Expression of Kir6.2-V59M in β-cells rapidly induces diabetes

We generated an inducible mouse model selectively expressing a gain-of-function K ATP channel mutation (Kir6.2-V59M) in pancreatic β-cells (βV59M mice). Expression was induced at 12 weeks of age by tamoxifen injection. This resulted in a rapid rise in the blood glucose concentration of free-fed mice that exceeded 20 mM within 2 days and was sustained throughout the next 4 weeks (Fig. 1a). Free-fed (Supplementary Fig. 1a) and fasted (Supplementary Fig. 1b) plasma insulin levels were significantly reduced (~60%) by Kir6.2-V59M gene expression. Plasma glucagon levels did not change (Supplementary Fig. 1c,d).

Figure 1: Gene induction results in rapid diabetes that is normalized by insulin and SU therapy. (a) Blood glucose levels for 12-week-old βV59M (open circle, black star) and control (black circle, n=41) mice. Mice were injected with tamoxifen (Tx) as indicated by the arrow to induce Kir6.2-V59M expression. Some Tx-injected mice were subsequently implanted with a subcutaneous slow-release placebo pellet at time zero (black star, n=35), whereas others were not (open circle, n=31). (b,c) Blood glucose levels measured in βV59M mice injected with Tx (arrow) and subsequently implanted (arrow) with an insulin pellet (b; open square, n=6) or glibenclamide pellet (c; open triangle, n=19). Control mice (black circle) and Tx-induced untreated βV59M mice (open circle) are the same data as in Fig. 1a. (d) βV59M mice injected with Tx (arrow) and subsequently implanted (arrow) with a glibenclamide pellet (Glib) after 4 weeks of diabetes (open diamond, n=6). Control littermates (black circle, n=6) were sham injected with Tx. Data are mean values±s.e.m. Full size image

We examined the effects of insulin or glibenclamide therapy using slow-release pellets implanted subcutaneously once blood glucose had risen above 20 mM. Free-fed blood glucose levels were not significantly different in βV59M mice implanted with a placebo pellet (containing no drug) from those that did not receive a pellet (Fig. 1a).

Implantation of an insulin pellet (releasing 0.2–0.3U per day) caused a rapid fall in blood glucose from 20.3±1.4 mM (n=6) on the day of implantation to 8.2±3.2 mM 2 days later (Fig. 1b). However, insulin therapy failed to maintain normoglycaemia consistently throughout the 4-week period, and sporadic episodes of hypoglycaemia (<2 mM) and hyperglycaemia (>20 mM) were common.

Glycaemic control was greatly improved in mice treated with glibenclamide pellets (Fig. 1c). However, the dose needed to control the blood glucose level varied with the duration of hyperglycaemia. When glibenclamide was administered immediately following a rise in blood glucose to >20 mM, a dose of 17 mg kg−1 per day was sufficient to produce normoglycaemia (Fig. 1c), whereas a dose of 34–95 mg kg−1 per day was necessary to normalize glycaemia in mice that had been diabetic for 4 weeks (Fig. 1d).

Glibenclamide therapy caused a rapid fall in free-fed blood glucose: 2 days after drug implantation, glucose concentrations were 4.6±0.2 mM (n=19; Fig. 1c) in mice previously exposed to 24 h of hyperglycaemia and 4.6±0.4 mM (n=6; Fig. 1d) in those exposed to 4 weeks of hyperglycaemia. These values are close to those measured in the same animals before gene induction (5.6±0.1 mM; n=25) or in control mice (5.9±0.2 mM; n=41). Blood glucose levels were stable throughout the 4 weeks of glibenclamide treatment.

Effect of long-term hyperglycaemia on islet morphology

Hyperglycaemia for 4 weeks led to marked changes in islet morphology. There was a dramatic decrease in insulin-positive (ins+) cells and a concomitant increase in glucagon-positive (glu+) cells, which were no longer confined to the mantle but populated the core of the islet (compare Fig. 2c,d with Fig. 2a,b). Quantitative analysis revealed a marked reduction (~70%) in the area of the islet staining for insulin and an equivalent increase in the area staining for glucagon (Fig. 3a,b). A reduction in the percentage area of the pancreas staining for insulin (a surrogate for β-cell mass) and an increase in the area staining for glucagon was also observed (Fig. 3c,d). Chronic hyperglycaemia did not produce a statistically significant change in the percentage area of the pancreas occupied by islets (Supplementary Fig. 2a) or in islet density (islet number per cm2 pancreas) (Supplementary Fig. 2b,c).

