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Am J Physiol Endocrinol Metab 286: E834-E843, 2004. First published January 21, 2004; doi:10.1152/ajpendo.00292.2003
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Restitution of defective glucose-stimulated insulin release of sulfonylurea type 1 receptor knockout mice by acetylcholine

Nicolai M. Doliba,1 Wei Qin,1 Marko Z. Vatamaniuk,1 Changhong Li,1 Dorothy Zelent,1 Habiba Najafi,1 Carol W. Buettger,1 Heather W. Collins,1 Richard D. Carr,2 Mark A. Magnuson,3 and Franz M. Matschinsky1

1The Diabetes Research Center and Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; 2Pharmacological Research 1, Novo Nordisk, Bagsvaerd, Denmark; and 3Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Submitted 26 June 2003 ; accepted in final form 15 January 2004


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Inhibition of ATP-sensitive K+ (KATP) channels by an increase in the ATP/ADP ratio and the resultant membrane depolarization are considered essential in the process leading to insulin release (IR) from pancreatic {beta}-cells stimulated by glucose. It is therefore surprising that mice lacking the sulfonylurea type 1 receptor (SUR1–/–) in {beta}-cells remain euglycemic even though the knockout is expected to cause hypoglycemia. To complicate matters, isolated islets of SUR1–/– mice secrete little insulin in response to high glucose, which extrapolates to hyperglycemia in the intact animal. It remains thus unexplained how euglycemia is maintained. In recognition of the essential role of neural and endocrine regulation of IR, we evaluated the effects of acetylcholine (ACh) and glucagon-like peptide-1 (GLP-1) on IR and free intracellular Ca2+ concentration ([Ca2+]i) of freshly isolated or cultured islets of SUR1–/– mice and B6D2F1 controls (SUR1+/+). IBMX, a phosphodiesterase inhibitor, was also used to explore cAMP-dependent signaling in IR. Most striking, and in contrast to controls, SUR1–/– islets are hypersensitive to ACh and IBMX, as demonstrated by a marked increase of IR even in the absence of glucose. The hypersensitivity to ACh was reproduced in control islets by depolarization with the SUR1 inhibitor glyburide. Pretreatment of perifused SUR1–/– islets with ACh or IBMX restored glucose stimulation of IR, an effect expectedly insensitive to diazoxide. The calcium channel blocker verapamil reduced but did not abolish ACh-stimulated IR, supporting a role for intracellular Ca2+ stores in stimulus-secretion coupling. The effect of ACh on IR was greatly potentiated by GLP-1 (10 nM). ACh caused a dose-dependent increase in [Ca2+]i at 0.1–1 µM or biphasic changes (an initial sharp increase in [Ca2+]i followed by a sustained phase of low [Ca2+]i) at 1–100 µM. The latter effects were observed in substrate-free medium or in the presence of 16.7 mM glucose. We conclude that SUR1 deletion depolarizes the {beta}-cells and markedly elevates basal [Ca2+]i. Elevated [Ca2+]i in turn sensitizes the {beta}-cells to the secretory effects of ACh and IBMX. Priming by the combination of high [Ca2+]i, ACh, and GLP-1 restores the defective glucose responsiveness, precluding the development of diabetes but not effectively enough to cause hyperinsulinemic hypoglycemia.

calcium

INHIBITION of ATP-sensitive K+ (KATP) channels by an increase in the ATP/ADP ratio and the resultant depolarization are considered essential for the physiological mechanisms that lead to insulin secretion from pancreatic {beta}-cells stimulated by high glucose (3, 8, 39). Functional KATP channels of the pancreatic {beta}-cell are a heterooctameric combination of K+ inward rectifiers (KIR6.2) and sulfonylurea receptors (SUR1) (1). These channels couple fuel metabolism to membrane electrical activity, which results in the [Ca2+]i accumulation necessary to promote exocytosis (1).

Mutations in human SUR1 or KIR6.2 cause a recessive form of persistent hyperinsulinemic hypoglycemia of infancy characterized by oversecretion of insulin despite severe hypoglycemia (10, 36). To understand the metabolic basis of this disorder and the role of KATP channels in glucose homeostasis, three mouse models were developed that involve the disruption of KATP channels: 1) expression of a dominant negative mutant KIR6.2 subunit that reduces or eliminates KATP channel activity (28); 2) KIR6.2 knockout (27); and 3) SUR1 (33, 35) knockout. It is noteworthy that transgenic mice with mutated KIR6.2G132S are only mildly hypoglycemic at birth but become hyperglycemic within 4 wk as a result of {beta}-cell destruction (28). The deletion of SUR1 (33, 35) or Kir 6.2 (27) from mouse pancreatic {beta}-cells has unexpectedly little or no effect on glucose homeostasis in contrast to the predicted hypoglycemic phenotype. SUR1 knockout mice exhibit normal insulin release in response to feeding but do not secrete insulin in response to parenteral glucose (35). Furthermore, {beta}-cells are unable to increase insulin release when stimulated with high glucose in a variety of extracorporeal test systems (33, 35). The lack of a glucose response should have caused hyperglycemia in the intact animal. The nature of the adaptations required to maintain euglycemia remains unexplained.

