Restitution of Defective Glucose-Stimulated Insulin Secretion in Diabetic GK Rat by Acetylcholine Uncovers Paradoxical Stimulatory Effect of
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《糖尿病学杂志》
1 Unitee Mixte de Recherche (UMR) 7059, National Center for Scientific Research (CNRS) and Paris University 7/D. Diderot, Paris, France
2 Department of Genetic Medicine and Development, University Medical Center, Geneva, Switzerland
ACh, acetylcholine; BIM, bisindolylmaleimide I; [Ca2+]i, intracellular free calcium concentration; dd-Ado, 2',5'-dideoxyadenosine; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide; InsP, inositol phosphate; IBMX, isobutyl methylxanthine; KRB, Krebs-Ringer buffer; mAChR, muscarinic ACh receptor; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PLC, phospholipase C; Rp-8-Br-cAMPS, 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer; SERCA, sarcoendoplasmic reticulum Ca2+-ATPase
ABSTRACT
Because acetylcholine (ACh) is a recognized potentiator of glucose-stimulated insulin release in the normal -cell, we have studied ACh’s effect on islets of the Goto-Kakizaki (GK) rat, a spontaneous model of type 2 diabetes. We first verified that ACh was able to restore the insulin secretory glucose competence of the GK -cell. Then, we demonstrated that in GK islets 1) ACh elicited a first-phase insulin release at low glucose, whereas it had no effect in Wistar; 2) total phospholipase C activity, ACh-induced inositol phosphate production, and intracellular free calcium concentration ([Ca2+]i) elevation were normal; 3) ACh triggered insulin release, even in the presence of thapsigargin, which induced a reduction of the ACh-induced [Ca2+]i response (suggesting that ACh produces amplification signals that augment the efficacy of elevated [Ca2+]i on GK exocytosis); 4) inhibition of protein kinase C did not affect [Ca2+]i nor the insulin release responses to ACh; and 5) inhibition of cAMP-dependent protein kinases (PKAs), adenylyl cyclases, or cAMP generation, while not affecting the [Ca2+]i response, significantly lowered the insulinotropic response to ACh (at low and high glucose). In conclusion, ACh acts mainly through activation of the cAMP/PKA pathway to potently enhance Ca2+-stimulated insulin release in the GK -cell and, in doing so, normalizes its defective glucose responsiveness.
Cholinergic muscarinic agonists, including the endogenous neurotransmitter acetylcholine (ACh) and the synthetic nonhydrolyzable analog carbachol, are known to enhance glucose-stimulated insulin secretion by the normal -cell (1). ACh is released by intrapancreatic vagal nerve endings during the preabsorptive and absorptive phases of feeding, and it acts mainly by activating phospholipase C (PLC)/protein kinase C (PKC) signaling pathways in the normal -cell (1). In insulin-resistant rodent models, such as C57BL/6J mice fed a high-fat diet (2) and preobese Lepob/Lepob mice (3), it is recognized that ACh induces exaggerated insulin release.
Whether a similarly altered cholinergic response is also found in type 2 diabetes is not so clear; it has been reported that chronic hyperglycemia increases the number of muscarinic binding sites in islets from streptozotocin-induced diabetic rats (4), whereas rat islets cultured at high glucose concentrations show a decrease in ACh-activated inositol phosphate (InsP) generation (5). However, we previously reported that in the basal state, the Goto-Kakizaki (GK) rat, a lean spontaneous model of type 2 diabetes, presented enhanced plasma insulin and islet blood flow that were totally abolished by vagotomy, whereas in nondiabetic control rats, vagotomy did not change these parameters (6), suggesting increased ACh activation of the GK islets. More recently, indications for enhanced insulin release in response to ACh (7) have been obtained in GK islets from the Stockholm GK colony, a subline whose islet phenotype is close to, but not identical to, that of our GK colony (Paris colony, GK/Par) (8).
These observations prompted us to explore interactions between ACh and glucose on insulin release and intracellular Ca2+ in freshly isolated GK/Par islets, as well as to examine the secondary messenger pathways involved.
RESEARCH DESIGN AND METHODS
All animal experimentation was conducted on fed age-matched male GK/Par rats and nondiabetic Wistar rats from our local colonies, in accordance with accepted standards of animal care as established by the French National Center for Scientific Research guidelines. The characteristics of the GK rats maintained in our colony at Paris University 7 have been described previously (9). Plasma glucose levels for Wistar and GK/Par rats at time of death were 6.6 ± 0.2 mmol/l (n = 30) and 9.6 ± 0.5 mmol/l (n = 30), respectively (P < 0.001).
Required chemicals and sources.
The 125I-labeled insulin was from DiaSorin. The 125I-labeled adenosine 3',5' cyclic-phosphoric acid 2'-O-succinyl-3, the myo-[2-3H]inositol and the radiolabeled InsP mixture [Ins(1,3,4)P3 + Ins(1,4,5)P3] were from Amersham. The Fura-2/AM was from Molecular Probes. Dowex AG1-X8 resin was supplied by Bio-Rad. BSA (fraction V) was from Roche Molecular Biochemicals. Purified rat insulin was supplied by Novo Research Institute (Copenhagen). 2',5'-dideoxyadenosine (dd-Ado) and bisindolylmaleimide I (BIM) were from Calbiochem. 8-bromoadenosine-3',5'-cyclic-monophosphorothioate, Rp-isomer (Rp-8-Br-cAMPS) was from Biolog. D-glucose, Tris, and the salts used to make the Hanks’ solution and Krebs-Ringer buffer (KRB) were from Merck. Other chemicals were from Sigma-Aldrich.
Islet isolation.
Rats (3eC4 months old) were killed by decapitation. For each experiment, three pancreata were digested with collagenase, and the islets were handpicked under a stereomicroscope.
Measurements of islet intracellular free calcium concentration and insulin release during perifusion procedure.
Freshly isolated islets were loaded 1 h with 5 e蘭ol/l Fura-2/AM at 37°C in KRB containing (in mmol/l) 115 NaCl, 5 KCl, 24 NaHCO3, 1 CaCl2, 1 MgCl2, and 5.5 glucose and 5 mg/ml BSA. After loading, eight islets at a time were allowed to attach on a polylysine-treated cover glass transferred to a perifusion chamber placed on the stage of an inverted fluorescent microscope (Nikon Diaphot, Champigny sur Marne, France). Cannulas feeding into the chamber were connected to a peristaltic pump and allowed a continuous superfusion of the islets at a flow rate of 1 ml/min with a 25 mmol/l HEPES-buffered medium maintained at 37°C containing (in mmol/l) 125 NaCl, 5.9 KCl, 1.28 CaCl2, 1.2 MgCl2, and 2.8 glucose and 1 mg/ml of BSA. Intracellular free Ca2+ concentration ([Ca2+]i) was determined as previously described (10). The perifusion fluid was collected from the chamber at 20-s intervals and stored at eC20°C for insulin radioimmunoassay.
Measurement of islet InsPs.