Figure 2: Chronic hyperglycaemia alters insulin and glucagon immunostaining in pancreatic islets. Representative serial sections of mouse pancreas immunostained for insulin (left) or glucagon (right) using DAB (brown). Control mouse pancreas (a,b). βV59M mouse pancreas 4 weeks after implantation with a placebo (c,d), insulin (e,f), or glibenclamide (g,h) pellet. (i,j) Islets from βV59M mice exposed to 4 weeks of hyperglycaemia and then 4 weeks of glibenclamide therapy (n=4). Results are representative of four (a–d,i–j) or three (g,h) mice. Scale bar, 50 μm (applies to all panels). Full size image

Figure 3: Effects of chronic hyperglycaemia on insulin and glucagon levels. Mean islet cross-sectional area immunostaining for insulin (a,c) or glucagon (b,d), expressed either as a percentage of the total islet cross-sectional area (a,b) or per cm2 of pancreas (c,d). Once plasma glucose exceeded 20 mM, βV59M mice were treated for 4 weeks with placebo (P), insulin (Ins) or glibenclamide (Glib); or, following 4 weeks of no therapy, with 4 weeks of glibenclamide (D+Glib). Data are mean±s.e.m. of three to six mice per genotype (five sections per mouse, 100 μm apart). (*P<0.05; **P<0.01, ***P<0.001 compared with placebo (P); one-way analysis of variance followed by post-hoc Bonferroni test). Insulin (e) and preproglucagon (f) mRNA levels determined by qPCR in islets isolated from control (C, black bars) and 4-week-diabetic (4wk, white bars) βV59M mice (n=6–7 per genotype). Insulin (g) and glucagon (h) protein content of islets determined by radioimmunoassay from control (C) and 4-week-diabetic (4wk) βV59M mice (*P<0.05; Mann–Whitney test; n=6–7 per genotype). Data are mean values±s.e.m. Full size image

Insulin therapy largely prevented the reduction in insulin staining and increase in glucagon staining when data were expressed relative to islet area (Fig. 2e,f and Fig. 3a,b) or per cm2 pancreas (Fig. 3c,d). Thus, the decline in ins+ cells (and corresponding increase in glu+ cells) is principally the result of hyperglycaemia per se and not K ATP channel hyperactivity.

Glibenclamide therapy also prevented the diabetes-induced changes in insulin (Fig. 2g) and glucagon (Fig. 2h) staining, and in the area of individual islets, or whole pancreas, composed of ins+ and glu+ cells (Fig. 3a–d). Drug treatment was slightly more effective than insulin, perhaps because it produced more stable control of blood glucose (compare Fig. 1b and c).

Remarkably, 4 weeks of glibenclamide therapy almost fully reversed the histological changes produced by 4 weeks of hyperglycaemia. Insulin staining was observed throughout the islet and glucagon staining was once more confined to the mantle (Fig. 2i,j). The islet area (Fig. 3a,b) and pancreas area (Fig. 3c,d) occupied by ins+ or glu+ cells were restored to levels similar to those found in control mice.

Consistent with the decrease in the percentage of the islet area showing insulin immunoreactivity, and the concomitant increase in glucagon immunoreactivity, insulin messenger RNA and insulin content were reduced (Fig. 3e,g), and preproglucagon mRNA and glucagon content were enhanced (Fig. 3f,h), in islets isolated from mice that had been hyperglycaemic for 4 weeks, when compared with control animals.

Effects of hyperglycaemia on islet cell ultrastructure

Exposure to chronic diabetes resulted in striking changes in β-cell ultrastructure. Compared with control littermates (Fig. 4a), the number of insulin granules was considerably reduced in mice exposed to diabetes for 4 weeks (Fig. 4b). Surprisingly, in many β-cells, large areas of cytoplasm were filled with a homogeneous unstructured substance that displaced the intracellular organelles. The shape of the nucleus was occasionally distorted, but there was no indication of any apoptotic changes. No morphological signs of cellular stress were detected: the mitochondria and endoplasmic reticulum were not swollen and appeared normal. Furthermore, transcript levels of the endoplasmic reticulum (ER) stress markers Chop and spliced Xbp1 were unaltered in islets isolated from 4-week diabetic mice compared with controls (Supplementary Fig. 3a). The ultrastructural changes were specific to β-cells, and not observed in adjacent α-cells or δ-cells (Supplementary Fig. 3b).

