Insulin Feedback Alters Mitochondrial Activity Through an ATP-sensitive K+ Channel–Dependent Pathway in Mouse Islets and ß-Cells

Craig S. Nunemaker, Min Zhang, and Leslie S. Satin

From the Department of Pharmacology and Toxicology, School of Medicine, Virginia Commonwealth University Medical Center, Richmond, Virginia


    ABSTRACT
 TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent work suggests that insulin may exert both positive and negative feedback directly on pancreatic ß-cells. To investigate the hypothesis that insulin modulates ß-cell metabolism, mouse islets and ß-cell clusters were loaded with rhodamine 123 to dynamically monitor mitochondrial membrane potential ({Delta}{Psi}m). Spontaneous oscillations in {Delta}{Psi}m (period: 218 ± 26 s) were observed in 17 of 30 islets exposed to 11.1 mmol/l glucose. Acute insulin application (100 nmol/l) hyperpolarized {Delta}{Psi}m, indicating a change in mitochondrial activity. The ATP-sensitive K+ (KATP) channel opener diazoxide or the L-type calcium channel blocker nifedipine mimicked the effect of insulin, suggesting that insulin activates KATP channels to hyperpolarize {Delta}{Psi}m by inhibiting calcium influx. Treatment with forskolin, which increases endogenous insulin secretion, also mimicked the effect of exogenous insulin, suggesting physiological feedback. Pretreatment with nifedipine or the KATP inhibitor glyburide prevented insulin action, further implicating a KATP channel pathway. Together, these data suggest a feedback mechanism whereby insulin receptor activation opens KATP channels to inhibit further secretion. The resulting reduction in ß-cell calcium increases the energy stored in the mitochondrial gradient that drives ATP production. Insulin feedback onto mitochondria may thus help to calibrate the energy needs of the ß-cell on a minute-to-minute basis.

Electrical activity plays a prominent role in ß-cell stimulus-secretion coupling in mouse islets (1,2). At low glucose concentrations (<3 mmol/l), ß-cells are electrically silent and secrete low or basal levels of insulin. In response to glucose stimulation (>5–7 mmol/l), metabolism and mitochondrial energy production increase (35). The resulting increase in the ATP/ADP ratio closes ATP-sensitive K+ (KATP) channels and depolarizes the ß-cell to initiate electrical activity and insulin secretion (2,6). ß-cell electrical activity typically follows a burst pattern consisting of slow oscillations or plateaus in membrane potential with superimposed fast calcium spikes (7). The resulting calcium influx induces insulin secretion and may activate several types of K+ channels to assist in terminating each burst (811).

Recent work suggests that this process may be regulated by insulin. Exogenous insulin has been found to modulate gene transcription and translation, intracellular signaling, intracellular calcium ([Ca2+]i), and insulin secretion itself (1219). We have previously shown that insulin acutely opens the KATP channel through a phosphatidylinositol (PI) 3-kinase-sensitive mechanism, leading to hyperpolarization of the ß-cell plasma membrane and a reduction in calcium influx as voltage-gated L-type calcium channels close (14). Insulin thus negatively feeds back to inhibit further insulin secretion via this pathway. Other studies, however, have suggested that insulin receptor activation results in increased [Ca2+]i and increased secretion, suggesting a positive feedback due to insulin receptor activation (15,16,18,19).

In the present study, we sought to determine whether insulin could also influence metabolic activity in ß-cells. Previous studies using the dye rhodamine 123 (Rh123) to dynamically measure mitochondrial membrane potential ({Delta}{Psi}m) as a marker of cell metabolic activity have established that {Delta}{Psi}m hyperpolarizes in response to increased glucose in ß-cells (2022), consistent with the known action of glucose metabolism to increase the proton gradient that drives mitochondrial ATP production. Oscillatory changes in Rh123 have been found to mirror oscillatory changes in [Ca2+]i (21,22), suggesting that increased [Ca2+]i in ß-cells decreases the ATP/ADP ratio, as discussed in the Keizer-Magnus model (23,24). To investigate the feedback effects of insulin on metabolism and their possible relation to plasma membrane ion channel activity, we measured Rh123 fluorescence in ß-cells while selectively blocking specific ß-cell ion channels to test the hypothesis that insulin modifies mitochondrial metabolism via ion channel activity in mouse islets and ß-cells.


    RESEARCH DESIGN AND METHODS
 TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Culture of islets and islet ß-cells.
Mouse islets were isolated by collagenase digestion from the pancreata of Swiss-Webster mice (ages 2–3 months, weight 25–35 g), as previously described (14,25). Islets were dissociated into single cells or clusters by gentle trituration in a low-calcium medium (26,27). Islets or islet cell suspensions were then plated on glass coverslips pretreated with 0.1% gelatin to facilitate attachment (Sigma-Aldrich, St. Louis, MO) and placed in 35-mm plastic Petri dishes for tissue culture. Islet tissue was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin-streptomycin (Gibco, Grand Island, NY) and incubated at 37°C in a 95% air/5% CO2 mixture. Cultures were fed every other day. Clusters of islet cells were studied after 2–5 days in culture, and islets were studied within 2–3 days.

