Haruo Hashimoto

Overexpression of Monocyte Chemoattractant Protein-1 in Adipose Tissues Causes Macrophage Recruitment and Insulin Resistance *

Adipose tissue expression and circulating concentrations of monocyte chemoattractant protein-1 (MCP-1) correlate positively with adiposity. To ascertain the roles of MCP-1 overexpression in adipose, we generated transgenic mice by utilizing the adipocyte P2 (aP2) promoter (aP2-MCP-1 mice). These mice had higher plasma MCP-1 concentrations and increased macrophage accumulation in adipose tissues, as confirmed by immunochemical, flow cytometric, and gene expression analyses. Tumor necrosis factor-α and interleukin-6 mRNA levels in white adipose tissue and plasma non-esterified fatty acid levels were increased in transgenic mice. aP2-MCP-1 mice showed insulin resistance, suggesting that inflammatory changes in adipose tissues may be involved in the development of insulin resistance. Insulin resistance in aP2-MCP-1 mice was confirmed by hyperinsulinemic euglycemic clamp studies showing that transgenic mice had lower rates of glucose disappearance and higher endogenous glucose production than wild-type mice. Consistent with this, insulin-induced phosphorylations of Akt were significantly decreased in both skeletal muscles and livers of aP2-MCP-1 mice. MCP-1 pretreatment of isolated skeletal muscle blunted insulin-stimulated glucose uptake, which was partially restored by treatment with the MEK inhibitor U0126, suggesting that circulating MCP-1 may contribute to insulin resistance in aP2-MCP-1 mice. We concluded that both paracrine and endocrine effects of MCP-1 may contribute to the development of insulin resistance in aP2-MCP-1 mice.

Quantitative and qualitative analysis of rat pup ultrasonic vocalization sounds induced by a hypothermic stimulus

Ultrasonic vocalizations (USVs) are essential communicative sounds used between rodent pups and their mother. Rat pups emit USVs in stressful situations, such as when they are cold or separated from the nest. We verified the ontogenetic changes in USVs emitted by infant rats isolated from their mother during the pre-weaning period. The number of calls, and the median frequency and first peak of frequency of the calls were measured at 1, 3, 5, 7, 10, 12, and 14 days postnatal age in Wistar-Imamichi rats. Pups were placed in a cold glass beaker and USVs were recorded for 5 min. The number of calls increased to a peak on day 5 and then gradually decreased. The median frequency of calls decreased slowly during the first 12 days, and then increased slightly on day 14. Similarly, the first peak frequency of calls was the highest on day 1, and then decreased gradually by day 12. A small increase was observed on day 14. These changes in frequency were correlated with the physical development of the pups, whose body weights increased significantly with age except during postnatal days 7-10.

Reconsideration of insulin signals induced by improved laboratory animal diets, Japanese and American diets, in IRS-2 deficient mice.

Current Japanese and American diets and Japanese diet immediately after the War were converted to laboratory animal diets. As a result, current laboratory animal diet (CA-1, CLEA) unexpectedly resembled the diet of Japanese after the War. This is considered to result in an under-evaluation of diabetes research using laboratory animals at present. Therefore, changes in insulin signals caused by current Japanese and American diets were examined using IRS-2 deficient mice ( IRS2(-/-) mice) and mechanisms of aggravation of type 2 diabetes due to modern diets were examined. IRS2(-/-) mice at 6 weeks of age were divided into three groups: Japanese diet (Jd) group, American diet (Ad) group and CA-1 diet [regular diet (Rd)] group. Each diet was given to the dams from 7 days before delivery. When the IRS2(-/-) mice reached 6 weeks of age, the glucose tolerance test (GTT), insulin tolerance test (ITT) and organ sampling were performed. The sampled organs and white adipose tissue were used for analysis of RNA, enzyme activity and tissues. In GTT and ITT, the Ad group showed worse glucose tolerance and insulin resistance than the Rd group. Impaired glucose tolerance of the Jd group was the same as that of the Rd group, but insulin resistance was worse than in the Rd group. These results were caused an increase in fat accumulation and adipocytes in the peritoneal cavity by lipogenic enzyme activity in the liver and muscle, and the increase in TNFalpha of hypertrophic adipocyte origin further aggravated insulin resistance and the increase in resistin also aggravated the impaired glucose tolerance, leading to aggravation of type 2 diabetes. The Japanese and American diets given to the IRS2(-/-) mice, which we developed, showed abnormal findings in some IRS2(-/-) mice but inhibited excessive reactions of insulin signals as diets used in ordinary nutritional management.

