Translating Animal Model Research: Does It Matter That Our Rodents Are Cold?
Shane K. Maloney, Andrea Fuller, Duncan Mitchell, Christopher Gordon and J. Michael Overton Physiology 29:413-420, 2014. doi:10.1152/physiol.00029.2014 You might find this additional info useful... This article cites 60 articles, 19 of which can be accessed free at: /content/29/6/413.full.html#ref-list-1 This article has been cited by 2 other HighWire hosted articles Rethinking Animal Models and Human Obesity Michael J. Joyner Physiology, November , 2014; 29 (6): 384-385. [Full Text] [PDF] Living Under Extreme Conditions Physiology, November , 2014; 29 (6): 386-387. [Full Text] [PDF]
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PHYSIOLOGY 29: 413– 420, 2014; doi:10.1152/physiol.00029.2014
Translating Animal Model Research: Does It Matter That Our Rodents Are Cold? Does it matter that rodents used as preclinical models of human biology are routinely housed below their thermoneutral zone? We compile evidence showing that such rodents are cold-stressed, hypermetabolic, hypertensive, sleep-deprived, obesity-resistant, fever-resistant, aging-resistant, and tumor-
Shane K. Maloney,1,2 Andrea Fuller,2 Duncan Mitchell,1,2 Christopher Gordon,3 and J. Michael Overton4 1 School of Anatomy Physiology and Human Biology, The University of Western Australia, Stirling Highway, Crawley, Australia; 2Brain Function Research Group, School of Physiology, Faculty of Health Sciences, University of the Witwatersrand, Parktown, South Africa; 3Toxicity Assessment Division, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina; and 4Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, Florida
prone compared with mice housed at thermoneutrality. The same genotype of mouse has a very different phenotype and response to physiological or pharmacological intervention when raised below or at thermoneutrality.
1548-9213/14 ©2014 Int. Union Physiol. Sci./Am. Physiol. Soc.
work on rodents because they are easy to study and have a short generation time, and we hope that such studies will reveal useful information on human physiology. Since 2003, the number of published studies on mice exceeded those on rats, driven primarily by the development of molecular tools to manipulate genes in mice. The international knockout mouse consortium has the laudable aim of knocking out individually each of the 20,000 or so protein-coding genes in the mouse genome (6). Studying the phenotypes of all of those new genotypes is expected to yield a wealth of information. Some of what we learn from mouse knockout studies will apply to humans, but mice are not humans. Environments that are unstressful to humans may well stress mice and alter their physiology and phenotype. In this review, we will discuss the way that ambient temperature (Ta) alters physiology and phenotype, focusing particularly on the mouse since it forms the basis of the current genetic revolution. It is increasingly recognized that we ignore those alterations in phenotype at our peril if we wish to translate the research to humans (12, 30, 43, 51).
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Since Galen, our knowledge of human biology has depended heavily on translating research from non-human animals (4). August Krogh summed up the field of comparative physiology with his eponymous principle: “for such a large number of problems there will be some animal of choice, or a few such animals, on which it can be most conveniently studied” (38). Differences between species are the primary interest for comparative physiologists trying to understand the variety of functional adaptations that shape those species. But those differences can create problems for translational research, where we use animal models to advance our understanding of human physiology and health. For example, we could not use dogs or cats as animal models to study the link between arrhythmia and the hyperkalemia that can arise from hemolysis during blood transfusions because, unlike humans, dogs and cats do not express the sodium-potassium pump on their erythrocytes, and therefore they do not have high intraerythrocyte potassium (24). Similarly, although the molecular mechanisms underlying depolarization in the human and murine heart are similar, the channels responsible for repolarization are different, meaning that genes or drugs that affect repolarization in murine models will have little or no effect in humans (58). Although there exists a general bauplan for mammals, there is vast variability in morphology and physiology between species. Often a finding in one species will be relevant to humans, but sometimes, as with hyperkalemia or ventricular repolarization, it will not. Fortunately, some of the differences between humans and the models we use to study human biology and disease are generalizable. In the last two decades, there has been an unprecedented increase in the number of publications on rodents (FIGURE 1). The vast majority of the studies have had a translational modus; we
The Scaling of Thermal Relations One problem in translating the results of studies in mice to human function arises from body size; humans are more than three orders of magnitude heavier than mice, and most traits do not scale proportionately (isometrically) with body mass. If a trait is plotted against body mass on a log-log plot, an isometric relationship has a slope of one. But if a mammal maintains the same body shape and doubles in length, its surface area increases fourfold (with the square of the length), whereas its mass increases eightfold (with the cube of the length). If bone scaled isometrically and an animal doubled in length, the cross-sectional area of the 413
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FIGURE 1. The number of publications from 1993 through to 2013 that have “mouse” or “rat” in the title Data from Science Direct (http://www.sciencedirect.com/; updated from Ref. 21). 414
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regulatory changes in metabolic heat production or evaporative heat loss” (25). By definition, an endotherm can be at BMR only when maintained at a Ta within its TNZ. Although mice generate more heat per gram than elephants, they also lose heat to a cool environment at a faster rate per gram than elephants. The outcome is that the LCT is higher for smaller than for larger mammals (10) (FIGURE 3). For a lightly clothed human, the LCT is ⬃21°C (31). For a mouse, it is ⬃30°C (21); for a rat it is ⬃28°C (20). An insulating body covering is important in influencing the LCT because removing the clothing from human, leaving them naked, increases the LCT to above 26°C (FIGURE 3). Factors other than body mass can influence the LCT of mice, including adiposity, the number of mice housed in a cage, and the amount of nesting material (insulation) provided (60). The U.S. National Research Council recommends housing mice at 20 –26°C (49); that is below LCT for even the largest mice strains. But mice usually are housed at 20 –24°C (19), within the TNZ of lightly clothed humans but well below the LCT for any mouse. In most studies, Ta does not appear to have been an important consideration for researchers. Based on the search terms “mouse,” “knockout,” and “disease” in Google Scholar, we looked at the first 50 papers listed when sorted by relevance. In two of those 50 papers, the conditions were “as recommended by the NRC,” and in one the Ta was specified as 21°C. The housing temperature was not even mentioned in the other 47 of those 50 papers. Does it matter that there are thousands of studies on knockout mice housed and studied below their LCT? Mice housed below their LCT are chronically cold-stressed (15), and if given a choice they will select a temperature within their TNZ, especially during the inactive phase of the daily activity cycle (21, 40). For nocturnal animals like rodents, the inactive phase occurs during the lights-on daytime. Mouse pups spend much of their time giving distress calls when Ta is below 27°C, but they rarely call when Ta is above 33°C (50). The cold stress is also reflected in the metabolic activity at all ages. The metabolism of mice is ⬃50% higher at 22°C than at 30°C (32, 61, 62, 66). When a mouse is raised at 22°C, its food consumption is ⬃50% higher than it is within the TNZ, its body mass is greater, and the liver, kidneys, and heart are relatively larger (67) (FIGURE 4). The increased food consumption occurs primarily in the inactive (light) phase, a time when mice exposed to thermoneutrality eat little (29). The larger body size helps to reduce heat loss, whereas the increased organ size helps to process the increased food intake and sustain the 50% higher metabolic rate at 22°C compared with 30°C. A mouse raised at
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leg bones would increase fourfold, but the skeleton would have to support eight times the weight. Unsurprisingly, skeleton size scales allometrically (non-isometrically) with a slope ⬎1, such that larger species have disproportionately larger skeletal mass (55). Books have been compiled that relate how body size influences everything from heart rate to the size of a home range (11, 59). Probably the most famous scaling law is the mouse-to-elephant curve that describes the relationship between basal metabolic rate (BMR) and body mass. There is ongoing debate about the exact value of the exponent (1, 65), but what is unequivocal is that the allometric slope is ⬍1; larger animals have a lower metabolic rate per gram than do smaller animals (FIGURE 2). To survive, even at BMR, mice need to process much more energy per gram of body mass than do humans, and consequently mice generate much more metabolic heat per gram. To put this principle into perspective, if a 500-kg oxen had the massspecific metabolic rate of a mouse, its surface temperature would have to exceed 100°C for it to dissipate its resting heat production (33). Because the surface area-to-mass ratio changes with body size, so too does the exchange of heat between an animal and the environment. The normal core body temperature (Tb) of mammals varies by only a few degrees Celsius from mice to elephants, but both mice and elephants maintain Tb in environments tens of degrees lower than their Tb. Below a defined Ta, called the lower critical temperature (LCT), a mammal cannot maintain normal Tb without increasing metabolic heat production above BMR. The LCT demarcates the lower boundary of the thermoneutral zone (TNZ), defined as “the range of ambient temperature at which temperature regulation is achieved only by the control of sensible heat loss, i.e., without
REVIEWS conventional animal-house temperature is not the same phenotype, metabolically or thermally, as one raised at thermoneutrality (FIGURE 4). Because conventional housing temperature is a cold-stress for mice, another potential confounder in physiological studies is the development of torpor. Mice, like some other species of small mammals, respond to thermal and energetic challenges by abandoning the defence of the normal homeothermic Tb and enter a state known as torpor. In the torpid state, as well as Tb being much lower than normal, nearly all other physiological functions are similarly depressed. In mice housed at conventional animal-house Ta, torpor is induced by a single night of fasting or a few days of calorie restriction (52).