Figure 4: Chronic hyperglycaemia reversibly alters β-cell ultrastructure. Representative electron micrographs of pancreatic sections from control mice (a), βV59M mice exposed to hyperglycaemia for 4 weeks (b), βV59M mice treated with insulin for 4 weeks (c) and βV59M mice that were hyperglycaemic for 4 weeks and then treated with glibenclamide for 4 weeks (d). Scale bar, 2 μm (refers to all panels). Images are representative of: a, 9 mice/9 islets/150 β-cells; b, 3 mice/9 islets/134 β-cells; c, 2 mice/3 islets/100 β-cells; d, 3 mice/3 islets/105 β-cells. Full size image

No effect on β-cell ultrastructure was observed following Kir6.2-V59M gene induction and only 24 h of hyperglycaemia and β-cells remained well granulated (Supplementary Fig. 3c). This supports the view that K ATP channel activation acutely inhibits insulin release by preventing β-cell electrical activity12,14 rather by affecting insulin content.

Immediate insulin therapy prevented the ultrastuctural changes associated with diabetes (Fig. 4c). Even more remarkably, the ultrastructural changes associated with 4 weeks of diabetes were completely reversed after 4 weeks of glibenclamide therapy (Fig. 4d). In glibenclamide-treated islets, the majority of β-cells were densely packed with insulin granules, which had a size and morphology similar to those observed in control animals. Taken together, the data indicate that β-cell ultrastructural changes are due to hyperglycaemia and/or hypoinsulinaemia and—importantly—are reversible on restoration of euglycaemia.

Effect of chronic hyperglycaemia on islet cell identity

We next investigated the mechanism(s) underlying the alterations in islet cell composition in chronically diabetic βV59M mice. We first explored whether they were caused by changes in cell turnover. Because of marked β-cell degranulation in βV59M mice, we used electron microscopy to identify β-cells unequivocally and nuclear morphology (condensed chromatin) as a measure of apoptosis. We found 3.7% of β-cells (that is, cells containing typical insulin granules) from 4-week diabetic βV59M mice had apoptotic nuclei (Supplementary Fig. 3d); similar numbers were found in control islets (4.0%). Cell proliferation, determined by Ki67 positivity, was also unchanged in glu+ cells (Supplementary Fig. 4a,b). Thus, the changes we observe are unlikely to be due to β-cell death or α-cell proliferation.

We also examined transcript levels of green fluorescent protein (GFP), which is located downstream of Kir6.2-V59M in the transgene cassette and thus serves as a marker of gene induction12. A >30-fold increase in islet GFP mRNA was observed 24 h after establishment of a free-fed blood glucose of >20 mM by gene induction (Fig. 5a). Islet GFP mRNA levels were unchanged after 4 weeks of chronic hyperglycaemia (Fig. 5a), despite the reduction in islet insulin immunostaining (Fig. 2), insulin mRNA (~50%, Fig. 3e) and insulin content (~70%, Fig. 3g). This provides further support for the idea that the reduction in ins+ cells we observe is not due to β-cell death.

Figure 5: Insulin/glucagon double-positive cells are reversibly increased by hyperglycaemia. (a). GFP mRNA levels determined by qPCR in islets isolated from control mice and βV59M mice 24-h (24 h) and 4 weeks (4wk) after diabetes onset. Data are mean values±s.e.m., n=4 per genotype. (*P<0.05; one-way analysis of variance (ANOVA) followed by post-hoc Bonferroni test). (b) Dual ins+/glu+ cells expressed as a percentage of the total number of ins+ cells. Control islets (C). Islets from βV59M mice implanted with placebo (P) or insulin (Ins) for 4 weeks, or treated for 4 weeks with glibenclamide after 4 weeks of hyperglycaemia (P+Glib). Data are mean values±s.e.m., n=2,600–7,700 ins+ cells; n=92–127 islets; n=3–4 mice per genotype. (*P<0.05 compared with placebo (P); one-way ANOVA followed by post-hoc Bonferroni test). (c) Representative example of immunofluorescence staining for insulin (green), glucagon (pink), DAPI (4′,6-diamidino-2-phenylindole; blue) and merged data (white) in control islets and 4-week βV59M diabetic islets. White arrowheads indicate cells positive for both insulin and glucagon. Scale bars, 50 μm control; 10 μm 4-week βV59M diabetic islets. Full size image

Chronic hyperglycaemia was associated with a ~20-fold increase in cells positive for both insulin and glucagon (ins+/glu+ cells) in islets from 4-week-diabetic βV59M mice (Fig. 5b,c). However, in mice treated with insulin for 4 weeks immediately after establishment of diabetes, or treated with glibenclamide following 4 weeks of hyperglycaemia, the number of ins+/glu+ cells was not significantly different from control animals (Fig. 5b). This argues the increase in ins+/glu+ cells is caused by hyperglycaemia/hypoinsulinaemia and is reversed when blood glucose is normalized.