Rhodamine 123 loading and fluorescence measurements.
Cultures were loaded with 5 µmol/l Rh123 (Molecular Probes, Eugene, OR) in RPMI and incubated for 10–15 min at 37°C. Cultures were then washed and incubated in Rh123-free RPMI before each experiment. Shards of coverslips containing mouse islets or single cells were placed in a recording chamber and perfused with external solution containing (in mmol/l): 11.1 glucose, 130.5 NaCl, 3 CaCl2, 5 KCl, 2 MgCl2, 10 HEPES (pH 7.3). All chemicals were obtained from Sigma-Aldrich, unless otherwise indicated. A Minipulse II peristaltic pump (Gilson, Villiers Le Bel, France) perfused various solutions through the recording chamber at 32–35°C (±0.3°C intra-experimental range) using an in-line heater (Cell Micro Controls, Virginia Beach, VA).

Rh123 fluorescence was measured using an Olympus BX61WI upright laser scanning confocal microscope and Fluoview image acquisition and analysis software (Olympus America, Melville, NY). Rh123 has been used in numerous studies (28,29), including several using ß-cells (2022), to monitor changes in {Delta}{Psi}m. Hyperpolarization of {Delta}{Psi}m leads to increased mitochondrial Rh123 uptake, Rh123 concentration, and self-quenching of fluorescence within the mitochondria. In contrast, as {Delta}{Psi}m diminishes, the relative depolarization of {Delta}{Psi}m leads to dye efflux from the mitochondria into the cytosol as oxidation decreases, causing fluorescence to increase. This explains why mitochondrial inhibitors, such as NaN3 and fluoro-carbonyl cyanide phenylhydrazone (FCCP), consistently increase Rh123 fluorescence. Rh123 was excited at 488 nm, and its emission was collected at 535 nm. Cell-free calibration experiments carried out using different concentrations of free dye confirmed a linear relation between dye concentration and fluorescence intensity within the range monitored, and rhodamine-coated Teflon beads (6 µm diameter; Molecular Probes) confirmed the stability of the fluorescence measurements (data not shown). For each experiment, the fluorescence of single ß-cells, ß-cell clusters, or islets was recorded in 11.1 mmol/l glucose for 10–20 min, with images acquired at 3-s intervals to establish baseline fluorescence and detect possible oscillations. This control period was followed by a 5-min treatment period with one of several ion channel-modulating drugs (100 nmol/l glyburide, 250 µmol/l diazoxide, 50 µmol/l nifedipine) or a 10-min exposure to insulin to test for concomitant changes in mitochondrial activity. In some studies, several drugs were given in series or in combination, as detailed in the results. In studies using high KCl, extracellular NaCl was lowered accordingly to maintain osmolarity. The mitochondrial poison NaN3 (5 or 50 mmol/l) or the mitochondrial uncoupler FCCP (5 µmol/l) was applied for 5 min at the end of each of the experiments to confirm that Rh123 acted appropriately. Clusters and islets that did not respond to NaN3 or FCCP were not included for further analysis.

Data analysis.
To determine mean Rh123 fluorescence, rectangular regions of interest (ROIs) were drawn around the images of each islet, cell, or cell cluster after each experiment (Fig. 1AC). (We refer to clusters of cultured islet cells as ß-cell clusters from here onward based on prior work demonstrating that >80% of cultured islet cells are typically ß-cells [25].) In the case of islets, ROIs were drawn around the central portion of the islet for comparison (Fig. 1C). Rh123 fluorescence could be observed several cell layers below the surface of the islet. This allowed fluorescence measurements to be made in the interior of the islet, where endogenous changes in Rh123 fluorescence were often greater. Because the center of each optical section represented the interior of the islet, this region was expected to have a higher proportion of ß-cells (30). Treatment-induced changes in Rh123 fluorescence were typically greater in whole-islet versus central ROIs (likely due to the somewhat slow diffusion of drugs to the interior), so whole-islet ROIs were preferred for analysis. The mean fluorescence of all pixels within each ROI was calculated for each image taken at 3-s intervals and then plotted versus time using arbitrary units of fluorescence. To prevent photobleaching of Rh123, laser intensity was set to 3% of the maximum power. Decreases in mean fluorescence of <1% could be established between two consecutive control periods of 5 min each using this approach (n = 30 islets). In the core of the islets, a mean decrease of <0.05% could even be observed. Changes in fluorescence attributed to endogenous oscillations in mitochondrial activity were assessed by collecting images during control periods of 10–20 min in 11.1 mmol/l glucose. The periodicity of Rh123 oscillations was determined visually by identifying two or more peaks in fluorescence having amplitudes >5% of the baseline signal, and then calculating the time interval between the peaks observed for each recording. Comparisons between successive treatment phases were made by averaging the fluorescence of each ROI during the last 2–3 min of drug exposure. Comparisons between control and drug treatment phases were carried out using a two-tailed t test of paired mean fluorescence, with significance at P < 0.05. One-way ANOVA was used to compare multiple treatments. Data are presented as the percent of control fluorescence. Note that although some results were presented as "responders" (>5% change) and "nonresponders" (<5% change) to provide more detailed information, all statistical comparisons were carried out using means for the entire treatment group.



