Obesity increases circulating levels of the bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P). Here, we identify adipocyte-expressed sphingosine kinase 2 (SPHK2), an enzyme that produces S1P, as a regulator of adipose tissue browning. Mice with adipocyte-specific deletion of Sphk2 were protected from Western diet-induced obesity concomitantly with elevated energy expenditure and browning of subcutaneous fat and enhanced expression of Ucp1 and key thermogenic genes, resulting in reduced adiposity and hepatic steatosis, improved glucose tolerance and insulin sensitivity. Mechanistically, obesogenic diet promoted nuclear localization of SPHK2 in adipocytes, where it cooperated with transcription factors to suppress the thermogenic transcriptome. Adipocyte SPHK2 was responsible for maintaining homeostatic circulating S1P levels and driving the pathogenic rise of S1P in response to obesogenic diet. Our findings reveal a distinct role of adipocyte SPHK2 in regulation of the thermogenic program important for whole-body metabolic homeostasis and energy expenditure and highlights SPHK2 as a potential therapeutic target for treatment of metabolic disorders.

Sphingolipid analysis
Sphingolipids were quantified by liquid chromatography electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS; API 5500 QTRAP; ABSciex) from 10 µL of whole blood, 10 µL plasma, 10 mg liver or 5 mg adipose. Homogenised tissue, blood or plasma samples were added to 13x100 mm borosilicate tubes (#53283-800, VWR) with phenolic-lined PTFE-Faced/14B white rubber liners (#60826-304, VWR) containing ice-cold HPLC-grade 3 mL of CH₃OH, 100 µL ddH₂O (#JT9831-3, VWR) and 10 µL of in-house internal standard cocktail. Internal standards contained 250 pmol of each species of unnaturally occurring 17-carbon chain length sphingoid bases (C17-sphingosine (#860640P, Avanti); C17-dihydrosphingosine (#860654P, Avanti); C17-sphingosine-1-phosphate (#860641P, Avanti); C17-dihydrosphingosine-1-phosphate (#860655P, Avanti)) and 12-carbon chain length fatty acid N-acyl sphingolipids analogs (C12-Ceramide (#860512P, Avanti); C12-sphingomyelin (#860583P, Avanti); C12-glucosylceramide (#860543P, Avanti); and C12-Lactosyl(ß)Ceramide (#860545P, Avanti)) in a total volume of 10 μL [7:2:1; EtOH:MeOH:H20].

Each sample was thoroughly sonicated for 30 seconds at room temperature, and then 1.5 mL of CHCl₃ (#CX1054-1, Sigma) added, vortexed for 5 seconds and sonicated for a further 30 seconds before the single-phase mixture was incubated overnight at 48°C. Extracts were centrifuged at 4200 xG for 10 min at 4°C, the supernatant transferred to fresh glass tubes (#40-4000-003, Colonial Scientific) and lipid extracts reduced to dryness using a speed vac before reconstituting in 600 µL of the starting mobile phase solvent ([MeOH:H2O] 1:1). Reconstituted lipids were sonicated for 30 seconds at room temperature, centrifuged at 4200 xG for 10 min at 4°C and the supernatant transferred to autoinjector vials (#10781-890A, VWR) with PTFE/silicone caps (#89239-020A, VWR) for LC-ESI-MS/MS analysis. Sphingolipids were separated by reverse-phase HPLC using a Supelco 2.1 (i.d.) x 50 mm Ascentis Express C18 (2.7 µm) column (#53822-U, Sigma) with a binary solvent system at a flow rate of 0.5 mL/min with a column oven set at 35°C. Prior to sample injection, the column was first equilibrated for 30 seconds with a solvent mixture of 95% mobile phase A1 (CH₃OH/H₂O/HCOOH, 58/41/1, v/v/v, with 5 mM ammonium formate) and 5% mobile phase B1 (CH₃OH/HCOOH, 99/1, v/v, with 5 mM ammonium formate). After sample injection (40 µL), the A1/B1 ratio was maintained at 95/5 for 135 seconds, followed by a linear gradient to 100% B1 over 90 seconds, and then held at 100% B1 for 330 seconds, followed by a 30 second gradient return to 95/5 A1/B1. After each run the column was again equilibrated with 95:5 A1/B1 for 30 seconds. The Shimadzu Nexera LC-30 AD binary pump system coupled to a SIL-30 AC autoinjector and DGU20A_5R degasser (HPLC component) was coupled to an AB Sciex 5500 quadrupole/linear ion trap (QTrap; SCIEX) operating in triple quadrupole mode. Q1 and Q3 were set to pass molecularly distinctive precursor and product ions (or a scan across multiple m/z in Q1 or Q3), using N₂ to collisionally induce dissociations in Q2 (which was offset from Q1 by 30–120 eV). The ion source temperature was set at 300°C. Peaks were integrated against internal standards using Analyst (ABSciex).

