Role of High-Fat Diet in Stress Response of Drosophila

Abstract

Obesity is associated with many diseases, one of the most common being obstructive sleep apnea (OSA), which in turn leads to blood gas disturbances, including intermittent hypoxia (IH). Obesity, OSA and IH are associated with metabolic changes, and while much mammalian work has been done, mechanisms underlying the response to IH, the role of obesity and the interaction of obesity and hypoxia remain unknown. As a model organism, Drosophila offers tremendous power to study a specific phenotype and, at a subsequent stage, to uncover and study fundamental mechanisms, given the conservation of molecular pathways. Herein, we characterize the phenotype of Drosophila on a high-fat diet in normoxia, IH and constant hypoxia (CH) using triglyceride and glucose levels, response to stress and lifespan. We found that female flies on a high-fat diet show increased triglyceride levels (p<0.001) and a shortened lifespan in normoxia, IH and CH. Furthermore, flies on a high-fat diet in normoxia and CH show diminished tolerance to stress, with decreased survival after exposure to extreme cold or anoxia (p<0.001). Of interest, IH seems to rescue this decreased cold tolerance, as flies on a high-fat diet almost completely recovered from cold stress following IH. We conclude that the cross talk between hypoxia and a high-fat diet can be either deleterious or compensatory, depending on the nature of the hypoxic treatment.

Full text

Role of High-Fat Diet in Stress Response of
Drosophila
Erilynn T. Heinrichsen
1
, Gabriel G. Haddad
1,2,3
*
1Department of Pediatrics (Division of Respiratory Medicine) and Biomedical Sciences Graduate Program, University of California San Diego, La Jolla, California, United
States of America, 2Department of Neurosciences, University of California San Diego, La Jolla, California, United States of America, 3Rady Children’s Hospital, San Diego,
California, United States of America
Abstract
Obesity is associated with many diseases, one of the most common being obstructive sleep apnea (OSA), which in turn
leads to blood gas disturbances, including intermittent hypoxia (IH). Obesity, OSA and IH are associated with metabolic
changes, and while much mammalian work has been done, mechanisms underlying the response to IH, the role of obesity
and the interaction of obesity and hypoxia remain unknown. As a model organism, Drosophila offers tremendous power to
study a specific phenotype and, at a subsequent stage, to uncover and study fundamental mechanisms, given the
conservation of molecular pathways. Herein, we characterize the phenotype of Drosophila on a high-fat diet in normoxia, IH
and constant hypoxia (CH) using triglyceride and glucose levels, response to stress and lifespan. We found that female flies
on a high-fat diet show increased triglyceride levels (p,0.001) and a shortened lifespan in normoxia, IH and CH.
Furthermore, flies on a high-fat diet in normoxia and CH show diminished tolerance to stress, with decreased survival after
exposure to extreme cold or anoxia (p,0.001). Of interest, IH seems to rescue this decreased cold tolerance, as flies on a
high-fat diet almost completely recovered from cold stress following IH. We conclude that the cross talk between hypoxia
and a high-fat diet can be either deleterious or compensatory, depending on the nature of the hypoxic treatment.
Citation: Heinrichsen ET, Haddad GG (2012) Role of High-Fat Diet in Stress Response of Drosophila. PLoS ONE 7(8): e42587. doi:10.1371/journal.pone.0042587
Editor: Fanis Missirlis, Queen Mary University of London, United Kingdom
Received February 10, 2012; Accepted July 9, 2012; Published August 1, 2012
Copyright: ß2012 Heinrichsen, Haddad. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported in part by National Institutes of Health (NIH) grant PO1 HL098053 and in part by the UCSD Genetics Training Program
through an institutional training grant from the National Institute of General Medical Sciences, T32 GM008666. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: ghaddad@ucsd.edu
Introduction
Over 60% of the population in the United States is estimated to
be obese or overweight, a number that has dramatically increased
in recent decades and continues to climb [1]. A multitude of social
and economic factors have contributed to the rise in obesity, not
the least of which is an abundance of processed foods high in
saturated fat and simple carbohydrates. With obesity comes many
disease complications, including sleep apnea, hypoxia, atheroscle-
rosis, cardiovascular diseases and stroke [2].
In obstructive sleep apnea (OSA), one of the most common
associated diseases, the upper airway collapses repeatedly during
sleep, causing chronic intermittent hypoxia [3]. OSA patients
therefore are often challenged with both obesity and intermittent
hypoxia conditions. In order to investigate the potential interac-
tion of obesity and hypoxia, we needed a model that would allow
us to study metabolic changes and be able to survive hypoxic
challenges. Furthermore, such a model would need to lend itself to
molecular analysis to appreciate the basis for such phenotypic
changes.