View larger version (121K):
[in this window]
[in a new window]
 
FIG. 1. Rhodamine fluorescence from islets and clusters. A and B: Images of a small cluster of cells during 11.1 mmol/l glucose control conditions (A) and after depolarizing {Delta}{Psi}m with NaN3 (B). C: Image of an islet during 11.1 mmol/l glucose control conditions. Scale bar in lower left corner denotes size. Boxes (ROIs) represent the area analyzed to produce plots of fluorescence intensity. D: Plot of fluorescence intensity versus time for an islet incubated for 8 min in 2.8 mmol/l glucose, followed by 22 min in 11.1 mmol/l glucose and then 3 min in 50 mmol/l NaN3 (representative of eight islets tested).

 

    RESULTS
 TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Confirmation of the relation between Rh123 fluorescence and {Delta}{Psi}m.
Before investigating the effects of insulin treatment on mitochondrial activity, we first confirmed that Rh123 reported changes in {Delta}{Psi}m appropriately, as in other ß-cells studies (2022). At high magnification, many round or elongated fluorescent structures could be observed in confocal images, indicative of individual mitochondria (Fig. 1A) (5,22). As shown in Fig. 1B, the pattern of fluorescence we observed became much more uniform after treatment with the mitochondrial poison NaN3, which dissipates the mitochondrial gradient and causes the release of Rh123 into the cytosol (Fig. 1B). In both clusters and islets under all experimental conditions, Rh123 was clearly excluded from the cell nucleus (see Fig. 1AC).

By monitoring Rh123 fluorescence levels over time, changes in {Delta}{Psi}m were ascertained in response to changes in metabolic fuel availability and after the addition of mitochondrial poisons. Specifically, switching from a solution containing 2.8 mmol/l glucose to one containing 11.1 mmol/l glucose caused Rh123 levels to decrease by 9 ± 2% (Fig. 1D; representative of eight islets tested), consistent with hyperpolarization of {Delta}{Psi}m (20). In contrast, Rh123 fluorescence markedly increased (31 ± 4%; n = 8) in response to the mitochondrial poison NaN3, which is consistent with a reduction or dissipation of {Delta}{Psi}m, resulting in more visible dye in the cytosol. Similar results were obtained with the mitochondrial uncoupler FCCP (5 µmol/l; data not shown). These findings confirm those of previous studies (2022) and support the validity of the technique to measure the metabolic activity of mitochondria.

Endogenous oscillations in {Delta}{Psi}m.
At stimulatory glucose levels, endogenous oscillations in {Delta}{Psi}m were detected in a majority of islets and clusters. Islets were maintained in 11.1 mmol/l glucose for 10–20 min to establish their baseline fluorescence. (Note that all remaining studies were performed using 11.1 mmol/l glucose.) During this control period, 17 of 30 islets displayed spontaneous oscillations in Rh123 fluorescence. The peak-to-peak amplitude of the oscillations was >5% of the total fluorescence signal (Fig. 2). The estimated period of the {Delta}{Psi}m oscillations was 218 ± 26 s (~3.6 min); oscillations ranged from 120 to 440 s (~2–7 min), consistent with previous reports of slow metabolic oscillations evoked by glucose stimulation in islets (21,22). Some ß-cell clusters displayed similar patterns, but these were not examined in detail because the signal-to-noise ratio was generally lower in clusters and single ß-cells due to the reduced area of their ROIs.



View larger version (18K):
[in this window]
[in a new window]
 
FIG. 2. Endogenous oscillations in {Delta}{Psi}m in steady-state glucose. AC: Plot of Rh123 fluorescence intensity versus time of sequential images from three islets displaying oscillations in fluorescence signal recorded in 11.1 mmol/l glucose. *Visually identified peaks in signal used to estimate the periodicity of activity. (Note that x- and y-axes differ among panels.)

 
Insulin hyperpolarizes {Delta}{Psi}m.
Previous work from this laboratory has demonstrated that insulin opens plasma membrane KATP channels in ß-cells, resulting in membrane hyperpolarization and an inhibition of islet calcium oscillations (14). In the present study, we wished to examine whether an identical insulin treatment also altered mitochondrial metabolic activity. Application of solutions containing 100 nmol/l insulin caused a clear hyperpolarization of {Delta}{Psi}m in both islets and ß-cell clusters, suggesting that insulin increased mitochondrial metabolism. As illustrated by the representative example shown in Fig. 3A, insulin reduced Rh123 fluorescence. This was the case in over half of the islets tested (n = 8 of 13; 12 ± 3% mean decrease in responders), whereas in the remaining islets, the effects of insulin were small (2 ± 1% decrease in fluorescence). The mean response among all islets was an 8 ± 2% decrease in fluorescence (n = 13; P < 0.004). Islets exposed to NaN3 after insulin treatment responded with a brisk increase in fluorescence in all cases (29 ± 7% increase; n = 9). The effects of insulin on {Delta}{Psi}m were not reversed by washing the cells for 5 min (n = 4). Similar results were observed using small clusters of islet cells (<30 µm diameter), with 15 of 22 clusters responding with a mean fluorescence decrease of 13 ± 1% (Fig. 3B). Of the remaining clusters, six demonstrated a change of <4% in Rh123 fluorescence in response to insulin and one exhibited an anomalous 17% increase in fluorescence. The mean response among all clusters was a 9 ± 1% decrease in fluorescence (n = 22; P < 0.00025).



