Paper - analysis of primary literature

A Critical Analysis of “Physical activity: benefit or weakness in metabolic adaptations in a
mouse model of chronic food restriction?”
Laura Patriarca
BIO 312
April 22, 2015

2A Critical Analysis
INTRODUCTION
In their study entitled, “Physical activity: benefit or weakness in metabolic adaptations in
a mouse model of chronic food restriction?” Mequinon and colleagues (Mequinon et al. 2015)
used a mouse model to address the impact of physical activity on various physiological measures
in cases of restrictive-type anorexia nervosa (AN). AN is one of several DSM-5 defined eating
disorders and shows the highest rate of mortality of all psychiatric disorders (Kinzig et al. 2007).
From a psychological standpoint, AN is characterized by intense fear of weight gain and
excessive desire for thinness, aberrant feeding behavior or restricted intake, and distorted body
image (Marzola et al. 2013). Physiologically, AN can be described as a disruption of hunger and
satiety cues associated with dysregulation of orexigenic and anorexigenic signaling. Both
metabolic and endocrine dysfunctions are implicated in the disorder. Phobia-like physiological
reactions toward food are also present in AN, namely reduced salivation and heightened
autonomic activation in response to food (Kinzig et al. 2007).
Important to the study by Mequinon and colleagues is the symptom of hyperactivity,
observed in thirty to eighty percent of individuals diagnosed with AN (Kostrzewa et al. 2013).
Hyperactivity, i.e. excessive physical activity, while highly prevalent among AN sufferers, is
poorly defined. Varying definitions include different combinations of criteria including degree of
restlessness, hours per week of exercise, intensity of exercise, body shape-control as motivation
for exercise, extreme distress when unable to exercise, etc. (Keyes et al. 2015; Kostrzewa et al.
2013; Zunker et al. 2011). Regardless of definition, excessive physical activity is associated with
increased treatment dropout, poorer treatment outcomes, higher relapse rates, and lower BMIs
upon recovery from AN (Keyes et al. 2015; Ghoch et al. 2013). While regular, moderate physical
activity is understood to have many positive implications for health – including lower blood

pressure, decreased risk for diabetes, cardiovascular disease and osteoporosis, and improvements
in mood disorders like depression and anxiety – the excessive physical activity observed in AN
can have deleterious consequences (Keyes et al. 2015, Mequinion et al. 2015). Sufferers of AN
often remain committed to an excessive exercise regimen despite illness or injury (Kostrzewa et
al. 2013). This excessive exercise is dangerous, because as a consequence of nutritional deficits,
AN individuals are already at risk for a host of medical issues including osteoporosis, bone
fractures, electrolyte imbalances, and even sudden death. Engaging in extreme amounts or
intensities of physical activity increases these risks (Zunker et al. 2011).
In treating AN, the primary goal is to stabilize the patient’s crucial medical condition by
refeeding; weight restoration and nutritional rehabilitation are key (Marzola et al. 2015), along
with the reestablishment of physical strength and healthy bone density (Zunker et al. 2011). The
impact of incorporating health-promoting exercise into AN treatment plans is not well-studied.
Despite the established federal guidelines for physical activity to promote health within the
general population, no such guidelines exist for exercise intervention programs for AN. The few
existing studies on the topic have shown varied and discrepant results regarding the duration,
intensity, and type of exercise that should be employed in AN treatment to promote health while
avoiding detrimental effects from over-exercise (Zunker et al. 2011). Mequinon and colleagues
acknowledged the present lack of understanding of how or whether to incorporate exercise into
AN interventions; they sought to gain insight into the most beneficial protocol to be adopted by
clinicians when treating AN sufferers. Thus, in their study, they addressed the impact of physical
activity on body weight, metabolism, body composition, and the estrous cycle in a chronic food
restricted mouse model of AN compared to control groups (Mequinon et al. 2015).

