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Journal of Stress Physiology & Biochemistry, Vol. 8 No. 2 2012, pp. 39-50 ISSN 1997-0838 Original Text Copyright © 2012 by Zhu, Yang, Cai, Meng and Wang
ORIGINAL ARTICLE
Effects of photoperiod on body mass, thermogenesis and body composition in Eothenomys miletus during cold exposure
Wan-long Zhu, Shengchang Yang, Jin-hong Cai, Lihua Meng, Zheng-kun Wang*
School of life Science of Yunnan Normal University, Engineering research center of sustainable development and utilization of biomass energy Ministry of Education, The Key Laboratory of Biomass Energy and Environmental Biotechnology in Yunnan Province, Kunming; 650092, China
*E-mail: zwl_8307@163. com
Received February 11 2012
Many small mammals respond to seasonal changes in photoperiod by altering body mass and adiposity. These animals may provide valuable models for understanding the regulation of energy balance. In present study, we examined the effect on body mass, rest metabolic rate, food intake and body composition in cold-acclimated (5 °C) in Eothenomys miletus by transferring them from a short (SD, 8h :16h L: D) to long day photoperiod (LD, 16h: 8h L:D). During the first 4 weeks of exposure to SD, E. miletus decreased body mass. After the next 4 weeks of exposure to LD, which the average difference between body masses of LD and SD voles was 4.76 g. This 14.74% increase in body mass reflected significant increases in absolute amounts of body components, including wet carcass mass, dry carcass mass and body fat mass. After correcting body composition and organ morphology data for the differences in body mass, only livers, kidney, and small intestine were enlarged due to photoperiod treatment during cold exposure. E. miletus increased RMR and energy intake exposure to LD, but maintained a stable level to SD after 28 days. Serum leptin levels were positively correlated with body mass, body fat mass, RMR as well as energy intake. All of the results indicated that E. miletus may provide an attractive novel animal model for investigation of the regulation of body mass and energy balance at organism levels. Leptin is potentially involved in the photoperiod induced body mass regulation and thermogenesis in E. miletus during cold exposure.
Key words: Eothenomys miletus; thermogenesis; body composition; serum leptin
ORIGINAL ARTICLE
Effects of photoperiod on body mass, thermogenesis and body composition in Eothenomys miletus during cold exposure
Wan-long Zhu, Shengchang Yang, Jin-hong Cai, Lihua Meng,
Zheng-kun Wang*
School of life Science of Yunnan Normal University, Engineering research center of sustainable development and utilization of biomass energy Ministry of Education, The Key Laboratory of Biomass Energy and Environmental Biotechnology in Yunnan Province, Kunming; 650092, China
E-mail: zwl_8307(u. 163. com
Received February 11 2012
Many small mammals respond to seasonal changes in photoperiod by altering body mass and adiposity. These animals may provide valuable models for understanding the regulation of energy balance. In present study, we examined the effect on body mass, rest metabolic rate, food intake and body composition in cold-acclimated (5 °C) in Eothenomys miletus by transferring them from a short (SD, 8h : 16h L: D) to long day photoperiod (LD, 16h: 8h L:D). During the first 4 weeks of exposure to SD, E. miletus decreased body mass. After the next 4 weeks of exposure to LD, which the average difference between body masses of LD and SD voles was 4.76 g. This 14.74% increase in body mass reflected significant increases in absolute amounts of body components, including wet carcass mass, dry carcass mass and body fat mass. After correcting body composition and organ morphology data for the differences in body mass, only livers, kidney, and small intestine were enlarged due to photoperiod treatment during cold exposure. E. miletus increased RMR and energy intake exposure to LD, but maintained a stable level to SD after 28 days. Serum leptin levels were positively correlated with body mass, body fat mass, RMR as well as energy intake. All of the results indicated that E. miletus may provide an attractive novel animal model for investigation of the regulation of body mass and energy balance at organism levels. Leptin is potentially involved in the photoperiod induced body mass regulation and thermogenesis in E. miletus during cold exposure.
Key words: Eothenomys miletus; thermogenesis; body composition; serum leptin
Phenotypic flexibility is the ability of organisms to reversibly modulate traits in response to environmental challenge (Aneta et al., 2009). In recent years, the magnitude of this modulation has been extensively quantified at different levels of biological organization (Sabat and Bozinovic, 2000; Villarin et al., 2003; Kristan and Hammond, 2006).