View larger version (21K):
[in this window]
[in a new window]
 
FIG. 3. Insulin hyperpolarizes {Delta}{Psi}m in mouse islets and ß-cells. Plot of fluorescence versus time for an islet (A) and a small cluster of cells (B) recorded in 11.1 mmol/l glucose and treated with insulin followed by NaN3. Arrows indicate the start of drug treatments.

 
Insulin acts on mitochondria through KATP and calcium channels.
Based on our previous findings that insulin inhibits islet electrical activity and reduces [Ca2+]i, we hypothesized that changes in KATP channel activity and concomitant changes in calcium channel activity were responsible for the effects of insulin on {Delta}{Psi}m. We confirmed that the activation of KATP channels could, in principle, mediate changes in {Delta}{Psi}m using the KATP channel opener diazoxide. Thus the application of diazoxide (250 µmol/l) had a similar hyperpolarizing effect on {Delta}{Psi}m in islets as did insulin (n = 8) (Fig. 4A), although the effect was smaller (mean decrease of 6 ± 1% in eight islets; P < 0.01). To determine if closing KATP channels produced the opposite effect, the KATP channel blocker glyburide (100 nmol/l) was tested. Glyburide had little or no effect on Rh123 fluorescence levels (mean increase of 1 ± 2% in 18 clusters); however, this may have been due, in part, to a majority of KATP channels being closed in 11.1 mmol/l glucose.



View larger version (17K):
[in this window]
[in a new window]
 
FIG. 4. The KATP channel opener diazoxide or the calcium channel blocker nifedipine mimics the effects of insulin. A: Plot of fluorescence versus time for an islet in 11.1 mmol/l glucose treated with 250 µmol/l diazoxide, followed by NaN3. Similar results were observed in five of eight islets. B: Plot of fluorescence versus time for an islet treated with 50 µmol/l nifedipine, followed by NaN3. Similar results were observed in five of five islets. Arrows indicate the start of drug treatments.

 
Because the opening of KATP channels causes calcium channels to subsequently close, we hypothesized that pharmacologically blocking calcium should mimic insulin modulation of {Delta}{Psi}m. Exposure of ß-cells to the L-type channel blocker nifedipine (50 µmol/l) resulted in an insulin-like hyperpolarization of {Delta}{Psi}m in islets (10 ± 1% decrease; n = 5; P < 0.002) (Fig. 4B) and ß-cell clusters (15 ± 8% decrease; n = 20; P < 0.005), thus supporting our hypothesis. These results suggest that changes in {Delta}{Psi}m brought about by opening KATP channels or closing calcium channels are mediated by {Delta}{Psi}m by reducing [Ca2+]i.

From these observations, we hypothesized that increased [Ca2+]i would have the opposite effect on {Delta}{Psi}m; therefore, islets were treated with 30 mmol/l KCl to depolarize the plasma membrane. Because KATP channels on mitochondria can be the direct targets of KATP channel drugs (31,32), diazoxide was also included in this study to test whether the hyperpolarization of {Delta}{Psi}m by diazoxide was due to the opening of KATP channels on the plasma membrane (thus reducing calcium) or to direct effects on the mitochondria (by modulating mitochondrial enzymes or mitochondrial KATP channels). Figure 5 is representative of the depolarizing effect of 30 mmol/l KCl + 250 µmol/l diazoxide observed in six islets (mean depolarization 7 ± 2%). This result was consistent with actions of increased calcium influx and not of direct hyperpolarizing actions of diazoxide on mitochondria. We further demonstrated that insulin had no additional effect on {Delta}{Psi}m under these conditions. These findings support the interpretation that changes in {Delta}{Psi}m are a consequence of changes in plasma membrane potential and [Ca2+]i.



View larger version (18K):
[in this window]
[in a new window]
 
FIG. 5. Induced calcium influx depolarizes {Delta}{Psi}m and prevents insulin action. Plot of fluorescence versus time for a representative islet in 11.1 mmol/l glucose treated with 30 mmol/l KCl + 250 µmol/l diazoxide, 100 nmol/l insulin + 30 mmol/l KCl + 250 µmol/l diazoxide, and washed. Representative of effects on six islets.

 
To further demonstrate that KATP channel activation mediates the actions of insulin on mitochondria, insulin was applied to islets or ß-cell clusters after first blocking KATP channels with glyburide. By closing KATP channels before insulin treatment, the KATP-dependent pathway of insulin feedback onto mitochondria should have been blocked. As shown in Fig. 6, inhibiting KATP channels prevented the effects of insulin on Rh123 fluorescence in islets (n = 11) (Fig. 6A) and ß-cell clusters (n = 12) (Fig. 6B). The mean fractional change in fluorescence among control, glyburide, and glyburide plus insulin phases for islets did not significantly differ (P > 0.10 for each comparison). This confirmed that insulin effects on mitochondria are indeed mediated by changes in KATP channel activity. Similar results were obtained when ß-cell clusters were first exposed to nifedipine before insulin application. Among eight ß-cell clusters tested, a mean decrease in Rh123 fluorescence of 20 ± 5% occurred during nifedipine pretreatment, followed by little or no change in {Delta}{Psi}m during insulin plus nifedipine treatment.