Mequinon and colleagues did not articulate a clear hypothesis regarding the impact of
physical activity in a restrictive-type AN mouse model. Rather, they posed the question of
whether such activity would be beneficial or detrimental to a wide variety of metabolic and
endocrine parameters relevant to the phenotypes observed in humans with AN (Mequinon et al.
2015). By opting not to formulate a hypothesis, the researchers eliminated potential biases; they
did not expect particular findings and thus avoided a temptation to evaluate their data in such a
way as to favor support of a proposed hypothesis. The lack of a hypothesis does call into
question, though, the observations upon which the researchers based their question. Hypotheses
ordinarily reflect observations that engender particular expectations in the observers. However,
in this instance, Mequinon and colleagues noted both a paucity of previous research, as well as
conflicting and varied findings in the minimal existing literature, on the topic of the benefits or
weaknesses of physical activity in AN. This legitimized the decision to forgo hypothesizing
particular outcomes of the study.
METHOD
Mice served as the study animal in the experiments performed by Mequinon and
colleagues. While AN is a disorder specific to humans, humans are an incredibly difficult
population on which to perform controlled experimentation because of enormous variations
background, environment, lifestyle, etc., and because of general ethical limitations in human-
subject research. Animal models have proven to be powerful in the study of a variety of human
psychological and neurobiological disorders (Kim 2012). In complex disorders like AN, it is
impossible to mimic all aspects of the disorder in animal models, but various models have been
created that are effective at causing a few select symptoms of such disorders, contributing to the
understanding of underlying physiological mechanisms that might be at work to produce the

phenotypes observed in humans. These models have been developed in rats and mice (Kim
2012). Rodents are easy to breed, house, maintain, and control or manipulate, making them
excellent research subjects.
Multiple rodent models of AN have been previously characterized, a common one of
which is the activity-based anorexia (ABA) model. The ABA model was developed by housing
male rats in activity wheels and subjecting them to a restricted feeding schedule, inducing in the
rats a period of heightened activity before feeding time and an unexpected reduction in food
intake despite excessive activity (Routtenberg and Kuznesof 1967). While the ABA model can
cause reproduction of many AN symptoms, it has significant drawbacks, including alterations to
the observed anorectic phenotype resultant from a change in the breed, sex, or age of the rodent
subject to the paradigm (Gelegen et al. 2007 in Mequinon et al. 2015; Klenotich and Dulawa
2012 in Mequinon et al. 2015) and an unnaturally ephemeral lifespan of the rodents due to
extreme weight reduction and energy depletion.
In the present study, Mequinon and colleagues developed a novel mouse model of AN to
avoid the drawbacks of the ABA model. They utilized female instead of male mice to better
reflect the fact that human females are more commonly afflicted with AN than human males.
Additionally, instead of limiting the feeding time, the researchers limited the feeding amounts,
implementing 30 percent food restriction for three days followed by a 50 percent food restriction
for the remainder of the protocol. Importantly, no mice died in these imposed conditions
(Mequinon et al. 2015). It is difficult to assess whether the cognitive/psychological symptoms of
AN can be mimicked in any non-human animal model, but the researchers addressed this
concern, noting that elements like self-starvation and negative body image are not necessary in
studying the physiological consequences of food restriction paired with physical activity

(Mequinon et al 2015). Thus, the aim of the study was narrow enough to justify the validity of
the findings despite an incomplete representation of the total AN pathology.
The researchers took precaution against various confounds that could arise as a result of
study design. They allowed one week of acclimation time during which the mice were handled
daily and had free access to water and a standard diet. Additionally, they housed the mice in
pairs to prevent them from developing isolation stress. After the acclimation week, the
researchers divided the mice into four groups. Mice in the experimental group, designated FRW
(food restriction and wheel), were provided with a free running wheel and were subjected to the
aforementioned quantitative food restriction. The researchers wisely designed three control
groups – FR (food restriction only), ALW (ad libitum feeding and wheel), and AL (ad libitum
feeding, no wheel) – to be able to separate the variables of activity and food in the experiment.
Ensuring environmental consistency, the researchers used mouse cages that were all pathogen-
free and had a dark-light cycle of 12:12 hours (Mequinon et al. 2015).
Mequinon and colleagues measured in their mice a myriad of variables related to the
known physiological consequences of excessive physical activity in humans with AN. To
address both short- and long-term effects of activity and food restriction, the researchers
designed two protocols: a fifteen-day short-term protocol and a fifty-five-day long-term protocol
(Mequinon et al. 2015). Because some of the measurements required sacrificing the mice, the
researchers did not have the option to follow a single protocol in which they took both short- and
long-term measurements.
In both protocols, the researchers measured the body weight, cumulative food intake, and
cumulative water intake of all mice on a daily basis. Because the mice were housed in pairs, the

