Spontaneous Occurrence of Various Types of Hepatocellular Adenoma in the Livers of Metabolic Syndrome-Associated Steatohepatitis Model TSOD Mice.

Male Tsumura-Suzuki Obese Diabetes (TSOD) mice, a spontaneous metabolic syndrome model, develop non-alcoholic steatohepatitis and liver tumors by feeding on a standard mouse diet. Nearly 70% of liver tumors express glutamine synthetase (GS), a marker of hepatocellular carcinoma. In contrast, approximately 30% are GS-negative without prominent nuclear or structural atypia. In this study, we examined the characteristics of the GS-negative tumors of TSOD mice. Twenty male TSOD mice were sacrificed at 40 weeks and a total of 21 tumors were analyzed by HE staining and immunostaining of GS, liver fatty acid-binding protein (L-FABP), serum amyloid A (SAA), and beta-catenin. With immunostaining for GS, six (29%) tumors were negative. Based on the histological and immunohistological characteristics, six GS-negative tumors were classified into several subtypes of human hepatocellular adenoma (HCA). One large tumor showed generally similar findings to inflammatory HCA, but contained small atypical foci with GS staining and partial nuclear beta-catenin expression suggesting malignant transformation. GS-negative tumors of TSOD mice contained features similar to various subtypes of HCA. Different HCA subtypes occurring in the same liver have been reported in humans; however, the diversity of patient backgrounds limits the ability to conduct a detailed, multifaceted analysis. TSOD mice may share similar mechanisms of HCA development as in humans. It is timely to review the pathogenesis of HCA from both genetic and environmental perspectives, and it is expected that TSOD mice will make further contributions in this regard.

Neonatal monosodium glutamate treatment causes obesity, diabetes, and macrovesicular steatohepatitis with liver nodules in DIAR mice.

BACKGROUND AND AIM: Non-alcoholic steatohepatitis (NASH) is the hepatic manifestation of metabolic syndrome (MS). Monosodium glutamate (MSG)-treated ICR mice is a useful model of MS and NASH, but it shows the different patterns of steatosis from human NASH. Because inbred aged DIAR (ddY, Institute for Animal Reproduction) mice spontaneously show the similar pattern of steatosis as NASH, we analyzed their liver pathology after administering MSG. METHODS: MSG-treated DIAR mice (DIAR-MSG) and untreated DIAR mice (DIAR-controls) were sacrificed and assessed histopathologically at 29, 32, 40, 48, and 54 weeks of age. The NASH activity score, body mass index, blood glucose level, and oral glucose tolerance test were also assessed. RESULTS: The body mass index and blood glucose levels of DIAR-MSG were significantly higher than controls. The oral glucose tolerance test revealed a type 2 diabetes pattern in DIAR-MSG. The livers of DIAR-MSG mice showed macrovesicular steatosis, lobular inflammation with neutrophils, and ballooning degeneration after 29 weeks. At 54 weeks, mild fibrosis was observed in 5/6 DIAR-MSG and 2/5 DIAR-control mice. In imaging mass spectrometry analysis, cholesterol as well as triglyceride accumulated in the liver of DIAR-MSG mice. Atypical liver nodules were also observed after 32 weeks in DIAR-MSG, some with cellular and structural atypia mimicking human hepatocellular carcinoma. The NASH activity score of DIAR-MSG after 29 weeks was higher than that of control mice, suggesting the development of NASH. CONCLUSIONS: DIAR-MSG had NASH-like liver pathology and liver nodules typically associated with MS symptoms. DIAR-MSG provides a valuable animal model to analyze NASH pathogenesis and carcinogenesis.

Establishment of repeated liver biopsy technique in experimental mice.

