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Endocrinology Vol. 147, No. 5 2155-2162
Copyright © 2006 by The Endocrine Society

Insulin Regulates Islet {alpha}-Cell Function by Reducing KATP Channel Sensitivity to Adenosine 5'-Triphosphate Inhibition

Yuk M. Leung, Ishtiaq Ahmed, Laura Sheu, Xiaodong Gao, Manami Hara, Robert G. Tsushima, Nicholas E. Diamant and Herbert Y. Gaisano

Departments of Medicine and Physiology (Y.M.L., I.A., L.S., X.G., R.G.T., N.E.D., H.Y.G.), University of Toronto, Toronto, Canada M5S 1A8; Department of Physiology (Y.M.L.), China Medical University, Taichung 404, Taiwan, R.O.C.; and Department of Medicine (M.H.), University of Chicago, Chicago, Illinois 60637

Address all correspondence and requests for reprints to: Drs. Yuk M. Leung and Herbert Y. Gaisano, Room 7226, Medical Sciences Building, 1 King’s College Circle, University of Toronto, Toronto, Ontario, Canada M5S 1A8. E-mail: yukman.leung{at}utoronto.ca or herbert.gaisano{at}utoronto.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glucose regulates pancreatic islet {alpha}-cell glucagon secretion directly by its metabolism to generate ATP in {alpha}-cells, and indirectly via stimulation of paracrine release of ß-cell secretory products, particularly insulin. How the cellular substrates of these pathways converge in the {alpha}-cell is not well known. We recently reported the use of the MIP-GFP (mouse insulin promoter-green fluorescent protein) mouse to reliably identify islet {alpha}- (non-green cells) and ß-cells (green cells), and characterized their ATP-sensitive K+ (KATP) channel properties, showing that {alpha}-cell KATP channels exhibited a 5-fold higher sensitivity to ATP inhibition than ß-cell KATP channels. Here, we show that insulin exerted paracrine regulation of {alpha}-cells by markedly reducing the sensitivity of {alpha}-cell KATP channels to ATP (IC50 = 0.18 and 0.50 mM in absence and presence of insulin, respectively). Insulin also desensitized ß-cell KATP channels to ATP inhibition (IC50 = 0.84 and 1.23 mM in absence and presence of insulin, respectively). Insulin effects on both islet cell KATP channels were blocked by wortmannin, indicating that insulin acted on the insulin receptor-phosphatidylinositol 3-kinase signaling pathway. Insulin did not affect {alpha}-cell A-type K+ currents. Glutamate, known to also inhibit {alpha}-cell glucagon secretion, did not activate {alpha}-cell KATP channel opening. We conclude that a major mechanism by which insulin exerts paracrine control on {alpha}-cells is by modulating its KATP channel sensitivity to ATP block. This may be an underlying basis for the proposed sequential glucose-insulin regulation of {alpha}-cell glucagon secretion, which becomes distorted in diabetes, leading to dysregulated glucagon secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PANCREATIC ISLET {alpha}- and ß-cells secrete glucagon and insulin, respectively, which play counter-regulatory roles in the fine-tuned glucose homeostasis (1, 2). Stimulation of ß-cell insulin secretion is via glucose metabolism and ATP production, subsequently leading to ATP-sensitive K+ (KATP) channel inhibition, membrane depolarization and exocytosis (3). Less is known about how glucose regulates {alpha}-cell glucagon secretion, and whether such regulation involves {alpha}-cell KATP channels has remained controversial (4, 5, 6, 7, 8). Both {alpha}- and ß-cell KATP channels have the same molecular composition (sulfonylurea receptor SUR1, and inward rectifier K+ channels Kir6.2) (9, 10, 11) and were found to be functionally equivalent in their high-affinity ATP blockade when examined by inside-out patch configuration (9, 12). However, KATP channel responses to high glucose in intact {alpha}-cells appear to be variable and not consistent with that in intact ß-cells (3, 4, 5, 6, 7, 9). This differential regulation may be attributed to possible differences in ATP sensitivity of the KATP channels (13) and/or differences in glucose metabolism (14) in these two islet cells.

