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The role of type I diacylglycerol kinases (DGKs) in the regulation of insulin secretion was investigated in MIN6 β-cells. In intracellular Ca2+ concentration ([Ca2+]i) measurement experiments, 1 μM R59949, a type I DGK inhibitor, and 10 μM DiC8, a diacylglycerol (DAG) analog, amplified 22.2 mM glucose-induced [Ca2+]i oscillations in a protein kinase C (PKC)-dependent manner, whereas 10 μM R59949 and 100 μM DiC8 decreased [Ca2+]i independent of PKC. High concentrations of R59949 and DiC8 attenuated voltage-dependent Ca2+ channel currents. According to these results, 22.2 mM glucose-stimulated insulin secretion (GSIS) was potentiated by 1 μM R59949 but suppressed by 10 μM of the same. The DGKα inhibitor R59022 showed a similar dual effect. Conversely, DiC8 at 10 and 100 μM potentiated GSIS, although 100 μM DiC8 decreased [Ca2+]i. These results suggest that DAG accumulated through declined type I DGK activity shows a dual effect on insulin secretion depending on the degree of accumulation; a mild DAG accumulation induces a PKC-dependent stimulatory effect on insulin secretion, whereas an excessive DAG accumulation suppresses it in a PKC-independent manner, possibly via attenuation of VDCC activity. Keywords: Diacylglycerol, Diacylglycerol kinase, Insulin secretion, Intracellular Ca2+ concentration, Voltage-dependent Ca2+ channel

Although an aberrant reduction in pancreatic β-cell mass contributes to the pathogenesis of diabetes, the mechanism underlying the regulation of β-cell mass is poorly understood. Here, we show that diacylglycerol kinase δ (DGKδ) is a key enzyme in the regulation of β-cell mass. DGKδ expression was detected in the nucleus of β-cells. We developed β-cell-specific DGKδ knockout (βDGKδ KO) mice, which showed lower blood glucose, higher plasma insulin levels, and better glucose tolerance compared to control mice. Moreover, an increased number of small islets and Ki-67-positive islet cells, as well as elevated cyclin B1 expression in the islets, were detected in the pancreas of βDGKδ KO mice. DGKδ knockdown in the β-cell line MIN6 induced significant increases in bromodeoxyuridine (BrdU) incorporation and cyclin B1 expression. Finally, we confirmed that streptozotocin-induced hyperglycemia and β-cell loss were alleviated in βDGKδ KO mice. Thus, suppressing the expression or enzymatic activity of DGKδ that functions as a suppressor of β-cell proliferation could be a novel therapeutic approach to increase β-cell mass for the treatment of diabetes.

The number of patients with type 2 diabetes mellitus (T2DM) has rapidly increased, especially in East and Southeast Asia. In these areas, in general, people have especially vulnerable β-cells and insulin secretion deficiency and reduced β-cell mass are the primary cause of T2DM. Therefore, the alleviation of such β-cell dysfunction would provide therapeutic approaches to prevent the development of T2DM. Nobiletin, a polymethoxylated flavonoid found in citrus fruits, has been shown to improve obesity and insulin resistance in T2DM model mice. We focused on β-cells and investigated the effects of nobiletin on insulin secretion and β-cell apoptosis. In β-cell line INS-1, nobiletin increased glucose-induced insulin secretion (GSIS) in a concentration-dependent manner, which was inhibited by an Epac inhibitor. In addition, nobiletin at 10 μM inhibited thapsigargin-induced apoptosis, which was inhibited by a PKA inhibitor. Nobiletin also suppressed thapsigargin-induced increases in cleaved caspase-3 and phosphorylated JNK. Thus, nobiletin is suggested to promote GSIS and prevent ER stress-induced β-cell apoptosis, which are mediated via Epac and PKA-dependent pathways, respectively. In summary, nobiletin is suggested to exhibit insulinotropic and anti-apoptotic effects on β-cells, which are one of the causes of its anti-diabetic effect. Moreover, nobiletin seems to be able to alleviate the development of T2DM by protecting β-cells from apoptosis.

To determine the mechanism underlying anti-diabetic effects of nobiletin, a citrus flavonoid, the effects of nobiletin on insulin secretion and β-cell apoptosis were investigated in the INS-1D β-cell line. Nobiletin increased glucose-induced insulin secretion (GSIS), and inhibited thapsigargin-induced apoptosis. Nobiletin also potentiated forskolin-induced cAMP accumulation. The insulinotropic effect was significantly suppressed by the EPAC inhibitor ESI-09, but not by the PKA inhibitor KT-5720. Conversely, the anti-apoptotic effect was inhibited by KT-5720, but not by ESI-09. Nobiletin also inhibited thapsigargin-induced increases in cleaved caspase-3 and JNK phosphorylation, but had no effect on the expression of CHOP. Similar insulinotropic and anti-apoptotic effects were also observed with the extract from Ponkan peels containing nobiletin. These results suggest that nobiletin exerts anti-diabetic effects by increasing GSIS and preventing β-cell apoptosis via cAMP-dependent mechanisms; the former is primarily mediated by EPAC, whereas the latter is due to PKA-induced alleviation of JNK-mediated ER stress.

