Yutaka Kato

Effects of Aryl Hydrocarbon Receptor Signaling on the Modulation of Th1/Th2 Balance

An orally active antiallergic agent, M50367, skews the Th1/Th2 balance toward Th1 dominance by suppressing naive Th cell differentiation into Th2 cells in vitro. Administration results in the suppression of IgE synthesis and peritoneal eosinophilia in vivo. In this report, we determined that M50354 (an active metabolite of M50367) was a ligand for the aryl hydrocarbon receptor (AhR); the immunological effects of this compound on in vitro Th1/Th2 differentiation from naive Th cells and Th1/Th2 balance in vivo were manifested through binding to AhR. These effects were completely abolished in AhR-deficient mice. AhR expression in the naive Th cell was significantly up-regulated by costimulation of TCR and CD28. Suppression of naive Th cell differentiation into Th2 cells via binding of M50354 to AhR was associated with inhibition of GATA-3 expression in Th cells. In addition, forced expression of a constitutively active form of AhR or activation of AhR by the addition of representative ligands suppressed naive Th cell differentiation into Th2 cells. Based on these results, we conclude that AhR functions as a modulator of the in vivo Th1/Th2 balance through activation in naive Th cells.

The Inhibitory Action of Sodium Arsenite on Lipopolysaccharide-Induced Nitric Oxide Production in RAW 267.4 Macrophage Cells: A Role of Raf-1 in Lipopolysaccharide Signaling

The effect of sodium arsenite (SA) on LPS-induced NO production in RAW 267.4 murine macrophage cells was studied. SA pretreatment of LPS-stimulated RAW cells resulted in a striking reduction in NO production. No significant difference in LPS binding was observed between RAW cells pretreated with SA and control untreated RAW cells, suggesting that SA might impair the intracellular signal pathway for NO production. SA inhibited LPS-induced NF-κB activation by preventing loss of IκB-α and -β. Furthermore, SA blocked phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2), but not phosphorylation of p38 and c-Jun N-terminal kinase. SA treatment resulted in the disappearance of Raf-1, suggesting that it might cause the inhibition of the Erk1/2 mitogen-activated protein (MAP) kinase pathway. The SA-mediated loss of Raf-1 also abolished LPS-induced NF-κB activation as well as the Erk1/2 pathway. The dominant negative mutant of MAP kinase kinase 1 inhibited both NO production and NF-κB activation in LPS-stimulated RAW cells. Taken together, these results indicate that the inhibitory action of SA on NO production in LPS-stimulated macrophages might be due to abrogation of inducible NO synthase induction, and it might be closely related to inactivation of the NF-κB and Erk1/2 MAP kinase pathways through loss of Raf-1.

The inhibitory action of butyrate on lipopolysaccharide-induced nitric oxide production in RAW 264.7 murine macrophage cells

The effect of butyrate, a natural bacterial product of colonic bacterial flora, on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 murine macrophage cells was studied. Butyrate significantly reduced NO production in LPS-stimulated RAW cells. The inhibition was abolished by the removal of butyrate. Butyrate also inhibited the expression of inducible type NO synthase (iNOS) in LPS-stimulated RAW cells. Furthermore, butyrate prevented the activation of nuclear factor (NF)-κB through the stabilization of IκB-α and IκB-β. Butyrate did not affect the phosphorylation of mitogen-activated protein (MAP) kinases by LPS. It was, therefore, suggested that butyrate down-regulated LPS-induced NO production in RAW cells through preventing the expression of iNOS, and that it was due to the inhibitory action of butyrate on the activation of NF-κB.

Augmentation of Nitric Oxide Production by Gamma Interferon in a Mouse Vascular Endothelial Cell Line and Its Modulation by Tumor Necrosis Factor Alpha and Lipopolysaccharide

ABSTRACT The effect of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and lipopolysaccharide (LPS) on nitric oxide (NO) production in the mouse vascular aortic endothelial cell line END-D was examined. LPS, TNF-α, and a low concentration of IFN-γ inhibited NO production in END-D cells, while a high concentration of IFN-γ definitely enhanced it. The NO production induced by a high concentration of IFN-γ was further augmented by using IFN-γ in combination with LPS or TNF-α. In sequential incubations of LPS and IFN-γ, the enhancement of NO production required prior treatment with IFN-γ. Stimulation of END-D cells with a high concentration of IFN-γ led to the expression of inducible NO synthase (iNOS). The augmentation of NO production by IFN-γ alone or in combination with LPS or TNF-α was completely blocked by several inhibitors of iNOS. It was strongly suggested that a high concentration of IFN-γ itself enhanced NO production in END-D cells through inducing the expression of iNOS. LPS and TNF-α exclusively modulated the activity of iNOS once its expression was triggered by IFN-γ. On the other hand, a low concentration of IFN-γ, LPS, and TNF-α reduced NO production through down-regulating constitutive NOS (cNOS). The differential regulation of cNOS- and iNOS-mediated NO production by IFN-γ, TNF-α, and LPS is discussed.

Cytoskeletal Alterations in Lipopolysaccharide-Induced Bovine Vascular Endothelial Cell Injury and Its Prevention by Sodium Arsenite

ABSTRACT Morphological changes, especially cytoskeletal alterations, in lipopolysaccharide (LPS)-induced vascular endothelial cell injury were studied by using LPS-susceptible bovine aortic endothelial cells (BAEC). BAEC in cultures with LPS showed cell rounding, shrinking, and intercellular gap formation. In those cells, LPS caused the disorganization of actin, tubulin, and vimentin. LPS also induced a reduction in the F-actin pool and an elevation in the G-actin pool. Cytoskeletal disorganization affected transendothelial permeability across the endothelial monolayer. Pretreatment of BAEC with sodium arsenite (SA) prevented alterations in LPS-induced BAEC injury. However, posttreatment with SA had no protective effect on them. SA upregulated the expression of heat shock protein in the presence of LPS. The role of SA in prevention of LPS-induced BAEC injury is discussed.