In addition, autocrine and paracrine secetory products of these islets cells add complexity to regulation of islet cell secretion (15, 16, 17, 18, 19, 20). In fact, two recent reports have shown that high glucose, in contrast to its inhibition on {alpha}-cell glucagon secretion in whole-islet, stimulates glucagon secretion in single {alpha}-cells (7, 19). This observation reveals the overriding negative paracrine control of {alpha}-cell glucagon release by ß-cell secretory products in intact islets. In particular, insulin has emerged as a major candidate in mediating such a paracrine control, and appears to achieve this by activating {alpha}-cell KATP channels (19). In further support, insulin receptors are found not only in ß-cells (21), but also in {alpha}-cells (22). How insulin enhances {alpha}-cell KATP channel opening, however, remains unknown.

In this work, we examined how insulin activates {alpha}-cell KATP channel opening. We also examined whether glutamate, an autocrine inhibitor of {alpha}-cell, would also affect {alpha}-cell KATP channel activities. We have created the MIP-GFP mice (CD1 background), which have genetically targeted GFP expression into the islet ß-cells, allowing high efficiency and reliability in identifying and examining ß-cells (green) and non-ß-cells (non-green) (13, 23, 24). Using whole-cell configuration to examine intact cells, we have just shown that KATP channel densities are comparable in {alpha}- and ß-cells of MIP-GFP mice (13). Interestingly, {alpha}-cell KATP channels are much more sensitive to ATP inhibition than ß-cell KATP channels (13). In the present work, we found that insulin, in a phosphatidylinositol-3 (PI-3) kinase-dependent manner, stimulated islet {alpha}- and ß-cell KATP channel opening by decreasing their sensitivity to ATP block. On the other hand, we found no modulation of {alpha}-cell KATP channel opening by glutamate. We suggest that insulin exhibits negative autocrine and paracrine regulation by activating islet cell KATP channels, thus reducing cell excitability.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Antibodies against insulin (guinea pig) and glucagon (mouse), glucagon (rabbit), and somatostatin (mouse) were purchased from DakoCytomation (Glostrup, Denmark), Sigma (St. Louis, MO), and GeneTex (San Antonio, TX), respectively. Rabbit anti-SUR1 antibody was a gift from J. Ferrer (Barcelona, Spain). Tetraethylammonium-Cl, glutamate, wortmannin, and Mg-ATP were from Sigma. Insulin was from Novo Nordisk Pharmaceutical Industries (Toronto, Ontario, Canada) and Sigma.

Islet isolation
Mouse pancreatic islets were isolated by collagenase digestion as described previously (25), and promptly dispersed into single cells with 0.05% trypsin (Sigma) in Ca2+- and Mg2+-free PBS. Islet cells were plated on glass coverslips in 35-mm dishes and cultured in RPMI 1640 medium containing 1 mM pyruvate, 11 mM glucose, 0.2% NaHCO3, 10% fetal bovine serum, 10 mM HEPES, and 100 U/ml penicillin G sodium, 100 µg/ml streptomycin sulfate. Islet cells were cultured overnight before electrophysiological recordings.

Cell culture
Human embryonic kidney (HEK) 293 cells were grown in MEM (Invitrogen Canada Inc., Burlington, Ontario, Canada) containing 1 g/liter glucose and supplemented with 10% fetal bovine serum (Cansera, Rexdale, Ontario, Canada). BA8 cells stably expressing Kir6.2/SUR1 were cultured in DMEM containing 4.5 g/liter glucose and supplemented with 10% fetal bovine serum, 0.6 mg/ml G418 (Sigma), and 44 µg/ml hygromycin (Sigma).

Recording of K+ currents
Mouse islet cells were voltage-clamped in the whole-cell configuration using an EPC-9 amplifier and Pulse software (HEKA Electronik, Lambrecht, Germany) as we previously described (25). Pipettes tip resistances ranged from 3–5 M{Omega} when filled with intracellular solutions. The intracellular solution for measurement of A-type K+ currents contained (mM): 140 KCl, 1 MgCl2, 1 EGTA, 10 HEPES, and 5 MgATP (pH 7.25 adjusted with KOH). For KATP current measurements, the intracellular solution was the same as above except that the concentration of MgATP was varied as indicated. Each individual cell was tested with a single concentration of ATP dialyzed through the recording pipette. The bath solution for KATP current measurements contained (mM): 140 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 2 D-glucose, and 10 HEPES (pH 7.4 adjusted with NaOH). The bath solution for A-type K+ current measurement was the same as above with the addition of 20 mM tetraethylammonium-Cl to block delayed rectifier K+ currents.

In a typical recording, the ß-cells are identified as green cells (13, 23, 24), and the {alpha}-cells are identified by being non-green and having A-type K+ currents (4, 13). After a whole-cell configuration was established in {alpha}-cells, the cell was held at –80 mV and a test pulse of –30 mV (500 msec) was given immediately to test the presence of A-type K+ currents. Subsequently, the cell was then stimulated by a –140 mV hyperpolarizing voltage step (500 msec) every 10 sec. Once KATP currents reached maximum, the cell was subject to a series of voltage pulses from –140 to –40 mV (500 msec) at 20-mV increments to obtain current-voltage (I-V) relationship. In experiments with low pipette ATP concentrations, KATP currents developed rapidly but also ran down very quickly. Therefore, to test the modulating effect of insulin on KATP channel, we pretreated cells with insulin for 10–20 min (before break-in) and then observed maximum KATP currents developed, rather than adding insulin at the peak of KATP current development. In one particular set of experiments to observe the immediate effects of insulin, we challenged the {alpha}-cells with insulin while KATP currents were slowly developing in the presence of 1 mM intracellular ATP. All experiments were performed at room temperature (~22 C). Concentration-response curves for ATP inhibition of KATP channels are fit by the Hill equation:

Formula
where I is the current in the presence of a certain pipette [ATP], Imax is the maximum current, [ATP] is the concentration of ATP inside the pipette, Kd is the apparent dissociation constant, and n is the Hill coefficient.

Confocal immunofluorescence microscopy
Laser confocal immunofluorescence microscopy was performed as previously described (13, 25). Dispersed pancreatic islet cells, HEK293 cells, and BA8 cells were plated on glass coverslips and incubated overnight. The cells were then fixed in 2% formaldehyde for 30 min, treated with 5% goat serum and 0.1% saponin for 1 h, and finally immunolabeled with mouse antiglucagon (1:2000), rabbit antiglucagon (1:100), guinea pig antiinsulin (1:100), mouse monoclonal antisomatostatin (1:100), or rabbit SUR1 (1:250) for 2 h at room temperature. The coverslips were rinsed with 0.1% saponin in PBS and then incubated with appropriate fluorescent-labeled secondary antibodies (either Texas red or Cy-5) for 1 h at room temperature. After rinsing once more, coverslips were mounted on slides in a fading retarder (0.1% p-phenylenediamine in glycerol) and examined using a laser scanning confocal imaging system (LSM 510; Carl Zeiss, Oberkochen, Germany).

Statistics
Results are presented as means ± SEM. ANOVA was used for multiple group comparisons and statistical significance was determined by Student-Newman-Keuls test. A value of P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Visualization of ß-, {alpha}-, and {delta}-cells and the presence of SUR1 in {alpha}-cells
Figure 1AGo shows a bright-field image of dispersed islet cells. The thin solid arrows point to the ß-cells, which were green in the confocal image as they expressed GFP (Fig. 1BGo). The empty arrow in Fig. 1AGo indicates an {alpha}-cell, which was labeled with glucagon antibody and appears in white (pseudocolor for Cy-5 fluorescence) (Fig. 1CGo). The thick arrow points to a {delta}-cell at the center of Fig. 1AGo, which was labeled by antisomatostatin and appears in red (Fig. 1DGo). Note that ß-cells were larger than {alpha}- or {delta}-cells, as has been previously reported (5, 13) (Fig. 1AGo). Figure 1EGo shows a bright-field image of ß-cells, which were green in the confocal image (Fig. 1FGo), and appeared red when they were probed with insulin antibody (Fig. 1GGo), confirming that green cells were insulin-secreting ß-cells.


