Faster Response to High-Fat Diet in Body Mass Regulation from Lower Altitude Population in Eothenomys miletus from Hengduan Mountain Regions

Gong Xue-na1, Jia Ting2, Zhang Di2 and Zhu Wanlong1* 1Key Laboratory of Ecological Adaptive Evolution and Conservation on AnimalsPlants in Southwest Mountain Ecosystem of Yunnan Province Higher Institutes College, School of Life Sciences, Yunnan Normal University; Engineering Research Center of Sustainable Development and Utilization of Biomass Energy Ministry of Education; Key Laboratory of Yunnan Province for Biomass Energy and Environment Biotechnology, Kunming, 650500. 2Yunnan College of Business Management, Kunming, 650106, China Article Information Received 11 February 2020 Revised 30 March 2020 Accepted 23 April 2020 Available online 10 June 2021


INTRODUCTION
E fficiency of energy intake and expenditure were essential for survival in small mammals (Mitchell et al., 2016;Hu et al., 2018). Food quality was an important environmental factor, which affected survival and reproduction in the wild small mammals (Delciellos et al., 2018). Previously studies showed that fat content was closely related to body fat mass accumulation, high-fat (HF) diet increased energy intake and body mass in animals (Yoo et al., 2006). However, different animals had different body mass regulation for HF diet. For example, rats and O n l i n e

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with body mass, and higher leptin levels inhibited animals' food intake Zhu et al., 2017). Therefore, leptin could inhibit NPY/AgRP pathway and stimulated POMC/CART pathway to regulate energy balance (Pérez-Maceira et al., 2016). Resting metabolic rate (RMR) refers to the minimum number of calories needed basic functions, including breathing and circulation (Splinter and Wilson, 2019), which is an important physiological indicator for field small mammals to respond to different environmental pressures (Zhang et al., 2019). Activity behavior of animals had great significance for adapting environmental changes (Kingsbury et al., 2019). Mammals usually increased the activity behavior under low temperature or food restriction. For example, food restriction increased activity significantly in Mus musculus (Blank and Desjardins, 1985). HF diet reduced activity behavior significantly in rats (Chen et al., 2014).
Eothenomys miletus belongs to the genus Eothenomys is an inherent species in Hengduan mountain regions. E. miletus was a nocturnal rodent, and its main food was fresh pulp plants and roots of grass. Hengduan Mountain regions is located the boundary between the Palaearctic region and the Oriental region (Zhu et al., 2017), which altitude and climate changes dramatically, and small mammals in different locations may have different physiological characteristics (Ren et al., 2020). Previous studies showed that changes of RMR and relative fatness in E. miletus form different regions were related to environmental temperature and food resources (Mu et al., 2015); temperature and food restriction had significant effects on physiological characteristics in E. miletus (Zhu et al., 2010(Zhu et al., , 2014. In the present study, we selected E. miletus from two areas (Jianchuan, JC and Xianggelila, XGLL) with significant phenotypic differentiation (Zhang et al., 2019). XGLL has higher altitude, lower annual average temperature (5.5°C), less crops and vegetations, so food conditions in winter were relatively poor; while JC has lower altitude, higher average annual temperature (13.9°C), higher vegetations and almost no snow cover, so food resources in winter were relatively better, so the present study was to investigate the effects of HF diet on physiological and behavioral plasticity, body mass, food intake, RMR, activity behavior, serum leptin levels, hypothalamic neuropeptide expressions and body compositions were measured. We hypothesized that E. miletus would respond to HF diet by changing body mass, serum leptin levels, activity behavior and adjusting the hypothalamic neuropeptides genes expressions. We predicted that E. miletus may change the physiological responses to regulate body mass, and there were regional differences in physiological changes in E. miletus in response to high-fat diet and refeeing.