We used lineage tracing to test whether β-cells start to express glucagon when exposed to chronic hyperglycaemia. Although GFP was located downstream of Kir6.2-V59M in the transgene cassette, expression was too weak to be visualized by immunofluorescence. Thus, a Rosa26RFP reporter was crossed into RIPII-CreER mice with (βV59M-RFP mice) or without (β-RFP control mice) the floxed Kir6.2-V59M transgene, to label all β-cells and their progeny with red fluorescent protein (RFP) after tamoxifen injection (Fig. 6a and Supplementary Fig. 5a,b).

Figure 6: Chronic hyperglycaemia induces glucagon expression in β-cells. (a) Schematic illustrating how Rip-CreER+/+ (i), RosaRFP/− (ii) and RosaV59M/− (iii) were used to generate RosaRFP/V59M mice (βV59M-RFP mice). β-Cells were selectively and irreversibly labelled following tamoxifen injection, by crossing an inducible rat insulin promoter Cre line (i; β) with a floxed tdRFP reporter line in which tdRFP expression was driven by the endogenous ROSA promoter (ii; RFP). These β-RFP control mice were then crossed with an inducible Kir6.2-V59M line (iii; V59M) to create βV59M-RFP mice (iv). (b) Representative examples of immunofluorescence staining for insulin (green), glucagon (pink) and RFP (red) in control (top panel, β-RFP) and 4-week-diabetic βV59M-RFP (middle and bottom panels) isolated islet cells. White arrows, RFP+/glu+ cells. White arrowhead, RFP+/glu+/ins+ cell. Scale bar, 10 μm. (c,d) Islet cells from β-RFP and 4-week-diabetic βV59M-RFP mice were FAC-sorted into RFP+ and RFP− populations, and analysed for preproglucagon mRNA by qPCR (c) and glucagon protein (d); n=4 mice per genotype. Data are mean values±s.e.m. *P<0.05; Mann–Whitney test. Full size image

In β-RFP mice, 67% of ins+ cells labelled with RFP (this indicates the recombination frequency). In βV59M-RFP mice exposed to chronic hyperglycaemia for 4 weeks, 7% of RFP+ cells (that is, cells of β-cell lineage) contained both insulin and glucagon, and 8% expressed glucagon alone (Fig. 6b and Supplementary Fig. 5c). Approximately 60% of RFP+ cells expressed insulin and 24% did not detectably express either insulin or glucagon. Fluorescence-assisted cell (FAC) sorting by RFP fluorescence (Supplementary Fig. 5a,b) revealed RFP+ cells from diabetic βV59M-RFP mice had more preproglucagon mRNA (Fig. 6c) and glucagon protein (Fig. 6d), and less insulin content (Supplementary Fig. 5d), than β-RFP littermate controls. No significant change in glucagon content was observed in RFP− cells (Supplementary Fig. 5e).

Expression of several β-cell (Pdx-1, MafA, Nkx6.1 and Glut2) and α-cell (Arx, Pax6 and MafB) markers was examined to determine whether the molecular identity of islet cells was altered by exposure to chronic hyperglycaemia. Immunofluorescence microscopy of intact islets from 4-week-diabetic βV59M mice identified expression of Pdx-1 (Fig. 7a), Glut2 (Fig. 7b) and MafA (Fig. 7c) in ins+ cells. Ins+/glu+ cells retained expression of Pdx-1 and Glut2, but also expressed MafB (Fig. 7d). All glu+ cells expressed MafB but a few also contained Pdx1. Nkx6.1 protein was undetectable by immunofluorescence in 4-week diabetic islets (compare Supplementary Fig. 6a with 6b). Quantitative PCR (qPCR) analysis revealed reduced expression of the β-cell markers Pdx-1 (P=0.05; Mann-Whitney test), Nkx6.1, MafA and Glut2 in intact islets from 4-week-diabetic βV59M mice (Fig. 7e). Although the α-cell marker MafB appeared elevated, Arx and Pax6 mRNA levels were unchanged (Fig. 7e).

