Bear sample collection
A total of 28 free ranging subadult brown bears (Ursus arctos) from Dalarna and Gävleborg counties, Sweden, were included in this study, including 4 bears captured two consecutive years. All samples and data were collected under protocols approved by the Swedish Ethical Committee on Animal Experiment (applications Dnr C3/2016 and Dnr C18/2015), the Swedish Environmental Protection Agency (NV-00741-18), and the Swedish Board of Agriculture (Dnr 5.2.18-3060/17). All procedures complied with Swedish laws and regulations.
As described previously [10,77], blood, subcutaneous adipose tissue, and muscle tissue (vastus lateralis) samples were collected at two time points, in February during winter hibernation (W) and in June during summer-active period (S). Blood samples were collected from the jugular vein into 8 ml dry tubes for serum (Vacuette® Z serum Sep Clot Activator, Greiner Bio-One GmbH, Kremsmünster, Austria) or into 10 ml EDTA-coated tubes (BD Vacutainer®, FisherScientific, Illkirch, France) for plasma.
The analyses were performed on samples coming from different subsets of bears as described in Supplementary Table S1.
Lipid extraction and analysis
To perform serum lipidomic analysis, serum mixes were prepared as followed: for a given year, 50 µl of summer serum from each bear of the year was pooled to obtain the summer mix. In parallel, 50 µl of winter serum from the same bears was pooled to obtain the winter mix. A total of 6 summer and winter paired mixes were obtained (Supplementary Table S1). Lipids were extracted and analyzed as previously described . After addition of an internal standard (tri-17:0 triacylglycerol), total lipids were extracted twice from bear serum mixes with ethanol/chloroform (1:2, v/v). The organic phases were dried under nitrogen and lipids were transmethylated. Briefly, samples were treated with toluene-methanol (1:1, v/v) and boron trifluoride in methanol (14%). Transmethylation was carried out at 100 °C for 90 min in screw-capped tubes. Then 1.5 mL K2CO3 in 10% water was added and the resulting fatty acid methyl esters were extracted by 2 mL of isooctane and analyzed by gas chromatography (GC) with a HP6890 instrument equipped with a fused silica capillary BPX70 SGE column (60 x 0.25 mm). The vector gas was hydrogen. Temperatures of the Ross injector and the flame ionization detector were set to 230 °C and 250 °C, respectively. Data were expressed in mmol/L for total or individual fatty acids (FAs) concentration or molar percentage of total lipids for individual FAs. Detailed lipidomic results are presented in supplementary Table S2 (serum fatty acid concentrations) and S3 (serum fatty acid relative proportions).
For quantification of circulating endocannabinoids, analysis was performed on 500 µl of plasma collected at the two time points (S and W) from 8 individual animals (see supplementary Table S1). Standard endocannabinoids (eCBs), i.e.- PEA, PEA-d5, OEA, OEA-d4, AEA, AEA-d4, 2AG, and 2AG-d5, were purchased from Cayman (Bertin BioReagent, Saint-Quentin en Yvelines, France). Mass spectrometry quality grade solvents were purchased from Fischer Scientific (Illkirch, France). Tissue samples (adipose and muscle tissues); c.a 100 mg) were crushed in an Omni Bead Ruptor 24 apparatus (Omni International, Kennesaw, USA) with circa twenty 1.4 mm OD zirconium oxide beads (S=6.95 m/s, T=30s, C = 3; D = 10s) and 900 µl of methanol/Tris-buffer (50 mM, pH=8) 1/1 containing 20 ng of PEA-d5, 2 ng OEA-d4, 10 ng AEA-d4, and 20 ng 2AG-d5. Then, each homogenate was added with 2 mL of CHCl3/MeOH (1:1, v/v) and 500 µL of Tris (50 mM, pH=8), vortexed and centrifuged 10 min at 3000g. The organic layer was recovered and the upper aqueous phase was extracted twice with chloroform (1mL). Finally, organic phases were pooled and evaporated under vacuum.