Plasma analysis
After chow or WD feeding for 18 weeks, mice were fasted for 6 hours (7AM-1PM). After euthanasia, the plasma fraction was collected for downstream analysis by centrifuging whole blood at 2000 xG for 15 minutes at 4°C. Fed and fasted blood glucose was measured from a nick in the tail vein using Nipro TRUEtrack Blood Glucose Meter (#B0727V3XQX, Amazon). Fasted circulating concentrations of plasma free fatty acids, total cholesterol, insulin, leptin and adiponectin were quantified by the Baylor College of Medicine, Mouse Metabolism and Phenotyping Core.

Whole-body metabolic phenotyping
After 16 weeks of WD feeding, mice were individually housed and acclimatized in metabolic chambers (PhenoMaster, TSE Systems) for 48 hours under regulated humidity (50%), temperature (23°C) and 12-hour dark/light cycles (70% light intensity). Measurements of water consumption, food intake, energy expenditure, VO₂, VCO₂, and respiratory exchange ratio (RER) for dark and light cycles were measured during 4 subsequent days. Exported raw data was analysed using CalR (https://CalRapp.org/).

Glucose tolerance test
After chow or WD feeding for 15 weeks, mice were fasted either for 6 hours (7AM-1PM) for glucose tolerance testing. Baseline blood glucose was measured from the tail vein using the Nipro TRUEtrack Blood Glucose Meter (#B0727V3XQX, Amazon). 1.5 mg/kg glucose (#D16-10, Fisher) in PBS (#10010023, Fisher) was filter sterilized (#SLGP033RS, Sigma) and injected intraperitoneally for glucose tolerance testing. Blood glucose was measured at 15, 30-, 60-, 90- and 120-minutes post-injection for both assays.

Insulin tolerance test
After chow or WD feeding for 15 weeks, mice were fasted either for 4 hours (7AM-11AM) for insulin tolerance testing. Baseline blood glucose was measured from the tail vein using the Nipro TRUEtrack Blood Glucose Meter (#B0727V3XQX, Amazon). 0.75 U/kg insulin (#I9278, Sigma) in PBS (#10010023, Fisher) was injected intraperitoneally for insulin tolerance testing. Blood glucose was measured at 15, 30-, 60-, 90- and 120-minutes post-injection for both assays.

Oil red O staining
For oil red O staining, 10 µm liver cryosections were left to thaw for 30 minutes before submerging in 60% isopropanol (#383920010, Fisher). Slides were then incubated with 0.3% oil red O solution (#O0625, Sigma) for 15 minutes at room temperature before sequential submersions in 60% isopropanol and distilled H2O. Sections were counterstained with haematoxylin for 2 minutes, and submerged in H2O before blueing reagent (#H-3502, Vector Laboratories) was added for 15 seconds. Sections were then thoroughly washed by submerging 10 times in H2O and mounted with CC/mount (#C9368, Sigma).

Tissue RNA extraction and quantitative PCR
Samples are from WT or SphK2ΔAdipo sWAT of mice fed CD or WD for 18 weeks. RNA was extracted from equal weights of frozen tissue or cell densities using TRIzol (#15596026, Fisher). Tissues were homogenized in 1 mL TRIzol for 1 minute until visibly homogenized using a Tissue Tearor (#985370-07, BioSpec products). The homogenates were placed at room temperature for 10 minutes, centrifuged at 12000 xG for 10 minutes at 4°C and on ice the TRIzol phase below the insoluble lipid phase transferred to a new tube. 200 µL chloroform (#C2432, Sigma) was added, the samples vigorously shaken and vortexed for 15 seconds each.

