Overexpressed syntaxin 1A/HPC-1 inhibits insulin secretion via a regulated pathway, but does not influence glucose metabolism and intracellular Ca2+ in insulinoma cell line beta TC3 cells

We have previously established a stable beta TC3 cell line that overexpresses syntaxin 1A, designated beta TC-hpc1 cells, in which glucose-stimulated insulin release was decreased. Using beta TC-hpc1 cells, we aimed to determine whether syntaxin 1A functions in the regulatory or constitutive pathway of insulin release. We therefore examined the secretion of phorbol-12-myristate-13-acetate (TPA)-stimulated newly synthesized proinsulin/insulin and total immunoreactive insulin, beta TC3 and beta TC-hpc1 cells were simultaneously pulse-labeled with H-3-leucine for 30 min in 11 mM glucose and chased for 1 h in one of a number of different concentrations of TPA in 11 mM glucose. Total immunoreactive insulin release (IRI) by both cell types during the chase period was markedly increased by the addition of TPA in a dose-dependent manner; however, the IRI from beta TC-hpc1 cells was lower than that from beta TC3 cells, The secretion of newly synthesized proinsulin/insulin from both cell types, which in beta TC3 cells is thought to occur via a constitutive pathway, was in the same range under any condition. Thus, the evidence indicates that syntaxin 1A preferentially functions in the regulated insulin release pathway in beta TC3 cells, In order to clarify the effect of overexpressed. syntaxin 1A on glucose metabolism and intracellular Ca-2+ we analyzed the glucose transport system, glucose phosphorylation activity, and cytosolic concentration of free Ca2+ ([Ca2+](i)). 2-Deoxy-glucose uptake and the content of GLUT1 protein in the plasma membrane fractions of beta TC-hpc1 cells were not different from those of beta TC3 cells, Radiometric assays of glucose phosphorylation activity showed that there were no differences in hexokinase activity and glucokinase activity between beta TC3 and beta TC-hpc1 cells. [Ca2+](i) measured by using fura 2 demonstrated that there was no difference in [Ca2+](i) between beta TC3 and beta TC-hpc1 cells under glucose-stimulated conditions, The present experiments indicate that syntaxin 1A plays a central role in a late step of the regulatory insulin release pathway without a change in glucose metabolism and [Ca2+](i) in beta TC3 cells. (C) 1997 Academic Press.