Batches of 150 freshly isolated islets were loaded with myo-[2-3H]inositol (16 e藽i) during 150 min at 37°C in 0.8 ml KRB-BSA containing 2.8 mmol/l glucose. After washing, prelabeled islets were incubated for 15 min at 37°C in 0.5 ml KRB-BSA containing 2.8 mmol/l glucose, 10 mmol/l LiCl (to inhibit phosphatase), and 1 mmol/l unlabeled myo-inositol, in the absence or presence of 1 mmol/l ACh. Free myo-inositol, glycerophosphoinositol, and InsP were eluted in a stepwise manner by anion-exchange chromatography, as previously described (11). The accumulation of radiolabeled InsP was taken as an index of islet cell phosphoinositide breakdown.
Measurements of islet cAMP content and insulin release during static incubation.
cAMP content was measured in islet pellets, and insulin release was determined in the incubation buffer from the same batches of islets, using radioimmunoassays as described previously (12). Briefly, after a 60-min preincubation period, groups of 20 freshly isolated islets were incubated for 20 min in 1 ml KRB-BSA medium containing 2.8 mmol/l glucose alone or supplemented with 100 e蘭ol/l isobutyl methylxanthine (IBMX), 100 e蘭ol/l dd-Ado, and 100 e蘭ol/l SQ 22536, as specified. In ACh experiments, all islet samples were incubated with the above-mentioned test substances 10 min before time 0 stimulation with 1 mmol/l ACh. Islets’ cAMP content and insulin release were determined after up to 10 min of incubation.
Islet DNA content.
In some experiments, at least two batches of 20 islets from control or GK rats were collected to determine DNA by fluorometric assay, as previously described (11).
Microarray analysis.
RNA was extracted from 4-month-old Wistar and GK islets using an RNeasy total RNA isolation kit (Qiagen). Then, 2 e蘥 of RNA was used to synthesize double-stranded cDNA using the Superscript Choice system (Invitrogen, Groningen, the Netherlands). In vitro transcription was carried out on 6 e蘬 cDNA using Bioarray High Yield RNA transcript labeling reagents (Enzo Diagnostics). Reactions yielded 50eC70 e蘥 biotin-labeled cRNA, which was purified on RNeasy affinity columns (Qiagen) and fragmented at 94°C for 35 min in fragmentation buffer, as previously described (13). Next, 15eC20 e蘥 fragmented cRNA was hybridized to Affymetrix RG-U34A oligonucleotide microarrays representative of 7,000 rat genes. Arrays were scanned, and the data obtained were analyzed using Genespring 6 (Silicon Genetics). The microarray experiments were performed using the Genomics Platform (NCCR Frontiers, Geneva).
Data presentation and statistical methods.
The kinetic variations of [Ca2+]i are presented as percentage changes in 340-to-380 fluorescence ratios. To quantify the [Ca2+]i responses, both integrated elevation above baseline (Ca, in arbitrary units) and percent elevation of [Ca2+]i above basal at peak value (PCa) were calculated. Integrated elevation above baseline value (Ins, in ng per eight islets) were used for insulin response quantitation. All data are presented as the means ± SE. Statistical analysis was performed using unpaired Student’s t test or ANOVA (Fisher test) as appropriate. Differences were considered significant at P < 0.05.
RESULTS
ACh restores blunted glucose-stimulated insulin secretion by GK pancreatic islets.
Whereas in Wistar islets stimulation with 16.7 mmol/l glucose resulted in a typical [Ca2+]i response (Fig. 1A), it produced a monophasic and significantly less pronounced (P < 0.001) change in [Ca2+]i in the GK islets (Ca7eC27min 24.3 ± 3.7 vs. 10.9 ± 1.1). ACh in the presence of 16.7 mmol/l glucose (Fig. 1B) similarly induced a biphasic [Ca2+]i pattern in both GK and Wistar islets. Nevertheless, although the initial peak was not significantly different, the following prolonged rise was significantly less pronounced (P < 0.05) in the GK islets (Ca7eC27min 19.8 ± 4.7 vs. 33.7 ± 3.0).
We found that 16.7 mmol/l glucose triggered biphasic insulin secretion (attenuated pattern attributable to averaging) in the Wistar islets (Fig. 1C), whereas only a small amount of insulin was released by the GK islets (Ins7eC27min 28.2 ± 5.1 vs. 13.5 ± 1.6 ng per eight islets, P < 0.001). ACh potentiation of 16.7 mmol/l glucoseeCinduced insulin secretion was observed in both Wistar and GK islets (Fig. 1D), and, remarkably, the total amount of insulin secreted during the 20-min period became no longer significantly different between GK and Wistar islets (Ins7eC27min 122 ± 14.8 vs. 107.3 ± 12.8 ng per eight islets), despite very different [Ca2+]i elevations.
ACh acts as a glucose-independent triggering signal in the GK islet.
In the presence of a nonstimulatory glucose concentration (2.8 mmol/l), ACh similarly increased [Ca2+]i in GK and Wistar islets (Ca7eC27min 8.4 ± 1.8 vs. 7.4 ± 1, and PCa 35.9 ± 6.6 vs. 27.1 ± 3.7%) (Fig. 2A). By contrast, this ACh-induced [Ca2+]i signal triggered a significant (P < 0.02) release of insulin only in GK islets (Ins7eC27min 4.9 ± 1.3 ng per eight islets) (Fig. 2B). Perifusion of the GK islets with a glucose-free medium did not prevent ACh from triggering insulin secretion (data not shown). Additionally, the efficacy of ACh in the GK islet cannot be ascribed to the high concentration used because we obtained the same secretory and [Ca2+]i responses with 50 e蘭ol/l carbachol (data not shown). Moreover, 20 e蘭ol/l atropine prevented these effects of ACh in the GK islets (data not shown), indicating that they were mediated by muscarinic receptors.
ACh-induced PLC activation and inositol triphosphate accumulation are not enhanced in GK islets.
In GK islets, the basal production of [3H]InsP was increased 1.6-fold (P < 0.05) compared with Wistar islets (Fig. 2C). In response to 1 mmol/l ACh, the levels of labeled InsP1, -2, and -3 and total InsP increased significantly above baseline in both GK and Wistar groups (Fig. 2C). The level of ACh-induced total InsP was increased 1.4-fold (P < 0.05) compared with Wistar islets (Fig. 2C). This effect was abolished by 20 e蘭ol/l atropine (data not shown). However, when expressed as a percentage of the paired basal value, ACh similarly stimulated [3H]InsP production in the GK and Wistar groups (517 and 581%, respectively). Additionally, total PLC activity, as measured in vitro in GK and Wistar islet homogenates by hydrolysis of exogenous [3H]phosphatidylinositol in the presence of 1 mmol/l Ca2+, was found to be similar in both groups (Fig. 2D).
ACh-induced insulin release by GK islets brings into operation a Ca2+ mobilization from intracellular Ca2+ stores and an increased efficacy of Ca2+ on exocytosis.
As shown in Fig. 2, there existed a tight temporal parallelism between the rise in [Ca2+]i and insulin release induced by ACh in the GK islets. Thapsigargin, an inhibitor of the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) pump, elicited a short-lived rise of [Ca2+]i in both Wistar and GK islets (Fig. 3A). [Ca2+]i response to ACh was significantly decrease in the GK and Wistar islets treated with thapsigargin (Ca22eC27min 0.6 ± 0.1 and 0.4 ± 0.1 vs. Ca7eC12min 3.4 ± 0.4 and 2.6 ± 0.4, respectively, in GK and Wistar islets with or without thapsigargin) (Fig. 3A), suggesting that the intracellular ACh-sensitive Ca2+ pool was largely emptied. Whereas in the Wistar islets addition of ACh in the presence of thapsigargin did not affect significantly insulin release (Fig. 3B), in the GK islets ACh still triggered a significant release of insulin (Ins22eC27min 4.0 ± 1.1 vs. Ins7eC12min 2.4 ± 0.5 ng per eight islets for GK islets with or without thapsigargin) (Fig. 3B).
