Cortex from E14.5 C57BL/6 embryos were harvested in ice-cold complete HBSS (HEPES pH 7.4, 2.5 mM, D-Glucose 30 mM, CaCl2 1 mM, MgSO4 1 mM, NaHCO3 4 mM in Hanks Buffered Salt Solution) to remove the meninges. Cortical tissue was dissociated (250 μg/mL trypsin, 10 μg/mL DNase I, 10 mM HEPES, 200 μg/mL EDTA in Ca 2+ /Mg 2+ -free Hanks Buffered Salt Solution) for 5 min at 37°C and then quenched with 140 μg/mL soybean trypsin inhibitor in HEPES-buffered MEM. The dissociated tissue was centrifuged (1,000 rpm, 5 min, 4°C), the supernatant discarded and the tissue mechanically dissociated in 0.2% (w/v) BSA PBS at 4°C. The cell suspension was filtered with a 40 μm cell strainer and cells pelleted (1,000 rpm, 5 min, 4°C) and resuspended in serum-free Neurobasal medium (1% (v/v) B27, 25 mM KCl, 1X GlutaMAX, 3 g/L glucose, penicillin/streptomycin).

To generate Agrp-Ires-Cre;Ptpn2(AgRP-TC), Ptpn2(C57BL/6) mice () were bred with Agrp-Ires-Cre mice (). To generate AgRP-TC;Npy-GFP mice AgRP-TC mice were mated with Npy-hrGFP reporter mice (). AgRP-TC mice were mated with Insr1mice () to generate Agrp-Ires-Cre;Ptpn2;Insr(AgRP-TC-IR) mice. Pomc-eGFP (), Ptpn2) and Nestin-Cre;Ptpn2) mice have been described previously. All transgenic mice were on a C57BL/6 background.

Aged-matched adult male mice were used for experiments. Mice were maintained on a 12 h light-dark cycle in a temperature-controlled high barrier facility with free access to food and water. Mice were fed a chow diet (8.5% fat; Barastoc, Ridley AgriProducts, Australia) or a high-fat diet (23% fat; 45% energy from fat) diet as indicated. Experiments were approved by the Monash University School of Biomedical Sciences Animal Ethics Committee.

Method Details

Genotyping Tong et al., 2008 Tong Q.

Ye C.P.

Jones J.E.

Elmquist J.K.

Lowell B.B. Synaptic release of GABA by AgRP neurons is required for normal regulation of energy balance. Cowley et al., 2001 Cowley M.A.

Smart J.L.

Rubinstein M.

Cerdán M.G.

Diano S.

Horvath T.L.

Cone R.D.

Low M.J. Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus. van den Pol et al., 2009 van den Pol A.N.

Yao Y.

Fu L.Y.

Foo K.

Huang H.

Coppari R.

Lowell B.B.

Broberger C. Neuromedin B and gastrin-releasing peptide excite arcuate nucleus neuropeptide Y neurons in a novel transgenic mouse expressing strong Renilla green fluorescent protein in NPY neurons. fl/fl ( Loh et al., 2011 Loh K.

Fukushima A.

Zhang X.

Galic S.

Briggs D.

Enriori P.J.

Simonds S.

Wiede F.

Reichenbach A.

Hauser C.

et al. Elevated hypothalamic TCPTP in obesity contributes to cellular leptin resistance. Wiede et al., 2011 Wiede F.

Shields B.J.

Chew S.H.

Kyparissoudis K.

van Vliet C.

Galic S.

Tremblay M.L.

Russell S.M.

Godfrey D.I.

Tiganis T. T cell protein tyrosine phosphatase attenuates T cell signaling to maintain tolerance in mice. fl/fl allele (ΔPtpn2): forward primer 5′GTA ATT ATG CTT TAA GAA CAG C’3, reverse primer 5′CAG AGT GGT TAA GAG CAC TGG’3 ( Wiede et al., 2011 Wiede F.

Shields B.J.

Chew S.H.

Kyparissoudis K.

van Vliet C.

Galic S.

Tremblay M.L.

Russell S.M.

Godfrey D.I.

