1753-P: Alteration of Splice Type in Type 2 Ryanodine Receptor Causes Impaired Insulin Secretion in Mice

Increase of intracellular Ca²⁺ concentration is essential for glucose-induced insulin secretion. Type 2 ryanodine receptor (RyR2) is a Ca²⁺ release channel that mobilizes Ca²⁺ from the endoplasmic reticulum to the cytosol. Unlike RyR2 mRNA expressed in heart, RyR2 mRNA expressed in pancreatic β cells lacks exon 75, probably due to a unique splice donor site “gg” of intron 75 in RyR2 gene. We previously reported that the alteration of splicing in RYR2 caused impaired insulin biosynthesis in human 1.1B4 β cells. Here, to further investigate the significance of RyR2 alternative splicing in vivo, especially glucose-induced insulin secretion, we generated the homozygous genome-modified mice, named RyR2^{Ex75KI/Ex75KI}, in which exon 75-containing RyR2 is expressed systemically including pancreatic β cells by using CRISPR/Cas9 system. Immunohistochemical analysis of pancreas from 15-week-old RyR2^{Ex75KI/Ex75KI} and control wild-type (WT) mice revealed that the size of islets in RyR2^{Ex75KI/Ex75KI} mice was almost the same as that in WT mice, while the insulin-positive area per islet was significantly decreased (0.44-fold, P=0.005). Isolated islets from 9- to 12-week-old mice of each genotype were stimulated by low (2.8 mmol/L) or high (20 mmol/L) glucose for 1 h and secreted insulin was measured by ELISA. Insulin secretion after low glucose treatment from RyR2^{Ex75KI/Ex75KI} islets was significantly higher than that from WT islets (5.27-fold, P=0.019), and insulin secretion after high glucose stimulation was not increased (0.77-fold relative to low glucose stimulation). Intraperitoneal glucose tolerance test (2 g/kg) for 120 min on 14- to 19-week-old mice revealed that blood glucose levels did not differ between genotypes and serum insulin levels at 60-120 min after the glucose challenge were slightly higher in RyR2^{Ex75KI/Ex75KI} mice. Taken together, our results suggest that the WT splice variant of RyR2 would have a crucial role in glucose-induced insulin secretion in vivo.