The PKC pathway is not involved in the ACh-triggered insulin release by the GK islets.
The PKC pathway is considered to play a major role in the response of the normal -cell to ACh for sensitizing the secretory machinery to Ca2+ (1). The treatment of GK islets with 1 or 2.5 e蘭ol/l BIM, a PKC inhibitor (14), did not significantly change the ACh-activated [Ca2+]i response (PCa 37.1 ± 6 and 26.1 ± 4.8%, respectively) (Fig. 4AeCB) or the basal rate of insulin release (Fig. 4CeCD). The addition of ACh to 1 e蘭ol/l BIM produced an increased insulin release (Ins17eC27min 4.0 ± 1.1 ng per eight islets) similar to that obtained in the absence of BIM (Ins7eC17min 3.7 ± 0.2 ng per eight islets) (Fig. 4C). In the presence of 2.5 e蘭ol/l BIM, the secretory response to ACh became even more pronounced (Ins17eC27min 13.3 ± 1.7 ng per eight islets) (Fig. 4D).
ACh activates adenylyl cyclase, cAMP generation, and the cAMP-dependent protein kinase pathway in the GK islets.
Because the ACh-triggered insulin release by the GK islets was found independent of PKC, and because we have previously reported that some adenylyl cyclase (adenylyl cyclase-2 and -3) and the Gs protein Golf are overexpressed in GK/Par islets (15), we tested the hypothesis that stimulation of GK islets by ACh, at low glucose levels, has an effect on cAMP generation and the cAMP-dependent protein kinase (PKA) pathway.
First, we evaluated PKA inhibitors. Although both 250 e蘭ol/l Rp-8-Br-cAMPS (16) and 1 e蘭ol/l N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H-89) (17) decreased PCa induced by ACh in the GK islets, by 39% (P < 0.05) and 36%, respectively, they did not significantly affect the integrated Ca2+ values (Ca17eC27min 3.3 ± 0.4 and 4.9 ± 1 vs. Ca7eC17min 4.8 ± 0.7 for ACh-stimulated GK islets without inhibitors) (Fig. 5AeCB). However, these inhibitors significantly reduced ACh-activated insulin release (Ins17eC27min 0.3 ± 0.5 and 1.2 ± 0.6 ng per eight islets with Rp-8-Br-cAMPS and H-89, respectively) (Fig. 5CeCD), indicating that activation of the PKA signal transduction pathway is involved.
We then evaluated the possibility that cAMP is directly implicated. First, treatment of GK islets with dd-Ado, an adenylyl cyclase inhibitor (18), also decreased by 45% (P < 0.05) the PCa induced by ACh. It had no significant effect on Ca17eC27min (3.9 ± 0.2) (Fig. 7A), but it completely suppressed the ACh-activated insulin release at 2.8 mmol/l glucose (I17eC27min 0.2 ± 0.3 ng per eight islets) (Fig. 6B). Second, in Wistar islets, under static incubation at 2.8 mmol/l glucose, ACh in the presence of IBMX significantly decreased cAMP production by 35% (P < 0.05) below basal level, whereas it failed in the absence of IBMX (Fig. 7AeCB). Under these conditions, decreased cAMP generation was correlated with a significant 29% reduction in insulin release (P < 0.001) (Fig. 7D).
In GK islets, cAMP generation was quite different. In the basal condition (2.8 mmol/l glucose without IBMX), the GK islet cAMP level was significantly increased (by 67%, P < 0.001) compared with the Wistar level (Fig. 7A). In response to ACh, we were unable to detect any cAMP accumulation in the absence of IBMX. However, in the presence of IBMX, ACh increased cAMP generation by 44% (P < 0.001) (Fig. 7B), and this was associated with an increase in insulin release of 60% (P < 0.001), despite the low concentration of extracellular glucose (Fig. 7D). Remarkably, these effects of ACh on cAMP generation and insulin release were completely suppressed by the specific adenylyl cyclase inhibitors dd-Ado or SQ 22536 (Fig. 7D). We used 100 e蘭ol/l dd-Ado because we worked with freshly isolated islets, and we determined the effect of inhibition of adenylyl cyclase after acute pretreatment (10 min). Lower doses are usually chosen for chronic treatment of isolated cells (19). However, German et al. (20) have used 1 mmol/l dd-Ado with rat islet cells incubated for 48 h.
The restitution of the glucose responsiveness of GK islets by ACh is also cAMP mediated.
To study the potential cAMP dependence of the Ach potentiation of 16.7 mmol/l glucoseeCinduced insulin secretion by GK islets, we blocked adenylyl cyclase activity. dd-Ado decreased PCa by 37.4% but did not significantly affect the integrated [Ca2+]i value in response to 16.7 mmol/l glucose + ACh (Ca17eC37min 10.6 ± 1.4 vs. 13.0 ± 4.2 in GK islets with or without dd-Ado) (Fig. 8A). By contrast, it significantly (P < 0.001) lowered the early increase of insulin release as well as the late phase obtained in response to a stimulation by 16.7 mmol/l glucose + ACh (Ins17eC37min 13.1 ± 2.7 vs. 87.8 ± 6.2 ng per eight islets, respectively, with or without dd-Ado) (Fig. 8B). In addition, dd-Ado did not affect significantly the [Ca2+]i patterns and the insulin release obtained in response to 16.7 mmol/l glucose alone (data not shown). Therefore, blocking ACh-activated cAMP generation in the GK islets eliminated the ability of ACh to reactivate their glucose-induced insulin release.
DISCUSSION
In the first part of this study, we confirmed that poor glucose-induced insulin secretion by GK islets is correlated to dysfunctions in glucose-induced [Ca2+]i handling. Both of these defects have been identified in the GK/Par line (10) as well as the GK/Stockholm line (21), a GK line with no decreased -cell density and insulin content at variance with the GK/Par line phenotype (8).
Because their effects on insulin secretion are glucose dependent, cholinergic agonists might theoretically be helpful to improve insulin secretion in type 2 diabetes. For this reason we studied the effect of ACh on glucose-induced insulin secretion in freshly isolated GK/Par islets. We found that ACh was able to restore the insulin secretory glucose competence of the GK -cell to such a degree that the insulin release became indistinguishable from those of normal Wistar islets, despite different [Ca2+]i elevations. Our conclusion related to insulin output is consistent with the previous report in the GK/Stockholm line in that carbachol normalized the insulin secretion at high glucose levels (7). Furthermore, our group previously reported that ACh also normalized glucose-stimulated insulin secretion in n0-STZ (streptozotocin) rats (another recognized model of rat diabetes) (22), suggesting that restoration by ACh of glucose responsiveness in the diabetic -cell is not restricted to the GK model but is probably a more general mechanism.
Under a low glucose concentration or even in its absence, we find that ACh, through muscarinic receptor activation, stimulates insulin release by GK islets with a clear first phase of insulin secretion and a faint but sustained second phase. This result suggests that ACh triggers -cell granule mobilization in the GK islets from a reserved pool to a releasable pool, despite the low glucose concentration. This obviously contrasts with the lack of insulin response in the Wistar islets.
