Critical role of gap junction coupled KATP channel activity for regulated insulin secretion

Abstract

Pancreatic beta-cells secrete insulin in response to closure of ATP-sensitive K+ (KATP) channels, which causes membrane depolarization and a concomitant rise in intracellular Ca2+ (Cai). In intact islets, beta-cells are coupled by gap junctions, which are proposed to synchronize electrical activity and Cai oscillations after exposure to stimulatory glucose (>7 mM). To determine the significance of this coupling in regulating insulin secretion, we examined islets and beta-cells from transgenic mice that express zero functional KATP channels in approximately 70% of their beta-cells, but normal KATP channel density in the remainder. We found that KATP channel activity from approximately 30% of the beta-cells is sufficient to maintain strong glucose dependence of metabolism, Cai, membrane potential, and insulin secretion from intact islets, but that glucose dependence is lost in isolated transgenic cells. Further, inhibition of gap junctions caused loss of glucose sensitivity specifically in transgenic islets. These data demonstrate a critical role of gap junctional coupling of KATP channel activity in control of membrane potential across the islet. Control via coupling lessens the effects of cell-cell variation and provides resistance to defects in excitability that would otherwise lead to a profound diabetic state, such as occurs in persistent neonatal diabetes mellitus.

Figure 1. Whole-Islet Ca_i and Insulin Responses

(A) Fluorescence (488-nm excitation and 620–680-nm emission band-pass filter) image of a Fura Red–labeled Kir6.2[AAA] islet. As with labeling of intact WT islets, the fluorescence intensity of Kir6.2[AAA] islets shows increased intensity in the nucleus and relatively little intensity variance across the tissue. (B) Fluorescence (488-nm excitation and a 540/20-nm band-pass filter) image of the same islet. Note that in (A) the islet is uniformly labeled and there is no indication of GFP fluorescence bleed-through. In (B), 16 regions that were either positive (eight regions) or negative (eight regions) for Kir6.2[AAA]-GFP transgene expression, are outlined in red. (C) The Ca_i responses in 6 mM glucose from groups of cells (outlined in [B]) that are either Kir6.2[AAA]-positive (filled black circle) or -negative (open red circle) demonstrated identical oscillatory behaviors, indicating that both are in synchrony. (D) A comparison of glucose-stimulated Ca_i responses measured using Fluo-4 from WT (left) and Kir6.2[AAA] (right) transgenic islets. Islets were exposed to the indicated glucose concentrations for greater than 10 min by changing the reagent well solution. Images were then collected at 1.5-s intervals over a period of 125 s. In contrast to the traces found in (C), each trace in each panel (solid, dot, and dashed) represents a whole single islet. These islet traces are done to show the dose -response of individual islets rather than synchrony across the tissue. WT islets showed no oscillatory behavior until treated to 8 mM glucose or greater (responses of three different islets are shown). Kir6.2[AAA] islets were also quiescent at low glucose concentrations; however, these islets showed Ca_i oscillations at 6 mM glucose or above (n = 5 islets). (E) Glucose-stimulated insulin secretion from WT and Kir6.2[AAA] islets (mean ± SEM, n = 17 and 15, respectively). Islets were also exposed to 16.7 mM glucose + glibenclamide (Glib) to achieve maximum possible insulin secretion. The difference in means ± 95% confidence interval is shown in Figure S2A.

Figure 2. Dispersed Cell Ca_i and Insulin Responses

(A) Dispersed Kir6.2[AAA] cells labeled with Fura-2 (340-nm image). These are mainly β-cells, with the expected minority of non–β-cells. (B) GFP image of the same field of view of (E). A fraction of cells (four of eight) are GFP positive. (C) The glucose-stimulated Ca_i response of dispersed cells from WT (black square) and Kir6.2[AAA] (red circle) islets measured using Fura-2 fluorescence microscopy. Kir6.2[AAA] cell preparations were a combination of GFP-labeled and non-labeled cells. The percentage of Ca_i-active cells was measured in 2, 6, and 10 mM glucose on three separate days in WT and Kir6.2[AAA] dispersed cells, respectively (mean ± standard error of the mean [SEM], n = 3). The WT cells show a strong glucose dependence, whereas the Kir6.2[AAA] cells show similar numbers of Ca_i active cells at all glucose concentrations. (D) Insulin secretion from dispersed WT and Kir6.2[AAA] cells (mean ± SEM, n = 4) from static 1-h incubations. This incubation time shows K_ATP channel–dependent insulin secretion, but does not encompass K_ATP channel–independent insulin secretion shown to occur in knockout islets beyond 3 h of incubation [13]. The difference in means ± 95% confidence interval for (C) and (D) are shown in Figure S2B and S2C, respectively).