The fruit fly, Drosophila melanogaster, has served as a useful genetic
model system in many situations, including development and
human disease states. We undertook this investigation to
determine whether we can develop a model in the fly that would
be helpful for understanding the effects of hypoxia and obesity and
their potential interactions. We believe that the fly could be an
important model for various reasons, including the facts that a)
many of the metabolic and signaling pathways involved in fat
metabolism and insulin signaling in flies are conserved in humans
[4] and b) Drosophila have many organ systems analogous to
humans that control uptake, storage and metabolism; one of these
is the fat body, which functions like human liver and white adipose
tissue, metabolizing nutrients and storing reserves of glycogen and
lipids [4]. The adult fat body is subject to diet-induced lipid
overload, making Drosophila even more appealing as a genetic
model in which to study obesity [5–7]. Furthermore, many human
disease genes (.70%) have been found to exist in flies [8].
Since the early 1990s, our laboratory has used Drosophila
melanogaster to investigate the effects of hypoxia on metabolism,
gene regulation, and the role of gene regulation in resistance or
susceptibility to injury. More recently, we have studied the effects
of both intermittent (IH) and constant hypoxia (CH) [9–11], since
each of these conditions occurs in many different diseases and
causes significant stress on the organism. Additionally, we have
shown that the two separate paradigms of IH and CH result in a
differential change in gene expression [12], making it all the more
important to individually evaluate and compare the two stresses.
With the ability to put flies on a diet high in saturated fat while
simultaneously exposing them to either normoxic or hypoxic
conditions, we were able to investigate three different aims. These
involved assessing the effects of a high-fat diet, the effects of
intermittent hypoxia, and their potential interaction on the
phenotype of Drosophila. We hypothesized that intermittent
hypoxia alters lipid metabolism, leading to changes in stress
tolerance in Drosophila, and the results show clear evidence of this
interaction.
PLoS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e42587

Materials and Methods
Fly Rearing and Collection
All stocks were maintained on standard cornmeal Drosophila
medium in an incubator at 25uC and 30–50% humidity. Adult
flies were collected at 0–3 days and transferred to a separate vial of
the standard cornmeal medium in room air. After aging for 3
more days, male and female flies were separated and only the
females were transferred to the experimental diet and oxygen
condition. In all experiments, unless otherwise noted, w
1118
flies
were used in order to facilitate future work with genetically
mutated lines, since this is a common background of flies with P-
element and UAS insertions. When adipose
60
mutants were studied,
the wildtype strain used was Oregon-R in order to control for
genetic background. The stocks of Oregon-R,Canton-S and w
1118
were obtained from Bloomington Stock Center, while adipose
60
was
a gift from Dr. Tania Reis.
Experimental Diets: Regular food and Coconut food
Jazz Mix Drosophila food from New Horizon Foods was prepared
as directed and placed in plastic vials as the Regular food (RF).
The Coconut food (CF) diet was based on a recipe developed by
Dr. Sean Oldham at the Sanford/Burnham Institute, adding
coconut oil to the regular food as a source for increased saturated
fat in the diet [7]. The recipe has been specialized for the current
model, with the coconut food diet consisting of 5, 10 or 20%
weight per volume of food-grade coconut oil, with the last two
higher than the maximum 7% saturated fat recommended by the
American Heart Association. The 5% diet did not have an obvious
enough effect, and no significant difference was found between the
10 and 20% supplemented food, so only results from the 20% diets
are reported. For verification of results, we also tested a diet
supplemented with palm oil in the same manner as described
above for coconut oil.
Experimental Oxygen Conditions: Normoxia, Intermittent
and Constant Hypoxia
At age 3–6 days, female flies were sorted onto the diets and
immediately placed in normoxia, intermittent hypoxia or constant
hypoxia, with approximately 25 flies per vial. All three conditions
were at room temperature (22–24uC) and in a similar environ-
ment.
In normoxic conditions, flies were kept in room air (21% O
2
).
