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Biomedical subjects

I Niki

Publications and source records attributed to I Niki.

At least 19 recordsLinked to original sources
PubMed2001

Characterization of secretory and morphologic properties of primary cultured endocrine cells from porcine pancreata.

We characterized morphologic and secretory properties of porcine pancreatic endocrine cells in primary culture obtained by autolytic preparation without any exogenous proteolytic enzymes. The endocrine cells exhibited a neuron-like shape, and insulin granules were accumulated at the terminal of the processes. Thus derived endocrine cells survived in culture medium containing nicotinamide and remained sensitive to glucose for at least 6 weeks after preparations. The cells responded well to physiologic concentrations of glucose, and high K+ depolarization and the antidiabetic sulfonylureas, tolbutamide, and glibenclamide also elicited the release. With high glucose, insulin release was markedly potentiated by forskolin, glucagon, glucagon-like peptide-1, and arginine and inhibited by somatostatin, the Ca2+ channel blocker nitrendipine, and the ATP-sensitive K+ channel opener diazoxide. Epinephrine had dual effects on the release by glucose; enhanced within a low nanomolar range and inhibited at 1 micromol/L. However, the cells were unresponsive to leucine. Such secretory sensitivities to nutrients, hormones, and pharmacologic agents, and long survival rate (as long as 5-6 weeks) of these cells suggest to us therefore that derived endocrine cells may be useful for xenotransplantation of pancreatic beta cells for treatment of insulin-dependent diabetes mellitus.

Animals↗
PubMed2001

Lack of effect of incretin hormones on insulin release from pancreatic islets in the bile duct-ligated rats.

Hyperglycemia associated with obstructive jaundice seriously affects the prognosis of patients with hepatobiliary diseases. We investigated secretory properties of isolated islets from bile duct-ligated (BDL) rats. Pancreatic islets from BDL rats lost their secretory responses to glucagon-like peptide-1 (GLP-1), although their responses to glucose were normal. Loss of potentiation of insulin release was also observed in glucagon and glucose-dependent insulinotropic peptide (GIP), whereas modulation of the release by forskolin, dibutyryl cAMP, or epinephrine remained unaffected. cAMP production by BDL islets was not increased by these insulinotropic hormones. Serum levels of glucagon, but not GIP, were increased in BDL rats. GLP-1 levels were also elevated, although they did not reach statistical significance. Immunoblotting of trimeric G protein subunits demonstrated that G(s)alpha L and G(s)alpha S, but not G(i)alpha 1/2 and G(i)alpha 3/o alpha, were less expressed in BDL islets. Therefore, unresponsiveness of the beta-cell to cAMP-raising hormones is involved in glucose intolerance under cholestasis. It results from diminished expression of alpha-subunits of the relevant G protein, G(s), and desensitization of receptors of these hormones.

Animals↗
PubMed2000

Annexin XI may be involved in Ca2+ - or GTP-gammaS-induced insulin secretion in the pancreatic beta-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 beta-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-gammaS, 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 beta-cells.

Animals↗
PubMed2000

[Regulation of insulin release: analysis of the insulin secretory cascade and possible contribution to novel anti-diabetic drug development].

Insulin is the only 'hypoglycemic' hormone synthesized in and secreted from the pancreatic beta cell. Type 2 diabetes results from both secretory failure in the beta cell and insulin resistance in the target tissues for insulin. Attempts to develop anti-diabetic drugs that induce insulin secretion from residual beta cells in type 2 diabetic patients originate from the serendipitous discovery of sulphonylureas as hypoglycemic agents 60 years ago. Generally, secretion is carried out by sequential processes such as granule formation, intracellular traffic, granule docking/priming and the final step, exocytosis (secretory cascade). In the beta cell, recent progress in cell biology enables us to analyze each step in the secretory cascade and to reveal controlling mechanisms. This review describes regulatory mechanisms of insulin release by distinct nutrients, hormones and neurotransmitters, and roles of second messengers and protein phosphorylation in the insulin secretory cascade. Possible development of insulinotropic drugs for the treatment of type 2 diabetes has been also discussed.

