Peroxisomal metabolism of branched fatty acids regulates power homeostasis

Mouse fashions and animal experiments

All animal protocols had been authorised by the Washington University Institutional Animal Care and Use Committee. C57BL/6J WT mice had been obtained from the Jackson Laboratory (pressure no. 000664). FAS^Lox/Lox mice had been beforehand described in ref. ³⁴. To generate mice with brown-adipose-specific knockout of FAS (FAS-BKO), FAS^Lox/Lox animals had been crossed with Ucp1-Cre mice obtained from the Jackson Laboratory (inventory no. 024670). FAS^Lox/Lox mice with out Cre had been used as a management for FAS-BKO mice. ACOX2^−/− mice have been beforehand described in ref. ³⁶ and had been generated utilizing two TALEN pairs concentrating on starting and finish of exon 4 that led to deletion of 176 base pairs masking your complete exon 4, leading to a frameshift. Founder mice had been generated by cytoplasmic injection of TALENs into C57BL/6N mouse zygotes after which backcrossed into the WT C57BL/6J background. ACOX2^Lox/Lox mice on the C57BL/6J background had been generated utilizing the CRISPR–Cas9 system. CRISPR-mediated mutagenesis was performed by the Genome Engineering and IPSC Center at Washington University. To generate mice with adipose-specific knockout of ACOX2 (ACOX2-AKO), ACOX2^Lox/Lox animals had been crossed with adiponectin-Cre mice obtained from the Jackson Laboratory (inventory no. 028020). ACOX2^Lox/Lox mice with out Cre had been used as a management for ACOX2-AKO. To generate mice with adipose-specific overexpression of ACOX2 (ACOX2^Adipo-OE), murine ACOX2 was cloned downstream of a beforehand characterised 5.4-kilobase adiponectin promoter⁵¹. The adiponectin promoter plasmid was a beneficiant reward from P. Scherer (University of Texas Southwestern Medical Center). The transgenic assemble was microinjected into the pronucleus of a newly fertilized egg from a C57BL/6J × CBA hybrid mouse and implanted right into a pseudopregnant feminine by the Mouse Genetics Core at Washington University. The founders had been backcrossed greater than seven generations into the WT C57BL/6J genetic background.

UCP1^−/− mice had been obtained from the Jackson Laboratory (inventory no. 003124). To generate mice with adipose-specific overexpression of ACOX2 on the UCP1^−/− background, ACOX2^Adipo-OE mice had been crossed with UCP1^−/− mice. An inbred pressure of those mice on the C57BL/6J genetic background was developed and used for all experiments. Mice had been fed both regular chow weight loss plan (Purina 5053) or a HFD (D12492, Research Diets). All animals had been randomly allotted into totally different teams. For metabolic phenotyping research, each female and male animals had been studied, and all knowledge had been disaggregated by intercourse. Mice had been maintained underneath fixed temperature (23–25 °C), circulating air and humidity (45–65%) with a 12-h mild/darkish cycle and offered advert libitum entry to meals and water. Body composition (fats and lean mass) was measured utilizing an EchoMRI system.

To assess glucose homeostasis, intraperitoneal glucose tolerance exams and intraperitoneal insulin tolerance exams had been carried out primarily based on physique weight or lean physique mass, as indicated. Mice had been injected intraperitoneally with glucose (2.5 g kg⁻¹ physique weight or 2 g kg⁻¹ lean mass) or insulin (0.75 U kg⁻¹ physique weight or 1 U kg⁻¹ lean mass) after being fasted for six h or 4 h, respectively. Blood glucose ranges had been measured at 0 min, 15 min, 30 min, 60 min and 120 min after injection by a glucometer.