In normal animals, feeding elicits acetylcholine (ACh) release by intrapancreatic nerve endings (13) and glucagon-like peptide-1 (GLP-1) release from the intestine (16). On the basis of this basic physiology and in light of the ability of the islet to respond to feeding, we have evaluated the effect of ACh, GLP-1, and IBMX on insulin release and free intracellular Ca2+ of freshly isolated or cultured islets of SUR1–/– knockout mice and B6D2F1 controls (SUR1+/+). We found that pancreatic islets from SUR1 knockout mice are hypersensitive to ACh, as demonstrated by a marked increase of insulin release even in the absence of glucose. ACh also restitutes glucose-induced insulin secretion, and its effect is potentiated by GLP-1. The ability of SUR1 knockout mice to maintain normoglycemia may be explained by neuroendocrine modification of {beta}-cell function.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal. SUR1 knockout mice were generated as described (35). The SUR1–/– mice contained a mutation that was made in RW4 ES cells (129/SvJ origin). They had been backcrossed three times into C57Bl/6. The mice were maintained on a 12:12-h light-dark cycle and were fed a standard rodent chow diet. B6D2F1 mice (SUR+/+; Jackson Labs) were used as controls.

Islet isolation. Mouse islets were isolated using collagenase (EC 3.4.24.3 [EC] ; Serva, 17449) digestion in Hanks' buffer followed by separation of islets from exocrine tissue in a Ficoll (Sigma, F-9378) gradient. Isolated islets were used fresh or cultured for 4 days in RPMI 1640 medium (GIBCO BRL, Grand Island, NY) containing 10% fetal bovine serum, 10 ml/l penicillin-streptomycin-amphotericin B solution (GIBCO BRL), and 10 mM glucose (34).

Perifusion of islets and insulin release experiments. Freshly isolated islets were placed on a nylon filter in a plastic perifusion chamber (Millipore, Bedford, MA). The perifusion apparatus consisted of a computer-controlled fast-performance HPLC system (Waters 625 LC System) that allowed programmable rates of flow and glucose concentration in the perfusate, a water bath (37°C), and fraction collector (Waters Division of Millipore). The perifusate was a Krebs buffer (pH 7.4) containing 2.2 mM Ca2+ and 0.25% of bovine serum albumin equilibrated with 95% O2-5% CO2. In some studies, islets were preperifused with no substrate for 30 min followed by a glucose ramp with a slope of 0.8 mM glucose/min. The maximal islet response was tested at the end of experiment with 30 mM KCl after washout of glucose.

Glucokinase assay. The enzyme was measured using a spectrometric plate reader and was based on a coupled NAD+-dependent assay (38). Islets were homogenized in a buffer with 150 mM KCl, 2 mM dithiothreitol, and proteinase inhibitors and briefly spun at 1,000 g. With use of aliquots of the homogenate equivalent to ~20 islets per well, the Vmax, the glucose level at half-maximal activity of glucokinase, the ATP Km, and the Hill number indicating cooperativity with the substrate glucose were determined at 37°C with reaction progress curves extending to 90 min. Other hexokinases (primarily subtype I) were blocked by 40 µM 5-desoxy-5-fluoro-G-6-P.

Ca2+ measurement. Mouse islets, cultured for 4 days in 10 mM glucose, were loaded with fura-2 AM (Molecular Probes, Eugene, OR) during a 40-min pretreatment at 37°C in 2 ml of Krebs Ringer bicarbonate buffer (KRBB) supplemented with 1 mmol/l fura-2 AM. The loaded islets were transferred to the perifusion chamber and placed on the homeothermic platform of an inverted Zeiss microscope (12). Islets were perfused with KRBB at 37°C at a flow rate of 2 ml/min, while various treatments were applied to the islets. The microscope was used with a 40x oil immersion objective. The intracellular Ca2+ was determined by the ratio of the excitation of fura at 334 and 380 nm. Emission was measured at 520 nm by a Attofluor charge-coupled device camera and calibrated using the Attofluor Ratio Vision Software.

Insulin measurements. Insulin in the effluent was measured by radioimmunoassay with charcoal separation (17). Rat insulin from Linco Research served as standard, and Miles anti-insulin antibody from ICN was the primary antibody.