A versatile technique for the in vivo imaging of human tumor xenografts using near-infrared fluorochrome-conjugated macromolecule probes.

Here, we present a versatile method for detecting human tumor xenografts in vivo, based on the enhanced permeability and retention (EPR) effect, using near-infrared (NIR) fluorochrome-conjugated macromolecule probes. Bovine serum albumin (BSA) and two immunoglobulins—an anti-human leukocyte antigen (HLA) monoclonal antibody and isotype control IgG2a—were labeled with XenoLight CF770 fluorochrome and used as NIR-conjugated macromolecule probes to study whole-body imaging in a variety of xenotransplantation mouse models. NIR fluorescent signals were observed in subcutaneously transplanted BxPC-3 (human pancreatic cancer) cells and HCT 116 (colorectal cancer) cells within 24 h of NIR-macromolecule probe injection, but the signal from the fluorochrome itself or from the NIR-conjugated small molecule (glycine) injection was not observed. The accuracy of tumor targeting was confirmed by the localization of the NIR-conjugated immunoglobulin within the T-HCT 116 xenograft (in which the orange-red fluorescent protein tdTomato was stably expressed by HCT 116 cells) in the subcutaneous transplantation model. However, there was no significant difference in the NIR signal intensity of the region of interest between the anti-HLA antibody group and the isotype control group in the subcutaneous transplantation model. Therefore, the antibody accumulation within the tumor in vivo is based on the EPR effect. The liver metastasis generated by an intrasplenic injection of T-HCT 116 cells was clearly visualized by the NIR-conjugated anti-HLA probe but not by the orange-red fluorescent signal derived from the tdTomato reporter. This result demonstrated the superiority of the NIR probes over the tdTomato reporter protein at enhancing tissue penetration. In another xenograft model, patient-derived xenografts (PDX) of LC11-JCK (human non-small cell lung cancer) were successfully visualized using the NIR-conjugated macromolecule probe without any genetic modification. These results suggested that NIR-conjugated macromolecule, preferably, anti-HLA antibody probe is a valuable tool for the detection of human tumors in experimental metastasis models using whole-body imaging.

Fulminant type 1 diabetes mellitus observed in insulin receptor substrate 2 deficient mice

The objective of this study was to characterise the fulminant type 1 diabetes mellitus (DM) accompanying abrupt hyperglycaemia and ketonuria observed in insulin receptor substrate 2 (IRS2)-deficient mice. IRS2-deficient mice backcrossed onto the original C57BL/6J:Jc1 background (B6J-IRS2−/− mice) for more than 10 generations were used. Eight male IRS2-deficient mice with ketonuria and abrupt increase in plasma glucose concentrations over 25 mmol/l were used as the fulminant type 1 diabetic mice (diabetic mice) and 8 male IRS2-deficient mice (8 weeks old) without glycosuria were used as the control mice. Plasma metabolite, immunoreactive insulin (IRI) and C-peptide concentrations, hepatic energy metabolism related enzyme activities and histopathological change in pancreatic islets were investigated. The diabetic mice showed significantly higher plasma glucose and cholesterol concentrations and lower plasma IRI and C-peptide concentrations than the control mice. In livers of the diabetic mice, glycolytic and malate-aspartate shuttle enzyme activities decreased significantly and gluconeogenic, lipogenic and ketone body synthesis enzyme activities increased significantly compared to those in the control mice. The pancreatic islets of the diabetic mice decreased significantly in size and number of β cells. The diabetic IRS2-deficient mice did not show the islet-related antibodies observed in the diabetic NOD mice in their sera. The characteristics of the diabetic IRS2-deficient mice resembled those of the human nonautoimmune fulminant type 1 DM. IRS2-deficient mice may be a useful animal model for studying the degradation mechanism of pancreatic β cells in the process of development of fulminant type 1 DM.

