Animals and Experimental Designs

Animals and experimental designs

Mongolian gerbils were the offspring of our breeding colony established by animals captured in Inner Mongolia. Subjects were singly housed in plastic cages (30 × 15 × 20 cm) with sawdust as bedding. They were maintained at the environmental temperature of 21 ± 1°C, under a photoperiod of 16L:8D (lights on at 04:00) with an access to food and water ad libitum. All procedures were conducted with the permit of the Animal Care and Use Committee of Institute of Zoology, the Chinese Academy of Sciences.

Experiment I

Adult gerbils (70-90 days of age, 38 males and 29 females) were divided into three groups randomly: LD group, in 16L:8D photoperiod for 10 weeks; SD group, in 8L:16D photoperiod for 10 weeks; transformation from SD to LD group (SL), in SD for 6 weeks to recover their photosensitivity and then were transferred back to LD condition for next 4 weeks. Body mass and food intake per day were monitored weekly (food intake per day was tested just in the first 8 weeks). After 10-weeks acclimation, gerbils were sacrificed in the morning by excessive CO2. Blood samples were collected and centrifuged at 4 oC at 4000 rpm for 30 min after 1-hour clotting. Then serum samples were collected and stored in -80 oC for the melatonin test.

Experiment II

Subadult gerbils (30-40 days of age, 16 males and 16 females) were acclimated under either LD or SD for 8 weeks. Body mass, dry matter intake per day, and RMR were measured once a week and NST was examined every two weeks. After acclimation, gerbils were sacrificed in the morning as Experiment I. Serum samples were acquired for the melatonin test. The retinal tissue in eyes and the iBAT were removed immediately, then separated carefully, quick froze with liquid nitrogen and stored in -80 oC for the western-blot of melanopsin and UCP1.

Metabolic trials

RMR was measured by a FoxBox O2 and CO2 analyzer (Sable Systems International Inc, NV, USA) at 30 ± 0.5 oC (within gerbils, thermal neutral zone, TNZ) between 0900 and 1700. The air entering and leaving the metabolic chamber (200 × 130 × 85 cm) were dried with anhydrous CaSO4 (W.A. Hammond Drierite Co. Ltd, USA) and ND-2 drier (Sable Systems International Inc, NV, USA) respectively. The flow rate of air entering the chamber was 600-800 mL/min, and gas throughout the chamber at 100 mL/min were subsampled, then O2 and CO2 concentration were recorded. The baseline of oxygen and carbon dioxide concentration was measured before and after each test. Each individual was placed into the metabolic chamber for 3 hr, and O2 and CO2 concentration were recorded at 5-min intervals. The O2 consumption was calculated based on the following formula (Chi et al., 2016; Hill, 1972; Li et al., 2010):

Where, FR = flow rate (mL/min), Fi = input fractional concentration (%), and Fe = output fractional concentration (%).

And RMR was calculated from two stable consecutive lowest O2 consumptions.

NST was induced by subcutaneous injection of norepinephrine (NE) (Shanghai Harvest Pharmaceutical Co Ltd, Shanghai, China) at 25 ± 1 oC near the lowest limit of TNZ. The dosage of NE was based on the equation of Heldmaier (Heldmaier, 1971): NE dosage (mg/kg) = 6.6 Mb.0.458, where Mb is body mass in gram. As mentioned above, O2 consumption was recorded for 60 min at 5 min intervals and two consecutive highest recordings were chosen to calculate the maximum NST (Wang, 1996; Wang et al., 1999).

Dry matter intake

Dry matter intake was measured in three days once a week. Animals were housed individually with a stainless steel wire mesh to separate food and feces. Food was provided regularly and quantitatively, and then the uneaten food and feces were collected after each test, oven-dried at 60 oC and separated manually. Dry matter intake was calculated from the difference between the food given and the food residue.

