Ichiro Niki

Increase in insulin release from rat pancreatic islets by quinolone antibiotics

1 The present study was undertaken to elucidate the mechanism(s) of hypoglycaemia caused by quinolone antibiotics. We investigated the effects of various quinolone antibiotics on insulin release in rat pancreatic islets. 2 At a non‐stimulatory concentration of 3 mM glucose, lomefloxacin (LFLX) or sparfloxacin at 1 mM and pipemidic acid (0.1‐1 mM) induced slight insulin release but tosufloxacin or enoxacin up to 100 μm did not. 3 At the stimulatory concentration of 10 mM glucose, all quinolones augmented insulin release in a dose‐dependent manner. LFLX (100 μm) shifted the dose‐response curve of glucose‐induced insulin release to the left without altering the maximal response. 4 At 10 mM glucose, LFLX (100 μm) increased insulin release augmented by forskolin (5 μm) or 12‐0‐tetradecanoyl phorbol‐13‐acetate (100 nM) but not by raising the K+ concentration from 6 to 25 mM. 5 Verapamil (50 μm) or diazoxide (50–400 μm) antagonized the insulinotropic effect of LFLX. 6 These data suggest that quinolone antibiotics may cause hypoglycaemia by increasing insulin release via blockade of ATP‐sensitive K+ channels.

H-series protein kinase inhibitors and potential clinical applications.

During the course of generating derivatives of N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide, a synthetic calmodulin inhibitor, we came across several analogues with shorter alkyl chains that exhibited inhibition of serine/threonine protein kinase activities in an ATP-competitive manner. Certain derivatives proved to be selective inhibitors of protein kinases useful for elucidation of relevant functions of the enzymes. One of them turned out to be a unique vasodilator that preferentially suppresses delayed cerebral vasospasm, a critical complication of subarachnoid hemorrhage, without significant changes in systemic blood pressure. The compound in question, 1-(5-isoquinolinesulfonyl)-homopiperazine, was identified from sequential development of protein kinase inhibitors with isoquinolinesulfonyl structures, which occupy the adenine pocket of the ATP-binding site of the enzyme. It recently has been proposed that the target kinase responsible for vasodilation by 1-(5-isoquinolinesulfonyl)-homopiperazine may be Rho-kinase, which regulates phosphorylation of myosin light chains and vasocontraction. Because protein phosphorylation plays important roles in regulation of various cellular functions, the foregoing is a good example of current progress in the development of protein kinase inhibitors with potential clinical applications.

Insulin release by glucose anomers in a rat model of non-insulin-dependent diabetes

SummaryThe effects of the α and β anomers of D-glucose on insulin release were studied in a rat model of non-insulin-dependent diabetes, which was induced by streptozotocin injection at 2 days of age. Glucose tolerance of the streptozotocin-treated rats at 8–10 weeks of age was mildly diabetic. Insulin release from the isolated perfused pancreas of the diabetic rats in response to 10 mmol/l α-D-glucose was markedly impaired, while insulin response to 10 mmol/l β-D-glucose in the diabetic pancreas was only slightly reduced as compared to that in the control pancreas.

Annexin XI may be involved in Ca2+- or GTP-γS-induced insulin secretion in the pancreatic β-cell

The aim of this study was to investigate possible involvement of annexin XI in the insulin secretory machinery. In fluorescence immunocytochemistry, annexin XI was found in the cytoplasm of pancreatic endocrine cells and a pancreatic β‐cell line, MIN6, in a granular pattern. MIN6 cells also possessed weak and diffused annexin XI immunoreactivity in the cytoplasm. Immunoelectron microscopy revealed annexin XI in the insulin granules. Insulin secretion from streptolysin‐O‐permeabilized MIN6 cells was inhibited by anti‐annexin XI antibody, when the release was stimulated by either Ca2+ or GTP‐γS, but not by a protein kinase C‐activating phorbol ester. Inhibition of insulin release by anti‐annexin XI antibody was reproduced in permeabilized rat islets. These findings suggest that annexin XI may be involved in the regulation of insulin secretion from the pancreatic β‐cells.