There are several mechanisms to account for the unusual triggering capacity of ACh in GK -cells. First, in the GK -cell, the early steps of ACh signaling may be increased; our study shows that this does not hold true because ACh exerts the same increasing effect on [Ca2+]i in Wistar and GK islets, mainly because of normal PLC-catalyzed inositol triphosphate production and normal intracellular ACh-sensitive Ca2+ pool mobilization. It is interesting to note that the normal amplitude of the ACh-dependent mobilization of intracellular Ca2+ stores was obtained in the GK islet despite a reduction of the expression of SERCA-3 (23), which we were able to confirm in the GK/Par line using an Affimetrix approach. We found that SERCA-3 mRNA (accession no. M30581 at) was decreased 1.71-fold (average of experiment 1: 1.80; and experiment 2: 1.61) compared with Wistar islets. In addition, inositol triphosphate receptor type 3 mRNA (accession no. L06096 g at) was increased 1.40-fold (average of experiment 1: 1.22; and experiment 2: 1.60) in GK islets. This is in line with the previous proposal that SERCA-3 is not involved in the replenishment of inositol triphosphateeCsensitive and eCinsensitive Ca2+ pools in the endoplasmic reticulum (24).
A second option is that ACh produces amplification signals that augment the efficacy of elevated [Ca2+]i on exocytosis by the GK -cell, whereas the normal Wistar -cell lacks these ACh-dependent signals. This is indeed supported by the observation that ACh triggered release of insulin by the GK islets equally well in the presence or absence of thapsigargin, despite very different [Ca2+]i elevations.
In the normal -cell, the most important ACh-dependent amplifying mechanism is considered to involve PKC (1). As shown in our experiments using the PKC inhibitor BIM, ACh-dependent PKC activation at low glucose, if any, is not involved in the ACh-triggered insulin release by the GK islets.
cAMP was demonstrated to induce insulin secretion independent of a glucose stimulus (25), and cAMP-dependent PKA activation was reported to activate exocytosis distal to [Ca2+]i changes in normal -cell (26eC29). Although ACh is not usually recognized as a stimulator of cAMP production at low glucose concentrations by the normal islet (30eC32), a recent report suggests that such is the case (33). Using Wistar islets incubated at low glucose concentrations, we were unable to confirm this observation. By contrast, under the same experimental conditions (low glucose, presence of IBMX), ACh triggered activation of adenylyl cyclase and accumulation of cAMP in the GK islets. In addition, at low glucose and in the absence of ACh, the basal cAMP accumulation was found to be significantly elevated in the GK islets. This is supported by our previous findings that some adenylyl cyclase (adenylyl cyclase-2 and -3) and the Gs protein Golf are overexpressed in GK/Par islets (15). Similar adenylyl cyclase (including the adenylyl cyclase-1 and -8 isoforms) and Gs overexpressions have been reported in the islets from the GK/Stockholm line (34,35). However, in the GK/Stockholm islets, no elevation of cAMP under basal conditions was detectable (7,36) and carbachol failed to increase cAMP formation (7).
Our results suggest that ACh-stimulated cAMP generation, rather than increased basal cAMP, is instrumental in the ACh-triggered insulin release at low glucose by GK/Par islets. First, GK -cell secretion became no longer reactive to ACh when adenylyl cyclases were acutely blocked by dd-Ado or SQ 22536. In the absence of ACh, GK -cell insulin secretion and the cAMP level decreased when adenylyl cyclase was blocked by the adenylyl cyclase blocker dd-Ado. Second, normal Wistar -cell insulin secretion still remained inactivated by ACh under conditions that artificially (with IBMX) raised the cAMP level to a value identical to that in GK islets. Strikingly, under these conditions ACh exerted in fact a significant inhibitory effect on cAMP generation and insulin release by the normal -cell. This last observation complements the previous results of Miguel et al. (37) obtained in the -cell line BRIN BD11. They showed a paradoxical inhibitory effect of ACh on glucagon-like peptide-1eCstimulated cAMP production achieved through a novel pertussis toxineCand pirenzepine-sensitive M1 muscarinic receptoreCactivated pathway.
Modulation of adenylyl cyclase is not considered a primary function of the muscarinic ACh receptors (mAChRs) compared with coupling to Gq and stimulation of PLC isoenzymes, even if there are some reports in the literature indicating that activation of mAChR can increase cAMP (33,38). Our observation that inhibition of adenylyl cyclase by dd-Ado and SQ 22536 had such a marked effect on mAChR-mediated modulation of insulin release indicates an unusual functional cooperation between mAChR and cAMP production in the -cell. The intricate details of the underlying signaling system remain to be established, but the present study clearly demonstrates that this signaling pathway is altered under pathological conditions, as illustrated here in the GK model of type 2 diabetes.
How increased cAMP generation participates in ACh-triggered insulin secretion in the GK islet is not well understood. A number of reports have stressed the importance of both PKA-dependent phosphorylation (26,28) and PKA-independent activation (39,40) in the normal -cell. Exocytosis has been reported to be activated directly by cAMP (41,42), and interactions of the cAMP-binding protein cAMP-GEFII (cAMP-regulated guanine nucleotide exchange factor II, also referred to as Epac2 [exchange protein activated by cAMP]), with sulfonylurea receptor-1 and Rim2 have been identified (43,44). Our observation that the two PKA inhibitors H89 and Rp-8-Br-cAMPS markedly blunted the ACh-induced insulin output by the GK islet, whereas the ACh-induced [Ca2+]i response was not significantly affected, clearly demonstrates that the distal cAMP/PKA-dependent pathway is involved downstream of the [Ca2+]i elevation. It is unknown so far whether the cAMP-dependent PKA-independent pathway may also participate to the ACh-induced insulin release in the GK -cells. It could play a role because the PKA inhibitors did not completely block the ACh-triggered insulin release (Fig. 5D).
Finally, our data have demonstrated that activation of the cAMP pathway by ACh reset the GK -cell secretory competence for glucose. In support of such a role for cAMP, forskolin, db-cAMP (dibutyryl cAMP), or glucagon-like peptide-1 have been found to improve in vitro insulin release in response to glucose by islets from the GK/Stockholm line (36) or the GK/Par line (45).
In summary, we have identified the pathway on which ACh acts in vitro to enhance insulin secretion and that restores the defective glucose responsiveness in the GK/Par -cells. At variance with its effect on the normal -cell, ACh acts mainly through activation of the cAMP/PKA signaling pathway to potently enhance Ca2+-stimulated insulin release in the GK/Par -cell. These findings suggest that cholinergic stimulation could represent one mechanism to tentatively compensate for the severely impaired response to glucose, as observed in vitro and also in vivo in the GK rat model of type 2 diabetes. It remains to be determined whether GK/Par rats secrete increased amounts of insulin on meal ingestion compared with their very low insulin secretion in vivo in response to a glucose load (46), and whether they have an intact vagal activation in response to feeding.
ACKNOWLEDGMENTS
This work was supported in part by grants from the Centre National de la Recherche Scientifique and the Swiss National Science Foundation. M.D. is a recipient of doctoral fellowships from the Association de Langue Franaise pour l’Etude du Diabeete et des Maladies Meetaboliques/Lilly and the Socieetee Franaise d’Endocrinologie/Novo Nordisk Foundation for Medical Research.