We accomplished a divergent synthesis of sudachitin (2), a polymethoxyflavone isolated from citrus fruits, and six derivatives from acetophenone 9, which was an intermediate in our previous synthesis of nobiletin (1). Compound 2 enhanced glucose-induced insulin secretion in INS-1D cells, but was less potent than 1. Compared with 1, compound 2 and two derivatives were more potent inhibitors of cAMP-specific phosphodiesterase.

The appropriate secretion of insulin from pancreatic β-cells is essential for regulating blood glucose levels. Glucose-stimulated insulin secretion (GSIS) involves the following steps: Glucose uptake by pancreatic β-cells is metabolized to produce ATP. Increased ATP levels result in the closure of ATP-sensitive K(+) (KATP) channels, resulting in membrane depolarization that activates voltage-dependent Ca(2+) channels to subsequently trigger insulin secretion. In addition to this primary mechanism through KATP channels, insulin secretion is regulated by cyclic AMP and diacylglycerol (DAG), which mediate the effects of receptor agonists such as GLP-1 and acetylcholine. Glucose by itself can also increase the levels of these second messengers. Recently, we have shown an obligatory role of diacylglycerol kinase (DGK), an enzyme catalyzing the conversion of DAG to phosphatidic acid, in GSIS. Of the 10 known DGK isoforms, we focused on type-I DGK isoforms (i.e., DGKα, DGKβ, and DGKγ), which are activated by Ca(2+). The protein expression of DGKα and DGKγ was detected in mouse pancreatic islets and the pancreatic β-cell line MIN6. Depletion of these DGKs by a specific inhibitor or siRNA decreased both [Ca(2+)]i and insulin secretion in MIN6 cells. Similar [Ca(2+)]i responses were induced by DiC8, a membrane-permeable DAG analog. These results suggest that DGKα and DGKγ play crucial roles in insulin secretion, and that their depletion impairs insulin secretion through DAG accumulation. In this article, we review the current understanding of the roles of DAG- and DGK-signaling in pancreatic β-cells, and discuss their pathophysiological roles in the progression of type-2 diabetes.

Sialidase cleaves sialic acid residues from a sialoglycoconjugate: oligosaccharides, glycolipids and glycoproteins that contain sialic acid. Histochemical imaging of the mouse pancreas using a benzothiazolylphenol-based sialic acid derivative (BTP3-Neu5Ac), a highly sensitive histochemical imaging probe used to assess sialidase activity, showed that pancreatic islets have intense sialidase activity. The sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA) remarkably enhances glutamate release from hippocampal neurons. Since there are many similar processes between synaptic vesicle exocytosis and secretory granule exocytosis, we investigated the effect of DANA on insulin release from β-cells. Insulin release was induced in INS-1D cells by treatment with 8.3 mM glucose, and the release was enhanced by treatment with DANA. In a mouse intraperitoneal glucose tolerance test, the increase in serum insulin levels was enhanced by intravenous injection with DANA. However, under fasting conditions, insulin release was not enhanced by treatment with DANA. Calcium oscillations induced by 8.3 mM glucose treatment of INS-1D cells were not affected by DANA. Blood insulin levels in sialidase isozyme Neu3-deficient mice were significantly higher than those in WT mice under ad libitum feeding conditions, but the levels were not different under fasting conditions. These results indicate that DANA is a glucose-dependent potentiator of insulin secretion. The sialidase inhibitor may be useful for anti-diabetic treatment with a low risk of hypoglycemia.

Pathophysiologically, excessive accumulation of diacylglycerol (DAG), a lipid signal messenger, in pancreatic β-cells has been shown to lead to the insufficiency of insulin secretion. Thus, the regulation of intracellular DAG levels is expected to be an important factor in maintaining β-cell function. The intracellular DAG levels are strictly controlled by DAG kinases (DGKs). We have previously shown that DGKα and γ, type I DGK isoforms, are highly expressed in mouse β-cells, and that DAG accumulation resulting from dysfunction of these DGKs reduces insulin secretion. In the present study, we investigated the effect of type I DGK dysfunction on the regulation of intracellular Ca2+ concentration ([Ca2+]i), which is correlated with insulin secretion, in the β-cell line MIN6B. We also analyzed changes in expression levels of DGKα and γ in β-cells of type 2 diabetes model NSY mice. The amplitude of glucose-induced [Ca2+]i oscillations was increased by 1 μM R59949, a type I DGK inhibitor, and by 10 μM DiC8, a DAG analog. These effects were abolished by Ro31-8220, a protein kinase C (PKC) inhibitor. In contrast, 10 μM R59949 and 100 μM DiC8 suppressed the glucose-induced [Ca2+]i elevation, which was insensitive to Ro31-8220, and furthermore reduced voltage-dependent Ca2+ channel (VDCC) currents, which were measured by patch-clamp recording, implying that DAG accumulation due to type I DGK dysfunction suppresses VDCC, thereby reducing [Ca2+]i in β-cells. Real-time qPCR showed that the expression level of DGKα and γ was lower in islets from NSY mice compared with control mice. Taken together, the results obtained here suggest that DAG accumulation due to type I DGK dysfunction has contradictory dual effect in β-cells depending on the degree of accumulation; mild accumulation PKC-dependently induces a stimulatory effect on insulin secretion, whereas excessive accumulation suppresses it independently of PKC. DGK is likely a key molecule for the progression of type 2 diabetes. Disclosure T. Sawatani: None. Y.K. Kaneko: None. T. Ishikawa: None.

