GDP-bound Rab27a regulates clathrin disassembly through HSPA8 after insulin secretion.

Clathrin-dependent endocytosis is a key process for secretory cells, in which molecules on the plasma membrane are both degraded and recycled in a stimulus-dependent manner. There are many reports showing that disruption of endocytosis is involved in the onset of various diseases. Recently, it has been reported that such disruption in pancreatic ß-cells causes impaired insulin secretion and might be associated with the pathology of diabetes mellitus. Compared with exocytosis, there are few reports on the molecular mechanism of endocytosis in pancreatic ß-cells. We previously reported that GDP-bound Rab27a regulates endocytosis through its GDP-dependent effectors after insulin secretion. In this study, we identified heat shock protein family A member 8 (HSPA8) as a novel interacting protein for GDP-bound Rab27a. HSPA8 directly bound GDP-bound Rab27a via the ß2 region of its substrate binding domain (SBD). The ß2 fragment was capable of inhibiting the interaction between HSPA8 and GDP-bound Rab27a, and suppressed glucose-induced clathrin-dependent endocytosis in pancreatic ß-cells. The region also affected clathrin dynamics on purified clathrin-coated vesicles (CCVs). These results suggest that the interaction between GDP-bound Rab27a and HSPA8 regulates clathrin disassembly from CCVs and subsequent vesicle transport. The regulatory stages in endocytosis by HSPA8 differ from those for other GDP-bound Rab27a effectors. This study shows that GDP-bound Rab27a dominantly regulates each stage in glucose-induced endocytosis through its specific effectors in pancreatic ß-cells.

Apigenin Alleviates Endoplasmic Reticulum Stress-Mediated Apoptosis in INS-1 ß-Cells.

The improvement of type 2 diabetes mellitus induced by naturally occurring polyphenols, known as flavonoids, has received considerable attention. However, there is a dearth of information regarding the effect of the trihydroxyflavone apigenin on pancreatic ß-cell function. In the present study, the anti-diabetic effect of apigenin on pancreatic ß-cell insulin secretion, apoptosis, and the mechanism underlying its anti-diabetic effects, were investigated in the INS-ID ß-cell line. The results showed that apigenin concentration-dependently facilitated 11.1-mM glucose-induced insulin secretion, which peaked at 30 µM. Apigenin also concentration-dependently inhibited the expression of endoplasmic reticulum (ER) stress signaling proteins, CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) and cleaved caspase-3, which was elevated by thapsigargin in INS-1D cells, with peak suppression at 30 µM. This was strongly correlated with the results of flow cytometric analysis of annexin V/propidium iodide (PI) staining and DNA fragmentation analysis. Moreover, the increased expression of thioredoxin-interacting protein (TXNIP) induced by thapsigargin was remarkably reduced by apigenin in a concentration-dependent manner. These results suggest that apigenin is an attractive candidate with remarkable and potent anti-diabetic effects on ß-cells, which are mediated by facilitating glucose-stimulated insulin secretion and preventing ER stress-mediated ß-cell apoptosis, the latter of which may be possibly mediated by reduced expression of CHOP and TXNIP, thereby promoting ß-cell survival and function.

Development of Therapeutic Agents with a Novel Mechanism of Action Targeting Pancreatic ß-Cells for Diabetes.

Although diabetes is associated with an increased risk of various diseases, including cancer and infectious diseases, no definitive cure has yet been found. Long-term treatment for blood glucose control significantly reduces the QOL. Pancreatic ß-cells are the only cells that can lower blood glucose levels by secreting insulin. Therefore, maintaining insulin-secreting ß-cells is crucial in preventing the progression of diabetes and improving the QOL. We have investigated the mechanisms for the regulation of insulin secretion, the prevention of ß-cell apoptosis, and the increase in ß-cell mass. In particular, we have elucidated the involvement of type I diacylglycerol kinase (DGK) in the regulation of insulin secretion and the effects of nitric oxide (NO) signaling and natural products in suppressing ß-cell death. In addition, we have elucidated the function of DGKδ as a suppressor of ß-cell proliferation. This review introduces the findings of our study leading to development of novel anti-diabetic therapeutics that targets pancreatic ß-cells.

