Calcium-binding proteins in rat and human pancreatic islets as well as in INS1-cells

Introduction: Calcium is the central second messenger in the regulation of insulin release from the pancreatic β-cell. Transduction and efficacy of intracellular calcium signaling mainly depends on calcium-binding proteins (CaBPs) like calbindin-D28k (CALB), secretagogin (SCGN), calmodulin (CALM) and calreticulin (CALR). In pancreatic b-cells, the proposed functional role of these CaBPs includes the modulation of insulin secretion (CALB), influences on calcium influx and insulin transcription (SCGN), or modulation of insulin release via protein phosphorylation by calcium/calmodulin-dependent protein kinases (CALM). CALR is an endoplasmic reticulum (ER) luminal CaBP, a calcium storage protein and a molecular chaperone, which function in β-cells remains a subject for further inquiry. The present study dealt with the localization of these four CaBPs in the pancreatic tissue of humans and rats as well as in rat insulinoma β-cells (INS-1). To confirm their functional significance, the immunohistochemical distribution and their transcript levels were analyzed by comparing metabolically healthy Wistar rats with Goto-Kakizaki (GK) rats. The latter is an animal model of spontaneous non-insulin-dependent type 2 diabetes mellitus.

Materials and Methods: Pancreata were obtained from surgical specimens of patients (n=4), or from Wistar rats and diabetic GK rats (each n=6; approximately 6 weeks old). Immunofluorescence labelings were performed on pancreatic sections. The sections were examined with a CCD-camera or by confocal laser scanning microscopy. In addition, rat pancreatic tissue was treated with the immunogold technique. Real time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine transcript levels.

Results: Immunohistochemistry demonstrated that three of CaBPs (CALM, CALR and CALB), were located predominantly in the pancreatic islets (in both α- and β-cells) of humans and rats, showing weaker labeling of the exocrine tissue. SCGN was exclusively found within islets. All CaBPs were immunohistochemically also detected in INS1-cells. In addition, immunogold labeling revealed specific locations for individual CaBPs in the cell organelles or cellular matrix of β-cells. Immunohistochemical analysis demonstrated distinct differences in CaBP distribution when comparing the pancreatic tissue of type 2 diabetic GK rats with that of non-diabetic Wistar rats. RT-PCR demonstrated that transcripts of CaBPs displayed different expression levels in Wistar rat pancreatic tissue and in INS1-cells. The pancreatic tissue of GK rats showed significantly higher transcript levels of all CaBPs compared to those in Wistar rats.

Discussion: The functional significance of the CaBPs investigated in the pancreatic tissue is emphasized by their predominant location within islets. In addition, immunogold labeling signifies their unique functions in pancreatic islet physiology; for example SCGN immunogold particles were predominantly associated with secretory granules und with membranes of the rough ER. Comparing diabetic and non-diabetic rats, these findings indicate that CaBP transcripts and their cellular locations are associated with the pathological defects involved in type 2 diabetes and therefore may have important consequences regarding the maintenance of normal β-cell functions.