Critical Role of Gap Junction Coupled KATP Channel Activity for Regulated Insulin Secretion

1β-cells within the intact islet of Langerhans exhibit synchronous glucose-dependent bursts of electrical activity. Dissociated β-cells also show increased electrical activity at elevated glucose concentrations, but this activity is variable from cell to cell, with some cells being electrically “silent” and others continuously “bursting” at any given glucose concentration. Synchronization of electrical activity via gap junctions has long been argued as essential for normal glucose-dependent insulin secretion in the intact islet. In particular, islets devoid of gap junction–forming Connexin 36 (Cx36) exhibit limited synchronization of glucose-stimulated intracellular Ca2+ (Cai). To test the extent and role of this coupling in the islet electrical response, it would be desirable to examine within the intact islet the behavior of cells that would be silent or bursting as isolated cells. To date, it has not been possible to know which cell is which in the setting of the intact islet. However, we have generated transgenic mice containing two electrically distinct types of β-cells that uniquely permit us to track the behavior of specific individual cells within the intact islet. As previously shown, these mice express a β-cell–specific, dominant-negative Kir6.2[AAA]-GFP transgene, in which the pore-forming subunit of the ATP-sensitive K+ (KATP) channel is rendered nonfunctional. Mosaic expression of the Kir6.2[AAA]-GFP transgene results in a high-level expression in approximately 70% of the β-cells, and these expressing cells are distributed randomly throughout the islet. Thus, KATP channels are functionally “knocked out” of 70% of cells, yet the intact islets still show glucose-dependent insulin secretion, with an even steeper concentration dependence of insulin secretion than wild-type (WT) islets.

2To examine the behavior of individual cells within the intact islet, we developed a microfluidic device to hold pancreatic islets stationary while under continuous fluid flow (see Figure S1). We compared the glucose-dependent Cai responses of WT and Kir6.2[AAA] transgenic islets (Figure 1). Near the periphery of the islet, fluorescence from a Ca2+ sensor (Fura Red) was relatively uniform in all cells (Figure 1A), and spectrally separable from green fluorescent protein (GFP) fluorescence (Figure 1B). Both the GFP-positive and -negative populations of β-cells (KATP-absent and KATP-present cells, respectively) within a Kir6.2[AAA] transgenic islet showed identical Cai-responses (Figure 1C). WT islets were “Cai inactive” across the tissue below 8 mM glucose (i.e., Cai remained low and no oscillations were observed; Figure 1D), consistent with previous studies. Kir6.2[AAA] islets were also completely Cai inactive across the tissue at low glucose concentrations (2 and 4 mM), but they exhibited Cai activity at slightly lower glucose levels than WT (6 mM). This leftward shift in glucose dependence of Cai in Kir6.2[AAA] islets is consistent with the leftward shift in whole-islet glucose-dependent insulin secretion (Figure 1E). Kir6.2−/− and SUR1−/− mice, which completely lack KATP channels, show essentially no glucose dependence of electrical activity, Cai, or insulin secretion. Thus, the near-normal glucose response of Cai (Figure 1D) and insulin secretion (Figure 1E) by Kir6.2[AAA] islets are initially somewhat surprising, since KATP is functionally knocked out in approximately 70% of the β-cells. Paracrine signaling could influence the electrical synchronization between islet cells, for instance by δ-cell secretion of somatostatin. However, these data suggest that KATP channel activity in the residual 30% of cells is coupled through gap junction conductance that is strong enough to hyperpolarize the knockout cells. This suggestion, in turn, predicts that glucose dependence will be lost in dispersed Kir6.2[AAA]-GFP cells. Accordingly, we measured the glucose-stimulated Cai response and insulin secretion from dispersed cell preparations (Figure 2). Image fields of isolated Kir6.2[AAA] β-cells show uniform loading of the Ca2+ indicator dye (Fura-2; Figure 2A), and no overlap with GFP fluorescence (Figure 2B). Both the Cai response and insulin secretion were essentially glucose independent in dispersed Kir6.2[AAA] cells (Figure 2C and 2D). This is in striking contrast to the Cai response and insulin secretion from dispersed WT cells which are strongly glucose dependent, but is consistent with previous results from SUR knockout mice.

