Journal of Thermal Biology 57 (2016) 35–43

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Journal of Thermal Biology

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Leptin regulates energy intake but fails to facilitate hibernation in fattening Daurian ground ( dauricus)

Xin Xing a,c, Gang-Bin Tang a, Ming-Yue Sun b, Chao Yu b, Shi-Yi Song b, Xin-Yu Liu b, Ming Yang b,n, De-Hua Wang a,nn a State Key Laboratory of Integrated Management of Pest Insects and , Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang, 100101, b College of Life Science, Shenyang Normal University, 253 Huanghe North Street, Shenyang 110034, China c University of Chinese Academy of Sciences, Beijing 100049, China article info abstract

Article history: Body fat storage before hibernation affects the timing of immergence in Daurian ground squirrels Received 10 June 2015 (Spermophilus dauricus). Leptin is an adipose signal and plays vital role in energy homeostasis mainly by Accepted 26 January 2016 action in brain. To test the hypothesis that leptin plays a role in facilitating the process of hibernation, Available online 2 March 2016 squirrels were administrated with recombinant murine leptin (1 μg/day) through intracerebroventricular Keywords: (ICV) injection for 12 days during fattening. From day 7 to 12, were moved into a cold room Hibernation (571 °C) with constant darkness which functioned as hibernaculum. Energy intake, body mass and core Leptin body temperature (Tb) were continuously monitored throughout the course of experiment. Resting Fattening metabolic rate (RMR) was measured under both warm and cold conditions. At the end of leptin ad- Daurian ground squirrels (Spermophilus ministration, we measured the serum concentration of hormones related to energy regulation, mRNA dauricus) expression of hypothalamic neuropeptides and uncoupling protein 1 (UCP1) levels in brown adipose tissue (BAT). Our results showed that during leptin administration, the cumulative food intake and in-

crease of body mass were suppressed while Tb and RMR were unaltered. The proportion of torpid squirrels was not different between two groups. At the end of leptin administration, the expressions of hypothalamic neuropeptide Y and agouti gene-related protein were suppressed. There were no differ- ences in UCP1 mRNA expression or protein content in BAT between groups. Our data suggest that leptin can affect energy intake via hypothalamic neuropeptides, but is not involved in the initiation of hi- bernation in fattening Daurian ground squirrels. & 2016 Elsevier Ltd. All rights reserved.

1. Introduction fattening and hibernation have been suggested to be en- dogenously controlled processes in obligate hibernators (Drew Hibernation is an adaptive strategy used by a variety of mam- et al., 2007; Williams et al., 2014). However, previous studies mals to withstand long periods of harsh environmental conditions showed that the timing of immergence and emergence can be during winter (Lyman et al., 1982; Heldmaier et al., 2004; Geiser, affected by body fat reserves (mainly white adipose tissue, WAT) 2013). Fat-storing hibernators, such as ground squirrels (e.g. in some species (Vuarin et al., 2013; Bieber et al., 2014). Since the Spermophilus, , and , etc.) central nervous system (CNS) plays important roles in regulation and (e.g. Marmota), gain large amounts of body fat of energy balance and hibernation (Drew et al., 2007; Florant and through the sharp increase of food intake during pre-hibernation Healy, 2012), we supposed that the timing of immergence may be fattening period, then they enter into hibernation spontaneously influenced by the peripheral adipose signals though the action in even in the presence of food. The accumulated body fat is the CNS. As a primary energy storage organ, WAT also produces and primary fuel during hibernation (Boyer and Barnes, 1999; Con- secretes a variety of hormones related to various physiological cannon et al., 2001; Dark, 2005; Florant and Healy, 2012). Both functions (Kershaw and Flier, 2004; Vázquez-Vela et al., 2008). Leptin is a hormonal signal for adiposity providing the CNS with information about body fat stores and regulating energy home- n Correspondence to: College of Life Science, Shenyang Normal University, 253 ostasis in (Zhang et al., 1994; Flier, 2004; Morton et al., Huanghe North Street, Shenyang 110034, China. nn 2006). Leptin modulates food intake and energy expenditure Corresponding author. E-mail addresses: [email protected] (M. Yang), through the action on its receptors (ObR) in the hypothalamus [email protected] (D.-H. Wang). (Schwartz et al., 2000). High serum leptin concentration increases http://dx.doi.org/10.1016/j.jtherbio.2016.01.013 0306-4565/& 2016 Elsevier Ltd. All rights reserved. 36 X. Xing et al. / Journal of Thermal Biology 57 (2016) 35–43 the expression of anorectic peptides pro-opiomelanocortin the first two weeks of captivity. After acclimation, we only pro- (POMC) and cocaine- and amphetamine-regulated transcript vided rat chow and water ad libitum. Body mass was measured (CART) and suppresses the expression of orexigenic peptides once a week before the experiment for determining the fattening, neuropeptide Y (NPY) and agouti gene-related protein (AgRP) which was characterized by continuous body mass gain. The (Flier, 2004; Morton et al., 2006). Additionally, high leptin con- squirrels used in this study had similar body mass before leptin centration can also induce adaptive thermogenesis via increasing administration. procedures were approved by the Animal the expression of uncoupling protein 1 (UCP1) in brown adipose Care and Use Committee of the Institute of Zoology, Chinese tissue (BAT) (Cancello et al., 1998; Demas et al., 2002; Gullicksen Academy of Sciences. et al., 2002). Several lines of evidence showed that leptin cross- talks with the other hormones, such as insulin, thyroid hormones, 2.2. Experimental procedures obestatin and ghrelin, to regulate energy homeostasis and lipid metabolism (Baskin et al., 1999; Zimmermann-Belsing et al., 2003; The experiment was performed from July 18th to July 30th in Tups et al., 2004; Kim et al., 2004; Zhang et al., 2005; Martin, 2011. Eighteen fattening female ground squirrels were randomly 2008; Healy and Florant, 2012). assigned to either the leptin administration group (n¼10) or the The available data suggested that leptin might also provide a control group (n¼8). Leptin (1 μg/day) or artificial cerebrospinal signal of adiposity in fat-storing hibernators. It has been shown fluid (aCSF) was chronically delivered into the ICV via constant that, during the fattening period, serum leptin concentration in- infusion using an osmotic minipump for 12 days. The chosen dose creased and correlated positively with body mass in some fat- of leptin was shown to be effective on energy regulation in rats storing hibernators (Boyer and Barnes, 1999; Concannon et al., (Overton et al., 2001), as well as in Daurian ground squirrels ac- 2001; Florant et al., 2004; Healy and Florant, 2012). Peripheral cording to our pilot experiment. During the course of experiment, leptin administration had catabolic effects on body mass gain and energy intake, body mass, and core body temperature (Tb) were appetite drive in fattening arctic ground squirrels (Urocitellus continuously recorded. Body mass was expressed as percentage of parryii)(Ormseth et al., 1996; Boyer et al., 1997). individual baseline levels established before leptin administration. The Daurian ground (Spermophilus dauricus) is a typical To test the effects of leptin on torpor initiation, on day 7, all the fat-storing hibernator with a wide distribution in northern China squirrels were transferred from warm (2571 °C) and natural light (Zhang and Wang, 1998). This species usually doubles its body cycle condition to a cold (571 °C) and constantly dark room for mass during fattening, which is mainly due to the accumulation of another 6 days to facilitate the torpor. The resting metabolic rate WAT. The serum leptin concentration showed a seasonal cycle and (RMR) was measured under both warm and cold conditions. At the correlated positively with body mass. During the fattening period, end of the administration, the squirrels were euthanized using serum leptin concentration increased significantly during the late CO2. Trunk blood was collected from euthanized squirrels and phase of fattening and reached the peak value after fattening centrifuged at 2500g for 30 min at 4 °C. Serum was collected and (Chen et al., 2008; Xing et al., 2015). After fattening, Daurian stored at 80 °C until analyzed. The whole brain was removed ground squirrels decreased food intake, body mass and metabolic and slowly frozen by placing on dry ice, then stored at 80 °C rate gradually (Lv et al., 2014). In laboratory, body fat storage until subsequent analysis. Before analysis, the hypothalamus was during fattening correlated positively with overwinter survival carefully dissected under 20 °C condition as previously de- (Guan, 2009). After cold acclimation, the proportion of ground scribed (Glowinski and Iversen, 1966). The interscapular BAT was squirrels that entered torpor was significantly higher after fat- quickly dissected and stored at 80 °C until assayed. After dis- tening than that of during fattening (Guan, 2010). Thus, we hy- section of BAT, subcutaneous white adipose tissue (SWAT) and pothesized that leptin would function as an energy signal to fa- visceral white adipose tissue (VWAT) were carefully dissected and cilitate the immergence in fattening Daurian ground squirrels. To weighted (70.1 g). Total adipose tissue (TWAT) was calculated as test this hypothesis, we performed ICV leptin injection in fattening the sum of SWAT and VWAT. squirrels for 12-day. The squirrels were exposed to a cold and constant dark room to facilitate torpor (Yang et al., 2011) during 2.3. Surgery the second week of administration. We predicted that the squirrels would reduce their energy intake and show hypothermia after Under sterile conditions, squirrels were anesthetized by so- leptin administration. Increased leptin concentration in CNS might dium pentobarbital (50 mg/kg) for the implantation of tempera- facilitate the lipolysis and glycometabolism in which insulin, ture loggers, so-called iButtons (Thermocron DS1922 L, 3.3 g) and ghrelin and obestatin may be involved. ICV infusion cannulae (Alzets Brain infusion kit 2).

