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Am J Physiol Endocrinol Metab 291: E9-E14, 2006; doi:10.1152/ajpendo.00213.2005
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Adrenomedullin inhibits insulin exocytosis via pertussis toxin-sensitive G protein-coupled mechanism

Nobuo Sekine,1 Koji Takano,2 Nako Kimata-Hayashi,2 Takashi Kadowaki,1 and Toshiro Fujita2

Departments of 1Metabolic Diseases and 2Nephrology and Endocrinology, University of Tokyo Graduate School of Medicine, Tokyo, Japan

Submitted 11 May 2005 ; accepted in final form 23 December 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Direct effects of adrenomedullin on insulin secretion from pancreatic beta-cells were investigated using a differentiated insulin-secreting cell line INS-1. Adrenomedullin (1–100 pM) inhibited insulin secretion at both basal (3 mM) and high (15 mM) glucose concentrations, although this inhibitory effect was not observed at higher concentrations of adrenomedullin. The inhibition of glucose-induced insulin secretion by adrenomedullin was restored with 12-h pretreatment with 1 µg/ml pertussis toxin (PTX), suggesting that this effect could be mediated by PTX-sensitive G proteins. Cellular glucose metabolism evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide colorimetric assay was not affected by adrenomedullin at concentrations that inhibited insulin secretion. Moreover, electrophysiological studies revealed that 10 pM adrenomedullin had no effect on membrane potential, voltage-gated calcium currents, or cytosolic calcium concentration induced by 15 mM glucose. Finally, insulin release induced by cAMP-raising agents, such as forskolin plus 3-isobutyl-1-methylxanthine or the calcium ionophore ionomycin, was significantly inhibited by 10 and 100 pM adrenomedullin. In conclusion, adrenomedullin at picomolar concentrations directly inhibited insulin secretion from beta-cells. This effect is likely due to the inhibition of insulin exocytosis through the activation of PTX-sensitive G proteins.

insulin secretion; G protein

ADRENOMEDULLIN, originally isolated as a hypotensive peptide from human pheochromocytoma (10), is now known to be widely distributed throughout the body, exerting various biological effects such as vasodilation, diuresis, bronchodilation, regulation of hormone secretion, and cell proliferation (3). Adrenomedullin is structurally related to the calcitonin gene-related peptide (CGRP) and amylin (23), both of which are present in the endocrine pancreas and may be involved in the regulation of insulin secretion (1, 4). Expression of adrenomedullin in pancreatic islets has been demonstrated in the cells that produce pancreatic polypeptide (PP cells) (13), and the presence of adrenomedullin receptors has been identified and well characterized in insulin-producing cells (14). It is thus postulated that adrenomedullin is also involved in the physiological regulation of insulin secretion as a paracrine factor, as is the case for CGRP and amylin. Moreover, adrenomedullin has been shown to inhibit amylase secretion by pancreatic acini (22), further supporting the notion that adrenomedullin may serve as a local regulator of pancreatic function.

With respect to the effect of adrenomedullin on insulin secretion, conflicting results have been found. Specifically, adrenomedullin has been reported to stimulate (16) or inhibit (13) insulin secretion from isolated rat islets. However, these studies were performed using isolated pancreatic islets consisting of various cell types and thus may not be appropriate in evaluating the direct effect of adrenomedullin on beta-cell functions per se.

In the present study, we investigated the direct effect of adrenomedullin on insulin secretion from beta-cells by using a differentiated rat insulinoma cell line INS-1 (2). This cell line retains various differentiated features of native beta-cells (2, 19, 20) and is considered a suitable model for studying the physiological mechanisms of insulin secretion. Here, we show that adrenomedullin inhibits insulin secretion from beta-cells, presumably by inhibiting insulin exocytosis, and that this effect may be mediated by pertussis toxin (PTX)-sensitive G proteins.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents. RPMI 1640 and 2-mercaptoethanol were purchased from GIBCO (Rockville, MD), whereas other components of the culture medium, namely 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), PTX, forskolin, and 3-isobutyl-1-methylxanthine (IBMX) were from Sigma-Aldrich (St. Louis, MO). Rat adrenomedullin was purchased from Peptide Institute (Osaka, Japan).