The authors thank Emmanuelle Westrelin for her skillful participation in the experiments.
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2 Department of Genetic Medicine and Development, University Medical Center, Geneva, Switzerland
ACh, acetylcholine; BIM, bisindolylmaleimide I; [Ca2+]i, intracellular free calcium concentration; dd-Ado, 2',5'-dideoxyadenosine; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide; InsP, inositol phosphate; IBMX, isobutyl methylxanthine; KRB, Krebs-Ringer buffer; mAChR, muscarinic ACh receptor; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PLC, phospholipase C; Rp-8-Br-cAMPS, 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer; SERCA, sarcoendoplasmic reticulum Ca2+-ATPase
ABSTRACT
Because acetylcholine (ACh) is a recognized potentiator of glucose-stimulated insulin release in the normal -cell, we have studied ACh’s effect on islets of the Goto-Kakizaki (GK) rat, a spontaneous model of type 2 diabetes. We first verified that ACh was able to restore the insulin secretory glucose competence of the GK -cell. Then, we demonstrated that in GK islets 1) ACh elicited a first-phase insulin release at low glucose, whereas it had no effect in Wistar; 2) total phospholipase C activity, ACh-induced inositol phosphate production, and intracellular free calcium concentration ([Ca2+]i) elevation were normal; 3) ACh triggered insulin release, even in the presence of thapsigargin, which induced a reduction of the ACh-induced [Ca2+]i response (suggesting that ACh produces amplification signals that augment the efficacy of elevated [Ca2+]i on GK exocytosis); 4) inhibition of protein kinase C did not affect [Ca2+]i nor the insulin release responses to ACh; and 5) inhibition of cAMP-dependent protein kinases (PKAs), adenylyl cyclases, or cAMP generation, while not affecting the [Ca2+]i response, significantly lowered the insulinotropic response to ACh (at low and high glucose). In conclusion, ACh acts mainly through activation of the cAMP/PKA pathway to potently enhance Ca2+-stimulated insulin release in the GK -cell and, in doing so, normalizes its defective glucose responsiveness.
Cholinergic muscarinic agonists, including the endogenous neurotransmitter acetylcholine (ACh) and the synthetic nonhydrolyzable analog carbachol, are known to enhance glucose-stimulated insulin secretion by the normal -cell (1). ACh is released by intrapancreatic vagal nerve endings during the preabsorptive and absorptive phases of feeding, and it acts mainly by activating phospholipase C (PLC)/protein kinase C (PKC) signaling pathways in the normal -cell (1). In insulin-resistant rodent models, such as C57BL/6J mice fed a high-fat diet (2) and preobese Lepob/Lepob mice (3), it is recognized that ACh induces exaggerated insulin release.
Whether a similarly altered cholinergic response is also found in type 2 diabetes is not so clear; it has been reported that chronic hyperglycemia increases the number of muscarinic binding sites in islets from streptozotocin-induced diabetic rats (4), whereas rat islets cultured at high glucose concentrations show a decrease in ACh-activated inositol phosphate (InsP) generation (5). However, we previously reported that in the basal state, the Goto-Kakizaki (GK) rat, a lean spontaneous model of type 2 diabetes, presented enhanced plasma insulin and islet blood flow that were totally abolished by vagotomy, whereas in nondiabetic control rats, vagotomy did not change these parameters (6), suggesting increased ACh activation of the GK islets. More recently, indications for enhanced insulin release in response to ACh (7) have been obtained in GK islets from the Stockholm GK colony, a subline whose islet phenotype is close to, but not identical to, that of our GK colony (Paris colony, GK/Par) (8).
These observations prompted us to explore interactions between ACh and glucose on insulin release and intracellular Ca2+ in freshly isolated GK/Par islets, as well as to examine the secondary messenger pathways involved.
RESEARCH DESIGN AND METHODS
All animal experimentation was conducted on fed age-matched male GK/Par rats and nondiabetic Wistar rats from our local colonies, in accordance with accepted standards of animal care as established by the French National Center for Scientific Research guidelines. The characteristics of the GK rats maintained in our colony at Paris University 7 have been described previously (9). Plasma glucose levels for Wistar and GK/Par rats at time of death were 6.6 ± 0.2 mmol/l (n = 30) and 9.6 ± 0.5 mmol/l (n = 30), respectively (P < 0.001).
Required chemicals and sources.
The 125I-labeled insulin was from DiaSorin. The 125I-labeled adenosine 3',5' cyclic-phosphoric acid 2'-O-succinyl-3, the myo-[2-3H]inositol and the radiolabeled InsP mixture [Ins(1,3,4)P3 + Ins(1,4,5)P3] were from Amersham. The Fura-2/AM was from Molecular Probes. Dowex AG1-X8 resin was supplied by Bio-Rad. BSA (fraction V) was from Roche Molecular Biochemicals. Purified rat insulin was supplied by Novo Research Institute (Copenhagen). 2',5'-dideoxyadenosine (dd-Ado) and bisindolylmaleimide I (BIM) were from Calbiochem. 8-bromoadenosine-3',5'-cyclic-monophosphorothioate, Rp-isomer (Rp-8-Br-cAMPS) was from Biolog. D-glucose, Tris, and the salts used to make the Hanks’ solution and Krebs-Ringer buffer (KRB) were from Merck. Other chemicals were from Sigma-Aldrich.
Islet isolation.
Rats (3eC4 months old) were killed by decapitation. For each experiment, three pancreata were digested with collagenase, and the islets were handpicked under a stereomicroscope.
Measurements of islet intracellular free calcium concentration and insulin release during perifusion procedure.
Freshly isolated islets were loaded 1 h with 5 e蘭ol/l Fura-2/AM at 37°C in KRB containing (in mmol/l) 115 NaCl, 5 KCl, 24 NaHCO3, 1 CaCl2, 1 MgCl2, and 5.5 glucose and 5 mg/ml BSA. After loading, eight islets at a time were allowed to attach on a polylysine-treated cover glass transferred to a perifusion chamber placed on the stage of an inverted fluorescent microscope (Nikon Diaphot, Champigny sur Marne, France). Cannulas feeding into the chamber were connected to a peristaltic pump and allowed a continuous superfusion of the islets at a flow rate of 1 ml/min with a 25 mmol/l HEPES-buffered medium maintained at 37°C containing (in mmol/l) 125 NaCl, 5.9 KCl, 1.28 CaCl2, 1.2 MgCl2, and 2.8 glucose and 1 mg/ml of BSA. Intracellular free Ca2+ concentration ([Ca2+]i) was determined as previously described (10). The perifusion fluid was collected from the chamber at 20-s intervals and stored at eC20°C for insulin radioimmunoassay.
Measurement of islet InsPs.
Batches of 150 freshly isolated islets were loaded with myo-[2-3H]inositol (16 e藽i) during 150 min at 37°C in 0.8 ml KRB-BSA containing 2.8 mmol/l glucose. After washing, prelabeled islets were incubated for 15 min at 37°C in 0.5 ml KRB-BSA containing 2.8 mmol/l glucose, 10 mmol/l LiCl (to inhibit phosphatase), and 1 mmol/l unlabeled myo-inositol, in the absence or presence of 1 mmol/l ACh. Free myo-inositol, glycerophosphoinositol, and InsP were eluted in a stepwise manner by anion-exchange chromatography, as previously described (11). The accumulation of radiolabeled InsP was taken as an index of islet cell phosphoinositide breakdown.