Our previous study using apoptosis analysis suggested that Ca(2+) release through inositol 1,4,5-trisphosphate (IP3) receptors and the subsequent Ca(2+) influx through store-operated channels (SOCs) constitute a triggering signal for H2O2-induced β-cell apoptosis. In the present study, we further examined the obligatory role of early Ca(2+) responses in β-cell apoptosis induction. H2O2 induced elevation of the cytosolic Ca(2+) concentration ([Ca(2+)]c) consisting of two phases: an initial transient [Ca(2+)]c elevation within 30 min and a slowly developing one thereafter. The first phase was almost abolished by 2-aminoethoxydiphenylborate (2-APB), which blocks IP3 receptors and cation channels including SOCs, while the second phase was only partially inhibited by 2-APB. The inhibition by 2-APB of the second phase was not observed when 2-APB was added 30 min after the treatment with H2O2. 2-APB also largely inhibited elevation of the mitochondrial Ca(2+) concentration ([Ca(2+)]m) induced by H2O2 when 2-APB was applied simultaneously with H2O2, but not when applied 30 min after H2O2 application. In addition, 2-APB inhibited the release of mitochondrial cytochrome c to the cytosol induced by H2O2 when 2-APB was applied simultaneously with H2O2 but not 30 min post-treatment. H2O2-induced [Ca(2+)]m elevation and cell death were not inhibited by Ru360, an inhibitor of the mitochondrial calcium uniporter (MCU). These results suggest that the H2O2-induced initial [Ca(2+)]c elevation, occurring within 30 min and mediated by Ca(2+) release through IP3 receptors and subsequent Ca(2+) influx through SOCs, leads to [Ca(2+)]m elevation, possibly through a mechanism independent of MCU, thereby inducing cytochrome c release and consequent apoptosis.

Diacylglycerol (DAG) is a lipid signal messenger and plays a physiological role in β-cells. Since defective glucose homeostasis increases de novo DAG synthesis, DAG may also contribute to β-cell dysfunction in type 2 diabetes. Although the primary function of DAG is to activate protein kinase C (PKC), the role of PKC in insulin secretion is controversial: PKC has been reported to act as both a positive and negative regulator of insulin secretion. In addition to the PKC pathway, DAG has also been shown to mediate other pathways such as the Munc-13-dependent pathway in β-cells. The intracellular levels of DAG are strictly regulated by diacylglycerol kinase (DGK); however, the role of DGK in β-cells and their involvement in β-cell failure in type 2 diabetes remain to be fully elucidated. We have recently reported the roles of type I DGK, DGKα and γ, in insulin secretion from β-cells. DGKα and γ were activated by glucose or high K(+) stimulation in β-cells, and the inhibition of the DGKs by a type I DGK inhibitor or by knockdown with small interfering RNA (siRNA) decreased insulin secretion. Thus, DGKα and γ are suggested to be activated in response to elevated [Ca(2+)]i in β-cells and to act as positive regulators of insulin secretion. In this article, we review the current understanding of the roles of DAG and DGK in β-cell function and their involvement in the development of β-cell dysfunction in type 2 diabetes.

Background & Aims : One of the roles of the nurse who comes into contact with a patient at the end of life is to attend to a person who, although living, faces imminent death. Purposeful touch is an example of an active support method. This means that the nurse’s act of touching is intended to help the patient become more comfortable, even if only slightly, and has the objective of mentally and physically healing the patient. To date, no studies concerning how patients experience touch have been reported. The aim of the present study was to clarify the process of end-of-life cancer patients finding meaning in receiving touch intervention. Methods : The study participants comprised 12 end-of-life cancer patients who underwent touch intervention by nurses for 20 minutes, two or three times per week. A semi-structured interview was carried out following each intervention, and the results of the patients’ responses were analyzed according to the modified grounded theory approach. Salivary secretory immunoglobulin A (s-IgA) concentration and a visual analogue scale (VAS) on comfort level were evaluated both before and after the series of planned interventions. Results : End-of-life cancer patients passively accepted touch intervention, and subsequently rated touch to have value. They were also given a boost of power to live in comfort, an emotion that occurs as a result of continuous touch intervention. Finally, they experienced a series of processes to surrender to touch. No significant differences were found in IgA concentration in saliva taken before and after the series of planned interventions. The VAS on comfort level after the series of interventions was significantly higher than that before the interventions. Conclusions : Continuous purposeful touch intervention may positively affect patients’ psychological comfort. Furthermore, patients’ intentions to allow themselves to undergo touch intervention by nurses might positively affect their will to survive.