Statistical analysis. Data are presented as the means ± SE of 4–7 experiments. In appropriate cases, significant differences between groups were determined by one-way analysis of variance (ANOVA) with post hoc analysis using Dunnett's multiple-comparison test. Values of P <= 0.05 were accepted as significant.


   RESULTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Hypersensitivity of SUR1–/– islets to ACh. Figure 1A shows insulin secretion of control and SUR1–/– islets during stimulation with glucose. In these experiments, the glucose concentration was increased progressively from 0 to 30 mM by 0.8 mM increment/min. Insulin release was not affected by glucose in SUR1–/– islets, in contrast to a significant increase in hormone secretion in pancreatic islets from control mice. To test whether this effect was caused by a change in glucose metabolism, we measured glucokinase activity as one of the most critical parameters in freshly isolated and cultured islets from both sets of mice. We found glucokinase activity and kinetics of freshly isolated islets and of islets cultured for 4 days at 10 mM glucose to be normal (Table 1). The data suggest that, with the assumption of normal oxidation, the lack of glucose responsiveness is not caused by reduced glucose metabolism. We found that glucose responsiveness may be regained when islets are pretreated with ACh, GLP-1, or IBMX, as exemplified in the following experiments. A gradual increase in the concentration of ACh from 0 to 500 µM in the presence of 16.7 mM glucose and 10 µM of the cholinesterase inhibitor neostigmine caused a burst of insulin secretion in SUR1–/– islets (Fig. 1B). Removal of ACh from the perifusate decreased the hormone release. In the next sets of experiments, 100 µM or 0.3 µM ACh was added to control and SUR1–/– islets during perifusion with substrate-free media (Fig. 1, C and D). In contrast to controls, SUR1–/– islets responded to high and low concentrations of ACh by a significant increase in insulin release, even in the absence of glucose, indicating hypersensitivity of SUR1–/– islets to the cholinergic agonist. After 30 min of preperfusion with ACh, a glucose ramp (from 0 to 30 mM with an 0.8-mM increment/min) was applied to control and SUR1–/– islets. The graded rise of glucose in the perifusate led to graded increase in hormone secretion in SUR1–/– islets as well as in the control islets. However, the kinetics of the hormone release were different in the two groups of islets, an initial burst of insulin release in the controls as glucose rose from 0 to 10 mM contrasting with a gradual increase of insulin release in SUR1–/– islets as the glucose increased from 0 to 30 mM. High concentrations of ACh significantly lowered the threshold for glucose-stimulated insulin release in control islets, whereas at low concentrations of ACh, islets exhibited a normal threshold for insulin release at 5–6 mM glucose. Diazoxide, a KATP channel activator, reduced insulin release back to baseline in control islets in contrast to sustained hormone release in SUR1–/– islets. Detailed studies with 10 and 1 µM ACh demonstrated a clear dose dependency of action (data are not shown).



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  		Fig. 1. Effect of glucose, acetylcholine (ACh), and verapamil on insulin release in sulfonylurea type 1 receptor knockout (SUR1–/–) islets. A: insulin release of control and SUR1–/– islets during stimulation with glucose. The glucose concentration was increased progressively from 0 to 30 mM by an 0.8 mM increment/min ramp. Insulin release was not elicited by glucose (G) in SUR1–/– islets, in contrast to significant stimulation in controls. KRB, Krebs-Ringer bicarbonate buffer with no inhibitors/secretagogues. B: insulin release during an ACh ramp (0–500 µM). A gradual increase of the ACh concentration in the presence of 16.7 mM glucose and neostigmine, a cholinesterase inhibitor, led to a burst of insulin secretion in SUR1–/– islets. C and D: hypersensitivity of SUR1–/– islets to ACh. Concentrations of ACh were 100 µM in C and 0.3 µM in D. ACh significantly increased the insulin release in SUR1–/– islets in the absence of glucose, suggesting marked hypersensitivity to the transmitter. After 30 min of preperfusion with ACh, a glucose ramp (from 0 to 30 mM with an 0.8 mM increment/min rate) was applied to control and SUR1–/– islets. Increase in the glucose concentration in the perfusate led to an increase in hormone secretion in SUR1–/– islets as well as in controls. Diazoxide, an ATP-sensitive K+ (KATP) channel activator, immediately decreased insulin release to baseline in control but was unable to block hormone release in SUR1–/– islets. Each curve in A and B represents the mean ± SE of 5–7 perifusions. Data in C and D are means of 4–6 perfusions without SE.

