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The Correlation Between Agouti Related Protein Expression and Dietary Intake
within Zebrafish, Danio rerios
March 8, 2013
Kelly Giffey
W.F. West High School
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
Background - Agouti-Related Protein
• AgRP gene found in humans and vertebrates.
– Most production hypothalamus (brain)
– Regulates eating response
• Signals for hunger
Why We Feel Hunger?
HUNGRY!Melanocortin3 or 4
Receptor
AgRPNeuron
stomach Ghrelin
Why We Feel Satisfied?
Food Eaten Leptin Secreted from fat tissue
STOPAgRP Neuron
FULL!MC3R/MC4R
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 AgRPexpression (Pillot, 2011)
• After ghrelin injection to rats, AgRP mRNA levels rose (Kamegai, 2001)
Zebrafish Model Organism for AgRPGene Expression
• Danio rerios – contain similar organs as humans
• AgRP protein synthesis occurs in hypothalamus
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.
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.
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
Fish Euthanization
• End of feeding cycles, 5 fish removed from both tanks
– Placed into 2 separate beakers of water
Procedure Overview
Tissue Dissection
RNA Extraction
mRNA converted to cDNA (Reverse Transcription)
cDNA used in RT-PCR
Results
18S Control
AgRP Amplified
Product
AgRP Gene
Expression
18S Control Amp18S Control
AgRP
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
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
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.
Plate with Second Run AgRP
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
Acknowledgements
• My Parents
• My advisors Henri Weeks and Mike Stratton
• David Cogan and Trish from Applied Biosystems
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