Glucose dependence of insulinotropic actions of pituitary adenylate cyclase-activating polypeptide in insulin-secreting INS-1 cells

Abstract

The cAMP-elevating pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates insulin release in pancreatic B-cells. Here, we have investigated its potentiating action in rat insulinoma INS-1 cells. In intact cells, PACAP-27 (100 nM) stimulated glucose-induced insulin secretion by >60%. Using the patch-clamp technique with single-cell exocytosis monitored as increases in cell capacitance, we observed that at 10 mM and 20 mM extracellular glucose, PACAP-27 acted mainly by a >50% enhancement of depolarization-elicited Ca(2+) entry, whereas at low (3 mM) glucose, the predominant effect of the peptide was a twofold increase in Ca(2+) sensitivity of insulin exocytosis. The latter effect was mimicked by glucose itself in a dose-dependent fashion. PACAP-27 exerts a prolonged effect on insulin secretion that is dissociated from changes of cytoplasmic cAMP. Whereas an elevation of cellular cAMP content (135%) could be observed 2 min after addition of PACAP-27, after 30 min preincubation with the peptide, cAMP concentrations were not different from basal. Yet, such pretreatment with PACAP-27 stimulated subsequent insulin release by congruent with60%. This sustained action is likely to reflect an increased degree of protein-kinase-A-dependent phosphorylation, and inhibitors of the kinase largely prevented the PACAP-mediated effects.

Calcium Current Inactivation Rather than Pool Depletion Explains Reduced Exocytotic Rate with Prolonged Stimulation in Insulin-Secreting INS-1 832/13 Cells

“…Indeed, experiments performed on INS-1 cells has previously demonstrated that a depolarization of the same size and duration (300 ms) can give rise to large differences in Ca 2+ influx measured as charge ( Q ). Plotting ΔC m to Q in this case gave a linear relationship [35]. Our data showed a near-linear ΔC m to Q relation (Fig.…”

A Highly Ca2+-sensitive Pool of Granules Is Regulated by Glucose and Protein Kinases in Insulin-secreting INS-1 Cells

“…Similarly, Wan et al (2004) report a complete block of the forskolin-induced enhancement of HCSP and RRP pool sizes by Rp-cAMP. These findings are surprising because previous reports describe only a partial block of the cAMP-induced enhancement of depolarization-evoked exocytosis by PKA inhibitors in mouse β cells and INS-1 cells ( Renstrom et al, 1997 ; Rosengren et al, 2002 ). This has led to the hypothesis that cAMP enhances exocytosis through a PKA-independent pathway such as cAMP-GEFII ( Eliasson et al, 2003 ).…”

“…Glucose triggers insulin release from pancreatic β cells by closing ATP/ADP-gated K + channels, which leads to membrane depolarization, Ca 2+ influx through voltage-gated Ca 2+ channels, and [Ca 2+ ] i -triggered exocytosis of insulin-containing granules. Glucose also promotes exocytosis from β cells independently from effects on K + (ATP) channels ( Eliasson et al, 1997 ; Takahashi et al, 1999 ; Rosengren et al, 2002 ). For example, elevated ATP levels may increase the number of vesicles in a readily releasable state.…”

“…For example, elevated ATP levels may increase the number of vesicles in a readily releasable state. Activation of protein kinases such as cAMP-dependent protein kinase (PKA; Jones et al, 1986 ; Ammala et al, 1993a ; Gillis and Misler, 1993 ; Rosengren et al, 2002 ) and PKC ( Jones et al, 1985 ; Ammala et al, 1994 ) modulate Ca 2+ -triggered exocytosis from β cells and thus provides a mechanism for hormonal modulation of insulin secretion through second messengers (for review see Jones and Persaud, 1998 ).…”

“…Here we investigate the Ca 2+ sensitivity of exocytosis in the INS-1 insulin-secreting cell line. INS-1 cells, like primary β cells, undergo Ca 2+ -triggered exocytosis of insulin that is modulated by hormones, glucose, and protein kinases ( Asfari et al, 1992 ; Rosengren et al, 2002 ). We find that INS-1 cells have an HCSP in addition to the less Ca 2+ -sensitive readily releasable pool (RRP) that has previously been characterized.…”