2.3.1. Implantation of iButtons 2. Materials and methods The iButtons were calibrated by mixture of ice and water and sealed in a thin paraffin/Ethylene-Vinyl Acetate copolymer coating 2.1. Animals and sterilized using 75% alcohol and iodophor before implanted.

Daurian ground squirrels were trapped at farmland of Inner 2.3.2. Implantation of ICV infusion cannulas (122.5°E, 43.9°N) in May 2011 and brought to the la- We set the cross point of the ear rod connection and the sa- boratory of Shenyang Normal University. Squirrels were housed gittal suture as the zero point. Brain infusion cannulae were po- individually in plastic cages (48 35 20 cm3) with sawdust as sitioned in the left lateral cerebral ventricle (Coordinates from zero bedding. They were kept at room temperature (2571 °C) under a point: 8.6 mm anteroposterior, 1.7 mm lateral, 4.5 mm ventral, natural light cycle (provided by a closed glass window, the pho- these parameters were verified and determined according to our toperiod was roughly 15L:9D throughout the experiment) without pilot experiment by dye injection) and fixed to the skull with 3Ms artificial lighting. The commercial rat chow (crude protein 20.36%; cyanoacrylate gel. After surgery, squirrels were allowed a 1-week crude fat 3.75%; crude fiber 3.32%; ashes 6.39%; moisture 4.7%; recovery phase before the start of the experiments. Osmotic calcium 1.0–1.5%; phosphorus 0.5–0.8%; Shenyang QianMin Feed pumps (Alzets Osmotic Pumps 2002; flow rate¼0.570.1 ml/h) Co.) and water were provided ad libitum. The diet was supple- were attached to infusion cannulae via polyethylene catheters and mented with sunflower seeds, carrot pieces and cabbage during were implanted subcutaneously in the upper back when squirrels X. Xing et al. / Journal of Thermal Biology 57 (2016) 35–43 37

started to receive vehicle (aCSF) or the recombinant murine leptin 0.4–6.4 ng/ml for T3 and 7.5–120 ng/ml for T4. Above-mentioned (PeproTechs) which was dissolved in aCSF. Pumps were filled 6 h hormones were measured through the standard curves. Glucose, before implantation and primed with saline. triglyceride (TG) and free fatty acid (FFA) were tested by colori- metric method through the controls according to the manu- 2.4. Core body temperature and metabolic trails facturer’s protocols. Each sample was analyzed in single. The as-

says of TG, insulin, ghrelin, T3 and T4 employed 50 μl serum for μ Core Tb was recorded at 30 min intervals by the iButtons. Each each test; 100 l serum was used for leptin and obestatin assay μ group has 1 undetectable iButton, so we had 7 samples of Tb in and 200 l serum was used for FFA assay. control group and 9 samples in the leptin administration group. Ghrelin, obestatin and insulin kits (for rats) were purchased

We defined heterothermic events as when the Tb was at or below from Phoenix Pharm. Inc. (Mountain View, CA, USA). Glucose, TG 30 °C, which included hibernation (Tb o20 °C, duration more than and FFA (for rats) were purchased from Jian-Cheng Corp (Nanjing, 24 h) and daily torpor (Tb Z20 °C, duration less than 24 h) (Held- China). T3 and T4 kits (for rats) were purchased from Sun Biome- maier et al., 2004; Buck et al., 2008; Sheriff et al., 2012). dical Technology (Beijing, China).