researchers averaged the food and water intake measurements per cage between the two mice to
determine individual intakes; they excluded any data obtained from cages in which the mice
showed evident body weight differences indicating highly unequal food intake. At the beginning
of the short-term protocol, and around day 15 in both protocols, and at around day 45 in the long-
term protocol, the researchers isolated some subgroups of mice in metabolic cages (equipped
with a wheel for FRW and ALW groups) for a period of three days. The use of metabolic cages
allowed the researchers to measure food intake patterns, consumption of O2 and production of
CO2, and locomotor activity of individual mice in all four groups. From the VO2 and VCO2, the
researchers calculated the respiratory exchange ratio (RER), energy expenditure (EE), and fat
oxidation (FAO). Food intake data from the individual mice in metabolic cages served to
validate food intake data obtained from paired mice in home cages (Mequinon et al. 2015).
On D1, D15, and D55 of the long-term protocol, the researchers conducted CT scans on
mice from all groups to gather body composition data, i.e. bone, lean, and fat masses, visceral fat
mass, subcutaneous fat mass, and bone mineral composition. On D15 and D50 of the long-term
protocol, the researchers measured the glycemia of the mice in the morning. They also
administered an intraperitoneal glucose tolerance test (IPGTT) to mice from all groups. They
employed a control to keep all mice at comparable conditions of satiety by giving the same
quantity of food to all mice to be tested on the day before (i.e. D14 and D49) these tests. The
researchers carried out this same control on the day prior to euthanasia and tissue collection,
which they performed on all mice at the end of both protocols. They collected blood samples by
cardiac puncture. From these samples, they measured levels of plasma hormones: leptin,
corticosterone, ghrelin (total), acyl ghrelin (AG), and des-acyl ghrelin (DAG), as well as levels
of plasma metabolites: nonesterified fatty acids (NEFA), triglycerides, and beta-hydroxybutyrate

(ketone bodies). The researchers took the masses of the liver, gastrocnemius muscle, and uterus,
measured ovary width and length, and homogenized samples of the collected liver tissue to
assess hepatic glycogen.
To assess estrous cycle stages, the researchers performed vaginal smears on the mice
between the fifth day preceding the start of protocol and day 16 of the protocol and between days
48 and 55 of the protocol (Mequinon et al. 2015). The research article does not give a clear
indication of how frequently the researchers performed the vaginal smears, nor does it indicate
whether the smears were performed on mice in both protocols or only in mice in the long-term
protocol (the latter seems probable). This was a fault in the reporting of procedural methods.
A potential weakness of this study’s design was small sample size, which varied from six
to twenty-four mice per group depending on the experiment. The researchers clarified in their
paper that the variation in group size was a result only of specific experiments within the
procedures that could have affected, if only slightly and temporarily, the feeding or physical
activity of the mice. Such procedures included the collection of blood samples and of metabolic
data. The seemingly questionable variation in group size was thus actually a consequence of
careful control in the experimental design. Additionally, for many measures, the researchers
were able to report statistically significant results (p < 0.05) despite limited sample sizes, which
suggests quite powerful findings (Mequinon et al. 2015).

