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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 Islets¹

Department of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401

	Abstract

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To delineate the underlying mechanisms by which glucose augments both Ca²⁺-dependent and Ca²⁺-independent insulin release, the latter induced by the simultaneous activation of protein kinases A and C, we examined the effects of GTP depletion by mycophenolic acid (MPA), an inhibitor of GTP synthesis, on the augmentation of insulin release from rat pancreatic islets. MPA treatment reduced GTP content by 30–40% and completely abolished glucose-induced augmentation of Ca²⁺-independent insulin release. Thus, this pathway is extremely sensitive to a decrease in cellular GTP content. Complete inhibition was also observed in islets treated with MPA plus adenine, to maintain ATP levels, under which conditions GTP is selectively depleted. Provision of guanine, which increases the activity of a salvage pathway for GTP synthesis and normalizes GTP content, completely reversed the inhibitory effect of MPA. Neither glucose utilization nor glucose oxidation was affected by MPA. The augmentation of Ca²⁺-independent insulin release by several other metabolizable nutrients including -ketoisocaproic acid (KIC) was also inhibited by MPA. In sharp contrast, augmentation of Ca²⁺-dependent insulin release by KIC was resistant to GTP depletion, indicating that nutrient-induced augmentation of the Ca²⁺-dependent- and Ca²⁺-independent secretory pathways can be differentiated by GTP dependency. We interpret these data in accord with current knowledge concerning the two known stimuli for exocytosis, Ca²⁺ and GTP (independently of Ca²⁺). We propose that both Ca²⁺-dependent and Ca²⁺-independent augmentation occurs via one metabolic pathway acting upon Ca²⁺- and upon GTP-stimulated exocytosis. Activation of PKA and PKC stimulates the GTP-sensitive exocytosis.

	Introduction

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THE PRECISE mechanisms by which glucose stimulates insulin secretion have been only partially elucidated. Glucose enters the pancreatic ß-cell via glucose transporters (1) that allow rapid equilibration of extracellular glucose with the cell interior. Intracellular glucose is subsequently metabolized by a cascade of reactions that produce ATP. An increase in the ATP concentration, a decrease in ADP, and/or an increase in the ATP/ADP ratio appear to be involved in closure of the ATP-sensitive K⁺ channels (K⁺_ATP channels) leading to membrane depolarization (2, 3, 4). Membrane depolarization activates L-type voltage dependent Ca²⁺ channels and causes increased Ca²⁺-influx across the plasma membrane (5, 6). The resulting elevation of the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) triggers Ca²⁺-dependent exocytosis of insulin. The underlying mechanisms of exocytosis are still largely unknown. The fact that this "K⁺_ATP channel-dependent" pathway has a crucial role in glucose-induced insulin release is supported by the findings that Ca²⁺ channel blockers or activators of K⁺_ATP channels can almost completely abolish glucose-induced insulin release (7, 8). In 1992, it was found that glucose metabolism led to stimulation of insulin release by a pathway that was distinct from the K⁺_ATP channel and entered stimulus-secretion coupling at a point beyond the elevation of [Ca²⁺]_i (9, 10). In these studies, glucose enhanced insulin release even in the presence of a sufficient concentration of diazoxide to fully activate the K⁺_ATP channel, when [Ca²⁺]_i was elevated, for example, by a depolarizing concentration of KCl. This second pathway is now referred to as the K⁺_ATP channel-independent insulinotropic pathway of glucose action (11, 12, 13). This pathway, however, is still dependent on the elevation of [Ca²⁺]_i for activity and acts to augment the stimulatory effect of increased [Ca²⁺]_i on secretion.

In studying the mechanisms underlying the action of the K⁺_ATP channel-independent pathway, we recently found an unexpected insulinotropic effect of glucose. Glucose stimulated insulin release in the complete absence of extracellular Ca²⁺ and in the absence of any elevation of [Ca²⁺]_i (14). This stimulatory effect of glucose occurs under stringent Ca²⁺-deprived conditions when protein kinases A and C are activated simultaneously. The effect occurs over a physiological range of plasma glucose concentration and can be inhibited by norepinephrine. The mechanism of this Ca²⁺-independent insulinotropic action is unknown, except for the fact that glucose metabolism is essential.