O2 consumption was assessed using a FOXBOX O2 and CO2 analyzer (Sable systems, NV, USA) between 09:00 h and 19:00 h. 2.7. Analysis of mRNA levels and protein levels Non-torpid squirrels were placed in an airtight chamber (32 20 18 cm3) for 3 h. Fresh air was dried then passed through 2.7.1. Analysis of mRNA levels s the chamber at a mass flow rate of 1300–1400 ml/min. Gases Total RNA of hypothalamus and BAT was isolated using TRIzol leaving the chamber were subsampled and dried, then sent to the reagent (Cat. no. 15596-026, Invitrogen, USA). The RNA samples analyzer at approximately 100 ml/min (ND-2; Sable systems, NV, were treated with DNase I (Cat. no. D2215, TaKaRa, Japan) to re- USA). Analyzer outputs were recorded every 10 s. The rate of move the contaminating gDNA. We used phenol/chloroform/iso- oxygen consumption was calculated using the equations: amyl alcohol extraction to eliminate residual DNase I. The quality FR×( FiO22 − FeO )− FR × FeO 2 × ( FeCO 2 − FiCO 2 ) VO2 ¼ ;FR¼flow rate (stan- and quantity of total RNA were tested by agarose gel electro- 1FeO − 2 phoresis and A260/280 ratio using a UV-spectrophotometer dard temperature and pressure; ml/min), VO2 ¼rate of oxygen (ThermoFisher, NanoDrop 1000, USA). An equal amount (400 ng) consumption (ml/min), Fi¼input fractional concentration (O2, of total RNA was transcribed into first strand cDNA for each sample CO2) (%), Fe¼excurrent fractional concentration (O2,CO2) (%). RMR using a reverse transcription kit (Cat. no. RR037A, TaKaRa, Japan). was estimated as the mean of the stable and lowest values of The primers used in this study (Table 1) were proved available oxygen consumption over 5 min in normothermic squirrels (Li previously (Xing et al., 2015). Quantitative PCR was performed by et al., 2010; Chi and Wang, 2011). SYBR Green I qPCR kit (Cat. no. RR420A, TaKaRa, Shiga, Japan) in the Mx3005P quantitative PCR system (Stratagene, La Jolla, CA, 2.5. Energy intake USA). Real-time RT-PCR was carried out in 25 μl reaction agent comprised of 12.5 μl2 SYBR Premix EX Taq™master mix, 2 μl Foods were offered at pre-weighed quantity in excess on the cDNA templates and 0.2 μmol/L primers. Each sample was ana- first day of the experiment. The food residues and feces were lyzed in duplicate. Thermal cycling conditions were: 95 °C for collected after a 2-day period from a metabolic cage 3 min; 45 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 20s. (48 35 20 cm3) and separated manually after being dried at Melting curve analysis showed a single PCR product after am- 60 °C to constant mass. Cumulative food intake was calculated as plification of target genes and β-actin (the reference gene). Re- the sum of the food intakes of prior trails. At the beginning and lative mRNA expression was determined by the ΔΔ-C method end of each trial, body mass was measured with an electronic q (Bustin et al., 2009; Schmittgen and Livak, 2008). balance (70.1 g). The energy contents of the food and feces were measured using a Parr 1281 oxygen bomb calorimeter (Parr In- 2.7.2. Analysis of protein levels strument, USA). Gross energy intake (GEI), digestible energy intake BAT tissues were lysed in a modified RIPA buffer [1% Triton (DEI) and apparent digestibility of energy (hereafter referred to X-100, 158 mM NaCl, 5 mM EDTA, 10 mM Tris (pH 7.0), protease simply as digestibility) were calculated according to the following inhibitor cocktail (Sigma, St. Louis, Missouri, USA), 1 mM DTT, and equations (Liu et al., 2003; Grodzinski and Wunder, 1975): 1 mM phenylmethylsulfonyl fluoride] on ice for 30 min and then GEI()= kJ/d dry matter intake ()× g/d food caloric value () kJ/g ; centrifuged at 14,000g for 10 min in 4 °C. The concentration of protein in BAT was determined using the method of Lowry et al. DEI()=−[ kJ/d GEI mass of feces ()× g/d feces caloric value ()] kJ/g ; (1951). Proteins were separated by SDS-PAGE in ordinary 10% polyacrylamide gel (80 mg/lane). Proteins were transferred to s Digestibility()=( % DEI/GEI )× 100%; polyvinylidene difluoride (PVDF) membranes (Immobilon -P, Millipore). After transfer, the membrane was blocked in 5% skim- med milk in Tris-buffered saline-Tween for 1.5 h at room tem- 2.6. Serum assays perature and probed with the indicated antibodies [rabbit anti– UCP1 (ab10983, Abcam, Cambridge, MA, USA), diluted 1:3000] Serum leptin concentration was measured by radio- overnight at 4 °C. The immunoblot was visualized with appro- 125 immunoassay (RIA) with a I multi-species kit (Cat. No. XL-85K, priate horseradish peroxidase-conjugated secondary antibodies Linco Research Inc., St Charles, MO, USA). This kit has been pre- [goat anti-rabbit IgG (ZSGB-BIO Co., Beijing, China), diluted viously validated in Daurian ground squirrels (Chen et al., 2008; 1:3000] for 2 h and detected with ECL (Beyotime, China) and Xing et al., 2015). Intra- and inter-assay coefficients of variation quantified using Quantity One software (version 4.4.0, BioRad, were 3.6% and 8.7% respectively and the detection range of the Hercules, CA). Protein content was expressed as relative units (RU). assay kit was 1–50 ng/ml (Li and Wang, 2005a). Serum ghrelin, obestatin, insulin, tri-iodothyronine (T3) and thyroxine (T4) con- 2.8. Statistical analysis centrations were measured using ELISA kits according to the manufacturer’s protocols. The detection ranges of assay kits were Data were analyzed using SPSS 18.0 software (SPSS Inc., Chi- 0–100 ng/ml for ghrelin and obestatin, 1.5–24 mIU/l for insulin, cago, IL, USA). Temporal changes in body mass, food intake, DEI, 38 X. Xing et al. / Journal of Thermal Biology 57 (2016) 35–43