Cardiovascular Physiology Of Mice Housed At Different Ta
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Unsurprisingly, more than just the thermal and metabolic physiology of an animal is affected by being housed below the LCT. During the light phase of the daily cycle, the resting heart rate (HR) of a mouse exposed to 30°C is ⬃375 beats/min (61, 62, 66). The resting HR increases by ⬃25 beats/min for every 1°C decrease in Ta below 30°C (61, 62) (FIGURE 5). When both arms of the autonomic nervous system are blocked, the heart beats at its intrinsic rate (i.e., the rate in a heart isolated from its nervous supply). Such autonomic blockade in a resting human results in an increase in HR because, at rest, parasympathetic tone dominates the control of the heart rhythm. At 22°C, the autonomic blockade of mice results in a decrease in HR, implying that the “resting” HR is sustained by elevated sympathetic tone. When mice are tested at 30°C, autonomic blockade results in an increase in HR because resting HR is dominated by parasympathetic input, just as it is in resting thermoneutral humans (61, 62). Furthermore, the change in autonomic drive to the heart is reflected in the change in heart rate variability (FIGURE 5C), which decreases with Ta below the LCT, suggestive of increased sympathetic drive (61). The increase in HR below the LCT reflects the increase in metabolism that is required to maintain Tb. When humans turn over more energy, as mice do when they are housed below their TNZ, HR increases, and our blood pressure also rises. At 30°C, the mean arterial pressure (MAP) of a mouse during the light phase of the daily cycle is ⬃80 mmHg, and it increases 2 mmHg for every 1°C decrease in Ta below 30°C (61, 62, 66). Another factor associated with hypertension in mice, and in humans, is sleep deprivation. Mice spend much more time awake when they are housed at 20°C than when they are housed at 30°C
(42). It could be argued that nearly everything we know about the cardiovascular system of rats and mice comes from sleep-deprived and chronically hypertensive animals because we study them at a Ta that is comfortable for us as investigators but that is cold for rodents. Importantly, the observed hypertension is physiological, not pathological, meaning that any intervention that lowers MAP will be influencing the normal physiological regulation of MAP and will not necessarily alleviate pathology. An appreciation of the effects of Ta on cardiovascular physiology is critical if we wish to translate results of research on rodents to humans. A drug or a gene that reduces the influence of sympathetic nervous input to the heart would have little effect on the HR of a mouse within the TNZ but would reduce HR and MAP in a mouse housed in the cold. On the other hand, a gene or drug that decreases HR or MAP in a mouse at 22°C might
FIGURE 2. Mass-specific basal metabolic rate and logarithms of the same data A: mass-specific basal metabolic rate measured in 356 species of eutherian mammals with body mass up to 500 g (data taken from Ref. 64), showing the decrease in metabolism per gram as body mass increases. B: logarithms of the same data as shown in A. Thick line is the line of best fit for the data; broken line is the isometric line if BMR scaled proportionately to body mass.
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Fuelling an Elevated Metabolism Below the LCT
FIGURE 3. Relative metabolic rate as a function of ambient temperature for the mouse and the human Metabolic rate has been standardized to the metabolic rate measured above the LCT (usually resting metabolic rate as opposed to basal metabolic rate because of the confounder of torpor in fasted mice). The metabolic rate at several Ta from the literature was used to establish the relationships. Mouse data are the average of strains weighing from 23 to 55 g from Table 1 in Ref. 21. Human data are taken from Ref. 23. The inflection in each line is the lower critical temperature, the ambient temperature below which an endotherm increases heat production to balance heat loss. Note that the LCT of the mouse, and the relative metabolic response to Ta below the LCT, is higher than in a naked human. 416
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An attractive approach for targeting diabetes and obesity is to alter human metabolism using drug or genetic interventions. Almost inevitably, such interventions are, or will be, developed and tested in rodents. A potential problem is that, under chronic cold stress, mice not only increase their metabolic rate but also alter their metabolic substrate. When mice are moved acutely for 2 h from TNZ to 21°C, they metabolize more carbohydrate (15). Similarly, moving mice from 30°C to 5°C results in an increase in the respiratory quotient (RQ) for a few hours, indicative of elevated carbohydrate metabolism, but then the RQ decreases and becomes steady at a significantly lower value than at 30°C, indicating higher lipid oxidation than at thermoneutrality (45). The long-term exposure of mice to 22°C results in higher lipid uptake in heart, lungs, and brown adipose tissue (BAT) than at TNZ, with the result that plasma triglycerides and cholesterol are lower in mice housed at 22°C (28). A gene or drug that influences fat or cholesterol metabolism, therefore, will likely have different effects in a mouse at different Ta, just as would a gene or drug that affects carbohydrate metabolism. For many years, it was held that metabolism in the cold differed in mice and humans because of
BAT deposits in mice, a highly vascularized tissue that is dense with mitochondria (53). The primary site of nonshivering thermogenesis in rodents exposed to cold is BAT, and BAT stores increase when a rodent is exposed to cold (12). In addition to thermoregulation, BAT also has been implicated in diet-induced thermogenesis, the increase in metabolism, and therefore heat production, that occurs with overfeeding (56). In BAT, uncoupling protein 1 (UCP-1), a protein resident on the inner mitochondrial membrane, uncouples oxidation from phosphorylation in the mitochondrion by dissipating the energy stored in the proton gradient across the membrane as heat instead of harnessing that energy to phosphorylate ADP to ATP (48). Until recently, the dogma was that adult humans did not possess BAT, which would mean that rodents could not be a good model for studying obesity in humans. That argument had to change in the last 10 years with the realization that active BAT exists in some adult humans (47), opening up potential new targets for treating obesity and related disorders (39), and leading to the use of the UCP⫺/⫺ knockout mouse to test the significance of BAT in diet-induced thermogenesis. It was predicted that UCP⫺/⫺ mice would be cold sensitive and prone to obesity because they would lack BAT thermogenesis. When the knockout mice first were tested, it was found that they indeed were cold sensitive but were not prone to obesity (16). And when they were placed on a high-fat diet, which should have exacerbated the effect of lacking dietinduced thermogenesis, the UCP⫺/⫺ mice were found to be resistant to obesity compared with the wild-type controls (41). Up to that point, the experiments had been done at the conventional animal house temperatures of 20 –22°C. When adult control and UCP⫺/⫺ mice were placed at 27°C, the UCP⫺/⫺ mice exhibited an acceleration of body mass gain, implying that Ta could be important to the phenotype (41). It emerges that, when control and UCP⫺/⫺ mice are housed at thermoneutrality from birth, the UCP⫺/⫺ mice express an obese phenotype, and the obesity is exacerbated when the animals are fed a high-fat diet (17). Similarly, D2KO mice lacking the type-2 deiodinase activity that is necessary for the activation of thyroid hormone in BAT have an impaired response to cold but are no more sensitive than controls to diet-induced obesity when studied at 22°C. When studied at 30°C, the D2KO mice develop an obese, glucose-intolerant phenotype with hepatic steatosis (13). The phenotype that results from these two genetic manipulations therefore is completely different at thermoneutrality compared with conventional animal-house Ta. Only UCP⫺/⫺ or D2KO
REVIEWS mice housed within their TNZ could serve as models for obesity-related disorders in humans. Similarly, an agent like bone morphogenic protein BMP7, which activates BAT and reduces body mass in mice with diet-induced obesity when those mice are housed at 21°C, but not when they are housed at 28°C, is an unlikely target for managing human obesity (5).