Mouse B1 cell line responds to lipopolysaccharide via membrane-bound CD14

The role of membrane-bound CD14 in the response of mouse B1 cell lines to lipopolysaccharide (LPS) was studied. The surface profile of mouse TH2.52 B cells was positive for CD5, IgM, B220, CD11b and F4/80, suggesting that TH2.52 cells carried the typical phenotype of B1 cells. Furthermore, TH2.52 B1 cells were found to express membrane-bound CD14, which plays a critical role in LPS recognition. TH2.52 B1 cells responded to a very low concentration of LPS and exhibited: (i) augmentation of DNA synthesis; (ii) activation of nuclear factor (NF)-κB; and (iii) phosphorylation of extracellular signal regulated kinase 1/2 (Erk1/2). They were markedly inhibited by anti-CD14 antibody. Therefore, the expression of membrane-bound CD14 was suggested to provide high sensitivity to LPS for TH2.52 B1 cells.

Intestine-Targeted DGAT1 Inhibition Improves Obesity and Insulin Resistance without Skin Aberrations in Mice

Objective Diacylglycerol O-acyltransferase 1 (DGAT1) catalyzes the final committed step in triglyceride biosynthesis. DGAT1 null mice are known to be resistant to diet-induced obesity, and more insulin sensitive relative to the wild-type; however, the mice exhibit abnormalities in the skin. This work determined whether the intestine-targeted DGAT1 inhibitor could improve obesity and insulin resistance without skin aberrations in mice. Design and Methods We synthesized 2 DGAT1 inhibitors: Compound A, described in the patent application from the Japan Tobacco, and Compound B (A-922500), reported by Abbott Laboratories. Both compounds were evaluated for inhibitory activities against DGAT1 enzymes and effects on the skin in mice in vivo. Compound B was further investigated for effects on obesity and insulin resistance in diet-induced-obese (DIO) mice. Results The 2 compounds comparably inhibited the DGAT1 enzyme activity and the cellular triglyceride synthesis in vitro, while they showed different distribution patterns in mice in vivo. Compound A, which distributed systemically, caused skin aberrations, while Compound B, which preferentially distributed to the intestine, improved obesity and insulin resistance without skin aberrations in DIO mice. Conclusions Our results suggest that the intestine is the key tissue in which DGAT1 plays a role in promoting obesity and insulin resistance.

A novel free fatty acid receptor 1 (GPR40/FFAR1) agonist, MR1704, enhances glucose‐dependent insulin secretion and improves glucose homeostasis in rats

Activation of G protein‐coupled receptor 40/Free fatty acid receptor 1 (GPR40/FFAR1), which is highly expressed in pancreatic β cells, is considered an important pharmacologic target for the treatment of type 2 diabetes mellitus. The aim of this study was to determine the effect of MR1704, a novel GPR40/FFAR1 agonist, on glucose homeostasis in rats. MR1704 is a highly potent and selective, orally bioavailable agonist with similar in vitro potencies among humans, mice, and rats. Treatment of rat islets with MR1704 increased glucose‐dependent insulin secretion. Augmentation of glucose‐dependent insulin secretion was abolished by adding a GPR40/FFAR1 antagonist. In mouse, insulinoma MIN6 cells, palmitic acid induced the activity of caspase 3/7 after a 72‐h exposure, while pharmacologically active concentrations of MR1704 did not. In an oral glucose tolerance test in normal Sprague‐Dawley rats, orally administered MR1704 (1–10 mg·kg−1) reduced plasma glucose excursion and enhanced insulin secretion, but MR1704 did not induce hypoglycemia, even at 300 mg·kg−1, in fasted Sprague‐Dawley rats. In addition, orally administered MR1704 reduced plasma glucose excursion and enhanced insulin secretion in diabetic Goto‐Kakizaki rats. Oral administration of MR1704 once daily to Goto‐Kakizaki rats reduced their blood glucose levels during a 5‐week treatment period without reducing pancreatic insulin content; as a result, hemoglobin A1C levels significantly decreased. These results suggest that MR1704 improves glucose homeostasis through glucose‐dependent insulin secretion with a low risk of hypoglycemia and pancreatic toxicity. MR1704 shows promise as a new, glucose‐lowering drug to treat type 2 diabetes mellitus.

Exacerbation of Vascular Endothelial Injury in the Generalized Shwartzman Reaction by the Administration of Anti-E-Selectin Antibody

Previously, we reported that the consecutive administration of lipopolysaccharide (LPS) into LPS‐sensitized mice for the generalized Shwartzman reaction (GSR) induced systemic injury of vascular endothelial cells. The aim of this study was to investigate the participation of vascular adhesion molecules in the vascular endothelial injury of GSR. The administration of anti‐E‐selectin antibody in GSR‐induced mice resulted in massive apoptosis of vascular endothelial cells and congestion in blood vessels. Further, marked hemorrhage was found in the pulmonary alveoli of those mice. GSR, especially lung injury, was definitely exacerbated by the administration of anti‐E‐selectin antibody. On the other hand, the administration of anti‐VCAM‐1 antibody did not induce such injury of vascular endothelial cells. The possible role of E‐selectin in the exacerbation of vascular endothelial injury in GSR is discussed.