Cell culture. INS-1 cells were cultured in RPMI 1640 medium supplemented with 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, and 50 µM 2-mercaptoethanol, as previously reported (2).

Static incubation for determination of insulin secretion and MTT reduction. INS-1 cells (~3 x 105 cells/well) were seeded in multiwell plates coated with poly-L-ornithine (Sigma) and cultured for 3 days prior to the experiment. After a 30-min preincubation in modified Krebs-Ringer bicarbonate-HEPES buffer (KRBH) containing 3 mM glucose, these cells were incubated in the same buffer containing test substances as indicated for 30 min at 37°C. Insulin secreted in the incubation buffer was measured by RIA, using rat insulin as standard. After additional incubation, an MTT colorimetric assay was performed by incubating the cells for an additional 30 min in the presence of test substances. Tetrazolium salt MTT is reduced by the reaction of FAD- or NAD-linked dehydrogenases, reflecting nutrient metabolism in the cell (8). Generation of the reduced form of MTT during incubation was determined using a kit (Boehringer Mannheim) according to the manufacturer's instructions.

Electrophysiological studies. The perforated whole cell clamp technique was used (7) along with the standard patch electrode solution that contained the following (in mM): 95 K aspartate, 47.5 KCl, 1 MgCl2, 0.1 EGTA [tetramethylammonium (TMA) salt], and 10 HEPES (TMA salt, pH 7.2). The standard external solution comprised the following (in mM): 128 NaCl, 5 KCl, 1 MgCl2, 2.5 CaCl2, and 10 HEPES (Na salt, pH 7.4). To analyze the voltage-gated Ca2+ current, Ba2+ ion was used as a charge carrier. Voltage-gated Na+ channels were blocked by 1 µM tetrodotoxin, and K+ currents were blocked by intracellular Cs+ and extracellular Ba2+. The standard patch electrode solution for the analyses of voltage-gated Ca2+ current contained the following (in mM): 95 Cs aspartate, 47.5 CsCl, 1 MgCl2, 0.1 EGTA (TMA salt), and 10 HEPES (TMA salt, pH 7.2). The standard external solution contained the following (in mM): 129 NaCl, 5 KCl, 1 MgCl2, 1 BaCl2, and 10 HEPES (Na salt, pH 7.4). During the experiments the extracellular solution was continuously perfused using a peristaltic pump. Agonists were applied by changing the extracellular solution. Approximately 2 min were required to change the bath solution in this perfusion system. The liquid junction potentials between the standard extracellular solution and other solutions used (internal and external) were measured using a 3 M KCl electrode as reference, and all data were corrected for the liquid junction potential. A List EPC-7 amplifier was used to record the membrane current and potential. All experiments were performed at room temperature (22–25°C). Glass capillaries 1.5 mm in diameter and equipped with a filament were used to make patch electrodes. The resistance of patch electrodes was between 5 and 8 M{Omega}. Current clamp recordings were started after the series resistance fell below 50 M{Omega}. Voltage clamp recordings were made after the series resistance fell below 20 M{Omega}. Because the current amplitude was less than 250 pA, errors caused by series resistance were considered negligible.