Table 1 There was no significant difference in RMR between two Species specific primers used in real-time PCR. groups under either warm (t¼0.413, df¼14, P 40.05) or cold condition (t¼0.644, df¼14, P 40.05; Table 2). Primers Oligonuncleotide sequence (5′ to 3′) PCR product size (bp) 3.2. Energy intake, body mass and body fat ObR forward AGATGGTGCCAGCAACTATGGTC 204 ObR reverse CATCCGTTTCGCTTGACTTGGA There was no significant difference in food intake (t¼0.736, AgRP forward GCAGAACAATCAGAAGAGGCT 178 df¼14, P 40.05; Fig. 2A), GEI (t¼0.736, df¼14, P 40.05; Fig. 2B) AgRP reverse CGTTGAAGAAGCGGCAGTAG NPY forward TCG CTCTGTCCCTGCTCGTGTG 161 and DEI (t¼0.667, df¼14, P 40.05; Fig. 2C) between two groups NPY reverse TCTCTTGCCGTATCTCTGCCTGGTG before leptin administration. However, cumulative food intake POMC forward TCGTGTTATAGCCGCTCAGG 196 (t¼2.175, df¼14, P o0.05), GEI (t¼2.175, df¼14, P o0.05) and DEI POMC reverse GCTGCTCGTCGCCATTGCC (t¼2.235, df¼14, P o0.05) in the leptin administrated squirrels CART forward CCTGCTACTGATGCTACCTCT 193 fi CART reverse GACTTGCCCGTACTTCTTCTC were signi cantly lower than those in the control squirrels on day UCP1 forward GCGTTGTAGGTTCCAGTGTAG 195 12. Digestibility was similar between the two groups (F1,12 ¼3.754, UCP1 reverse CTATGATTCTGTCCAGGAGTTC P40.05; Fig. 2D). Under warm condition, body mass did not show β-actin GACTCGTCGTACTCCTGCTT 223 significant difference between groups (F1,13 ¼3.857, P40.05). forward During cold exposure, leptin-treated squirrels exhibited greater β-actin reverse AAGACCTCTATGCCAACACC weight loss than the controls (F1,14 ¼6.476, Po0.05; Fig. 2E). Note: ObR, leptin receptor; AgRP, agouti gene-related protein; NPY, neuropeptide Y; However, the masses of SWAT (t¼0.726, df¼14, P 40.05), VWAT POMC, pro-opiomelanocortin; CART, cocaine- and amphetamine-regulated tran- (t¼0.008, df¼14, P 40.05) and TWAT (t¼0.464, df¼14, P 40.05) script; UCP1, uncoupling protein 1. showed no differences between two groups (Table 3).

3.3. Hormones

GEI, digestibility and Tb throughout the experiment were assessed Serum TG concentration decreased (t¼2.676, df¼11, by repeated measures ANOVA, followed by LSD post-hoc test. Po0.05) but FFA concentration increased (t¼2.226, df¼12, Differences in RMR, hormone level, WAT mass, mRNA expression Po0.05) significantly after 12 days of leptin administration. Both and UCP1 protein content between two groups were analyzed serum T (t¼2.108, df¼14, P ¼0.05) and T (t¼2493, df¼14, using independent sample t-test. Proportions of heterothermia 3 4 Po0.05) concentrations decreased after leptin administration. and hibernation were analyzed using χ-square. Data were ex- Serum obestatin concentration in leptin administrated squirrels pressed as mean7standard error (SE) and the level of statistical significantly higher than that in controls (t¼3.044, df¼14, significance was set at Po0.05. Po0.01) at the end of the study, but no significant difference was Due to the fact that there were two squirrels hibernating in found on serum ghrelin concentration (t¼0.077, df¼14, P 40.05) leptin administration group after the cold treatment, they were or the ratio of ghrelin/obestatin (t¼0.077, df¼14, P 40.05). Serum expelled from statistics of energy intake, digestibility, serum hor- leptin (t¼1.015, df¼14, P 40.05), insulin (t¼1.536, df¼14, mone assay, mRNA expression and protein contains analyses to P40.05) and glucose (t¼1.582, df¼12, P 40.05) concentrations remove the possible influence of hibernation. Due to the missing remained stable in leptin administrated squirrels (Table 4). BAT tissues, there were 6 samples in the control group and 5 samples in the leptin group for UCP1 protein assay; there were 3.4. mRNA expressions in hypothalamus 6 samples each in the control and leptin groups for the UCP1 mRNA assay. Due to the running out of serum samples, some The mRNA expressions of hypothalamic NPY (t¼2.34, df¼13, hormone analyses used fewer animals, the sample size was noted Po0.05; Fig. 3A) and AgRP (t¼2.59, df¼13, P o0.05; Fig. 3B) de- in the note of Table 4. creased by 30% and 52% respectively at the end of leptin admin- istration. No significant differences in mRNA expressions of hy- pothalamic ObR (t¼0.64, df¼13, P 40.05; Fig. 3C), POMC 3. Results (t¼0.633, df¼13, P 40.05; Fig. 3D) or CART (t¼1.12, df¼13, P40.05; Fig. 3E) was detected between groups. 3.1. Body temperature and metabolic rate 3.5. UCP1 levels in BAT There was no statistical difference in daily maximal Tb (F1, ¼ 4 ¼ 4 14 0.039, P 0.05), minimal Tb (F1, 14 0.933, P 0.05) and delta At the end of administration, no significant difference of UCP1 Tb (the differences between maximal and minimal Tb)(F1, mRNA expression (t¼1.58, df¼10, P 40.05; Fig. 4A) or protein ¼ 4 14 0.683, P 0.05) between control and leptin administration content (t¼0.86, df¼9, P40.05; Fig. 4B) were detected in BAT groups. The daily maximal Tb remained constant throughout the between groups. course of experiment (F11, 154 ¼2.337, P40.05). However, the daily minimal Tb decreased (F11, 154 ¼4.992, Po0.05) and the delta Tb ¼ o fi increased (F11, 154 35.924, P 0.05) signi cantly during the cold 4. Discussion exposure in both groups (Fig. 1A). Neither control squirrels nor leptin administrated squirrels showed In fat-storing hibernators, serum leptin concentration is cor- heterothermia under the warm condition. During the subsequent cold related positively with body mass and body fat mass (Concannon exposure, there were two squirrels showed deep hibernation (the et al., 2001; Chen et al., 2008; Healy and Florant, 2012). Florant minimal Tb was 5 °Cand4°C respectively), but none in control group. et al. (2004) proposed leptin resistance hypothesis to explain this Two squirrels and three squirrels showed daily torpor in leptin group paradox phenomenon. Our data showed that 6-day ICV leptin and control group respectively. Rest of the animals kept euthermic administration decreased the body mass, and the subsequent cold during cold exposure (Fig. 1B). There were no statistical differences in exposure can further decreased the body mass in leptin group the ratio of daily torpor (P40.05) and hibernation (P40.05; Fig. 1C). during fattening in Daurian ground squirrels. Similar results were X. Xing et al. / Journal of Thermal Biology 57 (2016) 35–43 39

Fig. 1. Effects of leptin on body temperature of fattening Daurian ground squirrels. A: Effects of 12-day ICV infusion of leptin (1 μg/day) on changes of daily maximal body temperature (Tb max), minimal body temperature (Tb min) and the differences between maximal and minimal body temperature (delta Tb) in fattening Daurian ground squirrels. The dash line marks the day when the animals were transferred from warm (25 °C) natural lighting room into the cold (5 °C) constant dark room. B: Traces of body temperature in Dauria ground squirrels with ICV leptin administration (L; n¼9) and control administration (C; n¼7). The bouts of daily torpor are marked with red asterisks. The dash line marks the day when the animals were transferred from warm (25 °C) natural lighting room into the cold (5 °C) constant dark room. C: Effects of 12-day ICV infusion of leptin (1 μg/day) on the proportions of heterothermic squirrels during fattening period. Data were expressed as mean7SE (n¼7 for control group, n¼9 for leptin group). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 2 regulation of food intake (Schwartz et al., 2000; Flier, 2004; The resting metabolic rate of fattening Daurian ground squirrels during leptin Morton et al., 2006), suggesting these two orexigenic neuropep- administration. tides may be involved in leptin-mediated decrease of food intake. These results also showed that the fattening Daurian ground aCSF Leptin P squirrel retained the sensitivity to centrally administered leptin.