 

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  		Table 1. Effect of the SUR1–/– condition on pancreatic islet glucokinase

 
Effect of verapamil on ACh-stimulated insulin release. To evaluate the role of extracellular Ca2+ in ACh-mediated insulin release, the Ca2+ channel blocker verapamil was added to perifusate containing 16.7 mM glucose. Fifteen minutes later the acetylcholine ramp was applied. Verapamil decreased but did not abolish ACh-stimulated insulin release (compare Fig. 1B and Fig. 2), indicating partial dependence on Ca2+ influx and a prominent role of intracellular Ca2+ in stimulus-secretion coupling.



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  		Fig. 2. Effect of verapamil on ACh-stimulated insulin release. Verapamil, a Ca2+ channel blocker, was added to perifusate containing 16.7 mM glucose 15 min before the ACh ramp was applied. Verapamil partially decreased but did not abolish ACh-stimulated insulin release. Results are means ± SE of 4 perifusions.

 
GLP-1 potentiates the ACh effect on insulin release. In view of the supposition that pancreatic islets in vivo could be simultaneously stimulated by glucose and neuroendocrine factors, we tested the effect of GLP-1 on insulin release in SUR1–/– (Fig. 3A) and control (Fig. 3B) islets. The effect of GLP-1 was significantly smaller in SUR1–/– islets compared with control. Most striking is the finding that, in all experiments, GLP-1 potentiated the effect of ACh on insulin release two- to threefold (Fig. 3A) to a level that is comparable to that of normal islets perfused with high glucose, GLP-1, and ACh.



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  		Fig. 3. Synergistic effect of glucagon-like peptide-1 (GLP-1) and ACh on insulin release in SUR1–/– (A) and control (B) islets. A: SUR1–/– islets were pretreated with 16.7 mM glucose. At 30 min, GLP-1 was added to one set of islets while the second set of islets continued to be perfused with 16.7 mM glucose alone. Ten minutes later, ACh was added to both perifusions. B: experiments with control islets were done in the same order as in A with GLP-1. Each curve is the mean ± SE of 6–7 perifusions.

 
Changes in intracellular Ca2+ concentration. To evaluate the role of Ca2+ in ACh-stimulated insulin release, we recorded the changes of cytosolic Ca2+ by use of fura-2 AM. Basal free cytosolic Ca2+ concentration ([Ca2+]i) was markedly higher in SUR1–/– islets than in controls (Fig. 4). ACh at 1 µM increased the [Ca2+]i in SUR1–/– islets even in substrate-free medium (Fig. 4), indicating that SUR1–/– islets are hypersensitive to the cholinergic transmitter. After ACh pretreatment, 16.7 mM glucose was added to control and SUR1–/– islets. In contrast to a burst increase of [Ca2+]i in control islets, a sharp dip of [Ca2+]i followed by a sustained phase of relatively low cytosolic [Ca2+]i was observed in SUR1–/– islets (Fig. 4A). Verapamil decreased [Ca2+]i and abolished oscillations in [Ca2+]i but did not completely prevent a subsequent ACh-induced increase in [Ca2+]i (Fig. 4B). ACh in the presence of 16.7 mM glucose caused a dose-dependent increase in [Ca2+]i (at 0.1–1 µM ACh; Fig. 5, A-C) or biphasic changes, an initial sharp increase in [Ca2+]i followed by a sustained phase of low [Ca2+]i, at 1–100 µM ACh (Fig. 5, D and F). High glucose transiently decreased [Ca2+]i in all cases in SUR1–/– islets, with a return of [Ca2+]i levels to slightly higher than baseline.



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  		Fig. 4. Changes in intracellular Ca2+ concentration ([Ca2+]i) in islets of SUR1–/– mice. Changes in [Ca2+]i were recorded using fura-2 acetoxymethylester (AM). A: thin line, control islets; bold line, SUR1–/– islets. In contrast to control islets, ACh significantly increased [Ca2+]i in SUR1–/– islets, even in the absence of glucose. B: experiments were done in the same order as in A except that verapamil was added 10 min before ACh. Typical traces are presented (n = 4).

 


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  		Fig. 5. Effect of different concentrations of ACh on intracellular Ca2+ in SUR1–/– islets. ACh concentrations: A, 0.1 µM; B, 0.3 µM; C, 0.8 µM; D, 1 µM; E, 10 µM; F, 100 µM. In the presence of 16.7 mM glucose, ACh caused a dose-dependent increase in intracellular Ca2+ (at 0.1–1 µM) or biphasic changes (a spike of Ca2+ followed by new decreased basal level of Ca2+) at 1–100 µM. Typical experiments are shown (n = 3–4).