Tiganis T. T cell protein tyrosine phosphatase attenuates T cell signaling to maintain tolerance in mice. fl/fl allele: 5′GAT GTG CAC CCC ATG TCT G’3, 5′CTG AAT AGC TGA GAC CAC AG’3 and 5′GGG TAG GAA ACA GGA TGG’3. Genotyping was performed by PCR on DNA extracted from tail biopsies using primers previously described for the Agrp-Ires-Cre (), Pomc-eGFP (), Npy-hrGFP () and Ptpn2) alleles. The following primers were used to monitor the recombined Ptpn2allele (ΔPtpn2): forward primer 5′GTA ATT ATG CTT TAA GAA CAG C’3, reverse primer 5′CAG AGT GGT TAA GAG CAC TGG’3 (). The following primers were used to monitor the Insrallele: 5′GAT GTG CAC CCC ATG TCT G’3, 5′CTG AAT AGC TGA GAC CAC AG’3 and 5′GGG TAG GAA ACA GGA TGG’3.

Feeding We maintained mice on a 12 h light-dark cycle from 7 pm (lights off) to 7 am (lights on). Mice were fed a chow diet (8.5% fat; Barastoc, Ridley AgriProducts, Australia) or a high-fat diet (23% fat; 45% energy from fat; SF024-027; Specialty Feeds) as indicated. For ad libiutm fed mice, measurements were performed at 11 am. For ‘fed’ mice, food was restricted 4 h prior to lights off to ensure uniform satiety between groups. Mice then received access to food from the start of the dark cycle (7 pm) for 4 h until satiated. For ‘food-restricted’ mice food was withheld from 6.30 pm onward. For ‘fasted’ mice food was removed at lights off for 6 h, 12 h or 24 h. Unless otherwise indicated ‘fasted’ mice were fasted for 24 h. For ‘re-fed’ mice, food was removed at lights off (7 pm) for 24 h and mice then allowed access to food for 4 h (starting at 7 pm the following night). When necessary, experiments were undertaken under reverse light cycle conditions (lights off, 7 am) with mice acclimatised for 10-12 days prior to any intervention. Diurnal feeding in 8-week-old male C57BL/6 mice was assessed using BioDAQ E2 cages (Research Diets, NJ). Mice were singly housed and food intake measured every second over a 24 h time period and grouped into 15 min time bins.

Immunohistochemistry For brain immunohistochemistry, mice were anaesthetized with 5% isoflurane (Concord Pharmaceuticals, Essex, UK) in oxygen (1 l/min) and perfused transcardially with heparinized saline [10,000 units/l heparin in 0.9% (w/v) NaCl] followed by 4% (w/v) paraformaldehyde in phosphate buffer (0.1 M, pH 7.4). Brains were post-fixed overnight and then kept for two days in 30% (w/v) sucrose in 0.1 M phosphate buffer to cryoprotect the tissue, before freezing on dry ice. 30 μm sections (120 μm apart) were cut in the coronal plane throughout the entire rostral-caudal extent of the hypothalamus. For detection of eGFP and TCPTP, or mCherry alone, sections were subjected to antigen-retrieval in citrate acid buffer [10 mM Sodium Citrate, 0.05% (v/v) Tween 20, pH 6.0] at 85°C for 20 min. Sections were then incubated at room temperature for 1 h in blocking buffer [0.1M phosphate buffer, 0.2% (v/v) Triton X-100, 10% (v/v) normal goat serum (Sigma, St. Louis, MO); for TCPTP staining blocking buffer also contained unlabelled Mouse IgG (1:500, Vector, Burlingame, CA)] and then overnight at 4°C in either chicken anti-eGFP (1/1000; Abcam, Cambridge, UK) and mouse anti-TCPTP (1:200; 6F3 from Medimabs, Quebec, Canada) or rabbit anti-dsRed (1:2500, Clontech) in blocking buffer. After washing with PBS, sections were incubated with goat anti-chicken Alexa Fluor 488- and goat anti-mouse Alexa Fluor 568- conjugated secondary antibodies or goat anti-rabbit Alexa Fluor 568-conjugated secondary antibodies (1/1000, Life Technologies, VIC, Australia) in blocking buffer for 2 h at room temperature. Sections were mounted with Mowiol 4-88 mounting media and visualized using an Olympus Provis AX70 microscope. Images were captured with an Olympus DP70 digital camera and processed using ImageJ and AnalySIS software (Olympus, Notting Hill, VIC, Australia). Brightness and contrast in the merged color images were adjusted to aid in the analysis of co-incidence. Dodd et al., 2015 Dodd G.T.

Decherf S.

Loh K.

Simonds S.E.

Wiede F.

Balland E.

Merry T.L.