3It was previously shown that dispersed GFP-positive Kir6.2[AAA] β-cells are continuously depolarized in nonstimulatory glucose concentrations. To examine whether cell–cell coupling reconstitutes glucose-dependent membrane potentials in intact islets, we imaged intact islets using a membrane potential–sensitive dye (Figure 3A). At 2 mM glucose, a few cells on the islet periphery were noticeably more depolarized than others. These bright cells were never seen to co-localize with GFP fluorescence (not shown), and were likely α-cells. The rest of the islet cells showed relatively dim uniform fluorescence, consistent with uniformly polarized membranes, and all of these cells brightened (depolarized) upon glucose stimulation. GFP-expressing and non-expressing cells had indistinguishable membrane polarization (Figure 3A). Thus, β-cells across the Kir6.2[AAA] islet have similar resting membrane polarization, and all depolarize with increased glucose concentrations.

4In contrast to electrical activity, we have not observed metabolic coupling of β-cells, although there may be feedback between electrical activity and metabolic response. The Kir6.2[AAA] islets provide a unique opportunity to examine this potential feedback. We used intrinsic NAD(P)H fluorescence as a measure of cellular metabolic status. In comparison to WT, Kir6.2[AAA] islets showed no difference in baseline NAD(P)H intensity, but showed a slight (∼1 mM) leftward shift in the (glucose) response (Figure 3B). This shift is comparable to that of glucose-stimulated Cai response and insulin secretion (see Figure 1D and 1E), suggesting a feedback modulation of metabolism during electrical activity. Such feedback could result from Cai influx or insulin signaling since both likely modulate β-cell metabolism and occur when the tissue is electrically active. We further compared the baseline and stimulated responses of dispersed Kir6.2[AAA] cells (Figure 3C). Baseline NAD(P)H intensity was significantly elevated in GFP-positive cells (1.3-fold vs. GFP negative at 2 mM glucose), but was similar upon glucose stimulation. The elevated basal metabolism in GFP-positive cells with no difference in glucose-stimulated metabolism is again consistent with a modulating effect of Cai or insulin. Thus, even though metabolism is not directly coupled between cells in the islet, entrained electrical activity and shifted Cai responses can influence metabolic response.

5The above results suggest a model whereby the Kir6.2[AAA] islet response is due to coupling of KATP channel activity, presumably through gap junctions. This model predicts that inhibition of gap junctions would recapitulate the uncoupled Cai activity found in dispersed Kir6.2[AAA] cells. Thus, we examined glucose-stimulated Cai responses in the presence of a gap junction inhibitor, 18α-glycyrrhetinic acid (αGA). Cai measurements of WT islets (Figure 4A) showed no β-cell oscillations at sub-threshold glucose levels in the presence or absence of either 10 or 50 μM αGA. In contrast, spontaneous Cai oscillations were observed in the presence of 50 μM αGA in four of five Kir6.2[AAA] islets at 2 mM glucose, and in six of six Kir6.2[AAA] islets at 4 mM glucose (Figure 4B, Videos S1–S3). Unlike the synchronous oscillations across the entire islet that are seen in the absence of αGA (Figure 4A, bottom trace, and Figure 1C), these oscillations occurred asynchronously in small groupings of cells (Figure 4A, middle trace). The resulting asynchronous Cai activity in portions of Kir6.2[AAA] islets is consistent with partial decoupling of the KATP channel activity by the inhibition of gap junction conductance.

6The data presented here indicate that glucose control of membrane potential, Cai, and insulin secretion all depend critically on the gap junctional coupling of KATP channel activity across neighboring cells within the islet (Figure 5). It has previously been shown that knockout of Cx36 in islets results in reduction of glucose-stimulated electrical synchrony. Our results provide additional experimental evidence for gap junctional coupling between islet β-cells. Furthermore, our results demonstrate the mechanism by which the coupling of KATP channel activity via gap junctions results in near-normal glucose-dependent electrical and Cai responses in the intact islet, even when a majority of β-cells are essentially glucose insensitive. More specifically, this coupling allows β-cells in low glucose with normal KATP channel activity to clamp the membrane potential of neighboring cells. The electrical properties of isolated β-cells are very variable. This variability along with our results suggests that the rise in basal insulin secretion from Cx36 knockout islets is due to an inability of quiescent cells to clamp the membrane potential of other β-cells with low stimulation threshold. This may also explain an evolutionary drive to organize these secretory cells into a unique electrical syncytium. Such a mechanism provides a protective effect against hypoglycemia if individual cell properties change under pathological or physiological stimuli. Factors that reduce KATP channel density, such as channel subunit trafficking mutants, could do so to the point where individual cells become glucose insensitive. However, the remaining glucose-sensitive cells in the islet with sufficient residual KATP channel activity will maintain a glucose response of the whole islet. Such cell coupling would help resist the development of persistent hypoglycemic hyperinsulinemia (PHHI), unless compounded by additional defects, as may be the case in human PHHI.