Figure 1
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  		FIG. 1. Visual identification of islet cells. A, Bright-field image of islet cells. Both {alpha}- and ß-cells were labeled by thin empty and solid arrows, respectively. The thick arrow points to a {delta}-cell. B, ß-Cells expressing GFP (C and D) {alpha}- and {delta}-cells with immnuofluorescence labeling (E–G) show GFP-expressing ß-cells are indeed insulin-secreting cells after being labeled with antiinsulin antibody. Bright-field image (H) and SUR1 labeling (I) of BA8 cells. Bright-field image (J), glucagon labeling (K), and SUR1 labeling (L) of two {alpha}-cells.

 
The KATP channel density was previously shown to be very low in mouse {alpha}-cells (5), but we recently found it to be high when mouse {alpha}-cells were dialyzed with low intracellular ATP concentrations (13). To confirm the presence of KATP channels in islet {alpha}-cells, we probed the SUR1 subunit of the KATP channel with a specific antibody. BA8 cells, stably expressing Kir6.2/SUR1, were used as a positive control for SUR1 staining [Fig. 1HGo, bright-field image; Fig. 1IGo, confocal image showing SUR1 staining (Texas Red)]. Untransfected HEK293 cells did not show such staining (data not shown). Two {alpha}-cells in Fig. 1JGo showed labeling with antiglucagon (Fig. 1KGo) and anti-SUR1 antibodies (white, pseudocolor for Cy-5 fluorescence; Fig. 1LGo), confirming that {alpha}-cells expressed SUR1-containing KATP channels.

Insulin decreased {alpha}- and ß-cell KATP channel sensitivity to ATP block
In our recent report (13), we showed that the densities of KATP channels in mouse islet {alpha}- and ß-cells are comparable when examined at low intracellular concentrations of ATP. In that report, maximal KATP current densities were determined to be 142 pA/pF for {alpha}-cells and 99 pA/pF for ß-cells at the optimal ATP concentrations of 0.05 mM and 0.3 mM ATP, respectively. Of note, the sensitivity of KATP channel to ATP block was five times higher in {alpha}-cells (IC50 = 0.18 mM) than in ß-cells (IC50 = 0.84 mM). The cause for this differential sensitivity is so far unknown.

Insulin is now known to be inhibitory to {alpha}-cell secretory function (16, 19, 20), but the precise molecular substrates acted upon by insulin in the {alpha}-cell are unclear. We have postulated that the underlying inhibitory cellular action of insulin may be on the {alpha}-cell KATP channel. We therefore tested whether insulin (3 µM) would activate {alpha}-cell KATP channel opening. Insulin was used at a high concentration (3 µM) as intraislet insulin levels, particularly between adjacent {alpha}- and ß-cells, are expected to be high. In fact, micromolar, but not nanomolar concentrations, of exogenous insulin are required to blunt glucopenia-induced glucagon secretion in perfused rat pancreas (26). Figure 2Go, B and C, shows the KATP currents in {alpha}-cells dialyzed with 0.3 mM ATP or 1 mM ATP, respectively, in the absence (top cell) or presence (bottom cell) of insulin (3 µM insulin pretreatment for 10–20 min). In both ATP concentrations, insulin pretreatment induced greater KATP currents. However, when {alpha}-cells were dialyzed with a very low ATP concentration (0.05 mM ATP; Fig. 2AGo), which is the optimal ATP concentration that elicits maximal KATP currents in these {alpha}-cells (in Ref.13), or with a high ATP concentration (5 mM ATP; Fig. 2DGo), insulin pretreatment did not enhance KATP currents. Figure 2EGo shows the summary of these results quantified as an increase in current density (at –140 mV stimulation) and plotted against the different ATP concentrations dialyzed into the cells. Taken together, these results suggest that insulin did not cause an acute recruitment or translocation of KATP channels to the plasma membrane. Insulin pretreatment also did not enhance KATP currents when the KATP channels were completely inhibited by 5 mM ATP, suggesting that insulin did not activate any K+ conductance other than KATP channels. When the current density is normalized (Fig. 2FGo), we noted that insulin pretreatment caused the ATP sensitivity curve (no insulin control, IC50 = 0.18 mM) to shift to the right (insulin pretreated, IC50 = 0.50 mM) by 2.8-fold, indicating that insulin can effectively decrease the ATP sensitivity of {alpha}-cell KATP channels.