Samples were left at room temperature for 3 minutes before being centrifuged at 12000 xG for 15 minutes at 4°C. On ice, the aqueous phase was kept, 1 µL glycogen (#10901393001, Sigma) and 500 µL isopropanol (#A416, Fisher) added, then inverted 15 times. After incubating for 10 minutes at room temperature, the mixture was centrifuged at 12000 xG for 10 minutes followed by 22000 xG for 1 minute at 4°C to sequentially remove all supernatant. On ice, 1 mL 75% ethanol (#BP2818, Fisher) was added to the pellet, inverted 15 times and kept overnight at -20°C. The sample was then centrifuged at 7500 xG for 5 minutes followed by 22000 xG for 1 minute at 4°C to sequentially remove all ethanol before being left to air dry on ice for 30 minutes. RNA was resuspended in 20-40 µL ddH2O, DNase I treated (#E1011, Zymo Research) and concentration measured using a NanoDrop 2000 (#ND2000, Fisher). 2 µg was converted to cDNA with High-Capacity cDNA Reverse Transcription Kit (#4368814, Fisher) and mRNA expression determined by mixing 20 ng cDNA, SYBR (#4367659, Fisher) and primers specified in table 1. SYBR products were measured using a CFX Opus 96 Real-Time PCR Detection System (#12011319, BioRad) and relative fold changes in gene expression were determined by 2^-∆∆Ct normalized to PPIA.

Primary adipocyte RNA extraction and quantitative PCR
Mature WT or SphK2ΔAdipo primary adipocytes. RNA was extracted from equal cell densities using TRIzol (#15596026, Fisher). Cells were directly scraped into 1 mL TRIzol. The cells were placed at room temperature for 10 minutes, centrifuged at 12000 xG for 10 minutes at 4°C and on ice the TRIzol phase below the insoluble lipid phase transferred to a new tube. 200 µL chloroform (#C2432, Sigma) was added, the samples vigorously shaken and vortexed for 15 seconds each. Samples were left at room temperature for 3 minutes before being centrifuged at 12000 xG for 15 minutes at 4°C. On ice, the aqueous phase was kept, 1 µL glycogen (#10901393001, Sigma) and 500 µL isopropanol (#A416, Fisher) added, then inverted 15 times.

After incubating for 10 minutes at room temperature, the mixture was centrifuged at 12000 xG for 10 minutes followed by 22000 xG for 1 minute at 4°C to sequentially remove all supernatant. On ice, 1 mL 75% ethanol (#BP2818, Fisher) was added to the pellet, inverted 15 times and kept overnight at -20°C. The sample was then centrifuged at 7500 xG for 5 minutes followed by 22000 xG for 1 minute at 4°C to sequentially remove all ethanol before being left to air dry on ice for 30 minutes. RNA was resuspended in 20-40 µL ddH2O, DNase I treated (#E1011, Zymo Research) and concentration measured using a NanoDrop 2000 (#ND2000, Fisher). 2 µg was converted to cDNA with High-Capacity cDNA Reverse Transcription Kit (#4368814, Fisher) and mRNA expression determined by mixing 20 ng cDNA, SYBR (#4367659, Fisher) and primers specified in table 1. SYBR products were measured using a CFX Opus 96 Real-Time PCR Detection System (#12011319, BioRad) and relative fold changes in gene expression were determined by 2^-∆∆Ct normalized to PPIA.

Body temperature
Body temperature measured with a lubricated rectal probe (#20250-92, Cole-Parmer).

