Nucleotide-mediated insulin secretion

Several putative messengers, or additive signals, have been proposed to participate in metabolism-secretion coupling. Among them, nucleotides are possible candidates.

ATP

Intracellular ATP has two functions. On the one hand, an increase in the ATP/ADP ratio is necessary for KATP-channel closure. On the other hand, ATP has a role in priming granules for fusion with the plasma membrane and the exocytotic release of insulin. However, at non-stimulatory Ca²⁺-concentrations, ATP does not induce insulin release in permeabilized cells [64]. In contrast, in the presence of stimulatory Ca²⁺-concentrations, ATP potentiates exocytosis [64, 65]. In the absence of extracellular calcium glucose-stimulated ATP generation did not promote insulin secretion [66]. However, when the KATP-channel is kept in an open state using the inhibitor diazoxide and [Ca²⁺]i is raised

independently of K_ATP-channel activation was observed. These results were taken to suggest that ATP stimulates insulin exocytosis in a KATP-independent manner [66].

GTP

The nucleotide GTP is formed in the mitochondria during TCA cycle activity. It was proposed that GTP triggers insulin exocytosis via GTPases [67, 68]. Evidence in support of a role for mitochondrial GTP production in GSIS involves experiments with siRNA-mediated suppression of the GTP-generating form of succinyl-CoA synthetase (SCS-GTP).

Suppression of this enzyme in clonal β-cells and rat islets resulted in impaired GSIS and reduced GTP levels [69]. In contrast, repression of the ATP-generating form of this enzyme (SCS-ATP) enhanced insulin release at stimulatory glucose concentrations [69].

Furthermore, the same study demonstrated that suppression of SCS-GTP impairs glucose-stimulated increases in [Ca²⁺]i whereas downregulation of SCS-ATP had the opposite effect. In general, GTP is not leaving the mitochondria, but the nucleotide can also be generated from ATP in the cytosol through the action of nucleoside diphosphate kinase. In contrast to ATP, GTP is able to initiate insulin secretion in a Ca²⁺-independent manner in native [68, 70] and in clonal β-cells [64, 68, 71]. This pathway is completely and selectively blocked by reducing GTP levels in the islet [72, 73]. It is not known if GTP acts via monomeric or heteromeric G-proteins to control insulin exocytosis [71, 74].

Cyclic AMP

It is well established that cyclic AMP (cAMP) potentiates Ca²⁺-dependent GSIS. Several hormones, including glucagon, GLP-1 (glucagon-like peptide 1) and glucose-dependent insulinotropic polypeptide (GIP), are known to increase intracellular cAMP levels. The action of these hormones involves the activation of G-protein coupled receptors (GPCR), followed by activation of adenylate cyclase (AC) and a rise in cAMP concentrations [75, 76]. Subsequently, cAMP can activate either proteinkinase A (PKA)–dependent or PKA-independent pathways in order to potentiate GSIS [77].

Figure 1.10: Protein kinase-dependent and –independent pathways implicated in the regulation of GSIS [77].

Several PKA-dependent pathways have been suggested by which cAMP increases [Ca²⁺]i. The phosphorylation of the K_ATP-channel subunit Kir6.2 (pore-forming subunit) and SUR1 (regulatory subunit) by PKA has been demonstrated to decrease KATP-channel activity [78]. Furthermore, PKA-mediated activation of voltage-dependent L-type Ca²⁺-channels (VDCC) caused an increase in Ca²⁺-influx and thereby a potentiation of insulin granule exocytosis [79]. The phosphorylation of the glucose transporter GLUT2 by PKA has also been shown to decrease glucose transport activity by GLP-1 in purified β-cells and thus affect GSIS [80]. Another PKA-dependent pathway that has been suggested is the mobilization of Ca²⁺-ER-stores [81, 82] and the activation of Ca²⁺-activated nonselective ion channels [83].

Cyclic AMP can also stimulate insulin exocytosis via PKA-independent mechanisms. It enhances the translocation of granules to the plasma membrane (active zone) and increases the size of the ready releasable pool as well as the rate of replenishment [84, 85]. The protein cAMP-GEFII (GTP-exchange factor) has been described as direct target of cAMP in the regulation of insulin exocytosis [86]. Cyclic AMP-GEFII-mediated exocytosis of insulin secretory granules requires direct interaction of the factor with the (active zone) protein Rim2. It also interacts with the active zone protein piccolo [87]

The exact mechanism by which cAMP binding to cAMP-GEFII affects insulin exocytosis has still to be elucidated.

NADPH

There is strong evidence in support of an important role of the pyridine nucleotide NADPH in the regulation of insulin secretion. NADPH is generated during glucose metabolism (pentose phosphate pathway, malic enzyme & isocitrate dehydrogenase). Experiments from toadfish islet cells propose that NADPH stimulates insulin release from secretory granules [88]. Generation of NADPH is an early event in β-cell activation preceding the rise in [Ca²⁺]i, a prerequisite for triggering nutrient-mediated insulin release [89, 90].

Several studies demonstrated an increase in the NADP/NADP ratio in proportion to glucose concentrations and GSIS, whereas this relationship does not exist for the NADH/NAD⁺ ration and GSIS [91, 92]. The elevation of NADPH levels occurs more rapidly in the cytosol than in the mitochondria [93]. Conversely, if the NADPH/NADP ratio was decreased through molecular manipulations GSIS was impaired [92, 94, 95].

Furthermore, addition of NADPH but not NADH to patch-clamped β-cells increased cell capacitance and thereby insulin exocytosis [91]. Two targets have been proposed for NADPH. The NADPH-dependent glutathione reductase has been suggested which catalyzes the formation of reduced glutathione from glutathione. Reduced glutathione is then converted to glutaredoxin (GRX), which participates in posttranslational modifications of proteins, including exocytosis-regulating t-SNARE proteins [96]. The administration of GRX to patch-clamped β-cells caused a potentiation of NADPH-induced exocytotic activity [91]. The second potential target of NADPH that has been proposed is the voltage-dependent K⁺-channel (Kv-channel) [97]. Kv-channels might serve as negative regulators of insulin secretion as they repolarize glucose-stimulated action potentials and inhibit Ca²⁺-influx through voltage-gated calcium channels. It has been shown that an increase in the cytosolic NADPH/NADP ratio in patch-clamped β-cells was associated with an increased rate of Kv-channel inactivation [98]. Additonally, NADPH oxidase seems also play an important role in the regulation of insulin secretion through the generation of reactive oxygen species (ROS) [99].