Rodent Longevity
In contrast to its effects in B6 mice, thermoneutrality does not attenuate the effect of CR on longevity in the MRL strain of mice (35). What is different? The B6 strain is prone to developing lymphoma, whereas the MRL strain tends to develop autoimmune disease (35). CR is known to suppress cellular proliferation and enhance apoptosis, changes that decrease the likelihood of tumor growth (34), but that effect is lost at 30°C. Housing mice below their LCT decreases the probability of lymphoma development or progression (34). In all likelihood, the Ta, and an animal’s response to it, differently affects various aspects of tumor initiation and development (such as the rate of cell division in various organs) and the response of the immune system. The ability of mouse dendritic cells to regulate T cells is influenced profoundly by the Ta at which the mice are housed (37), and BALB/c and C57BL/6 mice are more susceptible to cancer when they are housed at 21°C compared with 28°C (36). Clearly, as far as cancer and the immune system are concerned, mild cold
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The rodent models that have attracted public attention, perhaps more than any others, are those demonstrating artificially enhanced longevity. Engineering a mouse’s hypothalamus to overproduce uncoupling protein 2 results in the local production of heat in the hypothalamus. Because the hypothalamus is the most thermosensitive region in the mammalian body and provides the majority of the input signal for thermoregulation (26), the local heating results in a lower than normal Tb everywhere else in the body. Those engineered mice also live longer than normal (14), providing evidence of an association between Tb, energetics, and longevity (3). There are other mouse genotypes that live longer than average. These genotypes produce dwarf strains, and their LCTs are higher than those of the average mouse. They tend to have lower Tb than normal mice, even at Ta of 26°C (7). The hypopituitary Ames and Snell strains live for ⬃1,150 days compared with 720 days for a normal mouse (8). In dwarf mice, the longevity appears to be related to increased metabolic rate (3). The long-lived growth hormone-resistant (GHR-KO) strain has a metabolic rate higher than that of wild-type mice when housed at 23°C but not when housed at 30°C (3), implying that enhanced longevity would not be evident in the TNZ. That implication is manifested in the C57Black-6 (B6) strain of mouse that lives for ⬃785 days (35) when its lifespan is extended by calorie restriction (CR). Placing B6 mice onto a CR diet, where they receive 60% of their normal daily energy requirement, extends their life to ⬃1,148 days at 21°C (35). If the B6 mice are fed a CR diet at thermoneutrality, they live for ⬃810 days, not significantly different from the 785 days of the control-fed mice (35). So CR does not extend lifespan in B6 mice in their TNZ. Koizumi (35) thought that the explanation lay in the restricted use of torpor at the higher Ta, but Bartke (3) reports that the longlived genotypes do not use torpor and thus maintain a high metabolism when they are exposed to 21°C. There is clearly much we do not understand about the interactions between Ta, Tb, metabolism, and longevity.
Fever and Disease
FIGURE 4. Morphological and physiological differences in mice at Ta ranging from 18 to 30°C
A: body mass at 9 wk of age of JCL-ICR mice bred and reared at each respective Ta (average of males and females is shown; data from Ref. 67). B: oxygen consumption measured in conscious and unrestrained female NIH Swiss mice, measured as Ta was lowered from 30°C and held at each Ta (data from Ref. 62). C: daily food intake at 8 wk of age in JCL-ICR mice bred and reared at each respective Ta (average of males and females is shown; data from Ref. 67). D: heart mass as a percent of body mass at 24 wk of age in JCL-ICR mice bred and reared at each respective Ta (average of males and females is shown; data from Ref. 67).
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FIGURE 5. Cardiovascular and hematological data from mice at Ta ranging from 18 to 30°C
Mean arterial blood pressure (A), heart rate (B), and heart rate variability (C) assessed as the standard deviation of the interbeat interval, measured by telemetry in conscious and unrestrained mice at each respective Ta (data from Refs. 61, 62, 66). D: leukocyte count at 10 wk of age of JCL-ICR mice bred and reared at each respective Ta (average of males and females is shown; data from Ref. 67). 418
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performed within the TNZ, but below the TNZ the same dose induces hypothermic shock, and Tb falls below 34°C (57). LPS stimulates different hypothalamic nuclei in rats housed within their TNZ compared with below the TNZ (63). Testing the immune response of rodents might be misleading if the tests are conducted at normal animal house or laboratory Ta.
