Shigeko Kato

High-fat and high-cholesterol diet rapidly induces non-alcoholic steatohepatitis with advanced fibrosis in Sprague–Dawley rats

The development of fibrosis is considered an important phase in the progress of non‐alcoholic steatohepatitis (NASH) towards the end stage of liver disease, including cirrhosis. However, few small animal models can display NASH‐associated fibrosis. We aimed to establish a dietary model of NASH with rapid progression to fibrosis using genetically normal rats.

Phycocyanin prevents hypertension and low serum adiponectin level in a rat model of metabolic syndrome.

Endothelial dysfunction is associated with hypertension, atherosclerosis, and metabolic syndrome. Phycocyanin is a pigment found in the blue-green algae, Spirulina, which possesses antihypertensive effect. In this study, we hypothesized that phycocyanin derived from Spirulina exerts antihypertensive actions by improving endothelial dysfunction in metabolic syndrome. Spontaneously hypertensive/NIH-corpulent (SHR/NDmcr-cp) rats were divided into 4 groups then fed a normal diet with or without phycocyanin (2500-, 5000-, or 10,000-mg/kg diet) for 25 weeks. At 34 weeks of age, although systolic blood pressure was not significantly different among groups, phycocyanin-fed groups exhibited a dose-dependent decrease in blood pressure. Serum levels of adiponectin and messenger RNA levels of adiponectin and CCAAT/enhancer-binding protein α in the adipose tissue of rats fed diets containing phycocyanin tended to be higher than those of rats fed a normal diet, but the differences were not statistically significant. Immunohistochemistry analysis showed a significant and positive correlation between aortic endothelial nitric oxide synthase (eNOS) expression levels, a downstream target of the adiponectin receptor, and serum adiponectin levels, although there were no significant differences in eNOS expression among groups. There was also no significant correlation between eNOS expression levels and systolic blood pressure. These results suggest that long-term administration of phycocyanin may ameliorate systemic blood pressure by enhancing eNOS expression in aorta that is stimulated by adiponectin. Phycocyanin may be beneficial for preventing endothelial dysfunction-related diseases in metabolic syndrome.

Olive leaf extract prevents spontaneous occurrence of non-alcoholic steatohepatitis in SHR/NDmcr-cp rats

Aims: Oxidative stress may play an important role in the pathogenesis of non‐alcoholic steatohepatitis (NASH). Oleuropein, the active constituent of olive leaf, possesses anti‐oxidant, hypoglycaemic, and hypolipidaemic activities. We aimed to investigate the preventive effects of olive leaf extract on hepatic fat accumulation in a rat model of NASH. Methods: Spontaneously hypertensive/NIH‐corpulent rats were fed a diet of AIN‐93G with or without olive leaf extract (500, 1000, 2000 mg/kg diet, and control; 5 rats each) for 23 weeks. Serological and histopathological findings, anti‐oxidative activity, and the alteration of fatty acid synthesis in the liver were evaluated. Results: Histopathologically, a diet of AIN‐93G containing more than 1000 mg/kg olive leaf extract had a preventive effect for the occurrence of NASH. Thioredoxin‐1 expression in the liver was more evident in rats fed this diet, and 4‐hydroxynonenal expression in the liver was less evident in these rats. There were no significant differences in the activities of hepatic carnitine palmitoyltransferase, fatty acid synthase, malic enzyme, and phosphatidic acid phosphohydrolase among the groups. Conclusions: Our data suggest that olive leaf extract may help prevent NASH, presumably through its anti‐oxidative activity.

Predictive Factors for the Development or Regression of Fatty Liver in Japanese Adults

Fatty liver is commonly associated with alcohol or metabolic syndrome. We aimed to examine the longitudinal aspects of fatty liver, and clarify the independent predictors for the development or regression of fatty liver. In the present study, the clinical features of 1578 Japanese adults (1208 men and 370 women; 35 to 69 years of age) who visited our center both in 2000 and 2007–2008 were recorded and compared, including liver status diagnosed by ultrasonography. Of the 1578 participants, 217 (13.8%) showed fatty liver development, and 74 (4.7%) showed fatty liver regression. Logistic regression analysis revealed that body mass index and percentage body fat were strongly associated with the development or regression of fatty liver. Metabolic syndrome-related disorders such as serum levels of total cholesterol, triglyceride, uric acid, and fasting blood glucose were also associated with clinical course to some degree. However, the history of alcohol intake, the presence of metabolic syndrome, blood pressure, and habitual physical exercise were not independent predictors for the development or regression of fatty liver. Our present data suggest that control of body weight in men and the percentage body fat in women are particularly important for the prevention or treatment of fatty liver.