 
Effect of IBMX on insulin release in SUR1–/– islets. It has been shown recently that carbamylcholine evokes a concentration-dependent increase in cAMP generation in normal rat {beta}-cells with a maximum increase >=4.5-fold above control (37). GLP-1 also increased cAMP level in pancreatic {beta}-cells (13). On the basis of these data, we hypothesized that an increase in cAMP might mimic the effect of ACh (or ACh plus GLP-1) on insulin release in SUR1–/– islets. Figure 6A shows that the phosphodiesterase inhibitor IBMX causes a dose-dependent increase in insulin release in the presence of 16.7 mM glucose in control and SUR1–/– islets, with a much higher increase seen in the control islets. In contrast to control islets, IBMX was able to increase insulin release in SUR1–/– islets in the absence of glucose (Fig. 6B). Increasing the glucose concentration from 0 to 30 mM in the presence of IBMX led to biphasic changes in insulin release in SUR1–/– islets. Initially, insulin secretion decreased, was then followed by a rapid increase at 5–6 mM glucose, and was maximal at ~10 mM glucose. Control islets exhibited a graded increase in insulin secretion after the glucose concentration reached the threshold of 5–6 mM. This glucose effect was insensitive to diazoxide in SUR1–/– islets, whereas control islets responded by a return of insulin release to baseline (Fig. 6B). The IBMX effect on insulin release in SUR1–/– islets was supported by Ca2+ data. IBMX caused higher frequency oscillations in [Ca2+]i and slightly increased basal levels in the absence of glucose in SUR1–/– islets, in contrast to no changes in control (Fig. 6C). Low glucose (2 mM) decreased both frequency and amplitude of oscillations, and high glucose (8 mM) transiently decreased [Ca2+]i even further in SUR1–/– islets, an effect followed by a sharp biphasic increase in [Ca2+]i (Fig. 6C).



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  		Fig. 6. Effect of IBMX on insulin release and intracellular Ca2+ in SUR1–/– islets. A: insulin release from control ({circ}) and SUR1–/– islets ({bullet}) during an IBMX ramp (0–1 mM). An IBMX ramp from 0 to 1 mM was applied at a 25 µM increment/min rate in the presence of 16.7 mM glucose. To increase clarity, SUR1–/– data are also shown in inset. B: IBMX restores glucose-stimulated insulin release. A glucose ramp from 0 to 30 mM was applied at an 0.8 mM increment/min rate in the presence of 100 µM IBMX. C: effect of IBMX on intracellular Ca2+ concentration in control (line 1) and SUR1–/– (line 2) islets. Each curve represents the mean ± SE of 3–5 perifusions. Ca2+ tracing is a typical example (n = 3).

 
Cell depolarization by glyburide increases sensitivity of islets to ACh. Because SUR1–/– islets are permanently depolarized, it is reasonable to speculate that this primary abnormality sensitized {beta}-cells to ACh. To test this hypothesis, an ACh ramp (from 0 to 100 µM) was applied to control islets, which were depolarized by the SUR1 inhibitor glyburide. Glyburide slightly increased insulin release, tripled cytosolic Ca2+ in glucose-free medium, and markedly sensitized islets to ACh (Fig. 7). The ACh response was biphasic: a sharp increase as ACh increased from 0 to 30 µM, followed by inhibition as the transmitter dose was raised further to 100 µM (Fig. 7A). {beta}-Cell depolarization by glyburide led to increased [Ca2+]i in glucose-free medium (Fig. 7B). Under these conditions, ACh induced a further increase in [Ca2+]i followed by a sustained phase of relatively low [Ca2+]i (Fig. 7B), supporting the hypothesis that cell depolarization may increase the sensitivity to ACh in normal islets.



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  		Fig. 7. Glyburide pretreatment sensitizes control islets to ACh. A: effect of glyburide and ACh on insulin release; B: changes in intracellular Ca2+ concentration. Each curve represents the mean ± SE of 4 perifusions. Ca2+ tracing is a typical example(n = 3).

 

   DISCUSSION
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results of the present study indicate that glucose is able to stimulate insulin release from pancreatic islets of SUR1 knockout mice provided that a combination of the following conditions is met (Fig. 8): an increased phosphorylation potential, which requires that glucokinase activity be normal (pathway 1); elevated basal cytosolic Ca2+ (pathways 2 and 4); activation of protein kinase C (pathway 3); elevated cAMP levels and activation of the cAMP-guanidine nucleotide exchange factor II (GEFII) pathway (pathway 5); and activation of protein kinase A (pathway 6). We discuss these signaling pathways in the following paragraphs on the basis of present results and data in the literature.