Diabetes Mellitus, Type 2↗
PubMed2000

Synergism of protein kinase A, protein kinase C, and myosin light-chain kinase in the secretory cascade of the pancreatic beta-cell.

Protein phosphorylation by myosin light-chain kinase (MLCK), protein kinase A, and protein kinase C (PKC) plays a positive role in insulin secretion from the pancreatic beta-cell. To investigate the underlying mechanisms, we examined intracellular distribution of the insulin granules and MLCK by immunofluorescence and immunoelectron microscopies and also investigated intracellular traffic of the granules in cultured beta-cells (MIN6) by video microscopy. Considerable parts of MLCK immunoreactivity were colocalized with the insulin granules. Subcellular fractionation of MIN6 cell extracts revealed that myosin light chain (MLC) may be distributed with the insulin-rich fractions, and immunofluorescence staining using specific antibodies against mono- and diphosphorylated MLCs depicted presence of phosphorylated MLCs in the cytoplasm, in part, with colocalization with the insulin granules. Activation of PKC by 12-O-tetradecanoyl-phorbol 13-acetate (TPA) caused a shift of both insulin granules and MLCK to the cell periphery, which was not reproduced by the adenylate cyclase activator, forskolin. In contrast, forskolin, but not TPA, increased the granule movement. Costimulation of the beta-cell by TPA and forskolin induced drastic translocation of insulin granules and MLCK to the cell periphery, resulting in enormous potentiation of insulin release. These findings suggest that these protein kinases increase insulin granules in the ready-releasable pool by acting on different steps in the secretory cascade.

Cell Line↗
PubMed1999

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

Ca2+ is the central second messenger in the regulation of insulin release from the pancreatic beta-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 beta-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 beta-cells.

Animals↗
PubMed1999

Ca2+ signaling and the insulin secretory cascade in the pancreatic beta-cell.

Recent progress in electrophysiological and microscopic techniques have enabled us to estimate exocytotic and pre-exocytotic events in the secretory machinery in single pancreatic beta-cells. We have been studying mechanisms involved in the regulation of insulin granule movement, which supplies release-ready granules, by direct visualization of granule traffic in living beta-cells and found the movement to be regulated by a mechanism different from that controlling exocytosis. From the obtained findings together with those from electrophysiological approaches, a new understanding of the role of the crucial second messenger Ca2+, and other second messengers, as well as resultant protein phosphorylation has been generated. The aim of this review is to describe a synergistic network for the control of insulin release by second messengers and protein kinases.

Animals↗
PubMed1998

Insulin secretion from isolated rat islets induced by the novel hypoglycemic agent A-4166, a derivative of D-phenylalanine.

A derivative of D-phenylalanine, A-4166, reportedly evokes a more rapid and short-lived hypoglycemic action in vivo than any of the currently available sulfonylureas. This novel oral hypoglycemic agent is structurally different from sulfonylureas. Therefore, studies were designed to elucidate the mechanisms by which A-4166 stimulates insulin secretion. Insulin release from incubated or perifused rat islets was dose-dependently stimulated by 10 to 200 mumol/l A-4166, in the presence of 2.8 mmol/l glucose. Both A-4166 and tolbutamide evoke a prompt rise in insulin secretion followed by a sustained gradually decreasing release from perfused islets in the presence of low glucose, although A-4166 appeared to be more sensitive than tolbutamide to subthreshold glucose concentration. Diazoxide abolished the initial release and blunted sustained release. Removing calcium from the perifusate abolished insulin release within 15 minutes. A-4166 inhibited [3H]-glibenclamide binding to HIT cell membranes and 86Rb efflux from ATP-depleted or diazoxide-treated cells. These results suggest that the insulin release induced by A-4166 is relevant to this agent occupying the tolbutamide binding sites. Therefore, one possible mechanism accounting for the more rapid and short-lived hypoglycemic action of A-4166 in vivo, as compared with tolbutamide, may involve the reported differences in the bioavailability of A-4166.

