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Journal of Stress Physiology & Biochemistry, Vol. 10 No. 1 2014, pp. 26-36 ISSN 1997-0838 Original Text Copyright © 2014 by Zhu, Yang, Gao, Zhang, Wang
ORIGINAL ARTICLE
Effect of Cold Temperature and Food Restriction on Energy Metabolism and Thermogenesis in Eothenomys miletus
Wan-Long Zhu#, Sheng-Chang Yang, Wen-Rong Gao#, Lin Zhang, Zheng-Kun Wang*
Institute of Zoology, School of life Science of Yunnan Normal University, Kunming 650500, China
#Zhu WL and Gao WR contributed equally to this work.
*E-Mail: zwl_8307@163.com
Received August 8, 2013
The aim of the present study was to examine the energy strategy in response to the cold temperature and food shortage. The survival rate, body mass, body fat content, serum leptin levels, basal metabolic rate (BMR) and nonshivering thermogenesis (NST) as well as masses and the morphology of visceral organs and the digestive tract were measured in Eothenomys miletus that was subjected to the cold temperature (5°C) and food restriction (80% of ad libitum food intake). The results showed that body mass, body fat content, serum leptin levels, brown adipose tissue (BAT) mass and dry mass of digestive tract in cold and food restriction group were lower than those in control group. In contrasts, BMR and NST in cold and food restriction group was significantly higher relative to control group. The rate of survival was 18.18% in E. miletus during cold and food restriction after 4 weeks acclimation. In addition, serum leptin levels were positively correlated with body mass and body fat content, and negatively correlated with BMR and NST. These results suggested that E. miletus apply physiological adjustments to adapt cold and food lacking external environment by reducing body mass, body fat content, and increasing energy metabolism. However, energy intake is insufficient to compensate for the increase in energy requirement due to cold, led to body mass decreased and mortality rate increased. Moreover, serum leptin may acts as a fat signals, and may be involved in the regulation of energy balance and body mass in E. miletus under the cold temperature and food restriction.
Key words: Cold acclimation, Food restriction, Serum leptin levels, Eothenomys miletus
ORIGINAL ARTICLE
Effect of Cold Temperature and Food Restriction on Energy Metabolism and Thermogenesis in Eothenomys miletus
Wan-Long Zhu#, Sheng-Chang Yang, Wen-Rong Gao#, Lin Zhang, Zheng-Kun Wang*
Institute of Zoology, School of life Science of Yunnan Normal University, Kunming 650500, China
# Zhu WL and Gao WR contributed equally to this work.
*E-Mail: zwl_8307@163.com
Received August 8, 2013
The aim of the present study was to examine the energy strategy in response to the cold temperature and food shortage. The survival rate, body mass, body fat content, serum leptin levels, basal metabolic rate (BMR) and nonshivering thermogenesis (NST) as well as masses and the morphology of visceral organs and the digestive tract were measured in Eothenomys miletus that was subjected to the cold temperature (5°C) and food restriction (80% of ad libitum food intake). The results showed that body mass, body fat content, serum leptin levels, brown adipose tissue (BAT) mass and dry mass of digestive tract in cold and food restriction group were lower than those in control group. In contrasts, BMR and NST in cold and food restriction group was significantly higher relative to control group. The rate of survival was 18.18% in E. miletus during cold and food restriction after 4 weeks acclimation. In addition, serum leptin levels were positively correlated with body mass and body fat content, and negatively correlated with BMR and NST. These results suggested that E. miletus apply physiological adjustments to adapt cold and food lacking external environment by reducing body mass, body fat content, and increasing energy metabolism. However, energy intake is insufficient to compensate for the increase in energy requirement due to cold, led to body mass decreased and mortality rate increased. Moreover, serum leptin may acts as a fat signals, and may be involved in the regulation of energy balance and body mass in E. miletus under the cold temperature and food restriction.
