Huddling relieves myocardial glycogen synthesis of Brandt’s voles in 1 mild cold environment

Background: Small mammals have limited glucose use and limited glycogen accumulation during 11 hypothermia. Huddling is a highly evolved cooperative behavioral strategy in social mammals, 12 allowing adaptation to environmental cooling. As yet, however, is not clear whether this behavior 13 affects the utilization of glycogen in cold environments. Here, we studied the effect of huddling on 14 myocardial glycogen content in Brandt’s voles ( Lasiopodomys brandtii ) under a mild cold 15 environment (15 °C). 16 Results: Results showed that (1) Compared to the control (22 °C) group (CON), the number of 17 glycogenosomes more than tripled in the cool separated group (CS) in both males and females; 18 whereas the number of glycogenosomes increased in females but was maintained in males in the 19 cool huddling group (CH). (2) The ratio of glycogen synthetase phosphorylation showed a similar 20 trend as the change in glycogenosome number in the three treatment groups in both males and 21 females. (3) Protein expression of glycogen phosphorylase remained stable in males in the three 22 treatment groups but increased in CS group females compared with the CON group. 23 Conclusion: These results indicate that huddling in voles alleviated the increase in myocardial glycogen content caused by the increase of glycogen synthesis under cool environments.


Introduction 28
Low temperature is a stress stimulus for mammals, especially for small mammals as their energy 29 requirements are high due to the large surface area to volume ratio. Moreover, when 30 environmental stressors persist for prolonged periods, small animal tissues and organs are more 31 vulnerable to the impact of external environmental temperature [1]. Altered carbohydrate 32 metabolism during hypothermia in mammals is accompanied by abnormalities in glucose 33 metabolism [2][3][4]. For example, in rats [5, 6] and rabbits [7], metabolism of both endogenously 34 and exogenously administered glucose is substantially reduced during hypothermia. Hypothermia 35 can lead to a slowed heart rate, decreased blood flow output, and decreased myocardial 36 contraction and relaxation function [8][9][10][11]. As above, the cardiac muscle of small mammals is 37 more susceptible to low external temperatures. In rats, for example, exposure to only 4 h of cold 38 temperature (15°C) can lead to an increase in myocardial glycogen content [12], suggesting that 39 the effects of hypothermia on cardiac muscle may involve the balance between glycogen synthesis 40 and degradation. 41 Glycogen synthase (GS), a key enzyme for synthesis, polymerizes UDP-glucose to form glycogen 42 granules, with phosphorylated GS (P-GS) being its active state [13][14][15]. Glycogen phosphorylase 43 (GYPL) is a rate-limiting enzyme that breaks down glycogen granules to glucose [16,17]. 44 Research on hibernating Daurian ground squirrels (Spermophilus dauricus) has shown that the 45 increase in glycogen content in skeletal muscle in winter is mainly due to the maintenance of 46 P-GS and decrease in GYPL protein expression [15]. Thus, studies on the above factors could help 47 reveal the mechanism related to changes in myocardial glycogen content under cool environments. 48 Huddling is a social thermoregulatory behavior, defined as the active aggregation of nestled 49 animals. It is a cooperative group behavior, permitting individuals involved in social 50 thermoregulation to minimize heat loss and thereby lower energy expenditure, possibly allowing 51 Experimental Animal Breeding Co., Ltd., China) and water were provided ad libitum and wood 94 shavings were used as bedding. 95 Based on body weight and degree of wear on the upper molars, a total of 24 male (28-50 g, 96 average 38 g) and 24 female adult voles (27-54 g, average 33 g) were randomly divided into three 97 groups, respectively. Voles in the control groups (CON) of each sex were housed individually in 98 cages at an ambient temperature of 22 ± 2°C, but with a changed light regime to a short day 99 pattern (8 h:16 h light⁄dark cycle; light on from 08:00 to 16:00). Two groups of each sex were 100 transferred to a cool cabinet under different grouping conditions (huddling and separated). Voles 101 in the cool separated groups (CS) were housed individually in cages, whereas voles in the cool 102 huddling groups (CH) were housed together in a cage (two males and two females). Group size 103 (four voles in each cage) ensured most animals remained inactive in a huddle (Sukhchuluun et al.,104 2018). The temperature in the cabinet was set to 15°C and the light regime was as same as the 105 CON group. Animal treatment started in late September and lasted eight weeks. 106