Münzberg H.

Zhang Z.-Y.

Kahn B.B.

et al. Leptin and insulin act on POMC neurons to promote the browning of white fat. For inguinal WAT immunohistochemistry animals were culled and inguinal fat immediately dissected and fixed in buffered formalin solution for 48 h. Tissues were embedded in paraffin and 4 μm sections of the entire block prepared. Every tenth to fourteenth tissue section was processed for immunohistochemistry to detect UCP-1 or tyrosine hydroxylase (TH) as described previously ().

Hypothalamic leptin and insulin signaling Mice were injected intraperitoneally with either vehicle or leptin (0.5 −1.0 μg/g; Peprotech, Rehovot, Israel) for p-STAT-3 (Y705) or human insulin (0.85 mU/g, Actrapid, Denmark) for p-AKT (Ser-473). Mice were transcardically perfused either 15 min (for p-AKT staining) or 45 min (for p-STAT3 staining) post-injection with a solution of 4% w/v paraformaldehyde. The brains were post-fixed overnight and then kept for two days in 30% (w/v) sucrose in 0.1 M phosphate buffer to cryoprotect the tissue, before freezing on dry ice. 30 μm sections (120 μm apart) were cut in the coronal plane throughout the entire rostro-caudal extent of the hypothalamus. Sections were pre-treated for 20 min in 0.5% (w/v) NaOH and 0.5% (v/v) H 2 O 2 in PBS, followed by immersion in 0.3% (w/v) glycine for 10 min. Sections were then placed in 0.03% (w/v) SDS for 10 min and then in blocking solution containing 4% (v/v) normal goat serum, 0.4% (v/v) Triton X-100 and 1% (w/v) BSA (fraction V) for 20 min before incubation for 48 h with rabbit anti-p-STAT3 (Y705) (1:1000; #9131, Cell Signaling Technology, Beverly, MA) or rabbit anti-p-AKT (Ser-473) (1:300; #4060, Cell Signaling Technology, Beverly, MA). p-STAT3- or p-AKT-positive cells were processed for immunoperoxidase staining using rabbit IgG VECTORSTAIN ABC Elite and DAB (3,30-diaminobenzidine) Peroxidase Substrate Kits (Vector Laboratories, UK); rostral-caudal hypothalamic p-STAT3 and p-AKT positive cells counted using bright field. Alternatively, sections were incubated for 2 h with goat anti-rabbit Alexa Fluor 568 at room temperature and processed for immunofluorescence microscopy using an Olympus Provis AX70 microscope.

c-Fos immunohistochemistry For the determination of fed and fasted AgRP c-Fos expression 8-10 week old AgRP-TC;Npy-GFP or Ptpn2fl/fl;Npy-GFP control mice were transcardially perfused in the ‘fed’ or ‘fasted’ state. For the determination of ghrelin-induced c-Fos expression 8-10 week old AgRP-TC;Npy-GFP or Ptpn2fl/fl;Npy-GFP were intraperitoneally administered vehicle or ghrelin (0.3 mg/kg, NeoMPS, Strasbourg, France) in the ‘fed’ state (11 pm). Upon administration, all food was removed from the cage and the mice were transcardially perfused 90 min post injection. 30 μm sections (120 μm apart) were cut in the coronal plane throughout the entire rostral-caudal extent of the hypothalamus. Sections were blocked in 10% (v/v) normal goat serum and incubated overnight (4°C) with rabbit c-Fos antibody (1:4000, sc-52, Santa Cruz, CA, USA) in 1% (v/v) blocking buffer. After washing with PBS sections were incubated for 2 h room temperature with goat anti-rabbit Alexa Fluor 568-conjugated secondary antibody (Life Technologies, VIC, Australia) in 5% (v/v) blocking buffer. Sections were mounted with Mowiol 4-88 mounting media and visualized using an Olympus Provis AX70 microscope. Images were captured with an Olympus DP70 digital camera and processed using ImageJ and AnalySIS software (Olympus, Notting Hill, VIC, Australia).

Cell culture Primary cortical neurons isolated from E14.5 C57BL/6 embryos were cultured on plates coated with 50 μg/ml poly-L-lysine and 20 μg/ml laminin at 37°C for 12 h and then treated with 200 μM dexamethasone for 48 h and processed for real-time PCR.