Intracellular calcium measurement. Cells were loaded with fura 2 by incubating with 2 µM fura 2-AM in Hanks' balanced salt solution containing 0.1% bovine serum albumin for 40 min at room temperature. Ca2+ measurements were performed using a Nikon Diaphot microscope (Nikon, Tokyo, Japan). Each cell was excited at 340 and 380 nm alternately at a frequency of 100 Hz with CAM220 (Nihonbunko, Tokyo, Japan). A band filter was used to monitor the fluorescent emission at 510 nm. The cytosolic free Ca2+ concentration ([Ca2+]i) was determined from the equation [Ca2+]i = K(R – Rmin)/(Rmax – R) (21). In this equation, K represents Kd(Fmin/Fmax), where Kd is the dissociation constant of fura 2 (130 nM at 25°C), and Fmax/Fmin is the ratio of Ca2+-free and Ca2+-bound fura 2 fluorescence at 380 nM. Rmin is the 340/380 fluorescence ratio of Ca2+-free fura 2, and Rmax is the 340/380 ratio of Ca2+-bound fura 2. Calibration was performed on every cell by permeabilizing the cell to Ca2+ with 2 µM digitonin. Cells were first permeabilized in Ca2+-free saline (5 mM EGTA, 150 mM KCl, and 10 mM HEPES, pH 7.2) for determination of Rmin and Fmin and then in high Ca2+ saline (2.5 mM CaCl2, 150 mM KCl, and 10 mM HEPES, pH 7.4) for determination of Rmax and Fmax. [Ca2+]i traces were filtered at a bandwidth of 1 Hz to reduce the noise. Agonists were applied by changing the bath solution with a peristaltic pump. In the [Ca2+]i measurement experiment, ~30 s were required to change the bath solution.

Statistics. Results are presented as means ± SE, and statistical significance was determined by unpaired Student's t-test. In case of multiple comparisons, data were evaluated by one-way ANOVA followed by post hoc analysis of Scheffé. Differences between experimental and control groups were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Adrenomedullin inhibits insulin secretion from INS-1 cells. Effects of adrenomedullin on insulin secretion were examined during 30 min of static incubation of INS-1 cells at basal (3 mM) and at stimulatory (15 mM) concentrations of glucose. There was a 2.2-fold stimulation of insulin secretion by 15 mM glucose compared with the basal secretion (Fig. 1). Adrenomedullin (10 and 100 pM) significantly inhibited insulin secretion at both 3 and 15 mM glucose (Fig. 1). Moreover, insulin release at 15 mM glucose was also inhibited by 1 pM adrenomedullin (Fig. 1). This inhibitory effect was, however, no longer observed at even higher doses (10 and 100 nM at 3 mM glucose, 100 nM at 15 mM glucose) of adrenomedullin, although there was still a small but significant decrease in insulin secretion by 10 nM adrenomedullin in the presence of 15 mM glucose (Fig. 1).


Figure 1
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  		Fig. 1. Effect of adrenomedullin on glucose (Glc)-induced insulin secretion in INS-1 cells. INS-1 cells were preincubated for 30 min in Krebs-Ringer bicarbonate-HEPES buffer (KRBH) containing 3 mM Glc and then incubated for an additional 30 min at 3 or 15 mM Glc in the presence or absence of various concentrations of adrenomedullin as indicated. Insulin secretion is expressed as %control, with the amount of insulin secreted at 3 mM Glc being 100%. Values are means ± SE from 3 independent experiments performed in quadruplicate. Statistical analysis by ANOVA: *P < 0.05 vs. 3 mM Glc; #P < 0.01; §P < 0.05 vs. 15 mM Glc.

 
PTX abolishes the inhibition of insulin secretion by adrenomedullin. Because adrenomedullin receptors are known to couple to G proteins (9, 15), the inhibitory effect of adrenomedullin on insulin secretion was believed to be mediated by a PTX-sensitive G protein (22). We therefore examined the effect of PTX on the inhibition of glucose-induced insulin secretion by adrenomedullin. Pretreatment of INS-1 cells with 1 µg/ml PTX for 12 h resulted in an enhancement of insulin- secretory response to 15 mM glucose (Fig. 2). On the other hand, the inhibitory effect of 10 pM adrenomedullin on glucose-induced insulin secretion was abolished by the pretreatment with PTX (Fig. 2), indicating that this inhibitory effect involved a PTX-sensitive G protein.


