Co-expression and regulation of connexins 36 and 43 in cultured neonatal rat pancreatic islets

Abstract

Fetal and neonatal pancreatic islets present a lower insulin secretory response as compared with adult islets. Prolonged culturing leads to an improvement of the glucose-induced insulin secretion response in neonatal pancreatic islets that may involve regulation of gap junction mediated cell communication. In this study, we investigated the effect of culturing neonatal islet cells for varying periods of time and with different glucose medium concentrations on the cellular expression of the endocrine pancreatic gap junction associated connexin (Cx) 36 and Cx43. We report here that the 7-d culture induced upregulation of the expression of these junctional proteins in neonatal islets in a time-dependent manner. A correlation was observed between the increased mRNA and protein expression of Cx36 and Cx43 and the increased insulin secretion following islet culturing. In addition, increasing glucose concentration within the culture medium induced a concentration-dependent enhancement of Cx36 islet expression, but not of Cx43 expression in cultured neonatal islets. In conclusion, we suggest that the regulation of gap junctional proteins by culture medium containing factors and glucose may be an important event for the maturation process of beta cells observed at in vitro conditions.

Beta cell coupling and connexin expression change during the functional maturation of rat pancreatic islets

“…Insulin secretion Five to 15 overnight-cultured islets from fetal, neonatal, young and adult rats were placed in 1 ml Eppendorf tubes and preincubated for 30 min at 37°C in 0.5 ml bicarbonate-buffered Krebs solution containing 3 mg/ml bovine serum albumin/ml and equilibrated to pH 7.4 with a mixture of 95% O 2 and 5% CO 2 (BBKS) [9] supplemented with 5.6 mmol/l glucose. The islets were then incubated for 1 h at 37°C in 0.5 ml BBKS supplemented with either 2.8 or 16.7 mmol/l glucose.…”

“…Aliquots of the supernatant fraction were stored at −20°C. Secreted insulin was determined by RIA and expressed as ng islet −1 h −1 [9].…”

“…The nature of this poor responsiveness is not fully understood, and has been variably attributed to many factors, including fuel metabolites, intracellular signals, hormones and other environmental conditions [1][2][3][8][9][10][11].…”

“…The synthesis and release of insulin are markedly altered after dispersion of beta cells in vitro, but rapidly recover after cell reaggregation, indicating that the secretory mechanism depends on intercellular interactions within the pancreatic islet [12,13]. Several in vivo and in vitro studies have demonstrated that insulin secretion stimulated by glucose and other secretagogues is associated with increased gap junction coupling of beta cells, whereas the pharmacological blockage of gap junction channels impairs the secretory function of beta cells [9,[14][15][16]. Thus, loss of connexin 36 after treatment of beta cell lines with antisense oligonucleotides or knocking out the gene (Cx36 [also known as Gjd2]) in mice resulted in loss of beta cell coupling, associated with a loss of synchronisation of intracellular calcium oscillations, as well as altered basal and glucose-stimulated insulin secretion [17][18][19][20].…”

Role of islet structure and cellular interactions in the control of insulin secretion

“…In addition to cell adhesion molecules, the connexins 36 and 43 are known to play pivotal roles in regulating normal pancreatic islet function. [40][41][42] Connexins are channels formed by the apposition of two transmembrane structures called connexons. Each connexon comprises six connexins containing both C-and N-termini in the cytoplasm, a cytoplasmic loop and two extracellular loops as shown in Figure 5.…”