Key words: Cold acclimation, Food restriction, Serum leptin levels, Eothenomys miletus
Energy metabolism plays an important role in distribution, richness and survival of species (Bozinovic, 1992). In the natural environment, the level of energy metabolism of wild animal is affected by many environmental factors, including
temperature (Abelenda et al., 2003) and food (Bacigalupe & Bozinovic, 2002). Due to low temperature in winter, energy consumption increased and the available food resource relative reduced for mammals, which are important
environmental factor affecting the metabolism and physiological action of energy of small mammals (Nagy et al., 1995). Leptin is a protein hormone secreted by fat cells, and plays an important role in the regulation of energy homeostasis and body mass of the animals, higher leptin concentration can inhibit the food intake, promote heat production and reduce body mass (Friedman and Halaas, 1998). Researches showed that the injection of exogenous leptin can inhibit energy intake, improve energy consumption (Abelenda et al., 2003). Abelenda (2003) studies showed that leptin is involved in the heat regulation and food intake under cold conditions. Many studies have showed that food restriction decreased the serum leptin concentration, and leptin was positively correlated with body mass, heat production ability (Rousseau et al., 2003), which indicated that serum leptin may be a hunger signals involved in adjustment of body mass and heat production under food deficient conditions (Zhao and Wang, 2007).
The Hengduan Mountains region is located the boundary between the Palaearctic region and the Oriental region. It is alpine with high mountains and gorges; the diversity and abundance of mammals is high and it is considered to be "the harbor in fourth ice age". Therefore small mammals may differ from those from other regions. Eothenomys were the proper genus in China, were a special group in Microtus, and were the typical animals in Hengduan Mountains region. E. miletus is the inherent species in Hengduan mountains region (Zhu et al., 2012a), has special status in Microtus. In the present study, we examined the effect on body mass, thermogenesis and body composition in cold-acclimated E. miletus under food restriction. We predicted that E. miletus change their body mass,
thermogenesis and body composition under simulated winter conditions, and leptin would be involved in the regulation of body mass and thermogenesis.
MATERIALS AND METHODS
Samples
E. miletus were captured in farmland (26°15 ~26°45N; 99°40~99°55E;altitude 2,590m) in
Jianchuan County, Yunnan province, 2011. Average temperature is 9.1°C each year, minimum average temperature was -4.0°C in January, maximal average temperature was 24.1°C in July.
E. miletus were breed for two generations in School of life Science of Yunnan Normal University, park in plastic box(260mmx160mmx150mm), one in a box without any bedding material, and were maintained at the room temperature of 25±1°C, under a photoperiod of 12L:12D (with lights on at 08:00). After 1 month stabilization, animas were randomly divided into two groups(n=41). Food intake were measured by the food balance (Rosenmann and Morrison, 1974), a total of 5 days, quantitative timing (before day 11: 00 - 13: 00, food 9.5 G - 10.5 g) for animal feeding, the collecting, weighing surplus food (each collection time were in 11: 00 - 13: 00), calculation of the 5 day average food intake as part of their normal daily intake. Then all animal randomly divided into control group: free feeding at room temperature
(temperature: 25 ± 1 °C) (n=8); experimental group: 80% normal food intake and low temperature (temperature: 5 ± 1 C) in (n=33) last for 28 days. The survival rate was measured daily animal, every 7 days to measure body mass, BMR and NST. The voles were sacrificed between 09:00 and 11:00 by puncture of the posterior vena cava. Blood was centrifuged at 4000 rpm for 30 min, and serum was
sampled and stored at -20°C for later measurement. All voles were dissected to evaluate organ morphology. All pregnant, lactating or young individuals were excluded.
Measurement of metabolic rates
Metabolic rates were measured by using AD ML870 open respirometer (AD Instruments, Australia) at 30°C within the TNZ (thermal neutral zone), gas analysis were using ML206 gas analysis instrument, the temperature was controlled by SPX-300 artificial climatic engine (±0.5°C), the metabolic chamber volume is 500 ml, flow is 200 ml/min. The tree shrew were stabilized in the metabolic chamber for at least 60 min prior to the RMR measurement, oxygen consumption was recorded for more than 60 min at 5 min intervals. Two stable consecutive lowest readings were taken to calculate RMR (Zhu et al., 2010). Calculate method of metabolic rate is detailed by Hills (1972) (Hill,
1972). All metabolic measurements were performed more than 400 hours.