Several studies have demonstrated that processes related to energy acquisition and expenditure is among the most sensitive to changes in environmental conditions (Naya et al., 2008). The responses to changing photoperiod can be readily induced in the laboratory by simulating the natural progression of day lengths or by acutely
transferring animals between summer (long) and winter (short) day lengths (Kryl et al., 2005). There were many studies about the transfer of small mammals from long day photoperiod (LD, 16-h:8-h L:D) to short day photoperiod (SD, 8-h:16-h L:D), they showed the changes of body mass, thermogenesis and body composition (Ebling, 1994; Klingenspor et al., 2000; Mercer and Speakman, 2001; Hunter and Nagy, 2002). Leptin is a protein which is synthesized primarily by adipose tissue and is secreted into the bloodstream (Silva, 2006). Leptin is considered to be an adipostatic signal linking energy metabolism, regulating food intake, and providing prompt feedback to brain areas involved in regulation of energy balance (Barb and Kraeling, 2004). It is also hypothesized to contribute to the maintenance of body mass by altering food intake and energy expenditure (Friedman and Hallas, 1998).
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., 2011a), has special status in Microtus. Now few studies about E. miletus were reported (Zhu et al., 2008; 2010a; 2010b; 2011b). In the present study, we examined the effect on body mass, thermogenesis and body composition in cold-acclimated E. miletus by transferring them from short to long day photoperiod. We predicted that E. miletus change their body mass, thermogenesis and body composition by transferring them from short to long day
photoperiod, 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°45'N; 99°40'~99°55'E; altitude 2,590m) in Jianchuan County, Yunnan province, 2011. Average temperature is 9.1oC each year, minimum average temperature was -4.0oC in January, maximal average temperature was 24.1oC 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=7 each group). Following the acclimation period, all animals were kept in SD for a further 24days to obtain baseline measurements of body mass and food intake, after which 7 voles were exposed to a long day photoperiod (LD, 16h: 8h L: D, lights on 04:00h) at 5°C, whereas 7 control voles remained in original SD conditions. 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 25oC within the TNZ (thermal neutral zone), gas analysis were using ML206 gas analysis instrument, the temperature was controlled by SPX-300 artificial climatic engine (±0.5oC), the
metabolic chamber volume is 500ml, flow is 200 ml/min. The tree shrew were stabilized in the metabolic chamber for at least 60 min prior to the BMR measurement, oxygen consumption was recorded for more than 60-min at 5-min intervals. Two stable consecutive lowest readings were taken to calculate BMR (Li and Wang, 2005). Calculate method of metabolic rate is detailed by Hills (Hills, 1972).
Energy budget
Energy budget were measured by food equity (Rosenmann and Morrison, 1974), one animal in a metabolic cage ( 20*15*15cm3 ) , had no nest materials. After adaptation one week, energy budget were measured. Animals were feeding fix quantify and time, next day weighted animal body mass, collected residual food, faeces. Residual food and faeces were dryness in vacuum airer until the mass were invariable, got sample (about 1g, precision: 0.0001g), the samples were measured by YX-ZR/Q automatism calorimeters instrument. The calorie of the diet fed to these animals were 18.0±0.8 kJ/g. Calculate the energy intake. The calculation was according to corresponding reports (Drozdz, 1975):
Energy intake (KJ/d) =food (g/d)* energy content (KJ/g)
Measurement of serum leptin level and thyroid hormones
Serum leptin levels were determined by radioimmunoassay (RIA) with the 125I Multispecies 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.
Carcass and body fat content
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 (Zhang 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, changes in body mass, BMR and energy intake were analyzed by a twoway analysis of covariance (ANCOVA) with body mass as a covariate. Body composition between SD and LD 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 energy intake. Results were presented as mean ± SEM, and P < 0.05 was considered to be statistically significant. RESULTS
Body mass and serum leptin
No group differences were found between acclimation voles (F=0.972, P>0.05, Fig 1) before acclimation. During the acclimation, both acclimation voles decreased body mass in the first 4 weeks, SD voles showed relatively stable body mass after 4 weeks(F=0.073, P>0.05, Fig 1),
whereas the LD voles showed a gradual increase over the next 4-week acclimation (F=2.449, P<0.05, Fig 1). Correlation analysis indicated that body mass was positively correlated with body fat mass (r = 0.836, P < 0.01; Fig 2A). There had positive correlation between serum leptin level and body mass (r = 0.960, P < 0.01; Fig 2B), between serum leptin level and body fat mass during the acclimation(r = 0.784, P < 0.01; Fig 2C).
