Zebrafish Non-alcoholic Fatty Liver Disease Models

Non-alcoholic fatty liver disease (NAFLD) is characterized by excess fat accumulation in the liver and is not associated with excessive alcohol consumption. It also has a broad disease spectrum, including steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. NAFLD is closely associated with insulin resistance, fatty diet, drug-induced liver injury and metabolic syndrome. Due to the increasing prevalence of obesity and type II diabetes, NAFLD has become the fastest-rising cause of chronic liver disease worldwide. NAFLD progresses from benign liver fat accumulation to liver inflammation and cirrhosis, and eventually to liver failure.

Although several rodent models have been established to study NAFLD, they have limitations including cost, rate of disease progression, key differences, and poor adaptability to pharmacological screens. Similar to humans, zebrafish respond to environmental triggers such as liver toxins, fasting and diets high in fat, cholesterol or fructose. Additionally, the benefits of using zebrafish to study NAFLD include low cost, rapid maturation, ease of genetic modification, and feasibility of experimental manipulation in terms of therapeutic paradigms. Zebrafish serve as a physiologically relevant model system for studying NAFLD due to their hepatic-pancreatic-biliary anatomy similar to human.

Fig. 1 Effect of high cholesterol diet on hepatic steatosis in larval zebrafish.

Our Zebrafish Non-alcoholic Fatty Liver Disease Models

Creative Biogene treated zebrafish larvae with a high-fat diet (HFD), causing fat to accumulate in their livers. During this process, as the larva consumes dietary lipids, these lipids are processed and transported to the hepatocytes resulting in a mild inflammatory state. Additionally, we offer chemically induced NAFLD models such as zebrafish induced by PFOS, thioacetamide, thapsigargin, and tributyltin. We can also generate mutant and transgenic zebrafish by reverse genetics to manipulate the function of known genes and study the molecular mechanism of hepatic steatosis. Our zebrafish NAFLD models would advance your understanding of the etiology of NAFLD by possessing human NAFLD-like phenotypes, including macrophage infiltration and reactive oxygen species (ROS)-mediated liver stress.

Table 1 Zebrafish Genetic Mutants

Mutant	Mutant	Liver phenotype
ffoie gras/trappc11	ER-to-Golgi trafficking	Hepatic steatosis; impaired hepatic function.
ducttrip/ahcy	Methionine metabolism	Hepatic steatosis; mitochondrial dysfunction; liver degeneration.
cdipt	Phospholipid synthesis	Hepatic steatosis due to ER stress and defects in de novo phosphatesitol synthesis.
sec63	ER-associated protein involved in translocation of glycoproteins	ER stress and hepatic steatosis.
gmps	Guanosine monophosphate synthetase	Steatosis due to down-regulated activity of the small GTPase Rac1 and ROS production.
stk11	Serine/Threonine kinase 11 regulating phosphorylation of the nutritional sensor AMP-kinase	Fasting-induced hepatic steatosis; glycol depletion.
red moon/ slc16a6a	β-hydroxybutyrate transporter required for hepatocyte secretion of ketone bodies during fasting.	Increased neutral lipids and induction of hepatic lipid biosynthetic genes when fasted.
red moon/ slc16a6a	β-hydroxybutyrate transporter required for hepatocyte secretion of ketone bodies during fasting.	Increased neutral lipids and induction of hepatic lipid biosynthetic genes when fasted.
slc7a3a	Arginine transporter required for arginine-dependent nitric oxide synthesis.	Fasting-induced steatosis.
mbtps1	Membrane-bound transcription factor peptidase site 1	The mutant is protected from chronic ER stress-induced steatosis.
cnr1 cnr2	Cannabinoid receptor	Adult cnr2 mutants are susceptible to hepatic steatosis, and show impaired methionine metabolism. cnr1 mutants are protected from steatosis.
socs1a	Suppressor of cytokine signaling-1a	Hepatic steatosis and insulin resistance linked to elevated Jak- STAT5 signaling

Table 2 Zebrafish Genetic Mutants

Transgenic line	Transgene expressed	Liver phenotype
Tg(-2.8fabp10a:HBV.HBx- GFP)	Hepatitis B virus X protein (HBx)	Hepatic steatosis, hepatitis, liver hypoplasia.
Tg(fabp10a:GFP-gank)	ankyrin repeat protein (gankyrin)	Hepatic steatosis, increased hepatocyte apoptosis and liver failure.
Tg(fabp10a:dnfgfr1- EGFP)	dominant-negative fibroblast growth factor receptor 1 (dnfgfr1)	Hepatic steatosis, cholestasis, ballooning degeneration of hepatocytes.
Tg(fabp10a:EGFP-YY1)	ubiquitous transcription factor yin yang 1 (yy1)	Hepatic steatosis.
Tg(fabp10a:Tetoff- CB1R:2A:eGFP)	cannabinoid receptor 1 (cb1r)	Hepatic steatosis.
Tg(actb2:EGFP-nr1h3) Tg(fabp2:EGFP-nr1h3)	Global (actb2 promoter) or intestinal (fabp2 promoter) expression of Liver X receptor (nrlh3)	Global overexpression of nrlh3 induces steatosis; Intestine- specific overexpression suppresses steatosis caused by high-fat diet.

Advantages

High-throughput gene and drug screening
Rapidly perform reverse and forward genetic screening
Whole body real-time monitoring
Labeling individual hepatocyte types using a transgenic fluorescent reporter strain, enabling real-time tracking of their morphology and behavior during development and injury

References

Kulkarni A, et al. A Novel 2-Hit Zebrafish Model to Study Early Pathogenesis of Non-Alcoholic Fatty Liver Disease. Biomedicines. 2022, 10(2):479.
Pham DH, Zhang C, Yin C. Using zebrafish to model liver diseases-Where do we stand?. Curr Pathobiol Rep. 2017, 5(2):207-221.
Ma J, et al. A Comprehensive Study of High Cholesterol Diet-Induced Larval Zebrafish Model: A Short-Time In Vivo Screening Method for Non-Alcoholic Fatty Liver Disease Drugs. Int J Biol Sci. 2019, 15(5):973-983.

For research use only. Not intended for any clinical use.

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