VO₂, VCO₂ and respiratory change ratio had been measured by oblique calorimetry utilizing a PhenoMaster (TSE Systems) metabolic cage system and analysed utilizing CalR web-based software program (v.1.3). For measurement of CL316,243-induced power expenditure, mice had been anaesthetized utilizing pentobarbital (90 mg kg⁻¹ intraperitoneally) and acclimated to the atmosphere for 60 min, permitting oxygen consumption to stabilize. The mice had been then injected with CL316,243 at a dose of 1 mg kg⁻¹, and knowledge had been collected for two h in mice housed at 23 °C or 30 °C as beforehand described⁵² To assess chilly tolerance, physique temperature was measured at time 0 and hourly for six h throughout chilly publicity utilizing implantable IPTT-300 temperature-sensitive transponders and a DAS-8007 programmable reader from Bio Medic Data Systems as beforehand described⁵².

Cell tradition and coverings

Immortalized mouse brown preadipocytes had been established and differentiated as beforehand described¹³. Immortalized human brown preadipocytes, kindly offered by Y.-H. Tseng (Joslin Diabetes Center), had been cultured and differentiated as beforehand described^53,54. For isolation of mature pig adipocytes from subcutaneous WAT, contemporary iWAT was digested in a collagenase buffer containing HBSS (GIBCO 14065-45), 12.6 mM CaCl₂, 4.9 mM MgCl₂, 2% BSA and 800 U g⁻¹ (3 mg g⁻¹ tissue) of kind 2 collagenase for roughly 40 min in a 37 °C water tub. The digested materials was handed by way of a 250-μm strainer and washed 3 occasions with a complete of 1 l of KRHB (1× KBH, 25 mM HEPES, 2 mM glucose, 2% BSA) to separate the floating mature adipocytes. The cells had been centrifuged at 50g for 3 min. The mature adipocytes (floating cells) had been collected and cultured with DMEM/F12 medium.

Mouse brown adipocytes and pig iWAT mature adipocytes had been cultured in Dulbecco’s modified eagle medium F12 (DMEM/F12) supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) penicillin/streptomycin, 1% (v/v) l-glutamine and 1% (v/v) sodium pyruvate. Human embryonic kidney 293T (HEK293T) cells and immortalized human brown adipocytes had been cultured in DMEM. All cells had been cultured in a humidified incubator at 37 °C with 5% CO₂ in air.

Palmitic acid, C15:0 OCFA, C17:0 OCFA and BCFA iso-C17:0 had been dissolved in ethanol, then diluted with serum-free medium containing 0.1% fatty acid-free BSA, then added into tradition medium at last concentrations of 0.2 μM, 1 μM or 2 μM primarily based on experimental want. Norepinephrine was first dissolved in 1× PBS and diluted into the tradition medium at last concentrations of 0.25 μM, 1 μM or 2 μM for various experiments. H₂O₂ was dissolved in 1× PBS and diluted into the tradition medium at a last focus of 500 μM. Isotope-labelled glucose (U-¹³C₆ glucose) was dissolved in 1× PBS and diluted into the tradition medium at a last focus of 17.5 mM.

Plasmid constructs

Genome-wide information RNA (gRNA) databases had been used to design gRNA oligonucleotides in opposition to ACOX2, CRAT, Bckdha, CAT and CKB. The oligonucleotides had been ordered from Integrated DNA Technologies and subcloned into lentiCRISPRv2 plasmid. ACOX2 and Hyper7 complementary DNA (cDNA) clones had been bought from transOMIC (BC021339) and Addgene (136466), respectively, and subcloned into pLJM1 lentiviral overexpression plasmid. Sequences of primers used for cloning are listed in Supplementary Table 1. Pexo-TEMP plasmid was generated by inserting PTS2-Sirius-T2A fragment by PCR utilizing primers beneath. Forward: CGG CGA CCG GTG CCA CCA TGC ACC GGC TGC AGG TGG TGC TGG GCC ACC TGG CCG GCC GGC CCG AGT CCT CCT CCG CCC TGC AGG CCG CCC CCT GCA GCT CGG ATC CCA CCA TG, and reverse: TCA CCA TGA GCT CGG GGC C and inserting mT-Sapphire fragment by PCR utilizing primers beneath. Forward: CGG CCG AGC TCA TGC ACC GGC TGC AGG TGG TGC TGG GCC ACC TGG CCG GCC GGC CCG AGT CCT CCT CCG CCC TGC AGG CCG CCC CCT GCG TGA GCA AGG GCG AGG AGC T, reverse: TGT GAT GGA TAT CTG CAG AAT TC primarily based on the gTEMP_pcDNA3 plasmid (Addgene no. 89583). To generate recombinant lentiviruses, the overexpression plasmid (pLJM1), gRNA plasmid (lentiCRISPRv2) or short-hairpin RNA plasmid (pLKO.1-puro) along with packaging plasmids (pMD2.G and psPAX2) had been cotransfected into HEK293T cells. Lentiviral particles had been collected 48 h after transfection and saved at −80 °C till they had been used to transduce immortalized BAT SVF cells.