The important role of cellular GTP in insulin release from rat pancreatic islets has been demonstrated, using inhibitors of the de novo synthesis of GTP such as mycophenolic acid (MPA) (15, 16, 17). The roles of GTP in mammalian cells have received intense scrutiny in recent years. The heterotrimeric G proteins and the low molecular weight G proteins are known to participate extensively in signal transduction and stimulus-secretion coupling in pancreatic ß-cells (18, 19, 20, 21, 22, 23, 24, 25). Actions of GTP that may be relevant to insulin release include effects on microtubule assembly (26), calcium mobilization (27), translocation of the nascent secretory products across the endoplasmic reticulum, and vesicular traffic (28, 29), leading to exocytosis, which can be stimulated by GTP (19). Selective depletion of GTP in islets by treatment with MPA inhibited not only glucose-induced K⁺_ATP channel-dependent insulin release, which is "Ca²⁺-induced" insulin release, but also the K⁺_ATP channel-independent insulinotropic action of glucose, i.e. augmentation of Ca²⁺-induced insulin release (17). Interestingly, however, augmentation of Ca²⁺-induced insulin release by mitochondrial fuels such as succinic acid monomethyl ester (SAME) and -ketoisocaproic acid (KIC), was resistant to the depletion of GTP by MPA (17). The authors of this study concluded that among the multiple sites of involvement of GTP in glucose stimulus-secretion coupling, is the step at which nutrients (glucose and mitochondrial fuels) close K⁺_ATP channels leading to membrane depolarization and increased Ca²⁺-influx, and the step(s) where metabolic signals generated through glycolysis increase GTP synthesis and potentiate insulin release in a GTP-dependent manner. Consequently, and because the augmentation of Ca²⁺-dependent and Ca²⁺-independent secretion share similar characteristics, including the time course and concentration dependence for glucose (9, 13, 14), it was of interest to determine whether the newly demonstrated Ca²⁺-independent augmentation also involves GTP-dependent steps. Therefore, in this study we examined the effects of MPA treatment and GTP depletion of rat pancreatic islets, on the Ca²⁺-independent nutrient-induced augmentation of insulin release.

	Materials and Methods

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Isolation and treatment of pancreatic islets
Male Sprague-Dawley rats weighing from 250–450 g were killed by CO₂ asphyxiation. Immediately after death, the pancreases were surgically removed and islets isolated by collagenase digestion (30). For the experiments with MPA, the islets were cultured exactly as described by Metz et al. (15). In brief, the islets were cultured for 20 h in RPMI 1640 medium containing 10% FBS, 11.1 mM glucose, 100 U/ml penicillin and 100 µg/ml streptomycin. MPA was dissolved in hot ethanol and diluted (0.05% ethanol) in the culture media to a final concentration of 25 µg/ml. The same amount of ethanol was added in the media for control experiments. In some experiments, either 150 µM adenine or 100 µM guanine was added in the culture media as indicated in the text.

Insulin release under Ca²⁺-deprived conditions
Insulin release was measured under both static incubation conditions and perifusion conditions. In static incubations, batches of five size-matched islets were used. They were washed with Ca²⁺-free KRB containing 129 mM NaCl, 5 mM NaHCO₃, 4.8 mM KCl, 1.2 mM KH₂PO_4, 1.2 mM MgSO₄, 1 mM EGTA, 2.8 mM glucose, 0.1% BSA, and 10 mM HEPES at pH 7.4 (Ca²⁺-free KRB/EGTA buffer). Then, the islets were incubated in 1 ml Ca²⁺-free KRB/EGTA buffer for 60 min at 37 C (preincubation). After the preincubation period, the incubation medium was removed by aspiration, and 1 ml of fresh Ca²⁺-free KRB/EGTA buffer containing test substances was introduced. Incubation under the test conditions was then continued for 60 min at 37 C. In experiments with MPA-treated islets, MPA was present not only during the culture periods, but also was included in any islet picking, wash, or preincubation steps to avoid attenuation of the effects of MPA over time. However, MPA was excluded from the final incubation period. At the end of the incubations, the medium was aspirated and kept at -20 C until RIA was performed for insulin. To measure the insulin content of the islets, the incubation media were removed, and 1 ml of ice-cold 1% Triton X-100 in 154 mM NaCl was added to the tube for extraction of insulin from the islets and insulin was measured after freeze-thaw vortex (31). Rat insulin was used as standard for the RIA. Insulin release from cultured islets was expressed as fractional release (% of content per 60 min) because treatment with MPA slightly, but significantly, reduced insulin content during the culture periods (insulin content; control: 52.4 ± 1.6 ng/islet, MPA-treated: 46.1 ± 1.5 ng/islet, n = 228, P < 0.005 by nonpaired Student’s t test). For perifusion experiments, the islets were cultured for 20 h in RPMI 1640 containing 25 µg/ml MPA plus 100 µM guanine or 150 µM adenine. After the culture period, 30–40 size-matched islets were placed in each 0.7 ml perifusion chamber and perifused with Ca²⁺-free KRB/EGTA buffer at 37 C with a flow rate of 1 ml/min (14, 21, 32). The experiments were started after a 60-min perifusion equilibration period. During the equilibration periods, the perifusate contained 25 µg/ml MPA, in this case, also with 100 µM guanine or 150 µM adenine, respectively. However, they were excluded during the stimulation period. Samples were collected every 2 min, and insulin in the perifusate was measured by RIA.