RMR and energy intake
Prior to acclimation, no group differences in RMR between acclimation voles (t=0.318, P>0.05). During the acclimation, both acclimation voles increased RMR in the first 4 weeks, SD voles showed relatively stable RMR after 4 weeks (F=0.125, P>0.05, Fig 3), whereas the LD voles showed a gradual increase over the next 4-week acclimation (F=3.245, P<0.05, Fig 3). Prior to acclimation, no group differences in energy intake between acclimation voles (t=0.364, P>0.05). During the acclimation, both acclimation voles increased energy intake in the first 4 weeks, SD
voles showed relatively stable energy intake after 4 weeks(F=0.296, P>0.05, Fig 4), whereas the LD voles showed a gradual increase over the next 4-week acclimation (F=2.985, P<0.05, Fig 4). There had positive correlation between serum leptin level and RMR (r = 0.956, P < 0.01; Fig 5A), between serum leptin level and energy intake during the acclimation(r = 0.676, P < 0.01; Fig 5B).
Body composition
At the end of the acclimation, wet carcass mass, dry carcass mass and body fat mass showed a significant differences between SD voles and LD voles (t=2.242, P<0.01, Table 1), (t=2.717, P<0.05, Table 1), (t=2.035, P<0.05, Table 1), respectively. No differences were found in water of carcass between the two groups (t=1.603; P>0.05, Table 1). By contrast, the wet organ masses of liver, kidney and small intestine showed significant between the LD and SD voles (Table 2), the dry organ masses of liver and kidney showed significant between the LD and SD voles (Table 3).
Table 1. Changes of body compositions exposed to short and long day photoperiods during cold exposure
E. miletus
Parameters SD LD P value
Body mass(g) 22.52±0.51 26.12±0.98 0.042
Wet carcass mass(g) 32.29±0.86 37.05±0.95 0.001
Dry carcass mass(g) 11.27±0.34 13.44±0.72 0.019
Water of carcass(g) 11.26±0.70 12.64±0.49 0.135
Body fat mass(g) 4.62±0.14 5.50±0.29 0.025
Table 2. Changes of mean wet organ masses exposed to short and long day photoperiods during cold exposure E. miletus
Parameters SD LD P value
Heart(g) 0.233±0.019 0.251±0.013 0.271
Lungs(g) 0.308±0.015 0.324±0.008 0.401
Liver(g) 1.525±0.071 1.848±0.078 0.012
Kidney(g) 0.179±0.010 0.218±0.014 0.049
Spleen(g) 0.020±0.002 0.020±0.003 0.896
Stomach(g) 0.405±0.031 0.414±0.013 0.071
Small intestine(g) 0.674±0.024 0.751±0.015 0.023
Cecum(g) 0.420±0.015 0.438±0.014 0.443
Large intestine(g) 0.325±0.013 0.335±0.013 0.608
Table 3. Changes of mean dry organ masses exposed to short and long day photoperiods during cold exposure E. miletus
Parameters SD LD P value
Heart(g) 0.043±0.002 0.045±0.002 0.610
Lungs(g) 0.066±0.002 0.070±0.001 0.131
Liver(g) 0.384±0.015 0.497±0.022 0.001
Kidney(g) 0.040±0.001 0.043±0.001 0.020
Spleen(g) 0.004±0.001 0.004±0.001 0.763
Stomach(g) 0.093±0.008 0.093±0.009 0.324
Small intestine(g) 0.029±0.001 0.027±0.001 0.676
Cecum(g) 0.041±0.003 0.041±0.002 0.981
Large intestine(g) 0.044±0.003 0.044±0.004 0.906
Figure 1 Effect of body mass exposed to short and long day photoperiods during cold exposure in E.
