Catherine Hambly

A guide to analysis of mouse energy metabolism

We present a consolidated view of the complexity and challenges of designing studies for measurement of energy metabolism in mouse models, including a practical guide to the assessment of energy expenditure, energy intake and body composition and statistical analysis thereof. We hope this guide will facilitate comparisons across studies and minimize spurious interpretations of data. We recommend that division of energy expenditure data by either body weight or lean body weight and that presentation of group effects as histograms should be replaced by plotting individual data and analyzing both group and body-composition effects using analysis of covariance (ANCOVA).

Animal models of obesity

Background Obesity stems from a prolonged imbalance between the levels of energy intake and expenditure, with the resultant surplus being stored as body lipids. Our understanding of the regulation of food intake and the physiological basis of differences in energy expenditure is owed, in large part, to studies made on animals. Moreover, animal models have been a cornerstone of studies of environmental effects, such as epigenetics, responses to high-fat and low-calorie diets and the identification and development of pharmaceuticals for obesity treatment. This review provides some examples of the animal work that has been performed, and focuses on the variation in approaches that have been taken and their potential, rather than aiming to be a comprehensive summary.

The contribution of animal models to the study of obesity

Summary Obesity results from prolonged imbalance of energy intake and energy expenditure. Animal models have provided a fundamental contribution to the historical development of understanding the basic parameters that regulate the components of our energy balance. Five different types of animal model have been employed in the study of the physiological and genetic basis of obesity. The first models reflect single gene mutations that have arisen spontaneously in rodent colonies and have subsequently been characterized. The second approach is to speed up the random mutation rate artificially by treating rodents with mutagens or exposing them to radiation. The third type of models are mice and rats where a specific gene has been disrupted or overexpressed as a deliberate act. Such genetically-engineered disruptions may be generated through the entire body for the entire life (global transgenic manipulations) or restricted in both time and to certain tissue or cell types. In all these genetically-engineered scenarios, there are two types of situation that lead to insights: where a specific gene hypothesized to play a role in the regulation of energy balance is targeted, and where a gene is disrupted for a different purpose, but the consequence is an unexpected obese or lean phenotype. A fourth group of animal models concern experiments where selective breeding has been utilized to derive strains of rodents that differ in their degree of fatness. Finally, studies have been made of other species including non-human primates and dogs. In addition to studies of the physiological and genetic basis of obesity, studies of animal models have also informed us about the environmental aspects of the condition. Studies in this context include exploring the responses of animals to high fat or high fat/high sugar (Cafeteria) diets, investigations of the effects of dietary restriction on body mass and fat loss, and studies of the impact of candidate pharmaceuticals on components of energy balance. Despite all this work, there are many gaps in our understanding of how body composition and energy storage are regulated, and a continuing need for the development of pharmaceuticals to treat obesity. Accordingly, reductions in the use of animal models, while ethically desirable, will not be feasible in the short to medium term, and indeed an expansion in activity using animal models is anticipated as the epidemic continues and spreads geographically.

Faecal corticosterone concentrations indicate that separately housed male mice are not more stressed than group housed males.

Mice account for over 80% of all animals used in experimentation. This study investigated how different housing conditions affected stress levels by measuring both corticosterone levels, using non-invasive faecal collection, and behaviour. Sixty outbred MF1 male mice were used which were separated into five different housing conditions at the beginning of the study, (A) individually housed, floor area 490 cm(2) per individual, (B) groups of three mice, floor area 163 cm(2) per individual, (C) groups of three mice, floor area 320 cm(2) per individual, (D) groups of six mice, floor area 160 cm(2) per individual, (E) groups of six mice, floor area 230 cm(2) with extra height per individual to allow visual contact. Mice in all housing conditions were provided with a basic enrichment of paper bedding and a plastic house. The results from this study showed that singly housed mice reduced their corticosterone levels over time after separation reaching a minimum from 14 days onwards. Groups of 6 mice housed together showed no difference over time. Also there was no significant difference in corticosterone levels between the different housing densities, with no differences for aggression or stereotypical behaviour suggesting that there is no ideal group density for this strain and sex of mouse. Providing additional enrichment to the cages caused a significant decrease in corticosterone levels for group housed mice, but individually housed mice remained unaffected by increasing their enrichment level. They spent significantly more time sleeping in the enhanced cage but without any reduction in stereotypical behaviour. For group housed mice, additional enrichment should be mandatory to reduce stress levels and therefore increase their welfare standards, while singly housed mice required only basic levels of enrichment and should be separated from their group for a minimum of 2 weeks before measurements are taken.