For intermittent hypoxia (IH) and constant hypoxia (CH), flies
were placed in specially designed chambers where the oxygen
levels are carefully controlled using a combination of oxygen and
nitrogen with the Oxycycler hydraulic system (Model A4460,
BioSpherix, Redfield, NY) and ANA-Win2 Software (Version
2.4.17, Watlow Anafaze, CA). In the chamber for IH, flies were
exposed to O
2
levels alternating between 4 minutes at 1% and
4 minutes at 21% O
2
. The total time for one IH cycle was
20 minutes, with a ramp time of 1 minute for 1%–21% O
2
and
around 10 minutes for 21%–1% O
2
. In the CH chamber, flies
were exposed to a constant oxygen level of 5% O
2
. Oxygen levels
were chosen based on previous observations in our laboratory in
order to allow flies to be mobile and still consume their diets.
Flies remained in their oxygen condition for one week if they
were to be assayed or as long as needed to examine lifespan. To
maintain consistent food conditions, flies were transferred to fresh
food of their respective diet every 3–4 days.
Metabolic Profile: Triglyceride, Glucose and Protein
Measurement
After one week in a specific oxygen condition, flies were
collected in groups of five female flies and placed in 1.5 ml
microcentrifuge tubes. Their live weight was determined and the
flies were frozen on dry ice. They were then homogenized using
the Precelly’s 24 homogenizer and prepared as described in
Gro¨nke 2003 [13] to measure absolute triglyceride levels using the
Thermo Infinity Triglyceride kit and protein levels using Pierce
BCA protein assay (protein levels not shown, as normalizing
triglycerides to protein was comparable with normalizing to
bodyweight). Differing levels of triglycerides reported in the
current literature can easily be accounted for by multiple factors,
including the sex and age of the flies, and the method of
extraction. To determine whole-body glucose, groups of five
female flies were homogenized in 1 ml deionized water. The
homogenates were centrifuged and the supernatants transferred to
a 96-well plate in triplicate. Glucose levels were quantified using
the Glucose GO assay kit (Sigma–Aldrich, Saint Louis, MO)
according to the manufacturer’s instructions. In brief, glucose is
oxidized to gluconic acid and hydrogen peroxide by glucose
oxidase. The hydrogen peroxide then reacts with o-dianisidine in
the presence of peroxide to form a colored product, and the
oxidized o-dianisidine reacts with sulfuric acid to form a more
stable colored product. The intensity of the color is proportional to
the original glucose concentration. The whole-body free glucose
was measured, although it is important to note that the major
carbohydrate in Drosophila is trehalose. Trehalose is the product of
two glycolytic intermediates, with the condensation of glucose-6-
phosphate and the glucose moiety of uridine diphosphoglucose
(UDP-glucose). Trehalose is then hydrolyzed into glucose by the
enzyme trehalase. This study measures the glucose already
available in the organism.
CAFE Assay
Flies were placed on regular food (RF) and in normoxia or IH
for 5 days. Following that time, groups of 5 flies were placed in a
plastic vial with only a piece of filter paper containing 500 ml
water. Through the top plug, a capillary tube was inserted
containing 5 ml liquid food (5% yeast, 5% sucrose) as described in
Ja et al [14]. The capillary tube was removed every 24 hours and
replaced with a new capillary tube containing 5 ml of food. The
flies were allowed to adjust to the new setup for the initial
24 hours, after which measurements were taken each time the
capillary was removed, measuring the difference in level of food (in
mm). Knowing the initial height (in mm) of the 5 ml, the calculated
ml/mm could be multiplied by the change in food level to
determine how much food (in ml) was consumed.
Stress Tolerance: Cold, Anoxia and Starvation
All assays were performed in normoxia, immediately following
the week of exposure to experimental diet and oxygen conditions.
Flies exposed to the different diets were assayed simultaneously
when possible (cold and starvation) or consecutively (anoxia).
Cold Stress. A25uC bath was made using water, ice and
salt. For each group, sets of 15–20 female flies were placed in
empty plastic vials and into the water bath. Flies fell unconscious
almost immediately and vials were checked to make sure all flies
were at the bottom and thus submerged in the cold bath. They
remained as such for 2 hours, with the temperature being checked
regularly throughout. At the end of the 2 hours, vials were
removed from the water bath, and flies transferred to regular food
and left to recover at room temperature. After 24 hours, survival
Effects of High-Fat Diet on Drosophila
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was recorded as the number of flies that had regained
consciousness.
Anoxic Stress. A specially designed chamber was used to
study flies under controlled O
2
levels [9]. Sets of 30 flies from a
particular group were placed in the chamber and exposed to
anoxic conditions (O
2
concentration = 0% with administration of
100% N
2
) for 2 hours. They were then returned to a vial with
regular food in room air, and the number of flies that regained
consciousness after 24 hours of recovery was counted as the
survival.