25 °C RMR (mLO2/h) 291.02718.46 276.34730.39 NS Leptin, ghrelin, obestatin and insulin are all involved in the ° 7 7 5 C RMR (mLO2/h) 588.29 43.38 538.19 64.64 NS regulation of energy balance in fat-storing hibernators (Florant et al., 1985; Healy et al., 2010, 2011; Healy and Florant, 2012). In Note: Values are means7SE. Values determined by independent t-tests (n¼8 for each group). the present study, no change was detected in serum ghrelin con- centration while the serum concentration of obestatin, which is encoded by ghrelin gene (Zhang et al., 2005), increased after leptin also reported for fattening arctic ground squirrels after 3 weeks of administration, suggesting that leptin and obestatin may have a peripheral leptin administration (Ormseth et al., 1996; Boyer et al., cooperative role in the regulation of food intake in squirrels. Ser- um insulin and glucose concentrations in leptin administrated 1997). Our previous study showed that the expression of hy- squirrels were not significantly different from those of controls, pothalamic NPY increased by 67% during fattening, in spite of the which indicated that 12-day ICV leptin administration has no ef- elevated serum leptin concentration (Xing et al., 2015). Schwartz fect on the glucose homeostasis in this species. et al. (2015) demonstrated that the expression of NPY did not ICV leptin administration decreased serum TG concentration change between the hyperphagic and hypophagic squirrels before and increased FFA concentration significantly, indicating the lipo- hibernation, though the serum leptin protein expression was sig- lytic role of leptin in fattening squirrels. We did not find the dif- fi ni cantly higher in hypophagic group. The time of the sampling ferences in body fat mass between the two groups. It has been can be responsible for the differences in NPY expression. However, shown that peripheral delivery of leptin did not reduce body fat in the present study, the mRNA expression of NPY and AgRP were mass in pre-hibernating arctic ground squirrels (Ormseth et al., suppressed after ICV leptin administration. Since several lines of 1996). The surgical body fat removal in the fattening golden- evidence showed that NPY and AgRP were involved in the mantled ground squirrels (Callospermophilus lateralis) was quickly 40 X. Xing et al. / Journal of Thermal Biology 57 (2016) 35–43

Table 4 Serum hormone concentrations of fattening Daurian ground squirrels after 12-day ICV infusion of leptin (1 μg/day) or artificial cerebrospinal fluid (aCSF).

Hormones aCSF Leptin P

Leptin (ng/ml) 1.72 70.37 2.2870.40 NS Insulin (mIU/l) 5.4770.30 4.8170.31 NS Ghrelin (ng/ml) 3.0370.26 3.0770.32 NS Obestatin (ng/ml) 1.11 70.05 1.4270.08 o0.01 Ghrelin/obestatin 2.7670.25 2.2670.30 NS Glucose (ng/ml) 7.2670.53 5.8070.68 NS Triglyceride (ng/ml) 0.9470.08 0.5770.11 o0.05 Free fatty acid (ng/ml) 340.2738.1 471.4744.9 o0.05

T3 (ng/ml) 1.4070.10 1.1070.07 0.05

T4 (ng/ml) 29.171.7 23.471.5 o0.05

Note: Values are means7SE. Values determined by independent t-tests. In ana-

lyses of leptin, insulin, ghrelin, T3 and T4, the sample number was 8 for each group. Due to the running out of serum samples, the other hormone analyses used fewer animals: there were 8 samples for control and 7 samples for leptin group in ana- lyses of obestatin and ghrelin/obestatin; there were 7 samples for each group for analyses of glucose and free fatty acid; there were 6 samples for control and 7 samples for leptin group in analysis of triglyceride.

expression and protein content in BAT to maintain the energy homeostasis (Cancello et al., 1998; Demas et al., 2002; Gullicksen et al., 2002). In several wild rodents, serum leptin level was usually negatively correlated with UCP1 content (Li and Wang, 2005b; Wang et al., 2006 a,b; Zhang and Wang, 2007; Chen et al., 2012). Such a relationship between serum leptin concentration and UCP1 was not detected in Dauiran ground squirrels (Xing et al., 2015). We also observed that 3-week cold acclimation sig- nificantly increased the UCP1 expression and protein contents (Xing et al. unpublished data). In this study, we did not observe any statistical difference in mRNA expression or protein content of UCP1 in BAT at the end of leptin administration, through the 6-day cold acclimation might increase the UCP1 level. We also did not detect the effect of leptin on RMR in fattening squirrels under ei- ther warm or cold conditions. These results suggest the dissocia- tion between leptin and thermogenic ability in the Daurian ground Fig. 2. Effects of 12-day ICV infusion of leptin (1 μg/day) on changes of cumulative food intake (CFI; A), cumulative gross energy intake (CGEI; B), cumulative digestible squirrels. energy intake (CDEI; C), digestibility (D) and body mass (E) in fattening Daurian Hibernation in obligate hibernators is spontaneous and non- ground squirrels. Data were expressed as mean7SE (n¼8 for each group). Asterisk opportunistic (Drew et al., 2007; French, 2008). It has been shown o (*) indicates a statistical difference between groups (P 0.05). The dash line marks that both short photoperiod and cold acclimation cannot induce the day when the animals were transferred from warm (25 °C) natural lighting room into the cold (5 °C) constant dark room. hypothermia in the summer active Daurian ground squirrels (Zhang et al., 1996; Huang et al., 2006). However, timing and hi- bernation patterns were influenced by the energy storage in some fat-storing hibernators (Bieber et al., 2014; Vuarin et al., 2013) and Table 3 food-storing hibernators (Otsu and Kimura, 1993; Humphries The masses of subcutaneous WAT, visceral WAT and total WAT in fattening Daurian et al., 2003). In the present study, 12-day leptin administration, a ground squirrels after leptin administration. supposed exogenous energy signal, did not significantly facilitate torpor in fattening squirrels in either warm or cold condition, aCSF Leptin P therefore our hypothesis was not supported. The thyroid hor- Subcutaneous WAT (g) 30.1375.18 27.8175.24 NS mones have significant calorigenic effect in mammals (Mullur 7 7 Visceral WAT (g) 13.51 3.10 15.10 3.71 NS et al., 2014). The serum T3 and T4 concentrations were significantly 7 7 Total WAT (g) 43.64 7.84 42.91 8.77 NS higher in hibernating season than that in summer active seasons

