Haruhisa Fujita

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.

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.

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.

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.

Mechanism of Action of Anti-Influenza Benzamidine Derivatives

Benzamidine derivatives exhibited a high antiviral effect in vivo against influenza virus strains A2/Adachi and B/Lee. The inhibiting properties of anti-influenza benzamidine derivatives on the virus-induced inflammation undoubtedly plays an important role in the clarification of the mechanism of action of these drugs.

Effects of 5-fluorouracil and 2α-methyldihydrotestosterone propionate on the growth of human breast carcinoma MCF-7 in vitro

5-Fluorouracil (5-FU) and 2 alpha-methyldihydrotestosterone propionate (MDTP) have effectively induced complete regressions of induced rat mammary carcinomas; in combination, regressions were additive and synergistic. Present aims were to determine whether similar antitumor effects were obtainable with a human mammary carcinoma, MCF-7, and to affirm the synergism of 5-FU and MDTP. After incubation in vitro for 3 days and exposure to drug for another 2 days, cell counts and/or determinations of total cell protein revealed growth inhibitions of 16-87% by 5-FU at 130-1300 micrograms/ml and 16-94% by MDTP at 0.36-360.5 micrograms/ml. Combinations of 5-FU and MDTP at the same inhibitory doses (ID) yielded approximately additive growth inhibitions. Algebraic and geometric (isobole) methods of analyses showed that these inhibitions were additive or synergistic, depending on the iso-effective dose used. Precursor incorporation into macromolecules also showed approximately additive effects for MCF-7 cells treated with 5-FU and MDTP, each at ID15. These data demonstrate significant additive growth-inhibitory activity of 5-FU and MDTP in combination against MCF-7 in vitro, thus affirming their antitumor effects in vivo.