Diacylglycerol kinase (DGK) catalyzes the conversion of diacylglycerol (DAG) to phosphatidic acid. This study investigated the expression and function of DGK in pancreatic β-cells. mRNA expression of type I DGK isoforms (α, β, γ) was detected in mouse pancreatic islets and the β-cell line MIN6. Protein expression of DGKα and DGKγ was also detected in mouse β-cells and MIN6 cells. The type I DGK inhibitor R59949 inhibited high K(+)- and glucose-induced insulin secretion in MIN6 cells. Moreover, single knockdown of DGKα or DGKγ by small interfering RNA slightly but significantly decreased glucose- and high K(+)-induced insulin secretions, and the double knockdown further decreased them to the levels comparable with those induced by R59949. R59949 and DiC8, a membrane permeable DAG analog, decreased intracellular Ca(2+) concentration elevated by glucose and high K(+) in MIN6 cells. Real-time imaging in MIN6 cells expressing green fluorescent protein-tagged DGKα or DGKγ showed that the DGK activator phorbol 12-myristate 13-acetate rapidly induced translocation of DGKγ to the plasma membrane, whereas high K(+) slowly translocated DGKα and DGKγ to the plasma membrane. R59949 increased the DAG content in MIN6 cells when stimulated with high KCl, whereas it did not increase the DAG content but decreased the phosphatidic acid content when stimulated with high glucose. Finally, R59949 was confirmed to inhibit high K(+)-induced insulin secretion from mouse islets and glucose-induced insulin secretion from rat islets. These results suggest that DGKα and DGKγ are present in β-cells and that the depression of these DGKs causes a decrease in intracellular Ca(2+) concentration, thereby reducing insulin secretion.

An involvement of inducible nitric oxide (NO) synthase (NOS) in pancreatic β-cell degeneration during the process of type 1 diabetes has been well discussed. Recently, there is growing evidence for pivotal roles of constitutive NOS (cNOS) in β-cells; the presence of NOS1 and NOS3 in pancreatic β-cells and the effects of low-concentration NO, which is assumed to be derived from cNOS, on β-cell functions have been reported. However, the roles of cNOS-derived NO in β-cells are still under debate. One of the reasons seems to be that NO has multiple biological activities, which are dependent on its concentration. In β-cells, NO has been shown to exert positive and negative regulation of insulin secretion and anti- and pro-apoptotic activities, which is likely to be dependent on concentrations. In this review article, we will describe the current understanding of the roles of NO in pancreatic β-cells, especially focusing on cNOS-derived NO and its differential roles depending on concentrations. Keywords:: nitric oxide, pancreatic islet, β-cell, insulin secretion, apoptosis

Pancreatic β-cell dysfunction is a major feature of type 2 diabetes. Therefore maintenance of β-cell function is essential to preventing the onset and progression of type 2 diabetes. To elucidate the mechanisms underlying the regulation of insulin secretion and β-cell survival, we particularly focused on the roles of gasotransmitters in pancreatic β-cells. Nitric oxide (NO) and hydrogen sulfide (H2S) are recognized as toxic gases. However, they are also vital physiological and pathophysiological mediators in various cell types. NO, generated from L-arginine by reactions catalyzed by NO synthases, is a well-known neurotransmitter and smooth muscle relaxation factor. In pancreatic β-cells, induction of nitric oxide synthase 2 (NOS2) by inflammatory cytokines generates a large amount of NO, which contributes to the impairment of β-cell function and induction of β-cell apoptosis, which are, in turn, involved in the development of type 1 diabetes. In contrast, a physiological level of NO, generated by constitutive NOS (cNOS), acts as a positive or negative regulator of insulin secretion and β-cell survival, depending on concentration. H2S generated from L-cysteine has been shown to play a role of neuromodulator, and this gas possesses cytoprotective properties. In pancreatic β-cells, H2S functions as a potent suppressor of insulin secretion. Furthermore, chronic exposure to high glucose induces H2S production by increasing the expression of a H2S-producing enzyme, cystathionine γ-lyase (CSE). H2S generated by CSE prevents β-cell apoptosis via an antioxidant mechanism. Here, we describe the current understanding of the function of gasotransmitters in regulating insulin secretion and pancreatic β-cell survival.

Recently, endothelial dysfunction induced by an uncoupling of vascular endothelial growth factor (VEGF) and nitric oxide has been implicated in the pathogenesis of diabetic nephropathy (DN). Investigating the pathogenesis of DN has been limited, however, because of the lack of animal models that mimic the human disease. In this report, pancreatic beta cell-specific calmodulin-overexpressing transgenic (CaMTg) mice, a potential new model of DN, are characterized with particular emphasis on VEGF and related molecules. CaMTg mice developed hyperglycemia at 3 wk and persistent proteinuria by 3 mo. Morphometric analysis showed considerable increases in the glomerular and mesangial areas with deposition of type IV collagen. Moreover, the pathologic hallmarks of human DN (mesangiolysis, Kimmelstiel-Wilson-like nodular lesions, exudative lesions, and hyalinosis of afferent and efferent arteries with neovascularization) were observed. In addition, increased VEGF expression was associated with an increased number of peritubular capillaries. Expression of endothelial nitric oxidase synthase was reduced and that of VEGF was markedly elevated in CaMTg mice kidney compared with nontransgenic mice. No differences in VEGF receptor-1 or VEGF receptor-2 expression were observed between CaMTg mice and nontransgenic kidneys. In summary, CaMTg mice develop most of the distinguishing lesions of human DN, and the elevated VEGF expression in the setting of diminished endothelial nitric oxide synthase expression may lead to endothelial proliferation and dysfunction. This model may prove useful in the study of the pathogenesis and treatment of DN.