7Our results also have implications for the understanding of diabetic phenotypes resulting from KATP channel gain of function or from other causes of hypoexcitability. It is now clear that overactive KATP channels cause neonatal diabetes due to inexcitability of β-cells and failure to secrete insulin. Our data demonstrate that cell–cell coupling can ensure maintained hyperpolarization in all cells if KATP channel activity in just a few reaches a threshold of inexcitability. However, this also suggests that the domination of KATP channel strength on neighboring cell membrane potentials, may permit elevated KATP channel activity in only a few β-cells to clamp the remainder at a hyperpolarized potential, thereby stopping secretion. Such a mechanism may underlie the profound diabetic state in persistent neonatal diabetes mellitus. Equivalent gain-of-function model systems and the experimental approach that we describe here should allow a critical test of the syncytial involvement in these types of neonatal diabetes.

8Islets were isolated as previously described and maintained in complete RPMI medium 1640 containing 10% fetal bovine serum and 11 mM glucose at 37 °C under 5% humidified CO2 for 24–72 h. Islets were dispersed to single cells as described previously. Insulin release was measured from pancreatic islets (ten per well) or dispersed cells (equivalent to 15 islets per well) in static 1-h incubations as described previously.

9Devices were fabricated using the elastomer polydimethylsiloxane (PDMS) as described elsewhere (see Figure S1 for details of fabrication and use). Islets were labeled with 4 μM of either Fluo-4, acetoxymethyl (AM) or Fura Red, AM at room temperature for 1 to 2 h in imaging buffer (125mM NaCl, 5.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 10 mM HEPES, 2 mM glucose, and 0.1% bovine serum albumin [pH 7.4]). One- and two-photon microscopy was performed on a LSM 510 microscope with a 20 × 0.75 NA Fluar objective lens (Carl Zeiss, Thornwood, NewYork, United States). The device was held on the microscope in a humidified temperature controlled stage (Carl Zeiss) for imaging at 37 °C. Fluo-4 (Molecular Probes, Eugene, Oregon, United States) was imaged using the 488-nm laser line and the long-pass 505-nm emission filter. NAD(P)H intensity was imaged with a 710-nm mode-locked Ti:Saph laser (∼3.5 mW at the sample) and fluorescence collection through a non-descanned detector with a custom 380- to 550-nm filter (Chroma, Rockingham, Vermont, United States). We collected four focal planes separated by 3-μm intervals. Fura Red (Molecular Probes) was imaged using the 488-nm laser line and a band-pass filter (620–680 nm). The GFP label of Kir6.2[AAA] islets was imaged using the 488-nm laser and band-pass (540/20) filter, with no noticeable bleed-through of the Fura Red signal. Membrane potential was measured with bis-(1,3-diethylthiobarbituric acid)trimethine oxonol (DiSBAC2(3); Molecular Probes). This dye accumulates in the cells in a Nernst-dependent manner. We flowed the indicated solutions with 500 nM of DiSBAC2(3). These solutions were allowed to equilibrate for approximately 30 min to allow dye penetration before imaging. DiSBAC2(3) images were collected by 543-nm excitation and a long-pass 560-nm filter. GFP fluorescence in these same islets was imaged separately using 488-nm excitation and a 500–530-nm band-pass filter.

10Prior to imaging, cells were labeled with 2 μM Fura-2, AM (Molecular Probes) for 30 min at room temperature and washed with imaging buffer containing 2 mM glucose. Imaging was done on a TE300 Eclipse microscope (Nikon, Melville, New York, United States) using the 40 × 1.3 NA DIC PlanFluor lens, side-mounted CoolSnap HQ camera (Roper Scientific, Tucson, Arizona, United States), and Fura-2 filter set (Chroma). The cells were placed on a stage enclosed in Plexiglas and kept in humidified air at 37 °C. The system was controlled with Metamorph 6.0 (Universal Imaging, Downington, Pennsylvania, United States). Image pairs were collected at 2-s intervals for 2 min after incubation for at least 10 min in 2, 6, and 10 mM glucose.

11Images were analyzed with MetaMorph 5.0 (Universal Imaging). For Fluo-4 analysis, islets/regions of islets were outlined and their intensities versus time were plotted. From these plots, the relative intensity change (F/F0) and frequencies were calculated. For Fura-2 analysis, individual cells were outlined for calculation of the background corrected 340:380-nm intensity ratio. Cells were categorized as “calcium active” if they had a ratio that was either more than two standard deviations larger than that observed for WT cells in 2 mM glucose or demonstrated a change of more than one standard deviation change during the 2-min observation period.