Note: Values are means7SE. Values determined by independent t-tests (n¼8 for in Daurian ground squirrels (Huang et al., 2003), similar results each group). were reported in other seasonal hibernators (reviewed in Lyman et al., 1982). The elevated thyroid hormones might be prepared for the thermogenesis capacity during the interbout arousal. In mice eliminated before hibernation (Dark et al., 1984). There might be and rats, T3 and T4 were stimulated by high leptin concentration compensatory recruitment in WAT in squirrels, because the WAT is via the central regulation (Zimmermann-Belsing et al., 2003). the primary fuel for hibernation. However, in Daurian ground squirrel, high thyroid hormones UCP1 in BAT is critical for non-shivering thermogenesis in during hibernation were coincident with the relative low leptin mammals during winter and cold exposure (Cannon and Ne- concentration resulted from consumption of body fat. In this study, dergaard, 2004). In laboratory rodents, increased leptin level can leptin administration decreased the serum T3 and T4 concentration stimulate thermogenic capacity by increasing UCP1 mRNA during fattening period. We suppose that, in Daurian ground X. Xing et al. / Journal of Thermal Biology 57 (2016) 35–43 41

Fig. 3. Relative changes in hypothalamic NPY (A), AgRP (B), ObR (C), POMC (D) and CART (E) mRNA expressions as the function of 12-day ICV leptin administration (1μg/day). Data were expressed as mean7SE (n¼8 for each group). Asterisk (*) indicates a statistical difference between groups (Po0.05). squirrels, leptin may have a different relationship with thyroid signal of starvation and energy stress, was supposed to be neces- hormones compared with that in rats. sary for evoking torpor in Siberian hamsters (Freeman et al., Contrary to obligate hibernators, facultative hibernators are 2004). The differences in physiological energetics before entering stimulated to enter hibernation by winter-like conditions, such hibernation between obligate and facultative hibernators imply as short photoperiod, cold exposure and/or food shortage the differences of energy regulation and the initiation of (Kirsch et al., 1991; Klingenspor et al., 2000; Grimpo et al., 2013). hibernation. Most facultative hibernators resist becoming torpid until they have lost body mass (Drew et al., 2007; French, 2008). Hibernation of Siberian hamsters (Phodopus sungorus) is induced by body Acknowledgments mass loss triggered by short-photoperiod (Heldmaier and Stein- lechner, 1981; Bartness and Wade, 1985; Ruf et al., 1993). We are grateful to all the members of Animal Physiological More than half of the body mass loss is due to reduction of body Ecology Group for their help for the experiments and the discus- fat (Klingenspor et al., 2000). Low leptin concentration, as a sions. We thank Doctor Teresa Valencak, Research Institute of 42 X. Xing et al. / Journal of Thermal Biology 57 (2016) 35–43

Fig. 4. Relative changes of UCP1 mRNA expression (A) and protein content (B) in BAT as the function of 12-day ICV leptin administration (1 μg/day). Data were expressed as mean7SE (n¼6 for each group in analysis of mRNA expression; there were 6 samples in control and 5 samples in leptin group for analysis of protein content).