これまで, リンパ浮腫への有効な治療法がなく, がん治療後の続発性リンパ浮腫に悩む患者が相当数いた. われわれは, 慢性的に経過しているリンパ浮腫患者に, フェルディー式の医療徒手リンパドレナージ (ML) 手技を含む複合的リンパ療法を行っている. この療法によって, 浮腫の改善と, それに伴うADLの改善が見られた. さらにボディーイメージの改善によって社会参加を取り戻しQOLの向上をもたらすことが出来た2事例を紹介する. 認定資格者によるML手技の適用と共に, 圧迫療法としての弾性包帯と弾性圧迫衣の活用, さらに自ら運動療法に取り組む過程で, セルフケア管理が出来るようになることを目指している. 継続的に自己管理が出来るようになると, 繰り返す蜂窩識炎を回避して, 家族と共に生活の質を維持していくことが可能である.

【背景・目的】 口腔がんの術後に生じる顔貌の変化や口腔機能障害は, 患者の日常生活に大きな影響を与えている. 本研究の目的は, 口腔がん術後患者における退院後の生活実態や患者の抱える問題について明らかにすることである. 【対象と方法】 対象は, 口腔がんの術後, 顔貌の変化や口腔機能障害をきたした歯科口腔外科通院中の患者5名. 面接から, 退院後の生活や患者の抱える問題について聴取し, K-J法により質的帰納的分析を行った. 【結 果】 患者の語りは, 共に強く関連しあう『からだの状態』『こころの状態』『くらしの状態』と, そのそれぞれに関連する『医療者からのサポート』に大きく分類された. 【結 語】 患者は顔貌の変化や口腔機能障害などのつらさを抱えているが, その障害と共に生きるための工夫や技術を身につけて適応している. 医療者の声掛けや傾聴, 親身で一生懸命な姿勢, 安心できる関係の構築, 専門的知識に裏づけられた十分な説明が心の支えになっていた. 適応や役割遂行のための援助が重要であることが示唆された.

The effects of green tea catechins on glucose-stimulated insulin secretion (GSIS) were investigated in the β-cell line INS-1D. Epigallocatechin gallate (EGCG) at 10 µM or gallocatechin gallate (GCG) at 30 µM caused significant inhibitory effects on GSIS, and each of these at 100 µM almost abolished it. In contrast, epicatechin (EC) or catechin (CA) had no effect on GSIS at concentrations up to 100 µM. We thus investigated the structure-activity relationship by using epigallocatechin (EGC) and gallocatechin (GC) containing a trihydroxyl group in the B-ring, and epicatechin gallate (ECG) and catechin gallate (CG) containing the gallate moiety. EGC, GC, and ECG caused an inhibition of GSIS, although significant effects were obtained only at 100 µM. At this concentration, EGC almost abolished GSIS, whereas GC and ECG partially inhibited it. In contrast, CG did not affect GSIS at concentrations up to 100 µM. EGCG also abolished the insulin secretion induced by tolbutamide, an ATP-sensitive K(+) channel blocker, and partially inhibited that induced by 30 mM K(+). Moreover, EGCG, but not EC, inhibited the oscillation of intracellular Ca(2+) concentration induced by 11.1 mM glucose. These results suggest that some catechins at supraphysiological concentrations have inhibitory effects on GSIS, the potency of which depends on their structure; the order of potency was EGCG>GCG>EGC>GC≈ECG. The inhibitory effects seem to be mediated by the inhibition of voltage-dependent Ca(2+) channels, which is caused, at least in part, by membrane hyperpolarization resulting from the activation of K(+) channels.

Hydrogen sulfide (H(2)S) was historically recognized as a toxic gas generated by natural resources. However, its enzymatic production from L-cysteine has recently been demonstrated in mammals. Cystathionine beta-synthase and cystathionine gamma-lyase, both of which can produce H(2)S, were expressed in mouse pancreatic islet cells and the beta-cell line, MIN6. L-cysteine and the H(2)S donor NaHS inhibited glucose-induced insulin release from islets and MIN6 cells. These inhibitory effects were reproduced when insulin release was stimulated by alpha-ketoisocaproate, tolbutamide, or high K+. L-cysteine and NaHS inhibited glucose-potentiated insulin release in the copresence of diazoxide and high K+. Real-time imaging of intracellular Ca2+ concentration ([Ca2+](i)) demonstrated that both L-cysteine and NaHS reversibly suppressed glucose-induced [Ca2+](i) oscillation in a single beta-cell without obvious changes in the mean value. These substances inhibited Ca2+ - or guanosine 5'-0-3-thiotriphosphate-induced insulin release from islets permeabilized with streptolysin-O. L-cysteine and NaHS reduced ATP production and attenuated glucose-induced hyperpolarization of the mitochondrial membrane potential. Finally, L-cysteine increased H(2)S content in MIN6 cells. We suggest here that L-cysteine inhibits insulin release via multiple actions on the insulin secretory process through H(2)S production. Because the activities of H(2)S-producing enzymes and the tissue H(2)S contents are known to increase under diabetic conditions, the inhibition may participate in the deterioration of insulin release in this disease.