View larger version (31K):
[in this window]
[in a new window]
 
FIG. 6. Pretreatment with glyburide prevents the action of insulin on {Delta}{Psi}m. Plot of fluorescence versus time for a representative islet (A) and a small cluster (B) in 11.1 mmol/l glucose treated with 100 nmol/l glyburide, 100 nmol/l glyburide + 100 nmol/l insulin, and then NaN3. Solid bars indicate duration of glyburide treatment, dotted lines indicate duration of insulin treatment, and arrows indicate the start of NaN3 treatment.

 
Effects of increasing endogenous insulin secretion on mitochondria.
Although the data presented thus far indicate that ß-cell exposure to exogenous insulin results in hyperpolarization of {Delta}{Psi}m, we have not addressed whether endogenously secreted insulin can have the same effect. The most direct way to measure effects of secreted insulin is to acutely block the insulin receptor; however, there are currently no commercially available insulin receptor antagonists. We instead attempted to potentiate insulin secretion by using forskolin, a direct activator of adenylate cyclase that acts to increase granule exocytosis without profoundly affecting electrical activity in elevated glucose (33). Forskolin treatment (4 µmol/l) induced an 8 ± 3% decrease in Rh123, consistent with the hypothesis that endogenous insulin can indeed alter {Delta}{Psi}m in a qualitatively similar fashion to exogenous insulin (n = 5 islets; P < 0.05) (Fig. 7). However, it should be noted that forskolin can increase calcium influx and alter other aspects of ß-cell function (34,35), which could in turn influence {Delta}{Psi}m independently of secreted insulin. Nevertheless, this is the strongest evidence thus far for an autocrine action of insulin on islet mitochondrial function.



View larger version (18K):
[in this window]
[in a new window]
 
FIG. 7. Forskolin, an activator of adenylate cyclase, mimics the effects of insulin. A: Plot of fluorescence versus time for an islet in 11.1 mmol/l glucose treated with 4 µmol/l forskolin, followed by NaN3. Arrows indicate the start of drug treatments. Representative of effects on five islets.

 
Time course of treatment effects on mitochondria.
To compare the time course of the agents studied, we compared their latencies under comparable experimental conditions (Fig. 8). The most rapidly acting agent was the mitochondrial poison NaN3, which depolarized {Delta}{Psi}m in <30 s. Modulators of ion channel activity were found to be slightly slower, followed by changes brought about by higher levels of glucose, and finally insulin. These latencies suggest that changes in plasma membrane ion channels are closely linked to changes in mitochondria, and that the relatively longer times observed for glucose, insulin, and forskolin may logically follow, given the presence of several intervening steps in signaling. Thus, for glucose, it is likely that its transport, phosphorylation, and subsequent tricarboxylic acid cycle activation (36,37) must occur before altering {Delta}{Psi}m. In the case of insulin, it is well known that its signal transduction pathway requires insulin receptor phosphorylation and subsequent phosphorylation of insulin receptor substrate proteins and PI 3-kinase activation, among other intermediate steps (38,39). Finally, forskolin must first stimulate cAMP production and downstream kinases before activation of the insulin receptor pathway can occur (34), thus explaining the long latency. The time course for mitochondrial effects is generally in agreement with the time of insulin-induced KATP channel opening (14) and the timing of the classic insulin receptor-mediated cascade in insulin target tissues as well as ß-cells (40,41).



View larger version (14K):
[in this window]
[in a new window]
 
FIG. 8. Latencies of treatments that affect mitochondrial membrane potential. The times shown correspond to the mean latency between the treatment first entering the recording chamber and the first detected change in fluorescence from islets.

 

    DISCUSSION
 TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, we found that mitochondrial membrane potential was depolarized by raising [Ca2+]i and hyperpolarized by decreasing calcium influx using the KATP channel opener diazoxide or the L-type calcium channel blocker nifedipine. Further, the application of exogenous insulin also hyperpolarized {Delta}{Psi}m, an effect that was abolished in the presence of the KATP channel blocker glyburide. This insulin effect is consistent with our earlier report that insulin opens KATP channels in the ß-cell plasma membrane, leading to hyperpolarization, a cessation of bursting activity, and reduced [Ca2+]i (14). The present results confirm that insulin receptor activation stimulates mitochondrial activity in islets and ß-cells by opening KATP channels and decreasing ß-cell [Ca2+]i.

The observation that mitochondrial activity is calcium dependent is consistent with the effects of changing [Ca2+]i on {Delta}{Psi}m in other reports using Rh123 (21,22) and in measures of the ATP/ADP ratio (42). This observation is also in line with studies showing that a rise in [Ca2+]i due to a burst of action potentials or brief exposures to elevated potassium can lead to KATP channel activation in ß-cells (10,43). Although the precise mechanism of [Ca2+]i-induced KATP channel activation is not known, it has been suggested that a decrease in mitochondrial respiration, reflected by a decrease in {Delta}{Psi}m, in turn decreases the ATP/ADP ratio sufficiently to cause KATP channels to open. Keizer and Magnus (23,24) proposed a mathematical model based on this idea, in which increased calcium influx resulted in reduced ATP production, and demonstrated that KATP channel activation due to a decreased ATP/ADP ratio was feasible as a repolarization mechanism during glucose-induced electrical bursting. Alternatively, changes in ß-cell [Ca2+]i could, in theory, modulate {Delta}{Psi}m by affecting ATP consumption rather than production. In this view, an increase in [Ca2+]i would result in increased ATP utilization via increased calcium ATPase activity to pump calcium, which would decrease {Delta}{Psi}m (4244). Either scenario would be consistent with KATP channel activation due to a drop in the ATP/ADP ratio.