Insulin release under Ca²⁺-containing conditions
KRB containing 129 mM NaCl, 5 mM NaHCO₃, 4.8 mM KCl, 1.2 mM KH₂PO_4, 1.2 mM MgSO₄, 2.5 mM Ca²⁺, 2.8 mM glucose, 0.1% BSA, and 10 mM HEPES at pH 7.4 was used throughout experiments. Insulin release was measured in static incubation experiments with the same protocol described above. Diazoxide (250 µM) was included in both preincubation and incubation periods for experiments for Fig. 8.

View larger version (32K): [in this window] [in a new window]   Figure 8. Comparison of the effects of MPA treatment on KIC-induced augmentation of Ca2+-induced insulin release and on KIC-induced augmentation of Ca2+-independent insulin release. Islets were collected from three rats and pooled in the same dish. The islets were randomly divided into two groups. One group of islets was cultured in RPMI 1640 with 150 µM adenine for 20 h. The other group of islets was cultured in RPMI 1640 with 150 µM adenine in the presence of 25 µg/ml MPA for 20 h. After the culture period, two different static incubation experiments using the same batch of islets were carried out in parallel. In one type of static incubation, KRB containing 2.5 mM Ca2+ was used throughout the experiments. Diazoxide (250 µM) was present both in preincubation (60 min) and experimental incubation periods (60 min). The second type of static incubations were performed using Ca2+-free KRB containing 1 mM EGTA. Basal glucose concentration was 2.8 mM. Values are mean ± SEM of eight determinations from a single experiment.

The method for the separation of nucleotides by HPLC described here was a modification of the procedure of Stocchi et al. (34). Nucleotides analyses were performed on a 3-µm Supelcosil LC-18-T column (15.0 cm length and 4.6 mm internal diameter; Supelco, Bellefonte, PA) protected with a guard column (2.0 cm length and 4.6 mm internal diameter) filled with 5-µm porous particle Supelguard (Supelco, Bellefonte, PA). The injection volume was 50 µl out of the 400 µl extract obtained from total 70–100 islets. Two solutions were used for the separation of nucleotide. Buffer A was 0.1 M KH₂PO₄ containing 4 mM tetrabutylammonium hydrogen sulfate; buffer B was 60% (vol/vol) CH₃OH in buffer A. The pH of both solutions was adjusted to 5.0.

The chromatographic conditions were 2.5 min at 100% of buffer A, 7.5 min at up to 50% of buffer B, and a hold of 5 min at 50% of B; the gradient was then returned to 100% of buffer A in 0.5 min (at 15.5 min from the beginning) and held for 12 min. The flow rate was 1 ml/min and detector wavelength was set at 254 nm. By this gradient protocol, GTP and ATP were easily determined with retention times of 12.6 and 13.4 min, respectively. Quantitative measurements were carried out by comparing the peak heights of samples with those of a standard solution, containing 0.05 µg of each nucleotide, injected on the day of the experiments. This procedure was assisted by the program of the above-mentioned software. Samples and standard solutions, which were stored at -70 C, were applied immediately after they thawed.