ATP-Binding Cassette Transporters↗
PubMed1998

An essential role of myosin light-chain kinase in the regulation of agonist- and fluid flow-stimulated Ca2+ influx in endothelial cells.

Cytosolic Ca2+ ([Ca2+]i) plays an important role in endothelial cell signaling. Although it has been suggested that the influx of Ca2+ can be triggered by depletion of intracellular Ca2+ stores, the mechanism (or mechanisms) underlying this phenomenon needs further elaboration. In the present study, involvement of myosin light-chain kinase (MLCK) in the regulation of Ca2+ signaling was investigated in agonist- and fluid flow-stimulated endothelial cells loaded with Ca2+-sensitive dyes. Bradykinin (BK) and thapsigargin caused an increase in [Ca2+]i followed by a sustained rise due to Ca2+ influx from extracellular space and shifted total myosin light-chain (MLC) from the unphosphorylated to the diphosphorylated form. ML-9 (100 microM), an inhibitor of MLCK, abolished Ca2+ influx and prevented MLC diphosphorylation in BK- and thapsigargin-treated cells, but did not affect Ca2+ mobilization from internal stores. Fluid flow stimulation (shear stress=5 dynes/cm2) increased [Ca2+]i and enhanced MLC phosphorylation. ML-9 also inhibited Ca2+ response and MLC phosphorylation in fluid flow-stimulated cells. The Ca2+ influx in response to BK was linearly correlated with the diphosphorylation of MLC in ML-9 treated cells. Effects of ML-5 and ML-7, analogs of ML-9, to inhibit Ca2+ influx paralleled their potencies to inhibit MLCK activity. These findings demonstrate that MLCK plays an essential role in regulating the plasmalemmal Ca2+ influx in agonist- and fluid flow-stimulated endothelial cells. This study is the first to report the close relationship between Ca2+ influx and MLC diphosphorylation.

Animals↗
PubMed1998

Acetylcholine activates intracellular movement of insulin granules in pancreatic beta-cells via inositol trisphosphate-dependent [correction of triphosphate-dependent] mobilization of intracellular Ca2+.

Intracellular movement of secretory granules is a proximal stage in the secretory cascade that ends in the release product from cells. We investigated mechanisms underlying the control of this movement by acetylcholine using an insulinoma cell line, MIN6, in which acetylcholine increases both insulin secretion and granule movement. The peak activation of movement was observed 3 min after an acetylcholine challenge. The effects were nullified by the muscarinic inhibitor atropine, phospholipase C (PLC) inhibitors (D 609 and compound 48/80), and pretreatment with the Ca2+ pump inhibitor, thapsigargin. Inhibitors of Ca2+-dependent phospholipase A2 (arachidonyl trifluoromethyl ketone and methyl arachidonyl fluorophosphate) also partially inhibited the movement caused by acetylcholine, but downregulation of protein kinase C by overnight incubation with the phorbol ester 12-o-tetradecanoylphorbol-13-acetate failed to exert any influence. Acetylcholine stimulation of granule movement was not reproduced by membrane depolarization with high K+. Phosphorylation of the endogenous myosin light chain in MIN6 cells was increased by addition of acetylcholine and decreased by the Ca2+ chelator BAPTA (1,2-bis[2-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid). The calmodulin inhibitor W-7 and the myosin light-chain kinase inhibitor ML-9 decreased the motile events in the beta-cells under both nonstimulated and acetylcholine-stimulated conditions. These findings led us to conclude that inositol trisphosphate [corrected] causes Ca2+ mobilization by muscarinic activation of PLC, leading to intracellular translocation of insulin granules to the ready-releasable pool in pancreatic beta-cells via Ca2+/calmodulin-dependent phosphorylation of myosin light chains.

Acetylcholine↗
PubMed1997

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.

Animals↗
PubMed1997

Myosin light-chain phosphorylation controls insulin secretion at a proximal step in the secretory cascade.