At What Temperature Should We Be Housing Mice? That the phenotype and physiology of a mouse housed at 21°C is different to that of one housed at 30°C is clear, raising the question of whether studies on mice below their LCT have any relevance to human physiology? Translating results from mice at their conventional housing temperature might not be a problem if humans did indeed spend much of their lives below the TNZ, but whenever possible, humans use any means available, such as added insulation or heating and air conditioning, to maintain a state of thermal comfort. Especially in the developed world, we spend little, if any, of our time in the cold. By 1997, 47% of households in the U.S. were air-conditioned; by 2006, so were 50% of houses in New South Wales, Australia (22), and by 2007, 82% of homes in Perth, Western Australia, were air-conditioned (44), whereas in the UK and U.S. home and bedroom temperatures have increased by 3– 4°C over the last 30 years (27). Workplace temperatures appear to be increasing similarly (27). Behavioral thermoregulatory studies in mice and other rodents confirm that, when given the opportunity, rodents also will seek and maintain a zone of thermal comfort, especially, but not limited to, their periods of inactivity (20, 21). Speakman and Keijer (60) have advocated that mice should be housed in the cold, arguing that the average daily metabolic rate of free-living humans is ⬃1.6 times BMR, and so exposing mice to 20 – 22°C, which induces a metabolic rate of 1.6 times BMR, makes them an appropriate model for the human condition. However, it is unlikely that the “activity metabolism” of free-living humans is either physiologically or genetically similar to the cold-induced metabolism of a sleep-deprived and hypertensive mouse exposed to 21°C. There are factors other than Ta to consider when housing mice and interpreting data (40). The provision of nesting material to mice increases pup survival, litter size, and weaning body mass, and nest building varies with Ta. Given a choice of thermal environments, mice with low activity levels prefer a Ta of 25 or 30°C more than 20°C, irrespective of whether they have nesting material (18). Animal behavior, age, sex, strain, and time of day all alter thermal preferences, making it difficult
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stress can have profound effects on the phenotype that results from a given genotype. It is not just in tumor resistance but also in the physiological response to infection that thermoneutrality matters. After discovering that the mortality of mice infected with Typhus rickettsiae increased when the environmental temperature decreased, Moragues and Pinkerton in 1944 (46) commented that “by controlling the environmental temperature, conditions may be created under which murine typhus will have any desired degree of mortality.” Housing mice below their TNZ results in lower concentrations of circulating leukocytes (67) (FIGURE 5D), suppresses the release of pro-inflammatory cytokines, and increases the release of anti-inflammatory cytokines (30). Additionally, at 21°C, the entry of the pro-inflammatory cytokine IL-1 into the brain, where it exerts its pyrogenic effect, is impeded (9). It seems that multiple steps in the immune response pathway are altered when mice are housed below their TNZ. It is probably these differences in cytokine release and action that results in clear differences in the acute phase fever response at different Ta. Injecting mice with 104 g/kg of lipopolysaccharide (LPS), the pyrogenic moiety of the cell wall of Gram-negative bacteria, results in a robust fever with a Tb rise of ⬃2°C when the experiment is
REVIEWS to generalize about housing temperatures for all mice (18). Other factors like the number of animals per cage, noise, light, and human interference also are likely to introduce stressors that alter the physiology of the laboratory-housed mouse. Although reducing the challenges to thermal homeostasis by housing mice within their TNZ (21), or allowing them to control Ta (18), is an approach more likely to accurately reflect the physiology and pathophysiology of the modern human, the infrastructure changes required to keep rodents within their TNZ makes the prospect a long-term challenge. As proposed by Lodhi and Semenkovich (43) when they suggested (with tongue firmly in cheek) that mice should be clothed, we nevertheless must be aware of the nuances of environmental stressors, and particularly Ta, when translating data from mice to men. 䡲
Hansen J, Hérault Y, Hicks G, Hörlein A, Houghton R, Hrabé de Angelis M, Huylebroeck D, Iyer V, Jong P, Kadin J, Kaloff C, Kennedy K, Koutsourakis M, Kent Lloyd KC, Marschall S, Mason J, McKerlie C, McLeod M, Melchner H, Moore M, Mujica A, Nagy A, Nefedov M, Nutter L, Pavlovic G, Peterson J, Pollock J, Ramirez-Solis R, Rancourt D, Raspa M, Remacle J, Ringwald M, Rosen B, Rosenthal N, Rossant J, Ruiz Noppinger P, Ryder E, Schick J, Schnütgen F, Schofield P, Seisenberger C, Selloum M, Simpson E, Skarnes W, Smedley D, Stanford W, Francis Stewart A, Stone K, Swan K, Tadepally H, Teboul L, Tocchini-Valentini G, Valenzuela D, West A, Yamamura Ki Yoshinaga Y, Wurst W. The mammalian gene function resource: the international knockout mouse consortium. Mamm Genome 23: 580 –586, 2012. 7.
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The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does the mention of trade names of commercial products constitute endorsement or recommendation for use. No conflicts of interest, financial or otherwise, are declared by the author(s). Author contributions: S.K.M. and D.M. conception and design of research; S.K.M. prepared figures; S.K.M. drafted manuscript; S.K.M., A.F., D.M., C.J.G., and M.O. edited and revised manuscript; S.K.M., A.F., D.M., C.J.G., and M.O. approved final version of manuscript.
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Boon MR, van den Berg SAA, Wang Y, van den Bossche J, Karkampouna S, Bauwens M, De Saint-Hubert M, van der Horst G, Vukicevic S, de Winther MPJ, Havekes LM, Jukema JW, Tamsma JT, van der Pluijm G, van Dijk KW, Rensen PCN. BMP7 activates brown adipose tissue and reduces diet-induced obesity only at subthermoneutrality. PLos One 8: e74083, 2013.
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We are grateful to Gary Sieck for the invitation to create and submit this article, and to Mike Joyner who started it all with an invitation to present a review at IUPS. Prof. Tobias Wang provided comments that helped to improve the manuscript.
20. Gordon CJ. Thermal biology of the laboratory rat. Physiol Behav 47: 963–991, 1990.
24. Hoffman JF. The red cell membrane and the transport of sodium and potassium. Am J Med 41: 666 – 680, 1966. 25. IUPS Thermal Commission. Glossary of terms for thermal physiology: third edition (reprinted from the Japanese Journal of Physiology). J Therm Biol 28: 75–106, 2003. 26. Jessen C. Temperature Regulation in Humans and Other Mammals. Berlin: Springer-Verlag, 2001, p. 193.
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36. Kokolus KM, Capitano ML, Lee CT, Eng JWL, Waight JD, Hylander BL, Sexton S, Hong CC, Gordon CJ, Abrams SI, Repasky EA. Baseline tumor growth and immune control in laboratory mice are significantly influenced by subthermoneutral housing temperature. Proc Natl Acad Sci USA 110: 20176 –20181, 2013. 37. Kokolus KM, Spangler HM, Povinelli BJ, Farren MR, Lee KP, Repasky EA. Stressful presentations: mild chronic cold stress in mice influences baseline properties of dendritic cells. Front Immunol . In Press. 38. Krebs HA. The August Krogh principle: “For many problems there is an animal on which it can be most conveniently studied”. J Exp Zool 194: 221–226, 1975. 39. Lee YH, Jung YS, Choi D. Recent advance in brown adipose physiology and its therapeutic potential. Exp Mol Med 46: e78, 2014.
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41. Liu X, Rossmeisl M, McClaine J, Kozak LP. Paradoxical resistance to diet-induced obesity in UCP1-deficient mice. J Clin Invest 111: 399 – 407, 2003.
56. Rothwell NJ, Stock MJ. A role for brown adipose tissue in diet-induced thermogenesis. Nature 281: 31–35, 1979.
42. Lo Martire V, Silvani A, Bastianini S, Berteotti C, Zoccoli G. Effects of ambient temperature on sleep and cardiovascular regulation in mice: the role of hypocretin/orexin neurons. PLos One 7: e47032, 2012. 43. Lodhi IJ, Semenkovich CF. Why we should put clothes on mice. Cell Metab 9: 111–112, 2009. 44. Maloney S, Forbes C. What effect will a few degrees of climate change have on human heat balance? Implications for human activity. Int J Biometeorol 55: 147–160, 2011. 45. Meyer CW, Willershäuser M, Jastroch M, Rourke BC, Fromme T, Oelkrug R, Heldmaier G, Klingenspor M. Adaptive thermogenesis and thermal conductance in wild-type and UCP1-KO mice. Am J Physiol Regul Integr Comp Physiol 299: R1396 –R1406, 2010. 46. Moragues V, Pinkerton H. Variation in morbidity and mortality of murine typhus infection in mice with changes in the environmental temperature. J Exp Med 79: 41– 43, 1944. 47. Nedergaard J, Bengtsson T, Cannon B. Unexpected evidence for active brown adipose tissue in adult humans. Am J Physiol Endocrinol Metab 293: E444 –E452, 2007. 48. Nedergaard J, Golozoubova V, Matthias A, Asadi A, Jacobsson A, Cannon B. UCP1: the only protein able to mediate adaptive non-shivering thermogenesis and metabolic inefficiency. Biochim Biophys Acta 1504: 82–106, 2001. 49. NRC. Guide for the Care and Use of Laboratory Animals. Washington, DC: National Research Council of the National Academy of Science, 2011. 50. Okon EE. The effect of environmental temperature on the production of ultrasounds by isolated non-handled albino mouse pups. J Zool Lond 162: 71– 83, 1970. 51. Overton JM. Phenotyping small animals as models for the human metabolic syndrome: thermoneutrality matters. Int J Obes 34: S53–S58, 2010. 52. Overton JM, Williams TD. Behavioral and physiologic responses to caloric restriction in mice. Physiol Behav 81: 749 –754, 2004. 53. Owens B. Cell physiology: the changing colour of fat. Nature 508: S52–S53, 2014. 54. Peloso ED, Florez-Duquet M, Buchanan JB, Satinoff E. LPS fever in old rats depends on the ambient temperature. Physiol Behav 78: 651– 654, 2003.