Plasma (500 µL) were mixed with 500 µL cold methanol containing 11 ng AEA. After protein precipitation at -20°C for 2 hours, endocannabinoids were extracted with methanol/chloroform (1:1, v/v) (5 ml) and saline (1.25 mL). The organic phase was recovered and the aqueous phase was extracted twice with chloroform (3mL). Organic phases were finally pooled and evaporated under vacuum.
Dried extracts were solubilized with methanol (200 µL) and centrifuged for 5 min at 20,000 g. Four microliters of the supernatant were injected into a 1200 LC system coupled to a 6460-QqQ MS/MS system equipped with an ESI source (Agilent technologies). Separation was achieved on Zorbax SB-C18 2.1x50 mm, 1.8 µm column (Agilent technologies) at a flow rate of 0.4 mL/min, 40°C, with a linear gradient of (solvent A) water containing 0.1 % formic acid and (solvent B) methanol containing 0,1% formic acid as follows: 10% of B for 1 min, up to 85% of B in 8 min, and then 100% B for 4.5 min. Acquisition was performed in positive Selected Reaction Monitoring (SRM) mode (source temperature: 350°C, nebulizer gas flow rate: 10 L/min, 40 psi, sheath gas flow 10 L/min, sheath gas temperature 350°C, capillary 4000 V, nozzle 1000 V).
Transitions used were: 2AG-d5 384.3→91.1 (frag 120V, CE 62V), 2AG 379.1→91 (frag 120V, CE 62V), AEA-d4 352.2→66.1 (frag 115V, CE 14V), AEA 348.2→62 (frag 120V, CE 14V), OEA-d4 330.2→66.1 (frag 120V, CE 14V), OEA 326.2→62 (frag 115V, CE 14V), PEA-d5 305.2→62 (frag 124V, CE 14V), and PEA 300.2→62 (frag 124V, CE 14V).
Endocannabinoids quantification in tissues was performed on tissue samples collected at the two time points (S and W) from 5 (muscle tissue) and 6 (adipose tissue) bears (Supplementary Table S1). eCBs from tissues were quantitated according to the isotope dilution method. Results are expressed as pg per mg of wet weight of tissue. eCBs from plasma were quantitated using calibration curves obtained with authentic standards extracted by the same method used for plasma samples. Linear regression was applied for calculations. Results are expressed as ng of endocannabinoid per mL of plasma.
Quantification of mRNAs by real-time RT-PCR
For mRNA quantification using RT-qPCR, total RNAs were obtained from muscle and adipose tissues collected at the two time points (S and W). For the muscle tissue, RNAs were extracted from 8 bears in summer and winter, while for adipose tissue, RNAs were extracted from 5 bears in summer and 13 bears in winter (Supplementary Table S1).
Muscle and adipose tissue total RNA was isolated using the TRIzol reagent (Invitrogen, Courtaboeuf, France) according to the manufacturer’s instructions. First-strand cDNAs were synthesized from 1 µg of total RNA using the PrimeScript RT kit (Ozyme, saint quentin en Yveline, France) with a mixture of random hexamers and oligo(dT) primers, and treated with 60 units of RnaseH (Ozyme). Real-time PCR assays were performed with Rotor-Gene 6000 (Qiagen, Courtaboeuf, France). The primers and real-time PCR assay conditions are listed in supplementary Table S4. The results were normalized by using TBP (TATA box binding protein) mRNA concentration, measured as reference gene in each sample.
Statistical analysis was performed using the R software environment v3.0.2 . For each set of values, distribution of the data was tested using the Shapiro-Wilk normality test, and using the p=0.01 threshold normal distribution was considered in all cases. Differences between summer and winter data were tested using paired Student t-test for lipidomic, endocannabinoid quantification in plasma and tissues, and mRNA level in muscle tissue. For mRNA level in adipose tissue, differences between summer and winter data were tested using unpaired Student t-test. For multiple comparison (lipidomic data), the Benjamini-Hochberg correction using the p.adjust function (Package stats version 4.0.0 of R studio) was applied. Data are presented as means ± SEM and individual values are plotted as grey and black dots for respectively summer and winter values. Means, SEM, fold change and associated p-values are reported in supplementary Tables S2 to S5. Statistical significance was considered with p values or adjusted p values lower than 0.05.