Western blot analysis of UCP1 and melanopsin

The homogenization of the iBAT and the retinal tissue were performed in RIPA buffer supplemented with protease inhibitor PMSF. The retinal tissues of two animals from the same group were combined due to the content of protein extracted from one animal is too low. And whole protein of the tissue was obtained by centrifuging at 4 oC at 14,000 rpm for 15 min. Then protein concentration was determined using Folin phenol method (Lowry et al., 1951). After separation by discontinuous SDS-PAGE gels, samples were blotted into polyvinylidene difluoride (PVDF) membranes followed by being blocked with 5% fat-free dry milk. Then, the PVDF membranes were incubated with rabbit anti-UCP1 antibody (diluted 1:5000, ab10983, Abcam, Cambridge, MA, USA), rabbit anti-OPN4 (diluted 1:2000, PA1780, Thermo Scientific Pierce, Waltham, MA USA) and mice anti-GAPDH (diluted 1:3000, 30201ES60, Yesen, Shanghai, China) as primary antibody respectively, and horseradish peroxidase-conjugated goat anti-rabbit IgG (diluted 1:5000, 111-035-003, Jackson ImmunoResearch Labs Inc, PA, USA) and horseradish peroxidase-conjugated goat anti-mice IgG (diluted 1:3000, CW0102, Cwbiotech, Beijing, China) as the secondary antibody. The immunoblot was detected by ECL (Beyotime, Shanghai, China), and the concentration of UCP1 and melanopsin were analyzed with Quantity One Software (version 4.40, BioRad, Hercules, CA). Results were expressed as relative units (RU).

Serum melatonin level assay

Serum melatonin levels were measured by enzyme-linked immunosorbent assay (ELISA) with a rat-melatonin ELISA kit (Shanghai Enzyme-linked Biological Technology Co. Ltd, Shanghai, China). The lower and upper limits of the assay kit were 1.5 pg/mL and 48 pg/mL. According to the standard curve, serum melatonin levels were determined in a single ELISA and expressed as pg/mL.

Statistical analyses

Statistical analyses were processed with SPSS 20.0 software (SPSS Inc, Chicago, IL, USA). Before analyses, data were examined for normality and homogeneity of variance using Kolmogorov-Smirnov and Levene tests, respectively. Body mass, dry matter intake, RMR, and NST during acclimation were analyzed by repeated measures ANOVA followed by Tukey post hoc test. To standardize the influence of body mass, analyses of dry matter intake, RMR, and NST were conducted by ANCOVA with basal body mass as a covariant. Group differences in UCP1, melanopsin and melatonin were assessed by two-way ANOVA (photoperiod and sex). Mean differences were considered significant if P ≤ 0.05. Results were expressed as mean ± SE.

Figure S1 The effects of photoperiod on the body mass and dry matter intake of adult gerbils. No photoperiodic differences were found in the body mass (A and B) and dry matter intake (C and D) of adult male and female gerbils during 10-weeks acclimation. Compared to females (B and D), however, males showed a higher body mass and a similar dry matter intake (A and C). LD, long day (16L:8D); SD, short day (8L:16D); SL, transformation from short day to long day. Results are mean ± SE.

Figure S2 Serum melatonin levels of adult gerbils acclimated in different photoperiod. No photoperiod-induced differences were found among three groups. However, compared with females, males had higher serum melatonin levels. LD, long day (16L:8D); SD, short day (8L:16D); SL, transformation from short day to long day. * P < 0.05. Results are mean ± SE.

Figure S3 Changes of RMR (A and B) and NST (C and D) in subadult male and female gerbils during 8 weeks of acclimation. RMR, resting metabolic rate; NST, nonshivering thermogenesis; LD, long day (16L:8D); SD, short day (8L:16D). Photoperiod did not affect the RMR or NST. Results are mean ± SE.

Figure S4 The contents of UCP1 in iBAT of subadult gerbils treated with different photoperiod. No photoperiod-induced changes were observed between two groups. UCP1, the uncoupling protein 1; iBAT, interscapular brown adipose tissue; LD, long day (16L:8D); SD, short day (8L:16D). Results are mean ± SE.