Effects of food restriction on energy metabolism in male Apodemus chevrieri from Hengduan mountain region of China

Adaptive regulation of energy metabolism is important for animals to cope with the natural environmental changing. In the natural environment, many animals are faced with seasonal changes in food resources (Kamalasundari and Premalatha 2014). Energy strategies for food quantity changes in non hibernating small mammals are roughly divided into two categories: one is the reduction of energy metabolism under food shortage conditions (Naseer et al., 2018), such as MF1 Mus musculus (Hambly and Speakman, 2005), Acomys russatus (Gutman et al., 2007); the other is that the level of energy expenditure remains unchanged and even increased under food restriction, such as KM Mus musculus and Cavia porcellus (Williams et al., 2002; Zhao et al., 2009). “Metabolism switch hypothesis” pointed that the key to whether animals can adapt to changes in food resources or if they have the abilities to regulate metabolic rates under food restriction, which is possible to adapt to the chronic food shortage environment by changing the metabolic rate and decreasing the metabolic levels (Merkt and Taylor, 1994). Moreover, the choice of adaptation strategies for animals is also influenced by their environmental conditions, as well as their living habits. Researches showed that with the food storage habits of Gerbillus dasyurus after being restricted to 50% of ad libitum food intake, which can only survive for 2 weeks, but under the same food restriction level, Acomys russatus that do not have food storage habits can live for at least 6 weeks or even longer (Gutman et al., 2006). Therefore, it is assumed that animals with food storage habits have lower tolerance to food restriction. Whether this hypothesis is universal and how the species with food storage habits respond to food resource scarcity depends on its ability to metabolize rate change, which is not yet clear.
        
Chevrier’s field mouse, Apodemus chevrieri (Mammalia: Rodentia: Muridae) is an inherent species in Hengduan mountain region of China, which has food storage behavior in winter and it is the host of rat epidemic disease in the Hengduan mountains region. Previous studies demonstrated the presence of a seasonal variation in body mass, thermogenesis and digestive tract morphology in A. chevrieri (Zhu et al., 2012). During cold exposure, A. chevrieri showed increased energy intake, thermogenesis and reduced body mass, and serum leptin levels (Zhu et al., 2011). Food restriction alone may also reduce body mass in A. chevrieri (Zhu et al., 2013). Random food deprivation decreased body fat mass and increased activity significantly (Zhu et al., 2016). It can be seen that the change of food quantity may play an important role in the evolutionary adaptation of thermogenesis in A. chevrieri. On  the basis of the above studies, body mass, resting metabolic rate (RMR), nonshivering thermogenesis (NST) and cytochrome c oxidase (COX) activity in A. chevrieri with different levels of food restriction were measured. The aim of the present study was to elucidate the relationship between the energy response of food shortage and its metabolic level, and to test “metabolism switch hypothesis”. We hypothesize that A. chevrieri will change their thermogenesis and body mass to cope with food restriction.

Animals and experimental design
 
A. chevrieri were obtained from a laboratory colony, founded by animals captured from farmland (26°15¢–26°45¢N; 99°40¢–99°55¢E; altitude 2,590m) in Jianchuan County, Yunnan province. Adult male A. chevrieri (120 days of age) were housed individually in plastic boxes (26×16×15cm3). Animals were kept in a room temperature of 25±1°C with a photoperiod of 12L:12D (with lights on at 08:00 h) and provided food (standard rabbit chow produced by Kunming Medical University, Kunming) and water ad libitum. All animal procedures were licensed under the Animal Care and Use Committee of School of Life Sciences, Yunnan Normal University (Permit No.: 13-0901-011).
 
Experiment 1
 
Effects of food restriction and cold temperature on energy metabolism. 20 adult weight-matched A. chevrieri were randomly assigned to the following two groups: control group (25±1°C) and cold group (5±1°C), each group consisted 10 animals. All the animals were acclimated for 4 weeks (d-28~d0). Then two groups were acclimated another 7 days under 80% of ad libitum food intake. Survival rate were recorded from day 0 to 7. On day 7, body mass, RMR, NST and COX activity in brown adipose tissue (BAT) were measured.
 
