Defects in inositol 1,4,5-trisphosphate receptor expression, Ca(2+) signaling, and insulin secretion in the anx7(+/-) knockout mouse

Abstract

The mammalian anx7 gene codes for a Ca(2+)-activated GTPase, which supports Ca(2+)/GTP-dependent secretion events and Ca(2+) channel activities in vitro and in vivo. To test whether anx7 might be involved in Ca(2+) signaling in secreting pancreatic beta cells, we knocked out the anx7 gene in the mouse and tested the insulin-secretory properties of the beta cells. The nullizygous anx7 (-/-) phenotype is lethal at embryonic day 10 because of cerebral hemorrhage. However, the heterozygous anx7 (+/-) mouse, although expressing only low levels of ANX7 protein, is viable and fertile. The anx7 (+/-) phenotype is associated with a substantial defect in insulin secretion, although the insulin content of the islets, is 8- to 10-fold higher in the mutants than in the normal littermate control. We infer from electrophysiological studies that both glucose-stimulated secretion and voltage-dependent Ca(2+) channel functions are normal. However, electrooptical recordings indicate that the (+/-) mutation has caused a change in the ability of inositol 1,4,5-trisphosphate (IP(3))-generating agonists to release intracellular calcium. The principle molecular consequence of lower anx7 expression is a profound reduction in IP(3) receptor expression and function in pancreatic islets. The profound increase in islets, beta cell number, and size may be a means of compensating for less efficient insulin secretion by individual defective pancreatic beta cells. This is a direct demonstration of a connection between glucose-activated insulin secretion and Ca(2+) signaling through IP(3)-sensitive Ca(2+) stores.

Figure 1

Gene targeting strategy. (A) Restriction map of the mouse anx7 gene, the targeting vector, and the predicted structure of the locus after recombination. The top line represents the structure and partial restriction map from exon 3 to 9 of the wild-type allele of anx7. Exons are shown as black boxes. The middle line is the targeting plasmid in which a 900-bp XhoI and XbaI fragment is replaced with a 1.8-kb phosphoglycerate kinase (PGK)-neo-PA cassette and linearized at the unique NotI site. The dashed lines show the relationship of the two anx7 fragments in KSBX-pPNT to the genomic locus. The boxes represent the neo and HSV-tk genes, as labeled. The horizontal arrows show the phosphoglycerate kinase-1 promoters and their direction of transcription. The lowest line is the predicted structure of the targeted allele. The line marked KXX represents the 500-bp XhoI–XbaI fragment used for detection of homologous recombinants. The expected sizes of XbaI fragments that will hybridize to this probe is shown at lower right. (B) Southern blot analysis. Genomic DNA was digested with XbaI, blotted, and hybridized with KXX probe. The 3.6-kb fragment is derived from the wild-type allele, and the 4.5-kb fragment seen in lanes 1–3, representing 10–30 μg of genomic DNA from ES cells, is diagnostic of homologous recombination. (C) PCR analysis of genomic DNA from yolk sac of anx7(+/+), anx7(+/−), and anx7(−/−) embryos. The DNA (2 μl) was subjected to PCR analysis by using antisense oligonucleotide from the inserted neo cassette and sense oligonucleotide from genomic sequence outside the 2.0-kb 5′-flanking sequence in the targeting vector. (D) Immunoblot of anx7 extracted from pancreas of anx7(+/−) and control (+/+) mice. Pancreas from anx7(+/−) mice was harvested, frozen on dry ice, and then homogenized in boiling SDS buffer. Aliquots containing identical amounts of protein were separated by SDS/PAGE and transblotted to nitrocellulose, and ANX7 was visualized by Western blot analysis by using rabbit anti-human ANX7 primary antibody.

Figure 2

Islet hyperplasia in anx7(+/−) mice. A composite picture comparing hematoxylin- and eosin-stained pancreatic tissue and islet of Langerhans from anx7(+/−) (A–C) and control anx7(+/+) (D–F) mice. (A and D are enlarged ×50.) Arrows show islets. (B, C, E, and F are enlarged ×200.) Data are representatives of five control and mutant animals each. Islet of Langerhans from anx7(+/−)mice are hyperplastic.

Figure 3

Electron microscopy–immunogold labeling of insulin in control (A) and mutant (B) β cells. Pancreas tissues taken from mice were processed for electron microscopy and labeled with antibodies to insulin as described in Materials and Methods (C) Statistical analysis of A and B.

Figure 4

Calcium-dependent secretion of insulin from control and mutant β cells. Hextuplicate sets of five collagenase-dissociated islets were distributed into reaction vessels and challenged with different Ca²⁺ concentrations as shown. Data are presented as the fraction of total insulin after subtracting basal values from values in 22 mM glucose. Baseline levels of secretion changed very little across the Ca²⁺ titration range. Time point is 60 min.

Figure 5

(A and B) Influence of carbachol (Cch) and loperamide (Lop) on cytosolic calcium transients in control and mutant β cells. β cells from wild-type control mice (A) and mutant anx7(+/−) mice (B) were cultured from collagenase-treated islet tissue. After 3 days in culture, cells were loaded with indo-1, and intracellular Ca²⁺ concentrations were measured. Carbachol and loperamide were applied as indicated. The glucose concentration used was 5.6 mM. (C and D) influence of thapsigargin on calcium transients in control and mutant β cells. β cells were isolated from wild-type and mutant pancreatic tissue, as described in A and in refs. and . Thapsigargin (Thapsi), carbachol, and loperamide were applied as shown. K.O., knockout.

Figure 6

Electron microscopy–Immunogold labeling of IP₃ receptor in control (Upper) and mutant (Lower) β cells. Pancreas tissues taken from mice were processed for electron microscopy and labeled with antibodies to IP₃ receptor. The anti-IP₃ receptor antibody (Calbiochem) is active against isoforms I, II, and III.

Figure 7

Stimulus-secretion coupling in the pancreatic β cell. Glucose metabolism leads to membrane depolarization and Ca²⁺ influx. The Ca²⁺ that enters the cell through voltage-gated Ca²⁺ channels, along with Ca²⁺ released from the ER, stimulates insulin secretion. Defective processes in the anx7(+/−) mutant are shown in parentheses. IP₃ R, IP₃ receptor; ACh, acetylcholine; SERCA, smooth endoplasmic reticulum calcium.