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57. Rudaya AY, Steiner AA, Robbins JR, Dragic AS, Romanovsky AA. Thermoregulatory responses to lipopolysaccharide in the mouse: dependence on the dose and ambient temperature. Am J Physiol Regul Integr Comp Physiol 289: R1244 –R1252, 2005. 58. Sabir IN, Killeen MJ, Grace AA, Huang CLH. Ventricular arrhythmogenesis: Insights from murine models. Progr Biophys Mol Biol 98: 208 –218, 2008. 59. Schmidt-Nielsen K. Scaling. Why is Animal Size so Important? Cambridge, UK: Cambridge Univ. Press, 1984, p. 245. 60. Speakman JR, Keijer J. Not so hot: optimal housing temperatures for mice to mimic the thermal environment of humans. Mol Metab 2: 5–9, 2013. 61. Swoap SJ, Li C, Wess J, Parsons AD, Williams TD, Overton JM. Vagal tone dominates autonomic control of mouse heart rate at thermoneutrality. Am J Physiol Heart Circ Physiol 294: H1581– H1588, 2008. 62. Swoap SJ, Overton JM, Garber G. Effect of ambient temperature on cardiovascular parameters in rats and mice: a comparative approach. Am J Physiol Regul Integr Comp Physiol 287: R391– R396, 2004. 63. Wanner SP, Yoshida K, Kulchitsky VA, Ivanov AI, Kanosue K, Romanovsky AA. Lipopolysaccharide-induced neuronal activation in the paraventricular and dorsomedial hypothalamus depends on ambient temperature. PLos One 8: e75733, 2013. 64. White CR, Seymour RS. Allometric scaling of mammalian metabolism. J Exp Biol 208: 1611– 1619, 2005. 65. White CR, Seymour RS. Mammalian basal metabolic rate is proportional to body mass2/3. Proc Natl Acad Sci USA 100: 4046 – 4049, 2003. 66. Williams TD, Chambers JB, Henderson RP, Rashotte ME, Overton JM. Cardiovascular responses to caloric restriction and thermoneutrality in C57BL/6J mice. Am J Physiol Regul Integr Comp Physiol 282: R1459 –R1467, 2002. 67. Yamauchi C, Fujita S, Obara T, Ueda T. Effects of room temperature on reproduction, body and organ weights, food and water intakes, and hematology in mice. Exp Anim 32: 1–11, 1983.
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35. Koizumi A, Wada Y, Tuskada M, Kayo T, Naruse M, Horiuchi K, Mogi T, Yoshioka M, Sasaki M, Miyamaura Y, Abe T, Ohtomo K, Walford RL. A tumor preventive effect of dietary restriction is antagonized by a high housing temperature through deprivation of torpor. Mech Ageing Dev 92: 67– 82, 1996.
40. Leon LR. The use of gene knockout mice in thermoregulation studies. J Therm Biol 30: 273–288, 2005.
Shane K. Maloney, Andrea Fuller, Duncan Mitchell, Christopher Gordon and J. Michael Overton Physiology 29:413-420, 2014. doi:10.1152/physiol.00029.2014 You might find this additional info useful... This article cites 60 articles, 19 of which can be accessed free at: /content/29/6/413.full.html#ref-list-1 This article has been cited by 2 other HighWire hosted articles Rethinking Animal Models and Human Obesity Michael J. Joyner Physiology, November , 2014; 29 (6): 384-385. [Full Text] [PDF] Living Under Extreme Conditions Physiology, November , 2014; 29 (6): 386-387. [Full Text] [PDF]
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PHYSIOLOGY 29: 413– 420, 2014; doi:10.1152/physiol.00029.2014
Translating Animal Model Research: Does It Matter That Our Rodents Are Cold? Does it matter that rodents used as preclinical models of human biology are routinely housed below their thermoneutral zone? We compile evidence showing that such rodents are cold-stressed, hypermetabolic, hypertensive, sleep-deprived, obesity-resistant, fever-resistant, aging-resistant, and tumor-
Shane K. Maloney,1,2 Andrea Fuller,2 Duncan Mitchell,1,2 Christopher Gordon,3 and J. Michael Overton4 1 School of Anatomy Physiology and Human Biology, The University of Western Australia, Stirling Highway, Crawley, Australia; 2Brain Function Research Group, School of Physiology, Faculty of Health Sciences, University of the Witwatersrand, Parktown, South Africa; 3Toxicity Assessment Division, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina; and 4Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, Florida
prone compared with mice housed at thermoneutrality. The same genotype of mouse has a very different phenotype and response to physiological or pharmacological intervention when raised below or at thermoneutrality.
1548-9213/14 ©2014 Int. Union Physiol. Sci./Am. Physiol. Soc.
work on rodents because they are easy to study and have a short generation time, and we hope that such studies will reveal useful information on human physiology. Since 2003, the number of published studies on mice exceeded those on rats, driven primarily by the development of molecular tools to manipulate genes in mice. The international knockout mouse consortium has the laudable aim of knocking out individually each of the 20,000 or so protein-coding genes in the mouse genome (6). Studying the phenotypes of all of those new genotypes is expected to yield a wealth of information. Some of what we learn from mouse knockout studies will apply to humans, but mice are not humans. Environments that are unstressful to humans may well stress mice and alter their physiology and phenotype. In this review, we will discuss the way that ambient temperature (Ta) alters physiology and phenotype, focusing particularly on the mouse since it forms the basis of the current genetic revolution. It is increasingly recognized that we ignore those alterations in phenotype at our peril if we wish to translate the research to humans (12, 30, 43, 51).
