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