Figure 3. Membrane Potential and Metabolic Response of Kir6.2[AAA] Islets and Cells

(A) The membrane potential of GFP-positive and -negative cells within a Kir6.2[AAA] islet. Islets were bathed in 500 nM of membrane potential dye (DiSBAC₂(3)) (n = 4). This oxanol dye enters cells in a membrane potential–dependent manner with increasing intensity as a cell becomes depolarized, and is spectrally distinct from GFP. The GFP-positive and -negative cells showed similar intensity (membrane potential) at 2 and 15 mM glucose stimulation, and had responses consistent with the responses observed in WT islets (data not shown). (B) The NAD(P)H glucose-dose response of WT (n = 9) and Kir6.2[AAA]-GFP (n = 10) islets. Device-trapped islets were treated to the indicated glucose concentrations for more than 5 min prior to image collection. The normalized change in NAD(P)H fluorescence intensity (ΔF/ΔF_T) is plotted versus glucose stimulation. The black and red dotted lines are the data fitted to a Hill equation with Km values of 9.0 ± 0.7 and 7.6 ± 0.7 mM for the WT and Kir6.2[AAA] islets, respectively. (C) The NAD(P)H responses of dispersed GFP-negative and -positive Kir6.2[AAA] cells. The cells were incubated in 2 mM glucose (open bars) prior to incubation for 5–10 min with 15 mM glucose (closed bars). GFP fluorescence was used for the distinction as GFP negative or positive. NAD(P)H-intensity measures were taken from cells in four fields of view from nine separate dishes of Kir6.2[AAA] dispersed cells (n = 9).

Figure 4. The Ca_i Responses of WT and Kir6.2[AAA] Islets in the Presence of αGA, a Gap Junction Inhibitor

(A) Fluo-4, AM–labeled WT and Kir6.2[AAA] islets in 2 mM (n = 4 and 5, respectively) or 4 mM (n = 5 and 6, respectively) glucose (glc) were exposed to 0, 10, or 50 μM αGA. The islets were imaged to provide relative Ca_i traces from the Fluo-4 response, and each panel shows the response in four different regions of a single islet. The representative traces shown are from a WT islet in 2 mM glucose with 50 μM αGA (WT: 2 mM glc + 50 μM αGA, n = 4), a Kir6.2[AAA] islet in 2mM glucose with 50 μM αGA (Kir6.2[AAA]: 2 mM glc + 50 μM αGA, n = 5), and a WT islet in 8 mM glucose (WT: 8 mM glc). The black bar in each panel indicates 30 s. (B) The oscillating (Ca_i active) area and total islet area were measured in each islet under each condition. The data are expressed as the fraction of oscillating area, calculated as the oscillating area divided by the total area (mean ± SEM) from WT islets in 2mM (n = 4) and 4 mM glucose (n = 5), and Kir6.2[AAA] islets in 2 mM (n = 5) and 4 mM glucose (n = 6).

Figure 5. Schematic Model of the Responses of β-cells in the Islet and Dispersed β-cells from Kir6.2[AAA] Transgenic Mice in Both Low (<6 mM) and High (>6 mM) Glucose Concentrations

(A) and (C) show the β-cell responses in the islet, and (B) and (D) show the dispersed β-cell responses. (A) A WT β-cell has normal K_ATP channel activity (white cell), which maintains plasma membrane potential (blue membrane). The GFP-tagged Kir6.2[AAA] cell (green cell) would normally be depolarized; however, the K_ATP current from the normal cell is coupled through gap junctions to maintain membrane polarity (blue membrane). (B) In the absence of coupling through gap junctions, the cell with normal K_ATP channel activity (white cell) maintains polarity (blue membrane), but the Kir6.2[AAA] cell depolarizes (red membrane) resulting in Ca_i influx through voltage-gated channels. (C) The addition of glucose raises the ATP/ADP ratio, which closes the K_ATP channel in the normal cell. The loss of this K⁺ current results in membrane depolarization (red membrane) in both cells, and Ca_i influx through voltage-gated Ca²⁺ channels. (D) In the dispersed cells, this rise in ATP/ADP ratio results in depolarization and Ca_i influx occurring in both cells.