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Since Galen, our knowledge of human biology has depended heavily on translating research from non-human animals (4). August Krogh summed up the field of comparative physiology with his eponymous principle: “for such a large number of problems there will be some animal of choice, or a few such animals, on which it can be most conveniently studied” (38). Differences between species are the primary interest for comparative physiologists trying to understand the variety of functional adaptations that shape those species. But those differences can create problems for translational research, where we use animal models to advance our understanding of human physiology and health. For example, we could not use dogs or cats as animal models to study the link between arrhythmia and the hyperkalemia that can arise from hemolysis during blood transfusions because, unlike humans, dogs and cats do not express the sodium-potassium pump on their erythrocytes, and therefore they do not have high intraerythrocyte potassium (24). Similarly, although the molecular mechanisms underlying depolarization in the human and murine heart are similar, the channels responsible for repolarization are different, meaning that genes or drugs that affect repolarization in murine models will have little or no effect in humans (58). Although there exists a general bauplan for mammals, there is vast variability in morphology and physiology between species. Often a finding in one species will be relevant to humans, but sometimes, as with hyperkalemia or ventricular repolarization, it will not. Fortunately, some of the differences between humans and the models we use to study human biology and disease are generalizable. In the last two decades, there has been an unprecedented increase in the number of publications on rodents (FIGURE 1). The vast majority of the studies have had a translational modus; we
The Scaling of Thermal Relations One problem in translating the results of studies in mice to human function arises from body size; humans are more than three orders of magnitude heavier than mice, and most traits do not scale proportionately (isometrically) with body mass. If a trait is plotted against body mass on a log-log plot, an isometric relationship has a slope of one. But if a mammal maintains the same body shape and doubles in length, its surface area increases fourfold (with the square of the length), whereas its mass increases eightfold (with the cube of the length). If bone scaled isometrically and an animal doubled in length, the cross-sectional area of the 413
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FIGURE 1. The number of publications from 1993 through to 2013 that have “mouse” or “rat” in the title Data from Science Direct (http://www.sciencedirect.com/; updated from Ref. 21). 414
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regulatory changes in metabolic heat production or evaporative heat loss” (25). By definition, an endotherm can be at BMR only when maintained at a Ta within its TNZ. Although mice generate more heat per gram than elephants, they also lose heat to a cool environment at a faster rate per gram than elephants. The outcome is that the LCT is higher for smaller than for larger mammals (10) (FIGURE 3). For a lightly clothed human, the LCT is ⬃21°C (31). For a mouse, it is ⬃30°C (21); for a rat it is ⬃28°C (20). An insulating body covering is important in influencing the LCT because removing the clothing from human, leaving them naked, increases the LCT to above 26°C (FIGURE 3). Factors other than body mass can influence the LCT of mice, including adiposity, the number of mice housed in a cage, and the amount of nesting material (insulation) provided (60). The U.S. National Research Council recommends housing mice at 20 –26°C (49); that is below LCT for even the largest mice strains. But mice usually are housed at 20 –24°C (19), within the TNZ of lightly clothed humans but well below the LCT for any mouse. In most studies, Ta does not appear to have been an important consideration for researchers. Based on the search terms “mouse,” “knockout,” and “disease” in Google Scholar, we looked at the first 50 papers listed when sorted by relevance. In two of those 50 papers, the conditions were “as recommended by the NRC,” and in one the Ta was specified as 21°C. The housing temperature was not even mentioned in the other 47 of those 50 papers. Does it matter that there are thousands of studies on knockout mice housed and studied below their LCT? Mice housed below their LCT are chronically cold-stressed (15), and if given a choice they will select a temperature within their TNZ, especially during the inactive phase of the daily activity cycle (21, 40). For nocturnal animals like rodents, the inactive phase occurs during the lights-on daytime. Mouse pups spend much of their time giving distress calls when Ta is below 27°C, but they rarely call when Ta is above 33°C (50). The cold stress is also reflected in the metabolic activity at all ages. The metabolism of mice is ⬃50% higher at 22°C than at 30°C (32, 61, 62, 66). When a mouse is raised at 22°C, its food consumption is ⬃50% higher than it is within the TNZ, its body mass is greater, and the liver, kidneys, and heart are relatively larger (67) (FIGURE 4). The increased food consumption occurs primarily in the inactive (light) phase, a time when mice exposed to thermoneutrality eat little (29). The larger body size helps to reduce heat loss, whereas the increased organ size helps to process the increased food intake and sustain the 50% higher metabolic rate at 22°C compared with 30°C. A mouse raised at
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leg bones would increase fourfold, but the skeleton would have to support eight times the weight. Unsurprisingly, skeleton size scales allometrically (non-isometrically) with a slope ⬎1, such that larger species have disproportionately larger skeletal mass (55). Books have been compiled that relate how body size influences everything from heart rate to the size of a home range (11, 59). Probably the most famous scaling law is the mouse-to-elephant curve that describes the relationship between basal metabolic rate (BMR) and body mass. There is ongoing debate about the exact value of the exponent (1, 65), but what is unequivocal is that the allometric slope is ⬍1; larger animals have a lower metabolic rate per gram than do smaller animals (FIGURE 2). To survive, even at BMR, mice need to process much more energy per gram of body mass than do humans, and consequently mice generate much more metabolic heat per gram. To put this principle into perspective, if a 500-kg oxen had the massspecific metabolic rate of a mouse, its surface temperature would have to exceed 100°C for it to dissipate its resting heat production (33). Because the surface area-to-mass ratio changes with body size, so too does the exchange of heat between an animal and the environment. The normal core body temperature (Tb) of mammals varies by only a few degrees Celsius from mice to elephants, but both mice and elephants maintain Tb in environments tens of degrees lower than their Tb. Below a defined Ta, called the lower critical temperature (LCT), a mammal cannot maintain normal Tb without increasing metabolic heat production above BMR. The LCT demarcates the lower boundary of the thermoneutral zone (TNZ), defined as “the range of ambient temperature at which temperature regulation is achieved only by the control of sensible heat loss, i.e., without
REVIEWS conventional animal-house temperature is not the same phenotype, metabolically or thermally, as one raised at thermoneutrality (FIGURE 4). Because conventional housing temperature is a cold-stress for mice, another potential confounder in physiological studies is the development of torpor. Mice, like some other species of small mammals, respond to thermal and energetic challenges by abandoning the defence of the normal homeothermic Tb and enter a state known as torpor. In the torpid state, as well as Tb being much lower than normal, nearly all other physiological functions are similarly depressed. In mice housed at conventional animal-house Ta, torpor is induced by a single night of fasting or a few days of calorie restriction (52).
Cardiovascular Physiology Of Mice Housed At Different Ta
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Unsurprisingly, more than just the thermal and metabolic physiology of an animal is affected by being housed below the LCT. During the light phase of the daily cycle, the resting heart rate (HR) of a mouse exposed to 30°C is ⬃375 beats/min (61, 62, 66). The resting HR increases by ⬃25 beats/min for every 1°C decrease in Ta below 30°C (61, 62) (FIGURE 5). When both arms of the autonomic nervous system are blocked, the heart beats at its intrinsic rate (i.e., the rate in a heart isolated from its nervous supply). Such autonomic blockade in a resting human results in an increase in HR because, at rest, parasympathetic tone dominates the control of the heart rhythm. At 22°C, the autonomic blockade of mice results in a decrease in HR, implying that the “resting” HR is sustained by elevated sympathetic tone. When mice are tested at 30°C, autonomic blockade results in an increase in HR because resting HR is dominated by parasympathetic input, just as it is in resting thermoneutral humans (61, 62). Furthermore, the change in autonomic drive to the heart is reflected in the change in heart rate variability (FIGURE 5C), which decreases with Ta below the LCT, suggestive of increased sympathetic drive (61). The increase in HR below the LCT reflects the increase in metabolism that is required to maintain Tb. When humans turn over more energy, as mice do when they are housed below their TNZ, HR increases, and our blood pressure also rises. At 30°C, the mean arterial pressure (MAP) of a mouse during the light phase of the daily cycle is ⬃80 mmHg, and it increases 2 mmHg for every 1°C decrease in Ta below 30°C (61, 62, 66). Another factor associated with hypertension in mice, and in humans, is sleep deprivation. Mice spend much more time awake when they are housed at 20°C than when they are housed at 30°C
(42). It could be argued that nearly everything we know about the cardiovascular system of rats and mice comes from sleep-deprived and chronically hypertensive animals because we study them at a Ta that is comfortable for us as investigators but that is cold for rodents. Importantly, the observed hypertension is physiological, not pathological, meaning that any intervention that lowers MAP will be influencing the normal physiological regulation of MAP and will not necessarily alleviate pathology. An appreciation of the effects of Ta on cardiovascular physiology is critical if we wish to translate results of research on rodents to humans. A drug or a gene that reduces the influence of sympathetic nervous input to the heart would have little effect on the HR of a mouse within the TNZ but would reduce HR and MAP in a mouse housed in the cold. On the other hand, a gene or drug that decreases HR or MAP in a mouse at 22°C might
FIGURE 2. Mass-specific basal metabolic rate and logarithms of the same data A: mass-specific basal metabolic rate measured in 356 species of eutherian mammals with body mass up to 500 g (data taken from Ref. 64), showing the decrease in metabolism per gram as body mass increases. B: logarithms of the same data as shown in A. Thick line is the line of best fit for the data; broken line is the isometric line if BMR scaled proportionately to body mass.
