Hideo Mogami

Sweet Taste Receptor Expressed in Pancreatic β-Cells Activates the Calcium and Cyclic AMP Signaling Systems and Stimulates Insulin Secretion

Background Sweet taste receptor is expressed in the taste buds and enteroendocrine cells acting as a sugar sensor. We investigated the expression and function of the sweet taste receptor in MIN6 cells and mouse islets. Methodology/Principal Findings The expression of the sweet taste receptor was determined by RT–PCR and immunohistochemistry. Changes in cytoplasmic Ca2+ ([Ca2+]c) and cAMP ([cAMP]c) were monitored in MIN6 cells using fura-2 and Epac1-camps. Activation of protein kinase C was monitored by measuring translocation of MARCKS-GFP. Insulin was measured by radioimmunoassay. mRNA for T1R2, T1R3, and gustducin was expressed in MIN6 cells. In these cells, artificial sweeteners such as sucralose, succharin, and acesulfame-K increased insulin secretion and augmented secretion induced by glucose. Sucralose increased biphasic increase in [Ca2+]c. The second sustained phase was blocked by removal of extracellular calcium and addition of nifedipine. An inhibitor of inositol(1, 4, 5)-trisphophate receptor, 2-aminoethoxydiphenyl borate, blocked both phases of [Ca2+]c response. The effect of sucralose on [Ca2+]c was inhibited by gurmarin, an inhibitor of the sweet taste receptor, but not affected by a Gq inhibitor. Sucralose also induced sustained elevation of [cAMP]c, which was only partially inhibited by removal of extracellular calcium and nifedipine. Finally, mouse islets expressed T1R2 and T1R3, and artificial sweeteners stimulated insulin secretion. Conclusions Sweet taste receptor is expressed in β-cells, and activation of this receptor induces insulin secretion by Ca2+ and cAMP-dependent mechanisms.

Glucagon-like peptide 1 activates protein kinase C through Ca2+-dependent activation of phospholipase C in insulin-secreting cells.

Although the stimulatory effect of glucagon-like peptide 1 (GLP-1), a cAMP-generating agonist, on Ca2+ signal and insulin secretion is well established, the underlying mechanisms remain to be fully elucidated. We recently discovered that Ca2+ influx alone can activate conventional protein kinase C (PKC) as well as novel PKC in insulin-secreting (INS-1) cells. Building on this earlier finding, here we examined whether GLP-1-evoked Ca2+ signaling can activate PKCα and PKCϵ at a substimulatory concentration of glucose (3 mm) in INS-1 cells. We first showed that GLP-1 translocated endogenous PKCα and PKCϵ from the cytosol to the plasma membrane. Next, we assessed the phosphorylation state of the PKC substrate, myristoylated alanine-rich C kinase substrate (MARCKS), by using MARCKS-GFP. GLP-1 translocated MARCKS-GFP to the cytosol in a Ca2+-dependent manner, and the GLP-1-evoked translocation of MARCKS-GFP was blocked by PKC inhibitors, either a broad PKC inhibitor, bisindolylmaleimide I, or a PKCϵ inhibitor peptide, antennapedia peptide-fused pseudosubstrate PKCϵ-(149–164) (antp-PKCϵ) and a conventional PKC inhibitor, Gö-6976. Furthermore, forskolin-induced translocation of MARCKS-GFP was almost completely inhibited by U73122, a putative inhibitor of phospholipase C. These observations were verified in two different ways by demonstrating 1) forskolin-induced translocation of the GFP-tagged C1 domain of PKCγ and 2) translocation of PKCα-DsRed and PKCϵ-GFP. In addition, PKC inhibitors reduced forskolin-induced insulin secretion in both INS-1 cells and rat islets. Thus, GLP-1 can activate PKCα and PKCϵ, and these GLP-1-activated PKCs may contribute considerably to insulin secretion at a substimulatory concentration of glucose.

Real-time analysis of platelet aggregation and procoagulant activity during thrombus formation in vivo

The exact mechanism of blood vessel thrombus formation remains to be defined. Here, we introduce a new approach to probe thrombus formation in blood vessels of living animals using intravital microscopy in green fluorescent protein (GFP)-transgenic mice to simultaneously monitor platelet aggregation and procoagulant activity. To this end, GFP-expressing platelets and annexin A5 labeled with a fluorescent dye were employed to visualize and analyze platelet aggregation and markers of procoagulant activity (platelet surface phosphatidylserine (PS)). Laser-induced thrombi increased and then decreased in size with time in vessels of living animals, whereas platelet surface PS initiated at the site of injury and then penetrated into the thrombus. PS-positive platelets were predominantly localized in the center of the thrombus, as was fibrin generation. The experimental system proposed here is a valuable tool not only for investigating mechanisms of thrombus formation but also to assess the efficacy of antithrombotic drugs within the vasculature.

