CARD9 Mediates Glucose‐Stimulated Insulin Secretion in Pancreatic‐β Cells

Abstract

Background- Glucose stimulated insulin secretion (GSIS) from the pancreatic beta cell involves a variety of metabolic events including generation of second messenger molecules, activation of small G-proteins culminating in vesicular transport to the plasma membrane for their fusion and secretion of insulin. Caspase recruitment domain containing protein 9 (CARD9) is a major signal transducer in innate and adaptive immune responses. CARD9 has also been implicated in the onset of metabolic diseases including insulin resistance. However, potential roles of this scaffolding protein in GSIS remains unknown. Therefore, we undertook the current investigations to determine its expression and assess the roles of CARD9 in glucose- induced Rac1 activation and insulin secretion in pancreatic beta cells. Methods and Materials- Western blotting was used to demonstrate protein expression. CARD9 expression was suppressed using Horizon/ Dharmacon siRNA and transfection reagents. Membrane and cytosolic fractions were isolated using Membrane Extraction Kit from ThermoFisher Scientific. Insulin secretion was quantified by using ALPCO Rat insulin ELISA kit. The degree of activation of Rac1 was determined by pull-down assay from Cytoskeleton Inc. Results - CARD9 was expressed in a variety of insulin-secreting cells, including human islets, rat islets and clonal INS-1 832/13 cells. Subcellular fractionation studies revealed that CARD9 is predominantly cytosolic (~75%) in distribution in INS1- 832/13 cells. Transfection of INS-1 832/13 cells with CARD9 siRNA (CARD9-si), resulted in a 70% reduction of CARD9 expression. Under these conditions, GSIS was markedly suppressed (~ 50%) in CARD9-depeleted INS-1 832/13 cells. Interestingly, glucose-induced activation of Rac1 (~3- fold), a small G-protein, which is a requisite for GSIS to occur, was unaffected in siRNA-CARD9 transfected sells, suggesting that CARD9-mediates GSIS in a Rac1-independent fashion. Furthermore, insulin secretion elicited by a membrane-depolarizing concentration of KCl (60 mM) remained resistant to CARD9 depletion in INS-1 832/13 cells. These findings imply that CARD9 might regulate insulin secretion in a calcium-independent signaling module. Conclusions- We provide the first evidence to indicate that CARD9 plays regulatory roles in GSIS via Rac1- and calcium-independent signaling mechanisms. Additional studies are underway to further assess the effector proteins that might be involved in CARD9-mediated GSIS.