Figure 2
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  		FIG. 2. Insulin decreased KATP channel sensitivity to ATP block in single {alpha}-cells. A, Upper panel, An {alpha}-cell was held at –80 mV and dialyzed with 0.05 mM ATP. The cell was then stimulated by a –140 mV hyperpolarizing voltage step (500 msec) every 10 sec. Once KATP currents reached maximum, the cell was subject to a series of voltage pulses from –140 to –40 mV (500 msec) at 20 mV increments to obtain I-V relationship. A, Lower panel, The same protocol as above was performed on another cell with 3 µM insulin pretreatment for 10–20 min. B–D, The same protocol as in panel A except that the pipette contained different concentrations of ATP as indicated. E, The increase in KATP current density (at –140 mV) are plotted against ATP concentrations for control and insulin-pretreatment groups. F, The increase in KATP current density (at –140 mV) are normalized to the maximum increase in KATP current density and plotted against ATP concentrations. Data are fit with a Hill equation. All values are mean ± SEM of three to nine cells. *, Significant difference from the control (P < 0.05).

 
We then examined whether insulin might have a similar effect on ß-cell KATP channels. Figure 3BGo shows the KATP currents in ß-cells dialyzed with 1 mM ATP in the absence (top cell) or presence (bottom cell) of insulin in the bath solution. Here, pretreatment with 3 µM insulin (10–20 min) induced greater KATP currents. However, insulin did not enhance KATP currents in ß-cells dialyzed with 0.3 or 5 mM ATP (Fig. 3Go, A and C). Results are quantified as an increase in current density (at –140 mV stimulation) and plotted against different concentrations of ATP dialyzed (Fig. 3DGo). When the current density is normalized (Fig. 3EGo), we note that insulin also shifted the ß-cell ATP sensitivity curve (control IC50 = 0.84 mM) to the right (insulin pretreated, IC50 = 1.23 mM), which is a 1.5-fold reduction in ATP sensitivity.


Figure 3
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  		FIG. 3. Insulin decreased KATP channel sensitivity to ATP block in single ß-cells. A, Upper panel, A ß-cell was held at –80 mV and dialyzed with 0.3 mM ATP. The cell was then stimulated by a –140 mV hyperpolarizing voltage step (500 msec) every 10 sec. Once KATP currents reached maximum, the cell was subject to a series of voltage pulses from –140 to –40 mV (500 ms) at 20 mV increments to obtain I-V relationship. A, Lower panel, The same protocol as above was performed on another cell with 3 µM insulin pretreatment for 10–20 min. B and C, The same protocol as in panel A except that the pipette contained different concentrations of ATP as indicated. D, The increase in KATP current density (at –140 mV) are plotted against ATP concentrations for control and insulin-pretreatment groups. E, The increase in KATP current density (at –140 mV) are normalized to the maximum increase in KATP current density and plotted against ATP concentrations. Data are fit with a Hill equation. All values are mean ± SEM of three to six cells. *, Significant difference from the control (P < 0.05).

 
Because PI-3 kinase is a major signaling pathway activated by insulin receptor activation (27), we examined whether the reduced sensitivity of KATP channels to ATP induced by insulin involved the PI-3 kinase signaling pathway. Here, we have used wortmannin, a selective PI-3 kinase inhibitor. In the presence of 0.3 and 1 mM intracellular ATP concentration, which yield partial and comparable KATP channel opening in {alpha}- and ß-cells, respectively, the enhancement of currents by insulin was completely prevented by 300 nM wortmannin in both {alpha}- (Fig. 4AGo) and ß-cells (Fig. 4BGo). Note that wortmannin on its own did not significantly affect KATP channel opening in the absence of insulin. These results indicate that the PI-3 kinase signaling pathway is the underlying mechanism by which insulin desensitizes the KATP channels in both {alpha}- and ß-cells to ATP.


Figure 4
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  		FIG. 4. Wortmannin prevented insulin-induced enhancement of KATP currents in {alpha}- and ß-cells. The same protocol as in Fig. 2Go was performed with {alpha}-cells (A) and ß-cells (B), and the pipette concentrations were used at 0.3 mM ({alpha}-cells) and 1 mM (ß-cells) to allow partial KATP channel opening. The increase in KATP current density (at –140 mV) is plotted for the control and insulin-/wortmannin-pretreatment groups. The preincubation time for both insulin (3 µM) and wortmannin (300 nM) was 10–20 min. All values are mean ± SEM of three to six cells. *, Significant difference from the control (P < 0.05).

