The influence of intercellular connections on beta cell functions

Abstract

Background Pancreatic beta cells cultured in 2D systems display similar characteristics to dysfunctional beta cells of patients with diabetes. This thesis explores the impact of cell connectivity on functional and metabolic characteristics of insulin secreting beta cells. Methods The MIN6 mouse beta cell line was cultured as 2D monolayers and 3D structures named pseudoislets. The insulin secretion response and metabolic function of 2D and 3D MIN6 cultures, along with islets from human donors were compared. Roles for cell-cell interactions in regulating metabolic changes were explored with focus on the gap junctional protein, connexin36, using an inducible knockdown MIN6 cell line. Results MIN6 pseudoislets displayed improved functional responses compared to monolayers with 7.4-fold and 1.5-fold increases in glucose-induced insulin secretion respectively. XFe24 seahorse bioanalyser data showed the improved glucose-stimulated pseudoislet response was fuelled by large increases in glycolytic flux and a more moderate increase in oxidative phosphorylation. Basal insulin secretion and basal oxidative phosphorylation were both higher in monolayers but there were no differences in basal glycolysis. Human islets displayed a similar phenotype to pseudoislets with high contributions of glycolysis to glucose-induced ATP production. Pseudoislets indicated some hypoxia through trends towards increased lactate dehydrogenase and phosphoinositide-dependent kinase-1 expression and increased glycolytic activity of phosphfructokinase-1 and glyceraldehyde 3-phosphate dehydrogenase but superior glucose sensing through decreased hexokinase I and increased GLUT2 expression and more active mitochondrial activities of pyruvate carboxylase, citrate synthase, α-ketoglutarate dehydrogenase, and malate dehydrogenase. Knockdown of connexin36 did not alter glucose-stimulated insulin secretion or metabolic flux. However, there was a trend towards increased basal insulin secretion and basal oxidative phosphorylation indicating a possible role for this connection in regulating basal metabolic flux. Conclusions The improved glucose-stimulated secretion conferred by pseudoislet configuration was accompanied by an increase in ATP production suggesting a role for alteration in metabolic flux in the improved functionality. Improved functional responses of beta cells in 3D structures was accompanied by a small increase in oxidative phosphorylation but a large increase in glycolysis that cannot be fully explained by hypoxia. Connexin36 may play a role in regulating the basal response but other connections are involved in regulating the glucose-stimulated response.