Although we have previously shown that depolarization-induced calcium influx in ß-cells primarily activates another potassium channel, KCa,slow (11), on occasion we observed an additional current that is activated after a burst of action potential-like depolarizations, which could reflect KATP channel activation (P. Goforth, unpublished observations). Indirect calcium-dependent activation of KATP channels may thus result from increased [Ca2+]i acting on mitochondria or calcium ATPases, thus mediating a negative feedback loop to help repolarize ß-cells (10,45).

A second way in which a rise in [Ca2+]i could open KATP channels is by means of an exocytotic release of insulin. Secreted insulin would then activate the KATP channels of neighboring ß-cells through a PI 3-kinase-dependent pathway, contributing to their repolarization (14). Our present data suggest that the decrease in [Ca2+]i that would follow insulin-induced KATP channel activation could ultimately increase mitochondrial activity. Insulin signaling to the mitochondria could potentially mediate an increase in cellular ATP, which could contribute to a subsequent cycle of ß-cell depolarization and/or secretion. The closure of KATP channels by this action would occur on a time scale of tens to hundreds of seconds, as compared with the calcium-induced opening of KATP channels described earlier (<10 s), as evidenced by the much longer latency of insulin to mitochondrial changes compared with drugs that act directly on ion channels or mitochondria (Fig. 8). Although insulin is therefore unlikely to play a pacemaker role in islet burst firing patterns on the order of seconds, secreted insulin may instead act as a modulator of electrical activity on a minute-to-minute basis.

A general objection to the hypothesis that secreted insulin functionally modulates glucose-induced bursting via KATP channel activation is the observation that inhibiting islet insulin secretion by cooling islets from 37 to 20–27°C does not abolish glucose-induced bursting (46,47). This suggests that secreted insulin must therefore not be required for islet bursting to occur. However, when islets are cooled, there is a decrease in burst frequency and an increase in the fraction of time spent in the plateau or active phase of bursting. Although these changes may be due to the nonspecific effects of cooling, they would also, in fact, be expected if cooling inhibited KATP channel opening after the suppression of insulin secretion within the islet. We have quantitatively tested this hypothesis using a computer simulation in which the effect of cooling was modeled as an 8% reduction in maximal KATP conductance (R. Bertram and A. Sherman, unpublished observations) and found that indeed this resulted in qualitatively similar changes in bursting to those reported by Atwater et al. (46) and Debuyser et al. (47). Although this does not prove our hypothesis that secreted insulin activates KATP channels in mouse islets, these classic temperature experiments do not rule out the hypothesis.

In conclusion, our findings showed that insulin receptor activation by exogenous insulin can modulate metabolism in ß-cells, suggesting that released insulin might thus be coupled to the cellular ATP/ADP ratio to calibrate the energy needs of the ß-cell on a minute-to-minute basis. Further study will be required to determine if secreted insulin indeed plays a role in modulating mitochondrial, calcium, and/or electrical activities in islets. Alterations in these feedback processes may contribute to the dysfunctional insulin secretion, and especially abnormal insulin pulsatility, observed in patients with type 2 diabetes (48,49).


    ACKNOWLEDGMENTS
 
This study was supported by National Institutes of Health Grants DK-46409 (L.S.S.) and F32-DK-065462-01 (C.S.N.).

We thank Sophia Gruszecki and Heather Strange for assistance in tissue preparation, and Drs. Richard Bertram, Arthur Sherman, Paulette Goforth, Keith Tornheim, and Glenn VanTuyle for editorial comments and helpful discussions. We especially thank Drs. Sherman and Bertram for their generosity in modeling earlier studies on the effects of temperature on islet electrical activity and secretion. We also thank Todd Yaklin for electrical and communications assistance.

Address correspondence and reprint requests to Dr. Leslie S. Satin, Department of Pharmacology and Toxicology, Virginia Commonwealth University, P.O. Box 980524, Richmond, VA 23298. E-mail: lsatin{at}hsc.vcu.edu

Received for publication March 10, 2004 and accepted in revised form April 13, 2004

{Delta}{Psi}m, mitochondrial membrane potential; FCCP, fluoro-carbonyl cyanide phenylhydrazone; KATP channel, ATP-sensitive K+ channel; PI, phosphatidylinositol; Rh123, rhodamine 123; ROI, region of interest