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REVIEWS have no effect at all in a mouse exposed to 30°C. Are the results at 22°C or 30°C more likely to reflect the response in a human?
Fuelling an Elevated Metabolism Below the LCT
FIGURE 3. Relative metabolic rate as a function of ambient temperature for the mouse and the human Metabolic rate has been standardized to the metabolic rate measured above the LCT (usually resting metabolic rate as opposed to basal metabolic rate because of the confounder of torpor in fasted mice). The metabolic rate at several Ta from the literature was used to establish the relationships. Mouse data are the average of strains weighing from 23 to 55 g from Table 1 in Ref. 21. Human data are taken from Ref. 23. The inflection in each line is the lower critical temperature, the ambient temperature below which an endotherm increases heat production to balance heat loss. Note that the LCT of the mouse, and the relative metabolic response to Ta below the LCT, is higher than in a naked human. 416
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An attractive approach for targeting diabetes and obesity is to alter human metabolism using drug or genetic interventions. Almost inevitably, such interventions are, or will be, developed and tested in rodents. A potential problem is that, under chronic cold stress, mice not only increase their metabolic rate but also alter their metabolic substrate. When mice are moved acutely for 2 h from TNZ to 21°C, they metabolize more carbohydrate (15). Similarly, moving mice from 30°C to 5°C results in an increase in the respiratory quotient (RQ) for a few hours, indicative of elevated carbohydrate metabolism, but then the RQ decreases and becomes steady at a significantly lower value than at 30°C, indicating higher lipid oxidation than at thermoneutrality (45). The long-term exposure of mice to 22°C results in higher lipid uptake in heart, lungs, and brown adipose tissue (BAT) than at TNZ, with the result that plasma triglycerides and cholesterol are lower in mice housed at 22°C (28). A gene or drug that influences fat or cholesterol metabolism, therefore, will likely have different effects in a mouse at different Ta, just as would a gene or drug that affects carbohydrate metabolism. For many years, it was held that metabolism in the cold differed in mice and humans because of
BAT deposits in mice, a highly vascularized tissue that is dense with mitochondria (53). The primary site of nonshivering thermogenesis in rodents exposed to cold is BAT, and BAT stores increase when a rodent is exposed to cold (12). In addition to thermoregulation, BAT also has been implicated in diet-induced thermogenesis, the increase in metabolism, and therefore heat production, that occurs with overfeeding (56). In BAT, uncoupling protein 1 (UCP-1), a protein resident on the inner mitochondrial membrane, uncouples oxidation from phosphorylation in the mitochondrion by dissipating the energy stored in the proton gradient across the membrane as heat instead of harnessing that energy to phosphorylate ADP to ATP (48). Until recently, the dogma was that adult humans did not possess BAT, which would mean that rodents could not be a good model for studying obesity in humans. That argument had to change in the last 10 years with the realization that active BAT exists in some adult humans (47), opening up potential new targets for treating obesity and related disorders (39), and leading to the use of the UCP⫺/⫺ knockout mouse to test the significance of BAT in diet-induced thermogenesis. It was predicted that UCP⫺/⫺ mice would be cold sensitive and prone to obesity because they would lack BAT thermogenesis. When the knockout mice first were tested, it was found that they indeed were cold sensitive but were not prone to obesity (16). And when they were placed on a high-fat diet, which should have exacerbated the effect of lacking dietinduced thermogenesis, the UCP⫺/⫺ mice were found to be resistant to obesity compared with the wild-type controls (41). Up to that point, the experiments had been done at the conventional animal house temperatures of 20 –22°C. When adult control and UCP⫺/⫺ mice were placed at 27°C, the UCP⫺/⫺ mice exhibited an acceleration of body mass gain, implying that Ta could be important to the phenotype (41). It emerges that, when control and UCP⫺/⫺ mice are housed at thermoneutrality from birth, the UCP⫺/⫺ mice express an obese phenotype, and the obesity is exacerbated when the animals are fed a high-fat diet (17). Similarly, D2KO mice lacking the type-2 deiodinase activity that is necessary for the activation of thyroid hormone in BAT have an impaired response to cold but are no more sensitive than controls to diet-induced obesity when studied at 22°C. When studied at 30°C, the D2KO mice develop an obese, glucose-intolerant phenotype with hepatic steatosis (13). The phenotype that results from these two genetic manipulations therefore is completely different at thermoneutrality compared with conventional animal-house Ta. Only UCP⫺/⫺ or D2KO
REVIEWS mice housed within their TNZ could serve as models for obesity-related disorders in humans. Similarly, an agent like bone morphogenic protein BMP7, which activates BAT and reduces body mass in mice with diet-induced obesity when those mice are housed at 21°C, but not when they are housed at 28°C, is an unlikely target for managing human obesity (5).
Rodent Longevity
In contrast to its effects in B6 mice, thermoneutrality does not attenuate the effect of CR on longevity in the MRL strain of mice (35). What is different? The B6 strain is prone to developing lymphoma, whereas the MRL strain tends to develop autoimmune disease (35). CR is known to suppress cellular proliferation and enhance apoptosis, changes that decrease the likelihood of tumor growth (34), but that effect is lost at 30°C. Housing mice below their LCT decreases the probability of lymphoma development or progression (34). In all likelihood, the Ta, and an animal’s response to it, differently affects various aspects of tumor initiation and development (such as the rate of cell division in various organs) and the response of the immune system. The ability of mouse dendritic cells to regulate T cells is influenced profoundly by the Ta at which the mice are housed (37), and BALB/c and C57BL/6 mice are more susceptible to cancer when they are housed at 21°C compared with 28°C (36). Clearly, as far as cancer and the immune system are concerned, mild cold
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The rodent models that have attracted public attention, perhaps more than any others, are those demonstrating artificially enhanced longevity. Engineering a mouse’s hypothalamus to overproduce uncoupling protein 2 results in the local production of heat in the hypothalamus. Because the hypothalamus is the most thermosensitive region in the mammalian body and provides the majority of the input signal for thermoregulation (26), the local heating results in a lower than normal Tb everywhere else in the body. Those engineered mice also live longer than normal (14), providing evidence of an association between Tb, energetics, and longevity (3). There are other mouse genotypes that live longer than average. These genotypes produce dwarf strains, and their LCTs are higher than those of the average mouse. They tend to have lower Tb than normal mice, even at Ta of 26°C (7). The hypopituitary Ames and Snell strains live for ⬃1,150 days compared with 720 days for a normal mouse (8). In dwarf mice, the longevity appears to be related to increased metabolic rate (3). The long-lived growth hormone-resistant (GHR-KO) strain has a metabolic rate higher than that of wild-type mice when housed at 23°C but not when housed at 30°C (3), implying that enhanced longevity would not be evident in the TNZ. That implication is manifested in the C57Black-6 (B6) strain of mouse that lives for ⬃785 days (35) when its lifespan is extended by calorie restriction (CR). Placing B6 mice onto a CR diet, where they receive 60% of their normal daily energy requirement, extends their life to ⬃1,148 days at 21°C (35). If the B6 mice are fed a CR diet at thermoneutrality, they live for ⬃810 days, not significantly different from the 785 days of the control-fed mice (35). So CR does not extend lifespan in B6 mice in their TNZ. Koizumi (35) thought that the explanation lay in the restricted use of torpor at the higher Ta, but Bartke (3) reports that the longlived genotypes do not use torpor and thus maintain a high metabolism when they are exposed to 21°C. There is clearly much we do not understand about the interactions between Ta, Tb, metabolism, and longevity.