Culture and differentiation of murine primary adipocytes
Subcutaneous white inguinal adipose and axial brown adipose depots were excised from 5-week-old male mice (N=1 indicates 2 fat pads combined from 1 mouse per culture), briefly washed in HBSS (#14175095, Fisher) containing 1% antibiotic-antimycotic (#A5955, Sigma), finely minced and digested in 4 mL HBSS digestion buffer containing 1% antibiotic-antimycotic (#A5955, Sigma), 5 mM CaCl₂ (#349610250, Fisher), 100 mM HEPES (#H0887, Sigma), 3 mg/mL collagenase (#C6885, Sigma), and 15 mg/mL BSA (#A7906, Sigma) for 1 hour at 37°C with agitation. Once a homogenous slurry formed, the collagenase was neutralized by adding equal volumes of DMEM/F12 [1:1] (#10-090-CV, Corning) containing 1% antibiotic-antimycotic (#A5955, Sigma), 10% fetal bovine serum (#S11150H (lot J22204), R&D systems), 1% GlutaMax (#35050061, Gibco) (stromal vascular cell culture media), inverted 5 times and filtered through a 100 µm cell strainer to remove debris and undigested tissue (#CLS431752, Corning). The cell strainer was rinsed with a further 4 mL of stromal vascular cell culture media and the flowthrough centrifuged at 1200 xG for 5 minutes. The supernatant containing mature adipocytes was carefully aspirated and the pellet resuspended in 5 mL of stromal vascular cell culture media before filtering through a 40 µm cell strainer (#CLS431750, Corning). The strainer was rinsed with a further 2 mL media and the flowthrough centrifuged at 1200 xG for 5 minutes. The supernatant was aspirated, and the stromal vascular fraction single-cell suspension containing adipose-derived stem cells was resuspended in 6 mL of stromal vascular cell culture media (1 mouse plated across 6 wells of a 24-well plate achieved confluency after 7 days) and plated on 0.2% gelatine-coated plates, replacing media every 2 days for a total of 7 days until confluent at 37°C and 5% CO₂.

Once adipose-derived stem cells reached confluence, media was changed to induction media for 48 hours depending on originating depot. For white adipocytes (from subcutaneous white inguinal adipose) induction media contained 10 µg/mL insulin (sc-360248, Santa Cruz Biotechnology), 1 µM dexamethasone (#D4902, Sigma), 0.5 µM isobutylmethylxanthine (#BMLPD1401000, Enzo) and 1 µM rosiglitazone (#71740, Cayman Chemical) in stromal vascular cell culture media. After 48 hours, induction media was changed to maintenance media containing 10 µg/mL insulin and 1 µM rosiglitazone for 6 days, replacing media every 2 days. After 48 hours, induction media was changed to maintenance media containing 5 µg/mL insulin and 1 nM T3 for 6 days, replacing media every 2 days. For palmitate treatments, mature WT or SphK2ΔAdipo primary adipocytes were treated with vehicle or palmitate (500 µM) for 24 hours.

Immunochemistry staining of primary adipocytes
For Figures 6B, 6C, and 6D: Stromal vascular cells isolated from sWAT of WT and adipocyte-specific Sphk2 deleted (SphK2ΔAdipo) mice were cultured in vitro and differentiated into primary white adipocytes. Mature WT or SphK2ΔAdipo primary adipocytes cultured on Millicell EZ Slides (#PEZGS0816, Sigma) were pre-incubated, where indicated, with 3.8 µM BODIPY 493/503 (#D3922, Sigma) diluted in maintenance media for 15 minutes at 37°C and 5% CO2 for identification of lipid droplets. Adipocytes, including those for BODIPY 493/503 experiments, were washed with ice-cold PBS, fixed with 4% (w/v) paraformaldehyde (#043368.9M, Fisher) in PBS (#10010023, Fisher) for 15 minutes before permeabilization with 0.1% Triton X-100 (#AAA16046AE, Fisher) in PBS for 3 minutes, all at room temperature. Adipocytes were incubated with blocking buffer containing 5% fetal bovine serum and 1% BSA in PBS for 45 minutes at room temperature and subsequently incubated with primary antibodies diluted in blocking buffer overnight at 4°C in a dark/humidified chamber. Cells were then washed 3 times in ice-cold PBS for 10 minutes each and subsequently incubated with fluorescent secondary antibodies for 1 hour at room temperature in a dark/humidified chamber. Adipocytes were then washed 3 times in ice-cold PBS for 10 minutes each and mounted with VECTASHIELD Vibrance antifade mounting medium containing DAPI (#H-1800, Vector Laboratories).

