In vitro binding assays are useful in the initial screening of endocrine disrupting chemicals. Such assays should be applied to the estrogen receptors (ER) of not only humans but also wildlife. As a system for birds is yet to established, we expressed the ligand binding domain (LBD) of quail ERα and ERβ as a fusion protein with glutathione S-transferase and using these proteins, developed two systems (a competitive enzyme immunoassay and a fluorescence polarization assay) for assaying the capacity to bind ERs in vitro. Moreover, 20 test chemicals selected by Ministry of the Environment of Japan were evaluated in terms of binding ability. Both systems worked well, the competitive enzyme immunoassay proving especially powerful, since it needs no special equipment. This system is applicable to other species including fish, amphibians and reptiles when information on the LBD of ER is available.Key words ---endocrine disrupting chemicals, estrogen receptor, enzyme immunoassay, quail, endocrine disruptor, in vitro binding assay completed. 5)For the initial screening in vitro, the receptor binding assay is often utilized. The yeast two-hybrid assay is particularly useful, since it is easy and relatively cheap to perform, and also no special equipment is needed. 6,7) This method is based on the interaction between the ligand binding domain (LBD) in the hormone receptor and the coactivator in the ligand-dependent manner. 6) Recently, it was proposed that the effect of EDCs should be considered not only in humans but also in wildlife, and indeed adverse effects on humans and wildlife were reported. However, information on coactivators is restricted to specific species, such as humans, rats and mice.

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Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is the only enzyme that converts inactive glucocorticoids to active forms and plays an important role in the regulation of glucocorticoid action in target tissues. JTT-654 is a selective 11β-HSD1 inhibitor and we investigated its pharmacological properties in cortisone-treated rats and non-obese type 2 diabetic Goto-Kakizaki (GK) rats because Asians, including Japanese, are more likely to have non-obese type 2 diabetics. Systemic cortisone treatment increased fasting plasma glucose and insulin levels and impaired insulin action on glucose disposal rate and hepatic glucose production assessed by hyperinsulinemic-euglycemic clamp, but all these effects were attenuated by JTT-654 administration. Cortisone treatment also reduced basal and insulin-stimulated glucose oxidation in adipose tissue, increased plasma glucose levels after administration of the pyruvate, the substrate of gluconeogenesis, and increased liver glycogen content. Administration of JTT-654 also inhibited all of these effects. Cortisone treatment decreased basal and insulin-stimulated 2-deoxy-D-[1-3 H]-glucose uptake in 3T3-L1 adipocytes and increased the release of free fatty acids and glycerol, a gluconeogenic substrate, from 3T3-L1 adipocytes, and JTT-654 significantly attenuated these effects. In GK rats, JTT-654 treatment significantly reduced fasting plasma glucose and insulin levels, enhanced insulin-stimulated glucose oxidation in adipose tissue, and suppressed hepatic gluconeogenesis as assessed by pyruvate administration. These results demonstrated that glucocorticoid was involved in the pathology of diabetes in GK rats, as in cortisone-treated rats, and that JTT-654 ameliorated the diabetic conditions. Our results suggest that JTT-654 ameliorates insulin resistance and non-obese type 2 diabetes by inhibiting adipose tissue and liver 11β-HSD1.

Peripheral artery disease (PAD) is defined as peripheral blood flow impairment, especially in the legs, caused by atherosclerotic stenosis. The disease decreases quality of life because of intermittent claudication or necrosis of the leg. The hindlimb ischaemia model, in which ischaemia is induced by femoral artery ligation, is often utilized as a PAD model. In the hindlimb ischaemia model, nonmetabolic syndrome animals are mainly used. In this study, we investigated the usefulness of Spontaneously Diabetic Torii Leprfa (SDT fatty) rats, a new model for obese type 2 diabetes, as a new PAD animal model. We found that hindlimb blood flow in SDT fatty rats was significantly lower than that in Sprague–Dawley (SD) rats under nonischaemic conditions. Furthermore, SDT fatty rats showed a significantly higher plasma nitrogen oxide level, shorter prothrombin time, and shorter activated partial thromboplastin time than SD rats. In addition, we found that the change in blood flow 7 days after induction of hindlimb ischaemia and the number of Von Willebrand factor‐positive vessels in gastrocnemius muscles were significantly lower in SDT fatty rats than in SD rats. These results suggest that excess production of reactive oxygen species and coagulation activation could be involved in lower blood flow in non‐ischaemic rats and that decreased angiogenesis could be involved in the poor recovery of blood flow in SDT fatty rats with hindlimb ischaemia. Taken together, our results suggest that SDT fatty rats might be useful as a new model for PAD with metabolic syndrome.

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