Abstract
Pulsatile insulin release from glucose-stimulated beta-cells is driven by oscillations of the Ca(2+) and cAMP concentrations in the subplasma membrane space ([Ca(2+)](pm) and [cAMP](pm)). To clarify mechanisms by which cAMP regulates insulin secretion, we performed parallel evanescent wave fluorescence imaging of [cAMP](pm), [Ca(2+)](pm), and phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) in the plasma membrane. This lipid is formed by autocrine insulin receptor activation and was used to monitor insulin release kinetics from single MIN6 beta-cells. Elevation of the glucose concentration from 3 to 11 mm induced, after a 2.7-min delay, coordinated oscillations of [Ca(2+)](pm), [cAMP](pm), and PIP(3). Inhibitors of protein kinase A (PKA) markedly diminished the PIP(3) response when applied before glucose stimulation, but did not affect already manifested PIP(3) oscillations. The reduced PIP(3) response could be attributed to accelerated depolarization causing early rise of [Ca(2+)](pm) that preceded the elevation of [cAMP](pm). However, the amplitude of the PIP(3) response after PKA inhibition was restored by a specific agonist to the cAMP-dependent guanine nucleotide exchange factor Epac. Suppression of cAMP formation with adenylyl cyclase inhibitors reduced already established PIP(3) oscillations in glucose-stimulated cells, and this effect was almost completely counteracted by the Epac agonist. In cells treated with small interfering RNA targeting Epac2, the amplitudes of the glucose-induced PIP(3) oscillations were reduced, and the Epac agonist was without effect. The data indicate that temporal coordination of the triggering [Ca(2+)](pm) and amplifying [cAMP](pm) signals is important for glucose-induced pulsatile insulin release. Although both PKA and Epac2 partake in initiating insulin secretion, the cAMP dependence of established pulsatility is mediated by Epac2.
Highlights
Using a new technique for single-cell measurements of cAMP, we recently found that glucose triggers pronounced oscillations of the cAMP concentration beneath the plasma membrane ([cAMP]pm) and that these changes are important for regulating the kinetics of insulin secretion [21]
The Protein kinase A (PKA)-independent amplification of insulin secretion by cAMP is probably mediated by Exchange protein directly activated by cAMP 2” (Epac2), which has been proposed to act via the small GTPase Rap-1 [24], the regulatory SUR1 subunit of the KATP channel [27] as well as via SNAP-25 [32], Rim2, and Piccolo [33], which are proteins involved in the exocytosis machinery
The present study shows that cAMP is important for both initiating and maintaining pulsatile insulin release from glucose-stimulated MIN6 -cells
Summary
Using a new technique for single-cell measurements of cAMP, we recently found that glucose triggers pronounced oscillations of the cAMP concentration beneath the plasma membrane ([cAMP]pm) and that these changes are important for regulating the kinetics of insulin secretion [21]. Studies of insulin granule exocytosis based on cell membrane capacitance measurements have indicated that cAMP amplification of insulin secretion involves both PKA-dependent and PKAindependent mechanisms [25,26,27,28]. CAMP Effectors in Pulsatile Insulin Secretion insulin granules have been identified as targets for PKA phosphorylation [25], inhibitors of this kinase have surprisingly small effects on glucose-induced insulin secretion [29, 30] Such modest effects may be expected if PKA only amplifies first phase secretion as indicated by recent imaging of exocytosis with fluorescent tracers [31]. PKA seems important for establishing pulsatile insulin release by promoting concomitant initial elevation of the subplasma membrane Ca2ϩ concentration ([Ca2ϩ]pm) and [cAMP]pm, but the kinase is not required for maintaining already manifested pulsatile insulin secretion from glucosestimulated -cells
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