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Diabetes 51:S443-S447, 2002
© 2002 by the American Diabetes Association, Inc.

Section 5: Beta-Cell Stimulus-Secretion Coupling: Hormonal and Pharmacological Modulators

Glucagon-Like Peptide-1 Receptor Activation Antagonizes Voltage-Dependent Repolarizing K+ Currents in ß-Cells

A Possible Glucose-Dependent Insulinotropic Mechanism

Patrick E. MacDonald1, Anne Marie F. Salapatek1, and Michael B. Wheeler1,2

1 Department of Physiology, University of Toronto, Toronto, Ontario, Canada
2 Department of Medicine, University of Toronto, Toronto, Ontario, Canada


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glucagon-like peptide-1 (GLP-1) acts through its G-protein-coupled receptor to enhance glucose-stimulated insulin secretion from pancreatic ß-cells. This is believed to result from modulation of at least two ion channels: ATP-sensitive K+ (KATP) channels and voltage-dependent Ca2+ channels. Here, we report that GLP-1 receptor signaling also regulates the activity of ß-cell voltage-dependent K+ (KV) channels, themselves potent glucose-dependent regulators of insulin secretion. GLP-1 receptor activation with exendin 4 (10-8 mol/l) in rat ß-cells antagonized KV currents by 43.3 ± 6.3%, whereas the GLP-1 receptor antagonist exendin 9-39 had no effect. The effect of GLP-1 receptor activation on KV currents could be replicated (current reduction of 55.7 ± 6.0%) by G-protein activation with GMP-PNP (10 nmol/l). The cAMP pathway antagonist Rp-cAMPS (100 µmol/l) prevented current inhibition by exendin 4, implicating cAMP signaling in GLP-1 receptor modulation of ß-cell KV currents. Finally, exendin 4 (10-8 mol/l) increased the amplitude (130 ± 5.7%) and duration (285 ± 15.9%) of the ß-cell depolarization response to current injection, independent of any effect on KATP or Ca2+ channels. The present results demonstrate that GLP-1 receptor signaling can antagonize ß-cell repolarization by reducing voltage-dependent K+ currents, an effect likely to contribute to GLP-1’s glucose-dependent insulinotropic effect.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The glucoincretin hormone glucagon-like peptide-1 (GLP-1) and its analogs have been widely studied because of their glucose-dependent insulinotropic effect (1,2) and because of a recently reported ability to promote ß-cell mass expansion (3–5). Currently, GLP-1 is thought to potentiate glucose-stimulated insulin secretion (GSIS) through several mechanisms, including antagonism of ATP-sensitive K+ (KATP) channels, potentiation of voltage-dependent Ca2+ channels (VDCCs), release of intracellular Ca2+ stores, and activation of nonspecific cation channels (NSCCs), in addition to direct effects on exocytosis (1). The most well-characterized signal transduction pathway of GLP-1 involves G-protein-mediated elevations of cAMP and activation of protein kinase A (1), although other potential signaling mechanisms have been identified, including the phospholipase C (6), mitogen-activated protein kinase (7), and cAMP-regulated guanine nucleotide exchange factor II (8,9) pathways.

Recently, we have shown that dominant-negative antagonism of the voltage-dependent K+ (KV) channels expressed in ß-cells enhances GSIS (10). Briefly, KV channels, notably Kv2.1, are thought to open in response to membrane depolarization caused by glucose-induced KATP channel closure (11). The resulting outward K+ current would effectively repolarize the ß-cell, closing VDCCs and limiting Ca2+ influx and insulin secretion. Because we have shown that KV channels are important regulators of insulin secretion, we postulate that physiological secretagogues, such as GLP-1, may enhance ß-cell excitability in part through antagonism of repolarizing K+ currents.

We now show that GLP-1 receptor activation antagonizes voltage-dependent outward K+ currents in rat ß-cells. Intracellular signaling through cAMP is required for this effect. Additionally, GLP-1 receptor activation enhanced the amplitude and duration of the depolarization response to current injection independent of any effect on KATP or Ca2+ channels. The present data indicate that the insulinotropic effect of GLP-1 may in part be mediated by an inhibition of ß-cell KV channels dependent on cAMP signaling, leading to enhanced action potential amplitude and duration.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Western blotting.
A volume of 50 µg of protein from each sample was loaded and separated on a 10% polyacrylamide gel. Protein was transferred to a PVDF-Plus membrane (Fisher Scientific, Nepean, Ontario, Canada) and incubated with primary antibody (Kv1.4, -1.6, -2.1, and -4.2; Alomone Labs, Jerusalem, Israel) or antibody-antigen solutions (diluted as per suppliers’ instructions) for 1.5 h, followed by a subsequent incubation with secondary antibodies (donkey anti-rabbit, 1:7,500; Amersham Pharmacia Biotech, Baie d’Urfe, Quebec, Canada) for 1 h at room temperature. Visualization was by chemiluminescence (ECL; Amersham Pharmacia Biotech) and exposure to Kodak film (Eastman Kodak, Rochester, NY) for 5 s to 10 min.