Biochemical analyses Mouse tissues were dissected and immediately frozen in liquid N 2 . For mediobasal hypothalamic micro-dissections, brains were snap frozen in liquid N 2 then 160 μm sections were cut in the coronal plane throughout the entire rostral-caudal extent of the hypothalamus. After each section was cut, the mediobasal hypothalamus (MBH) was microdissected using microdissection scissors. To obtain sufficient protein for detection, all mediobasal hypothalamic sections from one brain were pooled into one sample, snap frozen in liquid N 2 and stored at −80°C for subsequent processing. 2 , 1 mM EGTA, 50 mM NaF, leupeptin (5 mg/ml), pepstatin A (1 mg/ml), 1 mM benzamadine, 2 mM phenylmethysulfonyl fluoride, 1 mM sodium vanadate) and clarified by centrifugation (100, 000 x g for 20 min at 4°C). Tissue lysates were resolved by SDS-PAGE and immunoblotted as described previously ( Tiganis et al., 1998 Tiganis T.

Bennett A.M.

Ravichandran K.S.

Tonks N.K. Epidermal growth factor receptor and the adaptor protein p52Shc are specific substrates of T-cell protein tyrosine phosphatase. Tissues were mechanically homogenized in 5-20 volumes of ice cold RIPA lysis buffer (50 mM HEPES [pH 7.4], 1% (v/v) Triton X-100, 1% (v/v) sodium deoxycholate, 0.1% (v/v) SDS, 150 mM NaCl, 10% (v/v) glycerol, 1.5 mM MgCl, 1 mM EGTA, 50 mM NaF, leupeptin (5 mg/ml), pepstatin A (1 mg/ml), 1 mM benzamadine, 2 mM phenylmethysulfonyl fluoride, 1 mM sodium vanadate) and clarified by centrifugation (100, 000 x g for 20 min at 4°C). Tissue lysates were resolved by SDS-PAGE and immunoblotted as described previously ().

Metabolic measurements Insulin tolerance tests, glucose tolerance tests and pyruvate tolerance tests were performed on 4 h, 6 h and 6 h fasted conscious mice respectively by injecting human insulin (0.5-0.65 mU insulin/g body weight), D-glucose (2 mg/g body weight), or sodium pyruvate (1-2 mg/g body weight) into the peritoneal cavity and measuring glucose in tail blood immediately before and at 15, 30, 45, 60, 90 and 120 min after injection using a Accu-Check glucometer (Roche, Germany). Plasma insulin and corticosterone levels were determined using a Rat insulin RIA kit (Merck Millipore, MA) and a Mouse Corticosterone ELISA (Arbor Assays, MI) according to the manufacturer’s instructions. For the determination of fed and fasted blood glucose and corresponding plasma insulin levels, blood was collected by retro-orbital bleeding after a 6 h fast. Body composition [lean, fat, and bone mineral density (BMD)] was measured by dual energy X-ray absorptiometry (DEXA; Lunar PIXImus2; GE Healthcare) and analyzed using PIXImus2 software; the head region was excluded from analyses. Alternatively, body composition was assessed using EchoMRI (Echo Medical Systems, Houston, TX). Mice were acclimated for 24 h and then monitored for 48 h in an environmentally controlled Comprehensive Lab Animal Monitoring System (CLAMS; Columbus Instruments, Columbus OH) or using a Promethion Metabolic Screening System (Sable Systems International, NV) fitted with indirect open circuit calorimetry and food consumption and activity monitors to measure activity, food intake, and energy expenditure. Where indicated food was restricted at 6.30 pm. Energy expenditure and the respiratory exchange ratio (RER = VCO 2 /VO 2 ) were calculated from the gas exchange data; data were smoothed to plus/minus one data point.

