Choline-deficient Diet-induced NAFLD Animal Model Recaptures Core Human Pathophysiology With Similar Gene Co-expression Networks.

BACKGROUND/AIM: Nonalcoholic fatty liver disease (NAFLD) is a wide spectrum of liver disorders ranging from simple steatosis to nonalcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma. Recently, the prevalence of NAFLD has dramatically increased, and treatment is urgently needed. Animal models are often used to understand the molecular mechanisms of disease development and progression, but their relevance to human diseases has not been fully understood. This study aimed to establish the usefulness of the animal model for preclinical research, we evaluated its relevance to human disease by gene expression analysis. MATERIALS AND METHODS: We performed weighted gene co-expression network analysis of liver tissues from a choline-deficient L-amino acid-defined (CDAA) diet-induced NAFLD animal model. In addition, module preservation analysis was conducted to evaluate similarity across species. RESULTS: Several modules were identified to be associated with disease severity, and their gene co-expression network was found to be preserved in the human NAFLD datasets. Of note, module brown (immune cell clusters involved in inflammatory responses) was positively associated with disease severity, and its gene co-expression network was highly preserved in the human datasets. Tyrobp, Laptm5 and Lgals3 were identified as hub genes in the brown module, and their increased expression was confirmed in the human datasets. CONCLUSION: CDAA diet-induced NAFLD animal model recaptured key aspects of human pathophysiology (especially immune cell functions) and is thought to be a powerful tool for understanding the molecular mechanisms of NAFLD development and progression.

Establishment of a new nonalcoholic steatohepatitis model; Ovariectomy exacerbates nonalcoholic steatohepatitis-like pathology in diabetic rats.

An increasing number of patients worldwide are being diagnosed with nonalcoholic fatty liver disease and nonalcoholic steatohepatitis (NAFLD/NASH) because of the growing prevalence of obesity and metabolic disorders. The incidence of NAFLD is higher in postmenopausal women than in premenopausal women. The decline in the level of female hormones might have an effect on the deterioration of metabolism. In the present study, we investigated the potential of Spontaneously Diabetic Torii (SDT) fatty rats as a new animal model for NAFLD. We created a menopausal model by ovariectomy (OVX) in female rats. Sprague-Dawley (SD) rats, SDT rats, and SDT-fatty rats were divided into sham and OVX groups and maintained until 40 weeks of age. The results showed that OVX-induced weight gain was observed in SD and SDT rats. In addition, OVX-induced hepatic triglyceride accumulation was increased in all strains, and there was a significant increase in hepatic triglyceride levels in OVX-SDT fatty rats compared to those in Sham-SD rats. Furthermore, liver fibrosis was worsened in the OVX-SDT fatty rats. In addition, OVX-induced increase in blood ALT level was observed in SDT-fatty rats. Gene expression analysis showed OVX-induced upregulation of Srebp1 expression and downregulation of Pemt and Mttp in OVX rats. These results indicate that OVX-SDT fatty rats exhibit NASH with more severe hepatic fibrosis than untreated animals, suggesting that OVX-induced estrogen reduction may have enhanced lipid synthesis in the liver. It is also possible, although hypothetical, that OVX may decrease VLDL secretion, which may more strongly induce NASH.

JTP-109192, a novel G protein-coupled receptor 119 agonist, prevents atherosclerosis by improving hypercholesterolaemia in congenic spontaneously hyperlipidaemic mice.

G protein-coupled receptor 119 (GPR119) expression in pancreatic ß-cells and intestinal L-cells is a potential therapeutic target for the treatment of type 2 diabetes. Previously, we have reported that the GPR119 agonist JTP-109192 improves glucose metabolism with single and repeated administration. Conversely, overexpression of the Gpr119 gene reportedly regulates cholesterol transporter expression in animal models, and a natural GPR119 agonist, oleoylethanolamide (OEA), improves atherosclerosis. Therefore, improving dyslipidaemia is considered a possible feature of GPR119 agonists. In the present study, the lipid-lowering effect of JTP-109192 was examined in BALB/c background spontaneously hyperlipidaemic (SHL) mice with repeated administration, once daily for 12 weeks. On repeated administration, JTP-109192 revealed a cholesterol-lowering effect and improved atherosclerosis following histopathological examination. With further investigation, the cholesterol-lowering effect and subsequent antiatherosclerotic effect of JTP-109192 was attributed to changes in intestinal cholesterol metabolism gene expression. Based on these results, JTP-109192 represents a new potential antihypercholesterolaemic agent for the treatment of dyslipidaemia.