An inverted Zeiss LSM880 confocal laser-scanning microscope equipped with a 63x PlanApo oil immersion lens (numerical aperture 1.4) was used to obtain micrographs of in vitro and in vivo immunofluorescence staining. For each experiment, the laser intensity remained constant, with the confocal pinhole set to 1 Airy unit for an emission wavelength of 520 nm, and the confocal pinhole for the other captured emission wavelengths (channels) within that experiment set to the same size to achieve optical sectioning. DAPI fluorescence was excited using a 405 nm laser diode, AlexaFluor 488 and BODIPY 493/503 excited with a 488 nm argon-ion laser, and AlexaFluor 555 excited using a 561 nm diode-pumped solid-state laser. DAPI, AlexaFluor 488 and and BODIPY 493/503, and AlexaFluor 555 emissions were detected in the wavelengths of 410-470 nm, 490-550 nm and 560-625 nm, respectively. 16-bit micrographs were captured using Zen Black (Carl Zeiss Microscopy) and quantified using ImageJ.

For in vitro quantification of lipid droplets, the 16-bit images of BODIPY 493/503 were equally threshold, and their size and number calculated automatically by particle analysis after selecting a region of interest around each cell. The 16-bit images of AlexaFluor 555 emissions detecting UCP1 were equally threshold and a region of interest drawn around each cell to calculate % area stained per cell. For in vivo quantification, 16-bit images of AlexaFluor 555 emission was equally thresholded to detect UCP1. % area per field (63x) of UCP1 around each DAPI-positive nucleus was measured.

Tissue histology and adipocyte size quantification
Excised adipose and liver sections were fixed in 10% neutral buffered formalin (#245684, Fisher) for 24 hours with agitation at 4°C, embedded in paraffin, and stained with haematoxylin-eosin by the Virginia Commonwealth University Tissue and Data Acquisition and Analysis Core.
Immunohistochemical brightfield micrographs were captured using a BZ-X810 fluorescence microscope (#BZ-X810, Keyence). Adipocyte size was automatically calculated from 20x brightfield micrographs of haematoxylin-eosin stained adipose using the Adiposoft plugin for ImageJ.

Immunochemistry staining in tissue sections
Excised adipose sections were fixed in 10% neutral buffered formalin (#245684, Fisher) for 24 hours with agitation at 4°, embedded in paraffin, and 5 micrometer sections were transferred to glass slides. For immunohistochemistry, adipose paraffin sections were dewaxed and rehydrated by sequential 5-minute-long submersions at room temperature: twice in 100% Histo-clear (#5089990147, Fisher) and Histo-clear/ethanol [90:10], twice in 100% ethanol, twice in 95% ethanol, twice in 80% ethanol and twice in 70% ethanol. Sections were then washed twice in H2O and then submerged in 98°C HIER antigen-retrieval buffer (#ab208572, Abcam), both for 20 minutes each. Slides were then washed 3 times in PBS, fixed in ice-cold methanol at -20°C for 15 minutes and washed a further 3 times in PBS for 10 minutes each. Sections were blocked and permeabilised in blocking/permeabilization buffer, consisting of PBS containing 5% normal goat serum (#31872, Fisher), 1% BSA (#A7906, Sigma) and 0.4% Triton X-100 (#AAA16046AE, Fisher) for 1 hour at room temperature in a humidified chamber. Sections were subsequently incubated with primary antibodies all diluted in blocking/permeabilization buffer overnight in a humidified chamber at 4°C.

Sections were washed 3 times in PBS for 10 minutes each and incubated with fluorescent secondary antibodies diluted in blocking/permeabilization buffer for 1 hour in a dark/humidified chamber and mounted with VECTASHIELD Vibrance antifade mounting medium containing DAPI (#H-1800, Vector Laboratories). The 16-bit confocal images of AlexaFluor 555 emission was equally thresholded to detect UCP1. % area per field (40x) of UCP1 was measured. For in vivo SphK2 quantification, 16-bit images of AlexaFluor 488 were thresholded to detect SPHK2. % area of SPHK2 covering the nucleus by selecting a region of interest around each DAPI-positive nucleus was measured. For UCP1 and PPARγ, the 16-bit images of AlexaFluor 488 and 555 emissions were equally threshold to detect UCP1 or PPARγ, respectively. % area per field (40x) of UCP1 and PPARγ was measured.

Beta-adrenergic receptor activation in vivo
10-week-old WT and Sphk2∆Adipo chow-fed littermates were injected with vehicle (#64938-009-01, SALJET) or CL316243 (#1499, Tocris) intraperitoneally at a dose of 1 mg/kg/day at 9AM daily for 7 consecutive days. On the 7th day, mice were fasted for 6 hours (7AM-1PM) while continuing to receive treatment at 9AM, and fasted glucose and rectal temperature measured before euthanasia by 5% isoflurane inhalation.