Extraction and mass spectrometric evaluation of fatty acids

BAT or brown adipocytes homogenate containing 50 μg of whole protein content material was hydrolysed in an acid hydrolysis buffer (CH₃CN:37% HCl, 4:1) in 90 °C water tub for two h. Then, hexane was used to extract the fatty acids. The samples had been dried underneath a stream of nitrogen and redissolved in chloroform, methanol, H₂O and 25% NH₄OH (50:45:5:0.01). After extraction, the electrospray ionization-mass spectrometry photos of the fatty acids within the adipose tissues and adipocytes had been obtained by a Thermo Fisher LTQ Orbitrap Velos within the negative-ion mode scanning from 200 m/z to 600 m/z with a decision of 100,000 (at m/z 400 Da). Data had been processed by built-in Xcalibur software program as beforehand described¹⁴ and the exogenous docosanoic-22, 22, 22-D₃ acid added to samples earlier than extraction was used as an inside normal for quantitation. To confirm the iso-form of the fatty acid buildings, dried fatty acids had been derivatized to the N-(4-aminomethylphenyl) pyridinium derivatives, which had been subjected to higher-energy collisional dissociation tandem mass spectrometry for structural identification as described beforehand⁵⁵. Data had been analysed utilizing R (v.4.2.1).

Carbon flux tracing and lipidomic evaluation

To assess incorporation of ¹³C-label into mmBCFA, BAT SVF cells had been differentiated into adipocytes in regular DMEM/F12 medium after which cultured in DMEM/F12 wherein glucose was changed with [U¹³C₆]-glucose, as beforehand described in ref. ⁹. Briefly, after differentiation, sgACOX2 or management adipocytes had been cultured within the presence of [U¹³C₆]-glucose in DMEM/F12 medium missing glutamine and pyruvate containing 10% FBS for an additional 3 days, adopted by 6 h of therapy with 10 nM CL316,243 or automobile in a traditional DMEM/F12 media and the lipids with labelled mmBCFA had been extracted and subjected to excessive decision electrospray ionization-mass spectrometry evaluation as described above.

Immunofluorescence evaluation

Frozen sections or cell samples had been mounted with ethanol or 4% paraformaldehyde, adopted by main antibody and the corresponding secondary antibody incubation. Nuclei had been counterstained with 4′6-diamidino-2-phenylindole. Samples had been subjected to immunofluorescence evaluation utilizing rabbit polyclonal anti-ACOX2 antibody (1:100), rabbit polyclonal anti-PMP70 antibody (1:100), rabbit polyclonal anti-FASN antibody (1:100) and rabbit polyclonal anti-CRAT antibody (1:100). Slides had been imaged utilizing a Nikon A1Rsi Confocal Microscope. Images had been analysed utilizing NIS (v.5.21). Fluorescence depth and colocalization had been calculated utilizing ImageJ (v.1.53) and Colocalization Finder (v.1).

Oil Red O staining

Adipocytes had been mounted with 10% formalin in a single day after which washed twice with 60% isopropanol. Oil Red O working answer was added, and the cells had been incubated for 10 min at room temperature. The Oil Red O answer was eliminated, and the wells had been washed 4 occasions with diH₂O. The stained cells had been photographed underneath a ×2 goal lens utilizing mild microscopy.