Fever and Disease
FIGURE 4. Morphological and physiological differences in mice at Ta ranging from 18 to 30°C
A: body mass at 9 wk of age of JCL-ICR mice bred and reared at each respective Ta (average of males and females is shown; data from Ref. 67). B: oxygen consumption measured in conscious and unrestrained female NIH Swiss mice, measured as Ta was lowered from 30°C and held at each Ta (data from Ref. 62). C: daily food intake at 8 wk of age in JCL-ICR mice bred and reared at each respective Ta (average of males and females is shown; data from Ref. 67). D: heart mass as a percent of body mass at 24 wk of age in JCL-ICR mice bred and reared at each respective Ta (average of males and females is shown; data from Ref. 67).
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FIGURE 5. Cardiovascular and hematological data from mice at Ta ranging from 18 to 30°C
Mean arterial blood pressure (A), heart rate (B), and heart rate variability (C) assessed as the standard deviation of the interbeat interval, measured by telemetry in conscious and unrestrained mice at each respective Ta (data from Refs. 61, 62, 66). D: leukocyte count at 10 wk of age of JCL-ICR mice bred and reared at each respective Ta (average of males and females is shown; data from Ref. 67). 418
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performed within the TNZ, but below the TNZ the same dose induces hypothermic shock, and Tb falls below 34°C (57). LPS stimulates different hypothalamic nuclei in rats housed within their TNZ compared with below the TNZ (63). Testing the immune response of rodents might be misleading if the tests are conducted at normal animal house or laboratory Ta.
At What Temperature Should We Be Housing Mice? That the phenotype and physiology of a mouse housed at 21°C is different to that of one housed at 30°C is clear, raising the question of whether studies on mice below their LCT have any relevance to human physiology? Translating results from mice at their conventional housing temperature might not be a problem if humans did indeed spend much of their lives below the TNZ, but whenever possible, humans use any means available, such as added insulation or heating and air conditioning, to maintain a state of thermal comfort. Especially in the developed world, we spend little, if any, of our time in the cold. By 1997, 47% of households in the U.S. were air-conditioned; by 2006, so were 50% of houses in New South Wales, Australia (22), and by 2007, 82% of homes in Perth, Western Australia, were air-conditioned (44), whereas in the UK and U.S. home and bedroom temperatures have increased by 3– 4°C over the last 30 years (27). Workplace temperatures appear to be increasing similarly (27). Behavioral thermoregulatory studies in mice and other rodents confirm that, when given the opportunity, rodents also will seek and maintain a zone of thermal comfort, especially, but not limited to, their periods of inactivity (20, 21). Speakman and Keijer (60) have advocated that mice should be housed in the cold, arguing that the average daily metabolic rate of free-living humans is ⬃1.6 times BMR, and so exposing mice to 20 – 22°C, which induces a metabolic rate of 1.6 times BMR, makes them an appropriate model for the human condition. However, it is unlikely that the “activity metabolism” of free-living humans is either physiologically or genetically similar to the cold-induced metabolism of a sleep-deprived and hypertensive mouse exposed to 21°C. There are factors other than Ta to consider when housing mice and interpreting data (40). The provision of nesting material to mice increases pup survival, litter size, and weaning body mass, and nest building varies with Ta. Given a choice of thermal environments, mice with low activity levels prefer a Ta of 25 or 30°C more than 20°C, irrespective of whether they have nesting material (18). Animal behavior, age, sex, strain, and time of day all alter thermal preferences, making it difficult
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stress can have profound effects on the phenotype that results from a given genotype. It is not just in tumor resistance but also in the physiological response to infection that thermoneutrality matters. After discovering that the mortality of mice infected with Typhus rickettsiae increased when the environmental temperature decreased, Moragues and Pinkerton in 1944 (46) commented that “by controlling the environmental temperature, conditions may be created under which murine typhus will have any desired degree of mortality.” Housing mice below their TNZ results in lower concentrations of circulating leukocytes (67) (FIGURE 5D), suppresses the release of pro-inflammatory cytokines, and increases the release of anti-inflammatory cytokines (30). Additionally, at 21°C, the entry of the pro-inflammatory cytokine IL-1 into the brain, where it exerts its pyrogenic effect, is impeded (9). It seems that multiple steps in the immune response pathway are altered when mice are housed below their TNZ. It is probably these differences in cytokine release and action that results in clear differences in the acute phase fever response at different Ta. Injecting mice with 104 g/kg of lipopolysaccharide (LPS), the pyrogenic moiety of the cell wall of Gram-negative bacteria, results in a robust fever with a Tb rise of ⬃2°C when the experiment is
REVIEWS to generalize about housing temperatures for all mice (18). Other factors like the number of animals per cage, noise, light, and human interference also are likely to introduce stressors that alter the physiology of the laboratory-housed mouse. Although reducing the challenges to thermal homeostasis by housing mice within their TNZ (21), or allowing them to control Ta (18), is an approach more likely to accurately reflect the physiology and pathophysiology of the modern human, the infrastructure changes required to keep rodents within their TNZ makes the prospect a long-term challenge. As proposed by Lodhi and Semenkovich (43) when they suggested (with tongue firmly in cheek) that mice should be clothed, we nevertheless must be aware of the nuances of environmental stressors, and particularly Ta, when translating data from mice to men. 䡲
Hansen J, Hérault Y, Hicks G, Hörlein A, Houghton R, Hrabé de Angelis M, Huylebroeck D, Iyer V, Jong P, Kadin J, Kaloff C, Kennedy K, Koutsourakis M, Kent Lloyd KC, Marschall S, Mason J, McKerlie C, McLeod M, Melchner H, Moore M, Mujica A, Nagy A, Nefedov M, Nutter L, Pavlovic G, Peterson J, Pollock J, Ramirez-Solis R, Rancourt D, Raspa M, Remacle J, Ringwald M, Rosen B, Rosenthal N, Rossant J, Ruiz Noppinger P, Ryder E, Schick J, Schnütgen F, Schofield P, Seisenberger C, Selloum M, Simpson E, Skarnes W, Smedley D, Stanford W, Francis Stewart A, Stone K, Swan K, Tadepally H, Teboul L, Tocchini-Valentini G, Valenzuela D, West A, Yamamura Ki Yoshinaga Y, Wurst W. The mammalian gene function resource: the international knockout mouse consortium. Mamm Genome 23: 580 –586, 2012. 7.
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The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does the mention of trade names of commercial products constitute endorsement or recommendation for use. No conflicts of interest, financial or otherwise, are declared by the author(s). Author contributions: S.K.M. and D.M. conception and design of research; S.K.M. prepared figures; S.K.M. drafted manuscript; S.K.M., A.F., D.M., C.J.G., and M.O. edited and revised manuscript; S.K.M., A.F., D.M., C.J.G., and M.O. approved final version of manuscript.
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We are grateful to Gary Sieck for the invitation to create and submit this article, and to Mike Joyner who started it all with an invitation to present a review at IUPS. Prof. Tobias Wang provided comments that helped to improve the manuscript.
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