1. The Correlation Between Agouti Related
Protein Expression and Dietary Intake
within Zebrafish, Danio rerios
March 8, 2013
Kelly Giffey
W.F. West High School

2. Relevance
• Obesity Epidemic in the United States
– Two/thirds population overweight
• 8.3% of population has diabetes
– 25.8 million people in the US
• Leading cause of death
– More than smoking
• Leads to complications:
– Heart disease
– Stroke
– High blood pressure
– Kidney Disease
– Nervous system disease

3. Background - Agouti-Related Protein
• AgRP gene found in humans and vertebrates.
– Most production hypothalamus (brain)
– Regulates eating response
• Signals for hunger

4. Why We Feel Hunger?
stomach Ghrelin
AgRP
Neuron
Melanocortin
HUNGRY!
3 or 4
Receptor

5. Why We Feel Satisfied?
Food Eaten Leptin Secreted from fat tissue
AgRP Neuron
STOP
FULL!
MC3R/MC4R

6. Effects of AgRP Overexpression
• High AgRP levels
– Leads to hyperphagia
• Excessive overeating
– Overeating overtime causes obesity
• Leads to other health complications
• Obese people and mice have higher levels of
AgRP (Pan, 2005)
• Mice fed 20% less protein diet have higher AgRP
expression (Pillot, 2011)
• After ghrelin injection to rats, AgRP mRNA levels
rose (Kamegai, 2001)

7. Zebrafish Model Organism for AgRP
Gene Expression
• Danio rerios – contain similar organs as
humans
• AgRP protein synthesis occurs in
hypothalamus

8. Purpose and Hypothesis
-To determine whether AgRP expression
increases in fish experiencing induced
starvation.
-Starved Zebrafish would have elevated
AgRP mRNA expression in the brain
compared normally fed fish.

9. Research Design Overview
• Two identical aquariums
– Control Tank: 15 fish fed
normal diet.
– Experimental Tank: 15 fish
fed with induced starvation.
• Manipulated Variable: Diet
• Responding Variable: AgRP expression levels
as measured by qPCR.

10. Research Design
• The Study - over 9 days
• Three 3 day intervals
– 5 fish taken from each tank at end of intervals
– Fish were to be fed
• Experimental - 2% of mass (tank 1)
• Control - 4% of mass (tank 2)
– Average fish mass 500 mg in both tanks

11. Fish Euthanization
• End of feeding cycles, 5 fish removed from
both tanks
– Placed into 2 separate beakers of water

12. Procedure Overview
Tissue Dissection
RNA Extraction
mRNA converted to cDNA (Reverse
Transcription)
cDNA used in RT-PCR

13. Results
18S Control Amp
18S Control
18S Control AgRP
AgRP Amplified
Product
AgRP Gene
Expression

14. What do my Findings Indicate?
• RNA extraction and conversion to
cDNA was successful
• Overall, AgRP expression in brain
tissue low
• 18S highly expressed in brain

15. Conclusions
• It was originally hypothesized that the starved
fish would have higher mRNA levels for AgRP.
• At this time results do not directly support the
hypothesis
• However, additional research focus could
eventually show that AgRP expression levels
are directly related to food intake

16. Further Research
• If hypothalamus possible to dissect
– AgRP could closely be examined
– Evidence could result in determining role AgRP on
regulating body weight
• 1st run, AgRP didn’t begin amplification until
cycle 30
– Very low level of transcript
– Repeated gene expression experiment with higher
DNA concentration.

17. Plate with Second Run AgRP

18. Further Research
• Even with a higher concentration it still
yielded the same results.
• More severely starve the fish
– Fish closer extreme starvation levels
• Time period increase
• Tissue Cell culture

19. Acknowledgements
• My Parents
• My advisors Henri Weeks and Mike Stratton
• David Cogan and Trish from Applied
Biosystems