Measurements of islet cAMP content and insulin release during static incubation.
cAMP content was measured in islet pellets, and insulin release was determined in the incubation buffer from the same batches of islets, using radioimmunoassays as described previously (12). Briefly, after a 60-min preincubation period, groups of 20 freshly isolated islets were incubated for 20 min in 1 ml KRB-BSA medium containing 2.8 mmol/l glucose alone or supplemented with 100 e蘭ol/l isobutyl methylxanthine (IBMX), 100 e蘭ol/l dd-Ado, and 100 e蘭ol/l SQ 22536, as specified. In ACh experiments, all islet samples were incubated with the above-mentioned test substances 10 min before time 0 stimulation with 1 mmol/l ACh. Islets’ cAMP content and insulin release were determined after up to 10 min of incubation.
Islet DNA content.
In some experiments, at least two batches of 20 islets from control or GK rats were collected to determine DNA by fluorometric assay, as previously described (11).
Microarray analysis.
RNA was extracted from 4-month-old Wistar and GK islets using an RNeasy total RNA isolation kit (Qiagen). Then, 2 e蘥 of RNA was used to synthesize double-stranded cDNA using the Superscript Choice system (Invitrogen, Groningen, the Netherlands). In vitro transcription was carried out on 6 e蘬 cDNA using Bioarray High Yield RNA transcript labeling reagents (Enzo Diagnostics). Reactions yielded 50eC70 e蘥 biotin-labeled cRNA, which was purified on RNeasy affinity columns (Qiagen) and fragmented at 94°C for 35 min in fragmentation buffer, as previously described (13). Next, 15eC20 e蘥 fragmented cRNA was hybridized to Affymetrix RG-U34A oligonucleotide microarrays representative of 7,000 rat genes. Arrays were scanned, and the data obtained were analyzed using Genespring 6 (Silicon Genetics). The microarray experiments were performed using the Genomics Platform (NCCR Frontiers, Geneva).
Data presentation and statistical methods.
The kinetic variations of [Ca2+]i are presented as percentage changes in 340-to-380 fluorescence ratios. To quantify the [Ca2+]i responses, both integrated elevation above baseline (Ca, in arbitrary units) and percent elevation of [Ca2+]i above basal at peak value (PCa) were calculated. Integrated elevation above baseline value (Ins, in ng per eight islets) were used for insulin response quantitation. All data are presented as the means ± SE. Statistical analysis was performed using unpaired Student’s t test or ANOVA (Fisher test) as appropriate. Differences were considered significant at P < 0.05.
RESULTS
ACh restores blunted glucose-stimulated insulin secretion by GK pancreatic islets.
Whereas in Wistar islets stimulation with 16.7 mmol/l glucose resulted in a typical [Ca2+]i response (Fig. 1A), it produced a monophasic and significantly less pronounced (P < 0.001) change in [Ca2+]i in the GK islets (Ca7eC27min 24.3 ± 3.7 vs. 10.9 ± 1.1). ACh in the presence of 16.7 mmol/l glucose (Fig. 1B) similarly induced a biphasic [Ca2+]i pattern in both GK and Wistar islets. Nevertheless, although the initial peak was not significantly different, the following prolonged rise was significantly less pronounced (P < 0.05) in the GK islets (Ca7eC27min 19.8 ± 4.7 vs. 33.7 ± 3.0).
We found that 16.7 mmol/l glucose triggered biphasic insulin secretion (attenuated pattern attributable to averaging) in the Wistar islets (Fig. 1C), whereas only a small amount of insulin was released by the GK islets (Ins7eC27min 28.2 ± 5.1 vs. 13.5 ± 1.6 ng per eight islets, P < 0.001). ACh potentiation of 16.7 mmol/l glucoseeCinduced insulin secretion was observed in both Wistar and GK islets (Fig. 1D), and, remarkably, the total amount of insulin secreted during the 20-min period became no longer significantly different between GK and Wistar islets (Ins7eC27min 122 ± 14.8 vs. 107.3 ± 12.8 ng per eight islets), despite very different [Ca2+]i elevations.
ACh acts as a glucose-independent triggering signal in the GK islet.
In the presence of a nonstimulatory glucose concentration (2.8 mmol/l), ACh similarly increased [Ca2+]i in GK and Wistar islets (Ca7eC27min 8.4 ± 1.8 vs. 7.4 ± 1, and PCa 35.9 ± 6.6 vs. 27.1 ± 3.7%) (Fig. 2A). By contrast, this ACh-induced [Ca2+]i signal triggered a significant (P < 0.02) release of insulin only in GK islets (Ins7eC27min 4.9 ± 1.3 ng per eight islets) (Fig. 2B). Perifusion of the GK islets with a glucose-free medium did not prevent ACh from triggering insulin secretion (data not shown). Additionally, the efficacy of ACh in the GK islet cannot be ascribed to the high concentration used because we obtained the same secretory and [Ca2+]i responses with 50 e蘭ol/l carbachol (data not shown). Moreover, 20 e蘭ol/l atropine prevented these effects of ACh in the GK islets (data not shown), indicating that they were mediated by muscarinic receptors.
ACh-induced PLC activation and inositol triphosphate accumulation are not enhanced in GK islets.
In GK islets, the basal production of [3H]InsP was increased 1.6-fold (P < 0.05) compared with Wistar islets (Fig. 2C). In response to 1 mmol/l ACh, the levels of labeled InsP1, -2, and -3 and total InsP increased significantly above baseline in both GK and Wistar groups (Fig. 2C). The level of ACh-induced total InsP was increased 1.4-fold (P < 0.05) compared with Wistar islets (Fig. 2C). This effect was abolished by 20 e蘭ol/l atropine (data not shown). However, when expressed as a percentage of the paired basal value, ACh similarly stimulated [3H]InsP production in the GK and Wistar groups (517 and 581%, respectively). Additionally, total PLC activity, as measured in vitro in GK and Wistar islet homogenates by hydrolysis of exogenous [3H]phosphatidylinositol in the presence of 1 mmol/l Ca2+, was found to be similar in both groups (Fig. 2D).
ACh-induced insulin release by GK islets brings into operation a Ca2+ mobilization from intracellular Ca2+ stores and an increased efficacy of Ca2+ on exocytosis.
As shown in Fig. 2, there existed a tight temporal parallelism between the rise in [Ca2+]i and insulin release induced by ACh in the GK islets. Thapsigargin, an inhibitor of the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) pump, elicited a short-lived rise of [Ca2+]i in both Wistar and GK islets (Fig. 3A). [Ca2+]i response to ACh was significantly decrease in the GK and Wistar islets treated with thapsigargin (Ca22eC27min 0.6 ± 0.1 and 0.4 ± 0.1 vs. Ca7eC12min 3.4 ± 0.4 and 2.6 ± 0.4, respectively, in GK and Wistar islets with or without thapsigargin) (Fig. 3A), suggesting that the intracellular ACh-sensitive Ca2+ pool was largely emptied. Whereas in the Wistar islets addition of ACh in the presence of thapsigargin did not affect significantly insulin release (Fig. 3B), in the GK islets ACh still triggered a significant release of insulin (Ins22eC27min 4.0 ± 1.1 vs. Ins7eC12min 2.4 ± 0.5 ng per eight islets for GK islets with or without thapsigargin) (Fig. 3B).
