Characterizing the Role of the Neuroregulatory Protein Piccolo in Secretion of Insulin from Beta Pancreatic Islet Cells

Abstract

Secretion of insulin from pancreatic beta‐cells is required for glucose homeostasis; however, the molecular mechanisms of insulin secretion have not been fully elucidated. In pancreatic beta‐cells, glucose‐induced insulin secretion is synergistically potentiated through a calcium (Ca2+)‐mediated Epac2‐Rim2‐Piccolo signaling response mechanism. Both Piccolo and Rim2 were originally identified in neurons as active zone proteins that regulate neurotransmitter secretion. It has been demonstrated that Piccolo is downregulated in human pancreatic islet cells isolated from patients with type 2 diabetes (T2D). We hypothesize that Piccolo plays a key role in regulated insulin secretion and further study of Piccolo in pancreatic islets cells may provide valuable insights into the etiology of T2D. The C‐terminus of Piccolo contains a Ca2+‐binding C2A domain and we hypothesize that in pancreatic beta‐cells, Piccolo may regulate insulin secretion by serving as a Ca2+‐sensor. Alternative splicing within the coding region of the C2A domain results in an exon‐skipping event generating either a splice variant that lacks the 27‐nucleotide exon 16 (e.g., short C2A) or a variant that retains this exon (e.g., long C2A). Due to this splicing event, the short C2A domain lacks 9 amino acids resulting in enhanced Ca2+‐binding affinity. In this study, we sought to examine the differential roles of the Piccolo splice variants in Ca2+‐mediated insulin secretion using a rat pancreatic beta‐cell line (INS‐832/13). We demonstrate that INS‐832/13 cells express both variants containing either the short or long C2A domains. We also demonstrate that these cells express an additional splice variant called Piccolino previously thought to be only expressed in neurons associated with the eye and ear. To further assess the role of Piccolo in regulating insulin secretion, we plan to use a gene knockdown approach employing multiple shRNAs expressed in lentivirus that target Piccolo RNA sequences. To assess the effect of knockdown of Piccolo, we will stimulate ß pancreatic islet cells with increasing glucose concentrations to quantify changes in insulin secretion.