Islet isolation and cell culture.
Islets of Langerhans were isolated from male Wistar rats (250–300 g) by collagenase digestion as described previously (10). After isolation, islets were dispersed to single cells by treatment with 0.015% trypsin (Invitrogen Canada, Burlington, Ontario, Canada) in Ca2+- and Mg2+-free PBS at 37°C and 5% CO2 for 10 min. Islet cells were plated on glass coverslips in 35-mm dishes in RMPI media supplemented with 2.5 mmol/l glucose, 0.25% HEPES, 7.5% fetal bovine serum, 100 units/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate (penicillin-streptomycin from Invitrogen Canada) and cultured for 1–3 days before electrophysiological recordings.

Electrophysiological studies.
Cells were patch-clamped in the whole-cell configuration using an EPC-9 amplifier and Pulse software from HEKA Electronik (Lambrecht, Germany). Patch pipettes were prepared from 1.5-mm thin-walled borosilicate glass tubes using a two-stage Narishige (Tokyo) micropipette puller, and the pipettes had typical resistances of 3–6 megaohm (M{Omega}) when fire-polished and filled with intracellular solution. Intracellular solutions contained (in mmol/l): 140 KCl, 1 MgCl2 · 6H20, 1 EGTA, 10 HEPES, and 5 MgATP (pH 7.25) with KOH. Extracellular solutions contained (in mmol/l): 140 NaCl, 2 or 0 CaCl2 (current-clamp [C-clamp] recordings), 4 KCl, 1 MgCl2 · 6H2O, and 10 HEPES (pH 7.3) with NaOH. Outward currents were elicited with a series of 500-ms depolarizing pulses in 20-mV increments, to +70 mV from a holding potential of -70 mV. Sustained outward current was taken as the mean current during the final 25 ms of the depolarization. Membrane potential measurements were performed in the C-clamp mode of the EPC-9 amplifier/Pulse software. Cells were transiently depolarized with a current injection of 100 pA for 5 ms. All electrophysiological recordings were performed at 32–35°C and normalized to cell capacitance. Results were confirmed in rat ß-cells identified by their electrical responsiveness to 11.1 mmol/l glucose in the amphotericin B (100 µg/ml) perforated-patch configuration. Pharmacological compounds were applied via addition to either the intracellular solution or the bath solution by perfusion for at least 10 min before final current recording. Exendin 4 and exendin 9-39 were from Bachem (Torrance, CA). Guanosine 5'-[ß,{gamma}-imido]triphosphate (GMP-PNP) and the Rp-diastereomer of adenosine-3',5'-cyclic monophosphothioate (Rp-cAMPS) were from Sigma-Aldrich Canada (Oakville, Ontario, Canada). Sustained outward currents elicited with the maximal depolarization (to +70 mV) were compared using the Student’s unpaired t test (P < 0.05 considered significant). Time courses of current amplitudes were analyzed by one-way ANOVA and Dunnett’s posttest. Repolarization waveforms were fitted to an exponential function to give a single time constant using Pulsefit software (HEKA Electronik).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
KV channel expression in rat islets.
We have previously demonstrated protein expression of Kv1.4, -1.6, and -2.1 in insulin-secreting cells (10). Here, we confirm expression of these channels in rat islet protein lysates (50 µg) by Western blotting and further demonstrate expression of Kv4.2 (Fig. 1A). Rat brain protein lysates (50 µg) were used as a positive control, and specificity of the bands was demonstrated by competition with control antigen (not shown). Specific bands were not observed in rat islet lysates probed for Kv1.1, -1.3, -3.1, -3.3, and -3.4, whereas only a very weak band was observed for Kv1.2 (not shown). We have previously been unable to demonstrate mRNA expression of Kv1.5, -1.7, and -2.2 in purified rat islet total RNA by RT-PCR (10).



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  		FIG. 1. GLP-1 receptor activation antagonizes voltage-dependent outward K+ currents in ß-cells. A: Kv1.4, -1.6, -2.1, and -4.2 protein expression was detected in rat pancreatic islet (RI) and rat brain (RB) protein lysates by Western blot. Specificity of detected bands was determined by competition with control antigen (not shown). Rat ß-cells were voltage-clamped in the whole-cell configuration, and outward currents were elicited with a series of depolarizing pulses from a holding potential of -70 mV in 20-mV increments, to +70 mV. B: Representative traces are shown for control cells and those treated with exendin 4 (10-8 mol/l), exendin 9-39 (10-8 mol/l), and TEA (15 mmol/l). C: Current-voltage relationships are shown for control ({blacksquare}) cells and those treated with exendin 4 (10-8 mol/l; {circ}), exendin 9-39 (10-8 mol/l; {triangleup}), and TEA (15 mmol/l; •). **P < 0.01, ***P < 0.001 compared with control.

 
GLP-1 receptor activation antagonizes ß-cell voltage-dependent outward K+ currents, an effect dependent on cAMP signaling.
Voltage-dependent outward K+ currents were recorded from rat islet cells patch-clamped in the whole-cell configuration. Currents were similar to those described previously (10), but inactivated to a greater extent over 500 ms because of a higher bath temperature (33–35°C) than used previously (Figs. 1B and 2A). The majority of these outward K+ currents (82.3 ± 1.8%, n = 10, P < 0.001) were sensitive to the general KV and Ca2+-sensitive K+ (KCa) channel antagonist tetraethylammonium (TEA; 15 mmol/l) (Fig. 1). The GLP-1 receptor agonist exendin 4 (10-8 mol/l) decreased voltage-dependent outward K+ currents from rat islet cells by 43.3 ± 6.3% (n = 6, P < 0.01) without appreciably altering current kinetics (Fig. 1). Similarly, but not shown, rat GLP-1 (10-8 mol/l) also reduced the observed currents by 40.8 ± 4.1% (n = 9, P < 0.001). These results were not replicated by the GLP-1 receptor antagonist exendin 9-39 (10-8 mol/l), which had no significant effect on outward K+ currents (Fig. 1, n = 9).



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  		FIG. 2. The inhibitory effect of GLP-1 receptor activation is replicated by direct G-protein activation and is blocked by antagonism of cAMP signaling. Intracellular dialysis of the nonhydrolyzable GTP analog GMP-PNP (10 nmol/l) antagonized voltage-dependent outward K+ currents in ß-cells, similar to the effect of exendin 4. Representative traces are shown in A. B: Maximum outward K+ current is plotted against time after intracellular access. Perfusion of exendin 4 (10-8 mol/l; {circ}) or intracellular dialysis of GMP-PNP (10 nmol/l; •) resulted in a time-dependent decrease in maximum currents compared with control ({blacksquare}). C: Cells were pretreated with the cAMP pathway antagonist Rp-cAMPS (100 µmol/l). In the continued presence of 100 µmol/l Rp-cAMPS, exendin 4 (10-8 mol/l; {circ}) was no longer able to antagonize outward K+ currents compared with control ({blacksquare}). *P < 0.05, **P < 0.01 compared with time = 0.

 
The inhibitory effect of exendin 4 or GLP-1 could be replicated by inclusion in the intracellular pipette solution of the nonhydrolyzable GTP analog GMP-PNP (10 nmol/l), a G-protein activator (current reduction of 55.7 ± 6.0%; n = 8, P < 0.01) (Fig. 2A). The time course for current reduction by GMP-PNP (Fig. 2B) reflects the time necessary for intracellular dialysis of the compound and subsequent G-protein signaling. Also shown for comparison is the time course for current reduction by exendin 4 (10-8 mol/l) (Fig. 2B), which reflects the time necessary for exendin 4 to equilibrate in the bath solution by perfusion in addition to receptor activation and signaling. Antagonism of the cAMP signaling pathway by preincubation of cells with Rp-cAMPS (100 µmol/l, 30 min) and inclusion of Rp-cAMPS (100 µmol/l) in the bath solution blocked the inhibitory effect of exendin 4 (10-8 mol/l) on outward K+ currents (n = 5) (Fig. 2C). The above results indicate that G-protein signaling is sufficient to antagonize voltage-dependent outward K+ currents in rat ß-cells and that cAMP signaling is required for the effect of GLP-1 receptor activation.

