All laboratory procedures were carried out at The University of Nottingham under the United Kingdom code of laboratory practice (COSHH: SI NO 1657, 1988).

Cell culture

Mouse mesenchymal cells (D1, ATCC CRL-12424) were grown and maintained routinely in standard medium containing low glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% L-glutamine and 1% non-essential amino acids. When cells reached 80% confluence, adipogenic differentiation was induced with adipogenic (‘AD’) medium prepared with standard medium supplemented with 1 μM dexamethasone (Cayman Chemicals, USA), 100 µM isobutylmethylxanthine (IBMX) (Sigma-Aldrich, UK), 1 μM rosiglitazone (Cayman Chemicals, USA), and 10 μg/ml insulin (Sigma-Aldrich, UK). All supplements were prepared according to the manufacturer’s guidelines. AD medium was used throughout the treatment period, as previously described55,56. Experimental cultures were treated for 9 days in two groups: cells differentiated under standard cell culture thermal conditions (37 °C), and cells differentiated at 32 °C57,58,59. Matching undifferentiated control cells maintained in standard medium were also grown at 37 and 32 °C. The same protocol was applied to human adipose-derived stem cells (hADSCs) (ThermoFisher UK, Cat. No R7788-115) and primary mouse BM-derived stem cells. For the analyses performed in this study, 3 batches of cells were analysed.

Cell Viability Assay

Cell viability and metabolic activity was evaluated using the resazurin-based PrestoBlue Cell Viability kit, according to the manufacturer’s instructions. Cells were seeded in 24-well plates in three culture replicates and treated with standard and AD medium in appropriate temperature conditions. At the end of experiment, medium was replaced with a working solution of 10% v/v Presto Blue stock solution and incubated for 40 min. The fluorescent signals of 100 μl samples were measured at 560 nm excitation and 590 nm emission in triplicate using an Infinite M200 PRO plate reader and i-control software (Tecan, Switzerland). The measured fluorescent signal was expressed as arbitrary units (AU).

Oil Red O staining

Cell monolayers were rinsed with phosphate buffered saline (PBS, pH 7.4), fixed with 4% paraformaldehyde, and analysed by ORO staining. After rinsing in PBS, cells were stained with 60% working ORO solution for 10 min, washed and subsequently counterstained with hematoxylin. Cells were imaged before incubation with 100% isopropanol to extract the incorporated stain. Absorption of extracted dye in triplicate was measured at 510 nm on an Infinite M200 PRO plate reader and i-control software (Tecan, Switzerland). The measured absorbance signal was expressed as AU. For all calculations and measurements, ImageJ software was used60.

Morphometric and stereological analysis

The percentage of differentiated cells was estimated by dividing the total number of differentiated cells by the total number of all cells, using 50 randomly selected micrographs per condition (three biological replicates) at a final magnification of 400. The same images were used to evaluate Maximum Feret’s Diameter (MFD), the longest distance between any two points of the object. Ferret’s diameter is generally used in optical microscopy to measure the size of irregularly shaped particles61. In order to analyse LDs distribution, 500 LDs per group were measured. For all calculations and measurements, ImageJ software was used.

Mitochondrial staining

Cells cultured for 9 days on coverslips were stained with MitoTracker Orange CM-H 2 TMRos (100 nM), according to the manufacturer’s protocol, and applied to cells in culture at 37 and 32 °C for 45 minutes before fixation. After PBS wash, coverslips were mounted on microscopic slides with Vectashield medium containing DAPI (VectorLabs, UK) and examined with a Zeiss fluorescence microscope. Mitochondria numbers were assessed using 20 randomly selected images per condition (three biological replicates). Relative fluorescence intensity was measured applying identical instrument settings. In addition, relative fluorescence intensity was measured around 50 LDs per group at 3 µm from the LDs.

Calcium staining

Cells differentiated at 37 °C and 32 °C were incubated for 40 minutes with 2 µM Fluo-4 solution (Molecular Probes, F14201) as previously described62. Afterwards, cells were washed three times, fixed with 4% PFA and counterstained with DAPI. Images were taken with Nikon Eclipse 90i microscope.

Immunocytochemistry

For immunostaining, cells seeded and differentiated on coverslips, were fixed with 4% ice-cold paraformaldehyde. Before immunostaining, cells were washed with PBS, permeabilised using 0.1% Triton X-100 (Sigma Aldrich, UK) in PBS and incubated with 5% normal goat serum or fetal bovine serum in PBS (depending on secondary antibody host) for 60 min at room temperature. Samples were then incubated in the appropriate primary antibody for 90 min and washed in PBT (PBS + 0.1% Tween20, Sigma Aldrich) before incubation with the appropriate fluorophore-conjugated secondary antibodies for 60 min. After extensive washing in PBT, samples were mounted with Vectashield medium containing DAPI (VectorLabs) and examined with Zeiss Elyra PS.1 microscope. If biotinylated secondary antibodies (VectorLabs) were used, signal was visualized with 3,3′-diaminobenzidine (DAB) as the chromogen (VectorLabs), samples were mounted on slides using DPX and examined with a Nikon microscope. All antibodies used in this study are presented in Table 1 (see Supplementary information).