Oral ingestion of Shiikuwasha extract suppresses diabetes progression in db/db mice by preserving ß-cell mass.

Introduction: Nobiletin is a polymethoxyflavonoid abundant in citrus peels and has been reported to have various bioactive effects. We have previously reported that nobiletin inhibits endoplasmic reticulum stress-induced apoptosis in the pancreatic ß-cell line INS-1 and that continuous subcutaneous administration of nobiletin suppresses the progression of diabetes by protecting ß-cells in type 2 diabetic db/db mice. In the present study, we investigated effects of oral ingestion of Shiikuwasha extract rich in nobiletin on the pathogenesis of type 2 diabetes in db/db mice. Materials and methods: A Shiikuwasha extract was dissolved in MediDrop sucralose. Twenty-four mice were equally divided in three groups and fed with vehicle or low or high dose of Shiikuwasha extract for 4 weeks. Blood glucose levels, pancreatic ß-cell mass, serum insulin levels, pancreatic insulin content, and other biomarkers were measured and compared between the groups. Results: The group that freely ingested the Shiikuwasha extract containing higher concentration of nobiletin (Shiikuwasha H) showed lower blood glucose levels. At the end of the experiment, the Shiikuwasha H group exhibited improved glucose tolerance, lower serum glycoalbumin levels, and an increase in ß-cell area per pancreas compared with the control group. Body weight, food intake, and serum biomarkers related to liver function and lipid metabolism of the Shiikuwasha H group were not different from those of the control group, although water intake of the former was significantly decreased than that of the latter. Conclusion: Our results suggest that the oral ingestion of Shiikuwasha extract preserves pancreatic ß-cell mass in diabetic mice, which might be attributed to ameliorating the progression of diabetes.

Asymmetric dimethylarginine accumulation under hyperglycemia facilitates ß-cell apoptosis via inhibiting nitric oxide production.

Low concentrations of nitric oxide (NO) produced by constitutive NO synthase (cNOS) has been shown to suppress apoptosis in pancreatic ß-cells. In the present study, the influence of asymmetric dimethylarginine (ADMA), the major endogenous inhibitor of NOS, on the apoptosis-suppressive effect of NO was investigated. The expression of dimethylarginine dimethylaminohydrolase 2 (DDAH2), an ADMA-metabolizing enzyme, in INS-1 ß-cells and in mouse pancreatic islets was drastically reduced by in vitro exposure to high-concentration glucose (20 mM) and by in vivo treatment of mice with the insulin receptor blocker S661, which resulted in hyperglycemia, respectively. In line with this, a higher ADMA level was observed in INS-1 cells exposed to 20 mM glucose. The treatment of INS-1 cells with ADMA, similarly to with the NOS inhibitor NG-nitro-L-arginine methyl ester, significantly facilitated 20 mM glucose-induced increase in cleaved caspase-3 protein expression. Furthermore, increased protein expression of cleaved caspase-3 and CHOP was observed in INS-1 cells with knockdown of DDAH2. These results suggest that ADMA accumulation through a decrease in DDAH2 expression in ß-cells, which is induced under hyperglycemic conditions, facilitates ß-cell apoptosis through suppression of cNOS-mediated NO production.

[Involvement of Diacylglycerol Kinase on the Regulation of Insulin Secretion in Pancreatic ß-Cells during Type 2 Diabetes].