Ambient temperature shapes reproductive output during pregnancy and lactation in the common vole (Microtus arvalis): a test of the heat dissipation limit theory

SUMMARY The heat dissipation limit theory suggests that heat generated during metabolism limits energy intake and, thus, reproductive output. Experiments in laboratory strains of mice and rats, and also domestic livestock generally support this theory. Selection for many generations in the laboratory and in livestock has increased litter size or productivity in these animals. To test the wider validity of the heat dissipation limit theory, we studied common voles (Microtus arvalis), which have small litter sizes by comparison with mice and rats, and regular addition of wild-caught individuals of this species to our laboratory colony ensures a natural genetic background. A crossover design of ambient temperatures (21 and 30°C) during pregnancy and lactation was used. High ambient temperature during lactation decreased milk production, slowing pup growth. The effect on pup growth was amplified when ambient temperature was also high during pregnancy. Shaving fur off dams at 30°C resulted in faster growth of pups; however, no significant increase in food intake and or milk production was detected. With increasing litter size (natural and enlarged), asymptotic food intake during lactation levelled off in the largest litters at both 21 and 30°C. Interestingly, the effects of lactation temperature on pup growth where also observed at smaller litter sizes. This suggests that vole dams trade-off costs associated with hyperthermia during lactation with the yield from investment in pup growth. Moreover, pup survival was higher at 30°C, despite lower growth, probably owing to thermoregulatory benefits. It remains to be seen how the balance is established between the negative effect of high ambient temperature on maternal milk production and pup growth (and/or future reproduction of the dam) and the positive effect of high temperatures on pup survival. This balance ultimately determines the effect of different ambient temperatures on reproductive success.

Comparison of the cost of short flights in a nectarivorous and a non-nectarivorous bird

SUMMARY Although most birds are accustomed to making short flights, particularly during foraging, the flight patterns during these short periods of activity differ between species. Nectarivorous birds, in particular, often spend time hovering, while non-nectarivorous birds do not. The cost of short flights is likely therefore to differ between nectarivorous and non-nectarivorous birds because of the different energetic contributions of different flight types to the behaviour. The 13C-labelled bicarbonate technique was used to measure the energy cost of short flights in the nectarivorous Palestine sunbird Nectarinia osea (mean mass 6.17±0.16 g, N=8) and the non-nectarivorous starling Sturnus vulgaris (mean mass 70.11±1.11 g, N=9). The technique was initially calibrated in five individuals for each species at temperatures ranging from 1 to 35°C, by comparing the isotope elimination rate to the metabolic rate measured simultaneously by indirect calorimetry. The cost for short intermittent flight was then measured by encouraging birds to fly between two perches at either end of a narrow corridor (perch distance for sunbirds, 6 m; for starlings, 5 m), and measuring the amount of isotope eliminated during the flight. The isotope elimination rate was interpolated onto the calibration equation to predict flight cost, as a direct calibration could not be performed during flight. Mean energy expenditure during flight was 1.64±0.32 W in sunbirds, while in starlings the flight costs averaged 20.6±0.78 W. Energy cost of flight relative to basal metabolic rate was substantially greater in the starling than the sunbird. Phylogenetic analysis of different modes of flight in these and additional species suggests that differences in flight behaviour may cause these elevated costs in slow flying non-nectarivores such as starlings, compared to birds that are more prone to short intermittent flights like the sunbirds.

The energy cost of loaded flight is substantially lower than expected due to alterations in flight kinematics

SUMMARY The effect of experimentally increased wing loading on the energy cost of flight was examined in cockatiels Nyphicus hollandicus. Five individuals were flown for periods of approximately 2 min, while carrying additional payload mass amounting to between 5 and 20% of unloaded body mass. The energy cost of flight was measured using the 13C-labelled bicarbonate technique, which was also calibrated in a separate experiment on resting birds, by comparing the elimination rate of 13C in breath with a simultaneous measurement of oxygen consumption by indirect calorimetry. It was not possible to perform a similar calibration during flight when energy costs were higher, so we extrapolated the relationship from the resting calibration to predict flight cost. Flight cost in the pre-manipulated individuals averaged 16.7±1.8 W. Flight cost in the pre-manipulated birds was significantly related to the interaction between downstroke duration and flight speed. There was no significant increase in flight cost with increases in payload mass. The birds responded to payload masses between 5 and 15% of their unloaded body mass by decreasing flight speed relative to unloaded birds, while maintaining wing beat frequency (Fb). At a payload mass equivalent to 20% of body mass, however, the birds flew at higher speeds than unloaded controls, and had a significantly higher Fb, generated by a reduction in both the upstroke and downstroke durations. Wing amplitude was unaffected by the increase in loading. Using the measured flight parameters, the effect of loading was not significantly different than predicted using aerodynamic models.

