Anagha Rama Varma

20 January, 2017

Science Research

Research Plan and Post Project Summary

Abstract

Recovery from cold stress—or exposure to low temperatures for extended periods of

time—in Drosophila melanogaster is modulated by genes that are responsible for homeostasis.

Research has shown that Lisinopril, a drug used to treat high blood pressure in humans, may be

interacting with genes responsible for maintaining homeostasis in D. melanogaster. To better

understand the impact of Lisinopril’s effect on gene expression, Lisinopril-treated and untreated

flies of three different genetic lines will be tested using a cold stress assay, and their recovery

times will be compared.

Rationale

Lisinopril is a drug approved by the FDA for use in treating high blood pressure in

humans. It works by restricting the Angiotensin Converting Enzyme (ACE) in the human renin-

angiotensin system (RAS). ACE converts the hormone angiotensin type 1 (Ang I) to angiotensin

type 2 (Ang II). Ang II is a vasoconstrictor; restricting ACE lowers the production of Ang II, and

thus lowers blood pressure (Abadir, n.d.; Abadir et. al, 2011; Krcobic, 2016; “Lisinopril”, n.d.;

“Renin-angiotensin system – Homo sapiens”, n.d.). In addition to treatment of high blood

pressure, the drug Lisinopril is associated with many positive side effects, such as improved

speed and reduced frailty. Frailty is defined as syndrome in which three or more of the following

trends occur: unintentional weight loss, fatigue, weakness, decreased walking speed, and less
physical activity (Fried et. al., 2001; Xue, 2011). Lisinopril has been shown to increase weight

retention among older adults (Schellenbaum et. al., 2005) and improve walking speed among

older adults with exercise (Buford et. al., 2012). Many studies have credited these effects to the

drug’s interaction with the human RAS. However, work in the Leips lab has shown that

Lisinopril-treated Drosophila melanogaster experience effects similar to those observed in

humans despite lacking RAS. D. melanogaster does contain genes “homologous” to the ACE

gene in humans: the angiotensin converting enzyme related gene Acer and angiotensin

converting enzyme gene Ance (Fournier et. al). The link between Lisinopril and the many

positive side effects in humans is not yet fully understood, and it is possible that Lisinopril’s

effects may not act solely through the RAS pathway. This study attempts to ascertain whether

genotype plays a significant role in drug response.

Hypothesis/Expected Outcomes

Lisinopril has been shown to have many positive side effects, such as improved strength

and longevity, which can be observed in lisinopril-treated Drosophila melanogaster. It is

hypothesized that Lisinopril may act upon genes regulating aspects of homeostasis, and the cold

stress assay will help determine whether Lisinopril has an impact on the genes modulating cold

stress.

Research Questions

The research questions that will be addressed by this work are how Lisinopril affects cold

stress recovery; what, if any, genetic mechanism might be underlying Lisinopril’s side effects
and interaction with RAS; and whether there is variation in cold stress recovery and Lisinopril’s

effect on this trait between genetic lines.

Procedures

This project will use D. melanogaster of genetic lines 229, 304, and 73 which will be

obtained from Bloomington Stock Center. Each of the lines will be expanded to about 80 vials

per line. The adults will then be removed, and a waiting period will ensue until such time that the

larvae eclose from their pupae cases. Virgin D. melanogaster will then be collected. Virgin

males will be separated from virgin females; about eighty males should be collected per line.

These will be maintained in six cages, with forty flies per cage, separated by line and treatment,

until experimentation.

The preliminary experiment will be conducted to compare recovery time of non-drugged

flies exposed to cold stress with that of non-drugged flies, and to compare the cold stress

recovery of the three different genetic lines when the flies are one week old, until which time

they will have been maintained in cages.

Cold Stress Assay

After one week has passed with the flies in cages, a respirator will be used to remove the

flies from the cages. CO2 will be used to render them unconscious, and they will be then moved

to the empty vials. Ten flies will be placed in each vial, labeled and sealed with cotton. A 30

minute period will be allowed for the flies to recover from the CO2, after which the vials will be

placed in buckets of ice. The vials will be placed such that they are mostly submerged, but the

tops of the vials will remain above the ice. The buckets will then be stored in a controlled

temperature room at 4 oC. The vials will be removed from the ice after 3 hours, and placed in a
room at 23 oC to allow for recovery. The flies will be monitored at 5-second intervals, and the

times at which the individuals regain consciousness will be recorded.

Risk and Safety

This project does not pose any risk to the researchers.

Data Analysis

Once data has been collected, it will be analyzed to find the overall trend. Average

duration of life will be calculated for the various test groups. Using this information, graphs

and/or other visuals will be created to better understand and observe the overall trend.

Additionally, categorical analysis using SAS software. Possible causes for the resultant trend will

be examined and researched, and will lead to the next step of the project.

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