The aim of this study was to investigate how insulin secretion is controlled by phosphorylation of the myosin light chain (MLC). Ca2+-evoked insulin release from pancreatic islets permeabilized with streptolysin O was inhibited by different monoclonal antibodies against myosin light-chain kinase (MLCK) to an extent parallel to their inhibition of purified MLCK. Anti-MLCK antibody also inhibited insulin release caused by the stable GTP analog guanosine 5'-O-(3-thiodiphosphate), even at a substimulatory concentration (0.1 microM) of Ca2+. Free Ca2+ increased MLC peptide phosphorylation by beta-cell extracts in vitro. In contrast to the phosphorylation by purified MLCK or by calmodulin (CaM) kinase II, the activity partially remained with the beta-cell under nonstimulatory Ca2+ (0.1 microM) conditions. The MLCK inhibitor ML-9 inhibited the activity in the beta-cell with both substimulatory and stimulatory Ca2+, whereas KN-62, an inhibitor of CaM kinase II, only exerted an influence in the latter case. ML-9 decreased intracellular granule movement in MIN6 cells under basal and acetylcholine-stimulated conditions. We propose that MLC phosphorylation may modulate translocation of secretory granules, resulting in enhanced insulin secretion.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

Modulation of tyrosine kinase activity has multiple actions on insulin release from the pancreatic beta-cell: studies with lavendustin A.

We investigated the role of tyrosine kinases in the regulation of insulin release from a hamster beta-cell line, HIT T15, using selective tyrosine kinase inhibitors. Genistein increased the insulin release induced by glucose, but herbimycin A, tyrphostins and the erbstatin analogue failed to change the release. Lavendustin A at 0.1 nM-1 microM caused a concave-shaped inhibition of the insulin release stimulated by 7 mM glucose. The inhibitory effect of lavendustin A was overcome by higher concentrations of glucose. Lavendustin B, the negative control analogue, had no effect on the release. Lavendustin A at a nanomolar range progressively inhibited insulin release by high K+ (50 mM)-depolarization, whereas the inhibitor did not change the insulin release by Ca2+ ionophore (A23187). On the contrary, lavendustin A at 10 nM significantly increased insulin release when glucose-induced insulin release was enhanced by either 5 microM forskolin or 162 nM 12-O-tetradecanoylphorbol 13-acetate. Lavendustin A failed to influence the Ca(2+)-induced insulin release from HIT cells permeabilized with streptolysin-O. These findings suggest that tyrosine kinases may play versatile roles in the control of insulin release from the pancreatic beta-cell.

Animals↗
PubMed1996

Identification and purification of Ca2+/calmodulin-dependent protein kinase V from human gastric carcinoma.

We previously purified a novel Ca2+/calmodulin-dependent protein kinase (CaM kinase) V, which has proven to be a member of the CaM kinase I family. Immunohistochemical staining of surgically-resected specimens from human subjects using specific antibody which reacts with CaM kinases I and V demonstrated heterogeneous distribution of CaM kinase I/V in normal gastric mucosa. The kinase was located mainly at the bottom of foveoral epithelium and in the gastric gland (< 25% immunopositive). In contrast, this kinase was abundant in various types of gastric carcinomas (> 75%), but not in gastric adenomas. Preferential and consistent presence of this kinase was confirmed by immunoblot analysis of gastric carcinoma and human gastric cancer cell lines, Kato-III and MKN-45. CaM kinase I/V was co-purified with CaM kinase II from resected gastric carcinoma using anion-exchange chromatography followed by calmodulin-affinity chromatography. The two kinases were finally separated by HPLC-based gel filtration. Purified CaM kinase I/V from gastric carcinoma did not possess detectable autophosphorylating activity, in contrast to CaM kinase II. The findings suggest CaM kinase I/V may possess abnormal biochemical properties in human gastric carcinoma, and the kinase could participate in cell growth of the carcinoma.

Adenocarcinoma↗
PubMed1996

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.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