Figure 2
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  		Fig. 2. Effect of pertussis toxin (PTX) on the inhibition of insulin secretion by adrenomedullin on Glc-induced insulin secretion. For the PTX-treated group, INS-1 cells were incubated for 12 h in culture medium containing 1 µg/ml PTX. After culture medium was removed, cells were preincubated for 30 min in KRBH containing 3 mM Glc and then incubated for another 30 min at 3 or 15 mM Glc in the presence or absence of 10 pM adrenomedullin as indicated. Insulin secretion is expressed as %control, with the amount of insulin secreted at 3 mM Glc being 100%. Values are means ± SE of 8 observations from 2 independent experiments performed in quadruplicate. Statistical analysis by ANOVA: *P < 0.05 vs. 3 mM Glc without PTX treatment; §P < 0.05 vs. 3 mM Glc with PTX treatment; #P < 0.05 vs. 15 mM Glc + adrenomedullin without PTX treatment.

 
Effect of adrenomedullin on glucose metabolism in INS-1 cells. In the next series of experiments, we investigated the effect of adrenomedullin on the factors implicated in glucose-induced insulin secretion. To examine whether adrenomedullin inhibits glucose-induced insulin secretion by affecting glucose metabolism, we evaluated cellular glucose metabolism in parallel with insulin secretion using the MTT colorimetric assay. The stimulatory glucose concentration (15 mM) demonstrated a 60% increase in MTT reduction compared with 3 mM glucose (Fig. 3). Adrenomedullin (10 pM), which inhibited glucose-induced insulin secretion (Fig. 1), did not significantly affect MTT reduction that had been stimulated by 15 mM glucose (Fig. 3).


Figure 3
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  		Fig. 3. Effect of adrenomedullin on 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction in INS-1 cells. INS-1 cells were incubated as described for the determination of insulin secretion followed by another 30 min incubation in the presence of 0.5 mg/ml MTT. Reduction of MTT was colorimetrically determined. There was no significant difference between 3 mM Glc alone and 3 mM Glc + 10 pM adrenomedulin, nor between 15 mM Glc alone and 15 mM Glc + adrenomedullin (10 pM, 100 pM, and 100 nM). Values are means ± SE from 3 independent experiments performed in quadruplicate. Statistical analysis by ANOVA: *P < 0.05 vs. 3 mM Glc.

 
Effects of adrenomedullin on membrane potential and membrane currents. Another possible mechanism underlying the adrenomedullin-induced inhibition of insulin secretion is the inhibition of membrane excitability by way of changing membrane potential or inhibiting voltage-gated calcium channels (VGCC). The effect of adrenomedullin on the membrane potential of INS-1 cells was examined under the current clamp. The resting potential was defined as the potential needed to set the whole cell current at zero under the voltage clamp. In the control experiment, the resting potential was –58 ± 3 mV at 3 mM glucose (n = 8). Increasing the glucose concentration from 3 to 15 mM depolarized the cell to –50 ± 5 mV (n = 8). However, this membrane potential did not change significantly (–49 ± 4, n = 8) even when 10 pM adrenomedullin was applied to the cell.

To investigate the effect of adrenomedullin on the membrane current, a ramp pulse was used to evoke a membrane current, which was compared before and after adrenomedullin application in 15 mM glucose. The membrane current evoked thus did not alter significantly upon the application of 10 pM adrenomedullin (Fig. 4A). Similar results were obtained in seven other cells.