 
We next examined the immediate effects (kinetics) of insulin activation as we challenged the {alpha}-cells with insulin while KATP currents were slowly developing in the presence of 1 mM intracellular ATP. In these experiments, insulin was used at a concentration much lower (100 nM) than that used in Fig. 2Go (3 µM). As shown in Fig. 5AGo, KATP currents slowly developed over a time course of 12 min, with a lag of 4–5 min. Addition of insulin augmented the rate and magnitude of KATP currents developed. In contrast to a recent report showing the transient nature of insulin activation of KATP currents (decaying after 6 min of insulin addition) in rat {alpha}-cells (19), the activation effect of insulin on our mouse {alpha}-cells was more persistent because there appeared no sign of decay 11 min after insulin challenge. Maximum increase in KATP currents observed 11–12 min after insulin treatment was about 2-fold of that in the untreated group (Fig. 5BGo). These data indicate that insulin could activate {alpha}-cell KATP currents at 100 nM. Because commercially available insulin may contain Zn2+ at a high level, and that a previous report has shown that Zn2+ could activate rat {alpha}-cell KATP channel (19), we also examined whether the insulin effect on mouse {alpha}-cell KATP channel could be due to Zn2+. Using the protocol as shown in Fig. 5Go to study the acute effects of Zn2+, we found that Zn2+ (50 µM) did not activate {alpha}-cell KATP currents in the presence of 1 mM intracellular ATP [increases in current density were 10.5 ± 2.8 pA/pF (n = 7) and 10.3 ± 1.7 pA/pF (n = 3) in control and Zn2+-challenged groups, respectively]. Therefore, the activating effect of insulin on mouse {alpha}-cell KATP channel was unrelated to Zn2+.


Figure 5
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  		FIG. 5. Insulin augmented KATP current development in {alpha}-cells. A, {alpha}-Cells were dialyzed with 1 mM ATP through the recording pipette to allow slow development of KATP currents. Cells were held at –80 mV, and once a whole-cell configuration was established and A-type K+ currents detected, they were stimulated by a –140 mV hyperpolarizing voltage step (500 msec) every 10 sec. Representative recordings are shown where control buffer or insulin (100 nM; Sigma) was added as indicated by the arrow. The dashed line indicates zero-current level. Maximum increase in KATP current density 11–12 min after insulin/control treatment was shown in (B). All values are mean ± SEM of five to eight cells. *, Significant difference from the control (P < 0.05).

 
Insulin did not affect {alpha}-cell A-type K+ channel opening
We then examined whether insulin could also affect the opening of other channels in {alpha}-cells. {alpha}-Cells possess A-type K+ channels, which are functional markers to distinguish {alpha}-cells from other islet cells (4, 13). Figure 6Go, A and B, shows the representative traces of A-type K+ currents triggered in control and 3 µM insulin-pretreated {alpha}-cells. Figure 6CGo is the I-V summary showing that insulin pretreatment did not affect A-type K+ channel opening or gating.


Figure 6
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  		FIG. 6. Insulin did not affect A-type K+ currents in single {alpha}-cells. A, A-type K+ currents were triggered when an {alpha}-cell was held at –80 mV and stimulated by a series of voltage pulses from –70 to +70 mV (500 msec) at 10 mV increments to obtain I-V relationship. B, The same protocol as in panel A was performed in another cell with 3 µM insulin pretreatment for 10–20 min. C, Currents are normalized with cell size to yield current density and plotted against voltage. All values are mean ± SEM of 12 cells.