Starvation. For each group, sets of 10–15 female flies were
placed in a plastic vial with no food. A small circular filter paper
was placed in the bottom of the vial with 75 ul of water to prevent
dehydration and this was replenished with water every 16 hours or
as needed. Survival was recorded every 4–8 hours as the number
of flies alive in each vial.
Lifespan
Flies were placed on experimental diets and in oxygen
conditions as described for previous assays. The lifespan of the
flies was observed by recording the number of flies alive each day
in that oxygen condition. Flies were transferred to fresh food every
3–4 days. The experiment was concluded when there were no flies
remaining in a group or when one group was below 50% survival
and there was a clear difference between groups.
Statistical Analysis
Graphpad Prism was used for statistical analysis. A t-test was
used to determine significance between RF and CF results in the
metabolic and stress assays. To determine interaction between
diets and oxygen a 2-way ANOVA was performed. In lifespan and
starvation assays, the significance was determined by comparing
the survival curves with a log-rank (Mantel-Cox) test.
Results
Phenotypic Profile of Flies on High-fat Diet
To evaluate the effect of a diet high in fat on Drosophila,we
placed female w
1118
flies on either a regular diet or food
supplemented with coconut oil (rich in saturated fats) as 20% of
the diet (see methods). After a week on these diets in normoxia, the
flies on coconut oil supplemented food (CF) showed a large
increase in whole-body triglyceride levels (p,0.0001) compared to
those fed regular food (RF) (Figure 1A). To evaluate whether this
increase seen with the CF diet was specific to the strain of fly or
type of food, we also assayed the triglyceride levels of Canton-S and
Oregon-R flies on RF and CF diets, as well as tested the effect of
palm oil in the diet (PF) rather than coconut oil. Both fly strains
responded in the same way as w
1118
flies, with increased
triglyceride levels when on the CF diet, and a high-fat PF diet
resulted in similar increases as CF (Figures 1B,C). Additionally we
measured the whole-body free glucose levels in the w1118 flies, as
it is well known that obesity also affects glucose metabolism
[15,16]. Flies on the CF diet had significantly increased glucose
levels (p,0.001) compared to those on the RF diet (Figure 1D).
The elevated triglyceride levels seen in obesity are often
correlated with a decreased lifespan, both in humans [17] and
Drosophila [18,19]. We found that the high-fat diet had a
detrimental effect on lifespan, as the lifespan of w1118 flies on a
CF diet was significantly shortened compared to RF flies
(p,0.0001) when given full access to food (Figure 2). This
significant decrease was also seen in the Oregon-R flies on CF diets
(p,0.0001) and Canton-S flies on CF (p,0.001) or PF (p,0.0001)
diets when compared to RF flies (Figure 2). Not surprisingly, under
starvation conditions the w1118 CF flies were more resistant to the
stress of starvation than flies on RF diet and survived longer
(p,0.05) (Figure S1A).
Since lifespan can be an indication of the ability of an organism
to tolerate stress [20], we also tested stress resistance in these flies
to cold and to anoxia (Figure 3A). The flies on the CF diet showed
Figure 1. Changes in metabolic profile of flies on high-fat diet
compared with regular diet. A–C) Triglyceride levels (per mg live
weight) in whole-body homogenate of 10–12 day old adult female (A)
w
1118
, (B) Oregon-R and (C) Canton-S flies after one week on diets in
room air. Flies were on regular food (RF), high-fat coconut food
(supplemented with 20% coconut oil, CF) or high-fat palm food
(supplemented with 20% palm oil, PF). D) Glucose levels (per mg live
weight) in whole-body homogenate of 10–12 day old adult female
w
1118
flies, after one week on RF and CF diets in room air. Flies were
homogenized in groups of five females: (A) n = 31 groups (155 flies), (B)
n = 17 groups (85 flies), (C) RF n = 11 groups (55 flies), (D) n = 24 groups
(120 flies). Error bars show SEM; * = p,0.05, ***= p ,0.001,
**** = p,0.0001.
doi:10.1371/journal.pone.0042587.g001
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a marked decrease in tolerance to these two acute stresses. After
2 hours in anoxia (0% oxygen), nearly 90% of flies on the RF diet
were able to recover during the subsequent 24 hours, compared
with less than 60% of flies on the CF diet (Figure 3B). After
exposure to 2 hours in 25uC, flies on a CF diet survived
significantly less than their RF counterparts, with just a quarter of
the RF survival rate (Figure 3C).