Biopsy is a commonly used method for determining pathological diagnoses by directly using human tissues and cells. Biopsies are widely used to determine disease progression and treatment efficacy. Although organs and tissues are usually obtained by sacrifice during animal experiments, it is theoretically possible to use the same biopsy techniques in humans. In the present study, we examined the feasibility of performing four repeated liver biopsies in a spontaneous metabolic syndrome mouse model. Even though a small number of mice died accidently, most mice were able to undergo four liver biopsies without significant adverse events. We also performed three liver biopsies in mouse liver tumor carcinogen models at 4, 8, and 12 weeks of age. In addition to the sample collected at 16 weeks of age during sacrifice, we successfully collected four liver samples from the same mice at different stages of disease progression. The application of this liver biopsy technique might make it possible for direct evaluation of pathological conditions in the same individual over time, thereby reducing the number of experimental animals.

Neonatal streptozotocin treatment rapidly causes different subtype of hepatocellular carcinoma without persistent hyperglycemia in 4CS mice fed on a normal diet.

Although diabetes mellitus (DM) is a well-known risk factor for hepatocellular carcinoma (HCC), the underlying mechanisms have not yet to be defined. We previously reported that DIAR mice fed with standard murine diet developed type 1 diabetes and HCC at age of 16 weeks old with a neonatal streptozotocin treatment (n-STZ). Because DIAR mice did not manifest obesity nor develop steatohepatitis, hyperglycemia with streptozotocin trigger or streptozotocin alone might turn on the hepato-carcinogenesis. An insulin-recruitment to DIAR-nSTZ mice showed an increased frequency of HCC during the first 12 weeks of age, although the diabetic indications notably improved. To elucidate the role of hyperglycemia in hepato-carcinogenesis, we performed a head-to-head comparative study by using 4CS mice and DIAR mice with n-STZ treatment. Newborn 4CS mice and DIAR mice were divided into STZ treated group and control group. The blood glucose levels of DIAR-nSTZ mice increased at age of eight weeks, while that of 4CS-nSTZ mice were maintained in the normal range. At eight weeks old, three out of five DIAR-nSTZ mice (60%) and one out of ten 4CS-nSTZ mice (10%) developed multiple liver tumors. At age of 12 weeks old, all eight of DIAR-nSTZ mice (100%) and two of 10 4CS-nSTZ mice (20%) developed multiple liver tumors. At 16 weeks old, all animals of DIAR-nSTZ and 4CS-nSTZ mice occurred liver tumors. DIAR-nSTZ showed hyperglycemia and HCC, and 4CS-nSTZ developed HCC without hyperglycemia. These results were interpreted that the onset of HCC maybe not related to the presence or absence of hyperglycemia but nSTZ treatment. On the other hand, since the carcinogenesis of 4CS-nSTZ is delayed compared to DIAR-nSTZ, hyperglycemia may play a role in the progression of carcinogenesis. Histologically, the liver tumor appeared irregularly trabecular arrangements of hepatocytes with various degrees of nuclear atypia. By immunohistochemical analyses, all liver tumors showed positive staining of glutamine synthetase (GS), an established human HCC marker. The expression pattern of GS was divided into a strong diffuse pattern and weak patchy pattern, respectively. The liver tumor showing the weak GS-patchy pattern expressed biliary/stem markers, EpCAM, and SALL4, partially. Because 4CS-nSTZ mice did not show any metabolic complications such as gaining body weight or high blood glucose level, it is a unique animal model with a simple condition to investigate hepatic carcinogenesis by excluding other factors.

Evaluation of LEXF/FXLE rat recombinant inbred strains for genetic dissection of complex traits.

Recombinant inbred (RI) strains are formed from an outcross between two well-characterized inbred stains followed by at least 20 generations of inbreeding. RI strains can be utilized for the analysis of many complex phenotypic traits. The LEXF/FXLE RI strain set consists of 34 RI strains derived by reciprocal crossing of LE/Stm and F344/Stm. Here we report on genetic dissections of complex traits using this RI set and their parental strains. We have developed strain distribution patterns for 232 informative simple sequence length polymorphism markers. The framework map covers the rat genome except for chromosome Y. Seventy-six phenotype parameters, which included physiological and behavioral traits, were examined for these RI lines. Quantitative trait locus (QTL) analysis of these parameters revealed 27 significant and 91 suggestive QTLs, illustrating the potential of this RI resource for the detection of underlying gene functions for various phenotypes. Although this RI set was originally developed to study susceptibility to chemical-induced tumors, it has been shown to be equally powerful for a wide spectrum of traits. The LEXF/FXLE RI strains have been deposited at the National Bio Resource Project for the Rat in Japan and are maintained under specific pathogen-free conditions. They are available at http://www.anim.med.kyoto-u.ac.jp/nbr.