    REFERENCES
 TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Dean PM, Matthews EK: Electrical activity in pancreatic islet cells. Nature219 :389 –390,1968[Medline]
  2. Ashcroft FM, Rorsman P: Electrophysiology of the pancreatic beta-cell. Prog Biophys Mol Biol54 :87 –143,1989[Medline]
  3. Hellerstrom C: Effects of carbohydrates on the oxygen consumption of isolated pancreatic islets of mice. Endocrinology81 :105 –112,1967[Medline]
  4. Hutton JC, Malaisse WJ: Dynamics of O2 consumption in rat pancreatic islets. Diabetologia18 :395 –405,1980[Medline]
  5. Duchen MR: Contributions of mitochondria to animal physiology: from homeostatic sensor to calcium signalling and cell death. J Physiol516 :1 –17,1999[Abstract/Free Full Text]
  6. Ashcroft FM, Harrison DE, Ashcroft SJ: Glucose induces closure of single potassium channels in isolated rat pancreatic beta-cells. Nature312 :446 –448,1984[Medline]
  7. Henquin JC, Jonas JC, Gilon P: Functional significance of Ca2+ oscillations in pancreatic beta cells. Diabetes Metab24 :30 –36,1998[Medline]
  8. Rorsman P, Bokvist K, Ammala C, Eliasson L, Renstrom E, Gabel J: Ion channels, electrical activity and insulin secretion. Diabetes Metab20 :138 –145,1994
  9. Gopel SO, Kanno T, Barg S, Eliasson L, Galvanovskis J, Renstrom E, Rorsman P: Activation of Ca(2+)-dependent K(+) channels contributes to rhythmic firing of action potentials in mouse pancreatic beta cells. J Gen Physiol114 :759 –770,1999[Abstract/Free Full Text]
  10. Kanno T, Rorsman P, Gopel SO: Glucose-dependent regulation of rhythmic action potential firing in pancreatic beta-cells by K(ATP)-channel modulation. J Physiol545 :501 –507,2002[Abstract/Free Full Text]
  11. Goforth PB, Bertram R, Khan FA, Zhang M, Sherman A, Satin LS: Calcium-activated K+ channels of mouse beta-cells are controlled by both store and cytoplasmic Ca2+: experimental and theoretical studies. J Gen Physiol120 :307 –322,2002[Abstract/Free Full Text]
  12. Kahn CR, Goldfine AB: Molecular determinants of insulin action. J Diabetes Complications7 :92 –105,1993[Medline]
  13. Leibiger IB, Leibiger B, Berggren PO: Insulin feedback action on pancreatic beta-cell function. FEBS Lett532 :1 –6,2002
  14. Khan FA, Goforth PB, Zhang M, Satin LS: Insulin activates ATP-sensitive K+ channels in pancreatic ß-cells through a phosphatidylinositol 3-kinase-dependent pathway. Diabetes50 :2192 –2198,2001[Abstract/Free Full Text]
  15. Aspinwall CA, Lakey JR, Kennedy RT: Insulin-stimulated insulin secretion in single pancreatic beta cells. J Biol Chem274 :6360 –6365,1999[Abstract/Free Full Text]
  16. Aspinwall CA, Qian WJ, Roper MG, Kulkarni RN, Kahn CR, Kennedy RT: Roles of insulin receptor substrate-1, phosphatidylinositol 3-kinase, and release of intracellular Ca2+ stores in insulin-stimulated insulin secretion in beta -cells. J Biol Chem275 :22331 –22338,2000[Abstract/Free Full Text]
  17. Persaud SJ, Asare-Anane H, Jones PM: Insulin receptor activation inhibits insulin secretion from human islets of Langerhans. FEBS Lett510 :225 –228,2002[Medline]
  18. Roper MG, Qian WJ, Zhang BB, Kulkarni RN, Kahn CR, Kennedy RT: Effect of the insulin mimetic L-783,281 on intracellular Ca2+ and insulin secretion from pancreatic ß-cells. Diabetes51 (Suppl. 1) :S43 –S49,2002
  19. Westerlund J, Wolf BA, Bergsten P: Glucose-dependent promotion of insulin release from mouse pancreatic islets by the insulin-mimetic compound L-783,281. Diabetes51 (Suppl. 1) :S50 –S52,2002
  20. Duchen MR, Smith PA, Ashcroft FM: Substrate-dependent changes in mitochondrial function, intracellular free calcium concentration and membrane channels in pancreatic beta-cells. Biochem J294 :35 –42,1993[Medline]
  21. Krippeit-Drews P, Dufer M, Drews G: Parallel oscillations of intracellular calcium activity and mitochondrial membrane potential in mouse pancreatic B-cells. Biochem Biophys Res Commun267 :179 –183,2000[Medline]
  22. Kindmark H, Kohler M, Brown G, Branstrom R, Larsson O, Berggren PO: Glucose-induced oscillations in cytoplasmic free Ca2+ concentration precede oscillations in mitochondrial membrane potential in the pancreatic beta-cell. J Biol Chem276 :34530 –34536,2001[Abstract/Free Full Text]
  23. Magnus G, Keizer J: Model of beta-cell mitochondrial calcium handling and electrical activity. I. Cytoplasmic variables. Am J Physiol274 :C1158 –C1173,1998
  24. Magnus G, Keizer J: Model of beta-cell mitochondrial calcium handling and electrical activity. II. Mitochondrial variables. Am J Physiol274 :C1174 –C1184,1998