GLP-1 receptor activation antagonizes ß-cell repolarization.
GLP-1 receptor activation has been implicated in the regulation of ß-cell membrane potential through its effects on KATP channels and VDCCs (1). We therefore wanted to determine whether regulation of voltage-dependent outward K+ currents by GLP-1 receptor activation affects membrane repolarization separately from the modulation of KATP or Ca2+ channels. To accomplish this, C-clamp experiments were performed in the presence of high intracellular ATP (5 mmol/l) to close the KATP channels and in the absence of extracellular Ca2+ to eliminate the Ca2+ currents. In the absence of current (I = 0), resting membrane potentials were -56.9 ± 3.3 mV (n = 11), reflecting the closure of KATP channels. Because ß-cell action potentials do not occur in the absence of Ca2+ (12,13), a transient depolarization was generated by a 5-ms current injection (100 pA). As expected under Ca2+-free conditions, transient depolarizations generated in this manner resulted only from current injection and not from activation of voltage-dependent channels, since no evoked action potentials could be elicited at intermediate levels of current injection (10–200 pA) or by a current injection ramp from 0 to 200 pA (not shown). Current injection (100 pA for 5 ms) depolarized cells to -0.5 ± 3.3 mV (n = 11), and repolarization occurred with a time constant of 4.6 ± 1.0 ms (n = 11) (Fig. 3). In the presence of the general KV channel antagonist TEA (15 mmol/l), which blocks rat ß-cell-repolarizing K+ currents by >80%, the level of depolarization increased slightly (7.9 ± 7.03 mV, n = 6, P = 0.12), and the repolarization time constant increased significantly (16.54 ± 3.21 ms, n = 6, P < 0.001) (Fig. 3). This effect could be completely washed out (not shown). Similarly, GLP-1 receptor activation with exendin 4 (10-8 mol/l) increased the level of depolarization (16.6 ± 7.8 mV, n = 6, P < 0.05) and prolonged the repolarization time constant significantly (13.1 ± 2.08 ms, n = 6, P < 0.001). Neither treatment with TEA nor treatment with exendin 4 significantly affected the resting membrane potential in the absence of current injection. These results suggest that GLP-1 receptor-mediated reductions in repolarizing K+ currents may lead to enhanced (i.e., larger and prolonged) action potentials.



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  		FIG. 3. Amplitude and duration of transient depolarization generated by current injection are increased in ß-cells by GLP-1 receptor activation. C-clamp experiments were performed in cells by whole-cell patch-clamp with high intracellular ATP (5 mmol/l) and no Ca2+ to eliminate the contribution of KATP and Ca2+ currents to membrane potential. Treatment of cells with the general KV channel antagonist TEA (15 mmol/l) or with exendin 4 (10-8 mol/l) increased the amplitude and repolarization time constant of transient depolarizations generated by injection of a 100-pA current for 5 ms. Representative waveforms are shown in A. Maximum depolarization (B) and repolarization time constants (C) are plotted for control ({blacksquare}) cells and those treated with TEA (15 mmol/l; ) and exendin 4 (10-8 mol/l; {square}). *P < 0.05, ***P < 0.001 compared with control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The ability of GLP-1 to directly enhance GSIS from pancreatic ß-cells has been attributed to GLP-1 receptor activation leading to enhanced depolarization and increases in the intracellular concentration of Ca2+ as well as direct effects on insulin exocytosis (1). The electrogenic effects of GLP-1 have in the past been attributed to its ability to inhibit KATP channels and to augment Ca2+ influx through VDCCs (1,14). In the present study, we provide two novel observations. First, GLP-1 receptor activation antagonizes rat ß-cell KV channels, an effect dependent on cAMP signaling. Second, antagonism of KV channels by GLP-1 leads to a slowing of membrane repolarization after a depolarizing stimulus independent of KATP channels or VDCCs, suggesting a contribution by KV channels to the electrogenic effects of GLP-1.

The GLP-1 receptor is expressed in the islets of several species, including rat, from which it was originally cloned (15). The current reduction observed in the present study is unlikely to be a nonspecific effect because both GLP-1 and the GLP-1 receptor agonist exendin 4 antagonized outward K + currents in ß-cells, but the truncated peptide (exendin 9-39) had no effect (Fig. 1). Furthermore, the ability of the G-protein activator GMP-PNP to replicate the effect of exendin 4 implicates G-proteins in the intracellular signal transduction machinery leading to the reduction in voltage-dependent K+ current.