The PKC pathway is not involved in the ACh-triggered insulin release by the GK islets.
The PKC pathway is considered to play a major role in the response of the normal -cell to ACh for sensitizing the secretory machinery to Ca2+ (1). The treatment of GK islets with 1 or 2.5 e蘭ol/l BIM, a PKC inhibitor (14), did not significantly change the ACh-activated [Ca2+]i response (PCa 37.1 ± 6 and 26.1 ± 4.8%, respectively) (Fig. 4AeCB) or the basal rate of insulin release (Fig. 4CeCD). The addition of ACh to 1 e蘭ol/l BIM produced an increased insulin release (Ins17eC27min 4.0 ± 1.1 ng per eight islets) similar to that obtained in the absence of BIM (Ins7eC17min 3.7 ± 0.2 ng per eight islets) (Fig. 4C). In the presence of 2.5 e蘭ol/l BIM, the secretory response to ACh became even more pronounced (Ins17eC27min 13.3 ± 1.7 ng per eight islets) (Fig. 4D).
ACh activates adenylyl cyclase, cAMP generation, and the cAMP-dependent protein kinase pathway in the GK islets.
Because the ACh-triggered insulin release by the GK islets was found independent of PKC, and because we have previously reported that some adenylyl cyclase (adenylyl cyclase-2 and -3) and the Gs protein Golf are overexpressed in GK/Par islets (15), we tested the hypothesis that stimulation of GK islets by ACh, at low glucose levels, has an effect on cAMP generation and the cAMP-dependent protein kinase (PKA) pathway.
First, we evaluated PKA inhibitors. Although both 250 e蘭ol/l Rp-8-Br-cAMPS (16) and 1 e蘭ol/l N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H-89) (17) decreased PCa induced by ACh in the GK islets, by 39% (P < 0.05) and 36%, respectively, they did not significantly affect the integrated Ca2+ values (Ca17eC27min 3.3 ± 0.4 and 4.9 ± 1 vs. Ca7eC17min 4.8 ± 0.7 for ACh-stimulated GK islets without inhibitors) (Fig. 5AeCB). However, these inhibitors significantly reduced ACh-activated insulin release (Ins17eC27min 0.3 ± 0.5 and 1.2 ± 0.6 ng per eight islets with Rp-8-Br-cAMPS and H-89, respectively) (Fig. 5CeCD), indicating that activation of the PKA signal transduction pathway is involved.
We then evaluated the possibility that cAMP is directly implicated. First, treatment of GK islets with dd-Ado, an adenylyl cyclase inhibitor (18), also decreased by 45% (P < 0.05) the PCa induced by ACh. It had no significant effect on Ca17eC27min (3.9 ± 0.2) (Fig. 7A), but it completely suppressed the ACh-activated insulin release at 2.8 mmol/l glucose (I17eC27min 0.2 ± 0.3 ng per eight islets) (Fig. 6B). Second, in Wistar islets, under static incubation at 2.8 mmol/l glucose, ACh in the presence of IBMX significantly decreased cAMP production by 35% (P < 0.05) below basal level, whereas it failed in the absence of IBMX (Fig. 7AeCB). Under these conditions, decreased cAMP generation was correlated with a significant 29% reduction in insulin release (P < 0.001) (Fig. 7D).
In GK islets, cAMP generation was quite different. In the basal condition (2.8 mmol/l glucose without IBMX), the GK islet cAMP level was significantly increased (by 67%, P < 0.001) compared with the Wistar level (Fig. 7A). In response to ACh, we were unable to detect any cAMP accumulation in the absence of IBMX. However, in the presence of IBMX, ACh increased cAMP generation by 44% (P < 0.001) (Fig. 7B), and this was associated with an increase in insulin release of 60% (P < 0.001), despite the low concentration of extracellular glucose (Fig. 7D). Remarkably, these effects of ACh on cAMP generation and insulin release were completely suppressed by the specific adenylyl cyclase inhibitors dd-Ado or SQ 22536 (Fig. 7D). We used 100 e蘭ol/l dd-Ado because we worked with freshly isolated islets, and we determined the effect of inhibition of adenylyl cyclase after acute pretreatment (10 min). Lower doses are usually chosen for chronic treatment of isolated cells (19). However, German et al. (20) have used 1 mmol/l dd-Ado with rat islet cells incubated for 48 h.
The restitution of the glucose responsiveness of GK islets by ACh is also cAMP mediated.
To study the potential cAMP dependence of the Ach potentiation of 16.7 mmol/l glucoseeCinduced insulin secretion by GK islets, we blocked adenylyl cyclase activity. dd-Ado decreased PCa by 37.4% but did not significantly affect the integrated [Ca2+]i value in response to 16.7 mmol/l glucose + ACh (Ca17eC37min 10.6 ± 1.4 vs. 13.0 ± 4.2 in GK islets with or without dd-Ado) (Fig. 8A). By contrast, it significantly (P < 0.001) lowered the early increase of insulin release as well as the late phase obtained in response to a stimulation by 16.7 mmol/l glucose + ACh (Ins17eC37min 13.1 ± 2.7 vs. 87.8 ± 6.2 ng per eight islets, respectively, with or without dd-Ado) (Fig. 8B). In addition, dd-Ado did not affect significantly the [Ca2+]i patterns and the insulin release obtained in response to 16.7 mmol/l glucose alone (data not shown). Therefore, blocking ACh-activated cAMP generation in the GK islets eliminated the ability of ACh to reactivate their glucose-induced insulin release.
DISCUSSION
In the first part of this study, we confirmed that poor glucose-induced insulin secretion by GK islets is correlated to dysfunctions in glucose-induced [Ca2+]i handling. Both of these defects have been identified in the GK/Par line (10) as well as the GK/Stockholm line (21), a GK line with no decreased -cell density and insulin content at variance with the GK/Par line phenotype (8).
Because their effects on insulin secretion are glucose dependent, cholinergic agonists might theoretically be helpful to improve insulin secretion in type 2 diabetes. For this reason we studied the effect of ACh on glucose-induced insulin secretion in freshly isolated GK/Par islets. We found that ACh was able to restore the insulin secretory glucose competence of the GK -cell to such a degree that the insulin release became indistinguishable from those of normal Wistar islets, despite different [Ca2+]i elevations. Our conclusion related to insulin output is consistent with the previous report in the GK/Stockholm line in that carbachol normalized the insulin secretion at high glucose levels (7). Furthermore, our group previously reported that ACh also normalized glucose-stimulated insulin secretion in n0-STZ (streptozotocin) rats (another recognized model of rat diabetes) (22), suggesting that restoration by ACh of glucose responsiveness in the diabetic -cell is not restricted to the GK model but is probably a more general mechanism.
Under a low glucose concentration or even in its absence, we find that ACh, through muscarinic receptor activation, stimulates insulin release by GK islets with a clear first phase of insulin secretion and a faint but sustained second phase. This result suggests that ACh triggers -cell granule mobilization in the GK islets from a reserved pool to a releasable pool, despite the low glucose concentration. This obviously contrasts with the lack of insulin response in the Wistar islets.