Depression of lipid metabolism in ß-cells has been indicated to be one of the causes of impaired insulin secretion in type 2 diabetes. Diacylglycerol (DAG) is an important lipid mediator and is known to regulate insulin secretion in pancreatic ß-cells. Intracellular DAG accumulation is involved in ß-cell dysfunction in the pathogenesis of type 2 diabetes; thus, the regulation of intracellular DAG levels is likely important for maintaining the ß-cell function. We focused on diacylglycerol kinases (DGKs), which strictly regulate intracellular DAG levels, and analyzed the function of type I DGKs (DGKα, γ), which are activated by intracellular Ca2+ and expressed in the cytoplasm, in ß-cells. The suppression of the DGKα and γ expression decreased the insulin secretory response, and the decreased expression of DGKα and γ was observed in islets of diabetic model mice. In the pancreatic ß-cell line MIN6, 1 µM R59949 (a type I DGK inhibitor) and 10 µM DiC8 (a cell permeable DAG analog) enhanced glucose-induced [Ca2+]i oscillation in a PKC-dependent manner, while 10 µM R59949 and 100 µM DiC8 suppressed [Ca2+]i oscillation and voltage-dependent Ca2+ channel activity in a PKC-independent manner. These results suggest that the intracellular accumulation of DAG by the loss of the DGKα and γ functions regulates insulin secretion in a dual manner depending on the degree of DAG accumulation. The regulation of the insulin secretory response through DAG metabolism by type I DGKs may change depending on the degree of progression of type 2 diabetes.

Harmine suppresses collagen production in hepatic stellate cells by inhibiting DYRK1B.

Liver fibrosis is a major consequence of chronic liver disease, where excess extracellular matrix is deposited, due caused by the activation of hepatic stellate cells (HSCs). The suppression of collagen production in HSCs is therefore regarded as a therapeutic target of liver fibrosis. The present study investigated effects of harmine, which is a ß-carboline alkaloid and known as an inhibitor of dual-specificity tyrosine-regulated kinases (DYRKs), on the production of collagen in HSCs. LX-2 cells, a human HSC cell line, were treated with harmine (0-10 µM) for 48 h in the presence or absence of TGF-ß1 (5 ng/ml). The expression of collagen type I α1 (COL1A1) and DYRK isoforms was investigated by Western blotting, quantitative RT-PCR, or immunofluorescence. The influence of knockdown of each DYRK isoform on the COL1A1 expression was further investigated. The expression of COL1A1 was markedly increased by treating with TGF-ß1 for 48 h in LX-2 cells. Harmine (10 µM) significantly inhibited the increased expression of COL1A1. LX-2 cells expressed mRNAs of DYRK1A, DYRK1B, DYRK2, and DYRK4, although the expression of DYRK4 was much lower than the others. Knockdown of DYRK1B, but not DYRK1A or DYRK2, with siRNA significantly suppressed TGF-ß1-induced increase in COL1A1 expression. These results suggest that harmine suppresses COL1A1 expression via inhibiting DYRK1B in HSCs and therefore might be effective for the treatment of liver fibrosis.

Diacylglycerol kinase δ functions as a proliferation suppressor in pancreatic ß-cells.

Although an aberrant reduction in pancreatic ß-cell mass contributes to the pathogenesis of diabetes, the mechanism underlying the regulation of ß-cell mass is poorly understood. Here, we show that diacylglycerol kinase δ (DGKδ) is a key enzyme in the regulation of ß-cell mass. DGKδ expression was detected in the nucleus of ß-cells. We developed ß-cell-specific DGKδ knockout (ßDGKδ 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 ßDGKδ KO mice. DGKδ knockdown in the ß-cell line MIN6 induced significant increases in bromodeoxyuridine (BrdU) incorporation and cyclin B1 expression. Finally, we confirmed that streptozotocin-induced hyperglycemia and ß-cell loss were alleviated in ßDGKδ KO mice. Thus, suppressing the expression or enzymatic activity of DGKδ that functions as a suppressor of ß-cell proliferation could be a novel therapeutic approach to increase ß-cell mass for the treatment of diabetes.

The sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid is a glucose-dependent potentiator of insulin secretion.

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 ß-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.

Dual effect of reduced type I diacylglycerol kinase activity on insulin secretion from MIN6 ß-cells.

The role of type I diacylglycerol kinases (DGKs) in the regulation of insulin secretion was investigated in MIN6 ß-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.

TRPV2 channels mediate insulin secretion induced by cell swelling in mouse pancreatic ß-cells.

