Zebrafish as a Model for Diabetes

The zebrafish which are in good condition are selected and a chemical mutagen is used to induce random point mutation and genotype screens are performed in the progeny to identify the progeny fish carrying a point mutation in gene associated with the desired diseases.

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What is a Zebrafish?

Zebrafish is a freshwater fish majorly found in South Asia. It is a prominent model for vertebrates primarily used in scientific research such as pre-clinical or drug discovery and development. Using Zebrafish, many advances occurred in the fields of developmental biology, oncology, and toxicology so on. Zebrafish is 70% homologous to human body making it an excellent model organism for predicting human diseases.

Diabetes – The incurable threat to humankind

Diabetes is a condition where the body’s glucose level rises above normal due to the impaired production of insulin by the islet cells of pancreas. There are two types of diabetes namely,

Type 1 – an autoimmune disease, body immune system kills the cells which produce insulin, common among young adults and kids
Type 2 – onsets at around 40 years, insulin levels not enough or the body develops insulin resistance due to extreme levels of glucose

Diabetes effect on different organs in Zebrafish

Heart:

Diabetes can affect the cardiovascular metabolism of glucose combined effect of insulin resistance, hyperglycemia and hypertension. This causes micro vascular damage, macrovasculopathy, and cardiovascular disease.

Liver:

Due to hyperglycemia, the lipid, protein and carbohydrate metabolism is altered causing cirrhosis, carcinomas etc. The damage to liver is caused by oxidative stress and inflammatory responses.

Pancreas:

This organ helps in digestion using exocrine and endocrine contributions. The endocrine beta cells producing insulin is dysfunctional. Insulin promotes the uptake of glucose in zebrafish through skeletal muscle and adipose tissue which also helps in the glycolysis and glycogenesis activation in liver.

Experimental Design

Histopathology Imaging of Heart, Liver and Pancreas

Fish were euthanized with 2-4 °C water. The fish was dissected through an incision in the viscera and organs of interest were isolated by cutting the localized tissue with a dissection knife. Thin slice of the organ of interest is stained and imaged.

Glucose Tolerance Test

The fish is anesthetized by placing it on ice water for 5 min, intraperitoneally 0.5mg/weight of glucose is injected and allowed to recover for 30, 90, and 180 min after injection.Fish were allowed to recover in the water system for 30, 60, and 120 min after dosing, and blood samples were collected at each time point to determine the blood glucose levels.

Relative Blood Glucose Test

Glucose level is continuously monitored on everyday basis using glucometer to map out the levels over a period of time and to monitor the pancreatic activity.

Wound Healing Rate

The caudal fin of Zebrafish has the capacity to regenerate after amputation. Due to the compound activity, the fish heals. The rate at which the fish is able to regenerate is measured.

Lipid Profiling

The fish is anesthetized by placing it on ice water for 1 min. The levels of triglyceride (TG) and total cholesterol (TC) were measured using commercial assay kits.

Molecular Biomarkers

Cystatin C – Nephropathy

Cystatin C is a biomarker for renal impairment in type 2 diabetes, it reflects the decreased glomerular filtration rate and the increased albumin to creatinine ratio. Cystatin C assay is performed where the cystatin C binds to anti cystatin antibodies coated on latex particle, causing agglutination.

Brain Derived Neutrophic factor (BDNF) – Neuropathy

BDNF is a nerve growth factor found in CNS. By regulating cell survival, proliferation, and synaptic development in the developing CNS, this biomarker is critical in controlling memory-associated neuroplasticity.

Vcam I – Retinopathy

Vcam I , a protein adhesion molecule is used as a biomarker since endothelial dysfunction plays a major role in diabetic retinopathy. The serum levels of Vcam I is measured.

Thus Bright field microscopy, fluorescent dyes in combination with fluorimetry or imaging techniques, western blotting, and quantitative RT-PCR are commonly used to monitor behavioural or morphological changes as well as protein and gene expression levels in zebrafish for toxicity and antidiabetic testing.

Advantages

Main advantage of using larvae is comparatively ethical
Ease of access and care
Wild type models have shown promising results which makes it more easy and accessible

The method likely to be feasible is inducing insulin resistance in larvae using cAMP glucocorticoids and monitoring larval glucose level parallel to the uptake of fluorescent glucose analogue in response to insulin mimetic. Using this method, substances having the potential to lower hepatic glucose production, improve peripheral glucose uptake, and reduce insulin resistance should be able to be identified.

Conclusion

An approach combining target-directed in vitro screening approaches, such as enzyme and cell-based assays, followed by the appropriate zebrafish model such as larvae can provide a efficient base for preclinical drug development. This method can be used to analyse risk of toxicity, which are a key contributor to drug reduction. Expanding the base to include new in vitro technologies like quantitative cell analysis can increase risk assessment accuracy even further. Using wild-type zebrafish as a replacement to mammalian models of diabetes could lower research costs and boost throughput while also contributing to the three Rs principle of ethical animal experimentation and speeding up the development of anti-diabetic drugs.

Zebrafish Studies:

Toxicology study in adult zebrafish
Modeling in zebrafish study
Toxicology study in larvae
Infection model in zebrafish
zebrafish fertility study
zebrafish neurology model