High fat plus high cholesterol diet lead to hepatic steatosis in zebrafish larvae: a novel model for screening anti-hepatic steatosis drugs

Abstract

Background: Non-alcoholic fatty liver disease (NAFLD), characterized as excessive lipid accumulation within hepatocytes, is growing in prevalence. The exploitation of effective drugs for NAFLD has been proven challenging. Herein, we aimed to establish a dietary model of hepatic steatosis using transparent zebrafish larvae in which high-throughput chemical screens could be conducted.

Methods: Zebrafish larvae fed with high fat (HF) diet and high fat plus high cholesterol (HFC) diet were compared to the control. We analyzed intrahepatic lipid accumulation, biological indexes and various pathways including lipid metabolism, ER stress and inflammation. In addition, the effects of ezetimibe and simvastatin on HFC diet-induced steatosis were evaluated.

Results: Zebrafish larvae fed with HF and HFC diets developed steatosis for 7 and 10 days. The incidence and degree of steatosis were more severe in HFC diet-fed larvae compared with the control and HF diet-fed larvae, suggesting that adding cholesterol to the HF diet promotes the hepatic lipid accumulation. These data were confirmed by the pathological observation. Biological indexes, free cholesterol (FC), total cholesterol (TC) and triacylglycerol (TG) were elevated in the liver of HFC diet-fed larvae compared with the control and HF diet-fed larvae. Additionally, the expression levels of endoplasmic reticulum (ER) stress and lipolytic molecules (atf6, hspa5, hsp90b1, pparab, cpt1a and acox3) were significantly up-regulated in the liver of HF and HFC diets-fed larvae compared to the control, whereas the expression of lipogenic molecules (acaca, fasn, srebf2, hmgcs1 and hmgcra) were decreased in the liver of HF and HFC diets-fed larvae compared to the control. To validate the reliability of the HFC model and utility value for screening potential anti-steaotsis drugs, HFC-fed larvae were treated with two accepted lipid-lowing drugs (ezetimibe and simvastatin). The results showed that these drugs significantly ameliorated HFC-induced steatosis.

Conclusion: Our results demonstrate that the zebrafish larvae steatosis model established and validated in this study could be used for in vivo steatosis studies and drug screening.

Keywords: Drug screening; Hepatic steatosis; Lipid accumulation; Zebrafish.

Fig. 1

Effects of HF and HFC diets treatment on the survival rate and growth of zebrafish larvae. a Outline of the HF and HFC diets feeding protocol. b Larvae treated with control, HF diet, 2.5 % diet and 5.0 % HFC diet were scored for mortality, n = 600 from 6 tanks, error bars show standard deviation. c Standard length was measured from top to the end of the body. S.L.: standard length. d Body length and (e) body weight were expressed as the mean ± SD, n = 90 from 3 tanks. N.S.: no significant difference, **P < 0.01, ***P < 0.001, by one-way ANOVA and LSD post-hoc test

Fig. 2

HF and HFC diets lead to hepatic steatosis in zebrafish larvae. a Representative image of larvae defined positive for steatosis by whole-mount oil red O staining. Dotted line outlines the liver. b The percent of larvae with steatosis was scored in 3 tanks with at least n = 100 per group. c Numberous lipid droplets in the hepatocytes were observed by oil red O staining of frozen liver sections and H&E staining in HF group, 2.5 % HFC group and 5.0 % HFC group after 10 days of feeding. d FC, (e) TC and (f) TG levels in livers of larvae fed with control, HF, 2.5 % HFC and 5.0 % HFC diets for 10 days were normalized to total protein. Data are expressed as mean ± SD, N.S.: no significant difference, *P < 0.05, ***P < 0.001, by one-way ANOVA. (A, ×63 magnification; C, ×400 magnification; in: intestine)

Fig. 3

Genes changes in the livers of HF and HFC diets-fed zebrafish larvae. The expression levels of genes involved in (a) lipid metabolism, (b) ER stress and (c) inflammatory pathway in HF group, 2.5 % HFC group and 5.0 % HFC group were compared with the gene expression in control group. Gene expression analysis using cDNA prepared from pools of livers dissected from larvae (n =20-30) in each group after 10 days of feeding. Data are expressed as mean ± SD, N.S.: no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001, by one-way ANOVA

Fig. 4

Ezetimibe and simvastatin treatment ameliorate hepatic steatosis in 2.5 % HFC-treated zebrafish larvae. a Whole-mount oil red O staining, oil red O staining of frozen liver sections and H&E staining of paraffin liver sections in larvae exposure to control, 2.5 % HFC, 2.5 % HFC + ezetimibe and 2.5 % HFC + simvastatin diets. Clear cytoplasmic lipid droplets were seen in livers of larvae fed with 2.5 % HFC diet. Ezetimibe and simvastatin treatment decreased lipid droplets in 2.5 % HFC-treated zebrafish larvae. b FC, (c) TC and (d) TG levels in livers of larvae fed with control, 2.5 % HFC, 2.5 % HFC + ezetimibe and 2.5 % HFC + simvastatin diets for 10 days were normalized to total protein. Data are expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, by one-way ANOVA

Fig. 5

Effect of ezetimibe and simvastatin on genes and protein expression. The expression levels of (a) srebf2, hmgcs1 and hmgcra (b) pparab, cpt1a and acox3 (c) atf6, hspa5 and hsp90b1 in 2.5 % HFC group, 2.5 % HFC + ezetimibe group and 2.5 % HFC + simvastatin group were compared with the gene expression in control group. Gene expression analysis using cDNA prepared from pools of livers dissected from larvae (n = 20-30) in each group after 10 days of feeding. Fold change in GRP78/BiP protein levels normalizing to β-actin was examined by western blot in the livers of zebrafish larvae. Data are expressed as mean ± SD, N.S.: no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001, by one-way ANOVA