Sigma Xi 2013 Kelly Giffey

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