Glucose utilization under Ca²⁺-deprived conditions
Glucose utilization by the islets under Ca²⁺-deprived conditions was measured as previously reported (13, 35). In brief, 20 islets were incubated in 1 ml Ca²⁺-free KRB/EGTA buffer with or without 25 µg/ml MPA at 37 C for 60 min (preincubation). At the end of the preincubation, the medium was aspirated, and then 100 µl fresh Ca²⁺-free KRB/EGTA buffer containing 1 µCi D-[5-³H] glucose was introduced and incubated for another 60 min at 37 C. The reaction was stopped by adding HCl. ³H₂O was equilibrated with H₂O in the outer vessel as described, and was quantitated by liquid scintillation spectrometry.

Glucose oxidation under Ca²⁺-deprived conditions
Glucose oxidation was measured as reported (36) with minor modifications (13). In brief, 25 islets were first incubated in 1 ml Ca²⁺-free KRB/EGTA buffer with or without 25 µg/ml MPA at 37 C for 60 min (preincubation). At the end of the preincubation, the medium was aspirated and 100 µl Ca²⁺-free KRB/EGTA buffer containing 1 µCi D-[¹⁴C(U)] glucose and test substances were added and further incubated for 60 min at 37 C. At the end of the 60 min incubation, ¹⁴CO₂ was trapped by benzethonium hydroxide, after adding 200 µl 0.1 N HCl to the incubation mixture, and quantitated by liquid scintillation spectrometry.

Materials
Forskolin and PMA, both from Sigma Chemical Co. (St. Louis, MO), were dissolved in dimethyl sulfoxide (DMSO) (final concentration of DMSO, 0.02%). Control conditions had the same final concentrations of DMSO as the test conditions. MPA, EGTA, DMSO, KIC, glutamine, leucine, SAME, adenine, guanine, ATP, GTP, and benzethonium hydroxide were obtained from Sigma. CF50 membranes were purchased from Amicon. Methanol and tetrabutylammonium hydrogen sulfate were purchased from Fluka. D-[¹⁴C(U)] glucose (specific activity 10.6 mCi/mmol) and D-[5–3H] glucose (specific activity 15.7 Ci/mmol) were purchased from New England Nuclear (Boston, MA).

Data analysis
Data are presented as the mean ± SEM and statistical significance was evaluated using one-way ANOVA with pairwise comparison by Bonferroni method, or paired t test when designated. Differences were considered significant at P < 0.05.

	Results

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Effects of various nutrients on insulin release under Ca²⁺-deprived conditions
As shown in Fig. 1, various nutrients stimulated insulin release from freshly isolated rat pancreatic islets when the islets were stimulated with PMA and forskolin simultaneously under Ca²⁺-deprived conditions (Ca²⁺-free KRB/EGTA buffer). Glucose (11.1 mM) produced a 1.5-fold increase in insulin release. Other nutrients including glyceraldehyde, leucine, KIC, glutamine, and SAME, produced 1.0- to 1.5-fold increases in insulin release. None of these nutrients stimulated insulin release under Ca²⁺-deprived conditions in the absence of PMA and forskolin (data not shown).

View larger version (25K): [in this window] [in a new window]   Figure 1. Effects of various nutrients on insulin release during simultaneous stimulation with 100 nM PMA and 6 µM forskolin under Ca2+-deprived conditions. Insulin release was measured in the Ca2+-free KRB/EGTA buffer in static incubation experiments as described in Materials and Methods. PMA (100 nM), forskolin (6 µM) and the nutrients were included during experimental incubation periods. Values are mean ± SEM of five determinations from a representative experiment. *, P < 0.05 vs. basal condition (2.8 mM glucose). . P < 0.01 vs. the basal condition. §, P < 0.001 vs. the basal condition.

View larger version (15K): [in this window] [in a new window]   Figure 2. Scheme of the pathway for synthesis of the nucleoside triphosphates as described by Metz et al. (15). Several cofactors, modulators, and intermediate steps have been omitted for the sake of clarity. PRPP, Phosphoribosylpyrophosphate; UMP, uridine monophosphate; UTP, uridine triphosphate; IMP, inosine monophosphate; IMPDH, inosine monophosphate dehydrogenase; XMP, xanthosine monophosphate.