Because the majority of reports demonstrate GLP-1 modulation of KATP channels or VDCCs to be cAMP dependent (1), we studied whether ß-cell voltage-dependent K+ current modulation by GLP-1 receptor activation was also dependent on cAMP. Indeed, antagonism of the cAMP signaling pathway with Rp-cAMPS was sufficient to abolish the effect of GLP-1 receptor activation on voltage-dependent outward K+ currents. Hormone-mediated reductions in voltage-dependent K+ currents have been previously demonstrated in arterial smooth muscle (16), human megakaryocytes (17), human acromegalic somatotropes (18), and rat sensory neurons (19). In two of these studies, cAMP signaling was implicated in the effect (16,19). One recent study has demonstrated that the KVLQT KV channel association with a neuronal A kinase anchoring protein is essential for cAMP modulation of channel current in a heterologous system (20). It remains to be determined whether the GLP-1 receptor or ß-cell KV channels interact directly (or are colocalized on lipid-rich membrane patches) with the signal transduction or excitatory machinery.

KV channels open in response to membrane depolarization (whether generated by current injection or glucose) and are believed to participate in repolarization of the ß-cell (11). Reduction in KV channel activity by GLP-1 may be expected to result in prolonged action potential duration and enhanced electrical excitability (21). Because alterations in KATP channel and VDCC activity are expected to contribute to the electrogenic effects of GLP-1, we sought to determine the contribution of GLP-1 receptor modulation of KV channels to membrane repolarization separate from the contribution of other channels. KATP channels were blocked by high intracellular ATP, whereas VDCCs and KCa channels were neutralized by the absence of extracellular Ca2+. Under these conditions, action potentials will not be elicited by small current injections to bring the cell to a "threshold" potential. Indeed, using a multiple current pulse or current ramp protocol, we were unable to elicit evoked action potentials in rat islet cells in the absence of Ca2+ (not shown). Therefore, the depolarization phase of the waveforms shown (Fig. 3) result solely from current injection and not from opening of VDCCs or voltage-dependent Na+ channels, whereas the extent and speed of repolarization is determined largely by the activation of voltage-dependent K+ currents. In the absence of GLP-1 receptor activation, the ß-cells repolarized quickly upon current injection (time constant = 4.5 ms) and an after-hyperpolarization was observed, reflecting a lag in the inactivation of KV channels. After GLP-1 receptor activation with exendin 4, resting membrane potential was unchanged, whereas upon current injection, the depolarization amplitude and repolarization time constant was increased and the after-hyperpolarization disappeared.

These results suggest that reduction of KV channel activity by GLP-1 receptor activation will enhance ß-cell action potentials. KV channels are strict depolarization-dependent (and therefore nutrient-dependent) regulators of secretion. These channels, which are closed under basal (i.e., low-glucose) conditions, open in response to nutrient-induced depolarization and contribute to ß-cell repolarization, limiting Ca2+ influx and insulin secretion. Therefore, the nutrient dependence of GLP-1 may in part be explained by its ability to antagonize KV channels. In addition to nonelectrogenic targets, the insulinotropic effect of GLP-1 may presently be attributed to a number of electrogenic effectors, such as KATP channels, VDCCs, NSCCs, and now KV channels. Important future experiments must distinguish the contribution of each of these targets to GLP-1-stimulated electrical activity and insulin secretion.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Xiaofang Ha for technical assistance. The present work was supported by a research grant (MOP-49521) to A.M.F.S and M.B.W. from the Canadian Institutes of Health Research (CIHR). P.E.M. was supported by a doctoral studentship from the CIHR. M.B.W. was supported by a CIHR Scientist Award.


   FOOTNOTES
 
Address correspondence and reprint requests to Dr. Michael B. Wheeler, University of Toronto, Department of Physiology, 1 Kings College Circle, Rm. 3352, Toronto ON, Canada, M5S 1A8. E-mail: michael.wheeler{at}utoronto.ca.

Received for publication 1 March 2002 and accepted in revised form 17 May 2002.

C-clamp, current-clamp; GLP-1, glucagon-like peptide-1; GMP-PNP, guanosine 5'-[ß,{gamma}-imido]triphosphate; GSIS, glucose-stimulated insulin secretion; KATP channel, ATP-sensitive K+ channel; KCa channel, Ca2+-sensitive K+ channel; KV channel, voltage-dependent K+ channel; NSCC, nonspecific cation channel; Rp-cAMPS, the Rp-diastereomer of adenosine-3',5'-cyclic monophosphothioate; TEA, tetraethylammonium; VDCC, voltage-dependent Ca2+ channel.

The symposium and the publication of this article have been made possible by an unrestricted educational grant from Servier, Paris.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Diabetes Diabetes Care Clinical Diabetes Diabetes Spectrum