Sample preparation 107
All animals were anaesthetized with 50 mg kg -1 sodium pentobarbital between 08:00 and 11:00 on 108 the last day of the experimental period [19,30]. After the rapid removal of cardiac muscle, 109 portions of the ventricles were immediately cut off and fixed in glutaraldehyde. The rest of the 110 cardiac muscle was frozen in liquid nitrogen and stored at -80°C. Specimens were fixed in 1% 111 osmium tetroxide in the same buffer, dehydrated with a graded series of ethanol, and embedded in 112 epoxy resin. After surgical intervention, the animals were sacrificed with an overdose injection of 113 sodium pentobarbital. All procedures were carried out in accordance with the approved guidelines. 114

Transmission electron microscopy (TEM) 115
The cardiac muscle samples were cut into blocks and immersed in 3% 116 glutaraldehyde-paraformaldehyde. The blocks were then dehydrated in a graded series of ethanol 117 and embedded in epoxy resin, with TEM then performed as described previously and then examined via TEM (Hitachi, HT7800, Japan). Images were processed with NIH 122 Image-Pro Plus 6.0. Images were analyzed using the measurement tools provided by the software. 123 Glycogenosome densities were determined within a defined region (4 μm 2 area) at a minimum of 124 three locations within an image taken at 25 000× magnification. 125

Statistical analyses 148
The normality of data and homogeneity of variance were tested by Shapiro-Wilk and Levene tests, 149 respectively. All data exhibited normal distribution and homogeneous variance. Double-factor 150 variance analysis (two-way analysis of variance (ANOVA)) was used to compare differences 151 between treatment and sex. Results were significant at P < 0.05. Data are expressed as mean ± 152 standard deviation (Mean ± SD). All statistical analyses were conducted using SPSS 19.0. 153

Results 154
Ultrastructural changes in number of glycogenosomes 155 The glycogenosome clusters were observed, with each glycogenosome showing a diameter of~30 156 nm. Most glycogenosomes were distributed between the muscle filaments, with a small number 157 distributed around the mitochondria (Fig. 1). 158 Figure 2a shows the distribution of glycogenosomes at low magnification. In the CS group, the 160 number of glycogenosomes was more than triple that in the CON and CH groups (P < 0.05). In 161 addition, the number was significantly higher (P < 0.05) in females than in males (Fig. 2b). 162

Changes in protein expression of glycogen synthesis-related proteins 164
The GS and P-GS concentrations were detected by western blot analysis, as shown in Fig. 3. 165 Representative polyacrylamide gels of total protein are shown in Fig. 3b. 166 The relative protein expression levels of GS and P-GS showed the different trends among the 167 three treatment groups. Specifically, the protein expression levels of GS in the CS groups were 168 lower than the levels in the CH and CON groups, whereas protein expression levels of P-GS in the 169 CH and CS groups were higher than levels in the CON group (P < 0.05). Levels of GS and P-GS 170 were also higher (P < 0.05) in females than in males ( Fig. 3c and d). 171 The P-GS to GS ratio is one of the most direct indicators of glycogen synthesis. Here, the ratio 172 showed the trends among the three treatment groups as CON < CH < CS (P < 0.05). The ratio was 173 also higher (P < 0.05) in females than in males (Fig. 3e). 174

Changes in protein expression of glycogen decomposition-related proteins 176
The content of GYPL was detected by western blot analysis, as shown in Fig. 4. Representative 177 polyacrylamide gels of total protein are shown in Fig. 4b. 178 The relative protein expression of GYPL showed a slight change among the three treatment 179 groups. Specifically, levels were higher in CS group females than in CON group females (Fig. 4c). 180