Unique secretory dynamics of tissue plasminogen activator and its modulation by plasminogen activator inhibitor-1 in vascular endothelial cells

We analyzed the secretory dynamics of tissue plasminogen activator (tPA) in EA.hy926 cells, an established vascular endothelial cell (VEC) line producing GFP-tagged tPA, using total internal reflection-fluorescence (TIR-F) microscopy. tPA-GFP was detected in small granules in EA.hy926 cells, the distribution of which was indistinguishable from intrinsically expressed tPA. Its secretory dynamics were unique, with prolonged (> 5 minutes) retention of the tPA-GFP on the cell surface, appearing as fluorescent spots in two-thirds of the exocytosis events. The rapid disappearance (mostly by 250 ms) of a domain-deletion mutant of tPA-GFP possessing only the signal peptide and catalytic domain indicates that the amino-terminal heavy chain of tPA-GFP is essential for binding to the membrane surface. The addition of PAI-1 dose-dependently facilitated the dissociation of membrane-retained tPA and increased the amounts of tPA-PAI-1 high-molecular-weight complexes in the medium. Accordingly, suppression of PAI-1 synthesis in EA.hy926 cells by siRNA prolonged the dissociation of tPA-GFP, whereas a catalytically inactive mutant of tPA-GFP not forming complexes with PAI-1 remained on the membrane even after PAI-1 treatment. Our results provide new insights into the relationship between exocytosed, membrane-retained tPA and PAI-1, which would modulate cell surface-associated fibrinolytic potential.

Bimodal role of conventional protein kinase C in insulin secretion from rat pancreatic β cells

The present study was conducted to evaluate the role of conventional protein kinase C (PKC) in calcium‐evoked insulin secretion. In rat β cells transfected with green fluorescent protein‐tagged PKC‐α (PKC‐α–EGFP), a depolarizing concentration of potassium induced transient elevation of cytoplasmic free calcium ([Ca2+]c), which was accompanied by transient translocation of PKC‐α–EGFP from the cytosol to the plasma membrane. Potassium also induced transient translocation of PKC‐θ–EGFP, the C1 domain of PKC‐γ and PKC‐ɛ–GFP. A high concentration of glucose induced repetitive elevation of [Ca2+]c and repetitive translocation of PKC‐α–EGFP. Diazoxide completely blocked both elevation of [Ca2+]c and translocation of PKC‐α–EGFP. We then studied the role of conventional PKC in calcium‐evoked insulin secretion using rat islets. When islets were incubated for 10 min with high potassium, Gö‐6976, an inhibitor of conventional PKC, and PKC‐α pseudosubstrate fused to antennapedia peptide (Antp‐PKC19–31) increased potassium induced secretion. Similarly, insulin release induced by high glucose for 10 min was enhanced by Gö‐6976 and Antp‐PKC19–31. However, when islets were stimulated for 60 min with high glucose, both Gö‐6976 and Antp‐PKC19–31 reduced glucose‐induced insulin secretion. Similar results were obtained by transfection of dominant‐negative PKC‐α using adenovirus vector. Taken together, PKC‐α is activated when cells are depolarized by a high concentration of potassium or glucose. Conventional PKC is inhibitory on depolarization‐induced insulin secretion per se, but it also augments glucose‐induced secretion.

Activated protein C attenuates coagulation‐associated over‐expression of fibrinolytic activity by suppressing the thrombin‐dependent inactivation of PAI‐1

Summary.  Several activated coagulation factors have been reported to enhance fibrinolysis by neutralizing plasminogen activator inhibitor type 1 (PAI‐1) activity. We evaluated the physiological relevance of this mechanism using the euglobulin clot lysis time (ECLT) assay in the presence and absence of Ca2+, which is controlled by PAI‐1 and mimics physiological thrombolysis. We found that the ECLT (18.5 ± 0.6 h) was shortened by Ca2+ (5 mm) (6.6 ± 0.1 h). A significant difference was observed in thrombin generation by the presence of Ca2+ in the euglobulin fraction. Prothrombin was almost fully converted to thrombin within 15 min in the presence of Ca2+, whereas essentially no conversion was observed without Ca2+. The presence of activated protein C (aPC) suppressed thrombin generation, and attenuated the shortening of ECLT in a dose‐dependent manner, an effect enhanced by phospholipid and protein S. In the absence of Ca2+, aPC did not prolong the ECLT. After addition of biotin‐labeled recombinant PAI‐1 to the euglobulin fraction, PAI‐1 was cleaved to lower molecular weight forms only in the presence of Ca2+. This cleavage did not occur in the presence of aPC, suggesting that thrombin was the catalyst for PAI‐1 cleavage. The cleavage and inactivation of PAI‐1 by generated thrombin is proposed to be responsible for the shortening of ECLT by Ca2+ and for coagulation‐associated over‐expression of fibrinolysis. Under such conditions, aPC appeared to suppress thrombin generation and to normalize highly activated fibrinolysis.