Effects of Intermittent or Constant Hypoxia on Flies on a
High-fat Diet
Similar to the evaluation of flies on the RF and CF diets in
normoxia, flies were placed on the diets and immediately put in a
hypoxic chamber in either intermittent (IH) or constant hypoxia
(CH) for 7 days. IH and CH have been shown to have differential
effects [21–23] and alter expression of different genes [12], so it
was important to separately evaluate the response of the flies on
the diets in both hypoxia conditions.
The alterations in the metabolic profile seen in normoxia were
also present in both IH and CH, with increased TG levels in flies
on the coconut-supplemented food (p,0.001) (Figure 4A, C).
Although the difference in TG levels due to diet was persistent,
there was also a difference in TG levels between flies on the same
diet, depending on the oxygen condition. When triglyceride levels
were compared between hypoxic and normoxic conditions in flies
on the same diet, IH was associated with lower TG levels in the
flies on both the RF and the CF diet (Figure 4A). There was no
significant interaction between diet and oxygen level, so IH was
not causing the flies to respond differently to the diet in terms of
TG, but rather just causing an overall decrease regardless of diet.
On the other hand, CH provided a very different response. While
the TG levels in flies on the RF diet significantly decreased in CH
compared to normoxia, the TG levels in flies on the CF diet
increased (Figure 4C). The interaction between oxygen and diet
was significant (p,0.01), indicating that being in constant low
oxygen (i.e. in CH) affected flies on the CF diet differently than the
RF diet.
As in normoxia, glucose levels were significantly increased in
flies on the CF diet compared to the RF diet when exposed to IH
(Figure 4B). However, the flies in IH showed a much larger
increase than those in normoxia, indicating that there was a
significant interaction between diet and IH when the regulation of
glucose levels was considered. Flies exposed to CH showed no
significant difference in glucose levels between diets, although
there was a slight increase in glucose levels between normoxia and
CH in flies on the RF diet (Figure 4D). Similar to results in
normoxia, flies on the CF diet in IH were more resistant to
starvation than those on RF (p,0.0001) (Figure S1B) and had a
significantly shortened lifespan (p,0.0001) (Figure 5). A compa-
rable lifespan difference was seen in flies in constant hypoxia.
The response to stress also varied with the two hypoxic
treatments. Flies on a CF diet in IH and CH had decreased
survival after anoxic stress when compared to RF, similar to the
response seen in normoxia (Figure 6). In contrast, survival after
cold stress was very much affected by the hypoxia paradigm.
There was a marked increase in survival after cold stress even in
flies on the RF diet in IH compared to normoxia. Flies on the CF
diet in IH saw a remarkable change in response, with nearly full
survival in IH compared to just over 20% survival in normoxia
(Figure 7A, B). There was no difference in survival between diets
Figure 2. Altered lifespan due to high-fat diet. Adult female w
1118
,
Ore-R, and Canton-S flies (d3-5, n = 50 per diet) were placed on regular
(RF) and high-fat (CF) diets in room air. Additionally one group of
Canton-S flies was placed on a high-fat diet using palm oil (PF). Flies
were counted daily, with the number alive recorded. There was a
significant difference between the regular and high-fat diet curves in
each comparison; w
1118
RF vs. CF p,0.0001, Ore-R RF vs. CF p,0.0001,
Canton-S RF vs. CF p,0.001, RF vs. PF p,0.0001 (Log-rank test).
doi:10.1371/journal.pone.0042587.g002
Figure 3. Decreased tolerance to acute stress in flies on high-
fat diet. A) Diagram of experimental paradigm for stress assays. Adult
female w
1118
flies were placed on regular (RF) or high-fat (CF) diets in
room air for one week, after which they were stressed for 2 hours in B)
anoxia (0% oxygen) or C) extreme cold (25uC water bath). After
recovering for 24 hours on regular food in room air, survival was
counted as number of flies alive. Flies were tested in groups of 15
females per vial: (B) n = 18 groups (270 flies), (C) n = 16 groups (240
flies). Error bars show SEM; *** = p,0.001.
doi:10.1371/journal.pone.0042587.g003
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in the IH flies, but there was a highly significant interaction
(p,0.0001) in how the oxygen condition affected the flies on the
two diets. This difference is not seen in CH, as the flies responded
similarly as in normoxia, with a decreased survival in the CF flies
after cold stress (Figure 7C).
To further describe the effect of IH on cold stress recovery, it
was important to test an additional paradigm causing increased fat
storage. In order to do this, we utilized the well-characterized
genetic mutant adipose
60
(adp
60
). On a regular diet, these mutants
have significantly higher triglyceride levels than wildtype flies, and
even surpass the triglyceride level of wildtype flies on a high-fat
diet. These triglyceride levels drop slightly after IH, as they do in
the wildtype flies on both diets (Figure 8A). Importantly, IH almost
fully restores cold stress survival in the adp
60
mutant flies, as was
seen with wildtype flies on both diets (Figure 8B).