The roles of protein kinase C (PKC) isoforms in cholinergic potentiation of glucose-induced insulin secretion were investigated in rat pancreatic islets. Western-blot analysis showed the presence of PKC-alpha, betaII, delta, epsilon, eta, and zeta, but not PKC-betaI, gamma, or iota, in the islets. Carbachol (CCh) caused translocations of PKC-alpha, betaII, delta, and epsilon from the cytosol to the plasma membrane. CCh facilitated 7-mM glucose-induced insulin secretion from isolated rat islets. The CCh-stimulated insulin secretion was significantly suppressed by the generic PKC inhibitor chelerythrine. In contrast, Go 6976, an inhibitor of conventional PKC isoforms, had no effect on the insulin secretion stimulated by CCh, although it significantly inhibited that induced by phorbol 12-myristate 13-acetate. These results suggest that the novel PKC isoforms activated by CCh, i.e., PKC-delta and/or epsilon, participate in the stimulatory effect of CCh on insulin secretion.

Amplified genes are frequently localized on extrachromosomal double minutes (DMs) or in chromosomal homogeneously staining regions (HSRs). We previously showed that a plasmid bearing a mammalian replication initiation region could efficiently generate DMs and HSRs after transfection into human tumor cell lines. The Breakage-Fusion-Bridge (BFB) cycle model, a classical model that explains how HSRs form, could also be used to explain how the transfected plasmids generate HSRs. The BFB cycle model involves anaphase bridge formation due to the presence of dicentric chromosomes, followed by the breakage of the bridge. In this study, we used our plasmid-based model system to analyze how anaphase bridges break during mitosis. Dual-color fluorescence in situ hybridization analyses revealed that anaphase bridges were most frequently severed in their middle irrespective of their lengths, which suggests that a structurally fragile site exists in the middle of the anaphase bridge. Breakage of the chromosomal bridges occurred prior to nuclear membrane reformation and the completion of cytokinesis, which indicates that mechanical tension rather than cytokinesis is primarily responsible for severing anaphase bridges. Time-lapse observation of living cells revealed that the bridges rapidly shrink after being severed. If HSR length was extended too far, the bridge could no longer be resolved and became tangled depending on the tension. The unbroken bridge appeared to inhibit the completion of cytokinesis. These observations strongly suggest that anaphase bridges are highly elastic and that the length of the spindle axis determines the maximal HSR length.

The present study investigated the effects of cGMP on cytosolic Ca(2+) concentration ([Ca(2+)](c)) of isolated rat pancreatic beta-cells. In the presence of 7.0 mM glucose, NOC 7, a nitric oxide (NO) donor, caused an increase in [Ca(2+)](c) of the beta-cells, which was abolished by the soluble guanylate cyclase inhibitor ODQ. Similar [Ca(2+)](c) elevation was evoked by 8-bromo-cGMP. The [Ca(2+)](c) elevating responses to NOC 7 and 8-bromo-cGMP were abolished by nicardipine or in a Ca(2+)-free medium, but were not affected by thapsigargin, suggesting that they are produced by the Ca(2+) influx through L-type voltage-operated Ca(2+) channels. In contrast, NOC 7 and 8-bromo-cGMP decreased the [Ca(2+)](c) when it was raised in advance by the elevation of external K(+) concentration to 30 mM or by 4-aminopyridine. The pretreatment with thapsigargin almost abolished the [Ca(2+)](c) reduction induced by the agents, suggesting that the action is likely to be primarily attributable to an acceleration of the Ca(2+) sequestration into the endoplasmic reticulum. These results suggest that cGMP has two distinct effects on the [Ca(2+)](c) of rat pancreatic beta-cells: a facilitation of the Ca(2+) influx through L-type voltage-operated Ca(2+) channels and an acceleration of the Ca(2+) sequestration in the endoplasmic reticulum.

We generated a series of knockin mouse lines, in which the cytokine receptor gp130-dependent STAT3 and/or SHP2 signals were disrupted, by replacing the mouse gp130 gene with human gp130 mutant cDNAs. The SHP2 signal-deficient mice (gp130F759/F759 were born normal but displayed splenomegaly and lymphadenopathy and an enhanced acute phase reaction. In contrast, the STAT3 signal-deficient mice (gp130FXQ/FXXQ) died perinatally, like the gp130-deficient mice (gp130D/D). The gp130F759/F759 mice showed prolonged gp130-induced STAT3 activation, indicating a negative regulatory role for SHP2. Th1-type cytokine production and IgG2a and IgG2b production were increased in the gp130F759/F759 mice, while they were decreased in the gp130FXXQ/FXXQ immune system. These results indicate that the balance of positive and negative signals generated through gp130 regulates the immune responses.

Reactive oxygen species, including hydrogen peroxide (H(2)O(2)), are known to induce β-cell apoptosis. The present study investigated the role of Ca(2+) in H(2)O(2)-induced apoptosis of the β-cell line INS-1. Annexin V assay with flow cytometry and DNA ladder assay demonstrated that treatment of INS-1 cells with 100 µM H(2)O(2) for 18 h significantly increased apoptotic cells. A comparable level of apoptosis was also observed after 18 h when the cells were treated with 100 µM H(2)O(2) only for initial 30 min. The H(2)O(2)-induced apoptosis was abolished by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA/AM), a chelator of intracellular Ca(2+), by 2-aminoethoxydiphenylborate (2-APB), a blocker of inositol 1,4,5-trisphosphate (IP(3)) receptors and cation channels, and by xestospongin D, a blocker of IP(3) receptors, and was partially blocked by SKF-96365, a non-selective cation channel blocker. However, nicardipine, an L-type voltage-dependent Ca(2+) channel blocker, or N-(p-amylcinnamoyl)anthranilic acid (ACA), a TRPM2 blocker, had little effect on the apoptosis. The inhibitory effect of BAPTA/AM or 2-APB on the H(2)O(2)-induced apoptosis was largely attenuated when the drug was added 30 min or 1 h after start of the treatment with H(2)O(2). These results suggest that the initial intracellular Ca(2+) elevation induced by H(2)O(2), which is mediated via IP(3) receptors and store-operated cation channels, plays an obligatory role in the induction of β-cell apoptosis.