Efficient production of recombinant adeno-associated viral vector, serotype DJ/8, carrying the GFP gene

The purpose of this study was to establish an efficient method for the preparation of an adeno-associated viral (AAV), serotype DJ/8, carrying the GFP gene (AAV-DJ/8-GFP). We compared the yields of AAV-DJ/8 vector, which were produced by three different combination methods, consisting of two plasmid DNA transfection methods (lipofectamine and calcium phosphate co-precipitation; CaPi) and two virus DNA purification methods (iodixanol and cesium chloride; CsCl). The results showed that the highest yield of AAV-DJ/8-GFP vector was accomplished with the combination method of lipofectamine transfection and iodixanol purification. The viral protein expression levels and the transduction efficacy in HEK293 and CHO cells were not different among four different combination methods for AAV-DJ/8-GFP vectors. We confirmed that the AAV-DJ/8-GFP vector could transduce to human and murine hepatocyte-derived cell lines. These results show that AAV-DJ/8-GFP, purified by the combination of lipofectamine and iodixanol, produces an efficient yield without altering the characteristics of protein expression and AAV gene transduction.

Adiponectin deficiency-induced diabetes increases TNFα and FFA via downregulation of PPARα

Expression of peroxisome proliferator-activated receptor (PPAR) α was investigated in adiponectin knockout mice to elucidate the relationship between PPARα and adiponectin deficiency-induced diabetes. Adiponectin knockout (Adp−/−) mice were generated by gene targeting. Glucose tolerance test (GTT), insulin tolerance test (ITT), and organ sampling were performed in Adp−/− mice at the age of 10 weeks. PPARα, insulin, triglyceride, free fatty acid (FFA), and tumor necrosis factor α (TNFα) were analyzed from the sampled organs. Adp−/− mice showed impaired glucose tolerance and insulin resistance. Additionally, PPARα levels were decreased and plasma concentration of triglyceride, FFA and TNFα were increased. These data may indicate that insulin resistance in Adp−/− mice is likely caused by an increase in concentrations of TNFα and FFA via downregulation of PPARα.

Abstract B13: The prevention of lymphoproliferative lesions arising in patient-derived cancer xenografts by anti-graft-versus-host-disease agents

Xenografts derived from engrafting fresh surgical specimens directly onto immunodeficient mice have enabled the development of more relevant in vivo models for human cancers. Such patient-derived xenograft (PDX) models retain similar morphology, heterogeneities, and molecular signatures with the original cancers. We reported the rapid and efficient establishment of PDXs using super-immunodeficient NOG mice (PDX/NOG models). During the establishment process, a transplantable lymphoproliferative lesion (LPL) that replaces the original tumor cells sometimes occurs. The occurrence of LPL is the most problematic aspect of the establishment of PDX. This phenomenon may arise because of the Epstein-Barr virus due to the severely immunodeficient nature of the animal model; however, the mechanisms underlying LPL have not yet been elucidated in detail. In the present study, we histologically analyzed and tried to prevent the occurrence of LPL in PDX/NOG. Over 100 lines of cancer xenografts were established in our previous studies. Throughout these processes, we also established 10 LPL-PDX lines. Some LPL-PDX lines consist of B cells, T cells, and differentiated plasma cells. These LPL lymphocytes exhibited negligible proliferative potency in vitro. The invasions of LPL in the liver, lung, and pancreas were observed immunohistochemically. NOG mice bearing LPL-PDX (LPL-PDX/NOG) were treated with the anti-graft-versus-host-disease (anti-GVHD) drugs FK506 (1.0 mg/kg/day) and mycophenolate mofetil (MMF, 120 mg/kg/day) for 4 weeks using intraperitoneal injections. The LPL proliferations were significantly suppressed with FK506 (p=0.011) and MMF (p=0.014) compared with the control. The occurrence of LPL during establishment of PDX may be based on the GVHD-like mechanism. This phenomenon can be prevented by using anti-GVHD agents in the severely immunodeficient mice. The LPL-PDX/NOG models are also proposed for the utilization of GVHD mouse models with organic damage, particularly chronic GVHD mouse models. Citation Format: Tsuyoshi Chijiwa, Akira Noguchi, Daisuke Komura, Makoto Katayama, Mizuha Haraguchi, Haruo Hashimoto, Hiroshi Suemizu, Yoshiyasu Nakamura, Daisuke Furukawa, Takayuki Isagawa, Hiroto Katoh, Takashi Moriya, Shumpei Ishikawa, Masato Nakamura, Yohei Miyagi. The prevention of lymphoproliferative lesions arising in patient-derived cancer xenografts by anti-graft-versus-host-disease agents [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B13.

