E-cadherin interactions regulate beta-cell proliferation in islet-like structures.

Islet function is dependent on cells within the islet interacting with each other. E-cadherin (ECAD) mediates Ca(2+)-dependent homophilic cell adhesion between b-cells within islets and has been identified as a tumour suppressor. We generated clones of the MIN6 beta-cell line that stably over- (S) and under-express (alphaS) ECAD. Modified expression of ECAD was confirmed by quantitative RT-PCR, immunoblotting and immunocytochemistry. Preproinsulin mRNA, insulin content and basal rates of insulin secretion were higher in S cells compared to aS and control (V) cells. However, stimulated insulin secretory responses were unaffected by ECAD expression levels. ECAD expression did affect proliferation, with enhanced ECAD expression being associated with reduced proliferation and vice versa. Formation of islet-like structures was associated with a significant reduction in proliferation of V and S cells but not alphaS cells. These data suggest that ECAD expression levels do not modulate insulin secretory function but are consistent with a role for ECAD in the regulation of beta-cell proliferation.

A key role for beta-cell cytosolic phospholipase A(2) in the maintenance of insulin stores but not in the initiation of insulin secretion.

Cytosolic phospholipase A(2) (cPLA(2)) is a Ca(2+)-sensitive enzyme that has been implicated in insulin secretion in response to agents that elevate beta-cell intracellular Ca(2+) ([Ca(2+)](i)). We generated clones of the MIN6 beta-cell line that stably underexpress cPLA(2) by transfection with a vector in which cPLA(2) cDNA had been inserted in the antisense orientation. Reduced expression of cPLA(2) was confirmed by Western blotting. The insulin content of cPLA(2)-deficient MIN6 cells was reduced by approximately 90%, but they showed no decrease in preproinsulin mRNA expression. Measurements of stimulus-dependent changes in [Ca(2+)](i) indicated that reduced expression of cPLA(2) did not affect the capacity of MIN6 cells to show elevations in Ca(2+) in response to depolarizing stimuli. Perifusion experiments indicated that cPLA(2) underexpressing MIN6 pseudoislets responded to glucose, tolbutamide, and KCl with insulin secretory profiles similar to those of cPLA(2) expressing pseudoislets, but that secretion was not maintained with continued stimulus. Analysis of the ultrastructure of cPLA(2)-deficient MIN6 cells by electron microscopy revealed that they contained very few mature insulin secretory granules, but there was an abundance of non-electron-dense vesicles. These data are consistent with a role for cPLA(2) in the maintenance of insulin stores, but they suggest that it is not required for the initiation of insulin secretion from beta-cells.