Aim: The purpose of this study was to clarify the experiences of difficulty that patients with cancer faced in the learning process of progressive muscle relaxation (PMR) and to consider which kind of PMR guidance should be given to weak patients with cancer. Methods: The participants were instructed to practise PMR for 2 weeks when they were in good condition. The interview was conducted 1 week and 2 weeks after the PMR intervention. The 21 participants who experienced difficulty were analyzed. The analysis was completed by using the content analysis method. Results: Fifteen patients practised PMR by themselves one-to-two times per day. However, six patients practised PMR three-to-four times per week when they were in good condition. The interview answers fell into two categories: (i) experiences of difficulty that patients encountered in the learning process; and (ii) experiences in which patients had no feeling of change though they had practised PMR. The former category included the subcategories of experiences in which patients felt difficulty with PMR, experiences in which patients felt uncomfortable symptoms after PMR, and experiences in which patients did not feel it was good timing to learn PMR. Conclusion: It is easy for disease to progress in patients with cancer and those with poor strength, so it is necessary to match the functional status of individual patients. It also is important to support patients in practising PMR whenever they have the desire, by respecting their will.

We examined the expression of the major H 2 S-producing enzymes, cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). CBS was ubiquitously distributed in the mouse pancreas, but CSE was found only in the exocrine. Freshly isolated islets expressed CBS, while CSE was faint. However, high glucose increased the CSE expression in the beta-cells. l -Cysteine or NaHS suppressed islet cell apoptosis with high glucose, and increased glutathione content in MIN6 beta-cells. Pretreatment with l -cysteine improved the secretory responsiveness following stimulation with glucose. The CSE inhibitor dl -propargylglycine antagonized these l -cysteine effects. We suggest H 2 S may function as an ‘intrinsic brake’ which protects beta-cells from glucotoxicity.

Rab27a is involved in the control of membrane traffic, a crucial step in the regulated secretion. Typically, the guanosine triphosphate (GTP)-bound form has been considered to be active and, therefore, searching for proteins binding to the GTP-form has been attempted to look for their effectors. Here, we have identified the actin-bundling protein coronin 3 as a novel Rab27a effector that paradoxically bound guanosine diphosphate (GDP)-Rab27a in the pancreatic β-cell line MIN6. Coronin 3 directly bound GDP-Rab27a through its β-propeller structure. The most important insulin secretagogue glucose promptly shifted Rab27a from the GTP- to GDP-bound form. Knockdown of coronin 3 by RNAi resulted in the inhibition of phogrin (an insulin-granule-associated protein) internalization and the uptake of FM4-64 (a marker of endocytosis). Similar results were reproduced by disruption of the coronin-3–GDP-Rab27a interaction with the dominant-negative coronin 3, and coexpression of the GDP-Rab27a mutant rescued these changes. Taken together, our results indicate that interaction of GDP-Rab27a and coronin 3 is important in stimulus-endocytosis coupling, and that GTP- and GDP-Rab27a regulates insulin membrane recycling at the distinct stages.

The aim of this study was to investigate the in vivo and in vitro effects of exendin-4, a potent glucagon-like peptide 1 agonist, on the protection of the pancreatic β-cells against their cell death. In in vivo experiments, we used β-cell-specific calmodulin-overexpressing mice where massive apoptosis takes place in their β-cells, and we examined the effects of chronic treatment with exendin-4. Chronic and s.c. administration of exendin-4 reduced hyperglycemia. The treatment caused significant increases of the insulin contents of the pancreas and islets, and retained the insulin-positive area. Dispersed transgenic islet cells lived only shortly, and several endoplasmic reticulum (ER) stress-related molecules such as immunoglobulin-binding protein (Bip), inositol-requiring enzyme-1α, X-box-binding protein-1 (XBP-1), RNA-activated protein kinase-like endoplasmic reticulum kinase, activating transcription factor-4, and C/EBP-homologous protein (CHOP) were more expressed in the transgenic islets. We also found that the spliced form of XBP-1, a marker of ER stress, was also increased in β-cell-specific calmodulin-overexpressing transgenic islets. In the quantitative real-time PCR analyses, the expression levels of Bip and CHOP were reduced in the islets from the transgenic mice treated with exendin-4. These findings suggest that excess of ER stress occurs in the transgenic β-cells, and the suppression of ER stress and resultant protection against cell death may be involved in the anti-diabetic effects of exendin-4.