INSERT FIGURE 4 HERE 181
Discussion 182 We studied the effects of cool environment on the number of cardiac glycogenosomes in huddling 183 Brandt's voles, as well as the mechanism related to the regulation of glycogenosome number. An 184 obvious increase in the number of myocardial glycogenosomes was observed in both CS males 185 and females. In addition, this number remained stable in males but increased in females in the CH 186 groups compared with that in the CON groups. The P-GS to GS ratio was highest in the CS group 187 in both males and females. GYPL protein expression only showed a slight change among the three 188 treatment groups. of GYPL in the CS group was maintained at the same level as observed in the CON group, 206 whereas the phosphorylation ratio of GS was significantly increased, suggesting that high 207 glycogen synthesis may be one of the main reasons for the increase in glycogen content. In 208 females, the protein expression of GYPL and the phosphorylation ratio of GS increased 209 significantly in the CS group, but the increment between the two groups was significantly 210 different compared with the CON group. This indicated that the increase in glycogen synthesis 211 was higher than that of glycogen decomposition, which may be the reason why glycogen content 212 increased at this time. In general, the increase in glycogen synthesis may be a key factor for the 213 increase in myocardial glycogen content observed in the CS group in both male and female voles. 214 Surprisingly, compared with the CON group, the myocardial glycogen content and 215 phosphorylation rate of GS in CH male voles remained unchanged. Compared with the CON 216 group, the protein expression levels of GYPL also remained unchanged in the hearts of both male 217 and female voles in the CH group, indicating that maintenance of glycogen synthesis and 218 decomposition may have led to maintenance of glycogen content in the CH group. This suggests 219 that the effect of low temperature on glycogen synthesis can be significantly alleviated by 220 huddling behavior. In addition, the myocardial glycogen content and phosphorylation rate of GS in 221 CH female voles was significantly higher than that in the CON group, although the degree of 222 increase was much lower than that found in the CS group. This suggests that huddling behavior 223 partially alleviated the effect of low temperature on glycogen synthesis in female voles. In general, 224 huddling behavior completely or partially alleviated the increase in glycogen content caused by 225 the increase in glycogen synthesis in the myocardium of voles following cold environment 226 exposure. Normal glycogen metabolism is the basis of exercise in mammals [39,40]. Earlier 227 studies on Brandt's voles showed that activity is higher in huddling groups than separated groups 228 under cool environments [19]. Thus, we speculated that the results that the level of glycogen 229 metabolism in the myocardium of CH group was closer to the level of CON group than that of CS 230 group might be one of the reasons. 231 Interestingly, the biggest difference between the sexes was in myocardial glycogen content and 232 phosphorylation rate of GS, which increased in CH females but remained unchanged in CH males 233 compared to the CON groups. This may be because the average weight of males selected in this 234 study was larger than that of females. Higher body weight may mean more fat and substance 235 storage and therefore possible higher tolerance to lower temperatures. 236 In summary, we explored the regulatory mechanism related to the balance between glycogen 237 synthesis and degradation on the number in myocardial glycogenosomes of huddling and 238 separated Brandt's voles under cool environments. Results showed that a cool environment led to 239 an increase in myocardial glycogen content in voles, which could be alleviated by huddling 240 behavior, and may be a good consequence of the collective overwintering behavior of socialized 241 animals. An increase in glycogen synthesis is a common mechanism for changes in myocardial 242 glycogen level. The differences in glycogen content and related mechanism between the sexes 243 mainly existed in the CH group, which could be impacted by differences in sample size between 244 the two groups. In general, huddling behavior in voles alleviated the increase in myocardial 245 glycogen content caused by the increase in glycogen synthesis.

Consent for publication 264
Not applicable. 265

Competing interests 266
The authors declare that they have no competing interests. 267 control group; CH, cool huddling group; CS, cool separated group. Different letters identify 276 statistically significant differences among temperature treatment groups (P < 0.05). * , P < 0.05 277 significant differences between males and females. 278 CH, cool huddling group; CS, cool separated group. Different letters identify statistically 284 significant differences among temperature treatment groups (P < 0.05). * , P < 0.05 significant 285 differences between males and females. 286