Chronic treatment of JTP-109192, a novel G-protein coupled receptor 119 agonist, improves metabolic abnormalities in Zucker Fatty rats.

G-protein coupled receptor 119 (GPR119) expression in pancreatic ß-cells and intestinal L cells is a potential therapeutic target for treating type 2 diabetes. A natural GPR119 agonist oleoylethanolamide is well known to enhance a glucose-stimulated insulin secretion (GSIS) and glucagon-like peptide-1 (GLP-1) secretion by elevating intracellular cAMP levels. In the present study, a glucose lowering effect of the GPR119 agonist, JTP-109192 leading to improvement of insulin sensitivity was examined in Zucker Fatty (ZF) rats. We investigated the in vitro effects of JTP-109192 on GSIS in the rat pancreatic ß-cell line (INS1E) cells and on GLP-1 secretion in the murine enteroendocrine cell line (GLUTag) cells. We also investigated the effect of JTP-109192 on GSIS in Sprague-Dawley (SD) rats with single administration and its effect on glucose metabolism in ZF rats with repeated administration once daily for about 6 weeks. After repeated administration, the hyperinsulinaemic euglycaemic glucose clamp test was performed to evaluate insulin sensitivity. JTP-109192 increased intracellular cAMP levels (EC50 value: 3.6 nmol/L) and enhanced GSIS in the INS1E cells and GLP-1 secretion in GLUTag cells. In SD rats, a single administration of JTP-109192 enhanced GSIS at high blood glucose levels. The repeated administrations in ZF rats improved glucose metabolism without lack of drug efficacy (tachyphylaxis) and increased glucose infusion rates due to improvement of insulin sensitivity.

Hepatic lesions induced by feeding Western diets to Zucker fatty rats, an insulin-resistant model.

Metabolic diseases including nonalcoholic steatohepatitis develop due to various environmental factors. In particular, the westernization of food is closely related to the development of these diseases. In this study, we investigated pathophysiological changes in the livers of Zucker fatty (ZF) rats induced by feeding Western diets. Male ZF rats were fed a sucrose/fat/cholesterol-enriched diet (Western diet, WD) or standard diet (SD) for 18 weeks, from 7 to 25 weeks of age. Body weight, food intake, and biochemical parameters were periodically measured, histopathological analyses were performed at 25 weeks, and mRNA expression in the liver was determined. ZF rats fed the WD (ZF-WD rats) developed obesity, hyperinsulinemia, hyperglycemia, and hyperlipidemia, and their alanine aminotransferase and aspartate aminotransferase levels increased compared with those of ZF rats fed the SD (ZF-SD rats). Hepatic lesions including fibrosis and necrosis were observed in the ZF-WD rats at 25 weeks; however, fibrosis and necrosis were not observed in the ZF-SD rats. Oxidative stress markers also increased in the livers of ZF-WD rats. Hepatic mRNA expression related to inflammation and fibrosis increased in the ZF-WD rats; however, mRNA expression related to lipid synthesis decreased. Microsomal triglyceride transfer protein mRNA levels in the ZF-WD rats also decreased. In Zucker lean rats fed the WD, similar changes were observed in the liver; however, the hepatic changes were not serious compared with ZF-WD rats. In conclusion, hepatic lesions, such as inflammation, fibrosis, and necrosis, were observed in the ZF-WD rats. The sucrose/fat/cholesterol-enriched diet induced significant lipotoxicity in the livers of animals in this insulin-resistant model.

Investigation of pharmacological responses to anti-diabetic drugs in female Spontaneously Diabetic Torii (SDT) fatty rats, a new nonalcoholic steatohepatitis (NASH) model.

