Yoshiko Seto

In vitro antiviral activity of polyoxotungstate (PM-19) and other polyoxometalates against herpes simplex virus.

Polyoxotungstates with Keggin-type structure were found to demonstrate marked antiherpetic activity. K7[TiW10PO40].6H2O (PM-19) caused a decrease in plaque formation by several strains of herpes simplex virus (HSV) type 1, including acyclovir-resistant (thymidine kinase-negative) strains, at concentrations which were not toxic to the host cells. The 50% plaque-inhibiting concentration (EC50) for the different strains was between 20 and 50 micrograms/ml. Single-cycle HSV growth was also inhibited by PM-19. PM-19 inhibited viral DNA synthesis in HSV-infected cells at a concentration of 5 micrograms/ml but did not exhibit a virucidal effect, and pretreatment of the host cells with PM-19 did not provide resistance to herpes infection. Yet, virus adsorption to the cells was markedly affected at PM-19 concentrations higher than 25 micrograms/ml. PM-19 was also effective against human cytomegalovirus, but not against adenoviruses and varicella-zoster virus, although it did delay the development of the cytopathic effect of these viruses.

In vitro antiviral activity of polyoxomolybdates. Mechanism of inhibitory effect of PM-104 (NH4)12H2(Eu4(MoO4(H2O)16(Mo7O24)4) · 13H2O on human immunodeficiency virus type 1

A screening for inhibitors of human immunodeficiency virus type 1 (HIV-1) among various types of isopolyoxomolybdates and heteropolyoxomolybdates was carried out by using an in vitro assay system measuring the cytopathogenicity of HIV-1 in CD4+ human MT-4 cells. A novel heteropolyoxomolybdate named PM-104 with the chemical formula (NH4)12H2(Eu4(MoO4)(H2O)16(Mo7O24)4).13H2O was found to be associated with potent anti-HIV-1 activity. PM-104 interferes with virus infection at a very early step such as adsorption and/or penetration into the cells. In addition to the cytopathic effect of HIV-1 on MT-4 cells, syncytium formation between mock-infected MOLT-4 cells and MOLT-4 cells chronically infected with either HIV-1 or HIV-2 is suppressed by PM-104. PM-104 also blocks the replication of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The antiviral properties of PM-104 could be attributed to the combined effect of europium atoms and its peculiar three-dimensional anion structure.

Troglitazone ameliorates lipotoxicity in the beta cell line INS-1 expressing PPAR gamma

To elucidate the mechanisms by which troglitazone, which is a direct ligand for peroxisome proliferator-activated receptor (PPAR) gamma, ameliorates insulin resistance, we have demonstrated that PPAR gamma is expressed in a pancreatic beta cell line, INS-1, using reverse transcription-polymerase chain reaction (RT-PCR). We incubated the cells with 5 micromol/l troglitazone and 1 mmol/l of each major free fatty acid (FFA; palmitic acid, oleic acid, and linoleic acid), alone or in combination, for 48 h. After that, we evaluated glucose-stimulated insulin secretion (GSIS) and 25 mmol/l KCl-induced insulin secretion in the presence of diazoxide, which clamps membrane potential. Our results showed: (1) treatment with troglitazone for 48 h caused enhancement of GSIS, although troglitazone significantly suppressed cell viability assessed by MTT assay. (2) In cells co-treated with troglitazone and FFA, troglitazone ameliorated lipotoxicity due to FFA. (3) In the presence of 300 micromol/l diazoxide and 25 mmol/l KCl, troglitazone did not affect the recovery of GSIS in INS-1 cells. These results suggest that insulin secretion from the rat insulinoma cell line, INS-1, is modulated by troglitazone, acting somewhere in the ATP-sensitive K(+) channel pathway, possibly through PPAR gamma.