Facilitatory effect of insulin treatment on hepatocellular carcinoma development in diabetes.

BACKGROUND: To evaluate the effect of insulin treatment on the incidence and/or severity of hepatocellular carcinoma (HCC) in a mouse model of HCC based on diabetes. METHODS: We recently reported that neonatal streptozotocin (STZ) treatment causes type 1 diabetes and subsequent HCC in ddY, Institute for Animal Reproduction (DIAR) mice. Newborn male DIAR mice were divided into three groups based on STZ and insulin (INS) treatment. STZ was subcutaneously injected (60 mg/g) into the STZ-treated group (DIAR-nSTZ mice, N = 13) and the STZ/insulin-treated group (DIAR-nSTZ/INS mice, N = 20). A physiologic solution was injected into the control group (DIAR-control mice, N = 8) 1.5 days after birth. Insulin was subcutaneously injected into the DIAR-nSTZ/INS mice according to the following protocol: 2 IU/day at 4-5 weeks of age, 3 IU/day at 5-7 weeks of age, and 4 IU/day at 7-12 weeks of age. All mice were fed a normal diet and were subjected to physiological and histopathological assessments at 12 weeks of age. RESULTS: DIAR-nSTZ mice had significantly lower body weight and higher blood glucose levels than DIAR-control mice, whereas no significant differences were observed between DIAR-nSTZ/INS mice and control mice. At 12 weeks of age, lower weight of paratesticular fat and higher levels of total cholesterol, triglyceride, and free fatty acids were observed in DIAR-nSTZ mice compared to DIAR-control mice, whereas there were no significant differences between DIAR-nSTZ/INS mice and DIAR-control mice. In the livers of DIAR-nSTZ mice, HCC was observed in 15% of cases, and dysplastic nodules were observed in 77% of cases. In the livers of DIAR-nSTZ/INS mice, HCC was observed in 39% of cases and dysplastic nodules were observed in 61% of cases (p = 0.011). Moreover, the average tumor size was significantly larger in STZ/INS-treated mice than in STZ-treated mice. Immunohistochemical analysis demonstrated that the expression of ERK1/2, downstream substrates of insulin signaling that activate cell proliferation, was significantly higher in STZ/INS-treated mice compared to STZ-treated mice. CONCLUSIONS: Insulin treatment promoted, rather than inhibited, the progression of liver carcinogenesis in DIAR-nSTZ mice. Hyperinsulinemia rather than hyperglycemia can accelerate the progression of HCC via insulin signaling.

Histopathological characteristics of glutamine synthetase-positive hepatic tumor lesions in a mouse model of spontaneous metabolic syndrome (TSOD mouse).

We previously reported that Tsumura-Suzuki obese diabetic (TSOD) mice, a polygenic model of spontaneous type 2 diabetes, is a valuable model of hepatic carcinogenesis via non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). One of the characteristics of tumors in these mice is the diffuse expression of glutamine synthetase (GS), which is a diagnostic marker for hepatocellular carcinoma (HCC). In this study, we performed detailed histopathological examinations and found that GS expression was diffusely positive in >70% of the hepatic tumors from 15-month-old male TSOD mice. Translocation of ß-catenin into nuclei with enhanced membranous expression also occurred in GS-positive tumors. Small lesions (<1 mm) in GS-positive cases exhibited dysplastic nodules, with severe nuclear atypia, whereas large lesions (>3 mm) bore the characteristics of human HCC, exhibiting nuclear and structural atypia with invasive growth. By contrast, the majority of GS-negative tumors were hepatocellular adenomas with advanced fatty change and low nuclear grade. In GS-negative tumors, loss of liver fatty acid-binding protein expression was observed. These results suggest that the histological characteristics of GS-positive hepatic tumors in TSOD mice resemble human HCC; thus, this model may be a useful tool in translational research targeting the NAFLD/NASH-HCC sequence.