View larger version (17K): [in this window] [in a new window]   Figure 3. Effects of MPA (25 µg/ml) treatment on insulin release under Ca2+-deprived conditions. A, Islets were cultured in RPMI 1640 with or without MPA. B, Islets were cultured in RPMI 1640 in the presence of 150 µM adenine with or without MPA. C, Islets were cultured in RPMI 1640 in the presence of 100 µM guanine with or without MPA. After the culture period of 20 h, the islets were subjected to static incubation experiments as described in Materials and Methods. The data were accumulated from four independent experiments. In each experiment, the islets were collected from three to four rats and pooled in the same dish. After collecting islets, they were randomly divided in each experimental condition. All the 24 experimental conditions were examined in each experiment. Insulin release was expressed as fractional release. Values are mean ± SEM of 16–20 determinations. The islet insulin content was 49 ± 2 ng (n = 360).

Effects of MPA treatment and subsequent incubation under Ca²⁺-deprived conditions on GTP and ATP contents in islets
The effects of MPA treatment on the nucleotide content of islets were examined previously by Metz and co-workers (15, 16). Nevertheless, it was important to determine the effects of MPA treatment on nucleotide contents under our experimental conditions, where the MPA treatment completely abolished the glucose-induced augmentation of Ca²⁺-independent insulin release. We therefore measured nucleotide contents in islets exposed to the same conditions as in our secretion experiments, i.e. after 20-h culture under various conditions, and subsequent incubations with Ca²⁺-free KRB/EGTA buffer. In Fig. 4 are shown the effects of these various culture conditions on GTP content (A) and ATP content (B). The amount of these nucleotides present in the islet was comparable to a previous report (37) in which the authors employed TCA extraction and HPLC by using an NH₂-anion exchange column. As shown in Fig. 4, MPA treatment decreased GTP content by 31% (P < 0.001). ATP content was also decreased by the MPA treatment (P < 0.002). Provision of 150 µM adenine during the culture period did not affect the decrease in GTP content caused by the MPA treatment, whereas the provision of adenine prevented the effect of MPA treatment to decrease ATP content. Importantly, when 100 µM guanine was present during the culture period, MPA treatment had no effect on either GTP or ATP content.

View larger version (72K): [in this window] [in a new window]   Figure 4. GTP and ATP content of islets incubated in the absence and presence of MPA and in the absence and presence of adenine or guanine. Islet extraction was performed as described in Materials and Methods after 20 h culture under the various experimental conditions corresponding to the static incubation experiments shown in Fig. 3 and following a 60-min incubation with Ca2+-free KRB/EGTA buffer containing 2.8 mM glucose. In other words, the GTP and ATP contents relate to the start of the test period in the experiments of Fig. 3. In this Ca2+-deprived incubation, MPA was added whenever it was contained in the medium of the preceding culture period. Each figure was obtained from six independent experiments carried out in a paired fashion regarding the presence or absence of MPA. Statistical analysis was performed by paired t test. In A, P values of the left and middle panel were <0.001 and <0.005, respectively. In B, P value of the left panel was <0.002. None of the other pairs were statistically significant.

Effects of MPA treatment on the temporal profile of glucose-induced augmentation of insulin release under Ca²⁺-deprived conditions
To confirm these strong inhibitory effects of MPA treatment on glucose-induced augmentation of Ca²⁺-independent insulin release, and to determine the temporal profiles, we performed perifusion experiments. In Fig. 5A are shown the results from islets cultured in RPMI 1640 containing 25 µg/ml MPA and 100 µM guanine (under which conditions the NTP content remained normal). After a 60-min equilibration period, basal rates of insulin release were stable under Ca²⁺-deprived conditions. As shown by the open circles in Fig. 5A, the administration of 100 nM PMA and 6 µM forskolin from min 10 gradually increased the rates of insulin release for the initial 12 min of stimulation and produced a plateau level of release thereafter. In one set of chambers, the glucose concentration of the perifusate was raised from 2.8 mM to 16.7 mM simultaneously with the stimulation of PMA and forskolin. Under these conditions, the rates of insulin release rapidly increased, again for the initial 12 min of the stimulation (as indicated by the closed circles). The rate of increase was more than double that of the controls. Subsequently, a plateau of insulin release remained above control rates until 70 min when the experiment was ended. In Fig. 5B are shown the results from islets cultured in RPMI 1640 containing 25 µg/ml MPA and 150 µM adenine under which conditions the GTP content was selectively depleted by over 40% (see next section). PMA and forskolin evoked insulin release from the control islets that was indistinguishable from that released from the islets cultured with MPA and guanine. As anticipated from the results shown in Fig. 3, 16.7 mM glucose had no effect on the Ca²⁺-independent insulin release induced by PMA and forskolin in this case. These data confirmed that selective depletion of GTP in islets abolishes the augmentation effect of glucose on Ca²⁺-independent insulin release.