In isolated rat pancreatic β-cells, hypotonic stimulation elicited an increase in cytosolic Ca2+ concentration ([Ca2+]c) at 2.8 mM glucose. The hypotonically induced [Ca2+]c elevation was significantly suppressed by nicardipine, a voltage-dependent Ca2+ channel blocker, and by Gd3+, amiloride, 2-aminoethoxydiphenylborate, and ruthenium red, all cation channel blockers. In contrast, the [Ca2+]c elevation was not inhibited by suramin, a P2 purinoceptor antagonist. Whole cell patch-clamp analyses showed that hypotonic stimulation induced membrane depolarization of β-cells and produced outwardly rectifying cation currents; Gd3+ inhibited both responses. Hypotonic stimulation also increased insulin secretion from isolated rat islets, and Gd3+ significantly suppressed this secretion. Together, these results suggest that osmotic cell swelling activates cation channels in rat pancreatic β-cells, thereby causing membrane depolarization and subsequent activation of voltage-dependent Ca2+ channels and thus elevating insulin secretion.

Hydrogen sulfide (H(2)S) was historically recognized as a toxic gas generated by natural resources. However, its enzymatic production from L-cysteine has recently been demonstrated in mammals. Cystathionine beta-synthase and cystathionine gamma-lyase, both of which can produce H(2)S, were expressed in mouse pancreatic islet cells and the beta-cell line, MIN6. L-cysteine and the H(2)S donor NaHS inhibited glucose-induced insulin release from islets and MIN6 cells. These inhibitory effects were reproduced when insulin release was stimulated by alpha-ketoisocaproate, tolbutamide, or high K+. L-cysteine and NaHS inhibited glucose-potentiated insulin release in the copresence of diazoxide and high K+. Real-time imaging of intracellular Ca2+ concentration ([Ca2+](i)) demonstrated that both L-cysteine and NaHS reversibly suppressed glucose-induced [Ca2+](i) oscillation in a single beta-cell without obvious changes in the mean value. These substances inhibited Ca2+ - or guanosine 5'-0-3-thiotriphosphate-induced insulin release from islets permeabilized with streptolysin-O. L-cysteine and NaHS reduced ATP production and attenuated glucose-induced hyperpolarization of the mitochondrial membrane potential. Finally, L-cysteine increased H(2)S content in MIN6 cells. We suggest here that L-cysteine inhibits insulin release via multiple actions on the insulin secretory process through H(2)S production. Because the activities of H(2)S-producing enzymes and the tissue H(2)S contents are known to increase under diabetic conditions, the inhibition may participate in the deterioration of insulin release in this disease.

In isolated rat pancreatic β-cells, the nitric oxide (NO) donor NOC-7 at 1 μM reduced the amplitude of the oscillations of cytosolic Ca2+concentration ([Ca2+]c) induced by 11.1 mM glucose, and at 10 μM terminated them. In the presence of NG-nitro-l-arginine (l-NNA), however, NOC-7 at 0.5 and 1 μM increased the amplitude of the [Ca2+]coscillations, although the NO donor at 10 μM still suppressed them. Aqueous NO solution also had a dual effect on the [Ca2+]coscillations. The soluble guanylate cyclase inhibitor LY-83583 and the cGMP-dependent protein kinase inhibitor KT5823 inhibited the stimulatory effect of NO, and 8-bromo-cGMP increased the amplitude of the [Ca2+]coscillations. Patch-clamp analyses in the perforated configuration showed that 8-bromo-cGMP inhibited whole cell ATP-sensitive K+currents in the isolated rat pancreatic β-cells, suggesting that the inhibition by cGMP of ATP-sensitive K+channels is, at least in part, responsible for the stimulatory effect of NO on the [Ca2+]coscillations. In the presence ofl-NNA, the glucose-induced insulin secretion from isolated islets was facilitated by 0.5 μM NOC-7, whereas it was suppressed by 10 μM NOC-7. These results suggest that NO facilitates glucose-induced [Ca2+]coscillations of β-cells and insulin secretion at low concentrations, which effects are mediated by cGMP, whereas NO inhibits them in a cGMP-independent manner at high concentrations.

This study examined the effect of nitric oxide (NO) on the cytosolic free Ca(2+) concentration ([Ca(2+)](c)) of alpha-cells isolated from rat pancreatic islets. When extracellular glucose was reduced from 7 to 0 mM, about half of the alpha-cells displayed [Ca(2+)](c) oscillations. Nicardipine, a Ca(2+) channel blocker, terminated the oscillations, while thapsigargine, an inhibitor of Ca(2+)-ATPase on the endoplasmic reticulum, did not affect them, suggesting that the [Ca(2+)](c) oscillations were produced by periodic Ca(2+) influx via L-type voltage-operated Ca(2+) channels. NOC 7, an NO donor, did not cause any changes in [Ca(2+)](c) at 7 mM glucose, but reduced [Ca(2+)](c) or terminated [Ca(2+)](c) oscillations at 0 or 2.8 mM glucose. A similar inhibitory effect on [Ca(2+)](c) of alpha-cells was caused by 8-bromo-cGMP. When the [Ca(2+)](c) of alpha-cells was elevated by L-arginine in the presence of N(omega)-nitro-L-arginine, an NO synthase inhibitor, the subsequent application of NOC 7 and 8-bromo-cGMP reduced [Ca(2+)](c). As there is a direct relationship between [Ca(2+)](c) and glucagon release, these results suggest that the NO-cGMP system in rat pancreatic islets reduces glucagon release by suppressing [Ca(2+)](c) responses in alpha-cells.