Tackling a pandemic with Zebrafish: Obesity and Non-Alcoholic Liver Disease

The emerging use of zebrafish to model metabolic diseases

By Christian Cortés-Campos, Head of Drug Discovery Operations at ZeClinics.

Christian is a Biochemist from Chile with ample training in rodent and zebrafish research. He did his Ph.D. at the Universidad de Concepción (Chile), investigating  feeding behavior in rodents. Then, he moved to the Zebrafish field as a postdoctoral researcher, first at Universidad de Valparaiso (Chile) and later at Whitehead Institute (MIT–USA). At MIT he used zebrafish as a model to investigate human diseases such as Kallmann and Pitt–Hopkins syndromes. During that period he became an expert in OMICs analysis; expertise that allowed him to gain a senior research position at the Developmental Biology Unit (UPF-Spain). After his stay at UPF, he joined ZeClinics to contribute his knowledge in feeding behavior and OMICs. His current aim is to study metabolic-related diseases together with transcriptomics analysis.

Previously in this blog, we talked about how adopting new technologies to automate and optimize scientific processes can help us accelerate screening throughput. Now, we are going to explore the zebrafish as a tool to study obesity and metabolic-related diseases such as Non-Alcoholic Fatty Liver Disease (NAFLD).

The 21st century pandemic

Obesity is a pandemic disease that involves excessive body fat accumulation (Body Mass Index (BMI) > 30). This disease has shown a steady increase worldwide, both in adults and children, triplicating its rate from 1975 to 2016. Nowadays, obesity affects 13% of the adult world population, and disease projections indicate this number will double by 2030 (WHO 2017). Moreover, obesity increases the risk of suffering several metabolic-related diseases such as type 2 diabetes, cardiovascular diseases, and NAFLD [1].

NAFLD is a hepatic disease, characterized by increased liver fat (steatosis) (>5%), in absence of significant alcohol consumption or other secondary causes. NAFLD morbidity has increased in parallel to obesity. 80 to 90% of obese adults will develop hepatic steatosis (NAFLD); among them, 10 to 25% will progress to Non-Alcoholic Steatohepatitis, NASH [2,3]. Both NAFLD and NASH represent a huge burden to the health and economic systems (>€35 billion only in Europe).

Importantly, no effective treatments have been identified for either disease. Specifically, only behavioral therapy has been successful for obesity, while NAFLD patients only count on palliative treatments [4]. Indeed, this unmet medical need has resulted in an attractive research field, where Zebrafish is positioned strategically to help develop new drugs, and understand better obesity and NAFLD  pathogenesis.

Zebrafish as a model for obesity and metabolic diseases

Zebrafish could be key for understanding metabolic-related diseases. This model shares remarkable genetic, anatomic, and functional similarities with humans inlipid metabolism, adipogenic pathways, and the presence of all key organs required for lipid metabolism [5]. Due to these facts, several zebrafish models have been published for obesity, NAFLD, and other metabolic diseases. This has led to the identification of therapeutic target genes/pathways, in which pharmacological modulation has the potential to regulate lipid metabolism and fat accumulation [4].

At ZeClinics, diet-induced approaches have been developed to study obesity, NAFLD (in juvenile and larval zebrafish) and other metabolic-related diseases. These studies have validated the impact of diet on liver physiology, cholesterol levels, and animal growth. As such, hepatic steatosis can be evaluated in larval and juvenile fish (Figure 1) fed with different diets.

Figure 1. Hepatic steatosis in juvenile fish. Fish are fed from 20 to 30 days post fertilization (dpf) with a Control Diet (CD), or High Fat Carbohydrates Diet (HFCD). A) Specific fat staining for evaluation of liver steatosis. Fat accumulation in the liver is observed in fish fed with HFCD. B) Steatotic liver quantification. The amount of dye per fish and diet is measured as red mean intensity. Statistical analyses have shown differences between CD and HFCD. Means and error standards are plotted. *** p<0.001.

In the juvenile fish model, visceral fat accumulation can also be evaluated (Figure 2), which has been associated with metabolic disturbances and increased risk for cardiovascular disease and type 2 diabetes.

Figure 2. Lipid accumulation model in juvenile fish. Fish are fed from 20 to 30 days post fertilization (dpf) with a Control Diet (CD), or High Fat Carbohydrates Diet (HFCD). A) Staining of fish adipocytes with a fluorescent dye for evaluation of fat accumulation and distribution. Abdominal fat accumulation is observed in fish fed with HFCD. B) Abdominal fat quantification. Fluorescence intensity measurement was done in the individual under different diets. Statistical analyses have shown significant differences between CD and HFCD. Means and error standards are plotted. *** p<0.001.

ZeClinics has also developed assays to understand the impact of different diets in growth and obesity development through the Body mass Index calculation (BMI) in adult and juvenile fish (Figure 3).

Figure 3. Juvenile obesity model. Fish are fed from 20 to 30 days post fertilization (dpf) with a Control Diet (CD), or High Fat Carbohydrates Diet (HFCD). Fish fed a HFCD show higher body length, weight and BMI when compared to CD-fed fish. Means and error standards are plotted. *** p<0.001.

Moreover, Gonadal Index (GI), fish fertility and hepatic steatosis can also be evaluated in adult fish to test animal fitness. This platform can be used to study metabolic diseases, and interestingly to evaluate the impact of different diets in animal growth and reproduction; an important field for the veterinary and food industries.

To summarize, we have validated larval, juvenile and adult zebrafish models to understand the disease mechanism of obesity, NAFLD, and metabolic-related diseases. Moreover, in combination with our expertise in transgenesis and gene editing techniques, ZeClinics is deepening its expertise in this relevant disease field,  offering a powerful research platform to perform gene candidate screening, or drug screenings for obesity, NAFLD, and other metabolic diseases.

REFERENCES

[1] Brahe LK, Astrup A, Larsen LH. Can We Prevent Obesity-Related Metabolic Diseases by Dietary Modulation of the Gut Microbiota? Adv Nutr. 2016 Jan 15;7(1):90-101.

[2] Asaoka Y, Terai S, Sakaida I, Nishina H. The expanding role of fish models in understanding non-alcoholic fatty liver disease. Dis Model Mech. 2013 Jul;6(4):905-14. Epub 2013 May 29. Erratum in: Dis Model Mech. 2014 Mar;7(3):409.

[3] Godoy-Matos AF, Silva Júnior WS, Valerio CM. NAFLD as a continuum: from obesity to metabolic syndrome and diabetes. Diabetol Metab Syndr. 2020 Jul 14;12:60.

[4] Faillaci F, Milosa F, Critelli RM, Turola E, Schepis F, Villa E. Obese zebrafish: A small fish for a major human health condition. Animal Model Exp Med. 2018 Nov 21;1(4):255-265.

[5] Zang L, Maddison LA, Chen W. Zebrafish as a Model for Obesity and Diabetes. Front Cell Dev Biol. 2018 Aug 20;6:91.

Disease modelingDisease modelsmetabolicmetabolic diseasesobesitypreclinical researchtarget validation