View larger version (26K): [in this window] [in a new window]   Figure 5. Temporal profiles of insulin release measured by perifusion experiments under Ca2+-deprived conditions. A, Islets were cultured in RPMI 1640 in the presence of 25 µg/ml MPA and 100 µM guanine for 20 h. B, Islets were cultured in RPMI 1640 in the presence of 25 µg/ml MPA and 150 µM adenine for 20 h. Ca2+-free KRB with 1 mM EGTA was used throughout experiments. The islets were stimulated with 100 nM PMA and 6 µM forskolin from min 10 as indicated by horizontal bars; from here, no MPA, adenine, nor guanine was included. In one set of each pair of chambers, basal glucose concentration (2.8 mM) was raised to 16.7 mM from min 10 (closed circles). Values are mean ± SEM of four determinations from four separate experiments.

View larger version (54K): [in this window] [in a new window]   Figure 6. GTP and ATP contents of islets incubated in the presence of MPA and guanine, and MPA and adenine, and 2.8 mM or 16.7 mM glucose. Islet extraction was performed as described in Materials and Methods after 20-h culture in various experimental conditions followed by a preincubation (60 min) and an incubation (60 min) period corresponding to the perifusion conditions in Fig. 5, in both of which Ca2+ was eliminated (Ca2+-free KRB/EGTA buffer). These data on GTP and ATP contents correspond to the state of the islets at the end of the perifusion period in Fig. 5. In the preincubation, the buffer included 25 µg/ml MPA. Either 150 µM adenine or 100 µM guanine was also included. In the incubation period, they were excluded and 6 µM forskolin, 100 nM PMA, and 2.8 or 16.7 mM glucose were added to the Ca2+-free KRB/EGTA buffer. The data were obtained from six independent experiments carried out in a paired fashion regarding the presence of adenine or guanine. Statistical analysis was performed by paired t test. In A, P values between 2.8 mM glucose and 16.7 mM glucose conditions were <0.015 and <0.001, respectively. In B, none of the pairs were significantly different.

View larger version (23K): [in this window] [in a new window]   Figure 7. Effects of MPA treatment with adenine (A) or guanine (B) on KIC-induced augmentation of insulin release under Ca2+-deprived conditions. A, Islets were cultured in RPMI 1640 in the presence of 25 µg/ml MPA and 150 µM adenine for 20 h. B, Islets were cultured in RPMI 1640 in the presence of 25 µg/ml MPA and 100 µM guanine for 20 h. Insulin release was measured in Ca2+-free KRB with 1 mM EGTA in static incubations as described in Materials and Methods. Experimental incubations were performed in the absence of glucose. Values are mean ± SEM of 11–12 determinations from 2 experiments (A) and 9 determinations from 2 experiments (B).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In a previous study, we found that glucose strongly augmented insulin release from rat pancreatic islets under stringent Ca²⁺-deprived conditions when protein kinases A and C were activated simultaneously (14). The mechanisms behind this novel effect of glucose are unknown except for the fact that glucose metabolism is essential. The present studies were designed to examine the underlying mechanisms by which glucose and other nutrients augmented Ca²⁺-independent insulin release under the stringent Ca²⁺-deprived conditions imposed. The data presented here reveal that the content of GTP in the pancreatic islet cells has a crucial role in nutrient-induced augmentation of Ca²⁺-independent insulin release. The site of GTP involvement in the augmentation of the Ca²⁺-independent release appears to be distal to the generation of the metabolic signal via nutrient metabolism. It is likely that the signal exerts its effect on GTP-stimulated exocytosis.

Metz et al. (15) demonstrated an important role for GTP in insulin release. They examined the effects of MPA, an inhibitor of de novo GTP synthesis, on NTP contents and insulin release. MPA treatment decreased the GTP content of the islets and inhibited glucose-induced insulin release. Treatment with 25 µg/ml MPA for 20 h, which was identical to the condition used in this study, reduced the islet GTP and ATP contents by 81% and by 39%, respectively, and increased the islet UTP contents by 87%. Although MPA treatment decreased the ATP content, it did not affect the ATP/ADP ratio (17). On the other hand, the decrease in GTP content was concomitant with a decrease in the GTP/GDP ratio (17). Provision of guanine reversed all the abnormalities in NTP contents via activation of the salvage pathway for GTP synthesis (Fig. 2) (15). Provision of adenine, via activation of the salvage pathway for ATP synthesis, enhanced ATP synthesis and allowed a selective depletion of GTP content without affecting the ATP and UTP contents (15).