Sox17 is essential for proper formation of the marginal zone of extraembryonic endoderm adjacent to a developing mouse placental disk

Abstract In mouse conceptus, two yolk-sac membranes, the parietal endoderm (PE) and visceral endoderm (VE), are involved in protecting and nourishing early-somite-stage embryos prior to the establishment of placental circulation. Both PE and VE membranes are tightly anchored to the marginal edge of the developing placental disk, in which the extraembryonic endoderm (marginal zone endoderm: ME) shows the typical flat epithelial morphology intermediate between those of PE and VE in vivo. However, the molecular characteristics and functions of the ME in mouse placentation remain unclear. Here, we show that SOX17, not SOX7, is continuously expressed in the ME cells, whereas both SOX17 and SOX7 are coexpressed in PE cells, by at least 10.5 days postconception. The Sox17-null conceptus, but not the Sox7-null one, showed the ectopic appearance of squamous VE-like epithelial cells in the presumptive ME region, together with reduced cell density and aberrant morphology of PE cells. Such aberrant ME formation in the Sox17-null extraembryonic endoderm was not rescued by the chimeric embryo replaced with the wild-type gut endoderm by the injection of wild-type ES cells into the Sox17-null blastocyst, suggesting the cell autonomous defects in the extraembryonic endoderm of Sox17-null concepti. These findings provide direct evidence of the crucial roles of SOX17 in proper formation and maintenance of the ME region, highlighting a novel entry point to understand the in vivo VE-to-PE transition in the marginal edge of developing placenta. Summary Sentence The marginal extraembryonic endoderm adjacent to a developing placental disk continuously expresses SOX17 during mouse placentation; its aberrant formation was observed in Sox17-null but not Sox7-null concepti in the pregnant uterus.

Fulminant Type 1 Diabetes Mellitus in IRS-2 Deficient Mice

Type 1 diabetes mellitus (T1DM), one of two major forms of diabetes, results from nearly complete destruction of pancreatic beta (┚) cells. According to the classification of diabetes made by the American Diabetes Association, T1DM is divided into two subtypes: immunemediated (type 1A) and idiopathic (type 1B) (American Diabetes Association, 2008). Fulminant type 1 diabetes mellitus (FT1DM), which was first reported by Imagawa et al. in 2000, is thought to be a unique subtype of type 1B diabetes. The initial reports of FT1DM were exclusively in Japanese population and accounted for about 20% of their T1DM (Imagawa et al., 2000; 2003). Outside Japan, Cho et al. (2007) reported prevalence for FT1DM of 7.1% in the newly diagnosed Korean T1DM patients. However, epidemiological study of FT1DM is lacking in other Asian populations and its incidence and pathogenesis remain to be elucidated. While a search for FT1DM was reported to be negative in the Caucasian population, case reports on FT1DM had surfaced in different ethnic groups, predominantly from Asian origins (Jung et al., 2004; Taniyama et al., 2004; Moreau et al., 2008). However, the causative mechanism of FT1DM is currently unknown. On the other hand, insulin receptor substrate (IRS) disorders are associated with onset of insulin resistance and diabetes mellitus (Withers et al., 1998; Kido et al., 2000). A small population of male IRS-2 deficient mice showed hyperglycemia associated with markedly diminished pancreatic islet size, and these extremely hyperglycemic IRS-2 deficient mice exhibited 1) abrupt onset of diabetes and 2) very short duration of diabetic symptoms, such as polyuria, thirst, and body weight loss. These symptoms resembled the features of human nonautoimmune FT1DM (Hashimoto et al., 2006). Characteristics of abrupt onset of hyperglycemia associated with marked diminished islet mass in IRS-2 deficient mice were investigated to analyze the onset mechanism of FT1DM.