Peak distribution quantification for histone acetylation
In WT or SphK2ΔAdipo sWAT of mice fed WD for 18 weeks, peaks representing regions in the genome with histone H3 lysine 9 acetylation (H3K9ac) or histone lysine 27 acetylation (H3K27ac) were identified from analysis of data for CUT&Tag (Cleavage Under Targets and Tagmentation). For each mouse, the number of peaks identified for H3K9ac or H3K27ac were summed the regions distal, 5'UTR, Promoter, Exon, Intron, and 3'UTR. The percentage of the number of peaks within each region versus the total number of peaks across all regions was then calculated.

Total histone deacetylase (HDAC) and HDAC3 activities
Total HDAC activity was measured in sWAT nuclear extracts using the HDAC fluorometric cellular activity assay kit (#BML-AK503-0001, Enzo Life Sciences). Fluorescence signals were converted to absolute values using a standard curve generated with a deacetylated standard and corrected for background activity from lysis buffer controls.

HDAC3 activity was measured as previously described with some modifications (95). Briefly, 80–100 mg of adipose tissue were homogenized in HDAC cell lysis buffer (#BML-KI346-0020, Enzo Life Sciences) and incubated overnight at 4°C with 1 µg HDAC3 antibody (#85057S, Cell Signalling Technology) or 1µg normal rabbit IgG (#sc-2763, Santa Cruz Biotechnology) as a control. Immune complexes were captured with Protein A/G Plus-Agarose (#sc-2003, Santa Cruz Biotechnology) for 4 hours at 4°C with rotation.

Immunoprecipitants were collected by centrifugation (1,000 × g, 5 minutes, 4°C) and washed four times with PBS. Pellets were resuspended in HDAC assay buffer, and aliquots were used for activity measurements. Fluorescence signals were quantified against a deacetylated standard curve. HDAC3-specific activity was calculated by subtracting IgG control activity from HDAC3 immunoprecipitated activity for each sample.

Quantification of protein levels by western blotting
Equal weights of frozen tissue were homogenized in RIPA buffer containing 50 mM Tris-HCl; pH 7.4, 1 mM EDTA, 150 mM sodium chloride, 0.1% sodium dodecyl sulfate, 1% Triton X-100, 0.5% sodium deoxycholate, with HALT protease/phosphatase inhibitor cocktail (#1861281, Fisher). Tissues were placed in 1.5 mL Eppendorf tubes with 300 µL RIPA buffer, homogenized with a pestle (#K749521-1590, Fisher) and left on ice for 1 hour. Homogenates were centrifuged at 3000 xG for 5 minutes at 4°C, the phase beneath the fat cake placed in a fresh 1.5 mL tube and centrifuged again at 12000 xG for 15 minutes at 4°C. Protein concentration was measured in the remaining supernatant using the Piece BCA Protein Assay Kit (#23227, Fisher) and diluted with Laemmli buffer. Lysates containing equal amounts of protein were incubated at 1200 rpm for 1 hour at 55°C, separated by 10% SDS-PAGE and transferred to nitrocellulose (#1620112, BioRad) using the PierceG2 Fast Blotter (#62287, Fisher). Membranes were blocked for 1 hour in TBST containing 1.4 M NaCl, 27 mM KCl, 250 mM Tris base (pH 7.4), 0.1% Tween-20 (#BP337, Fisher) with 5% BSA (#A7906, Sigma) before incubating overnight with agitation at 4°C with primary antibodies diluted in TBST containing 5% BSA. Membranes were then washed 3 times in TBST for 10 minutes each before being incubated with agitation at room temperature with peroxidase-conjugated secondary antibodies diluted in TBST containing 5% BSA. Membranes were then washed a further 3 times in TBST for 10 minutes each. Protein bands were visualized using chemiluminescent substrate (#34578, Fisher), imaged in the linear range using an Azure Imager C600 (Azure Biosystems, Inc) and analyzed by ImageJ for densitometric quantification normalized to loading controls. Data are represented as fold change relative to the respective WT controls.