In our hands, MPA treatment of islets caused only a 30–40% reduction in GTP content, but in all other respects our data were qualitatively similar to those of Metz et al. (15, 16). Nevertheless, glucose-induced augmentation under Ca²⁺-deprived conditions was completely abolished by MPA treatment. The fact that the reduction in GTP content was only 30–40% emphasizes the high sensitivity of the Ca²⁺-independent glucose augmentation pathway to GTP.

These data show that the glucose-induced augmentation of Ca²⁺-independent insulin release is extremely dependent upon the islet content of GTP. The Ca²⁺-independent insulin release induced by the activation of protein kinases A and C was resistant to the MPA treatment, suggesting that the reduction of GTP content did not impede common exocytotic processes. It was only the specific glucose-induced augmentation of release that required GTP. This inhibition is different from physiological inhibition by norepinephrine, where not only glucose-induced Ca²⁺-independent augmentation but also the Ca²⁺-independent insulin release by the activation of protein kinases A and C is completely inhibited (14).

It is now clear that glucose acts on two different pathways to augment insulin release, one in the presence of Ca²⁺ and one in the absence of Ca²⁺. Augmentation of exocytosis under these two conditions has several similar characteristics (12, 13, 14, 38, 39, 40). These include: 1) similar concentration-response curves; 2) similar temporal profiles that resemble the second phase of normal glucose-induced insulin release; 3) a similar dependence on glucose metabolism; 4) several metabolizable nutrients other than glucose are able to stimulate under both conditions; and 5) the nutrients do not "initiate" insulin release but only augment or potentiate release.

Consequently, it is important to know whether there are two augmentation mechanisms, or only one augmentation mechanism acting on parallel routes to exocytosis. Meredith and co-workers (17) reported that the augmentation of Ca²⁺-induced insulin release (K⁺_ATP channel-independent pathway) induced by mitochondrial fuels such as SAME and KIC was not inhibited by MPA treatment and concluded that this augmentation pathway did not depend upon GTP. In contrast, our data show that KIC-induced augmentation of Ca²⁺-independent insulin release is clearly dependent upon GTP. It should be noted that while augmentation of the Ca²⁺-independent pathway is abolished by MPA treatment and clearly GTP dependent, the Ca²⁺-dependent pathway cannot be described as GTP-independent as discussed later. We do not believe that any form of stimulus-secretion coupling in the ß-cell is GTP independent. Rather, we think that we have uncovered a distinct difference in the requirement for GTP, i.e. the requirement of the Ca²⁺-independent pathway for higher concentrations of GTP. This distinction between the augmentation pathways suggests the possibilities that there are two independent pathways with separate mechanisms and/or separate targets, although they could still converge on one final common path at a point beyond the GTP dependence, or there is one common glucose augmentation mechanism which operates on two types of differently regulated exocytosis. These results indicate that mitochondrial metabolism generates the signal that augments two pathways of secretion, one of which is very sensitive to GTP depletion. This distinctive feature of augmentation suggests functional differences. The augmentation of Ca²⁺-induced insulin release is exerted in a seemingly GTP-insensitive manner. In contrast, the augmentation of Ca²⁺-independent insulin release, evoked by activation of protein kinases A and C, is exerted in a GTP-sensitive manner. It is well established by studies on permeabilized ß-cells (19, 41) and by capacitance measurements on single ß-cells (42), that insulin secretion can be stimulated by Ca²⁺ alone, and by GTP alone (or at least at concentrations of Ca²⁺ that are described as vanishingly low). It seems likely therefore that the distinction between the two augmentation pathways that we have found is relevant to the two mechanisms by which exocytosis can be stimulated, i.e. that there is only one mechanism by which glucose augments insulin secretion but that it operates on the two known types of exocytosis, one driven by Ca²⁺ and the other by GTP. In this connection, a recent report on GTP-sensitive- and GTP-insensitive exocytosis in mouse pancreatic ß-cells (42) provides evidence for the existence of two pools of insulin-containing granules, a readily releasable pool and a reserve pool, the granules of which are not releasable until they have been mobilized to the immediately releasable pool. Both GTP and Ca²⁺ are involved in the mobilization step. However, exocytosis of the immediately releasable pool can be achieved by two independent processes, either by GTP (in the absence of Ca²⁺) or by Ca²⁺ per se. Our data show that Ca²⁺-dependent glucose augmentation of insulin release is not affected by GTP depletion, whereas the PKA/PKC-induced Ca²⁺-independent glucose augmentation is eliminated. If the mechanism of augmentation was to stimulate the mobilization of the reserve pool, all forms of augmentation would be affected by GTP depletion. Therefore, it may be concluded that the site of entry into stimulus-secretion coupling of the Ca²⁺-independent glucose augmentation must be at the final stages of exocytosis, i.e. exocytosis of the readily releasable pool. It may be that the pure Ca²⁺-dependent augmentation pathway has an analogous late site of action on Ca²⁺-stimulated exocytosis by means of a common component in exocytosis. This hypothesis is illustrated in Fig. 9, where we suggest that the glucose signal is the same in both augmentation pathways (Ca²⁺-dependent, and Ca²⁺-independent), and that exocytosis is augmented by a mechanism that is common to the two means of stimulation of the readily releasable pool, by Ca²⁺ and by GTP. In addition, regarding the signal responsible for glucose augmentation, it is likely that the reported glucose-induced elevation of malonyl CoA leading to increased levels of long chain acyl CoA derivatives (43, 44) is involved in the route to augmentation. This issue is not addressed here.