RESULTS
(all from Mequinon et al. 2015 unless otherwise listed)
Body Weight
Over the entirety of both the short- and long-term protocols, the researchers saw that
FR/FRW mice had lower average body weight than AL/ALW mice. The researchers found an
interaction of food and activity (p > 0.001), at D15 where, when coupled with food restriction,
wheel running activity caused a negative weight gain (i.e. weight loss in FRW, less weight gain
in ALW) far greater in magnitude than that of activity coupled with ad libitum feeding. From
D42 to D45, the researchers found effects of both food (p > 0.001) and activity (p > 0.05) on
body weight but no interaction between the two variables, such that food restriction and wheel
activity, considered independently, caused weight loss, but activity did not cause a greater weight
loss in either food restriction or ad libitum feeding. A precipitous decline in body weight of FRW
mice – greater than that of FR mice (p > 0.001) – occurred from D6 to D22. Interestingly, from
D43 to D55, however, FRW mice showed weight regain (p > 0.001) which brought them to an
average weight greater than that of FR mice (p > 0.001) at D55.
Food Intake
The metabolic cage data on food intake during the D15 dark period showed a slower rate
of food intake in FRW mice than FR mice during the first two hours following meal distribution
(p > 0.05). At D45, however, these groups showed the same feeding pattern. On both D15 and
D45, ALW mice exhibited similar patterns of food intake to AL mice: slow and continuing
throughout the entire nighttime period. Although ad libitum mice reached higher cumulative
food intake by the end of the night (presumably due to the unrestricted nature of their meals),

food-restricted mice finished their allotted meals in a shorter time than ad libitum-fed mice
consumed this same amount of food.
Locomotor Activity
FRW and ALW mice showed similar home cage activity from D0 to D35, but FRW mice
showed lower home cage activity than ALW mice from D35 to D50 (p > 0.05). Average
locomotor activity measured in metabolic cages over a 24-hour period corroborated the home
cage findings. In addition to obtaining mean locomotor activity, the researchers analyzed this
metabolic cage data in two phases – night and day. FRW mice were the most active of all groups
in the daytime on D15, especially in the several hours before food distribution (p > 0.01). During
the night on both D15 and D45, ALW mice were constantly active and were more active than AL
and FRW mice (p > 0.05). Surprisingly, the aforementioned daytime activity observed in FRW
mice on D15 disappeared on D45 (p > 0.001); this caused a decrease in the average daily activity
of the FRW mice on D45 as compared to D15.
Body Composition
At D15, both lean and fat mass were lower in food-restricted mice than ad libitum-fed
mice (p > 0.001 and p > 0.05, respectively), and fat mass was lower in wheel-running mice than
in non-wheel-running mice (p > 0.001). Food restriction and wheel-running activity resulted in
decreased visceral fat mass (p > 0.05 and p > 0.001, respectively), while only activity affected
subcutaneous fat mass (p > 0.001). The researchers found an interaction of food and activity on
fat mass at D55 (p > 0.005) such that the activity-induced decrease in fat mass was of lesser
magnitude in food-restricted mice than in ad libitum-fed mice. Additionally, they found an
interaction of food and activity (like that on fat mass) on both visceral and subcutaneous fat

masses (p > 0.05 and p > 0.05) at D55. When compared to ad libitum feeding alone (AL), food
restriction alone (FR) resulted in a greater decrease in fat tissue than did food restriction
combined with activity (FRW) or activity alone (ALW).
No significant differences in bone mineral content (BMC) were present at any stage of
the protocol. However, the researchers observed a trajectory of increasing BMC between AL and
ALW mice; no such trajectory existed between FR and FRW mice.
Energy Metabolism
Energy Expenditure (EE), Respiratory Exchange Ratio (RER), and Fat Oxidation (FAO):
Metabolic cage data from D15 showed main effects of both food restriction and activity
on EE (in kcal/hr) as well as an interaction of food and activity (p > 0.001) during nighttime
hours. That is, FR and FRW mice exhibited lower EE than their respective control groups, and
the difference in EE was greater between FR and FRW mice than between AL and ALW mice.
Data from the daytime hours showed similar results but lacked a main effect of activity on EE.
Interestingly, over a period of 24 hours at D15, the EE of ALW mice mirrored their pattern of
locomotor activity, while the EE pattern of FRW mice was similar to that of FR mice even at
times when FRW mice were more active than FR mice. Data from D45 again showed an
interaction of food and activity on EE in the day (p > 0.001) and at night (p > 0.01). However,
the relationship between the EE of FRW and FR mice changed; FRW mice showed greater EE
than FR mice (p > 0.05) but still lower EE than ALW mice (p > 0.05).
The researchers calculated RER as the ratio of CO2 production to O2 consumption, levels
of which they obtained from metabolic cage data taken every fifteen minutes. They found that

