1252-P: Alteration of Alternative Splicing of RYR2 Modulates Insulin Biosynthesis in Pancreatic ß-Cells

In pancreatic β cells, intracellular Ca²⁺ is essential for insulin biosynthesis. Ryanodine receptors (Ryrs) mediate Ca²⁺ mobilization from the endoplasmic reticulum. Previously we found that type 2 Ryr (Ryr2; originally called cardiac-type) of three isoforms from separate genes was expressed in pancreatic β cells and that Ryr2 mRNA expressed in the pancreatic β cells was deficient in the region of exon 75, unlike that expressed in the heart. This alternative splicing occurred tissue-specifically, which is presumably due to a unique splice donor site “gg” in intron 75 of Ryr2 gene. In order to clarify the role of the tissue-specific splice variant of Ryr2 on insulin biosynthesis, we constructed the homozygous genome-modified pancreatic β cells which express cardiac RYR2 by changing the splice donor “gg” of RYR2 gene to “gt” in human 1.1B4 pancreatic β cells using the CRISPR/Cas9 system. Insulin content in the culture medium and insulin mRNA level were measured by ELISA and real-time RT-PCR, respectively. The results showed that both insulin and insulin mRNA levels were significantly lower in the genome-modified cells than those in parental 1.1B4 cells. We examined the effect of complementation of Ryr2 by introducing the rat cardiac-type or islet-type Ryr2 expression vector into each cell. Real-time RT-PCR analysis showed that the expression of insulin mRNA was significantly increased in the cells transfected with the islet-type Ryr2 expression vector, whereas that in the cells transfected with the cardiac-type Ryr2 expression vector was not increased. Furthermore, the result of luciferase assay showed that the promoter activity of insulin gene was not decreased in the genome-modified cells as compared with the parental 1.1B4 cells.

In conclusion, those results indicate that exon 75-deficient Ryr2 could be important in the post-transcriptional step of insulin biosynthesis via Ca²⁺ signaling in pancreatic β cells.