Estrous cycle stage-independent treatment of PMSG and hCG can induce superovulation in adult Wistar-Imamichi rats.

The estrous cycle influence on the number of ovulated eggs after injection of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) was investigated in 12, 18, and 24 weeks old adult female Wistar-Imamichi (WI) rats. PMSG (150 IU/kg) was injected at metestrus, diestrus, proestrus, or estrus, followed by hCG (75 IU/kg) 55 h later. Ovulation was induced at all ages and stages of the estrous cycle. The number of ovulated eggs was not affected by stage for similarly aged rats, however, the number of ovulated eggs obtained after treatment decreased with age. These results demonstrate that the PMSG/hCG treatment can induce ovulation at any stage of estrous cycle in WI rats and efficient superovulation at 12 weeks of age.

Strain difference in pituitary-adrenal axis between Wistar-Imamichi and Long Evans adult male rats.

In the present study, we used closed colony-Wistar-Imamichi (WI), inbred WI and Long Evans (LE) adult male rats to examine the secretion of ACTH and corticosterone in response to restraint stress. Blood (0.3 ml) was withdrawn through a jugular cannula at 0, 15, 30, 60 and 120 min after the onset of restraint stress. Plasma concentrations of ACTH and corticosterone increased after stress in all groups, but the responses of ACTH and corticosterone secretion were higher in LE rats than in WI rats. Present data suggest that the LE rat might be a good model as a high-response strain and the closed colony or the inbred WI rat might be a good model as a low-response strain in restraint stress experiments.

Fertilizability of Superovulated Eggs by Estrous Stage-independent PMSG/hCG Treatment in Adult Wistar-Imamichi Rats.

We investigated the fertilization and developmental ability of superovulated eggs obtained from adult Wistar-Imamichi (WI) rats, by using pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) treatment. Female WI rats, 11-13 weeks of age, were divided into four groups by estrous stage (metestrus [ME], diestrus [DE], proestrus [PE], or estrus [E]). PMSG (150 IU/kg) and hCG (75 IU/kg) were injected at an interval of 48 or 55 h and the female rats were mated with mature male rats. The ovulated eggs were collected 20, 24, and 27 h after hCG injection. Regardless of the estrous stage at the time of PMSG injection, the treated rats mated and ovulated similar to the untreated spontaneously ovulated rats (S group). Although the proportion of fertilized eggs in the E- and PE-treated groups was less than the S group 20 h after hCG injection, the proportion was not different among all treated and S groups 24 h after hCG injection. The proportion of fertilized eggs using in vitro fertilization and the proportion of offspring obtained from 2-cell stage embryo transfer did not differ among the treated and S groups. In comparison with PMSG/hCG-treated immature rats, mating and ovulation rate of adult rats were significantly higher. The proportion of fertilized eggs obtained from mated rats did not differ between immature and adult rats. These results demonstrate that adult WI rats are good egg donors for reproductive biotechnological studies using unfertilized or fertilized eggs.

Neonatal streptozotocin treatment causes type 1 diabetes and subsequent hepatocellular carcinoma in DIAR mice fed a normal diet.