neither restriction nor activity affected average RER in the D15 nighttime period. However,
when hourly (not average) RER was investigated, a clear effect of food became apparent (p >
0.05), where between hours 2100 and 0100, food-restricted mice showed elevated RER levels –
values reaching close to 1.0 – compared to ad libitum-fed mice. Between D15 daytime hours of
0700 and 1900, FRW mice showed similar RER levels to AL and ALW mice, while FR mice
interestingly exhibited a decrease in RER values to nearly 0.7. The RER data from the daytime
period also showed higher average RER for wheel-running mice than non-wheel-running mice (p
> 0.001). The only notable change observed in the patterns of RER on D45 as compared to D15
was a food effect (p > 0.001) during the daytime hours such that average RER values were lower
in food-restricted groups than ad libitum groups.
The examination of FAO data showed an interaction of food and activity (p > 0.001) in
the D15 nighttime period – the observed decrease in FAO values from ALW to FRW (p > 0.001)
mice was much larger than from AL to FR mice (p = 0.07). During the daytime, food-restricted
mice had lower FAO than ALW mice (p > 0.001), but no interaction of food and activity like that
seen in the nighttime was present. Nighttime data from D45 showed patterns of FAO similar to
those from the D15 nighttime period. Data from the D45 daytime showed a tendency toward an
interaction of food and activity (p = 0.06), where FRW mice showed the lowest FAO levels of all
groups (i.e. activity resulted in a tendency toward a greater FAO difference between FR and
FRW mice than between AL and ALW mice).
Metabolic Hormone Plasma Levels:
An interaction of food and activity was present on both D15 (p > 0.05) and D55 (p >
0.001) such that there was a greater decrease in plasma leptin levels between the AL and ALW

mice than between the FR and FRW mice. ALW and FR mice had lower leptin levels than AL
mice on both D15 and D55 (p > 0.05). Food restriction increased plasma corticosterone levels
(FR/FRW greater than their respective controls) on D15 (p > 0.001), but on D55, AL and FRW
mice showed lower corticosterone levels than FR mice (p > 0.01). Total ghrelin levels were
elevated in food restricted mice as compared to ad libitum mice on both D15 (p > 0.001) and
D55 (p > 0.05). The ratio of AG/DAG was higher in wheel running mice than non-wheel running
mice on both D15 (p > 0.01) and D55 (p > 0.001) and was also higher in ad libitium-fed mice
than in food-restricted mice on D55 (p > 0.05).
Reproductive Function
Food restriction, independent of activity, precipitated disruption of the estrous cycle as
assessed by vaginal smears. It also resulted in decreased uterus mass (p > 0.05) and ovary length
and width (p > 0.05) on both D15 and D55.
DISCUSSION
Conclusions
From the slew of results Mequinon and colleagues obtained from their various
experiments, they concluded that moderate physical activity could be beneficial in chronic food
restriction scenarios, at least in the long-term, by inducing stabilizing metabolic and endocrine
adaptations. They also reported, however, that physical activity did not protect against the loss of
lean and bone mass caused by food restriction. Reproductive dysfunctions were also not rescued
by physical activity in the food restricted mice.