miletus
DISCUSSION
Many small mammals respond to winter-associated environmental cues by reducing body or enhancing thermogenesis (Heldmaier et al., 1982; 1989), such as Microus brandti (Li and Wang, 2005), Djungarian hamster (Steinlechner et al., 1983). Moreover, photoperiod is one of the environmental cues triggering seasonal responses in small mammals. Some small mammals reduced body mass while increased thermogenesis in SD conditions (Geiser et al., 2007; Arnold et al., 2011), such as Siberian hamsters (Demas et al., 2002), Brandt's voles (Zhao and Wang, 2005). In the
present study, it was clear that E. miletus showed decreasing of body mass in short day photoperiod during cold exposure in the first 4 weeks. But when E. miletus transferred from a short (SD, 8h: 16h L: D) to long day photoperiod (LD, 16h: 8h L: D). Body mass of E. miletus increased exposure to long day photoperiod. After the next 4 weeks of exposure to LD, which the average difference between body masses of LD and SD voles was 4.76 g. This 14.74% increase in body mass reflected significant increases in absolute amounts of body components, including wet carcass mass, dry carcass mass and body fat mass. At the end of the acclimation, our present study showed that SD voles had lower body mass
and body fat mass compared with LD voles. As dry carcass mass in SD voles was also lower than in LD
while no differences were found in water of carcass mass between the two groups.
Figure 2 Correlation of body mass with body fat mass (A), serum leptin levels with body mass (B), body fat mass (C) in E. miletus
Acclimation time (weeks)
Figure 3 Effect of RMR exposed to short and long day photoperiods during cold exposure in E.
miletus
Acclimation time (weeks)
Figure 4 Effect of energy intake exposed to short and long day photoperiods during cold exposure in
E. miletus
Changes of photoperiod are considered to be an important cue affecting RMR in many small mammals (Haim et al., 1999). In the present study, LD voles after 4 weeks photoperiodic acclimation showed higher RMR than that in SD voles during the cold exposure. These data clearly indicated that RMR was affected by changes of photoperiod in E. miletus. Energy intake also showed the similar trend. The increases of RMR and energy intake were the cause of increased in body mass in E. miletus. In
addition, themorphology of the organs was found to be altered to meet the increase in energy intake. Livers, kidney, and small intestine were enlarged due to photoperiod treatment during cold exposure. Liver, as an important energy-expending organ, is considered to make a large contribution to RMR. In our study, kidney and small intestine in LD voles was significantly higher than that in SD voles, which was consistent with the changes in BMR and energy intake.
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Figure 5 Correlation of serum leptin levels with RMR (A), energy intake (B) in E. miletus
Leptin level may reflect the contents of fattiness, could serve as a signal to the brain, to regulate food intake and energy expenditure and resistance to obesity (Michael et al., 2000). There was a positive correlation between leptin level and body mass (Friedman and Hallas, 1998). Subsequent parabiosis experiments in rodents supported the tenets of the lipostasis theory in that the serum “satiety signal” was at a higher concentration in obese animals than in lean animals (Concannon et al., 2001). In our result, although SD voles showed relatively lower
serum leptin levels compared with LD voles. Further correlation analysis showed that serum leptin levels were positively correlated with body mass and body fat mass, indicating that leptin may act as "adiposity indicator" (Zhao and Wang, 2005). To regulate body mass, leptin influences energy balance via its effects on both food intake and energy expenditure (Concannon et al., 2001). Leptin administration is thought to increase energy expenditure, it has been reported that lower serum leptin levels were accompanied with higher energy intake in Brandt's
membranes precedes seasonally changing body temperature in a Hibernator. PLoS ONE 6(4), 18641.
voles, indicating that leptin acted as a starvation signal (Liu et al., 2007). Furthermore, correlation analysis showed that serum leptin levels were positively correlated with RMR and energy intake, it was opposite to our previous studies (Zhu et al., 2010a; 2011b). It may indicated that the anorectic and thermogenic effects of leptin may be dissociated in cold-acclimated (5 °C) in Eothenomys miletus by transferring them from a short (SD, 8h : 16h L: D) to long day photoperiod (LD, 16h: 8h L:D). It need further study.
In conclusion, E. miletus decreased body mass during the first 4 weeks of exposure to SD. After the next 4 weeks of exposure to LD, which the average difference between body masses of LD and SD voles was 4.76 g. This increase in body mass reflected significant increases in absolute amounts of body components, including wet carcass mass, dry carcass mass and body fat mass. Livers, kidney, and small intestine were enlarged due to photoperiod treatment during cold exposure. E. miletus increased RMR and energy intake exposure to LD, but maintained a stable level to SD after 28 days. Serum leptin levels were positively correlated with body mass and body fat mass. Leptin is potentially involved in the photoperiod induced body mass regulation and thermogenesis in E. miletus during cold exposure.
ACKNOWLEDGMENTS
The project was financially supported by National Science Foundation of China (No.31071925); Project of Basic research for application in Yunnan Province (No. 2011FZ082). REFERENCES
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