Sex differences in the effect of fish-oil supplementation on the adaptive response to resistance exercise training in older people: a randomized controlled trial

Background: Resistance exercise increases muscle mass and function in older adults, but responses are attenuated compared with younger people. Data suggest that long-chain n–3 polyunsaturated fatty acids (PUFAs) may enhance adaptations to resistance exercise in older women. To our knowledge, this possibility has not been investigated in men. Objective: We sought to determine the effects of long-chain n–3 PUFA supplementation on resistance exercise training–induced increases in muscle mass and function and whether these effects differ between older men and women. Design: Fifty men and women [men: n = 27, mean ± SD age: 70.6 ± 4.5 y, mean ± SD body mass index (BMI; in kg/m2): 25.6 ± 4.2; women: n = 23, mean ± SD age: 70.7 ± 3.3 y, mean ± SD BMI: 25.3 ± 4.7] were randomly assigned to either long-chain n–3 PUFA (n = 23; 3 g fish oil/d) or placebo (n = 27; 3 g safflower oil/d) and participated in lower-limb resistance exercise training twice weekly for 18 wk. Muscle size, strength, and quality (strength per unit muscle area), functional abilities, and circulating metabolic and inflammatory markers were measured before and after the intervention. Results: Maximal isometric torque increased after exercise training to a greater (P < 0.05) extent in the long-chain n–3 PUFA group than in the placebo group in women, with no differences (P > 0.05) between groups in men. In both sexes, the effect of exercise training on maximal isokinetic torque at 30, 90, and 240° s−1, 4-m walk time, chair-rise time, muscle anatomic cross-sectional area, and muscle fat did not differ (P > 0.05) between groups. There was a greater (P < 0.05) increase in muscle quality in women after exercise training in the long-chain n–3 PUFA group than in the placebo group, with no such differences in men (P > 0.05). Long-chain n–3 PUFAs resulted in a greater decrease (P < 0.05) than the placebo in plasma triglyceride concentrations in both sexes, with no differences (P > 0.05) in glucose, insulin, or inflammatory markers. Conclusion: Long-chain n–3 PUFA supplementation augments increases in muscle function and quality in older women but not in older men after resistance exercise training. This trial was registered at clinicaltrials.gov as NCT02843009.

Physiological and behavioral responses to intermittent starvation in C57BL/6J mice

The dual intervention point model states that body mass is controlled by upper and lower intervention points, above and below which animals (and humans) intervene physiologically to bring their body mass back into the acceptable range. It has been further suggested that the lower intervention point may be defined by the risk of starvation, while the upper intervention point may be defined by the risk of predation. The objective of the present study was to test whether the risk of starvation determines the lower intervention point and to examine the physiological and behavioral mechanisms that underpin the regulation of body mass, when the risk of starvation is increased. Sixty-four mice were exposed to random days of complete fasting or 50% food restriction and their body mass and fat mass responses were measured. Food intake, physical activity and body temperature were measured throughout the experiment. In addition, plasma leptin and insulin, triglyceride and non-esterified fatty acids, along with hypothalamic neuropeptides gene expression in the arcuate nucleus were assessed after 13 and 42 days of treatment. We found that C57BL/6J mice increased body mass and fatness in response to a short-term (13 days) intermittent fasting, which was restored to baseline as the treatment was prolonged. In contrast, intermittently 50% food restricted mice showed no significant changes in body mass or fatness. Over the first 13 days of treatment the data were consistent with the dual intervention point model as the mice showed both increased body mass and adiposity over this period. Over the more protracted period of 42 days the effect waned and was therefore inconsistent with the model. The body mass and fat mass gains in intermittently fasted mice were mainly accounted for by increased food intake. Elevated NPY gene expression after 13 days (three 24 h fasting events) may have driven the increase in food intake. However, no changes were observed in such neuropeptides as POMC, CART, AgRP, Ob-Rb and SOCS 3 or circulating levels of leptin, insulin, NEFA and TG. Hypothermia during fasting days may have also contributed to the increase in body mass. Over 42 days of treatment (nine 24 h fasting events) cumulative food intake was not affected by intermittent starvation. However physical activity, mainly activity during the light phase was lowered suggesting an adaptation to unpredictable starvation. Overall, mice exhibited different behavioral and physiological responses to intermittent starvation depending on the duration of treatment.

Limits to sustained energy intake. XVI. Body temperature and physical activity of female mice during pregnancy

SUMMARY Lactation is the most energy-demanding phase of mammalian reproduction, and lactation performance may be affected by events during pregnancy. For example, food intake may be limited in late pregnancy by competition for space in the abdomen between the alimentary tract and fetuses. Hence, females may need to compensate their energy budgets during pregnancy by reducing activity and lowering body temperature. We explored the relationships between energy intake, body mass, body temperature and physical activity throughout pregnancy in the MF1 mouse. Food intake and body mass of 26 females were recorded daily throughout pregnancy. Body temperature and physical activity were monitored every minute for 23 h a day by implanted transmitters. Body temperature and physical activity declined as pregnancy advanced, while energy intake and body mass increased. Compared with a pre-mating baseline period, mice increased energy intake by 56% in late pregnancy. Although body temperature declined as pregnancy progressed, this served mostly to reverse an increase between baseline and early pregnancy. Reduced physical activity may compensate the energy budget of pregnant mice but body temperature changes do not. Over the last 3 days of pregnancy, food intake declined. Individual variation in energy intake in the last phase of pregnancy was positively related to litter size at birth. As there was no association between the increase in body mass and the decline in intake, we suggest the decline was not caused by competition for abdominal space. These data suggest overall reproductive performance is probably not constrained by events during pregnancy.