Figure 7: Effects of hyperglycaemia on islet cell transcription factors and transporters. (a–d) Representative examples of immunofluorescence staining for (red) Pdx-1 (a), Glut2 (b), MafA (c) or MafB (d) in a 4-week-diabetic βV59M islet. Insulin (green), glucagon (pink). (a) Insets show (upper) glu+ and (lower) ins+/glu+ cells that express Pdx1. (b) Inset shows an ins+/glu+ cell that expresses Glut2. (c) Inset shows an ins+/glu+ cell that does not express MafA. (d) Inset shows an ins+/glu+ cell that expresses MafB. Scale bars, 50 μm. (e) Pdx-1, Nkx6.1, MafA, Glut2, Arx, Pax6 and MafB mRNA levels assessed by qPCR in islets isolated from control mice (black bars) and 4-week-diabetic βV59M mice (white bars). Data are mean values±s.e.m., n=6–7 mice per genotype. (*P<0.05; Mann–Whitney test). (f) Nkx6.1, MafA, Glut2, Arx and MafB mRNA levels in FAC-sorted RFP+ cells isolated from control mice (black bars) and 4-week-diabetic βV59M mice (white bars). Data are mean values±s.e.m., n=4 mice per genotype. (*P<0.05; Mann–Whitney test). Full size image

In contrast to what was observed in islets, mRNA levels of Nkx6.1, MafA and Glut2 were unaltered in FAC-sorted RFP+ β-cells (Fig. 7f). The difference between the effect of diabetes on mRNA expression in islets and FAC-purified RFP+ β-cells is probably due to loss of a specific β-cell subpopulation during FAC sorting: ~30% fewer RFP+ β-cells were FAC sorted from 4-week diabetic mice than from β-RFP mice. It is possible that the absent cells represent β-cells with large areas of unstructured cytoplasm (identified in electron microscopy), which may be more fragile and thus do not survive FAC sorting.

Expression of the α-cell transcription factors MafA and Arx was increased in RFP+ FAC-sorted β-cells (Fig. 7f), which explains the elevated glucagon content. Taken together, the data confirm that cells with a β-cell lineage express glucagon in response to chronic hyperglycaemia by increasing expression of α-cell transcription factors. In addition, the islet progenitor cell marker Ngn3 was elevated in both islets (Supplementary Fig. 6b) and FAC-sorted RFP+ β-cells from 4-week-diabetic βV59M mice (Supplementary Fig. 6c).

We also assessed the electrophysiological fingerprint of islet cells from control and βV59M mice following 4 weeks of diabetes. All cells were identified by immunolabelling for insulin and glucagon after patch clamping. In βV59M mice, the Cre-lox approach restricts expression of the mutant Kir6.2 subunit to cells expressing insulin at the time of induction. As the Kir6.2-V59M mutation markedly decreases K ATP channel inhibition by ATP, and thereby increases current amplitude12,15, large amplitude K ATP currents also serve as a lineage marker for β-cells. Cell-attached K ATP currents from control β-cells were small (5±2 pA; n=27), due to inhibition by intracellular ATP. In contrast, ins+ cells from βV59M mice had ~10-fold larger currents (49±6 pA; n=48). Large on-cell K ATP currents were also recorded from ins+/glu+ cells, and even from a number of cells that expressed glucagon alone (Fig. 8a,b).

Figure 8: Glucagon-expressing β-cells retain β-cell electrophysiological characteristics. (a) Mean±s.e.m. cell-attached K ATP currents from control β-cells (hatched bars; n=27) and from 4-week-diabetic βV59M islet cells that express insulin (black bars; n=48), insulin and glucagon (grey bars; n=5), or glucagon alone (white bars; n=6). (*P<0.05; one-way analysis of variance followed by post-hoc Bonferroni test). (b) Representative examples of cells exhibiting large K ATP currents obtained in cell-attached patches that expressed both insulin and glucagon (top panels) or glucagon alone (bottom panels). Scale bar, 10 μm. (c) Voltage-dependent inactivation of whole-cell Na+ currents in β-cells (closed circles; n=5) and α-cells (open circles; n=6) from control mice and in ins+/glu+ cells from 4-week-diabetic βV59M mice (crosses; n=6). The pulse protocol consisted of 1 ms depolarizations to 0 mV preceded by 200 ms conditioning pulses to membrane potentials between −180 and −5 mV. Data are mean values±s.e.m. The superimposed curves represent Boltzmann fits to the data. (d) Representative example of a patched cell (indicated by the white arrowhead) showing Na+ current inactivation characteristic of a β-cell and identified by infusion of biocytin (red, upper left panel) that expressed both insulin (green, upper right) and glucagon (pink, lower left). Merged images, lower right panel. Scale bar, 10 μm. Full size image