 
{alpha}-Cell KATP channel activities were not affected by glutamate
{alpha}-Cells have been shown to be subject to both paracrine and autocrine regulation (16, 17, 18, 19, 20). Glutamate, cosecreted with glucagon in response to low glucose, has been shown to be an inhibitory autocrine factor for {alpha}-cell glucagon secretion (17). Here we examined whether glutamate might be inhibiting the {alpha}-cells by activating KATP channels. Figure 7Go, A and B, show the KATP currents in two {alpha}-cells dialyzed with 1 mM ATP in the absence and presence of glutamate in the bath solution. Pretreatment with 1 mM glutamate did not enhance KATP currents at any voltage (Fig. 7CGo). This would indicate that glutamate inhibition of glucagon secretion is independent of any action on {alpha}-cell KATP channels.


Figure 7
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  		FIG. 7. {alpha}-cell KATP channel opening was not modulated by glutamate. A, An {alpha}-cell was held at –80 mV and dialyzed with 1 mM ATP. The cell was then stimulated by a –140 mV hyperpolarizing voltage step (500 msec) every 10 sec. Once KATP currents reached maximum, the cell was subject to a series of voltage pulses from –140 to –40 mV (500 msec) at 20 mV increments to obtain I-V relationship. B, The same protocol as in panel A was performed on another cell with 1 mM glutamate pretreatment for 10 min. C, Currents are normalized with cell size to yield current density and plotted against voltage. All values are mean ± SEM of three to six cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has become increasingly evident that islet cells act as a social unit within an intact islet influenced profoundly by complex paracrine and autocrine regulation (15, 16, 17, 18, 19, 20). A major paracrine mechanism that inhibits {alpha}-cell secretion is insulin secreted by ß-cells (16, 19, 20, 28, 29, 30). However, how insulin inhibits {alpha}-cell secretion has remained poorly understood. A very recent report showed that insulin augments {alpha}-cell K+ currents in the presence of a low concentration of intracellular ATP (0.3 mM) (19). Such increase in K+ conductance may serve to set the membrane potential below the activation thresholds of T-type Ca2+ channels and Na+ channels, hence dampening {alpha}-cell excitability. In that report (19), however, it was not entirely certain as to whether the insulin-induced increase in {alpha}-cell K+ currents were in fact KATP currents. Furthermore, if insulin indeed enhances {alpha}-cell KATP currents, does it involve KATP channel surface translocation or changes in its sensitivity to ATP block?

In the present study, we have determined that insulin specifically affected the KATP channels. This effect of insulin on the KATP channels is specific because insulin had no effect on the {alpha}-cell A-type K+ currents. Here we have further examined insulin action on {alpha}-cell KATP channel subject to inhibition by different cytosolic concentrations of ATP. Insulin only enhanced {alpha}-cell KATP currents in the presence of intermediate ATP concentrations (0.3 and 1 mM). Insulin, however, did not enhance currents in the presence of a low but optimal ATP concentration (0.05 mM), suggesting that insulin did not cause the enhancement in {alpha}-cell KATP current by acutely recruiting or translocating KATP channels to the plasma membrane, or simply increasing channel open probability. Insulin pretreatment also did not enhance currents when the KATP channels were completely inhibited by 5 mM ATP, suggesting that insulin did not activate conductances other than the KATP conductance. We therefore elucidated that insulin enhancement of {alpha}-cells KATP channels to be due to a reduction in ATP sensitivity of {alpha}-cell KATP channels. Because changes in ATP concentrations in {alpha}-cells are within a much narrower range than ß-cells (20, 31), this provides a mechanism by which ATP may be able to dynamically regulate {alpha}-cell KATP channels through insulin modulation of {alpha}-cell KATP channel sensitivity to ATP. In our experiments (whole-cell configuration), with the absence of ADP (physiological KATP channel activator) in the intracellular solution, we noticed that insulin could not overcome the suppressive effect of 5 mM ATP on {alpha}-cell KATP channels. However, in intact {alpha}-cells where ADP is present in the cytosol, insulin may be able to open KATP channels despite the presence of 5 mM intracellular ATP. This would explain how insulin hyperpolarizes and curbs {alpha}-cell firing in perforated-patch studies (19). Of importance, this intricate relationship between ATP levels in the {alpha}-cell (generated by glucose stimulation) and paracrine insulin-mediated changes in {alpha}-cell KATP channel sensitivity to ATP may be an underlying basis for the recently proposed sequential glucose-insulin regulation of {alpha}-cell glucagon secretion (28, 29, 30). A distortion of these sequential events might contribute to the reduced glucagon secretion in type 1 diabetes (28, 29). Conceivably, a persistently high intraislet insulin levels occurring during hyperinsulinemia in type 2 diabetes might also alter this glucose-insulin sequential regulation of {alpha}-cell KATP channels to alter glucagon secretion, perhaps in part by desensitizing the {alpha}-cell insulin receptors.