Roles of intracellular Ca2+receptors in the pancreatic β-cell in insulin secretion

Ca2+ is the central second messenger in the regulation of insulin release from the pancreatic β-cell; and intracellular Ca2+-binding proteins, classified into two groups, the EF hand proteins and the Ca2+/ phospholipid binding proteins, are considered to mediate Ca2+ signaling. A number of Ca binding proteins have been suggested to participate in the secretory machinery in the β-cell. Calmodulin, the ubiquitous EF hand protein, is the predominant intracellular Ca2+ receptor that modulates insulin release via the multiplicity of its binding to target proteins including protein kinases. Other Ca binding proteins such as calcyclin and the Ca2+/phospholipid binding proteins may also be suggested to be involved. Ca2+ influx from the extracellular space appears to be responsible for exocytosis of insulin via Ca2+-dependent protein/protein interactions. On the other hand, intracellular Ca2+ mobilization resulting in secretory granule movement may be controlled by Ca2+/calmodulin-dependent protein phosphorylation. Thus, Ca2+ exerts versatile effects on the secretory cascade via binding to specific binding proteins in the pancreactic β-cells.

Immunocytochemical detection and spatial distribution of myosin light‐chain kinase in preimplantation mouse embryos

As a follow-up to our previous study on the role of myosin light-chain kinase (MLCK), a Ca2+/calmodulin-dependent enzyme, in the development of preimplantation mouse embryos, we examined the presence and pattern of distribution of MLCK during preimplantation development of the mouse by whole-mount, indirect immunocytochemistry and by Western blotting, using a monoclonal antibody against MLCK. At all stages of preimplantation development, the nucleus was brightly stained with an unstained region around the nucleus, and regions near the cell membrane were also brightly stained. Using the optical sectioning capability of the confocal laser scanning microscope, we found that, up to the eight-cell stage, the regions of cell contact were mostly unstained, but along with the process of compaction, cell contact regions showed a clear staining pattern along with clearing of the cytoplasm. During formation of the blastocyst, a ring of immunofluorescence was found at the margin of the blastocoel. In the blastocyst, cells of the inner cell mass were less immunofluorescent than trophectoderm cells. These staining results appear to be due to specific immunoreaction between MLCK and the antibody, because the staining patterns were abolished when the antibody was preabsorbed by MLCK purified from chicken gizzard smooth muscle. In Western blotting of blastocysts, we found a band at 130 kD. We also show by immunoblotting and immunohistochemistry of various mouse tissues that the antibody used in this study has cross-reactivity to MLCK of various muscle and non-muscle tissues of the mouse. The presence and spatial distribution of MLCK at various stages of preimplantation development of the mouse suggest that it could play a crucial role in the regulation of the contractile events involved in the initial differentiation that occurs during formation of the mouse blastocyst.

Calcyclin, a Calcium-Binding Protein, which Regulates Insulin Secretion from the Permeabilized Pancreatic β-Cell

Although it is widely accepted that an increase in intracellular Ca2+ is a crucial event in the stimulus-secretion coupling in the pancreatic β-cell [1], we have limited knowledge on the later steps in the signal transduction pathway. Calcium binding proteins are considered to be involved in the control of Ca2+-dependent cellular functions including hormone release [2]. Various types of calcium-binding proteins such as calmodulin, calbindin and S-100b protein are identified in the islet cells biochemically or immunohistochemically [3,4]. Calmodulin, the best characterized calcium binding protein, was demonstrated to exist in the pancreatic islet cells by Sugden et al [5]. The pancreatic β-cell possesses the kinase activity which is dependent on Ca2+/calmodulin [6]. Involvement of calmodulin and calmodulin-dependent protein kinases in insulin release has been suggested mainly based on the inhibitory effect of calmodulin antagonists on the release [7,8,9], albeit there is the argument against this idea [10].

90-kDa S6 kinase is insufficient or not involved in the activation of glycogen synthase induced by insulin.