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  		Fig. 8. Signaling in SUR1–/– pancreatic {beta}-cells. Six distinct signaling pathways in pancreatic {beta}-cells are depicted (arrows 1–6), with bold letters a-g identifying the initial step in each pathway. GK, glucokinase (a); the KATP channel complex, consisting of KATP channels and SUR1 subunits (b); VDCC, voltage-dependent Ca2+ or L channels (c); ACh and muscarinic receptor type 3 (M3) (d); SOCC, store-operated calcium channels (e); GLP-1 and receptor (f); IBMX, a phosphodiesterase inhibitor (g). AC, adenylate cyclase; DAG, diacylglyceride; IP3, inositol triphosphate; GEFII, cAMP-guanidine nucleotide exchange factor II (or Epac2); {Delta}{psi}, cell membrane potential.

 
SUR1 deletion depolarizes the {beta}-cells because functional KATP channels are absent. Voltage-dependent Ca2+ channels are opened, and basal intracellular Ca2+ is markedly elevated. We show here that basal intracellular Ca2+ concentrations are significantly higher in SUR1–/– islets compared with controls, in agreement with previous studies (35). Yet these islets do not secrete insulin under these conditions. Eliasson et al. (11) conclude that the lack of the secretory responses in SUR1–/– {beta}-cells is not attributable to decreased insulin content, which averaged 280 ± 19 ng insulin/islet and 253 ± 10 ng/islet in wild-type and SUR1–/– mice, respectively (11). However, Shiota et al. (35) and Nakazaki et al. (30) reported reduction in actual insulin content in the SUR1–/– pancreas by ~60 and 43–55%, respectively, compared with the wild-type pancreas. The difference in results is not explained.

Depolarization elevates Ca2+ and may in turn sensitize the {beta}-cells to the action of ACh. It was indeed observed that SUR1 knockout islets are hypersensitive to the cholinergic agonist and that ACh stimulates insulin release even in the absence of glucose. This is in contrast with normal islets: here ACh does not augment insulin secretion, nor does it change intracellular Ca2+ concentrations in the absence of glucose (26). These data complement the results of Shiota et al. (35) obtained with perfused pancreata by use of carbachol and of Nakazaki et al. (30) with perifused islets by use of the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) or carbamylcholine. In both of these studies, cholinergic agonists or TPA stimulated insulin release at low (2.8–3 mM) glucose in wild-type and in SUR1–/– islets, with greater amplitude in SUR1–/– islets. The authors concluded that SUR1–/– mice remain responsive to cholinergic agonists; however, they did not stress the fact that the mice are hypersensitive to ACh.

The hypersensitivity to ACh can be reproduced in control islets by treatment with the SUR1 inhibitor glyburide as one might predict. These new observations expand and clarify previous studies with ACh in the SUR1 knockout mouse (30, 35).

Pretreatment with ACh leads to restitution of glucose-induced insulin secretion in perifused islets of SUR1 knockout mice. However, the insulin secretion dynamics are different from those of normal islets, indicating that the network of signaling pathways has been altered. Normal islets exhibit a threshold for insulin release at 5–6 mM glucose (both in the absence and the presence of 0.3 µM ACh); pretreated SUR1–/– islets have, however, a much lower glucose threshold and begin to respond to glucose at 1 mM or even less. Furthermore, insulin release in SUR1–/– islets does not rise as rapidly as in controls during the glucose ramp and is not sensitive to diazoxide, consistent with a situation in which the KATP channel has been eliminated.

Figure 8 (pathway 3) presents the mechanisms by which ACh may affect {beta}-cells. When ACh binds to M3 receptors, it activates several transduction pathways; one is phospholipase C, which generates inositol 1,4,5-trisphosphate and diacylglycerol, a potent protein kinase C activator (13, 37). ACh could also depolarize the plasma membrane of {beta}-cells by a Na+- or nonspecific cation-dependent mechanism, and possibly by a mechanism involving store-operated channels activated by intracellular Ca2+ pool emptying. This additional depolarization may increase the probability of the open state of voltage-dependent Ca2+ channels in the plasma membrane already depolarized by the SUR1 knockout. Verapamil, a calcium channel blocker, reduces ACh-stimulated insulin release in SUR1 knockout mice, suggesting partial dependence on Ca2+ influx in a situation in which a prominent role of intracellular Ca2+ stores in stimulus-secretion coupling has been clearly demonstrated.