ß-Cell swelling induces membrane depolarization, which has been suggested to be caused at least partly by the activation of cation channels. Here, we show the identification of the cation channels. In isolated mouse pancreatic ß-cells, the exposure to 30% hypotonic solution elicited an increase in cytosolic Ca2+ concentration ([Ca2+]c). The [Ca2+]c elevation was partially inhibited by ruthenium red, a blocker of several Ca2+-permeable channels including transient receptor potential vanilloid receptors [transient receptor potential cation channel subfamily V (TRPV)], and by nicardipine, but not by the depletion of intracellular Ca2+ stores with thapsigargin and caffeine. The hypotonic stimulation also increased insulin secretion from isolated mouse islets, which was significantly suppressed by ruthenium red. Expression of TRPV2 and TRPV4 was confirmed in mouse pancreatic islets and the MIN6 ß-cell line by RT-PCR, Western blot, and immunohistochemical analyses. However, neither 4α-phorbol 12,13-didecanoate nor GSK1016790A, TRPV4 activators, showed any apparent effect on [Ca2+]c in isolated mouse ß-cells or in MIN6 cells. In contrast, probenecid, a TRPV2 activator, induced an increase in [Ca2+]c in MIN6 cells, which was attenuated by ruthenium red. Moreover, the [Ca2+]c elevation induced by 30% hypotonic stimulation was significantly reduced by knockdown of TRPV2 with siRNA and by tranilast, a TRPV2 inhibitor. The knockdown of TRPV2 also decreased insulin secretion induced by the hypotonic stimulation. In addition, glucose-stimulated insulin secretion was also significantly reduced in the TRPV2-knockdown MIN6 cells. These results suggest that osmotic cell swelling activates TRPV2 in mouse ß-cells, thereby causing membrane depolarization and subsequent activation of voltage-dependent Ca2+ channels and insulin secretion.

Development and Analysis of Novel Therapeutic Targets to Improve Pancreatic ß-Cell Function in Type 2 Diabetes.

Pancreatic ß-cell dysfunction is a major feature of type 2 diabetes. Therefore maintenance of ß-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 ß-cell survival, we particularly focused on the roles of gasotransmitters in pancreatic ß-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 ß-cells, induction of nitric oxide synthase 2 (NOS2) by inflammatory cytokines generates a large amount of NO, which contributes to the impairment of ß-cell function and induction of ß-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 ß-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 ß-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 ß-cell apoptosis via an antioxidant mechanism. Here, we describe the current understanding of the function of gasotransmitters in regulating insulin secretion and pancreatic ß-cell survival.

[Regulation of Lipid Metabolism by Diacylglycerol Kinases in Pancreatic ß-cells].

The appropriate secretion of insulin from pancreatic ß-cells is essential for regulating blood glucose levels. Glucose-stimulated insulin secretion (GSIS) involves the following steps: Glucose uptake by pancreatic ß-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, DGKß, and DGKγ), which are activated by Ca(2+). The protein expression of DGKα and DGKγ was detected in mouse pancreatic islets and the pancreatic ß-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 ß-cells, and discuss their pathophysiological roles in the progression of type-2 diabetes.

Obligatory Role of Early Ca(2+) Responses in H2O2-Induced ß-Cell Apoptosis.

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 ß-cell apoptosis. In the present study, we further examined the obligatory role of early Ca(2+) responses in ß-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 Signaling Pathway in Pancreatic ß-Cells: An Essential Role of Diacylglycerol Kinase in the Regulation of Insulin Secretion.

Diacylglycerol (DAG) is a lipid signal messenger and plays a physiological role in ß-cells. Since defective glucose homeostasis increases de novo DAG synthesis, DAG may also contribute to ß-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 ß-cells. The intracellular levels of DAG are strictly regulated by diacylglycerol kinase (DGK); however, the role of DGK in ß-cells and their involvement in ß-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 ß-cells. DGKα and γ were activated by glucose or high K(+) stimulation in ß-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 ß-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 ß-cell function and their involvement in the development of ß-cell dysfunction in type 2 diabetes.

Structure-dependent inhibitory effects of green tea catechins on insulin secretion from pancreatic ß-cells.

The effects of green tea catechins on glucose-stimulated insulin secretion (GSIS) were investigated in the ß-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.