Quantitative real-time PCR evaluation

Total RNA was remoted utilizing PureLink RNA Mini Kit (Invitrogen, 12183018A) and a couple of μg of whole RNA was reverse transcribed into cDNA utilizing the iScriptcDNA Synthesis Kit (Bio-Rad) as beforehand reported²³. Quantitative real-time PCR was performed utilizing PowerUp SYBR Green Master Mix. Relative mRNA expression stage was decided utilizing the two^(−ΔΔCT) technique and L32 was used as an inside reference. Primers utilized in PCR analyses are listed in Supplementary Table 1.

Western blot and immunoprecipitation analyses

Cells or mouse tissue samples had been homogenized in RIPA buffer (Cell Signaling Technology, 9806S) or homogenization buffer (0.25 M sucrose, 20 mM HEPES in distilled H₂O) containing a protease and phosphatase inhibitor cocktail (Sigma, P8465 and P0001). Total protein was extracted and quantitated utilizing a bicinchoninic acid (BCA) package. Denatured protein was separated by SDS–PAGE electrophoresis and transferred onto polyvinyl difluroride or Nylon membrane, blocked with 5% non-fat milk, incubated in a single day with main antibodies and eventually incubated with horse radish peroxidase-conjugated secondary antibodies as reported. Tissue or cell lysates had been subjected to immunoblot evaluation utilizing rabbit polyclonal anti-ACOX2 antibody (1:1,000), rabbit polyclonal anti-FASN antibody (1:10,000), mouse monoclonal anti-catalase antibody (1:1,000), rabbit polyclonal anti-UCP1 antibody (1:1,000), rabbit polyclonal anti-CRAT antibody (1:500), rabbit polyclonal anti-Ap2 antibody (1:1,000), rabbit polyclonal anti-AKT (1:1,000), rabbit polyclonal anti-PMP70 antibody (1:1,000), mouse monoclonal anti-COX 4 antibody (1:1,000), mouse polyclonal anti-oxidative phosphorylation cocktail (1:1,000), rabbit polyclonal anti-pAMPK (T172) antibody (1:1,000), rabbit polyclonal anti-AMPK antibody (1:1,000), rabbit polyclonal anti-pACC (S79) antibody (1:1,000), rabbit monoclonal anti-ACC antibody (1:1,000), rabbit polyclonal anti-BCKDHA antibody (1:1,000), rabbit polyclonal anti-pAK(T473) antibody (1:1,000), rabbit polyclonal anti-AKT antibody (1:1,000) and mouse monoclonal anti-CKB antibody (1:10,000). Rabbit polyclonal anti-β actin (1:1,000), rabbit polyclonal anti-β Tubulin (1:1,000) and rabbit polyclonal anti-Vinculin (1,000) had been used as loading controls. For western blot evaluation of ACOX2, preliminary experiments used both a rabbit polyclonal antibody (Invitrogen; catalogue no. PA5-114814) or a goat polyclonal antibody (Novus; catalogue no. 06011). Subsequently, a rabbit polyclonal anti-ACOX2 antibody from Millipore Sigma (catalogue no. HPA038280) was used. All antibodies utilized in western blot analyses are listed within the Reporting abstract. Proteins had been detected with the Odyssey Infrared Imaging System (LI-COR Biosciences). Uncropped uncooked blots are offered in Supplementary Fig. 1.

Interaction between FASN and PEX7 was assessed by co-immunoprecipitation evaluation as described beforehand⁵⁶. Briefly, HEK293 cells transiently transfected with Flag-FASN had been homogenized utilizing RIPA Lysis Buffer System (Santa Cruz) supplemented with protease and phosphatase inhibitors. Cell lysates had been centrifuged at 13,000 rpm in a microcentrifuge for 10 min to take away unlysed cells. Supernatants had been collected and subjected to protein quantification utilizing the BCA assay. Cell lysates had been then incubated with Anti-Flag M2 Affinity gel (Sigma-Aldrich, A220) in a single day. Immunoprecipitates had been washed 3 times with Tris-buffered saline earlier than elution with SDS–PAGE pattern buffer and subjected to SDS–PAGE. Cell lysates had been subjected to immunoblot evaluation utilizing mouse monoclonal anti-HA antibody (1:1,000) and rabbit polyclonal anti-Flag antibody (1:1,000).

