Hyperglycemia is a persistent symptom of diabetes mellitus. The complex pathogenesis of type 2 diabetes involves the progressive development of insulin resistance and defective insulin secretion, which leads to overt hyperglycemia. 1)These pathological states are mostly attributable to life styles and genetic background, 2) and various animal models of diabetes have been developed and investigated to clarify the mechanisms by which hyperglycemia is induced. [3][4][5][6][7][8] We previously showed that sustained hyperglycemia could be induced in ddY mice by refeeding with standard chow pellets after fasting for 48 h. 9) We proposed that, in this case, hyperglycemia was induced by defects in insulin secretion and action, and maintained by the resulting insulin resistance. However glucose levels and the duration of hyperglycemia were variable in individual mice. Since then, we have attempted to select those mice with induced serious hyperglycemia by selective breeding based on serum glucose levels after refeeding. Two strains, namely, spontaneous insulin resistant mice (ddY-H) and none insulin resistant mice (ddY-L), have been isolated from ddY mice by inbreeding. In this study, we investigated the characteristics of these mice. MATERIALS AND METHODSAnimal Care Four-week-old male ddY mice were purchased from SLC Inc. (Hamamatsu, Japan), and ddY-H and ddY-L mice from our own colony were used. Mice were maintained on 12 h light/dark cycles with free access to standard chow pellets (MF diet, Oriental Yeast Co., Ltd., Tokyo, Japan) and water ad libitum until the experiments were carried out. Animal care and experiments were performed in accordance with the guidelines for the care and use of laboratory animals of the University of Shizuoka, Japan.Fasting and Refeeding Fasting commenced by removing the chow pellets from mouse home cages at 21:00. After 48 h, mice were refed standard chow pellets for 12 h and blood was obtained from the caudal vein for the determination of serum glucose level. In some experiments, mice were fasted for 12 h and refed for 1 h as described above.Oral Glucose Tolerance Oral glucose tolerance test was carried out as described previously.9) Briefly, mice were orally administered with glucose (3 g/kg) and blood was withdrawn from caudal veins 0, 30, 60 and 120 min later to measure serum glucose level.Measurement of Serum Glucose and Insulin Levels Serum glucose and insulin levels were determined using Glucose CII Test Wako (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and the Insulin ELISA kit (Morinaga Institute of Biological Sciences, Inc., Kanagawa, Japan), respectively, according to manufacturer's instructions.Insulin Sensitivity and Insulin Resistance Insulin sensitivity and insulin resistance were calculated from glucose and insulin levels in the serum of mice fasted for 12 h (21:00-9:00) as follows: glucose (mg/dl)/insulin (ng/dl) and glucose (mg/dl)ϫinsulin (ng/dl)/100, respectively. 10,11)Cross-Mating between ddY-H Mice and ddY-L Mice ddY-H mice and ddY-L mice at 10 weeks of age w...

We have isolated insulin resistant mice (ddY-H mice) which are spontaneously induced at 12-weeks of age even if fed with the standard chow pellets. Since accumulated evidences have suggested that an appearance of insulin resistance is associated with obesity and a state of inflammation in adipose tissue, the present study investigated an appearance of macrophages in adipose tissue of ddY-H mice. Although ddY-H mice were fed the standard chow pellets ad libitam, increases in body weight, adipose tissue mass, and fat cell size were observed. In adipose tissues of ddY-H mice, gene expression of monocyte chemoattractant protein-1 (MCP-1) elevated slightly at 5-weeks of age and was maintained at higher levels at 9-and 12-weeks of age, and MCP-1 content in adipose tissue increased 2-fold at 12-weeks of age. Also, increased gene expressions of CD68 and F4/80, markers of macrophage, in adipose tissue were observed at 9-weeks of age. In addition, F4/80 positive cells were histologically found in adipose tissue at 15-weeks of age but not at 7-weeks of age, suggesting an increased infiltration of macrophage into adipose tissue. In adipose tissue of ddY-H mice, gene expressions of CD11c and toll-like receptor 4 (TLR4), markers of proinflammatory macrophages (M1), markedly increased although those of CD163 and mannose receptor (MR), markers of anti-inflammatory macrophages (M2), did not change. These results suggest that proinflammatory (M1) macrophages infiltrate into enlarged adipose tissues of ddY-H mice, which is preceding spontaneous appearance of insulin resistance.

Halogenated pyrimidines are incorporated in place of thymidine in DNA of replicating cells, making the cells more susceptible to the effects of radiation. 5-Bromo-2Ј-deoxyuridine (BrdUrd) has been clinically used as a radiosensitizer to enhance the tumor killing effect of radiation therapy. [1][2][3][4] The radiosensitizing effect of halogenated pyrimidines on mammalian cells in vitro is closely associated with the amount of drug incorporated into DNA. 5) Alkylating agents, such as cyclophosphamide (CPA), are widely used to treat a broad spectrum of malignancies, including both solid tumors and hematological malignancies. 6,7) CPA is converted by hepatic cytochrome P450 metabolic enzymes via two major pathways. 8) The first involves 4-hydroxylation to the active metabolite, 4-hydroxycyclophosphamide (4-OH-CPA). This conversion is carried out predominantly by cytochrome P450 CYP2B6 in humans. 9,10) 4-OH-CPA exists in equilibrium with aldophosphamide, which is broken down to form the DNA cross-linking agent phosphoramide mustard and the toxic metabolite acrolein. [11][12][13] The alternative pathway involves a CYP3A4-mediated N-dechloroethylation of CPA to form the inactive metabolite 3-dechloroethylcyclophosphamide (DECP) and the toxic byproduct chloroacetaldehyde. 11,14,15) In humans, this pathway is secondary to the activation pathway and accounts for less than 10% of the dose. 13) Alkylating agents undergo a transformation to produce highly reactive and positively charged ions. These ions can then form covalent bonds with electron-rich sites on biological molecules, such as nucleic acids, proteins, and amino acids. 16) Irradiation may cause the ablation of both singleand double-stranded DNA, degeneration or loss of bases, and intercalation of DNA and/or chromosomal protein. The actions of alkylating agents appear to have some similarities to those of gamma ray radiation. A previous in vitro report showed that pretreatment with iododeoxyuridine (IdUrd) or BrdUrd enhanced the cytotoxicity of various antineoplasmic agents, such as melphalan, cisplatin, doxorubicin, and bleomycin. 17) However, the mechanism of halopyrimidine's chemosensitizing effect is still unclear. There have been few in vivo studies on the combined antitumor effects of halogenated pyrimidines and alkylating agents. We investigated here the combined effect of BrdUrd and CPA, a bifunctional alkylating agent, on life span in L1210 ascites tumor-bearing mice and cytotoxicity in cultured L1210 cells using an MTT assay. Life Prolongation Study Male BDF1 mice were purchased from Shionogi Pharmaceutical Co., Ltd. and kept at Mie University Animal Center under constant conditions (a 12 h light : 12 h dark regimen with Oriental Chow pellet food and water freely available). L1210 leukemia cells were obtained from Shionogi Pharmacological Laboratory, Osaka, Japan and maintained in BDF1 mice by weekly intraperitoneal (i.p.) passage as previously described. 18) Experimental mice received an i.p. injection of 1ϫ10 6 ascites tumor cells in saline on Day 0. Se...

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