In addition, the mostly used genes ACTB and GAPDH were more unstable in the fat and liver, the ACTB mRNA levels were increased in four adipose tissues, and the GAPDH mRNA levels were decreased in four adipose tissues and liver after HFD feeding. The data were analyzed by the GeNorm, NormFinder, BestKeeper, and Delta-Ct method, and the results showed that the most stable reference genes were different for a specific organ or tissue in a specific time point however, PPIA, RPLP0, and YWHAZ were the top three most stable reference genes in qPCR experiments on adipose, hepatic tissues, and muscles of mice in diet-induced obesity. In this study, using the high-fat diet (HFD)-induced obese mouse model, we assessed the expression of 10 commonly used reference genes to validate gene-expression stability in adipose tissue, liver, and muscle across different time points (4, 8, 12, and 16 weeks after HFD feeding) during the process of obesity. Quantitative PCR (qPCR), the most accurate and sensitive technique for quantifying mRNA expression, and choice of appropriate reference genes for internal error controlling in qPCR are essential to understanding the molecular mechanisms that drive the obesity epidemic and its comorbidities. 1Laboratory of Nutrition and Development, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, Beijing, China.These data introduce the obese diet-induced DIO-NASH and ob/ob-NASH mouse models with biopsy-confirmed individual disease staging as a preclinical platform for evaluation of novel NASH therapeutics.ĭiet-induced obesity Fibrosis Liver biopsy Nonalcoholic fatty liver disease Nonalcoholic steatohepatitis.Xiuqin Fan 1 Hongyang Yao 1 Xuanyi Liu 1 Qiaoyu Shi 1 Liang Lv 2 Ping Li 1 Rui Wang 1 Tiantian Tang 1 Kemin Qi 1 * Notably, fibrosis stage was significantly (P < 0.001) increased in ob/ob-NASH mice, when compared to DIO-NASH mice. Furthermore, fibrosis stage was significantly elevated for DIO-NASH mice (0 vs 1.2 ± 0.2, P < 0.05 compared to lean chow) and ob/ob NASH (0.1 ± 0.1 vs 3.0 ± 0.2, P < 0.001 compared to ob/ob chow). Histopathological scoring demonstrated significantly increased NAS of DIO-NASH mice (0 vs 4.7 ± 0.4, P < 0.001 compared to lean chow) and ob/ob-NASH mice (2.4 ± 0.3 vs 6.3 ± 0.2, P < 0.001 compared to ob/ob chow), respectively. During the eight week repeated vehicle dosing period, the metabolic phenotype was sustained in DIO-NASH and ob/ob-NASH mice in conjunction with hepatomegaly and increased hepatic lipids and collagen accumulation. Overall, the metabolic NASH phenotype was more pronounced in ob/ob-NASH vs DIO-NASH mice. The diet also induced clear histological features of NASH including hepatosteatosis and fibrosis. Steatosis and fibrosis were also quantified using percent fractional area.ĭiet-induction for 26 and 12 wk in DIO-NASH and ob/ob-NASH mice, respectively, elicited progressive metabolic perturbations characterized by increased adiposity, total cholesterol and elevated plasma liver enzymes. Non-alcoholic fatty liver disease activity score (NAS) (steatosis/inflammation/ballooning degeneration) and fibrosis were scored. Metabolic parameters, plasma liver enzymes and lipids (total cholesterol, triglycerides) as well as hepatic lipids and collagen content were measured by biochemical analysis. Global gene expression in liver tissue was assessed by RNA sequencing and bioinformatics.

Mice were then stratified into groups counterbalanced for steatosis score and fibrosis stage and continued on diet and to receive daily PO dosing of vehicle for 8 wk. After the diet-induction period, mice were liver biopsied and a blinded histological assessment of steatosis and fibrosis was conducted. A normal chow diet served as control in C57 mice (lean chow) and ob/ob mice (ob/ob chow). Male wild-type C57BL/6J (C57) mice (DIO-NASH) and male Lep(ob) /Lep(ob) (ob/ob) mice (ob/ob-NASH) were maintained on a diet high in trans-fat (40%), fructose (22%) and cholesterol (2%) for 26 and 12 wk, respectively. To characterize development of diet-induced nonalcoholic steatohepatitis (NASH) by performing liver biopsy in wild-type and genetically obese mice.

Gapdh western blot lean vs obese mice