The weight gain and stabilization exhibited by FRW mice at the end of the long-term
protocol led the Mequinon and colleagues to suggest a long-term protective effect of exercise.
While this conclusion seems logical, it is important to note that early in the protocol, FRW mice
showed a precipitous drop in body weight compared to FR mice, which could result in
detrimental effects that outweigh potential exercise-induced long-term benefits. The researchers
claimed that body weight did not stabilize in FR mice, but their graph of body weight evolution
showed a clear stabilization, just at a lower stable weight than FRW mice. Furthermore, the
researchers stated that the obtained body composition data “suggest that physical activity
accelerates the effects of food restriction on fat tissue without protecting muscle and bone mass,”
which does not at all suggest a protective effect of exercise.
Mequinon and colleagues concluded that FRW mice better adapted to chronic food
restriction than did FR mice not only because FRW mice showed increased body weight at the
end of the long-term protocol, but also because “FRW mice had lower corticosterone and acyl
ghrelin levels compared with D15, whereas they remained constant in the FR group” (Mequinon
et al. 2015). The researchers weakened their conclusion by including acyl ghrelin levels as
corroborating evidence; although AG levels did decrease from D15 to D55 in the FRW group,
the D45 AG value for FR mice was not significantly lower than that of FRW mice. Thus, the
decrease itself represents only that in a food restricted scenario, activity caused such grossly
elevated AG levels in the short-term that a larger decrease was necessary for FRW AG levels to
reach a levels similar to that of FR mice by D55. It does not show that activity allowed FRW
mice to reach healthier AG levels than FR mice by the end of protocol. Additionally, the
researchers noted that the functions of AG and DAG are not yet clearly understood and that the
ghrelin ratio is therefore a better indication of potential ghrelin activity. The ghrelin ratios

observed in the mice did not suggest a greater adaptation to chronic food restriction of FRW
mice than FR mice.
Another metabolic hormone that Mequinon and colleagues discussed at length is leptin.
Human AN patients exhibit decreased plasma leptin, which is associated with their reduced fat
mass (Misra et al. 2004 in Mequinon et al. 2015). The reduced leptin levels observed in ALW,
FR, and FRW mice compared to AL mice showed an association of fat mass reduction and
decreased plasma leptin like that in human AN patients. However, this does not suggest that
decreased leptin levels put an animal in danger, as ALW mice showed significantly lower leptin
than AL mice throughout protocol (p > 0.05) but were in acceptable health. FRW mice did not
show leptin levels different from FR mice or ALW mice, so leptin should not have been included
in the set of metabolic hormones that Mequinon and colleagues claimed were normalized in
FRW mice compared to FR mice. This unsubstantiated claim of metabolic hormone
normalization fails to support the conclusion that “voluntary and moderate physical activity at
the beginning of a protocol of food restriction might be beneficial in the long term since it is
associated with a normalization of metabolic hormones that may favor a better adaptation to
these drastic conditions” (Mequinon et al. 2015). In fact, corticosterone was the only metabolic
hormone whose levels suggested a better adaptation of FRW mice compared to FR mice.
The fact that the weight gain in FRW mice occurred in tandem with a decrease in their
activity levels seems not to indicate an adaptation to chronic food restriction, but rather to a
natural effect of decreased calorie burn. Interestingly, unlike ALW mice, whose EE predictably
reflected their activity levels, FRW mice displayed a consistent EE despite a decrease in
locomotor activity during the light period from D15 to D45, eliminating the possibility that their
weight gain could be attributed to a decreased EE. Mequinon and colleagues thereby concluded