GLP-1 alone has a small effect, but it significantly potentiates ACh-stimulated insulin release in the presence of high glucose (Fig. 3). Previously published data (30, 35) showed that GLP-1 stimulates insulin secretion from wild-type but not from SUR1–/– islets. The apparent lack of response of SUR1–/– islets to GLP-1 was not due to altered coupling of GLP-1 receptors with adenylyl cyclase, because GLP-1 increased the islet cAMP content in wild-type and SUR1–/– islets to a comparable degree (30). In addition, the impaired response to GLP-1 did not appear to result from a defect in exocytosis or altered protein kinase A (30). It was suggested that a defect might exist in the PKA-independent potentiation of insulin release by cAMP (30). The phosphodiesterase inhibitor IBMX and activator of adenylyl cyclase forskolin were also employed to manipulate the cAMP content in islets (30). It was found that the secretory response of SUR1–/– islets was impaired compared with wild-type islets, whereas the intracellular cAMP content was elevated similarly in both wild-type and SUR1–/– islets treated with forskolin and IBMX, alone or in combination. On the basis of these data, it was speculated that a reduced islet response to GLP-1 and impaired PKA-independent potentiation of insulin release by cAMP may prevent the hypoglycemia expected to occur in these knockout animals. However, Eliasson et al. (11) showed recently that GLP-1 and forskolin potentiate glucose-induced secretion from SUR1–/– islets 2.2- to 2.8-fold, which is only slightly less than the amount seen in normal animals. These data contrast with in vivo experiments on the same SUR1–/– mouse strain that suggest the complete loss of GLP-stimulated secretion (35). The authors (11) suggest that the PKA-independent action of cAMP on exocytosis plays an important role in incretin-stimulated insulin secretion in vivo.

Our data show that isolated SUR1–/– islets respond to IBMX by increasing insulin release in a dose-dependent manner but at a lower magnitude than normal islets. The threshold for IBMX-stimulated insulin release is 150 µM. However, the more striking finding is that SUR1–/– islets respond to IBMX in the absence of glucose, an effect not seen in normal islets. Pretreatment with IBMX restored glucose-stimulated insulin release in SUR1–/– islets with a threshold of ~5–6 mM. It is remarkable that low glucose decreased insulin release in the presence of IBMX. This effect, also seen in the Ca2+ fluorescence tracings, could have several explanations, which were not further explored. Nakazaki et al. (30) showed that forskolin, added together with a high concentration of IBMX, stimulates insulin release in SUR1–/– islets even at low (2.8 mM) glucose. However, IBMX (500 µM) or forskolin alone did not stimulate insulin release in SUR1–/– islets at high (16.7 mM) glucose. These data are different from ours, which show that a lower concentration of IBMX and high glucose had significant effects on insulin release in SUR1–/– islets. In support of our data, Eliasson et al. (11) reported recently that islets from SUR1–/– mice responded well to forskolin and GLP-1, although the magnitude of the responses was only 50% of that seen in wild-type islets.

The synergy of GLP-1 and ACh in insulin secretion may be due to simultaneous activation of PKA by GLP-1 (16) and of PKC by ACh (37) (Fig. 8, pathways 3 and 6). It has been shown before that glucose augments insulin release markedly, even in the absence of extracellular Ca2+, when PKA and PKC are activated simultaneously (23). Komatsu et al. (24) demonstrated that the combination of the pituitary adenylate cyclase activation peptide (PACAP), carbachol, and glucose stimulated insulin release in the absence of the elevation of [Ca2+]i. It should be remembered in this context that ACh sensitizes the secretory machinery to Ca2+ (14, 25).

cAMP promotes exocytosis in the pancreatic {beta}-cells by a PKA-independent mechanism (Fig. 8, pathway 5) as well as by a PKA-dependent mechanism (pathway 6) (21, 32). Kawasaki et al. (22) reported that the cAMP-binding protein cAMP-GEFII, also referred to as Epac2 (6, 9), is a direct target of cAMP, thus regulating exocytosis, and that cAMP-GEFII, interacting with Rim2 (a target of the small G protein Rab3), mediates cAMP-dependent, PKA-independent exocytosis in a reconstituted system (31). It seems that cAMP-GEFII mediates cAMP-dependent PKA-independent mobilization of Ca2+ from the endoplasmic reticulum Ca2+ stores through ryanodine receptor-regulated Ca2+ channels (19). Eliasson et al. (11) reported recently that {beta}-cells of islets isolated from SUR1–/– mice lacked the PKA-independent component of exocytosis, whereas both cAMP-GEFII and Rim2 are transcribed in the SUR1–/– {beta}-cells. This was not attributable to a reduced capacity of GLP-1 to elevate intracellular cAMP but was associated instead with the inability of cAMP to stimulate influx of Cl– into the granules, a step important for granule priming.

ACh has little or no effect on [Ca2+]i of normal {beta}-cells at nonstimulatory glucose concentration (~3 mM), but it causes a sustained [Ca2+]i rise in the presence of high glucose (14, 41, 42). This sustained response requires the presence of extracellular Ca2+ and the possibility of Ca2+ entering {beta}-cells through voltage-operated Ca2+ channels. In contrast, SUR1–/– islets respond to ACh by significantly increasing [Ca2+]i, even in the absence of glucose. This effect of ACh may be facilitated by d-myo-inositol 1,4,5-trisphosphate (IP3)-dependent release of Ca2+ from the endoplasmic reticulum induced by elevated Ca2+ levels in SUR1–/– islets. It has been shown that even a relatively small increase in the cytosolic calcium concentration has a faciliatory effect on IP3-dependent calcium release from endoplasmic reticulum (5, 15, 20).