Nonalcoholic steatohepatitis (NASH) is a progressive liver disease, and some patients develop hepatic cirrhosis/carcinoma. Animal models play key roles in the development of new therapies for NASH. In this study, the pharmacological effects of metformin and pioglitazone were investigated in female Spontaneously Diabetic Torii (SDT) fatty rats to verify the utility of this model. The anti-diabetic drugs were administered to SDT fatty rats fed a cholesterol-enriched diet from 4 to 25 weeks, and changes in food intake, body weight, and blood chemistry parameters were evaluated every 4 weeks. The hepatic lipid content, mRNA expression in relation to lipid synthesis, inflammation, and fibrosis, and histopathological analyses were performed at 25 weeks. Pioglitazone improved hyperglycemia, hyperlipidemia, and abnormalities in hepatic parameters. The insulin levels were lower than those in the control rats before 16 weeks. Plasma glucose levels in the metformin-treated rats were lower than those in the control rats, and plasma alanine aminotransferase levels temporarily decreased. The lipid content and some mRNA expression in relation to fibrosis in the liver decreased with pioglitazone treatment, and the mRNA expression of microsomal triglyceride transfer protein increased. Hepatic fibrosis observed in the SDT fatty rats improved with pioglitazone treatment; however, the effect with metformin treatment was partial. These results in both drugs are in line with results in the human study, suggesting that the SDT fatty rat is useful for developing new anti-NASH drugs that show potential to regulate glucose/lipid metabolism.

Maternal high-fat diet promotes onset of diabetes in rat offspring.

The onset and progression of type II diabetes is closely related to environmental factors, in particular dietary habit. Moreover, the environmental exposures very early in life can influence the risk for development of type II diabetes later in life. In this study, we investigated pathophysiological changes in the pups of maternal Spontaneously Diabetic Torii (SDT) rats that were fed a high-fat diet (HFD) throughout gestation and lactation. Maternal SDT rats were continued on HFD for 5 weeks, from day 8 of gestation to day 21 after birth, and biological analyses of the pups were performed from 2 to 22 weeks of age. Results of serum lipid levels in pups from dams fed HFD were higher than pups from dams fed a standard diet, and the onset of diabetes was significantly accelerated in pups from dams fed HFD. In pathological analyses, pups from dams fed HFD showed increases in liver weight and vacuolation of hepatic cells at 2 weeks of age. In conclusion, the metabolic disorder of lipids and glucose in SDT rats is closely related to the nutritional condition of dams during the periods of gestation and lactation.

Effects of unilateral nephrectomy on renal function in male Spontaneously Diabetic Torii fatty rats: a novel obese type 2 diabetic model.

The Spontaneously Diabetic Torii (SDT) fatty rat is a new model for obese type 2 diabetes. The aim of the present study was to investigate the effect of 1/2 nephrectomy (Nx) on renal function and morphology and on blood pressure in SDT fatty rats. Male SDT fatty rats underwent 1/2 Nx or a sham operation (Sham). Subsequently, animals were studied with respect to renal function and histological alterations. Induction of 1/2 Nx in SDT fatty rats led to functional and morphological damage to the remnant kidney and to hypertension, which are considered main characteristics of chronic kidney disease, at a younger age compared with the sham group. In conclusion, the SDT fatty rat is useful in investigations to elucidate the pathogenesis of human diabetic nephropathy and in new drug discovery.

Participation of epoxygenase activation in saikogenin D-induced inhibition of prostaglandin E(2) synthesis.

We examined the effect of saikogenin D on arachidonic acid metabolism in C6 rat glioma cells to clarify its anti-inflammatory mechanism. Incubation of C6 cells with saikogenin D for 20 min resulted in the inhibition of prostaglandin E(2) production and the accumulation of an arachidonic acid metabolite that was found to be 11,12-dihydroxyeicosatrienoic acid, a metabolite of 11,12-epoxyeicosatrienoic acid. C6 cells expressed rat epoxygenase mRNAs, CYP1A1, CYP2B1 and CYP2J3, which converted arachidonic acid to epoxyeicosatrienoic acids. 11,12-Epoxyeicosatrienoic acid inhibited A23187-induced prostaglandin E(2) production and SKF-525A, an inhibitor of epoxygenase, attenuated the saikogenin D-induced inhibition of prostaglandin E(2) production in C6 cells. Furthermore, 11,12-epoxyeicosatrienoic acid and 11,12-dihydroxyeicosatrienoic acid, but not saikogenin D, inhibited the activity of cyclooxygenase in a cell-free condition. These data suggest that saikogenin D activates epoxygenases that rapidly convert arachidonic acid to epoxyeicosanoids and dihydroxyeicosatrienoic acids, and then the metabolites secondarily inhibit prostaglandin E(2) production.