Wildlife Ecology, University of Veterinary Medicine Vienna, for her removal of adipose tissue in ground squirrels. Proc. Natl. Acad. Sci. USA 81, suggestions and help improving the English. This study was fi- 2270–2272. Demas, G.E., Bowers, R.R., Bartness, T.J., Gettys, T.W., 2002. Photoperiodic regulation nancially supported partly by grants of National Natural Science of gene expression in brown and white adipose tissue of Siberian hamsters Foundation of China to MY (No. 31170380), GBT (No. 30900140), (Phodopus sungorus). Am. J. Physiol. Regul. Integr. Comp. Physiol. 282, 114–121. Chinese Academy of Sciences (KSCX2-EW-N-005), and The State Drew, K.L., Buck, C.L., Barnes, B.M., Christina, S.L., Rasley, B.T., Harris, M.B., 2007. Key Laboratory of Integrated Management of Pest Insects and Central nervous system regulation of mammalian hibernation: implications for metabolic suppression and ischemia tolerance. J. Neurochem. 102, 1713–1726. Rodents (No. ChineseIPM1506; ChineseIPM1616). Flier, J.S., 2004. Obesity wars: molecular progress confronts an expanding epidemic. Cell 116, 337–350. Florant, G.L., Healy, J.E., 2012. The regulation of food intake in mammalian hi- bernators: a review. J. Comp. Physiol. B. 182, 451–467. References Florant, G.L., Lawrence, A.K., Willimas, K., Bauman, W.A., 1985. Seasonal changes in pancreatic B-cell function in euthermic yellow-bellied marmots. Am. J. Physiol. Regul. Integr. Comp. Physiol. 249, 159–165. Bartness, T.J., Wade, G.N., 1985. Photoperiodic control of seasonal body weight Florant, G.L., Porst, H., Peiffer, A., Hudachek, S.F., Pittman, C., Summers, S.A., Rajala, cycles in hamsters. Neurosci. Biobehav. Rev. 9, 599–612. Baskin, D.G., Figlewicz Lattemann, D., Seeley, R.J., Woods, S.C., Porte Jr, D., Schwartz, M.W., Scherer, P.E., 2004. Fat-cell mass, serum leptin and adiponectin changes fl M.W., 1999. Insulin and leptin: dual adiposity signals to the brain for the reg- during weight gain and loss in yellow-bellied marmots (Marmota aviventris). J. – ulation of food intake and body weight. Brain Res. 848, 114–123. Comp. Physiol. B 174, 633 639. Bieber, C., Lebl, K., Stalder, G., Geiser, F., Ruf, T., 2014. Body mass dependent use of Freeman, D.A., Lewis, D.A., Kauffman, A.S., Blum, R.M., Dark, J., 2004. Reduced leptin hibernation: why not prolong the active season, if they can? Funct. Ecol. 28, concentrations are permissive for display of torpor in Siberian hamsters. Am. J. – 167–177. Physiol. Regul. Integr. Comp. Physiol. 287, 97 103. Boyer, B.B., Barnes, B.M., 1999. Molecular and metabolic aspects of mammalian French, A., 2008. Patterns of heterothermy in rodents. In: Lovegrove, B.G., hibernation. Bioscience 49, 713–724. McKechnie, A.E. (Eds.), Hypometabolism in Animals Hibernation, Torpor and – Boyer, B.B., Ormseth, O.A., Buck, L., Nicolson, M., Pelleymounter, M.A., Barnes, B.M., Cryobiology. University of KwaZulu-Natal, Pietermaritzburg, pp. 337 352. – 1997. Leptin prevents posthibernation weight gain but does not reduce energy Geiser, F., 2013. Hibernation. Curr. Biol. 23, 188 193. expenditure in arctic ground squirrels. Comp. Biochem. Physiol. C Pharmacol. Glowinski, J., Iversen, L.L., 1966. Regional studies of catecholamines in the rat brain – – Toxicol. Endocrinol. 118, 405–412. I. J. Neurochem. 13, 655 669. Buck, C.L., Breton, A., Kohl, F., Toien, O., Barnes, B.M., 2008. Overwinter body tem- Grimpo, K., Legler, K., Heldmaier, G., Exner, C., 2013. That's hot: golden spiny mice perature patterns in free-living Arctic squirrels (Spermophilus parryii). In: display torpor even at high ambient temperatures. J. Comp. Physiol. B 183, – Lovegrove, B.G., McKechnie, A.E. (Eds.), Hypometabolism in Animals Hiberna- 567 581. tion, Torpor and Cryobiology. University of KwaZulu-Natal, Pietermaritzburg, Grodzinski, W., Wunder, B., 1975. Ecological energetics of small mammals. In: pp. 317–326. Golley, E.B., Petrusewiez, K., Ryszkowski, L. (Eds.), Small Mammals: Their Pro- Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., ductivity and Copulation Dynamics. Cambridge University Press, Cambridge, Nolan, T., Pfaffl, M.W., Shipley, G.L., 2009. The MIQE guidelines: minimum in- pp. 173–204. formation for publication of quantitative real-time PCR experiments. Clin. Guan, G., 2009. Effects of diet quality on energy budgets and hibernation patterns Chem. 55, 611–622. in Daurian Ground Squirrel (Spermophilus dauricus) and the Regulatory Me- Cancello, R., Zingaretti, M., Sarzani, R., Ricquier, D., Cinti, S., 1998. Leptin and UCP1 chanism. Shenyang Normal University, Shenyang, , China. genes are reciprocally regulated in brown adipose tissue. Endocrinology 139, Guan, S., 2010. The Study on the Relationship between Entering Hibernation and 4747–4750. the Energy Accumulation of Daurian Ground Squirrel (Spermophilus dauricus). Cannon, B., Nedergaard, J., 2004. Brown adipose tissue: function and physiological Shenyang Normal University, Shenyang, Liaoning, China. significance. Physiol. Rev. 84, 277–359. Gullicksen, P.S., Flatt, W.P., Dean, R.G., Hartzell, D.L., Baile, C.A., 2002. Energy me- Chen, J., Yang, M., Zhong, W., Wang, D.H., 2008. Seasonal changes in body mass, tabolism and expression of uncoupling proteins 1, 2, and 3 after 21 days of serum leptin concentration and UCP1 content in Dauiran ground squirrel recovery from Intracerebroventricular mouse leptin in rats. Physiol. Behav. 75, (Spermophilus dauricus). In: Lovegrove, B.G., McKechnie, A.E. (Eds.), Hypome- 473–482. tabolism in Animals Hibernation, Torpor and Cryobiology. University of Kwa- Healy, J.E., Bateman, J.L., Ostrom, C.E., Florant, G.L., 2011. Peripheral Ghrelin sti- Zulu-Natal, Pietermaritzburg, pp. 271–282. mulates feeding behavior and positive energy balance in a Sciurid hibernator. Chen, J.F., Zhong, W.Q., Wang, D.H., 2012. Seasonal changes in body mass, energy Horm. Behav. 59, 512–519. intake and thermogenesis in Maximowiczi's voles (Microtus maximowiczii) Healy, J.E., Florant, G.L., 2012. Ghrelin, leptin, and fatty acids in free-living Callo- from the inner Mongolian grassland. J. Comp. Physiol. B. 182, 275–285. spermophilus lateralis (golden-mantled ground squirrels). In: Ruf, T., Bieber, C., Chi, Q.S., Wang, D.H., 2011. Thermal physiology and energetics in male desert Arnold, W., Millesi, E. (Eds.), Living in a Seasonal World: Thermoregulatory and hamsters (Phodopus roborovskii) during cold acclimation. J. Comp. Physiol. B. Metabolic Adaptations. Springer, Heidelberg, pp. 519–530. 181, 91–103. Healy, J.E., Ostrom, C.E., Wilkerson, G.K., Florant, G.L., 2010. Plasma Ghrelin con- Concannon, P., Levac, K., Rawson, R., Tennat, B., Bensasoun, A., 2001. Seasonal centrations change with physiological state in a Sciurid hibernator (Spermo- Changes in serum leptin, food intake, and body weight in Photoentrained philus lateralis). Gen. Comp. Endocr. 166, 372–378. woodchucks. Am. J. Physiol. Regul. Integr. Comp. Physiol. 281, 951–959. Heldmaier, G., Ortmann, S., Elvert, R., 2004. Natural hypometabolism during hi- Dark, J., 2005. Annual lipid cycles in hibernators: integration of physiology and bernation and daily torpor in mammals. Respir. Physiol. Neurobiol. 141, behavior. Annu. Rev. Nutr. 25, 469–497. 317–329. Dark, J., Forger, N.G., Zucker, I., 1984. Rapid recovery of body mass after surgical Heldmaier, G., Steinlechner, S., 1981. Seasonal pattern and energetics of short daily X. Xing et al. / Journal of Thermal Biology 57 (2016) 35–43 43