Given the consistent decrease in triglyceride levels in flies on
both diets in IH, we felt it was important to determine whether this
change was due to an altered level of food consumption. We
adapted the CAFE set-up described in Ja et al [14] for normoxic
and IH conditions, using flies previously on RF in normoxia or IH.
After an initial decrease in food consumption in IH flies on the first
Figure 4. Changes in metabolic profile due to high-fat diet and
hypoxia. A, C) Triglyceride levels (per mg live weight) and B, D)
glucose levels (per mg live weight) were assayed in whole-body
homogenate of 10–12 day old adult female w
1118
flies. Measurements
were taken after one week on diets in normoxia (solid bars, A–D),
intermittent hypoxia (IH, pattern bars, A and B) or constant hypoxia (CH,
pattern bars, C and D). During that week, flies were on diets of either
regular (RF) or high-fat diet (CF). Flies were homogenized in groups of
five females: normoxia triglyceride (TG) n = 31 (155 flies), glucose n = 24
groups (120 flies); IH TG n = 17 (85 flies), glucose n = 18 groups (90 flies);
CH TG n = 20 (100 flies), glucose n = 12 groups (60 flies). Error bars show
SEM; * = p,0.05, ** = p,0.01, *** = p,0.001.
doi:10.1371/journal.pone.0042587.g004
Figure 5. Altered lifespan due to high-fat diet and hypoxia.
Adult female w
1118
flies (d3-5) were placed on regular (RF) and high-fat
(CF) diets in normoxia (also shown in Figure 2) or intermittent hypoxia
(IH) (n = 50 flies per diet). Flies were counted daily, with the number
alive recorded. There was a significant difference between the regular
and high-fat diet curves in each comparison, as well as between
normoxia and IH when flies were on a regular diet; w
1118
RF-Normoxia
vs. CF-Normoxia p,0.0001, w
1118
RF-IH vs. CF-IH p,0.0001, w
1118
RF-N
vs. RF-IH p,0.05 (Log-rank test).
doi:10.1371/journal.pone.0042587.g005
Figure 6. Anoxia tolerance unaltered by hypoxia exposure.
Effect of prior exposure to intermittent hypoxia (IH) or constant hypoxia
(CH) on survival of flies after 2 hours in anoxia (0% oxygen) was
measured. A) Diagram of experimental paradigm for anoxia assay
following hypoxia exposure. Adult female w
1118
flies (10–12 days old)
were placed on regular (RF) or high-fat (CF) diets in normoxia (B, C solid
bars), IH (B, pattern bars), or CH (C, pattern bars) for one week prior to
anoxia assay. Flies were removed from hypoxia and immediately
assayed for tolerance to 2 hours of acute anoxia. After recovering for
24 hours on RF in room air, survival was measured as number of flies
alive. Flies recovered in groups of 15 flies per vial: normoxia n = 18
groups (270 flies); IH n = 13 groups (195 flies); CH n = 17 groups (255
flies). Error bars show SEM; *** = p,0.001.
doi:10.1371/journal.pone.0042587.g006
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day of CAFE measurement, there appears to be no significant
difference between the slopes of the cumulative consumption, as
determined by a Deming (Model II) Linear Regression (Figure 9A)
and no significant differences on the 2
nd
and 3
rd
day in the daily
food consumption between flies in normoxia as compared to IH
(Figure 9B).
Discussion
We have generated a model showing the detrimental effects of a
high-fat diet on lifespan and stress tolerance in Drosophila. Flies on a
diet supplemented with coconut oil (or palm oil) significantly
increased their triglyceride and glucose levels and shortened their
lifespan. This diet, high in saturated fat, not only hindered flies
from living as long as others on the regular diet, but it also greatly
diminished their tolerance to acute stresses. When challenged with
either anoxia or severe cold, few flies on the high-fat diet survived,
while most of those on the regular diet did. Conversely, under
starvation conditions the flies on the high-fat diet showed
increased resistance, due to their increased triglyceride and energy
stores.
The link between hypoxia and metabolism has been demon-
strated in models from flies and mice to humans [24–28].
