{"_buckets": {"deposit": "13f02e55-e66e-4ecb-aee3-956a145777e7"}, "_deposit": {"created_by": 1, "id": "1171", "owners": [1], "pid": {"revision_id": 0, "type": "depid", "value": "1171"}, "status": "published"}, "_oai": {"id": "oai:ir.soken.ac.jp:00001171", "sets": ["22"]}, "author_link": ["0", "0", "0"], "item_1_biblio_info_21": {"attribute_name": "書誌情報（ソート用）", "attribute_value_mlt": [{"bibliographicIssueDates": {"bibliographicIssueDate": "2006-03-24", "bibliographicIssueDateType": "Issued"}, "bibliographic_titles": [{}]}]}, "item_1_creator_2": {"attribute_name": "著者名", "attribute_type": "creator", "attribute_value_mlt": [{"creatorNames": [{"creatorName": "畠山, 裕康"}], "nameIdentifiers": [{"nameIdentifier": "0", "nameIdentifierScheme": "WEKO"}]}]}, "item_1_creator_3": {"attribute_name": "フリガナ", "attribute_type": "creator", "attribute_value_mlt": [{"creatorNames": [{"creatorName": "ハタケヤマ, ヒロヤス"}], "nameIdentifiers": [{"nameIdentifier": "0", "nameIdentifierScheme": "WEKO"}]}]}, "item_1_date_granted_11": {"attribute_name": "学位授与年月日", "attribute_value_mlt": [{"subitem_dategranted": "2006-03-24"}]}, "item_1_degree_grantor_5": {"attribute_name": "学位授与機関", "attribute_value_mlt": [{"subitem_degreegrantor": [{"subitem_degreegrantor_name": "総合研究大学院大学"}]}]}, "item_1_degree_name_6": {"attribute_name": "学位名", "attribute_value_mlt": [{"subitem_degreename": "博士（理学）"}]}, "item_1_description_1": {"attribute_name": "ID", "attribute_value_mlt": [{"subitem_description": "2006056", "subitem_description_type": "Other"}]}, "item_1_description_12": {"attribute_name": "要旨", "attribute_value_mlt": [{"subitem_description": "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\u003cSUP\u003e+\u003c/SUP\u003e(K\u003cSUB\u003eATP\u003c/SUB\u003e) 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\u003e-50 m V results in activation of voltage-dependent Ca\u003csup\u003e2+\u003c/sup\u003e channels and an uncrease in [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e that triggers insulin exocytosis.The K\u003csub\u003eATP\u003c/sub\u003e channels and Ca\u003csup\u003e2+\u003c/sup\u003e-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.\u003cbr /\u003e     Exocytosis in many secretory cell types and neurons is regulated by both Ca\u003csup\u003e2+\u003c/sup\u003e and cAMP. Their reserch group has previously shown that cytosolic cAMP potentiates Ca\u003csup\u003e2+\u003c/sup\u003e-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\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e induced by photolysis of a caged-Ca\u003csup\u003e2+\u003c/sup\u003ecompound,thereby bypassing the K\u003cSUB\u003eATP\u003c/SUB\u003e channel-dependent mechanism. They found that CIE was augmented by \u003csub\u003ec\u003c/sub\u003eAMP 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.\u003cbr /\u003e     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(\u003c1 s) individual insulin exocytic events in intact islet preparations and also allows analysis of the dynamics of the fusion pore that mediates exocytosis.\u003cbr /\u003e     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.\u003cbr /\u003e     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 (\u003c7 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.\u003cbr /\u003e     He also measured the possible effects of PKA inhibitors and forskolin on glucose-induced increases in [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e in islets.  Increases in  [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e were measured with either the high-affinity Ca\u003csup\u003e2+\u003c/sup\u003e indicator fura-2(K\u003csub\u003ed\u003c/sub\u003e=0.18 μM)or the low-affinity Ca\u003csup\u003e2+\u003c/sup\u003e indicator fura-4F(K\u003csub\u003ed\u003c/sub\u003e=1.16 μM);the latter was used in case fura2 became saturated during the physiological increases in [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e.  he found that neither the onset nor the maximal value apparent within 250s of the glucoseinduced increases in [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e was affected by PKA inhibitors.