torpor in the Djungarian hamster Phodopus-sungorus. Oecologia 48, 265–270. Ruf, T., Stieglitz, A., Steinlechner, S., Blank, J.L., Heldmaier, G., 1993. Cold exposure Huang, C.X., Lin, L., Li, Q.F., 2003. Thermogenic Activeiy and Hormonal regulation of and food restriction facilitate physiological responses to short photoperiod in Daurian ground squirrel during hibernation and nonhibernation. J. Beijing Djungarian hamsters (Phodopus sungorus). J. Exp. Zool. 267, 104–112. Norm. Univ. (Nat. Sci.) 39 (5), 683–687. Schmittgen, T.D., Livak, K.J., 2008. Analyzing real-time PCR data by the comparative Huang, C.X., Lin, L., Li, Q.F., 2006. Short photoperiod induces the thermogenesis in CT method. Nat. Protoc. 3, 1101–1108. Daurian ground squirrels. Acta Ther. Sin. 26, 346–353. Schwartz, C., Hampton, M., Andrews, M.T., 2015. Hypothalamic gene expression Humphries, M.M., Kramer, D.L., Thomas, D.W., 2003. The role of energy availability underlying pre-hibernation satiety. Genes Brain Behav. 14 (3), 310–318. in mammalian hibernation: an experimental test in free-ranging eastern Schwartz, M.W., Woods, S.C., Porte Jr., D., Seeley, R.J., Baskin, D.G., 2000. Central chipmunks. Physiol. Biochem. Zool. 76, 180–186. nervous system control of food intake. Nature 404, 661–671. Kershaw, E.E., Flier, J.S., 2004. Adipose tissue as an endocrine organ. J. Clin. Endocr. Sheriff, M.J., Williams, C.T., Kenagy, G., Buck, C.L., Barnes, B.M., 2012. Thermo- Metab. 89, 2548–2556. regulatory Changes anticipate hibernation onset by 45 days: data from free- Kim, M.S., Namkoong, C., Kim, H.S., Jang, P.G., Kim Pak, Y.M., Katakami, H., Park, J.Y., living Arctic ground squirrels. J. Comp. Physiol. B 182, 841–847. Lee, K.U., 2004. Chronic central administration of ghrelin reverses the effects of Tups, A., Helwig, M., Khorooshi, R.M., Archer, Z.A., Klingenspor, M., Mercer, J.G., – leptin. Int. J. Obes. Relat. Metab. Disord. 28, 1264 1271. 2004. Circulating Ghrelin levels and central ghrelin receptor expression are Kirsch, R., Ouarour, A., Pevet, P., 1991. Daily torpor in the Djungarian hamster elevated in response to food deprivation in a seasonal (Phodopus (Phodopus sungorus): photoperiodic regulation, characteristics and circadian sungorus). J. Neuroendocrinol. 16, 922–928. – organization. J. Comp. Physiol. A 168, 121 128. Vázquez-Vela, M.E.F., Torres, N., Tovar, A.R., 2008. White adipose tissue as endo- Klingenspor, M., Niggemann, H., Heldmaier, G., 2000. Modulation of leptin sensi- crine organ and its role in obesity. Arch. Med. Res. 39, 715–728. tivity by short photoperiod acclimation in the Djungarian hamster Phodopus Vuarin, P., Dammhahn, M., Henry, P.Y., 2013. Individual flexibility in energy saving: – sungorus. J. Comp. Physiol. B 170, 37 43. body size and condition constrain torpor use. Funct. Ecol. 27, 793–799. Li, X.S., Wang, D.H., 2005a. Regulation of body weight and thermogenesis in sea- Wang, J.M., Zhang, Y.M., Wang, D.H., 2006a. Seasonal regulations of energetics, ’ sonally acclimatized Brandt s voles (Microtus brandti). Horm. Behav. 48, serum concentrations of leptin, and uncoupling protein 1 content of brown 321–328. adipose tissue in root voles (Microtus oeconomus) from the qinghai-tibetan Li, X.S., Wang, D.H., 2005b. Seasonal adjustments in body mass and thermogenesis plateau. J. Comp. Physiol. B 176, 663–671. in Mongolian gerbils (Meriones unguiculatus): the roles of short photoperiod Wang, J.M., Zhang, Y.M., WANG, D.H., 2006b. Seasonal thermogenesis and body and cold. J. Comp. Physiol. B 175, 593–600. mass regulation in plateau pikas (Ochotona curzoniae). Oecologia 149, 373–382. Li, Y.G., Yan, Z.C., Wang, D.H., 2010. Physiological and biochemical basis of basal Williams, C., Barnes, B., Kenagy, G., Buck, C.L., 2014. Phenology of hibernation and metabolic rates in Brandt's voles (Lasiopodomys brandtii) and Mongolian gerbils reproduction in ground squirrels: integration of environmental cues with en- (Meriones unguiculatus). Comp. Biochem. Physiol. A Mol. Integr. Physiol. 157, dogenous programming. J. Zool. 292, 112–124. 204–211. Xing, X., Yang, M., Wang, D.H., 2015. The expression of leptin, hypothalamic Neu- Liu, H., Wang, D.H., Wang, Z.W., 2003. Energy requirements during reproduction in ropeptides and UCP1 before, during and after fattening in the Daurian ground female Brandt's voles (Microtus brandtii). J. Mammal. 84, 1410–1416. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement squirrel (Spermophilus dauricus). Comp. Biochem. Physiol. A Mol. Integr. Physiol. – with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. 184, 105 112. Lv, Z., Song, S.Y., Yang, M., Peng, X., 2014. Body temperature,metabolic rate and Yang, M., Xing, X., Guan, S.J., Zhao, Y., Wang, Z.Y., Wang, D.H., 2011. Hibernation energetic characteristics of Daurian ground squirrels (Spermophilus dauricus) patterns and changes of body temperature in Daurian ground squirrels (Sper- – during preparation of hibernation. Acta Ther. Sin. 34, 348–355. mophilus dauricus) during hibernation. Acta Theriol. Sin. 31, 387 395. Lyman, C.P., Willis, J.S., Malan, A., Wang, L.C.H., 1982. Hibernation and Torpor in Zhang, J.V., Ren, P.G., Avsian-Kretchmer, O., Luo, C.W., Rauch, R., Klein, C., Hsueh, A. Mammals and Birds. Academic Press, NY. J., 2005. Obestatin, a peptide encoded by the Ghrelin gene, opposes Ghrelin's – Martin, S.L., 2008. Mammalian hibernation: a naturally reversible model for insulin effects on food intake. Science 310, 996 999. resistance in man? Diabetes. Vasc. Dis. Res. 5, 76–81. Zhang, S.Z., Li, Q.F., Huang, C.X., 1996. Adaptive thermogensis of Daurian ground Morton, G., Cummings, D., Baskin, D., Barsh, G., Schwartz, M., 2006. Central nervous squirrel (Citellus dauricus) during cold acclimation in summer. Acta Ther. Sin. system control of food intake and body weight. Nature 443, 289–295. 17, 67–72. Mullur, R., Liu, Y.Y., Brent, G.A., 2014. Thyroid Hormone regulation of metabolism. Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L., Friedman, J.M., 1994. Physiol. Rev. 94, 355–382. Positional cloning of the mouse obese gene and its human homologue. Nature Ormseth, O.A., Nicolson, M., Pelleymounter, M., Boyer, B.B., 1996. Leptin inhibits 372, 425–432. prehibernation hyperphagia and reduces body weight in arctic ground squir- Zhang, Z.B., Wang, Z.W., 1998. Ecology and Management of Pests in Agri- rels. Am. J. Physiol. Regul. Integr. Comp. Physiol. 271, 1775–1779. culture (In Chinese with English Summary). Ocean Press, Beijing. Otsu, R., Kimura, T., 1993. Effects of food availability and ambient-temperature on Zhang, Z.Q., Wang, D.H., 2007. Seasonal changes in thermogenesis and body mass hibernation in the Japanese dormouse, Glirulus japonicus. J. Ethol. 11, 37–42. in wild Mongolian gerbils (Meriones unguiculatus). Comp. Biochem. Physiol. A Overton, J.M., Willimas, T.D., Chambers, J.B., Rashotte, M.E., 2001. Central leptin Mol. Integr. Physiol. 148, 346–353. infusion attenuates the cardiovascular and metabolic effects of fasting in rats. Zimmermann-Belsing, T., Brabant, G., Holst, J.J., Feldt-Rasmussen, U., 2003. Circu- Hypertension 37, 663–669. lating leptin and thyroid dysfunction. Eur. J. Endocrinol. 149, 257–271.