Figure 4
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  		Fig. 4. Effect of adrenomedullin on the membrane current of INS-1 cells. A: membrane current elicited by a ramp pulse between –120 and –50 mV in 1 s is shown. Membrane current in 15 mM Glc did not differ significantly from that treated with adrenomedullin (10 pM) in 15 mM Glc; same results were obtained from 7 other cells. B: voltage-gated Ca2+ currents evoked by pulse steps to –60, –50, –40, and –30 mV from a holding potential of –70 mV (left) and those by pulse steps to –20, –10, and 0 mV from a holding potential of –30 mV before (middle) and after (right) application of nitrendipine (1 µM) are plotted. C: voltage-gated Ca2+ currents evoked by pulse steps to –50, –40, –30, and –20 mV from a holding potential of –70 mV before (left) and after (right) application of adrenomedullin (10 pM) are plotted. Application of adrenomedullin did not change the membrane current. D: effect of 3 adrenomedullin concentrations (10 pM, 100 pM, and 1 nM) on the peak amplitude of voltage-gated calcium channels (VGCC) is summarized (n = 10). Peak amplitude is expressed as %control at 15 mM Glc being 100%. There were no significant differences among currents (one-way ANOVA).

 
Because the VGCC are key components in the regulation of insulin secretion, the effect of adrenomedullin on these channels was examined in 15 mM glucose. At the holding potential of –70 mV, there were two components to the voltage-gated calcium current (Fig. 4B, left). One was a transient one whose rapid inactivation was followed by a sustained inward current. To examine which type of VGCC was activated, the current was observed at the holding potential of –30 mV. At this depolarized holding potential, the initial transient component was not observed, but a sustained current remained (Fig. 4B, middle), indicating that the initial transient component was a T-type VGCC. The sustained current was remarkably inhibited by application of nitrendipine, an L-type VGCC blocker, leaving only a minor current (Fig. 4B, right). These data indicate that the VGCC of INS-1 cells are mainly comprised of the T-type and L-type components with a minor residual current. When 10 pM adrenomedullin were applied to cells with 15 mM glucose, neither of the two components was affected (Fig. 4C, left and right). Similar results were observed when 100 pM and 1 nM adrenomedullin were applied (n = 10 for each concentration; Fig. 4D).

Effect of adrenomedullin on [Ca2+]i in INS-1 cells. In addition to the effect of adrenomedullin on calcium currents as described above, we also measured [Ca2+]i using fura 2 as the calcium indicator. When glucose concentration of the medium was changed from 3 to 15 mM, [Ca2+]i markedly increased from the basal level (~60 nM) to ~250 nM. Application of 10 pM adrenomedullin, however, did not change [Ca2+]i (Fig. 5A). Application of 100 pM adrenomedullin also did not change [Ca2+]i. Figure 5B summarizes the results of these experiments (n = 7).


Figure 5
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  		Fig. 5. Effect of adrenomedullin on glucose-induced cytosolic free Ca2+ concentration ([Ca2+]i) rise in INS-1 cells. A: when extracellular Glc was changed to 15 mM from 3 mM, the [Ca2+]i of the cell increased. Additional application of adrenomedullin (10 pM) did not change the [Ca2+]i any further. B: summary of the effect of adrenomedullin (10 pM) on [Ca2+]i. Cellular [Ca2+]i in 3 mM Glc, 15 mM Glc, 15 mM Glc + 10 pM adrenomedullin, and 15 mM Glc + 100 pM adrenomedullin are shown (n = 7). There were no significant differences among cellular [Ca2+]i in 15 mM Glc, 15 mM Glc and 10 pM adrenomedullin, and 15 mM Glc and 100 pM adrenomedullin (one-way ANOVA).

 
Adrenomedullin inhibits insulin release induced by forskolin plus IBMX or by ionomycin in INS-1 cells. Finally, to investigate the effect of adrenomedullin on insulin exocytosis, we examined whether adrenomedullin could inhibit insulin release induced by a rise in either cAMP or [Ca2+]i, both of which directly induce insulin exocytosis of insulin granules (24). The cAMP-raising agent forskolin in combination with IBMX increased insulin release from INS-1 cells 3.3-fold, whereas a much smaller but significant increase was observed with the Ca2+ ionophore ionomycin (Fig. 6). Adrenomedullin at both 10 and 100 pM significantly inhibited insulin release induced by these agents (Fig. 6).