There are several mechanisms to account for the unusual triggering capacity of ACh in GK -cells. First, in the GK -cell, the early steps of ACh signaling may be increased; our study shows that this does not hold true because ACh exerts the same increasing effect on [Ca2+]i in Wistar and GK islets, mainly because of normal PLC-catalyzed inositol triphosphate production and normal intracellular ACh-sensitive Ca2+ pool mobilization. It is interesting to note that the normal amplitude of the ACh-dependent mobilization of intracellular Ca2+ stores was obtained in the GK islet despite a reduction of the expression of SERCA-3 (23), which we were able to confirm in the GK/Par line using an Affimetrix approach. We found that SERCA-3 mRNA (accession no. M30581 at) was decreased 1.71-fold (average of experiment 1: 1.80; and experiment 2: 1.61) compared with Wistar islets. In addition, inositol triphosphate receptor type 3 mRNA (accession no. L06096 g at) was increased 1.40-fold (average of experiment 1: 1.22; and experiment 2: 1.60) in GK islets. This is in line with the previous proposal that SERCA-3 is not involved in the replenishment of inositol triphosphateeCsensitive and eCinsensitive Ca2+ pools in the endoplasmic reticulum (24).
A second option is that ACh produces amplification signals that augment the efficacy of elevated [Ca2+]i on exocytosis by the GK -cell, whereas the normal Wistar -cell lacks these ACh-dependent signals. This is indeed supported by the observation that ACh triggered release of insulin by the GK islets equally well in the presence or absence of thapsigargin, despite very different [Ca2+]i elevations.
In the normal -cell, the most important ACh-dependent amplifying mechanism is considered to involve PKC (1). As shown in our experiments using the PKC inhibitor BIM, ACh-dependent PKC activation at low glucose, if any, is not involved in the ACh-triggered insulin release by the GK islets.
cAMP was demonstrated to induce insulin secretion independent of a glucose stimulus (25), and cAMP-dependent PKA activation was reported to activate exocytosis distal to [Ca2+]i changes in normal -cell (26eC29). Although ACh is not usually recognized as a stimulator of cAMP production at low glucose concentrations by the normal islet (30eC32), a recent report suggests that such is the case (33). Using Wistar islets incubated at low glucose concentrations, we were unable to confirm this observation. By contrast, under the same experimental conditions (low glucose, presence of IBMX), ACh triggered activation of adenylyl cyclase and accumulation of cAMP in the GK islets. In addition, at low glucose and in the absence of ACh, the basal cAMP accumulation was found to be significantly elevated in the GK islets. This is supported by our previous findings that some adenylyl cyclase (adenylyl cyclase-2 and -3) and the Gs protein Golf are overexpressed in GK/Par islets (15). Similar adenylyl cyclase (including the adenylyl cyclase-1 and -8 isoforms) and Gs overexpressions have been reported in the islets from the GK/Stockholm line (34,35). However, in the GK/Stockholm islets, no elevation of cAMP under basal conditions was detectable (7,36) and carbachol failed to increase cAMP formation (7).
Our results suggest that ACh-stimulated cAMP generation, rather than increased basal cAMP, is instrumental in the ACh-triggered insulin release at low glucose by GK/Par islets. First, GK -cell secretion became no longer reactive to ACh when adenylyl cyclases were acutely blocked by dd-Ado or SQ 22536. In the absence of ACh, GK -cell insulin secretion and the cAMP level decreased when adenylyl cyclase was blocked by the adenylyl cyclase blocker dd-Ado. Second, normal Wistar -cell insulin secretion still remained inactivated by ACh under conditions that artificially (with IBMX) raised the cAMP level to a value identical to that in GK islets. Strikingly, under these conditions ACh exerted in fact a significant inhibitory effect on cAMP generation and insulin release by the normal -cell. This last observation complements the previous results of Miguel et al. (37) obtained in the -cell line BRIN BD11. They showed a paradoxical inhibitory effect of ACh on glucagon-like peptide-1eCstimulated cAMP production achieved through a novel pertussis toxineCand pirenzepine-sensitive M1 muscarinic receptoreCactivated pathway.
Modulation of adenylyl cyclase is not considered a primary function of the muscarinic ACh receptors (mAChRs) compared with coupling to Gq and stimulation of PLC isoenzymes, even if there are some reports in the literature indicating that activation of mAChR can increase cAMP (33,38). Our observation that inhibition of adenylyl cyclase by dd-Ado and SQ 22536 had such a marked effect on mAChR-mediated modulation of insulin release indicates an unusual functional cooperation between mAChR and cAMP production in the -cell. The intricate details of the underlying signaling system remain to be established, but the present study clearly demonstrates that this signaling pathway is altered under pathological conditions, as illustrated here in the GK model of type 2 diabetes.
How increased cAMP generation participates in ACh-triggered insulin secretion in the GK islet is not well understood. A number of reports have stressed the importance of both PKA-dependent phosphorylation (26,28) and PKA-independent activation (39,40) in the normal -cell. Exocytosis has been reported to be activated directly by cAMP (41,42), and interactions of the cAMP-binding protein cAMP-GEFII (cAMP-regulated guanine nucleotide exchange factor II, also referred to as Epac2 [exchange protein activated by cAMP]), with sulfonylurea receptor-1 and Rim2 have been identified (43,44). Our observation that the two PKA inhibitors H89 and Rp-8-Br-cAMPS markedly blunted the ACh-induced insulin output by the GK islet, whereas the ACh-induced [Ca2+]i response was not significantly affected, clearly demonstrates that the distal cAMP/PKA-dependent pathway is involved downstream of the [Ca2+]i elevation. It is unknown so far whether the cAMP-dependent PKA-independent pathway may also participate to the ACh-induced insulin release in the GK -cells. It could play a role because the PKA inhibitors did not completely block the ACh-triggered insulin release (Fig. 5D).
Finally, our data have demonstrated that activation of the cAMP pathway by ACh reset the GK -cell secretory competence for glucose. In support of such a role for cAMP, forskolin, db-cAMP (dibutyryl cAMP), or glucagon-like peptide-1 have been found to improve in vitro insulin release in response to glucose by islets from the GK/Stockholm line (36) or the GK/Par line (45).
In summary, we have identified the pathway on which ACh acts in vitro to enhance insulin secretion and that restores the defective glucose responsiveness in the GK/Par -cells. At variance with its effect on the normal -cell, ACh acts mainly through activation of the cAMP/PKA signaling pathway to potently enhance Ca2+-stimulated insulin release in the GK/Par -cell. These findings suggest that cholinergic stimulation could represent one mechanism to tentatively compensate for the severely impaired response to glucose, as observed in vitro and also in vivo in the GK rat model of type 2 diabetes. It remains to be determined whether GK/Par rats secrete increased amounts of insulin on meal ingestion compared with their very low insulin secretion in vivo in response to a glucose load (46), and whether they have an intact vagal activation in response to feeding.
ACKNOWLEDGMENTS
This work was supported in part by grants from the Centre National de la Recherche Scientifique and the Swiss National Science Foundation. M.D. is a recipient of doctoral fellowships from the Association de Langue Franaise pour l’Etude du Diabeete et des Maladies Meetaboliques/Lilly and the Socieetee Franaise d’Endocrinologie/Novo Nordisk Foundation for Medical Research.
The authors thank Emmanuelle Westrelin for her skillful participation in the experiments.
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