  25. Hopkins WF, Satin LS, Cook DL: Inactivation kinetics and pharmacology distinguish two calcium currents in mouse pancreatic B-cells. J Membr Biol119 :229 –239,1991[Medline]
  26. Lernmark A: The preparation of, and studies on, free cell suspensions from mouse pancreatic islets. Diabetologia10 :431 –438,1974[Medline]
  27. Zhang M, Goforth P, Bertram R, Sherman A, Satin L: The Ca(2+) dynamics of isolated mouse beta-cells and islets: implications for mathematical models. Biophys J84 :2852 –2870,2003[Abstract/Free Full Text]
  28. Ronot X, Benel L, Adolphe M, Mounolou JC: Mitochondrial analysis in living cells: the use of rhodamine 123 and flow cytometry. Biol Cell57 :1 –7,1986[Medline]
  29. Chen LB: Mitochondrial membrane potential in living cells. Annu Rev Cell Biol4 :155 –181,1988[Medline]
  30. Cook DL, Taborsky GJ Jr: ß-Cell Function and Insulin Secretion. New York, Elsevier, 1990
  31. Liu Y, Sato T, Seharaseyon J, Szewczyk A, O’Rourke B, Marban E: Mitochondrial ATP-dependent potassium channels: viable candidate effectors of ischemic preconditioning. Ann N Y Acad Sci874 :27 –37,1999[Abstract/Free Full Text]
  32. Grimmsmann T, Rustenbeck I: Direct effects of diazoxide on mitochondria in pancreatic B-cells and on isolated liver mitochondria. Br J Pharmacol123 :781 –788,1998[Medline]
  33. Henquin JC, Meissner HP: The ionic, electrical, and secretory effects of endogenous cyclic adenosine monophosphate in mouse pancreatic B cells: studies with forskolin. Endocrinology115 :1125 –1134,1984[Abstract]
  34. Ammala C, Ashcroft FM, Rorsman P: Calcium-independent potentiation of insulin release by cyclic AMP in single beta-cells. Nature363 :356 –358,1993[Medline]
  35. Yaekura K, Kakei M, Yada T: cAMP-signaling pathway acts in selective synergism with glucose or tolbutamide to increase cytosolic Ca2+ in rat pancreatic ß-cells. Diabetes45 :295 –301,1996[Abstract]
  36. Wollheim CB: Beta-cell mitochondria in the regulation of insulin secretion: a new culprit in type II diabetes. Diabetologia43 :265 –277,2000[Medline]
  37. Matschinsky FM: Banting Lecture 1995: A lesson in metabolic regulation inspired by the glucokinase glucose sensor paradigm. Diabetes45 :223 –241,1996[Abstract]
  38. Rhodes CJ, White MF: Molecular insights into insulin action and secretion. Eur J Clin Invest32 (Suppl. 3) :3 –13,2002[Medline]
  39. Borge PD, Moibi J, Greene SR, Trucco M, Young RA, Gao Z, Wolf BA: Insulin receptor signaling and sarco/endoplasmic reticulum calcium ATPase in beta-cells. Diabetes51 (Suppl. 3) :S427 –S433,2002
  40. Shemer J, Adamo M, Wilson GL, Heffez D, Zick Y, LeRoith D: Insulin and insulin-like growth factor-I stimulate a common endogenous phosphoprotein substrate (pp185) in intact neuroblastoma cells. J Biol Chem262 :15476 –15482,1987[Abstract/Free Full Text]
  41. White MF, Maron R, Kahn CR: Insulin rapidly stimulates tyrosine phosphorylation of a Mr-185,000 protein in intact cells. Nature318 :183 –186,1985[Medline]
  42. Detimary P, Gilon P, Henquin JC: Interplay between cytoplasmic Ca2+ and the ATP/ADP ratio: a feedback control mechanism in mouse pancreatic islets. Biochem J333 :269 –274,1998[Medline]
  43. Rolland JF, Henquin JC, Gilon P: Feedback control of the ATP-sensitive K+ current by cytosolic Ca2+ contributes to oscillations of the membrane potential in pancreatic ß-cells. Diabetes51 :376 –384,2002[Abstract/Free Full Text]
  44. Capito K, Formby B, Hedeskov CJ: Ca-ATPases in pancreatic islets. Horm Metab Res Suppl.10 :50 –55,1980[Medline]
  45. Satin LS, Tavalin SJ, Smolen PD: Inactivation of HIT cell Ca2+ current by a simulated burst of Ca2+ action potentials. Biophys J66 :141 –148,1994[Abstract]
  46. Atwater I, Goncalves A, Herchuelz A, Lebrun P, Malaisse WJ, Rojas E, Scott A: Cooling dissociates glucose-induced insulin release from electrical activity and cation fluxes in rodent pancreatic islets. J Physiol348 :615 –627,1984[Abstract]
  47. Debuyser A, Drews G, Henquin JC: The influence of temperature on the effects of acetylcholine and adrenaline on the membrane potential and 86Rb efflux in mouse pancreatic B-cells. Exp Physiol76 :553 –559,1991[Abstract/Free Full Text]
  48. Lang DA, Matthews DR, Burnett M, Turner RC: Brief, irregular oscillations of basal plasma insulin and glucose concentrations in diabetic man. Diabetes30 :435 –439,1981[Abstract]
  49. Polonsky KS, Given BD, Hirsch LJ, Tillil H, Shapiro ET, Beebe C, Frank BH, Galloway JA, Van Cauter E: Abnormal patterns of insulin secretion in non-insulin-dependent diabetes mellitus. N Engl J Med318 :1231 –1239,1988[Abstract]