Densitometric analysis of UCP1 positive area

Twelve fields per condition (three biological replicates) were randomly selected at x400 magnification and the UCP1 positive areas were calculated using the image processing software ImageJ. The images were converted to HSB stack with saturation settings for MinThreshold and MaxThreshold to segment UCP1 positive areas. The percentage of area within the threshold range was measured.

Super-resolution imaging in structured illumination mode

For super-resolution imaging, cells mounted with CitiFluor (CF3) medium (Agarscientific, UK) were imaged using a Zeiss Elyra PS.1 microscope, in structured illumination mode (SIM), with the following settings: objective Plan-Apochromat 63 x /1.4 Oil DIC M27, filter set LBF −488/561, cmos camera exposure time 20 ms. Two imaging tracks were set up in fast frame mode, which alternates the excitation lasers (solid state 488 nm and 561 nm, at 20 and 10% laser power settings, respectively). In the fast frame mode, images from the two channels were acquired almost simultaneously with an exposure time difference of 20 ms between the red and green channel. Additionally, channel alignment was performed by scanning 100 nm beads with the same settings to ensure the precise localization. Image processing and channel alignment was done using the manufacturer’s software (https://www.zeiss.com/microscopy/int/products/microscope-software/zen-lite.html).

Gene expression analysis

Total RNA from cells, WAT, BAT and liver tissue samples was extracted, normalized to 0.5 µg/µl and reverse transcribed to cDNA as previously described63. Quantitative PCR was performed using either SYBR Green Taq polymerase master mix or TaqMan universal master mix with murine-specific oligonucleotide primers (Eurofins) or FAM-MGB TaqMan probes against a cDNA gene standard curve to verify the efficiency of the reaction (≥95%) and with appropriate negative controls using the Step One Plus Q-PCR system and v.2.2 software (Applied Biosciences). Gene expression was determined using GeNorm normalization algorithm against two selected reference genes (stability value M = 0.47), TBP (TATA sequence binding protein) and acidic ribosomal protein subunit P0 (RPLP0) using GeNorm software version 3.5 (Primer Design Ltd). UCP1, PRDM16, COX8b, P2RX5, TRPV1, TRPV2 and TRPV4 gene expression level was determined using a TaqMan probe (BioRad TaqMan Gene Expression assays; assay qHsaCEP0050537, Mm00712556_m1, qMmuCIP0034367 and qRnoCIP0024301, qMmuCIP0031313, qMmuCIP0035343 and qMmuCIP0032629, respectively). Primers used in this study are listed in Table 2 (see Supplementary information).

Seahorse Assay

Cells were seeded into the XFe96 Microplates (Seahorse Bioscience) to measure the OCR, an indicator of mitochondrial respiration, and the ECAR, an indicator of glycolysis. Prior to the start of the Seahorse XF Cell Mito Stress Test Assay, cells differentiated at either 37 or 32 °C were washed 3 times and incubated in XF-Basal Medium supplemented with 10 mM glucose, 1 mM sodium pyruvate, and 2mM L-glutamine. OCR and ECAR readings were taken over time under basal conditions and after the addition of mitochondrial inhibitors (2.5 μM oligomycin, 0.8 μM FCCP and 1 μM rotenone/antimycin) at 37 and 32 °C, respectively. OCR was determined using the Seahorse Wave 2.4 XF-96 software and the data obtained for each condition were normalized to the cell number per well and expressed as the OCR in pmol/min/104 cells. OCR and ECAR were determined using the Seahorse Wave 2.4 XF-96 software and the data obtained for each condition were normalized to the cell number per well and expressed as the OCR in pmol/min/104 cells. Measures of mitochondrial respiration (basal respiration, ATP-linked respiration, proton leak, maximal and reserve capacity) were derived by the sequential addition of oligomycin, FCCP and rotenone/antimycin. First, the basal respiration rate was calculated by subtracting the residual OCR determined after the addition of rotenone/antimycin. ATP-linked respiration was determined from the difference between basal OCR and OCR following oligomycin addition. The difference in OCR between rotenone/antimycin and oligomycin represented the amount of oxygen consumed due to proton leak respiration. Maximal OCR was determined by subtracting the OCR after rotenone/antimycin addition from the OCR induced by FCCP. Last, the mitochondrial reserve capacity was calculated by subtracting basal respiration from maximal reserve capacity.

ECAR was measured simultaneously with OCR using the same conditions as described previously49 and glycolytic rate was determined by monitoring extracellular pH induced by the change in lactate. Similarly to OCR, basal ECAR refers to the ECAR measured before injection of rotenone/antimycin. To determine the influence of low temperature on oxidative phosphorylation and glycolysis, basal OCR was plotted against basal ECAR. Lastly, coupling efficiency, an indicator of the proportion of respiratory activity used to make ATP, was determined by calculating the percentage of OCR immediately following the oligomycin treatment with the final baseline value.

Data and software availability

All data were analysed by Student’s t test. For data that was not normally distributed the nonparametric Mann Whitney test was used to evaluate the statistical significance between two treatment groups. Statistical significance was accepted at p < 0.05, with *p < 0.05; **p < 0.01; ***p < 0.001. Errors bars plotted on graphs are presented as the mean ± SEM. Data were analysed using Graph Pad Prism Software (https://www.graphpad.com).