Subcellular fractionation of adipocytes

Adipocytes had been lysed in Peroxisome Extraction Buffer (Sigma) utilizing a Dounce homogenizer. Lysed cells had been centrifuged at 1,000g for 10 min to pellet the nuclear fraction. The supernatant was transferred to a brand new tube and centrifuged at 12,000g for 15 min to gather the mitochondrial pellet. The supernatant was transferred to a brand new tube and centrifuged at 21,000g for 40 min to gather the peroxisome pellet. The remaining supernatant was saved because the cytosol fraction.

OCR

OCR in cultured adipocytes was measured utilizing a Seahorse XFe Extracellular Flux Analyzer (Agilent) in a 24-well plate. For mitochondrial stress take a look at, cells had been handled with oligomycin (3 μM), FCCP (1.5 μM) and antimycin (2 μM) plus rotenone (1 μM). For measurement of fatty acid-induced respiration, cells had been handled with BSA-conjugated 1 mM C15:0, C17:0, iso-C17:0 or BSA alone.

Measurement of mitochondrial respiration in BAT

Mitochondrial respiration in mouse BAT was measured utilizing a beforehand described technique⁵⁷. Briefly, BAT depots from ACOX2^Adipo-OE and WT mice had been thawed in ice-cold PBS, minced in an Eppendorf tube and mechanically homogenized with 10–20 strokes utilizing Teflon-glass in MAS buffer (70 mM sucrose, 220 mM mannitol, 10 mM KH₂PO₄, 5 mM MgCl₂, 2 mM HEPES, 1.0 mM EGTA and 0.2% (w/v) fatty acid-free BSA). The homogenates had been centrifuged at 1,000g for 10 min at 4 °C. The supernatant was collected and loaded right into a Seahorse XF96 microplate in 20 µl of MAS (5 µg BAT). The loaded plate was centrifuged at 2,000g for five min at 4 °C. An further 130 µl of MAS buffer was added to every nicely. Substrate injections had been as follows: 5 mM succinate + 2 μM rotenone for port A, 2 μM rotenone + 4 μM antimycin for port B, 0.5 mM TMPD + 1 mM ascorbic acid for port C and 50 mM azide for port D.

Infrared thermal imaging

The floor temperature within the area surrounding the BAT was measured utilizing a high-speed mid-wave infrared digicam (Telops FAST M3k) geared up with a 50 mm lens (Telops) and a spacer ring that facilitates a protracted working distance, leading to a decision of 0.25 mm per pixel. The temperature distribution was captured from a top-down perspective at 30 frames per second and a decision of 320 × 256 pixels. The recorded thermal photos had been processed utilizing the Reveal-IR software program suite v.1.13.0 (Telops), assuming a fur emissivity of 0.75 (refs. ^58,59).

MTT assay

To decide whether or not β-oxidation of mmBCFA leads to oxidative stress resulting in cytotoxicity, cell viability was assessed utilizing the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay (Cayman Chemicals). Control and catalase knockout brown adipocytes had been handled with iso-C17:0, C16:0, H₂O₂ or automobile for two h. Cell viability was assessed in accordance with the producer’s directions.

Lipid peroxidation assay

To decide the impact of ACOX2 overexpression on oxidative stress, BAT samples had been collected from ACOX2^AdipoOE and WT mice. Lipid peroxidation (4-HNE) protein adducts had been quantified within the samples utilizing a 4-HNE assay package (Abcam) in accordance with the producer’s directions.