View larger version (20K): [in this window] [in a new window]   Figure 9. Glucose-induced augmentation in stimulus-secretion coupling of the pancreatic ß-cell. Shown is a current concept of the stimulation of insulin release by glucose, and our proposal for the mechanisms of augmentation. On the left is shown the K+ATP-channel dependent pathway for the elevation of intracellular Ca2+ ([Ca2+ ]i). Closure of the K+ATP channel and resultant depolarization causes activation of the L-type voltage dependent Ca2+ channels, increased Ca2+ entry and increased [Ca2+]i. The rise in [Ca2+]i gives a small increase in the rate of insulin release. Glucose augments this release via the K+ATP channel-independent pathway by means of an as yet unknown mechanism, but possibly involving a rise in malonyl CoA and increased cytosolic long chain acyl CoA derivatives (43, 44). The site of action of this Ca2+-dependent augmentation pathway is likely to be at the level of exocytosis of the immediately releasable insulin pool (42). Exocytosis can be stimulated by either Ca2+ or GTP and it is the latter form of exocytosis that is augmented by glucose independently of Ca2+. This mechanism of augmentation can be demonstrated under Ca2+-free conditions (as shown on the right side of the diagram) when PKA and PKC are activated simultaneously, as occurs post prandially due to vagal stimulation and the elevation of incretin levels. Augmentation of this route of exocytosis is thought to occur by the same mechanism by which pure Ca2+-stimulated exocytosis is augmented so that there is only one glucose or nutrient derived augmentation pathway. Several important branches of stimulus-secretion coupling and cross-talk between signals have been omitted for the sake of clarity.

In summary, glucose and nutrients augment Ca²⁺-independent insulin. The augmentation of Ca²⁺-independent insulin release was abolished by the selective depletion of GTP. It can be concluded from these data that nutrient-induced augmentation of Ca²⁺-independent insulin release requires GTP late in stimulus-secretion coupling. As the augmentation of Ca²⁺-induced insulin release by a mitochondrial fuel, KIC, was not inhibited by the selective depletion of GTP, the underlying mechanisms of the augmentation of Ca²⁺-induced insulin release may, or may not, be distinct from that of the augmentation of Ca²⁺-independent insulin release. Two possibilities exist: 1) glucose has two different augmentation pathways that control insulin secretion with independent mechanisms as shown by the GTP dependency; or 2) there is only one glucose augmentation mechanism that operates on two types of differently regulated exocytosis, i.e. one driven by Ca²⁺ and the other by GTP. We favor the idea of only one glucose augmentation mechanism operating on the two systems for the stimulation of exocytosis.

	Acknowledgments

 
The authors are grateful to Dr. Lauren Trepanier for assistance in setting up the HPLC assays.

	Footnotes

 
¹ This work was supported by Grant RO1-DK-42063 from the National Institutes of Health.

Received April 22, 1997.

	References

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