We showed that insulin could also reduce the ß-cell KATP channel sensitivity to ATP, consistent with an earlier report showing that insulin activates ß-cell KATP channel (15). This insulin effect provides a negative autocrine control of ß-cell function (15). It is of interest to note that insulin also hyperpolarizes hypothalamic glucose-responsive neurons by opening KATP channels, which may have implications in energy homeostasis such as food intake and body weight (32). Wortmannin, a PI3 kinase inhibitor, effectively prevented the insulin modulation of both {alpha}- and ß-cells KATP channel gating, suggesting a role of PI3 kinase in insulin signaling (also see Ref.15). However, Franklin et al. (19) reported that in rat {alpha}-cells, the insulin activation of KATP channel, was independent of PI3 kinase. The reason of this discrepancy is unknown, but could possibly be due to species difference. In line with this, it is of interest to note that the activating effect of insulin on KATP channel appeared to be more sustained in mouse {alpha}-cells (this work) than rat {alpha}-cells (19).

{alpha}-Cell excitability can also be attenuated by other paracrine factors under high glucose conditions. GABA secreted by ß-cells is known to be inhibitory to {alpha}-cell glucagon secretion by inducing Cl– influx and subsequent hyperpolarization (33). Zn2+ is cosecreted with insulin by ß-cells. The role of Zn2+ in regulating {alpha}-cell glucagon secretion is controversial (16, 19, 20). In rat, Zn2+ has been shown to inhibit glucagon secretion by hyperpolarizing {alpha}-cells via KATP channel activation (19). However, we found that Zn2+ did not activate mouse {alpha}-cell KATP channels, which is in agreement with the inability of Zn2+ to inhibit glucagon secretion in mouse islets (20). Somatostatin, a hormone secreted by {delta}-cells, hyperpolarizes {alpha}-cells by activating a low-conductance K+ channel coupled to inhibitory G proteins (34). In addition to paracrine control, {alpha}-cells have been known to be under positive and negative autocrine regulation by glucagon (17) and glutamate (16), respectively. Here, we also examined whether glutamate inhibition of {alpha}-cell secretion might involve activation of the KATP channels. We found that glutamate as high as 1 mM did not activate {alpha}-cell KATP channels, and therefore the inhibitory actions of insulin and glutamate on {alpha}-cell secretion are via distinct pathways. Of note, besides paracrine control, high glucose itself has been shown to suppress {alpha}-cells by causing a refill of an intracellular Ca2+ store, thus inhibiting a Ca2+ store-dependent depolarizing conductance (35).

In summary, this study demonstrates that insulin exerts paracrine and autocrine actions on islet {alpha}- and ß-cells, respectively, in part by modulating their KATP channel sensitivity to ATP block. These insulin effects are likely contributing factors that curb islet {alpha}- and ß-cell excitability in health and in diabetes.


   Footnotes
 
This work was supported by grants from the Juvenile Diabetes Research Foundation (no. 1-2005-1112 to H.Y.G.), the Canadian Institutes of Health Research (MOP-69083 to H.Y.G. and R.G.T.; and MOP-36499 to N.D. and H.Y.G.), and by an equipment grant from the Banting and Best Diabetes Center (University of Toronto). Y.M.L. is supported by Fellowship Awards from the Canadian Diabetes Association in honor of the late Evelyn J. Parker.

Y.M.L., I.A., L.S., X.G., M.H., R.G.T., N.E.D. and H.Y.G. have nothing to declare.

First Published Online February 2, 2006

Abbreviations: HEK, Human embryonic kidney; I-V, current voltage; KATP, ATP-sensitive K+ channel; MIP-GFP, mouse insulin promoter-green fluorescent protein; PI-3 kinase, phosphatidylinositol-3 kinase; SUR1, sulfonylurea receptor.

Received October 3, 2005.

Accepted for publication January 23, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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