that there was “an uncoupling between physical activity and global EE for FRW mice,
suggesting different specific metabolic adaptations related to activity” (Mequinon et al. 2015).
While the results generally support this conclusion, of note is the authors’ gratuitous use of the
word “global.” The researchers stated that FRW decrease in activity was the result of the loss
(from D15 to D45) of an FRW condition-specific activity, called food anticipatory activity
(FAA). This FAA occurred only in FRW mice and only during the daytime/light period, and the
unchanging RER values referenced by the researchers again applied only to the light period.
Additionally the average EEs of both FRW and FR mice were lower than those of the ad libitum-
fed mice throughout the protocol, even though the average locomotor activity of FR mice did not
change from D15 to D4, indicating metabolic adaptations for energy reservation in both food-
restricted groups. So, it might be more accurate to say that the uncoupling between EE and
activity in FRW mice was not global, but rather limited to their activity in the light period – that
is, to FAA in the light period.
Mequinon and colleagues rightfully suggested from the obtained RER, FAO, and IPGTT
data that FR and FRW mice adapted differently to their drastic conditions over the course of the
protocol. The energy derived from FAO was elevated by food restriction for FR mice, while in
carbohydrate metabolism was elevated for FRW mice; these differences were limited to the
short-term and the daytime. In direct contrast to these findings, however, the researchers also
concluded that physical activity allowed for a balance of carbohydrate and lipid use in food-
restricted mice only in the short term and did not have an effect on either carbohydrate or lipid
use in the long-term, and that FRW mice “did not adapt properly to their metabolism” in the
short-term (Mequinon et al. 2015). The conflicting ideas proposed here certainly weaken the

general conclusion the researchers reiterated throughout their discussion: that physical activity
has a positive effect on energy metabolism regulation in food-restricted mice.
In the broadest of their conclusions, the researchers acknowledged that it is unclear
whether the physical activity-related metabolic and endocrine physical adaptations observed in
their AN mouse model could lead to a better or worse outcome in humans recovering from AN.
They stated in their introduction that answering the question of whether the physical activity in
AN is harmful or serves as a protective mechanism would allow clinicians to decide whether to
incorporate physical activity into AN treatment programs (Mequinon et al. 2015). This
assumption is simply overreaching. It does not consider that hyperactivity – not moderate
physical activity like that in their mouse model – is observed in AN patients, nor does it consider
the implications on metabolism of the refeeding protocols in AN treatments. Additionally, FRW
mice showed increased weight and decreased activity at the end of the protocol, which is not
representative of humans with AN, whose cognitive drive to lose weight/avoid weight gain
promotes increasing food restriction and continued excessive physical activity even in the face of
weakness or injury. These points considered, it was wise of Mequinon and colleagues not to
apply the conclusions they drew from their findings in FR and FRW mice to humans with AN.
Merits and Flaws
Mequinon and colleagues were careful to implement adequate controls in their
experiments. The use of four groups of mice – AL, ALW, FR, and FRW – allowed for complete
isolation of the different food and activity conditions and thus for comparison of different
combinations of these conditions. An acclimation week for the mice was an extremely important
control measure to take, as the stress otherwise induced by testing could skew the results. As

another control of stress, the researchers housed the mice in pairs. To avoid intragroup variations
that could arise if the mice were in differently fasted conditions, the researchers fed all mice the
same amount of food the day before the IPGTT and glycemia measures as well as the day before
euthanization and blood sampling. Importantly, the researchers maintained the mice on their
respective protocol-determined feeding programs before and during testing in the metabolic
cages, as the aim was to investigate the effects of such feeding protocols on different metabolic
parameters. The researchers also gave greater validity to their findings by performing duplicate
analysis of all blood assays.
There is little to criticize in the study design and controls employed by Mequinon and
colleagues. Perhaps having consistency in the diameters of the running wheels in the home and
metabolic cages would have been a simple way to incorporate more control into the
experimentation. However, it is difficult to say how this would have added to the strength of the
findings. A last opportunity to control the experimentation would be for the researchers to
distribute food to all mice at the same times; it is not clear in the paper whether ad libitum-fed
mice were given food multiple times per day or, if like the food-restricted groups, were fed once
per day. If, because of meal distribution, the ad libitum-fed mice were subjected to more human
disruption than the food-restricted mice, this could have confounded the results.
While the researchers can hardly be censured for their choice of methods, their reporting of
their protocols and results clearly lacked careful review. Throughout the article, there were
conflicting timelines presented in the figures versus the text that made it difficult to determine at
what time point the researchers actually conducted some of their experiments. For example, the
text indicated that the IPGTT was performed on D15, while Figure 1 indicated that it was
performed on D17. Food and water intake graphs were labeled as D45, but the figure legend and