Serum alanine aminotransferase concentration as a predictive factor for the development or regression of fatty liver

Serum alanine aminotransferase (ALT) concentration is the most commonly used marker for hepatocellular injury. We investigated the suitable cutoff value of serum ALT for the diagnosis or prediction of fatty liver. In 1578 Japanese adults (1208 men, 370 women; 35–69 years of age) who visited our center both in 2000 and between April 2007 and March 2008 (2007–2008), serum ALT concentration was an independent predictor of fatty liver in men in 2000 and in both sexes in 2007–2008. A significant increase in the frequency of fatty liver was detected in participants with elevated serum ALT concentrations, and serum levels of ALT in 2000 were associated with fatty liver in 2007–2008 when the cutoff value was set at 30 IU/L in men and 19 IU/L in women. The frequency of fatty liver in 2007–2008 was significantly lower in participants without fatty liver in 2000 whose serum ALT decreased between 2000 and 2007–2008. Our results suggest that serum ALT might be not only an indicator of fatty liver but also a predictor of the regression of fatty liver, and cutoff values of serum ALT of 30 IU/L in men and 19 IU/L in women are suitable for the screening of fatty liver.

A possible rat model for non‐alcoholic steatohepatitis: Histological findings in SHR/NDmcr‐cp rats

Dear Sir, The frequent association of non-alcoholic fatty liver disease (NAFLD), which includes fatty liver and nonalcoholic steatohepatitis (NASH), with metabolic syndrome is now well known. Hitherto, several animal models with transgenic animals or particular diets have been reported. These situations are not common in patients with NASH; therefore, a model with metabolic syndrome will be useful for studying the pathogenesis and treatment of NASH. The spontaneously-hypertensive/NIH-corpulent (SHR/ NDmcr-cp [cp/cp]) rat has a genetic background from SHR and also has a genetic mutation in the leptin receptor gene. This strain is thought to be a suitable animal model of metabolic syndrome because it exhibits hyperphasia, hyperglycemia, hyperinsulinemia, hyperlipidemia, hypertension, obesity, and fatty liver. Thirteen-week-old male SHR/NDmcr-cp rats (n = 5) were purchased from Nippon SLC (Hamamatsu, Japan) and kept in individual cages for 15 weeks in a temperature-, humidity-, and light-controlled room with free access to water and standard rat chow (AIN93G). After 20-h fasting, all rats were killed under anesthesia (pentobarbital sodium). Blood samples were taken from the inferior vena cava or heart for the measurement of serum insulin, glucose, total cholesterol, triglyceride, leptin, aspartate aminotransferase (AST), and alanine aminotransferase (ALT). The livers were excised and fixed in 10% neutral-buffered formalin. Paraffin-embedded liver tissues were processed routinely for hematoxylin–eosin and Azan staining for histopathological examination. All procedures were performed in accordance with the Guidelines for Animal Experimentation and approved by the Animal Usage Committee of the Siebold University of Nagasaki (Nagasaki, Japan). SHR/NDmcr-cp rats at 28 weeks of age exhibited obesity (760.4 1 14.2 g, mean 1 SE) and systolic hypertension (174 1 5 mmHg). Serum glucose, insulin, total cholesterol, and triglyceride were elevated. Serum leptin, AST, and ALT levels were 19.3 1 0.3 ng/mL, 293 1 64 IU/L, and 281 1 104 IU/L, respectively (Table 1). Histopathological findings were scored using the NASH Clinical Research Network Scoring System based on four semiquantitative factors: steatosis (0–3), lobular inflammation (0–3), hepatocyte ballooning (0–2), and fibrosis (0–4). The NAFLD activity score (NAS) was defined as the unweighted sum of the scores for steatosis, lobular inflammation, and hepatocyte ballooning (range, 0–8). NAS scores 22 are considered not diagnostic of steatohepatitis and scores 35 are diagnostic of steatohepatitis. Moderate to severe microvesicular fatty changes were found in liver parenchymas, and were more represented in the pericentral region (zone 3) in all rats. Mild lobular inflammation and hepatocyte ballooning were also seen, but fibrosis was not observed in all rats. Of the five rats, three (60%) had NAS of 5 or greater, representing steatohepatitis (Table 1). Furthermore, characteristic pathological findings, including Mallory bodies, neutrophils aggregation, megamitochondria, and lipogranuloma, were frequently observed. While hepatic fibrosis was not observed, 28-week-old male SHR/NDmcr-cp rats had histopathological characteristics of NASH. This strain can be used as an experimental animal model for the spontaneous occurrence of NASH. It is difficult to produce fibrosis in the rodent liver, therefore, longer follow-up periods are required to determine whether this strain will develop hepatic fibrosis.

An SHR/NDmcr-cp Rat Model of Non-alcoholic Steatohepatitis with Advanced Fibrosis Induced by a High-fat, High-cholesterol Diet

Mayuko Ichimura1, Maiko Hatanaka1, Koichi Tsuneyama2, Shigeko Kato1 and Katsuhisa Omagari1* 1Division of Nutritional Sciences, Graduate School of Human Health Science, University of Nagasaki, Nagasaki, Japan 2Department of Diagnostic Pathology, University of Toyama, Toyama, Japan *Corresponding author: Katsuhisa Omagari MD, Professor, Division of Nutritional Sciences, Graduate School of Human Health Science, University of Nagasaki, 1-1-1 Manabino, Nagayo-cho, Nagasaki 851-2195, Japan, Tel: +81-95-813-5201; E-mail: omagari@sun.ac.jp