Studies on the N-[(trans-4-Isopropylcyclohexyl)-carbonyl]-d-phenylalanine (A-4166) receptor in HIT T-15 cells: Displacement of [3H]glibenclamide

A-4166 is a new type of oral hypoglycemic agent that does not contain a sulfonylurea moiety. To clarify the mechanism of insulin secretion by A-4166, a specific receptor for A-4166 was investigated in a hamster pancreatic beta cell line (HIT T-15), using [3H]A-4166 or [3H]glibenclamide as a ligand. The saturation binding of [3H]A-4166 to HIT cell membranes was not observed up to 10 microM. In the displacement study, unlabeled A-4166 inhibited [3H]A-4166 binding to HIT cell membranes, but glibenclamide did not. On the other hand, A-4166 inhibited [3H]glibenclamide binding to the sulfonylurea receptor (Ki = 248 nM). A-4166 inhibited 86Rb efflux from HIT cells (IC50 = 350 nM). The EC50 for insulin secretion by A-4166 was 20 microM in HIT cells when they were incubated for 30 min in Krebs-Ringer bicarbonate buffer containing 16 mM HEPES supplemented with 5 mg/mL BSA in the absence of glucose. These data demonstrate the possibility of the presence of two kinds of binding sites for A-4166: one of them is the sulfonylurea receptor, and the other might be a binding site specific for A-4166.

Stimulating Activity of A-4166 on Insulin Release in in situ Hamster Pancreatic Perfusion

Using the in situ hamster pancreatic perfusion system, the stimulating action of A-4166 on insulin release was examined in comparison with that of glibenclamide. Both antidiabetic agents stimulated insulin release, but its onset by A-4166 was faster than that by glibenclamide. In the presence of a basal glucose concentration (3 mmol/l), insulin releases induced by A-4166 and glibenclamide were inhibited by preexisting diazoxide. At higher glucose concentrations (5-16.7 mmol/l), however, A-4166 was able to reverse the inhibitory effect of diazoxide on the first and second phases of insulin release, while glibenclamide did not reverse the first-phase release. On the other hand, in the presence of 16.7 mmol/l of glucose A-4166 completely reversed the inhibitory action of diazoxide added simultaneously, but glibenclamide reversed it only partially. In the presence of 8 mmol/l of glucose, the stimulating action of A-4166 and glibenclamide on insulin release was hardly affected by inhibitors of ATP production. These results indicate that the stimulating action of A-4166 on insulin release is different from glibenclamide in response to the inhibitory action of diazoxide. These results also suggest that A-4166 is an effective agent for release of insulin by acting on the KATP channel, especially under an impaired function of pancreatic B cells.

Effects of α2-adrenergic agonism, imidazolines, and G-protein on insulin secretion in β cells

It is well known that α2-adrenergic agonism inhibits insulin secretion and stimulates glucagon secretion in both animal and human studies. Recently, α2-adrenergic blockers (DG-5128, MK-912, and SL 84.0418) have been studied as antihyperglycemic agents in human subjects. To clarify the action mechanism(s) of these agents, we investigated the effects of α2 agonists and antagonists (10−10 to 10−4 mol/L) and pretreatment by pertussis toxin (PTX) on glucose-stimulated insulin secretion using the hamster insulinoma cell line HiT-T15. The imidazoline-derivative α2-adrenoceptor agonists clonidine and oxymetazoline at concentrations as low as 10−8 mol/L significantly inhibited glucose-stimulated insulin secretion by 63% and 65%, respectively (P < .01 for both). These inhibitory effects were abolished by 20-hour preincubation of these cells with PTX 100 ng/mL. The imidazoline-derivative α2-adrenoceptor antagonist DG-5128 at a concentration of 10−4 mol/L doubled insulin secretion with or without pretreatment by PTX (P < .01 for both). Furthermore, both clonidine and oxymetazoline at a high concentration of 10−4 mol/L stimulated insulin secretion with pretreatment of the cells by PTX (P < .05 for both). These results indicate that glucose-stimulated insulin secretion is inhibited by α2-adrenoceptor agonists through PTX-sensitive G-protein in HIT-T15 cells. It is also suggested that imidazoline compounds at high concentrations directly stimulate insulin secretion.