PURPOSE: Hepatocellular carcinoma (HCC) represents a major health challenge because of its increasing morbidity and mortality. The establishment of useful models of HCC can significantly contribute to unveiling its pathophysiology. We developed a novel mouse model of HCC based on type 1 diabetes and reported its histopathological features. METHODS: Newborn male ddY, Institute for Animal Reproduction (DIAR) mice were divided into two groups on the basis of streptozotocin treatment, which induces type 1 diabetes. Streptozotocin was subcutaneously injected (60 mg/g) into the treated group (DIAR-nSTZ mice), whereas physiologic solution was injected into the control group (DIAR-control mice) at 1.5 days after birth. All mice were fed a normal diet and histopathologically assessed at 6, 8, 10, 12, 19, and 27 weeks of age. RESULTS: At 8 weeks, small hepatocytic nodules with mild to moderate cellular atypia were observed in the livers of DIAR-nSTZ mice, which progressed to large hepatocytic nodules with cellular atypia and infiltrating growth at 12 weeks, identical to those in well-differentiated human HCC. At 19 and 27 weeks, moderately differentiated HCC was observed in all DIAR-nSTZ mice. Conversely, no neoplastic findings were evident in DIAR-control mice. No steatosis or fibrosis was evident in either group. Immunohistochemical analysis revealed that all nodules observed in DIAR-nSTZ mice were positive for glutamine synthetase. CONCLUSIONS: In DIAR-nSTZ mice, the development of HCC with similarity to human HCC and high reproducibility can be achieved using a short and simple protocol. We believe that this model will be useful for studying liver carcinogenesis.

Identification of QTLs involved in the development of amygdala kindling in the rat.

Amygdala kindling is useful for modeling human epilepsy development. It has been known that genetic factors are involved in the development of amygdala kindling. The purpose of this study was to identify the loci that are responsible for the development of amygdala kindling. To achieve this, rat strains from a LEXF/FXLE recombinant inbred (RI) strain panel were used. The phenotypes of amygdala kindling-related parameters for seven RI strains and parental LE/Stm and F344/Stm strains were determined. They included the afterdischarge threshold (ADT), the afterdischarge duration (ADD), and the kindling rate, an incidence of development of kindling. Quantitative trait loci (QTL) analysis was performed to identify linkage relationships between these phenotypes and 1,033 SNP markers. Although no significant differences in pre-kindling ADT and ADD were observed, a significant difference in the kindling rate was found for the LEXF/FXLE RI strain. Two QTLs for the amygdala kindling rate (Agkr1 and Agkr2) were identified on rat chromosome 2. These findings clearly prove the existence of genetic influences that are involved in kindling development and suggest that substantial genetic components contribute to the progression of partial seizures into generalized seizures.

Effects of cyclophosphamide on the kaolin consumption (pica behavior) in five strains of adult male rats.

It is known that pica, the consumption of non-nutritive substances such as kaolin, can be induced by administration of toxins or emetic agents in rats. In the present study, we examined the effects of intraperitoneal (i.p.) administration of cyclophosphamide on pica behavior and on the concentration of 5-hydroxyindoleacetic acids (5HIAA) in cerebrospinal fluid (CSF) in the following five strains of adult male rats: Sprague Dawley (SD), Wistar, Fischer 344 (F344), Wistar-Imamichi (WI) and Long Evans (LE). Cyclophosphamide (25 mg or 50 mg/kg) was injected (i.p.) into the rats and kaolin and food intake were measured at 24 hr after injection. The animals were anesthetized with urethane (1 g/kg) at 3 hr after injection of cyclophosphamide, and CSF was collected from the cisterna magna. WI and LE rats clearly showed pica behavior as compared with the other strains. In LE rats, the concentration of 5HIAA in CSF also increased in a dose-dependent manner of cyclophosphamide. The pretreatment with ondansetron (5-HT(3) antagonist) restored both changes (kaolin consumption and 5HIAA levels) induced by cyclophosphamide. These results suggest that the LE rat is sensitive to cyclophosphamide, that pica induced by cyclophosphamide mimics many aspects of emesis including the serotonergic response in the central nervous system and that use of the pica model would be a practical method for evaluating the effects of antiemetic drugs in addition to the mechanism of emesis.

Effects of bezafibrate in nonalcoholic steatohepatitis model mice with monosodium glutamate-induced metabolic syndrome.