Figure 6
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  		Fig. 6. Effect of adrenomedullin on insulin release induced by either forskolin + 3 IBMX or ionomycin in INS-1 cells. INS-1 cells were preincubated for 30 min in KRBH containing 3 mM G and were incubated for an additional 30 min in the same buffer containing 1 µM forskolin plus 100 µM IBMX or 1 µM ionomycin in the presence or absence of 10 or 100 pM adrenomedullin as indicated. In the presence of 3 mM G, insulin release was significantly increased either by forskolin plus IBMX (P < 0.01) or by ionomycin (P < 0.05). Insulin secretion is expressed as %control, with the amount of insulin secreted at 3 mM G being 100%. Values are means ± SE from 3 independent experiments. Statistical analysis by ANOVA: *P < 0.05 vs. forskolin plus IBMX; #P < 0.05 vs. ionomycin.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrated that adrenomedullin directly inhibits insulin secretion from beta-cells and that the inhibitory effect of adrenomedullin can be ascribed to the inhibition of insulin exocytosis through a PTX-sensitive G protein. We also found that, in INS-1 cells, adrenomedullin inhibited insulin secretion from INS-1 cells at low concentrations (picomolar range of adrenomedullin) but was ineffective at higher concentrations.

The effect of adrenomedullin on insulin secretion from isolated rat pancreatic islets thus far has yielded conflicting results (13, 16). Martínez et al. (13) have reported that adrenomedullin inhibits insulin secretion at 10 nM to 1 µM in a concentration-dependent manner, although Mulder et al. (16) have shown that adrenomedullin at 10 and 100 nM stimulates insulin secretion from isolated rat islets. Interestingly, the report by Martínez et al. (13) further revealed that adrenomedullin inhibited insulin secretion while increasing cAMP concentrations in the islet cells. However, adrenomedullin-induced cAMP increase is incongruous with the inhibition of insulin secretion because the elevation of intracellular cAMP concentration by agents such as those by glucagon, glucagon-like peptide-1, or forskolin results in the potentiation of glucose-stimulated insulin secretion (24). In this context, the results of these two previous reports may give insights into the present finding that adrenomedullin failed to inhibit insulin secretion at the higher concentrations in INS-1 cells. We speculate that adrenomedullin at higher concentrations evokes a stimulatory component of insulin secretion, presumably via the elevation of cAMP, that cancels out the inhibitory component observed at lower concentrations. However, whether adrenomedullin stimulated insulin secretion through the cAMP elevation is not provided in the report by Mudler et al. (16).

To better understand the results, we investigated the mechanisms involved in the inhibitory effect of adrenomedullin on insulin secretion. Glucose is known to stimulate insulin secretion through its metabolic action on beta-cells by generating metabolic coupling factors that promote exocytosis of insulin granules (18). Increase in ATP levels or in ATP-to-ADP ratio through the mitochondrial metabolism of glucose leads to closure of the ATP-sensitive K+ channel, thereby causing membrane depolarization. Membrane depolarization facilitates the opening of the VGCC, inducing Ca2+ influx into the beta-cell and thus promoting insulin exocytosis. Several cellular properties may be attributed to this inhibitory effect: 1) glucose metabolism within the cell that is coupled to cellular excitability of the beta-cell; 2) cellular excitability itself, specifically the membrane potential or the voltage-gated calcium currents; 3) intracellular cAMP concentration; and 4) the exocytosis machinery of insulin granules. These intracellular events mediating glucose-induced insulin secretion are depicted in Fig. 7. Our results revealed that the former three components were not influenced by adrenomedullin, leaving its effects on the exocytosis machinery as the only possibility. It has been known that exocytosis of insulin granules is stimulated by IBMX plus forskolin through the elevation of cAMP and by ionomycin through the rise in [Ca2+]i (24). Therefore, the finding that adrenomedullin inhibited insulin release induced by IBMX plus forskolin or by ionomycin supports the notion that the action of adrenomedullin on insulin secretion induced by these agents is due to the inhibition of insulin exocytosis. The small increase in insulin secretion by ionomycin may be due to the low concentration (3 mM) of glucose, leaving the possibility that the effect might better be demonstrated at higher concentrations of glucose.


