sigma xi 2013 kelly giffey
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The Correlation Between Agouti Related Protein Expression and Dietary Intake
within Zebrafish, Danio rerios
March 8, 2013
Kelly GiffeyW.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!Melanocortin 3 or 4
Receptor
AgRP Neuron
stomach Ghrelin
Hungry!
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 AgRP expression (Pillot, 2011)
• After ghrelin injection to rats, AgRP mRNA levels rose (Kamegai, 2001)
Zebrafish Model Organism for AgRP Gene 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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