Nonshivering thermogenesis (NST) was induced by subcutaneous injection of norepinephrine (NE) (Shanghai Harvest Pharmaceutical Co. Ltd) and measured at 25°C. Two consecutive highest recordings of oxygen consumption more than 60 min at each measurement were taken to calculate the NST (Zhu et al., 2010).
Measurement of serum leptin level and thyroid hormones
Serum leptin levels were determined by radioimmunoassay (RIA) with the 125I Multi-species Kit (Cat. No. XL-85K, Linco Research Inc.).The lowest level of leptin that can be detected by this assay was 1.0 ng/ml when using a 100-^l sample size. And the inter- and intra-assay variability for leptin RIA were <3.6% and 8.7%, respectively.
Body composition
Gastrointestinal tract (stomach, small intestine, cecum, large intestine) and the heart, lungs, pancreas, spleen, kidneys were extracted and weighed (±1 mg). The stomach and intestines were rinsed with saline to eliminate all the gut contents, before being dried and weighed. The remaining carcass and all the organs were dried in an oven at 60 °C to constant mass (at least 72 h), and then weighed again to obtain the dry mass. The remaining carcass and all the organs were dried in an oven at 60°C to constant mass (at least 72 h), and then weighed again to obtain the dry mass. The difference between the wet carcass mass and dry carcass mass was the water mass of carcass. Total body fat was extracted from the dried carcass by ether extraction in a Soxhlet apparatus (Zhao and Wang, 2007).
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. Throughout the acclimation, animal survival rate, body mass, energy intake, body fat content, BMR, NST and body composition were analyzed by were analyzed by independent t-test. Pearson's correlation was performed to detect possible correlations among serum leptin and body mass, body fat mass, RMR and NST. Results were presented as mean ± SEM, and P < 0.05 was considered to be statistically significant.
RESULTS
Changes of survival rate, body mass and body fat content
Before the experiment, no significant difference of body mass between low temperature restriction
group and control group (t=-0.221, P>0.05, Table 1). It showed an significant difference between the two groups in 7 day (t=7.005, animal P<0.01), body mass in low temperature food restricted group were 13.44% lower than that in control group. It showed significant difference in 28 day (t=37.56, P<0.01), low temperature food restricted group were lower 33.55% than that in control group (fig 1). After the acclimation, food restricted group survival rate was 18.18% at low temperature, the control group had no death occurred, there also had an significant difference of body fat content between two groups (t=5.818, P<0.01) (Table 1). During acclimation, body mass in low temperature and food restricted group decreased significantly (F=60.51, P<0.01), but the control group remained stable (F=0.863, P>0.05) (fig 1).
RMR and NST
Before the experiment, no significant difference of RMR and NST were found between cold and food
restriction group and the control group (RMR: t =-1.219, P>0.05; NST: t=1.191, P>0.05). During the acclimation, RMR and NST had no significant change in control group (BMR: F=0.247, P>0.05; NST: F=0.347, P>0.05), but RMR increased
significantly in cold and food restriction group (F=7.139, P<0.01), RMR showed significant
difference between two groups (RMR: T, =-6.159, P<0.01), and increased by 82.25% than that of the control group; NST in cold and food restriction group increased significantly in 28 day (F=11.228, P<0.01), NST significantly higher than the control group in 7 day and 14 day (7 days: NST: t =-5.918, P<0.01; 14 days: NST: t =-6.328, P<0.01,
respectively), increased by 64.72% and 56.67% than that of the control group. It had no significant difference of NST in 21 days and 28 days (21 days: NST: t =-2.206, P>0.05; 28 days: NST: t =-1.19, P>0.05) (fig 2).