High glucose lowered [Ca2+]i in ACh-pretreated SUR1–/– islets. Others (14) have also found that high concentrations of ACh lower [Ca2+]i in {beta}-cells. This effect was clearly demonstrated in islets depolarized with high K+ (14). 45Ca2+ efflux measurements indicate that acceleration of Ca2+ efflux contributes to this effect. This acceleration may be ascribed to PKC stimulation, because phorbol esters also promote Ca2+ efflux (37–39) by activating the plasma membrane Ca2+-ATPase (40) or the Na+/Ca2+ exchanger (41).

We show here that responses to ACh in isolated SUR1–/– islets are different at low and high concentration of the agonist. At low concentration (0.1–1 µM), ACh increased [Ca2+]i in SUR1–/– islets probably due to PLC-catalyzed production of IP3 and stimulation of specific IP3 receptors of intracellular Ca2+ stores (see pathway 3 in Fig. 8). It is noteworthy that at concentrations higher than 1 µM, ACh did actually lower [Ca2+]i during the sustained second phase and that this effect was associated with stimulation of insulin release. Such an effect of ACh was observed in control islets by others (13) and is related to an increased efficacy of Ca2+ on exocytosis due to simultaneous activation of pathways 3 and 6, as presented in Fig. 8.

The present investigation underscores some of the shortcomings of widely held concepts and terminologies regarding stimulus-secretion coupling in pancreatic {beta}-cells. The terms "KATP channel-dependent" and "KATP channel-independent" pathways are used to characterize distinct dual glucose actions leading to insulin release. The first action depolarizes the cell, which results in the activation of the voltage-dependent Ca2+ channels, elevated Ca2+, and triggering of insulin release. The second augments the efficacy of the first by metabolic coupling factors of still unspecified chemical nature and mode of action. The intensive studies with the SUR1 knockout mouse (11, 30, 35), including the present results, demonstrate that this conceptual framework has limited explanatory power and needs modification.

The KATP channel-dependent or triggering pathway is constitutively activated by the SUR1 knockout, which is equivalent to channel inhibition and membrane depolarization and is manifested by high Ca2+. The KATP channel-independent or augmentation pathway is operative as indicated by normal glucokinase levels, suggesting normal glucose metabolism and ATP production. Glucose is, however, ineffective in SUR1 knockout islets, even though the critical conditions of high Ca2+ and normal sensing by glucokinase are both met.

We speculate that signaling pathways 3, 5, and 6 (see Fig. 8), which are normally activated by high glucose via Ca2+, are not operative in SUR1 knockout islets, possibly due to persistently high Ca2+, but that ACh and/or GLP-1 is capable of bypassing such a block and of restituting these essential requirements for glucose-stimulated insulin release. Much work is required to test this hypothesis.

It seems then reasonable to conclude that neural and endocrine regulation, in particular by ACh and GLP-1, could determine insulin secretion in mice lacking SUR1. Priming by the combination of high Ca2+, ACh, and GLP-1 restores a defective glucose responsiveness. The restitution of glucose-stimulated insulin release precludes the development of diabetes, but it is probable that it is not effective enough to cause the hyperinsulinemic hypoglycemia that was expected to develop as a result of the SUR1 knockout.

The present observations and considerations have practical implications for the widely used treatment of type 2 diabetes with SUR1 inhibitors. As shown here (i.e., Fig. 7 and by inference Figs. 3 and 6), SUR1 inhibitors sensitize the {beta}-cells to incretins and ACh, which play an important role in maintaining glucose homeostasis. This sensitizing effect could be very pronounced, as demonstrated here, and could contribute in a critical manner to the efficacy and long-term benefit of diabetes therapy with SUR1 inhibitors. Such aspects of SUR1 inhibitor pharmacology deserve increased attention.


   GRANTS
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 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-22122 (to F. M. Matschinsky), a grant from the American Diabetes Association (to N. M. Doliba), by the NIDDK Penn Diabetes and Endocrinology Research Center Grant (DK-19525), and a grant in aid from Novo Nordisk.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. Doliba, Univ. of Pennsylvania, Biochemistry/Biophysics, 501 Stemmler Hall, 36th & Hamilton Walk, Philadelphia, PA 19104-6015 (E-mail:nicolai{at}mail.med.upenn.edu).

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.


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 REFERENCES
 

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