Fecal lipid extraction and measurement

Feces had been collected for twenty-four h from every cage and dried in an incubator for 16 h at 42 °C. One hundred milligrams of feces had been homogenized with 1 M NaCl, adopted with a chloroform:methanol answer (2:1). Chloroform layers had been collected after centrifuge and evaporated underneath N₂ circulation till dry. The 2% Triton X-100 in chloroform was added and evaporated once more underneath N₂ circulation. The samples had been dissolved with ddH₂O and measured by a business package (FFA, Wako; HR Series NEFA-HA(2)).

Ex vivo lipolysis assay

BAT was collected from WT and ACOX2^{Adipo OE} mice and positioned right into a nicely of a 24-well plate containing 1 ml of prewarmed KRBH-BSA with 2% fatty acid-free BSA. The tissues had been handled with automobile or with 10 μM isoproterenol for two h at 37 °C. The glycerol ranges within the media had been measured utilizing a business package (Glycerol Assay Kit; Sigma, MAK117).

ATP assay

The ATP ranges in differentiated adipocytes had been measured utilizing a business package (ATP Detection Assay Kit; Cayman, 700410). Briefly, adipocytes had been homogenized within the ATP detection pattern buffer provided within the package and cell lysates had been transferred to prechilled polypropylene tube. The samples had been handled with Reaction Mix at room temperature for 15 min and the luminescence was learn in a plate reader.

Bile acid measurement

Bile acids had been measured by the Metabolomics Innovation Centre utilizing an LC-multiple-reaction monitoring-MS technique, as beforehand described⁶⁰, with crucial modifications. Tissue samples had been exactly weighed into safe-lock Eppendorf tubes, and water (2 μl per mg of tissue) was added. Samples had been homogenized utilizing two metallic beads at a shaking frequency of 30 Hz for two min on a MM 400 mill mixer. Acetonitrile (8 μl per mg of tissue) was then added, adopted by a second homogenization underneath the identical situations for 3 min. Samples had been additional subjected to ultrasonication for two min in an ice-water tub and centrifuged at 21,000g and 5 °C for 10 min. The clear supernatant (100 μl) was collected and blended with 75 μl of an inside normal answer containing 14 isotope-labelled bile acids. The mixtures had been dried underneath nitrogen gasoline, and the residues had been reconstituted in 75 μl of fifty% methanol. A normal answer containing all focused bile acids was ready in the identical inside normal answer and serially diluted to generate 10 calibration requirements. Aliquots (10 μl) of the calibration and pattern options had been injected into an Agilent 1290 UHPLC system coupled to a 6495B Agilent QQQ mass spectrometer. The mass spectrometer was operated in multiple-reaction monitoring mode with negative-ion detection. Chromatographic separation was carried out on a C18 column (150 × 2.1 mm, 1.7 μm) utilizing a binary-solvent gradient with cellular part A (0.01% formic acid in water) and cellular part B (0.01% formic acid in acetonitrile). Calibration curves for particular person bile acids had been generated utilizing linear regression, and bile acid concentrations within the samples had been decided by interpolating the calibration curves with the analyte-to-internal normal peak ratios obtained from pattern injections, making certain quantification inside the applicable focus vary.

Statistics and reproducibility

Data are reported as imply ± normal error of the imply (s.e.m.) except said in any other case. Statistical comparisons between two teams had been carried out through the use of unpaired t-test. Analysis of variance (ANOVA) was used for greater than two teams. A P worth of lower than 0.05 was thought-about statistically vital. Statistical evaluation and graphs had been generated utilizing GraphPad Prism software program (v.9). All in vitro and in vivo experiments had been repeated independently at the least twice, and key experiments had been carried out many occasions utilizing separate cohorts. All replication makes an attempt yielded constant outcomes. Representative immunoblot photos replicate two impartial experiments, and microscopy photos are consultant of n = 3 per group, except in any other case specified within the determine legends.

Reporting abstract

Further info on analysis design is out there within the Nature Portfolio Reporting Summary linked to this text.