Experiment 2
 
Effects of food restriction and metabolic level on energy metabolism. RMR of 30 adult A. chevrieri were measured, 10 animals with higher RMR as high RMR (HR) group and another 10 animals with lower RMR as lower RMR (LR) group were selected. Food intake was measured for both the groups, then two groups acclimated 14 days under 80% of ad libitum food intake. Immediately after sacrifice on day 14, liver and BAT were excised and weighed (±1 mg). The carcasses was recorded after removal of visceral organs and digestive tract. The remaining carcass was dried to constant mass in an oven at 60°C (for at least 72 h) and then weighed again to obtain a dry mass. Total body fat was extracted from the dried carcass by ether extraction in a Soxhlet apparatus (Zhao et al., 2014). On day 14, body mass, RMR, NST and COX activity in liver and BAT were measured.
 
Measurement of metabolic rates
 
Metabolic rates were measured using an AD ML870 open respirometer (AD Instruments, Australia) at 25°C within the thermal neutral zone, and gas analysis was performed using a ML206 gas analysis instrument (AD Instruments). The temperature was controlled using a SPX-300 artificial climatic incubator (±0.5°C) (Changsha, China), the metabolic chamber volume was 500ml and airflow rate was 200 ml/min. Animals were stabilized in the metabolic chamber for at least 60 min prior to the RMR measurement, and oxygen consumption was recorded for at least 120 min at 1 min intervals. Ten stable consecutive low readings were taken to calculate RMR following Li and Wang (2005), using the method for calculating the metabolic rate provides by Hills (1972).
        
NST was induced by a subcutaneous injection of norepinephrine (NE) (Shanghai Harvest Pharmaceutical Co. Ltd, China) and measured at 25°C. Two consecutive high oxygen consumption readings from each 60-min measurement were taken to calculate NST (Li and Wang, 2005). The doses of norepinephrine were approximately 0.8-1.0 mg/kg, according to dose-dependent response curves generated before the experiment and using the equation of Heldmaier (1971).
 
Measurement of protein content of mitochondria and enzyme activity
 
Liver and BAT were carefully and quickly removed and weighted (0.1mg) and their adhering tissues separated. The organs were blotted, weighed, and placed in ice-cold sucrose-buffered medium and then homogenized for the isolation of mitochondria (Cannon and Lindberg, 1979). The protein content of mitochondria was determined by the Folin phenol method with bovine serum albumin as standard (Lowry et al., 1951). The COX (EC 1.9.3.1) activity of BAT was measured with polarographic method using oxygen electrode (Hansatech Instruments LTD., England) (Sundin et al., 1987).
 
Statistical analysis
 
Data were analyzed using SPSS 15.0 software package. Prior to all statistical analyses, data were examined for assumptions of normality and homogeneity of variance, using Kolmogorov- Smirnov and Levene tests, respectively. Body mass, RMR, NST and COX activity were analyzed by one-way analysis of variance (ANOVA) and significant group differences were further evaluated by Tukey post hoc test. Results were presented as mean ± SE and P < 0.05 was considered to be statistically significant.

Effects of food restriction and cold temperature on energy metabolism
 
At room temperature, FR-80% of A. chevrieri were acclimated to 7 day, the survival rate was 100%. But FR-80% group under cold acclimation, A. chevrieri died on day 5 and survival rate was 60% on day 7. Temperature had significant effect on body mass in A. chevrieri under FR- 80% acclimation, body mass in FR-80% and warm group decreased 8.46% than that of on day 0 and body mass in FR-80% and cold group decreased 12.9% than that of on day 0 (Fig 1). Food intake, RMR and NST increased at 4 weeks after cold acclimation, which were significantly higher than that in warm temperature group (Food intake: F_1,18= 8.39, P<0.01; RMR: F_1,18= 3.69, P<0.05, Fig 2A; NST: F_1,18= 8.96, P<0.01, Fig 2B). After 7day of FR-80% acclimation, RMR and NST in cold group were also higher than that of warm group (RMR: F_1,14= 4.58, P<0.01, Fig 2A; NST: F_1,14= 9.54, P<0.01, Fig 2B). On day 7, the protein content of mitochondria and COX activity in BAT were significant higher than that of warm group (protein content of mitochondria: F_1,14= 3.21, P<0.05, Fig 2C; COX activity: F_1,14= 3.81, P<0.05, Fig 2D).
 

