Zebra fish models for diabetes

Fishing to cure diabetes!
Anuradha
2013PHXF423P
Dept. of Pharmacy
BITS Pilani, Rajasthan
1

What are zebrafish?
• Scientific name: Danio rerio, belonging to the minnow
family (Cyprinidae) of the order Cypriniformes
• The name “zebrafish”: horizontal stripes on each side of
their bodies
• Habitat: Tropical fresh-water fish, native to the Indian sub
continent, abundantly found in Himalayan region. It is a
popular aquarium fish, frequently sold under the trade
name Zebra danio
• Food habits: Omnivorous (zooplankton, phytoplankton,
insects and insect larvae, can also worms and
small crustaceans). In research: adult zebrafish are often
fed with brine shrimp or paramecia.
Trends in Endocrinology and Metabolism, 2014, Vol. 25, No. 10
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Why Zebra fish?
Genetic similarity with human being (about 70-80%)
Physiological similarity
Adults-small in size, easy to maintain
Breed readily (approximately every 10 days) and can produce as
many as 50 to 300 eggs at a time
Embryos laid and fertilized externally- easily manipulated
In vitro fertilization- performed conveniently
Easier to generate transgenic or knock-out zebrafish lines
Nature, 2013, Vol. 496, 498-503;
http://www.fishforscience.org/zebrafish/why-zebrafish/
498–503
4

Limitations of zebrafish as animal models?
• Human diseases caused by genes that do not exist in
zebrafish can’t be developed
• Not useful models for human diseases that mainly take
place in a tissue type or body part that zebrafish do not
have (e.g., prostate, mammary glands, lungs)
• Expertise may be required for handling the animal models
Nature Reviews Genetics, 2007, Vol. 8, 353-367
https://www.yourgenome.org/facts/why-use-the-zebrafish-in-research
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Aim of the study: Development of a Novel Zebrafish
Model for Type 2 Diabetes Mellitus 6

Animal model development
Zebrafish(wildtypeorins-
EGFP)
Control group
(20mg/day/fish)
Overfeeding group
(120mg/day/fish)
Metformin (20microM )
Glimeperide (100microM)
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Methodology
Glucose tolerance test
Ins-EGFP DIO zebrafish image analysis
Western blot analysis
RNA isolation, sequencing, library construction and high-
throughput sequencing
Statistical analysis
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Diet-induced obesity model of zebrafish with
high fasting blood glucose
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Glucose intolerance with insulin overproduction
in DIO zebrafish
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Glucose intolerance with insulin overproduction
in DIO zebrafish
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Anti-diabetic drugs reduce blood glucose levels
of DIO zebrafish
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Gene expression profiling of liver-pancreas
revealed pathways common to human T2DM
• Genes dysregulated in T2DM zebra fish- 83 (↑) and 64(↓)
• According to the Ensembl gene orthologs database these
147 zebrafish genes corresponded to 121 human
orthologs.
• Gene Ontology (GO) Enrichment Analysis of these altered
genes: related to circadian rhythm, response to
oxygen/stress, transcriptions, and nitrogen metabolism
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Gene expression profiling of liver-pancreas
revealed pathways common to human T2DM
• Gene sets common to human and zebrafish T2DM:
– pancreas: gene-sets related to atherosclerosis, nervous
system, immune response and vascularization, circadian
rhythm and melanoma-related proteins
– liver: gene-sets related to immune response, nervous system
and vascularization. In addition, MAPK activity, oxygen
transport and voltage-gated calcium channel activity were
also listed for the diabetic liver as gene-sets common to
human and zebrafish T2DM
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Conclusions
• A zebrafish model of T2DM using a method of overfeeding
was developed that exhibited increased fasting blood
glucose with glucose intolerance and insulin resistance.
• Advantages of T2DM zebrafish model: 1) rapid onset of
T2DM phenotypes compared with rodent models, 2)
positive response to human anti-diabetic drugs using
exposure or oral administration and 3) pathological
similarity in transcriptomic pathways to the human
platform.
• Thus, T2DM zebrafish are suitable for elucidating the
mechanisms of early phase T2DM and appears to be a
useful animal model for phenotype-driven drug discovery
against diabetes.
15

References
• Srinivasan, K. et al. Combination of high-fat diet-fed and low-dose
streptozotocin-treated rat: a model for type 2 diabetes and
pharmacological screening. Pharmacol Res 52, 313–320,
doi:10.1016/j.phrs.2005.05.004 (2005).
• Oka, T. et al. Diet-induced obesity in zebrafish shares common
pathophysiological pathways with mammalian obesity. BMC Physiol 10,
21, doi:10.1186/1472-6793-10-21 (2010).
• Shimada, Y. et al. Downregulation of Max dimerization protein 3 is
involved in decreased visceral adipose tissue by inhibiting adipocyte
differentiation in zebrafish and mice. Int J Obes (Lond) 38, 1053–1060,
doi:10.1038/ijo.2013.217 (2014).
• Zang, L. et al. A Novel, Reliable Method for Repeated Blood Collection
from Aquarium Fish. Zebrafish 10, 425–432, doi:10.1089/
zeb.2012.0862 (2013).
• Westerfield, M. The zebrafish book: a guide for the laboratory use of
zebrafish (Danio rerio) (Eugene: University of Oregon Press, 2007).
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Human diseases successfully modelled in
zebrafish??
• Duchenne muscular dystrophy
• Cancers: melanoma, leukaemia, pancreatic cancer and
hepatocellular carcinoma
• Neurological diseases (Alzheimer’s disease, anxiety,
depression)
• Cardiovascular diseases (blood clotting, blood vessel
development, heart failure, and congenital heart and
kidney disease)
• Infectious diseases (mycobacterial infections like
tuberculosis)
• Metabolic disorders (atherosclerosis, Non-alcoholic
fatty liver disease and diabetes)
Nature Reviews Genetics, 2007, Vol. 8, 353-367; Cancer Research, 2012, Vol. 72, 4017–27
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Zebra fish: animal models for diabetes mellitus
• Toxin-mediated ablation of β-cells
• ENU-induced mutagenesis screening
• Morpholino and gRNA/Cas9 mediated knockdown
techniques
• The diet-induced obesity (DIO) by overfeeding with
Artemia (brine shrimps)
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Zebra fish models for diabetes

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