Real-time PCR RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) and total RNA quality and quantity determined using a NanoDrop 3300 (Thermo Scientific, Wilmington, DE, USA). mRNA was reverse-transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) and processed for quantitative real-time PCR either using the TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA) or SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA). The following TaqMan gene expression assays were used: Ptpn2 (Mm00501226_m1), Ptpn1 (Mm00448427_m1), Pomc (Mm00475829_g1), Npy (Mm03048253_m1), Agrp (Mm00475829_g1), Gapdh (Mm99999915_g1), Srebf1 (Mm00550338_m1), Fasn (Mm00662319_m1), Pnpla2 (Mm00503040_m1). The following primers were used for SYBR green expression assays: Ucp-1 (f-ACTGCCACACCTCCAGTCATT, r-CTTTGCCTCACTCAGG ATTGG), Pgc1-α (f-AGCCGTGACCACTGACAACGAG, r-GCTGCATGGTTCTGAGTGCTAAG), Cd137 (f-CGTGCAGAACTCCTGTGATAAC, r-GTCCACCTATGCTGGAGA AGG), Cidea (f-TGCTCTTCTGTATCGCCCAGT, r-GCCGTGTTAAGGAATCTG CTG), Tbp (f-GAAGCTGCGGTACAATTCCAG, r-CCCCTTGTACCCTTC ACCAAT), Tmem26 (f-ACCCTGTCATCCCACAGAG, r-TGTTTGGTGGAGTCCT AAGGTC), Prdm16 (f-CAGCACGGTGAAGCCATTC, r-GCGTGCATCCGCTT GTG), Gapdh (f-ACCACAGTCCATGCCATCAC, r-CACCACCCTGTTGCTGTA GCC). Inguinal white adipose gene comparisons were made using TATA boxbinding protein (Tbp) as the housekeeping gene; all other gene expression was normalized to Gapdh. Relative quantification was achieved using the ΔΔCT method. Reactions were performed using a BioRad CFX 384 touch (Bio-Rad, Hercules, CA). For hypothalamic neuropeptide gene expression (Pomc, Agrp, Npy) 8-10 week old AgRP-TC or Ptpn2fl/fl control mice were fasted for 18 h and injected intraperitoneally with PBS, 1 μg/g leptin or 0.85 mU/g human insulin. Hypothalami were extracted 2 h post-injection, snap frozen in liquid N 2 and processed for quantitative (ΔΔCT) real-time PCR.

Sympathetic inguinal WAT denervation Chao et al., 2011 Chao P.T.

Yang L.

Aja S.

Moran T.H.

Bi S. Knockdown of NPY expression in the dorsomedial hypothalamus promotes development of brown adipocytes and prevents diet-induced obesity. Dodd et al., 2015 Dodd G.T.

Decherf S.

Loh K.

Simonds S.E.

Wiede F.

Balland E.

Merry T.L.

Münzberg H.

Zhang Z.-Y.

Kahn B.B.

et al. Leptin and insulin act on POMC neurons to promote the browning of white fat. Mice received 20 microinjections of 6-hydroxydopamine [6-OHDA (Sigma); 1 uL per injection, 9 mg/ml in 0.15 M NaCl containing 1% (w/v) ascorbic acid] as described previously () throughout the right (unilateral) or both (bilateral) inguinal fat pads. Sham-operated fat pads received an equal volume of vehicle. Body weights were monitored and mice were culled and ingWAT and BAT extracted and either formalin-fixed for histological/immunohistochemical assessment or frozen and processed for quantitative (ΔΔCt) real-time PCR.

DREADDs Krashes et al., 2011 Krashes M.J.

Koda S.

Ye C.

Rogan S.C.

Adams A.C.

Cusher D.S.

Maratos-Flier E.

Roth B.L.

Lowell B.B. Rapid, reversible activation of AgRP neurons drives feeding behavior in mice. Dodd et al., 2015 Dodd G.T.

Decherf S.

Loh K.

Simonds S.E.

Wiede F.

Balland E.

Merry T.L.

Münzberg H.

Zhang Z.-Y.

Kahn B.B.

et al. Leptin and insulin act on POMC neurons to promote the browning of white fat. 10-12-week-old AgRP-Ires-Cre:Npy-hrGFP mice were sterotaxically injected with rAAV-hSyn-DIO-hM4D(Gi)-mCherry [rAAV-hM4Di-Cherry ()] bilaterally into the ARC (coordinates, bregma: anterior-posterior, –1.40 mm; dorsal-ventral, –5.80 mm; lateral, +/–0.20 mm, 200 nl/side) as described previously (). Two weeks after rAAV delivery, mice were unilaterally denervated with 6-OHDA. One-week post-denervation, mice received daily injections of vehicle or CNO (1.5 mg/kg intraperitoneal, Sigma) for 14 days. Body weights and food intake were recorded. Mice were anaesthetized, ingWAT extracted, and mice perfused with paraformaldehyde for hypothalamic immunohistochemical assessment.