Insulin and growth factors increase glycogen synthesis via complex pathways including protein phosphorylation/dephosphorylation processes. We investigated the involvement of 90-kDa S6 kinase in the control of insulin- or epidermal growth factor (EGF)-stimulated glycogen synthase activation using newly synthesized compounds which selectively inhibit 90-kDa S6 kinase. HH-5709 (1-(5-hydroxynaphthalenesulfonyl)-1H-hexahydro-1,4-diazepine) inhibited 90-kDa S6 kinase at lower concentrations than observed for protein kinases A or C. The inhibition by HH-5709 was competitive with respect to ATP with a Ki value of 1.3 microM. H-7, an inhibitor of protein kinases A and C, and HA-1077 (1-(5-isoquinolinesulfonyl)-homopiperazine), where the naphthalene ring of HH-5709 was replaced with isoquinoline, also inhibited 90-kDa S6 kinase to a similar extent as HH-5709. In 3Y1 fibroblasts, H-7 and HA-1077 attenuated the activation of glycogen synthase. HH-5709, however, failed to affect the glycogen synthase activation by either insulin or EGF. These findings suggest that 90-kDa S6 kinase is unrelated or insufficient to mediate activation of glycogen synthase and that unidentified pathway(s) sensitive to H-7 or HA-1077 would be involved in the activation of glycogen synthase by insulin or EGF in 3Y1 fibroblasts.

Insulin release by D-glucose anomers in a non-insulin-dependent diabetes rat model.

The alpha anomer of D-glucose is more potent than the beta anomer in stimulating insulin release. We have studied the effects of D-glucose anomers on insulin release from the perfused pancreas isolated from a rat model of non-insulin-dependent diabetes (NIDD) induced by streptozotocin injection at 2 days of age. Insulin release from the pancreas of the diabetic rat in response to 10 mM alpha-D-glucose was markedly impaired, while insulin response to the same concentration of beta-D-glucose was only slightly reduced as compared to that in the control pancreas. Thus, the pancreatic B cell of the diabetic rat did not discriminate between the alpha and beta anomers of D-glucose. Insulin release induced by 5 mM D-glyceraldehyde was decreased in the diabetic pancreas, while insulin release induced by 0.15 mg/ml tolbutamide did not differ from that in the control pancreas. Glucose oxidation in the islets isolated from the diabetic pancreas was not lower than that in comparable control islets. Treatment of the diabetic pancreas with 5 microM forskolin or 0.15 mg/ml tolbutamide did not restore its defective discrimination between the two anomers, although forskolin potentiated insulin release more markedly in the diabetic pancreas. The findings may provide some insight into the pathophysiology of the pancreatic B cell in NIDD.

Regulation of Insulin Release Independent of Changes of Cytosolic Ca2+ Concentration

Although the central role of cytosolic Ca2+ in insulin release has been widely accepted mainly based on the 45Ca2+ flux studies20, it is not conclusive because of inability to measure changes of cytosolic Ca2+ concentration ([Ca2+]i) directly in the pancreatic β-cell. Recently, a method to estimate [Ca2+]i using a fluorescent Ca2+ indicator, quin 2, has been developed and applied to the pancreatic β-cell5,14,21,22 Some of the findings by this method do not accord with those observed in the 45Ca2+ flux studies22. Since quin 2 chelates calcium12 and may affect insulin release22, the data obtained using quin 2 should be interpreted with caution. Another approach to analyze the relationship between [Ca2+]i in the pancreatic β-cell and insulin release is to clamp [Ca2+]i at arbitrary levels. For this purpose, three methods have been developed in other cells2; i) microinjection of Ca or a suitable Ca-buffer into a cell, ii) intracellular dialysis or perfusion, and iii) permeabilization of the plasma membrane to permit extracellular Ca or Ca-EGTA buffers easy access to the intracellular environment. The first two methods are not suitable for small cells such as the pancreatic β-cell. The last method has been applied to pancreatic islets using high voltage electric discharge11,23 or digitonin treatment4,16. We describe here finding obtained using digitonin to permeabilize the cells in the islet.