Recently, we reported that monosodium glutamate-treated mice (MSG mice) developed severe hyperlipidemia and diabetes mellitus and several complications of obesity. MSG mice acquired fatty livers and subsequently underwent changes that are characteristic of nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH). In the present study, the effects of bezafibrate on obesity, diabetes mellitus, and NAFLD/NASH were examined in MSG mice. A single dose of MSG (4 mg/g) was administered subcutaneously to neonatal male mice within 24h of birth. Bezafibrate was mixed into the normal feed for 8 weeks. The weight and body mass index of MSG mice increased significantly despite the unchanged intake of food. Triglyceride and total cholesterol levels in blood, visceral adipose tissue, and interscapular adipose tissue rose significantly. In the livers of MSG mice, moderate centrilobular microvesicular steatosis, ballooning degeneration with Mallory bodies, and scattered infiltration of neutrophils and lymphocytes were observed. Centrilobular hepatocytes were 4-hydroxynonenal-positive in MSG mice. Bezafibrate ameliorated the severity of diabetes mellitus, hyperinsulinemia, and hyperlipidemia. Adiponectin and leptin concentrations in blood improved, and the accumulation of visceral fat was inhibited. The expression of acyl-CoA oxidase, a beta-oxidation gene, and carnitine palmitoyl transferase, which regulates lipid metabolism, increased markedly on administration of bezafibrate. The liver pathology in MSG mice also improved with bezafibrate; specifically, macro- and microvesicles in hepatocytes nearly disappeared, and NAFLD activity score improved. It is concluded that bezafibrate inhibits the accumulation of visceral fat, following amelioration of hyperlipidemia, in MSG-induced obese mice, due to improvements in diabetes mellitus, fatty liver, and NAFLD.

The different effects of i.c.v. injection of pituitary adenylate cyclase activating polypeptide (PACAP) on prolactin secretion in adult male and lactating rats.

The effects of intracerebroventricular (i.c.v.) administration of pituitary adenylate cyclase activating polypeptide38 (PACAP38) on prolactin (PRL) secretion and the activity of tyrosine hydroxylase (TH) were examined in adult male and lactating rats with or without suckling stimulus. In adult male rats and lactating rats with suckling stimulus, administration of PACAP38 (0.25 or 1 nmol) decreased PRL secretion and increased the activity of TH in the stalk-median eminence. On the other hand, the injection of PACAP38 did not affect PRL secretion and TH activity in lactating rats without sucking stimulus. Administration of PACAP6-38 (4 nmol), a specific receptor antagonist, also had no effect on PRL secretion and TH activity in adult male rats. These results suggest that i.c.v. administration of PACAP inhibits PRL secretion mediated by dopamine neuron within the hypothalamus, but the effects of PACAP differ depending on the physiological condition of animals. These observed effects of PACAP on PRL release may be pharmacological responses rather than physiological responses.

Dose dependent development of diabetes mellitus and non-alcoholic steatohepatitis in monosodium glutamate-induced obese mice.

AIMS: We have recently reported that monosodium glutamate (MSG) induces severe obesity with diabetes mellitus and/or non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) in Crj:CD-1(ICR) neonatal mice. In this study, we investigated the effects of varying the dose of MSG on the resulting obesity and diabetes mellitus. MAIN METHODS: Crj:CD-1(ICR) neonatal mice were administered MSG in one of several courses: once-daily subcutaneous injections of 2mg/g for 5 consecutive days (2 mg/g x 5 group), a single subcutaneous injection of 4 mg/g (4 mg/g x 1 group) and once-daily subcutaneous injections of 4 mg/g for 5 consecutive days (4 mg/g x 5 group). KEY FINDINGS: In all the MSG treatment groups, severe obesity developed by 29 weeks of age. The onset of diabetes mellitus and liver lesions (resembling those of human NAFLD/NASH) were observed before 54 weeks of age. The obesity, diabetes mellitus and liver lesions were most severe in the 4 mg/g x 1 group. In the 4 mg/g x 5 group, increases in body weight and body length were inhibited by MSG's severe toxicity. SIGNIFICANCE: A single 4 mg/g dose of MSG is the most suitable as the obese model and induces not only severe obesity and diabetes mellitus, but also liver changes resembling human NAFLD/NASH. A small amount of MSG in the newborn develops obesity and the other complications without hyperphagia after a long term.