Figure 1. Body mass in E. miletus during cold and food restriction. Different letters indicate significant differences (P<0.05) (**P<0.01)
Figure 2. BMR and NST in E. miletus during cold and food restriction. Different letters indicate significant differences (P<0.05) (**P<0.01)
2 3 4 5
Rest metabolic rate (mKVg-h)
Figure 3. Correlation of serum leptin levels with body mass (A), body fat content (B), BMR (C) and NST (D) in E. miletus
Table 1 : Effect of cold and food restriction on rate morphology of digestive organs in E. miletus. of survival, body fat content, organ mass, and
Cold and food restriction Control group t p
Rate of survival (%) 18.18 100 vs vs
Body mass (g)
Initial 41.44±0.52 41.28±0. 98 -0.221 0.829
Final 27.83±0.35 41.88±0.95 37.56 0.000
Leptin (ng/ml) 1.84±0.27 3.29±0.83 4.071 0.002
Food intake (g/d) 4.47±0.24 5.58±0.30 10.00 0.015
Body fat mass (g) 0.58±0.07 1.67±0.58 6.023 0.000
Body fat content (%) 5.72±0.87 14.55±5.7 5.819 0.000
Carcass
Wet mass (g) 21.33±0.44 34.06±1.11 9.698 0.000
Dry mass (g) 12.09±1.01 16.41±0.58 3.897 0.006
Water mass (g) 9.24±0.76 17.65±0.71 8.054 0.000
BAT mass (g) 0.24±0.038 0.64±0.08 4.84 0.001
Heart mass (g) 0.18±0.007 0.18±0.08 -0.377 0.715
Lungs mass (g) 0.23±0.02 0.19±0.05 -0.857 0.414
Liver mass (g) 1.70±0.16 1.73±0.38 0.086 0.933
Spleen mass (g) 0.047±0.008 0.068±0.005 2.125 0.63
Kidneys mass (g) 0.35±0.033 0.42±0.021 1.60 0.143
Digestive tract
Length (cm) 74.06±1.64 72.41±1.94 -0.624 0.552
Mass with contents (g) 5.74±0.51 5.92±0.76 0.204 0.843
Wet mass (g) 2.51±0.23 2.9±0.26 1.114 0.294
Dry mass (g) 0.38±0.043 0.62±0.041 3.957 0.003
Stomch
Length (cm) 2.66±0.09 2.81±0.04 1.64 0.176
Mass with contents (g) 1.32±0.15 1.78±0.29 1.487 0.171
Wet mass (g) 0.32±0.02 0.38±0.03 1.834 0.100
Dry mass (g) 0.07±0.005 0.12±0.009 4.459 0.002
Small intestine
Length (cm) 32.96±3.13 37.5±1.39 1.428 0.196
Mass with contents (g) 1.56±0.21 1.69±0.20 0.466 0.652
Wet mass (g) 1.11±0.16 1.48±0.19 1.489 0.171
Dry mass (g) 0.15±0.02 0.30±0.03 4.529 0.001
Caecum
Length (cm) 14.39±1.50 9.38±1.83 -2.142 0.061
Mass with contents (g) 2.08±0.30 1.63±0.36 -0.955 0.365
Wet mass (g) 0.60±0.13 0.51±0.09 -0.576 0.578
Dry mass (g) 0.09±0.027 0.10±0.019 0.266 0.796
Large intestine
Length (cm) 23.22±3.10 22.72±1.05 -0.14 0.892
Mass with contents (g) 0.78±0.11 0.81±0.16 0.184 0.858
Wet mass (g) 0.48±0.06 0.52±0.09 0.415 0.688
Dry mass (g) 0.07±0.008 0.10±0.018 1.708 0.122
Serum leptin levels lower than that of the control group (t=4.071,
After 4 weeks acclimation, serum leptin levels in P<0.01) (Table 1). Significant positive correlation of
cold and food restricted group was significantly serum leptin levels and body mass , body fat
content, and RMR, NST were significantly negatively correlated (r=0.802, weight: P<0.01; body fat content: r=0.598, P<0.05; RMR: r=-0.538 P<0.05; NST:, r=-0.546, P<0.05) (fig 3).
Body composition
After 4 weeks acclimation, carcass mass (t =9.698, P<0.01), BAT (t =4.84, P<0.01), gastric dry weight (t =4.459, P<0.01) and intestine dry weight (t =4.529, P<0.01) in cold and food restricted group was significantly lower than the control group (Table 1).
