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! 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.
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
Most of 18s transcripts began to amplify around cycle 15 where as the agrpmrna did not even begin to amplify until about cycle 35
Since ribosomes are so necessary in all cells, the gene must be highly expressed and up regulated. This control proves that the brains were dissected, rna extracted, and cunverted to cDNA successfully because of the amplification of the 18s gene expression.
If it was possible to specifically dissect the hypothalamus from the brain, AgRP might be able to be examined and analyzed more successfully. This could provide more conclusive evidence to determine the role of AgRP in regulating body weight.
Unfortunately, there still was not enough AgRP amplification to draw any conclusions on the role of AgRP on regulating body weight. There was some amplification, but those that did amplify were from both Tank one and Tank two so it was challenging to draw any conclusions. For further, if I could find a positive control with a piece of tissue that is known to have high levels of AgRP mRNA expression, I could test the custom assay to make sure it was designed properly. The hypothalamus is known to have the highest levels of AgRP of the organs.