Samples
E. miletus were obtained from farmland in XGLL (99°83´E, 27°90´N, altitude 3321 m) and JC county (99°75´E, 26°43´N, altitude 2590 m) in January 2019. E. miletus were maintained at a room temperature of 25±1°C, under a photoperiod of 12L:12D (with lights on at 08:00), food (standard mice chow pellets; produced by Kunming Medical University, Kunming) and water were provided ad libitum. All pregnant, lactating or young individuals were excluded in the present study. HF diet used in the experiment was prepared from the standard mice chow pellets with about 18% soybean oil, and low-fat (LF) diet was the standard mice chow pellets, the main food components were shown in Table I. All animal procedures were compliance with the Animal Care and Use Committee of School of Life Science, Yunnan Normal University. This study was approved by the Committee (13-0901-011).

Experiment 1
Effects of HF diet on body mass, food intake, RMR and activity behavior in E. miletus. Sixteen adult E. miletus of XGLL (n=8, ♀4 and ♂4) and JC (n=8, ♀4 and ♂4) were housed individually (were maintained at 12L: 12D (light on at 08:00am), 25±1ºC, respectively), and kept for 1 weeks to familiarize with the environment. After the acclimatizing period, the animals of XGLL and JC were acclimated to HF diet for 28 days, and then re-feeding LF diet for another 28 days, animals were acclimated for 8 weeks. Food intake was calculated as the mass of food missing from the hopper, subtracting orts mixed in the bedding. Body mass, food intake, RMR and activity behavior were measured every day.

Experiment 2
Effects of HF diet on body mass, serum leptin levels, hypothalamic neuropeptide genes expression, body compositions and gastrointestinal tract in E. miletus. 43 adult weight-matched E. miletus from two regions (XGLL: n=21, ♀11 and ♂10; JC: n=22, ♀10 and ♂12) were O n l i n e

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selected, which were maintained at 12L: 12D (light on at 08:00 am), 25±1ºC, respectively. Animals of one region were randomly assigned to a control group, and a HF diet and refeeding LF diet group (HF-LF). Controls were fed LF diet for 8 weeks, while HF-LF group were fed HF diet for 4 weeks, then fed LF diet for a further 4 weeks. On day 28, animals were randomly selected from HF-LF group for the measurement of body mass, serum leptin levels, body composition, gastrointestinal tract mass and length, and hypothalamic neuropeptide genes expression. These measurements were taken again from the remaining animals of each group (Control group and HF-LF group) on day 56. All animals were sacrificed between 09:00 h and 11:00 h by decapitation, and animals were dissected to evaluate organ morphology. Blood was centrifuged at 4,000 rpm for 30 min after a 30 min interval. Blood serum was collected and stored at −75 °C prior to hormone measurement.

Measurement of RMR, food intake and activity behavior
Body mass, RMR, food intake and activity behavior were measured using the metabolic system (BXY-R, Sable Systems). E. miletus were acclimated to calorimetry cages prior to 30 min the study and data collection (Zhu et al., 2010).

Measurement of morphology, serum leptin levels and hypothalamic neuropeptide gene expression
Measurements of morphology was details in Zhang et al. (2018). Serum leptin levels were determined by radioimmunoassay (RIA) with the 125I Multi-species Kit (Millipore), and leptin values were determined in a single RIA; the lowest level of leptin that can be detected by this assay is 1.0 ng/mL when using a 100-μL sample size (instructions for Multi-species Kit). The inter-and intraassay variabilities for leptin RIA were 3.6% and 8.7%, respectively. Measurements of hypothalamic neuropeptide gene expression was details in Ren et al. (2020).

Statistical analysis
Data were analyzed using the software package SPSS 15.0. Prior to all statistical analyses, data were examined for assumptions of normality and homogeneity of variance using Kolmogorov-Smirnov and Levene tests, respectively. Since no gender effects were found on almost all measured parameters, data from females and males were combined. Differences in body mass, food intake, RMR, activity behavior, serum leptin levels and hypothalamic neuropeptide genes expression were analyzed by twoway ANOVA, and differences in body compositions and gastrointestinal tract were analyzed by two-way ANCOVA with body mass as a covariate, followed by Tukey's post hoc test. Results are presented as means ± SE, and P<0.05 was considered to be statistically significant.