Surface-retained tPA is essential for effective fibrinolysis on vascular endothelial cells

In a previous study, we demonstrated unique secretory dynamics of tissue plasminogen activator (tPA) in which tPA was retained on the cell surface in a heavy chain-dependent manner after exocytosis from secretory granules in vascular endothelial cells. Here, we examined how retained tPA expresses its enzymatic activity. Retained tPA effectively increased the lysine binding site-dependent binding of plasminogen on the cell surface and pericellular area; this was abolished by inhibition of enzymatic activity of either tPA or plasmin, which suggests that de novo generation of carboxyl-terminal lysine as a consequence of degradation of surface/pericellular proteins by plasmin is essential. Retained tPA initiated zonal clot lysis of a fibrin network that had been formed on vascular endothelial cells, which was preceded by the binding of plasminogen to the lysis front. Our results provide evidence that secreted and retained tPA is essential for maintaining both high fibrinolytic activity and effective clot lysis on the vascular endothelial cell surface.

Evaluation of Silicon Nitride as a Substrate for Culture of PC12 Cells: An Interfacial Model for Functional Studies in Neurons

Silicon nitride is a biocompatible material that is currently used as an interfacial surface between cells and large-scale integration devices incorporating ion-sensitive field-effect transistor technology. Here, we investigated whether a poly-L-lysine coated silicon nitride surface is suitable for the culture of PC12 cells, which are widely used as a model for neural differentiation, and we characterized their interaction based on cell behavior when seeded on the tested material. The coated surface was first examined in terms of wettability and topography using contact angle measurements and atomic force microscopy and then, conditioned silicon nitride surface was used as the substrate for the study of PC12 cell culture properties. We found that coating silicon nitride with poly-L-lysine increased surface hydrophilicity and that exposing this coated surface to an extracellular aqueous environment gradually decreased its roughness. When PC12 cells were cultured on a coated silicon nitride surface, adhesion and spreading were facilitated, and the cells showed enhanced morphological differentiation compared to those cultured on a plastic culture dish. A bromodeoxyuridine assay demonstrated that, on the coated silicon nitride surface, higher proportions of cells left the cell cycle, remained in a quiescent state and had longer survival times. Therefore, our study of the interaction of the silicon nitride surface with PC12 cells provides important information for the production of devices that need to have optimal cell culture-supporting properties in order to be used in the study of neuronal functions.

Binding of Thrombin-Activated Platelets to a Fibrin Scaffold through αIIbβ3 Evokes Phosphatidylserine Exposure on Their Cell Surface

Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose phosphatidylserine (PS) and fibrin accumulate only in the center of the thrombus but not in its periphery. To address the question how exposure of platelet anionic phospholipids is regulated within the thrombus, an in-vitro experiment using diluted platelet-rich plasma was employed, in which the fibrin network was formed in the presence of platelets, and PS exposure on the platelet surface was analyzed using Confocal Laser Scanning Microscopy. Almost all platelets exposed PS after treatment with tissue factor, thrombin or ionomycin. Argatroban abrogated fibrin network formation in all samples, however, platelet PS exposure was inhibited only in tissue factor- and thrombin-treated samples but not in ionomycin-treated samples. FK633, an αIIbβ3 antagonist, and cytochalasin B impaired platelet binding to the fibrin scaffold and significantly reduced PS exposure evoked by thrombin. Gly-Pro-Arg-Pro amide abrogated not only fibrin network formation, but also PS exposure on platelets without suppressing platelet binding to fibrin/fibrinogen. These results suggest that outside-in signals in platelets generated by their binding to the rigid fibrin network are essential for PS exposure after thrombin treatment.

A technique for monitoring multiple signals with a combination of prism-based total internal reflection fluorescence microscopy and epifluorescence microscopy.

Physiological phenomena are regulated by multiple signal pathways upon receptor stimulation. Here, we have introduced a new technique with a combination of prism-based total internal reflection fluorescence microscopy (PBTIRFM) and epifluorescence microscopy (EPI) to simultaneously monitor multiple signal pathways. This instrumentation allows us to visualize three signal pathways, Ca2+, cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), and diacylglycerol (DAG)/protein kinase C (PKC) signals in living cells. Three fluorescent indicators were employed for this purpose: (1) Fura-2 AM as a calcium sensor; (2) Epac1-camp, a cyan fluorescent protein–yellow fluorescent protein fluorescence resonance energy transfer-based cAMP indicator, as a cAMP sensor; and (3) C1-tagged monomeric red fluorescent protein, a tandem DAG-binding domain of PKC γ, as a DAG sensor or myristoylated alanine-rich C kinase substrate-tagged DsRed for the PKC activation pathway. The DAG signal was monitored by PBTIRFM, whereas the Ca2+ and cAMP signals were monitored by EPI. Adenosine trisphosphate resulted in generation of all three second messengers in triple probe-loaded Cos-7 cells. The spectral overlap between these signal probes was evaluated by means of linear unmixing. Forskolin also evoked Ca2+, cAMP/PKA, and DAG/PKC signals whereas acetylcholine activated Ca2+ and DAG/PKC signals as well as inhibiting cAMP generation in triple probe-loaded insulin-secreting cells. Thus, the optical observation system combining PBTIRFM and EPI offers a great advance in analyzing interplay of multiple signaling pathways, such as these second messengers, upon G-protein-coupled receptor stimulation in living cells.