Examination of Piccolo Splice Variants in Regulated Insulin Secretion from Pancreatic β‐Cells

Insights regarding the regulated secretion of insulin from pancreatic β‐cells has implications for understanding diabetes and regulation of glucose metabolism. Piccolo is a high‐molecular weight protein and part of a multi‐molecular complex involved in insulin secretion. Interestingly, genetic variants of the Piccolo gene ( PCLO ) have been modestly associated with increased risk of early‐onset type II diabetes among Pima Indians. Piccolo may regulate insulin secretion through a novel mechanism by serving as a Ca 2+ sensor that interacts with L‐type voltage‐dependent Ca 2+ channels and other molecules associated with insulin secretory vesicles. The purpose of this study is to examine the role of Piccolo and its splice variants in insulin secretion. Piccolo was originally identified as a protein involved in regulation of neurotransmitter secretion at presynaptic active zones. In neurons, PCLO undergoes extensive alternative splicing. Two major splice variants have been found in both neurons and pancreatic cells: isoform‐1 (I‐1) and isoform‐2 (I‐2). I‐1 has two Ca 2+ ‐binding C2 domains, C2A and C2B, while I‐2 includes only C2A. Alternative splicing also leads to additional minor splice variants with altered Ca 2+ ‐binding properties. Specifically, an exon‐skipping event results in a splice variant that lacks exon 16, a 27‐nucleotide (nt) exon within the C2A domain. Exclusion of this exon (i.e. short variant) enhances the Ca 2+ ‐ and phospholipid binding affinity of C2A motif of rat Piccolo. In this study, we sought to examine the role of Piccolo and its splice variants in Ca 2+ ‐mediated insulin secretion in rat INS‐832/13 pancreatic β‐cells. We demonstrate that INS‐832/13 cells express both I‐1 and I‐2 and also the long and short variant of the C2A domain. In addition, we used CRISPR‐CAS gene editing to selectively target exon 1 of PCLO to examine how loss of function of Piccolo may affect glucose‐stimulated insulin secretion. Finally, we examined the effect of overexpression of the short or long C2A‐domain on insulin secretion. Support or Funding Information Research in this study was funded by NIH‐Maximizing Access to Research Careers Undergraduate Student Training in Academic Research (MARC U*STAR) award number T34GM092711, start‐up funds provided to Dr. Steven Fenster (Fort Lewis College), and funds received from SSNAP (Science Scholars the Native American Path) by SACNAS (Dr. Les Sommerville, Fort Lewis College). We would also like to thank Dr. Christopher Newgaard at Duke University for supplying the pancreatic cells and culturing procedure. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .