Animals

Six-week-old male and female C57BL/6N and C57BL/6J mice were purchased from Japan SLC (Shizuoka, Japan). Upon arrival, the mice were housed in standard cages (175 × 245 × 125 mm) in groups of four. They were fed a commercial diet (MF; Oriental Yeast, Tokyo, Japan) comprising crude protein (23.2%), crude fat (4.9%), crude ash (5.9%), crude fiber (3.3%), and moisture (8.1%), providing 355.7 kcal, and drinking water ad libitum. The cages were placed in a box in a room at 24 ± 1 °C. They were maintained under 12 h of light and 12 h of darkness (12 L:12D) for at least one week before the start of the experiments. The light intensity on the heads of the animals was approximately 70 lx. All animal experiments were conducted in accordance with the Guidelines for Animal Experiments of the Faculty of Agriculture at Kyushu University and the Law (No. 105) and Notification (No. 6) of the Japanese Government. All experiments were approved by the Animal Care and Use Committee of Kyushu University (approval numbers: A21-398 and A23-217).

CJL treatment

C57BL/6N mice (male: n = 32, female: n = 32) were housed in groups of four per cage. Half of the mice were designated as control mice and housed under 12 L:12D. The other half of the mice were designated as the CJL group, in which lights on and off times were advanced by 6 h every 2 days for 8 weeks, according to a published method [13]. The CJL schedule is shown in Fig. 1A. Body weight was measured weekly during treatment. Food intake was measured in the 5th, 6th, and 7th weeks. Computed tomography (CT) scan was performed at the beginning of CJL and at week 6 using Cosmo Scan FX (Rigaku, Tokyo, Japan) with the following parameters: tube voltage, 90 kV; tube current, 88 µA; exposure time, 2 min; resolution, 10 μm isotropic. Micro-CT images were evaluated in three dimensions. During scanning, the mice were anesthetized with isoflurane. The open field test (OFT) was performed in the 6th week to evaluate anxiety-like behavior. Briefly, each mouse was placed at the center of an open field box (40 × 40 × 40 cm, 40 lx, central area: 10 × 10 cm), and the behavior of each mouse was analyzed for 5 min using a video tracking system (ANY-maze; Stoelting, Illinois, United States). C57BL/6J mice (male: n = 8, female: n = 8) were separated into control and CJL groups in a similar manner as C57BL/6N mice, and their body weights were recorded every week.

Fig. 1

Sex-dependent changes in body weight gain under CJL in C57BL/6N and C57BL/6J mice. (A) Schematic illustration of light schedules in control and chronic jet lag (CJL) groups. White and gray colors represent the light and dark phases, respectively. (B) Body weight gain of C57BL/6N mice under CJL. Data are presented as means ± standard errors of the means (S.E.M) (n = 21 per group in each sex, combined data of mice subjected to four-time points sampling and nano tag recording). (C) Body weight gain of C57BL/6J mice under 6 weeks CJL treatment (n = 4 per group in each sex). (D, E) Comparison of body weight gain of C57BL/6N and C57BL/6J female (D) and male (E) mice. The gain on week 6 of CJL treatment is shown. Two-way ANOVA followed by Šidák’s multiple comparison test, #### p < 0.0001, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Eight weeks after CJL, all C57BL/6N mice were transferred to constant darkness (DD) for 2 days and then euthanized using isoflurane at 2:00, 8:00, 14:00, and 20:00 based on the lighting conditions prior to the transfer to DD (0:00 was defined as the light onset on the prior day, four animals in each group per time point). The timing was determined, as modest individual variations of circadian phases are expected within 3 days of DD following CJL [12]. Brains were frozen on dry ice and stored at − 80°C. Blood was centrifuged at 3,000 × g for 10 min at 4℃, and plasma was stored at − 80°C. Liver fragments and adrenal glands were collected using RNAlater reagent (Ambion, TX, USA). In female mice, vaginal cytology was performed after euthanasia to confirm the estrous phase, and no significant bias between the groups was confirmed.

Nano tag recording

Another cohort of C57BL/6N mice (male: n = 10, female: n = 10) was used to measure core body temperature and locomotor activity rhythms using a nano tag (Kissei Comtec Co., Matsumoto, Japan). The mice were maintained under control and CJL conditions (five animals in each group and sex) for eight weeks, followed by DD conditions. One day before week 8 of CJL treatment, a nano tag was implanted into the abdominal cavity under anesthesia using an isoflurane inhalation anesthesia device (Natsume Seisakusho Co., Ltd., Tokyo, Japan). After surgery, cephalexin (15 mg/kg) dissolved in saline was injected subcutaneously. Core body temperature and activity levels were recorded every 5 min for one week under CJL (week 8) and the following week under DD. The data for the LD and DD conditions were analyzed separately by chi-square periodogram analysis using ClockLab software (Actimetrics, Wilmette, IL, USA, version 6.1.15). The circadian period and rhythm robustness which is power of the chi-square periodogram (Qp value) were used to evaluate the effect of CJL on circadian rhythmicity.

qPCR

Total RNA was extracted from the tissues using ISOGEN II (Nippon Gene, Tokyo, Japan) according to the manufacturer’s protocol. cDNA was synthesized using 1 µg of total RNA and a Primer Script & RT reagent kit with gDNA Eraser (Takara, Kusatsu, Japan) according to the manufacturer’s protocol. qPCR was performed using Stratagene Mx3000P (Agilent Technologies, Santa Clara, USA) with a denaturation step at 95°C for 30 s, 40 cycles of amplification at 95°C for 5 s, and a primer-specific annealing temperature for 30 s. Primer sequences and annealing temperatures are listed in Supplementary Table 1. Each mRNA level was calculated using threshold cycles for the amplification of unknown samples and was compared with those of the four concentrations of standard cDNAs. The calculated levels were normalized to the expression levels of 36b4. Melting curve analysis was performed for each gene to validate the specificity of the PCR conditions.

Measurements of insulin and glucose in plasma, glycogen, and fat in liver

Plasma glucose and insulin concentrations were measured using a Glucose CII Test Wako Kit (Fujifilm Wako Pure Chemical Co., Osaka, Japan) and an Ultra-Sensitive Mouse Insulin ELISA Kit (Morinaga Institute of Biological Science, Yokohama, Japan), respectively, according to the manufacturer’s protocol.

Liver glycogen was stained using the periodic acid-Schiff staining (PAS) method using a commercially available kit (Muto Pure Chemicals, Tokyo, Japan). Briefly, liver tissues were embedded in Tissue-Tek O.C.T. compound (Sakura Tissue Tek, Tokyo, Japan) and frozen in liquid nitrogen. The sections were fixed in 4% paraformaldehyde and soaked sequentially in 1% periodic acid solution for 5 min, Schiff reagent for 30 min at 37°C, sulfurous acid solutions for 5 min, and Carrazi hematoxylin for 15 min. Liver fat accumulation was assessed using Oil Red O (Sigma-Aldrich, St. Louis, USA) staining of frozen liver sections that was performed according to a previously described protocol [31]. Briefly, the sections were fixed in 4% paraformaldehyde and soaked in 60% isopropanol for 15 s and then Oil Red O working solution for 30 min, rinsed in 60% isopropanol for 15 s, washed with distilled water, and mounted on slides. Microscope images (689.55 μm x 517.16 μm for PAS staining, 355.56 μm x 266.67 μm for Oil Red O staining) were acquired from sections. Optical densities for PAS staining and stained areas for Oil Red O staining were analyzed using ImageJ (National Institutes of Health). The data are expressed as the percentage of the maximum value of all animals.

Glucose and insulin tolerance tests

A cohort of C57BL/6N mice (male: n = 8, female: n = 8) was used to evaluate glucose and insulin tolerance under CJL, followed by confirmation of insulin tolerance using two other cohorts (male: n = 8, female: n = 8 for each cohort) with different doses of insulin. The fourth cohort (males, n = 6; females, n = 6) was used to evaluate insulin response to glucose. For all cohorts, male and female mice were separated into control and CJL groups and subjected to the respective light-dark cycles, as described above.

For the first cohort on the first day of week 7 under CJL, the mice in the control and CJL groups were fasted overnight one day before testing. On the day of the experiment, tail vein blood glucose levels were measured after starvation and recorded using a glucometer (Sanwa Kagaku Kenkyusho, Nagoya, Japan). Next, 2 g/kg glucose was intraperitoneally injected at 2:00, and tail vein blood glucose levels were measured and recorded sequentially for 90 min (15, 30, 60, and 90 min) using a glucometer. Using the temporal changes in glucose levels, the incremental area under the curve (iAUC) was calculated for each animal as a glucose tolerance parameter. For the insulin resistance test on the last day of week 7 under CJL, the mice were starved for 2 h, and the primary glucose level was measured at 6:00. Insulin (0.75 units/kg body weight) was injected intraperitoneally. Blood glucose levels in the tail vein were measured 20, 40, 60, and 120 min after insulin injection. For the second and third cohorts, insulin tolerance tests using different doses of insulin (0.5 or 1 unit/kg) were performed simultaneously as in the first cohort. Using temporal changes in glucose levels, the kITT was calculated from the linear slope of the plasma glucose concentration curve between 0 and 20 min as a parameter of insulin sensitivity. To evaluate the insulin response to glucose on the 8th week of CJL treatment, mice in the fourth cohort were fasted overnight one day before testing. Plasma was collected before and 20 min after glucose intraperitoneal injection (2 g/kg) and stored at − 80°C for insulin measurement.

Castration and testosterone replacement

Male C57BL/6N mice were acclimated for 7 days. Prior to CJL treatment, the animals were bilaterally castrated. Surgeries were performed under isoflurane anesthesia. The testes were externalized via laparotomy and removed after clamping the testicular artery. Silicone tubes (inner diameter = 2.0 mm, outer diameter = 3.0 mm; Kaneka, Osaka, Japan) were filled with 100% testosterone propionate or 100% cholesterol and sealed with silicone adhesive. The tubes were 12 mm long, with an additional 3 mm on each end of the adhesive. In this method, testosterone constantly leaks from the tubes [32]. One day before implantation, the tubes were washed with 70% ethanol, primed in a saline solution at 37 °C overnight, and then implanted subcutaneously under isoflurane anesthesia. Castrated mice implanted with testosterone- or vehicle-containing tubes were subjected to control (12 L:12D) or CJL treatment as described above for six weeks, followed by DD conditions. Body weight was recorded weekly. Nano tags were implanted at week 5, and body temperature and activity rhythms during the CJL and DD conditions were recorded. CJL light was restarted the following week, and a glucose tolerance test was performed two weeks later. One week after the glucose tolerance test, all mice were euthanized using isoflurane at 16:00. Trunk blood was collected in heparinized tubes and centrifuged at 3,000 × g for 10 min at 4 °C to obtain plasma samples for testosterone measurement.

Testosterone measurement

The plasma sample was transferred to a glass tube and spiked with an isotope-labelled internal standard solution containing testosterone-13C3. Testosterone was extracted with 4 mL of methyl tert-butyl ether. Following evaporation, the extract was dissolved in methanol (0.5 mL) and diluted with distilled water (1 mL). The sample was applied to an OASIS MAX cartridge that had been previously conditioned with methanol (3 mL) and distilled water (3 mL). The cartridge was then washed with 1 mL of distilled water, 1 mL of methanol/distilled water/acetic acid (45:55:1, v/v/v), and 1 mL of 1% pyridine solution. Finally, the steroids were eluted with 1 mL of methanol/ pyridine (100:1, v/v).

After evaporation, the residue was reacted with 50 µL of mixed solution (80 mg of 2-methyl-6-nitrobenzoic anhydride, 20 mg of 4-dimethylaminopyridine, 40 mg of picolinic acid, and 10 µL of triethylamine in 1 mL of acetonitrile) for 30 min. Subsequently, the sample was dissolved in 0.5 mL of ethyl acetate/hexane/acetic acid (15:35:1, v/v/v), and the mixture was applied to an InertSep SI cartridge that had been previously conditioned with 3 mL of acetone and 3 mL of hexane. The cartridge was washed with 1 mL of hexane and 2 mL of ethyl acetate/hexane (3:7, v/v). Testosterone was eluted with 2.5 mL of acetone/hexane (7:3, v/v). After evaporation, the residue was dissolved in 0.1 mL of acetonitrile/distilled water (2:3, v/v), and the solution was subjected to LC-MS/MS. The SRM transitions were m/z 394.3/253.2 for T and m/z 397.2/256.2 for T-13C3, respectively. The measurement range was 2.9–14,285 pg/mL.

Statistical analysis

Weekly changes in body weight were analyzed using a two-way ANOVA, followed by Šidák’s multiple comparison test. The effects of CJL were analyzed in each sex. The results of the behavioral tests were analyzed using one-way ANOVA or unpaired t-test. Values were considered significantly different at p < 0.05. In the castration and testosterone replacement experiments, body weight changes were analyzed using two-way ANOVA, followed by Šidák’s multiple comparison test.

Rhythmic variations of clock genes and metabolic gene expression in the liver and adrenal gland as well as plasma insulin, glucose, and liver glycogen levels were analyzed by two-way ANOVA based on treatment and sampling time points (2:00, 8:00, 14:00, 20:00). Cosinor analysis was also performed based on linear harmonic regression using CircWave software (Roelof Hut, University of Groningen, version 1.4) with 0.05 for an assumed period of 24 h. This analysis allowed us to estimate the significance of circadian variations, acrophases as centers of gravity, and the amplitude from the peak to the middle of the fitted cosinor wave. Although both control and CJL mice were euthanized for tissue sampling based on the lighting conditions prior to the transfer to DD, the onsets of free-running rhythms of locomotor activity differed between control and CJL. Thus, we determined the onset of locomotor activity using ClockLab software and defined the averaged time in a group as circadian time (CT) 12. In the control male and female groups, 2:00, 8:00, 14:00, and 20:00 correspond to CT 2, CT 8, CT 14, and CT 20, respectively. In the CJL female groups, these time points correspond to CT 7, CT 13, CT 19, and CT 1, respectively, while in the CJL male groups, they correspond to CT 18, CT 24, CT 6, and CT 12. The data are presented with CTs.