 

        
Temperature is one of the important environmental factors that affect metabolic rate (Wang et al., 2006). Small mammals domesticated in cold condition may increase their metabolic rate (Klingenspor, 2003; Tang et al., 2009; Chi and Wang, 2011). In the present study, cold acclimation increased RMR significantly in A. chevrieri, which was consistent with the increase of food intake. Despite the cold acclimation increased food intake, but body mass in FR-80% and cold group decreased 12.9% than that of on day 0, the survival rate was only 60%, suggesting that cold temperature acclimation increased metabolism levels, resulting in lower tolerance of animals to food restriction, which is consistent with the prediction that higher level of metabolic rate may be the main reason for low tolerance to food restriction in A. chevrieri. The reason why A. chevrieri exhibit low tolerance to food restriction is uncertain. At the interspecific level, the researchers compared the metabolic levels of A. chevrieri with other rodents, which was found that A. chevrieri had smaller size and higher metabolic level (Zhu et al., 2008). A large number of studies showed that higher metabolic rate means higher demand for energy, so they need to increase food intake to supplement the metabolic energy expenditure, compared with the larger animals, the metabolic rate is higher and more easily influenced by food shortages, higher metabolic rate in smaller mammals were more likely to be affected by food shortage (Zhao et al., 2012).
 
Effects of food restriction and metabolic level on energy metabolism
 
Before the experiment, food intake between HR and LR groups had significant differences, which was significantly higher in HR than that in LR (F_1,18=10.36, P<0.01). On day 0, RMR in HR was 22.13% higher that of LR (Table 1). FR-80% of A. chevrieri were acclimated to 14 d, A. chevrieri appeared died both in two groups, survival rate were 80% and 90% in HR and LR groups, respectively. However, FR-80% acclimation on day 14 had no effect on body mass, carcass mass, body fat mass, NST, the protein content of mitochondria and COX activity in BAT and liver. On day 14, RMR in HR was also significantly higher that of LR (Table 1).
 

        
There were interspecific differences in metabolic rates, and also had individual differences at the intraspecific level (Savsani et al., 2015). “Metabolism switch hypothesis” pointed that animals can regulate their metabolic rates under a chronic food shortage by reducing metabolic levels (Merkt and Taylor, 1994). Many studies have found that food shortage leads to a significant reduction in metabolism (Hambly et al., 2007; Zhao et al., 2012). In the present study, there were significant differences in RMR between individuals, food restriction decreased RMR significantly in HR group. Similar to A. chevrieri, food restriction reduced metabolic rate in Meriones crassus, which was consistent with “metabolism switch hypothesis” (Gutman et al., 2007). LR group had lower food intake, but food restriction did not decrease its RMR significantly, suggesting that food intake is not enough to compensate for energy expenditure, so A. chevrieri were in the state of negative energy balance.

Cold-exposed A. chevrieri increased RMR and NST significantly with decreased body mass and survival rate. Food restriction decreased RMR significantly in HR group, which suggested that A. chevrieri characterized by higher levels of metabolism were sensitive to periods of FR, providing a support for the “metabolism switch hypothesis”.

This research was financially supported by National Science Foundation of China (No. 31760118; 31560126) and Young and Middle-aged Academic and Technical Leaders Reserve Talents Project of Yunnan (2019HB013). We wish to thank Pro. Burkart Engesser at Historisches Museum Basel, Switzerland for correcting the English usage in the draft. Thank you for the anonymous reviewers and the editor of the journal for their valuable comments.