DISCUSSION
Phenotypic plasticity is the ability of an organism to change its phonotype in response to changes in the environment, such as temperature and photoperiod (Miner et al., 2005). Food quality was also an important factor affecting animals' phenotypic changes (Gao et al., 2013). HF diet increased body mass and body fat mass significantly in mice (Wang et al., 2008), but it has no significant effect on body mass in Cricetulus barabensis (Shi et al., 2017). Changes in organ mass and digestive tract morphology play important roles in energy balance (Hume, 2002). The present results showed that body mass in E. miletus in JC was larger than that of in XGLL, but total digestive tract in XGLL area was longer and lighter than that in JC area, which may be related to the different food resources in the two regions. During the HF diet acclimation, body mass increased in E. miletus in the two areas, and then returned to the control level on day 56, showing higher phenotypic plasticity. However, body mass changes in the two areas were different after re-feeding LF diet. Body mass loss in JC was significant, which might be related to smaller changes of food intake after re-feeding LF diet. Body mass in XGLL decreased gradually after re-feeding, which was due to the increasing of food intake, suggesting that E. miletus in XGLL was more sensitive to changes in food quality. Moreover, the initial food intake in XGLL was O n l i n e

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Phenotypic Differentiations in Eothenomys miletus in Response to High-fat Diet higher than that of JC, which mainly because the environment temperature was lower and food resources were poor in winter of XGLL, when food was sufficient, food intake would be increased. Behavioral regulation is an important adaptive strategy for wild small mammals to cope with environmental changes, especially for uncertainty of food resources . Rats reduced their foraging behavior by a HF diet (Chen et al., 2014). In the current research, activity behavior only varied between regions, which was related to their habitat temperature and food conditions. There was no significant change in activity behavior form two regions, indicating that E. miletus did not need to increase activity behavior when food quality is good. But activity behavior in XGLL fluctuated larger, probably because E. miletus in XGLL were more sensitive to food resources in winter. RMR is often used to study the adaptation of small mammals to extreme environments (Li and Huang, 1994). LF diet increased RMR in mammals (Camp et al., 2018), while HF diet has no significant effect on RMR and body compositions in Labrador (Yoo et al., 2006). In the present study, higher RMR in XGLL was related to lower temperature and cold temperature. RMR in JC remained unchanged under acclimation, which was consistent with its activity behavior. RMR in XGLL was more volatile, one reason is that it may be related to the fluctuation of their activity behavior, the other reason is that the environment in XGLL is more volatile in winter.
Leptin plays an important role in the regulation of body mass and energy metabolism (Abelenda et al., 2003). In the present study, serum leptin levels were positively correlated with body mass, supporting the hypothesis that leptin could be used as a lipid signaling molecule (Schneider et al., 2000). Moreover, there was a significant difference O n l i n e

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G. X-n et al. in leptin levels between two regions. Serum leptin levels in JC was higher than that of in XGLL, which was related to the higher body mass in JC. Hypothalamus controls food intake and energy expenditure mainly by regulating the expression of two types of neuropeptides (food promoters: NPY, AgRP; food inhibition: POMC, CART), which plays an important role in body mass regulation (Trayhurn and Bing, 2006). Previous studies have shown that high-quality food increased NPY expression significantly (Kaga et al., 2001). However, other studies have shown that HF diet has no effect on POMC expression (Marco et al., 2013). In our study, HF diet decreased NPY expression significantly, and NPY showed a significant negative correlation with leptin levels. But HF diet had no significant effect on the other three neuropeptides expressions, suggesting that leptin and NPY expression may play an important role in body mass regulation in E. miletus.
In conclusion, HF diet increased body mass in E. miletus, which returned to the control level when refeeding LF diet. Leptin and NPY play important roles in the regulation of body mass and food intake. There were differences in body mass regulation patterns in E. miletus form different regions when they were exposed to HF diet, which may be related to different environmental conditions in Hengduan mountain regions.

O n l i n e F i r s t A r t i c l e
Phenotypic Differentiations in Eothenomys miletus in Response to High-fat Diet