A Critical Analysis14

A Critical Analysis of Physical activity: benefit or weakness in metabolic adaptations in a mouse model of chronic food restriction?

Laura Patriarca

BIO 312April 22, 2015INTRODUCTIONIn 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 patients 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.METHODMice 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 studys 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 WeightOver 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 IntakeThe 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 ActivityFRW 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 CompositionAt 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 MetabolismEnergy 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 FunctionFood 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.DISCUSSIONConclusionsFrom 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 FlawsMequinon 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.ContributionsThe 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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