Mechanism of the protective effect of heteropolyoxotungstate against herpes simplex virus type 2

The effects of heteropolyoxotungstate (K7[PTi2W10O40]· 6H2O; PM-19) on the replication of herpes simplex virus type 2 (HSV-2) were examined using a semiquantitative polymerase chain reaction of intracellular viral DNA established by us and also other methods. Vero cells were infected with HSV-2 strains: either the standard strain 169, or the acyclovir-resistant strain YS-4C-1. PM-19 was added at various stages during the replication of HSV-2. PM-19 strongly inhibited the synthesis of viral genomic DNA when it was added at the time of infection. The addition of PM-19 60–90 min after viral inoculation time-dependently decreased the antiviral activity and increased the relative yield of viral DNA, and the addition of PM-19 was completely ineffective at times later than 90 min. These results suggested that PM-19 inhibited viral penetration but did not affect the synthesis of viral DNA. Furthermore, PM-19 strongly inhibited a second round of infection.

Effects of D-Phenylalanine- Derivative Hypoglycemic Agent A-4166 on Pancreatic α- and β-Cells: Comparative Study with Glibenclamide

We have reported that N-[(trans-4-isopropyl-cyclohexyl)-carbonyl]-D-phenylalanine (A-4166) stimulates insulin secretion in animal studies. To further elucidate the mechanisms underlying the actions of

Effects of N-[(trans-4-isopropylcyclohexyl)-carbonyl]-D-phenylalanine (A-4166) on insulin and glucagon secretion in isolated perfused rat pancreas.

N-[(trans-4-isopropylcyclohexyl)-carbonyl]-D-phenylalanine (A-4166) has a structure which differs from those of other known blood glucose-lowering agents including sulfonylureas. It has been shown that oral administration of A-4166 exerts blood glucose-lowering effects in animal in vivo studies. In the present study, we investigated the effects of A-4166 on insulin and glucagon secretion at several glucose concentrations using isolated perfused rat pancreas preparations. Both 3.0 and 30 mumol/l A-4166 significantly stimulated insulin secretion as compared with basal levels at glucose concentrations of 8.0 and 11.0 mmol (p < 0.01 and p < 0.05, respectively). In contrast, glucagon secretion was not affected by administration of A-4166 up to 30 mumol/l at these glucose concentrations. At a glucose concentration of 5.6 mmol/l, neither 0.3 nor 3.0 mumol/l A-4166 produced significant changes in insulin and glucagon secretion. However, A-4166 at 30 mumol/l significantly stimulated insulin secretion and inhibited glucagon secretion as compared with basal levels (p < 0.01 and p < 0.01, respectively). We conclude that A-4166 at 3.0 and 30 mumol/l directly stimulates insulin secretion but has little effect on glucagon secretion in isolated perfused rat pancreas at glucose concentrations of 8.0 and 11.0 mmol/l. these results, taken together with previously published data, suggest that oral administration of A-4166 could be a useful strategy for stimulating endogenous insulin secretion in non-insulin-dependent diabetic patients.

Palmitic acid-rich diet suppresses glucose-stimulated insulin secretion (GSIS) and induces endoplasmic reticulum (ER) stress in pancreatic islets in mice

Abstract The objective was to clarify whether dietary palmitic acid supplementation affects glucose-stimulated insulin secretion (GSIS) and the endoplasmic reticulum (ER) stress pathway in pancreatic islets in mice. Eight-week-old male C57BL/6J mice were randomly divided into three treatment diet groups: control diet, palmitic acid-supplemented diet (PAL) and oleic acid-supplemented diet (OLE). After 2 weeks of treatment, intraperitoneal glucose tolerance test and intraperitoneal insulin tolerance test were performed. GSIS was assessed by pancreatic perfusion in situ with basal (100 mg/dL) glucose followed by a high (300 mg/dL) glucose concentration. We measured mRNA levels of ER stress markers such as C/EBP homologous protein (CHOP), immunoglobulin heavy-chain binding protein (BIP) and X-box binding protein (XBP)-1 using real-time polymerase chain reaction (PCR) analyses in isolated islets. Immunohistochemical staining was also performed. Mice fed PAL showed significantly decreased glucose tolerance (p < 0.05). In the perfusion study, GSIS was significantly suppressed in the PAL group (p < 0.05). Semi-quantitative RT-PCR revealed that islet CHOP, BIP, and XBP-1 mRNA expression were significantly increased in the PAL group (p < 0.05). TUNEL-positive β-cells were not detected in all groups. Dietary palmitic acid-supplementation for 2 weeks might suppress GSIS and induce ER stress in pancreatic islets in mice, in the early stage of lipotoxicity.