DISCUSSION
In winter many small mammals reduced body mass or slow growth, which are considered to an energy strategy adapt to environment in the lack of food and under cold stress by reducing the total energy demand (Wang et al., 2006), such as Lasiopodomys brandtii, Apodemus chevrieri, and Microtus pennsylvanicus (Iverson and Turner, 1974; Li & Wang, 2005; Zhu et al., 2012b), decreased body mass in winter in order to reduce the total energy demand, so as to adapt to the low temperature and the lack of food resources in winter (Merritt et al., 2001). However, there are some mammals maintained body mass stability in winter or even increased (Klause et al., 1988), such as Dicrostonyx groenlandicus (Nagy, 1993), Mesocricetus auratus (Del Valle and Busch, 2003). In this study, similar in winter conditions (temperature and food restriction), E. Miletus reduced body mass in order to reduce the total energy demand and the use of body fat in response to low temperature and food shortage condition. However, decreased body mass will lead to unit body surface area (Surface-to-volume) increased, heat loss increased (Duarte et al., 2010), then the energy demand is high to supple the metabolic energy expenditure, and are
vulnerable to food shortages stress (Gutman et al., 2007). E. Miletus in the cold and food restriction group cannot compensate energy demand increasing, leading to body mass decreased significantly, and increased mortality.
RMR and NST are the main energy expenditure of small mammals, play important roles in the regulation of energy balance (Hambly and Speakman, 2005). In winter many animals increased BMR and NST to adapt to the cold conditions in winter, such as Brandt's vole (Li and Wang, 2005), Apodemus chevrieri (Zhu et al., 2012b) and tree shrew (Zhu et al., 2012c). But there is also some animals decreased RMR in winter (Blaxter, 1989). It showed that small mammals have different energy strategy in the winter, choose the low BMR and NST, can reduce the total energy demand, and can therefore have a high survival rate in winter; or is to maintain a high BMR and NST, adapt to long cold condition in winter (Speakman, 1996). In the present study, a significant increase of RMR in cold and food restriction conditions in E. miletus, which indicated that increased RMR is one of adaptive strategy for E. miletus in response to low temperature and food shortages. NST is a effective and economic way of heat production for small mammals to adapt to the cold environment (Jansky,
1973). E. miletus of NST was significantly higher than that in the 7 day and 14 day group, but had no significant difference in 21 day and 28 day compared with control group, this is probably low caloric restriction caused great stress on survival in E. miletus, resulting insufficient heat production in cold and food restriction, and exhibit weak ability of adaptation to low temperature, so that the mortality rate high, reaching to 81.82%.
Leptin plays an important role in regulating the maintenance of homeostasis and body mass
(Wozniak et al., 2009). Feedback loop hypothesis showed that leptin concentrations reflect the body fat storage, and positively correlated with body fat content. Leptin signaling molecules form blood circulation through the stimulation of the hypothalamus, and then by the hypothalamus regulates food intake and energy expenditure, keeping the energy balance (Friedman and Halaas, 1998). Body mass and fat mass of many small mammals have annual change obviously, and the level of circulating Leptin was positively related, such as Phodopus campbelli (Heldmaier and Steinlechner, 1981). Recently studies showed that animals decreased the serum leptin concentration in winter, and leptin was positively correlated with body mass and fat mass, and significantly negative correlation on heat production ability, such as root vole (Wang et al., 2006). In our study, serum leptin levels decreased in cold and food restriction group. There had significantly positive correlation between serum leptin levels and body mass, body fat content, and showed significantly negative correlation between serum leptin levels and BMR and NST, suggesting that serum leptin may participate in the regulation of body mass and heat production in winter. In the cold and food shortage, serum leptin levels may mainly used to promote heat production, reduce body mass in order to adapt to the winter environment.
In conclusion, under low temperature and food restriction conditions, E. miletus mainly through reduced body mass and the use of stored body fat as a supplementary energy source to maintain energy balance. However, low caloric restriction caused larger stress to E. miletus, showing excessive mortality. Moreover, leptin may be involved in the regulation of body mass and thermogenesis under low temperature food restriction conditions in E.
miletus.
ACKNOWLEDGMENTS
This research was financially supported by the National Key Technology Research and Development Program (No. 2014BAI01B00), International cooperation in science and technology project (SQS2014RR102), National Science Foundation of China (No. 31260097; 31360096), Basilic Project of Yunnan Province (No. 2013FA014; 2011FZ082).
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Э6
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