cAMP on the nutrient-induced TDP was not searched for in the studies (11, 12) in which agents that elevate cAMP were tested with a basal, nonstimulatory concentration of glucose. In another study (9) the time-dependent effect of cAMP was not demonstrated when IBMX was used to increase cellular cAMP in the presence of 8.3 mM glucose. However, 8.3 mM glucose per se did not exhibit TDP in this study (9). The absence of time-dependent cAMP action in the other study, in which theophyline was used in combination with a stimulatory concentration of glucose, may be related in part to the use of bicarbonate buffer without HEPES (10). We used KRB buffer containing HEPES throughout, and differential effects of bicarbonate buffer with and without HEPES on the islet ß-cell under certain experimental conditions were recently recognized (29).
Fehmann et al. (13) showed the TDP of insulin exocytosis by incretin using perfused rat pancreas. The pancreas was perfused with buffer containing 2.8 mM glucose and considerably low concentrations (10–1000 pM) of incretins for 10 min, then with buffer containing 2.8 mM glucose without incretin for 10 min, and finally with buffer containing 10 mM glucose for 44 min. Thus, they observed the TDP by incretin applied simultaneously with a nonstimulatory concentration of glucose. Their protocol was quite different from ours. Such a low concentration of incretin, for example, 10 nM CCK-8, did not cause TDP in isolated islets (30). Fermann et al. (13) hypothesized that collagenase-isolated islets were less sensitive to peptide secretagogues than islets in the perfused pancreas. The fact that we needed a relatively high concentration of GLP-1 (100 nM) for enhancement of glucose-induced TDP is in accord with this hypothesis.
cAMP enhancement of TDP was independent of mitochondrial metabolism and protein acylation because it occurred normally in the presence of azide (a disrupter of mitochondrial respiratory chain) and cerulenin (an inhibitor of protein acylation). Neither membrane depolarization nor Ca2+ entry was required for TDP by cAMP because the forskolin effect was not attenuated by diazoxide (a K+ATP channel activator), nor was it attenuated under stringent Ca2+-free conditions.
The molecular basis for the action of cAMP in pancreatic ß-cells is fairly well defined. cAMP transiently raises the cytoplasmic free Ca2+ concentration ([Ca2+]i) via effects on the K+ATP channels (31, 32, 33), voltage-dependent calcium channels (25, 34, 35), and nonselective cation channels (36, 37, 38) in the ß-cell membrane. Nevertheless, in the absence of stimulatory concentration of glucose, cAMP potentiates little if any insulin release triggered by the elevation of [Ca2+]i (15, 32, 39). The findings indicate that cAMP potentiation of insulin release occurs mostly at a distal site, beyond the elevation of [Ca2+]i (15, 25). At least part of such cAMP action is PKA independent (23, 24, 26, 27, 28). Interestingly, we found that the time-dependent stimulation of insulin exocytosis by cAMP was at least partly independent of PKA. As a possible molecular basis for PKA-independent cAMP action, activation of phosphatidyl inositol 3-kinase by GLP-1 has been demonstrated in the INS-1 cell line (40). A role for cAMP-guanine nucleotide exchange factors in cAMP stimulation of insulin exocytosis, independent from PKA, has also been implicated (27, 28).
There are limitations to the pharmacological approach. We could not rule out the possibility that there is a compartmentalization of cAMP in the cell, so that the concentration of cAMP in a certain subcellular domain remained elevated after removal of forskolin (41). Although 200 µM RpcAMP, which showed a maximum inhibition of PKA in the cell-free system (42), was employed in the current study, involvement of residual PKA activity for time-dependent cAMP action also cannot be completely ruled out.
So-called TDP may well be an expansion of a readily releasable pool of insulin granules (43). A stimulatory concentration of glucose causes both triggering of insulin release and the expansion of the readily releasable pool of insulin-containing granules (43). Incretin stimulation of insulin exocytosis may occur in part via this mechanism in combination with other agonists, such as acetylcholine (44). In conclusion, we hypothesize that a major action of cAMP in the ß-cell is the enhancement of nutrient-induced expansion of the readily releasable pool.
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Acknowledgments |
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Footnotes |
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Abbreviations: [Ca2+]i, Cytoplasmic free Ca2+ concentration; GLP-1, glucagon-like peptide-1; IBMX, 3-isobutyl-1-methylxanthine; -KIC, -ketoisocaproic acid; KRB, Krebs-Ringer bicarbonate; PKA, protein kinase A; Rp-cAMPS, adenosine 3',5'-cyclic monophosphothioate, Rp isomer; TDP, time-dependent potentiation.
Received April 3, 2002.
Accepted for publication July 11, 2002.
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, P < 0.01 vs. the control (by Mann-Whitney U test). Values are the mean ± SE from 18 (left panel; each of the glucose dose-response experiments), 26 (middle panel; each of the KIC experiments), and 30 (right panel; each of the dibutyryl cAMP experiments) determinations. B and C: *, P < 0.01 vs. corresponding controls; 
