Abnormalities of pancreatic islets by targeted expression of a dominant-negative KATP channel

Abstract

ATP-sensitive K+ (KATP) channels are known to play important roles in various cellular functions, but the direct consequences of disruption of KATP channel function are largely unknown. We have generated transgenic mice expressing a dominant-negative form of the KATP channel subunit Kir6.2 (Kir6.2G132S, substitution of glycine with serine at position 132) in pancreatic beta cells. Kir6.2G132S transgenic mice develop hypoglycemia with hyperinsulinemia in neonates and hyperglycemia with hypoinsulinemia and decreased beta cell population in adults. KATP channel function is found to be impaired in the beta cells of transgenic mice with hyperglycemia. In addition, both resting membrane potential and basal calcium concentrations are shown to be significantly elevated in the beta cells of transgenic mice. We also found a high frequency of apoptotic beta cells before the appearance of hyperglycemia in the transgenic mice, suggesting that the KATP channel might play a significant role in beta cell survival in addition to its role in the regulation of insulin secretion.

Figure 1

(A) Alignment of amino acid sequences of the H5 regions of K⁺ channel members. The residues identical to mouse Kir6.2 are shown by dots. The Gly (G)-Phe (F) [or Tyr (Y)]-Gly (G) motifs are boxed. The Gly-132 (G) of mouse Kir6.2 was mutated to Ser (S) to generate the mutant Kir6.2 (Kir6.2G132S). The sequences are mouse Kir6.2 (GenBank accession no. D50581), rat Kir6.1(GenBank accession no. D42145), rat Kir1.1a (GenBank accession no. X72341), mouse Kir2.1(GenBank accession no. X73052), mouse Kir3.2 (GenBank accession no. U11859), shaker (GenBank accession no. M17211), and hslo (GenBank accession no. U11717). (B) ⁸⁶Rb⁺ efflux in COS-1 cells transfected with wild-type Kir6.2 (WT) and the mutant Kir6.2G132S (MT) at various ratios together with SUR1. ⁸⁶Rb⁺ efflux in COS-1 cells transfected with SUR1 alone is used as the control efflux (▴). The ratio of WT to MT is 1:0 (○), 1:1 (•), 1:3 (□), 1:5 (▪), and 0:1 (▵). Some of the error bars are not seen because of very small standard errors.

Figure 2

(A) Blood glucose levels in Kir6.2G132S transgenic mice (line M45) (Tg, •, n = 19) and control mice (control, ○, n = 18) in neonates (day 0). The values below 30 mg/dl (minimum detectable value) were calculated as 30 mg/dl for statistical analysis. The bars indicate the mean values. (B) Relationship between blood glucose levels and serum insulin levels in transgenic mice (•, n = 13) and control mice (○, n = 9) in neonates. The correlation between blood glucose and serum insulin levels in control mice is indicated. The shaded area indicates values below the sensitivity of assay in A and B. (C) Blood glucose levels in the nonfasted state in transgenic (•) and control mice (○) at the indicated ages. Each point was determined by 10–19 mice. (D) Blood glucose and insulin responses to i.p. glucose load in transgenic (n = 4) and control (n = 4) mice at 8 weeks of age. Filled and open circles indicate blood glucose levels in transgenic mice and control mice, respectively. Filled and open squares indicate serum insulin levels in transgenic and control mice, respectively.

Figure 3

(A) Representative traces of whole-cell recordings of pancreatic beta cells from control and Kir6.2G132S transgenic mice (line M4). The holding potential was −70 mV and alternate voltage pulses of ±10 mV and 200-ms duration every 2 s were applied. Removal of intracellular ATP beginning at the time indicated (breakthrough) caused a progressive increase in K⁺ conductance in response to ±10 mV amplitude pulses, and addition of 0.5 mM tolbutamide promptly inhibited the conductance in a beta cell of a control mouse (control). There was only a slight increase in K⁺ conductance in a beta cell of a transgenic mouse (Tg) under the same conditions. (B) Normalized ATP-sensitive K⁺ conductance of beta cells in control and transgenic mice (lines M4 and M10). Because the membrane area of each beta cell varied, the K⁺ conductance was normalized by dividing by the membrane capacitance measured for each cell. Control mice, 2.29 ± 0.27 nS/pF (mean ± SE, n = 20); line M10 transgenic mice, 1.27 ± 0.29 nS/pF (n = 20); line M4 transgenic mice, 0.37 ± 0.25 nS/pF (n = 10). ∗, P < 0.02, ∗∗, P < 0.001 (comparing with control).

Figure 4

(A) Histological changes of pancreatic islets from Kir6.2G132S transgenic and control mice. (a-l) Immunostaining of islet cells at indicated ages: 0d, transgenic and control mice in neonates; 7d, 7-day transgenic mice; 4w, 4-week transgenic mice; 8w, 8-week transgenic and control mice. The insulin column shows staining with anti-insulin antibody (a-f); the glucagon column shows staining with anti-glucagon antibody (g-l). Control mice are shown in b, f, h, and l. Three independent transgenic lines (M7, M8, and M45) have been analyzed. Histological changes of these three lines are similar. Representative examples of M45 mice are presented. The average beta cell population (per islet) of transgenic and control mice are 60.8 ± 4.6% and 63.8 ± 4.7% at day 0, 44.8 ± 2.5% and 62.7 ± 4.9% at 1 week, 34.7 ± 3.6% and 75.2 ± 5.3% at 4 weeks, and 32.5 ± 3.7% and 72.7 ± 4.4% at 8 weeks, respectively. (B) Detection of apoptotic cells in the islets by TUNEL method. (a) Apoptotic beta cells (indicated by arrowheads) are present in the islets (indicated by L) of a transgenic mouse. (b) No apoptotic cells are detected in the islets of a control mouse. (Bar = 100 μm).