Yukiko Kaneko

Glucose-induced production of hydrogen sulfide may protect the pancreatic beta-cells from apoptotic cell death by high glucose

We examined the expression of the major H2S‐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 H2 S may function as an ‘intrinsic brake’ which protects beta‐cells from glucotoxicity.

The GDP-dependent Rab27a effector coronin 3 controls endocytosis of secretory membrane in insulin-secreting cell lines

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.

Overexpression of Calmodulin in Pancreatic β Cells Induces Diabetic Nephropathy

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.

Depression of Type I Diacylglycerol Kinases in Pancreatic β-Cells From Male Mice Results in Impaired Insulin Secretion

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.

Diacylglycerol Signaling Pathway in Pancreatic β-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 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.

Inhibition by nitric oxide of Ca2+ responses in rat pancreatic α-cells

Abstract This study examined the effect of nitric oxide (NO) on the cytosolic free Ca 2+ concentration ([Ca 2+ ] c ) of α-cells isolated from rat pancreatic islets. When extracellular glucose was reduced from 7 to 0 mM, about half of the α-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 α-cells was caused by 8-bromo-cGMP. When the [Ca 2+ ] c of α-cells was elevated by L-arginine in the presence of N ω -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 α-cells.

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

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.

Obligatory Role of Early Ca(2+) Responses in H2O2-Induced β-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 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.

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

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.