Intermittent hypoxia in particular has been strongly implicated
as having a role in altering glucose and lipid metabolism. In rats,
IH has been shown to lead to oxidative stress, lipid peroxidation,
neuronal apoptosis and up-regulation of stress responsive proteins
[29,30]. Chronic IH has been shown to cause insulin resistance
and glucose intolerance in obese leptin-deficient mice [25,31] as
well as inducing hyperlipidemia in lean mice [25]. Although
studies in humans are more limited, experimental evidence
supports a detrimental effect of hypoxia on metabolism, with
diminished insulin sensitivity in subjects after just 30 minutes of
hypoxia exposure [24]. Our model presents an essential first step
in understanding the Drosophila response to a high-fat diet and
modulation of this response by IH.
Figure 7. Cold survival altered by intermittent hypoxia, but not
constant hypoxia. Effect of prior exposure to intermittent hypoxia (IH)
or constant hypoxia (CH) on survival of flies after 2 hours in extreme cold
(25uC water bath) was measured. A) Diagram of experimental paradigm
for cold stress assay following hypoxia exposure. Adult female w
1118
flies
(10–12 days old) were placed on regular (RF) or high-fat (CF) diets in
normoxia (B, C solid bars), IH (B, pattern bars), or CH (C, pattern bars) for
one week prior to cold stress assay. Flies were removed from hypoxia and
immediately assayed for tolerance to acute cold stress. After recovering
for 24 hours on RF in room air, survival was measured as number of flies
alive. Flies were tested in groups of 15 flies per vial: normoxia n = 17
groups (255 flies); IH n= 14 groups (210 flies); CH n =22 groups (330 flies).
Error bars show SEM; ** = p,0.01, *** = p,0.001.
doi:10.1371/journal.pone.0042587.g007
Figure 8. Changes due to intermittent hypoxia also seen in genetic mutant model of obesity. A genetic mutant model of obesity,
adipose
60
(adp
60
), was assayed in normoxia andintermittent hypoxia (IH) with the Oregon-R (Ore-R) strain as control. Adult female adp
60
flies (10–12 days
old) were placed on the regular (RF) diet and adult Ore-R females were placed on RF and high-fat (CF) diets. The flies were kept in normoxia (solid bars) or
IH (pattern bars) for one week. Following that week, the flies were assayed for A) triglyceride levels (per mg live weight) in whole-body homogenate and
B) survival of flies after 2 hours in extreme cold (25uC water bath), measured 24 hours after return to room air. For triglyceride determination, flies were
homogenized in groups of five females, normoxia: Ore-R n= 17 groups (85 flies), adp
60
n =20 groups (100 flies); IH: Ore-R n = 13 groups (65 flies), adp
60
n =15 groups (75 flies). In the cold assay, flies were tested in groups of 15 flies per vial: normoxia Ore-R n = 13 groups (65 flies), adp
60
n = 12 groups (60
flies); IH Ore-R n = 8 groups (40 flies), adp
60
n = 11 groups (55 flies). Error bars show SEM; * = p,0.05, ** = p,0.01, *** = p,0.001, **** = p,0.0001.
doi:10.1371/journal.pone.0042587.g008
Effects of High-Fat Diet on Drosophila
PLoS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e42587

When investigating the phenotype of flies on a high-fat diet,
both independently and in conjunction with hypoxia, we made
two important observations and pose here two interesting
questions that arise from these results. First, since the coconut
oil diet led to a decreased survival following cold stress, our
question is: how does this high-fat diet cause decreased survival in
this stressful condition? Second, and of interest, IH led to rescuing
flies from decreased survival after a severe cold stress; how does IH
allow flies to fully recover from this stress?
Normally, cells convert excess non-esterified free fatty acids into
triglycerides, which is why the coconut oil diet led to increased
levels of TG in our model. With this excess storage of triglycerides,
we showed that flies had decreased tolerance to stresses such as
anoxia and cold. Keeping in mind the diminished recovery from
cold stress in flies fed coconut oil, it is likely that the high-fat diet
alters energy and metabolic pathways in such a way that flies are
unable to activate some of the key mechanisms needed to recover
from the cold stress. This assumes that genes are activated not only
during the cold exposure itself but also during recovery from cold.
Indeed, Clark and Worland have demonstrated just that [32]. For
example, it is known that heat shock proteins, which are well
characterized in their response to stress, are important in recovery.
Newly discovered genes in this area, such as starvin and frost [33–
36], have also been implicated. While its exact function in
recovery from cold stress is not known, starvin is up-regulated in
the recovery phase following cold stress and is believed to be a co-
chaperone regulating the Hsp70 complex during recovery from
cold. A mucin-like protein, frost is thought to play a role in
protecting against oxidative stress and maintaining membrane
integrity, thus aiding in the ability to recover from cold stress. We
therefore hypothesize that flies on the CF diet were unable to
activate mechanisms such as these, jeopardizing their recovery and
survival from cold.