\u003cbr /\u003e     Thus, he nest examined whether glucose and PKAmight directly potentiate CIE.  for these experiments, Ca\u003csup\u003e2+\u003c/sup\u003e-dependent mechanisms were saturated by large increases in [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e generated by phatolysis of the caged-Ca\u003csup\u003e2+\u003c/sup\u003e compound o-nitrophenyl-EGTA(NPE).  He confirmed that irradiation with UV light induced an abrupt increase in [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e of\u003e20 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.\u003cbr /\u003e     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\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e,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\u003ef glucose.\u003cbr /\u003e      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 \u003c 6 nm, and that the action of PKA is mediated, at least in part, at the level of the fusion reaction.\u003cbr /\u003e     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.", "subitem_description_type": "Other"}]}, "item_1_description_7": {"attribute_name": "学位記番号", "attribute_value_mlt": [{"subitem_description": "総研大甲第969号", "subitem_description_type": "Other"}]}, "item_1_select_14": {"attribute_name": "所蔵", "attribute_value_mlt": [{"subitem_select_item": "有"}]}, "item_1_select_16": {"attribute_name": "複写", "attribute_value_mlt": [{"subitem_select_item": "application/pdf"}]}, "item_1_select_8": {"attribute_name": "研究科", "attribute_value_mlt": [{"subitem_select_item": "生命科学研究科"}]}, "item_1_select_9": {"attribute_name": "専攻", "attribute_value_mlt": [{"subitem_select_item": "20 生理科学専攻"}]}, "item_1_text_10": {"attribute_name": "学位授与年度", "attribute_value_mlt": [{"subitem_text_value": "2005"}]}, "item_creator": {"attribute_name": "著者", "attribute_type": "creator", "attribute_value_mlt": [{"creatorNames": [{"creatorName": "HATAKEYAMA, Hiroyasu", "creatorNameLang": "en"}], "nameIdentifiers": [{"nameIdentifier": "0", "nameIdentifierScheme": "WEKO"}]}]}, "item_files": {"attribute_name": "ファイル情報", "attribute_type": "file", "attribute_value_mlt": [{"accessrole": "open_date", "date": [{"dateType": "Available", "dateValue": "2016-02-17"}], "displaytype": "simple", "download_preview_message": "", "file_order": 0, "filename": "甲969_要旨.pdf", "filesize": [{"value": "362.2 kB"}], "format": "application/pdf", "future_date_message": "", "is_thumbnail": false, "licensetype": "license_11", "mimetype": "application/pdf", "size": 362200.0, "url": {"label": "要旨・審査要旨", "url": "https://ir.soken.ac.jp/record/1171/files/甲969_要旨.pdf"}, "version_id": "773b99bc-4da4-4558-a90d-7a5a875a3859"}, {"accessrole": "open_date", "date": [{"dateType": "Available", "dateValue": "2016-02-17"}], "displaytype": "simple", "download_preview_message": "", "file_order": 1, "filename": "甲969_本文.pdf", "filesize": [{"value": "3.1 MB"}], "format": "application/pdf", "future_date_message": "", "is_thumbnail": false, "licensetype": "license_11", "mimetype": "application/pdf", "size": 3100000.0, "url": {"label": "本文", "url": "https://ir.soken.ac.jp/record/1171/files/甲969_本文.pdf"}, "version_id": "57c2ee4b-dc84-4f84-9a34-155ef7275bc6"}]}, "item_language": {"attribute_name": "言語", "attribute_value_mlt": [{"subitem_language": "eng"}]}, "item_resource_type": {"attribute_name": "資源タイプ", "attribute_value_mlt": [{"resourcetype": "thesis", "resourceuri": "http://purl.org/coar/resource_type/c_46ec"}]}, "item_title": "Rapid glucose sensing by protein kinase A for exocytosis in mouse pancreatic islets", "item_titles": {"attribute_name": "タイトル", "attribute_value_mlt": [{"subitem_title": "Rapid glucose sensing by protein kinase A for exocytosis in mouse pancreatic islets"}, {"subitem_title": "Rapid glucose sensing by protein kinase A for exocytosis in mouse pancreatic islets", "subitem_title_language": "en"}]}, "item_type_id": "1", "owner": "1", "path": ["22"], "permalink_uri": "https://ir.soken.ac.jp/records/1171", "pubdate": {"attribute_name": "公開日", "attribute_value": "2010-02-22"}, "publish_date": "2010-02-22", "publish_status": "0", "recid": "1171", "relation": {}, "relation_version_is_last": true, "title": ["Rapid glucose sensing by protein kinase A for exocytosis in mouse pancreatic islets"], "weko_shared_id": 1}