text reported D55. These criticisms extend not only to the article authors, but also to the
reviewers.
Contributions
The work of Mequinon and colleagues in this study contributed to the body of
physiological knowledge as a whole. In addressing the complexities of AN pathology, it drew
upon a broad range of physiological fields, including endocrinology, metabolism, respiratory
physiology, exercise physiology, and reproductive physiology. The researchers developed a new
AN model to be further investigated and compared to past models, promoting the evolution of
AN research. They proposed numerous explanations for their more unexpected findings,
incorporating and building upon knowledge from across the realm of physiology, and they even
noted areas of their research they believe should be further examined. In doing this, Mequinon
and colleagues opened their experimentation and reflective hypotheses to the entire physiological
scientific community.
Despite the aforementioned concerns regarding Mequinon and colleagues’ conclusions
and reporting, their work was truly original. Perhaps most significantly, the researchers made a
contribution to eating disorder physiology by their development of a novel mouse model of AN
that circumvented some disadvantages of past models. Their new model used female instead of
male mice – a more representative sample, as the majority of humans diagnosed with AN are
female (Mequinon et al. 2015). The self-starvation exhibited by mice in the ABA model can be
extinguished by humidifying the food pellets or raising the ambient temperature, suggesting that
this self-starvation is less related to a cognitive drive not to eat, but rather to maintain fluid
homeostasis and thermogenesis (Boakes 2007 in Mequinon et al. 2015; Boakes and Juraskova

2001 in Mequinon et al 2015). So, Mequinon and colleagues took a different approach to food
restriction; they restricted food by decreasing the meal volume, not by restricting the time during
which the mice were permitted to feed. Additionally, this novel AN model allowed the
researchers to study chronic food deprivation in the long-term, which was not possible in the
ABA model because the mice would die rapidly of undernutrition.
Even if it did not answer the question of whether physical activity would improve
treatment outcomes for AN patients, the researchers’ thorough investigation of varying
parameters gave insight into the mechanisms of AN pathology. They found that disruption of the
estrous cycle and reduction in ovary size occurred only with the combination of physical activity
and food restriction, and they detailed the associated endocrine changes that might contribute to
such reproductive dysfunction. Furthermore, they hypothesized the involvement of elevated
leptin and decreased corticosterone levels in the disappearance of FAA in FRW mice toward the
end of the long-term protocol. This builds on what is known about human AN, in which patients
show decreased leptin and increased corticosterone, like the FRW mice at the beginning of the
protocol. The researchers drew another association to the human AN pathology by hypothesizing
that the development of FAA in their FRW mice could be attributed to exercise addiction
promoted by wheel running. Because dopamine reward drives FAA in mice (Greenwood et al.
2011 in Mequinon et al. 2015; Verhagen et al. 2011), this hypothesis supports the idea that the
deleterious cycle of food restriction and activity seen in human AN patients might be attributed
to the known dysfunction of their neural reward systems.
It would be interesting to further connect the novel AN mouse model presented by
Mequinon and colleagues to the human experience of AN by mimicking not only the pathology
of the disorder, but also a treatment phase. Such experimentation would involve extending the

protocols to include a refeeding, or “treatment” program for a subset of the FRW mice. Within
this subset of FRW mice, two groups would be created: one group with access to a running
wheel, and one group without access. Both groups would be fed identically, with the intention of
promoting weight regain. As long as the physical activity of “treated” FRW mice remained at a
moderate level, the same set of physiological parameters addressed in the study by Mequinon
and colleagues could be assessed in these new groups. This would serve as an investigation of
whether physical activity serves as a benefit or a detriment to health during a refeeding program.
In extending the research in the proposed manner, the scientific community would become a step
closer to informing clinicians how to decide whether to restrict or promote moderate physical
activity in their recovering AN patients.

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