The inability of flies on the high-fat diet to recover from the
stress of cold was completely rescued when these flies had been in
intermittent hypoxia prior to the cold. Since this rescue of survival
did not occur in flies in CH, these data would indicate that IH
specifically, and not just hypoxia in general, is necessary for this
rescue phenotype. It appears that intermittent hypoxia alters
processes in such a way to override the negative effect of increased
triglycerides, whether from a high-fat diet or as a result of a genetic
mutation (adp
60
). One possible explanation could involve the
increase in glucose levels seen in flies on both RF and CF diets
following IH. Sugars such as glucose and trehalose are considered
cryoprotective molecules and have been implicated in mainte-
nance of cell function at low temperatures [37,38]. With this
accumulation of free glucose during IH, the fly may be better
prepared to survive the cold stress. Another potential mechanism
may be related to other alterations in gene expression. For
example, we have previously shown that IH induces expression of
genes important in transport and defense, including the high
affinity inorganic phosphate: sodium symporter, l(2)08717 [12].
Ion transport appears to play an important role in survival to
extreme cold, as lower temperatures can lead to decreased ion
pump activity, decreased membrane fluidity and inhibited ion
channel gating [32,39]. If exposure to IH up-regulated expression
of genes is important in ion transport, we speculate here that this
IH is tantamount to a pre-conditioning of the flies to stress,
allowing them to resist the effects of the high-fat diet in the survival
from cold stress.
In summary, we found that flies on a high-fat diet have a
drastically worsened phenotype, with decreased resilience to stress
and decreased survival compared to those on a regular diet, and
this phenotype is altered by exposure to hypoxia. With signifi-
cantly increased triglyceride and glucose levels in normoxic
conditions, flies on a high-fat diet have a shortened lifespan and
decreased tolerance to stress. While the triglyceride and glucose
levels are altered with hypoxia, the most interesting change was
seen when exposure to IH appeared to rescue survival after cold
stress. The detrimental effects of a high-fat diet are clear in this
Drosophila model, and there appears to be both deleterious and
compensatory cross talk occurring between hypoxia and the high-
fat diet. With the climbing global obesity levels and lack of
understanding of the interaction between obesity and hypoxia,
developing an animal model that lends itself to an investigation of
this interaction becomes vital.
Supporting Information
Figure S1 Altered starvation resistance due to high-fat
diet. Adult female w
1118
flies (3–5 days old) were placed on
Figure 9. Similar food consumption observed in normoxia and
intermittent hypoxia. Adult female w
1118
flies (3–5 days old) were
placed on regular (RF) for 5 days in normoxia or intermittent hypoxia
(IH). Following that time, flies were transferred to the CAFE vials and
returned to normoxia or IH. In the CAFE setup, flies had access to water
but had to obtain their food out of a capillary tube through the top of
the vial. Measurements of the change in liquid food level in the capillary
tube allowed for determination of the total food consumed A)
cumulatively over 3 days and B) on a daily basis. Flies were tested in
groups of 5, n = 14 groups (70 flies). Error bars show SEM; * = p,0.05.
doi:10.1371/journal.pone.0042587.g009
Effects of High-Fat Diet on Drosophila
PLoS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e42587

regular (RF) or high-fat (CF) diets in A) normoxia (N) or B)
intermittent hypoxia (IH) for one week (n = 110 flies per group).
Following that week, flies were transferred to plastic vials without
food, but with access to water. Flies were kept in room air and
counted every 4–6 hours, with the number alive recorded. There
was a significant difference between the survival curves; A)
p = 0.02, B) p,0.0001 (Log-rank test).
(TIFF)
Acknowledgments
We wish to thank Drs. Sean Oldham and Ryan Birse for advice and
discussion regarding diet composition. The adp
60
mutant fly was a kind gift
from Dr. Tania Reis. We are grateful to Dr. Priti Azad, Dr. Dan Zhou,
Ying Lu-Bo, Mary Hsiao and other members of the Haddad laboratory for
guidance in experimental protocols.
Author Contributions
Conceived and designed the experiments: EH GH. Performed the
experiments: EH. Analyzed the data: EH. Contributed reagents/
materials/analysis tools: EH GH. Wrote the paper: EH GH.
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Effects of High-Fat Diet on Drosophila
PLoS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e42587