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内容記述 |
Glucose is the most important physiological regulator of insulin secretion from βcells of islets of Langerhans. Islet βcells rapidly take up and metabolize glucose, resulting in an increase in the cytosolic concentration of ATP within 1 min. Such increases in ATP concentration induce the closure of ATP-sensitive K<SUP>+</SUP>(K<SUB>ATP</SUB>) channels and consequent depolarization of the cell membrane, again within a few minutes of glucose application. Depolarization of the cell membrane to a voltage of>-50 m V results in activation of voltage-dependent Ca<sup>2+</sup> channels and an uncrease in [Ca<sup>2+</sup>]<sub>i</sub> that triggers insulin exocytosis.The K<sub>ATP</sub> channels and Ca<sup>2+</sup>-dependent mechanism are thought to play a central role in glucose sensing for insulin exocytosis. Althogh additional merchanisms of glucose sensing have been proposed to coexist, their relative importance has remained unclear.<br /> Exocytosis in many secretory cell types and neurons is regulated by both Ca<sup>2+</sup> and cAMP. Their reserch group has previously shown that cytosolic cAMP potentiates Ca<sup>2+</sup>-dependent insulin exocytosis(CIE)in βcells(Takahashi N.et al.Proc.Natl.Acad.Sci.U.S.A.96,960,1999). In these studies,individual βcells were subjected to whole-cell patch clamping and stimulated with large increases in [Ca<sup>2+</sup>]<sub>i</sub> induced by photolysis of a caged-Ca<sup>2+</sup>compound,thereby bypassing the K<SUB>ATP</SUB> channel-dependent mechanism. They found that CIE was augmented by <sub>c</sub>AMP in a manner dependent on protein kinase A(PKA) and cytosolic ATP. It was not possible to study the action of extracellular glucose under the whole-cell clamp conditions,however,and it has remained unknown whether PKA contributes to glucose-induced insulin exocytosis (GIE). Inhibitors of PKA have been shown to have relatively small inhibitory effects on GIE in previous studies,in which exocytosis was measured over a long period without separation into the first and second phases.<br /> They therefore subsequently developed an approach based on twophoton excitation imaging to quantify insulin exocytosis in intact pancreatic islets(Takahashi N et al. Science297,1349, 2002). This approach has been designated TEP(twophoton extracellular polar-tracer) imaging and TEPIQ(TEP imaging-based quantification) analysis (Kasai H.et al.J.Physiol.568,891,2005).TEP imaging is able to monitor reliably and with a relatively high time resolution(<1 s) individual insulin exocytic events in intact islet preparations and also allows analysis of the dynamics of the fusion pore that mediates exocytosis.<br /> He first examined the participation of PKA in GIE with the use of TEP imaging,in which insulin exocytic events were visualized by twophoton imaging of islets immersed in an extracellular solution containing the polar fluorescent tracersulforhodamine B(SRB). He detected exocytic events as discrete spots of fluorescence,which reflected diffusion of SRB into individual insulin granules via the fusion pore. The intensities of the spots of SRB fluorescence were consistent with them reflecting exocytosis of large dense-core vesicles. Moreover,he could detect all the exocytic events in the region of interest by this approach,because the measured rate of GIE was similar to the value obtained for mouse islets by radioimmunoassay.<br /> When he pretreated the cells with various inhibitors of PKA and then stimulated with 20 mM glucose,he found that the PKA inhibitors markedly and selectively inhibited the initial period(～250 s) of the first phase (<7 min) of GIE. In contrast,forskolin,which increases the cytosolic concentration of cAMP by activating adenyly1 cyclase,increased the extent of secretion both during the first phase of GIE and during the second phase, consistent with their previous observations. These results thus provided the notion that PKAis required for the initial period of the first phase of GIE.<br /> He also measured the possible effects of PKA inhibitors and forskolin on glucose-induced increases in [Ca<sup>2+</sup>]<sub>i</sub> in islets. Increases in [Ca<sup>2+</sup>]<sub>i</sub> were measured with either the high-affinity Ca<sup>2+</sup> indicator fura-2(K<sub>d</sub>=0.18 μM)or the low-affinity Ca<sup>2+</sup> indicator fura-4F(K<sub>d</sub>=1.16 μM);the latter was used in case fura2 became saturated during the physiological increases in [Ca<sup>2+</sup>]<sub>i</sub>. he found that neither the onset nor the maximal value apparent within 250s of the glucoseinduced increases in [Ca<sup>2+</sup>]<sub>i</sub> was affected by PKA inhibitors.<br /> Thus, he nest examined whether glucose and PKAmight directly potentiate CIE. for these experiments, Ca<sup>2+</sup>-dependent mechanisms were saturated by large increases in [Ca<sup>2+</sup>]<sub>i</sub> generated by phatolysis of the caged-Ca<sup>2+</sup> compound o-nitrophenyl-EGTA(NPE). He confirmed that irradiation with UV light induced an abrupt increase in [Ca<sup>2+</sup>]<sub>i</sub> of>20 μM. The latency histogram for the discrete exocyic events was fitted by a probability density function with two exponential components,consistent with the characteristics of CIE studied by amperometry.<br /> When islets were exposed to a high glucose concentation (20 mM) for only 1 min before uncaging of NPE, during which time glucose alone did not increase [Ca<sup>2+</sup>]<sub>i</sub>,the extent of CIE was markedly increased. Also,the glucose action was not mimicked by 2deoxy-D-glucose(20 mM), indicating that it required a metabolite of glucose. The effect ofglucose on CIE was abolished by pretreatment of islets with inhibitors of PKA. Furthermore, forskolin did not significantly affect CIE at the low glucose concentration of 2.8 mMbut potentiated the effect of 20 mM glucose on CIE. These results suggested that cAMP is necessary but not sufficient for the rapid effect of glucose on CIE, and that a metabolite of glucose, such as ATP, is required for this action>f glucose.<br /> Finally, he probed the dynamics of the exocytic fusion pore by simultaneous imaging of two fluorescent tracers with different molecular sizes, SRB (～1.4 nm) and 10-kDa fluorescein dextran (～6 nm). Transient opening of the initial small pore was detected in 6.5 % of events. The frequency of such transient opening was reduced to 3.8% in the presence of forskolin and increased to about 10% in the presence of PKA inhibitors. These results suggested that PKA affects the fusion pore when its diameter is < 6 nm, and that the action of PKA is mediated, at least in part, at the level of the fusion reaction.<br /> In conclusion,with the use of TEP imaging,he has now shown that a PKA-dependent mechanism,operative at the basal level of PKA activity,is important for the initial period of the first phase of GIE in mouse pancreatic islets. Furthermore, he found that PKA mediates rapid enhancement of CIE in islets only in the presence of high glucose concentration, indicating that PKA plays a glucose sensing role, specifically in the first phase of GIE. Given that the first phase of GIE is reduced in many individuals with type 2 diabetes mellitus from an early stage of the disease, impairment of this mechanism may contribute to the pathogenesis of this condition. |
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