Figure 7
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  		Fig. 7. Intracellular events mediating glucose-induced insulin secretion and the site of action of adrenomedullin inhibiting insulin secretion in beta-cells. Glucose enters the beta-cell by a facilitative transport through a glucose transporter (GLUT) on the cell membrane. Following cellular metabolism of glucose in mitochondria, increase in cytosolic ATP or ATP-to-ADP ratio leads to closure of KATP channels, thereby causing membrane depolarization, which results in Ca2+ influx through VGCC. Rise in [Ca2+]i facilitates exocytosis of insulin granules while G proteins are also implicated in insulin exocytosis. Adrenomedullin is considered to inhibit insulin exocytosis through a PTX-sensitive G protein downstream of adrenomedullin receptor (AM-R) on the beta-cell membrane. {Psi}, membrane potential; R, G protein-coupled receptors, such as GLP-1, that couple to Gs.

 
With respect to the mechanisms underlying the effect of adrenomedullin on insulin exocytosis, the inhibition of glucose-induced insulin secretion by adrenomedullin was found to be reversed by pretreatment with PTX, suggesting that the action of adrenomedullin is mediated by PTX-sensitive G proteins such as Gi or Go. As has been shown in other experimental systems (11), pretreatment with PTX resulted in an enhancement of glucose-induced insulin secretion in INS-1 cells. This finding thus supports the idea that PTX-sensitive G proteins are implicated in the regulation of insulin secretion (18) and indicates that PTX treatment leads to simulation of insulin release by blocking constitutive Gi/Go activity in beta-cells. Similar results have been obtained by Tsuchida et al. (22) on the inhibitory effect of adrenomedullin on amylase secretion from pancreatic acini. In their report, adrenomedullin inhibited CCK-induced amylase secretion at low concentrations (1 pM to 1 nM) comparable with those of adrenomedullin on inhibiting insulin secretion in INS-1 cells, an effect that was also abolished by PTX-pretreatment (22). In pancreatic acinar cells, amylase secretion is stimulated by an increase in intracellular Ca2+ concentration. Tsuchida et al. (22) have shown that this inhibitory action of adrenomedullin did not result from the modulation of CCK-induced calcium response but rather from inhibiting the calcium-induced amylase secretion, suggesting that adrenomedullin inhibits amylase secretion directly by acting on the exocytosis machinery.

It should be noted that insulin exocytosis could be directly regulated by receptor-mediated activation of the heterotrimeric G proteins (12). With the use of streptolysin O-permeabilized insulin-secreting cells, Lang et al. (12) have shown that the activation of both the {alpha}2-adrenergic receptors and Gi/Go directly inhibit calcium-induced insulin exocytosis in a PTX-sensitive manner. We speculate that a similar mechanism is involved in the inhibitory action of adrenomedullin on calcium-induced insulin exocytosis, as was demonstrated by ionomycin in the present study.

Finally, from a clinical perspective, results of the present study provide the evidence that adrenomedullin could be involved in both the physiological regulation of insulin secretion as well as the pathogenesis of diabetes as a causative factor of impaired insulin secretion. It has been reported (5) that diabetic patients show increased plasma adrenomedullin concentrations reaching the picomolar range. In vitro studies (6) have indicated that hyperglycemia can lead to an increased vascular expression of adrenomedullin. It could therefore be speculated that adrenomedullin in the serum of diabetic patients may play a role in further impairing the insulin secretion.


   ACKNOWLEDGMENTS
 
We thank R. Odajima for excellent technical help and for editorial help.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Takano, Dept. of Nephrology and Endocrinology, Univ. of Tokyo Graduate School of Medicine, 7–3-1 Hongo, Bunkyo-ku, Tokyo 113–8655, Japan

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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