Intra-ARC rAAV injections Ptpn2fl/fl mice high-fat-fed for 12 weeks were sterotaxically injected with rAVV expressing Cre recombinase and GFP (rAAV-CMV-Cre-GFP) or GFP alone (rAAV-CMV-GFP; UNC Vector Core) bilaterally into the ARC (coordinates, bregma: anterior-posterior, –1.40 mm; dorsal-ventral, –5.80 mm; lateral, +/–0.20 mm, 100 nl/side). Mice were allowed to recover for 4 weeks post-surgery before experimentation and ARC targeting confirmed by post-mortem GFP immunohistochemistry.

Lateral ventricle cannulations Under 2% (v/v) isoflurane in 1 l/min oxygen 8-week-old AgRP-TC, Ptpn2fl/fl or C57BL/6 mice were implanted stereotaxically with guide cannulas into the right lateral ventricle (0.2 mm posterior, 1.0 mm lateral from bregma). The tip of the guide cannula was positioned 1 mm above the injection site (1 mm ventral to the surface of the skull). All mice were allowed 4-5 days recovery before experimental manipulation. Where indicated mice received ad libitum access to food at the start of the dark cycle (7 pm) for 4 h so that mice were satiated. Food was then removed and mice administered ICV vehicle (PBS), uridine 5′-diphosphate (UDP; 2 μL 30 μM, Sigma), ghrelin (0.2 μg, NeoMPS, Strasbourg, France), dexamethasone (2 μL 319 μM, Sigma) or dexamethasone (2 μL 319 μM) plus RU486 (1 μg/animal, Tocris Bioscience, Bristol, UK). Mice received a second injection 2 h later and after a further 2 h mice were perfused with paraformaldehyde for immunohistochemical analysis, or hypothalami extracted for quantitative real-time PCR. To assess the influence of the glucocorticoid antagonist RU486 on the hypothalamic expression of TCPTP, mice were fasted at the start of the dark cycle (7 pm) and 6 h (1 am) later ICV administered vehicle (ethanol) or RU486 (0.2 μg or 1 μg in 2 μl). Mice received a second injection 3 h later (4 am) and culled after a further 3 h and hypothalami extracted for quantitative real-time PCR. To assess the influence of the melanocortin circuit on the AgRP-mediated promotion of WAT browning, 8-week-old AgRP-TC male mice were ICV administered melanocortin receptor antagonist (HS014, 2.4 nmol/animal, twice daily for 2 consecutive days), culled and inguinal WAT extracted for quantitative real-time PCR and immunohistochemistry. To assess the influence of the proteasome on feeding-induced TCPTP degradation, 8-week-old male re-fed mice were ICV administered the proteasome inhibitor MG132 (2 μL 50 μM, in 70% DMSO, Sigma) with repeat administration 2 h before hypothalamic extraction and processing for immunoblotting.

18F-FDG PET-CT imaging To assess the 18F-FDG (18F-fluoro-2-deoxy-D-glucose) uptake in the ingWAT and BAT of AgRP-TC and Ptpn2fl/fl mice 8-10 week-old male mice were fasted overnight, intravenously injected with 15 MBq of 18F-FDG (Cyclotek, Victoria, Australia) in a total volume of 0.1 mL and then immediately anaesthetised with isoflurane. Imaging was performed on anesthetize mice using a docked Inveon PET-CT (positron emission tomography-computed tomography) multimodal system (Siemens, Munich, Germany). To assess the 18F-FDG uptake in the ingWAT and BAT in fed versus food restricted mice, 7-9 week-old C57BL/6 male mice were intravenously injected with 15 MBq of 18F-FDG 4-6 h post lights off and imaging performed on anesthetize mice as described above. After 30 min of anesthesia mice were placed on a heated mouse imaging bed with the hind legs extended and secured to aid exposure of the inguinal fat pad. The CT and PET field of view was 10 cm x 10 cm and data were acquired for 10 min/mouse. Both CT and PET datasets were co-registered, attenuation corrected and analyzed using the Inveon Research Workplace (IRW) software (Siemens, Munich, Germany). Interscapular BAT was identified and quantified by viewing attenuation corrected PET images and drawing a Region of Interest (ROI) window and then manually contouring the 18F-FDG-positive volume of interest (VOI) using the IRW contouring tool. The area containing the ingWAT was manually segmented from the CT-image and transferred to the PET image. 18F-FDG update in ingWAT and BAT was quantified by determining the standard uptake value (SUV) = FDG uptake (kBq/ml) in the VOI/[injected dose (kBq) x animal weight (g)]. For ingWAT the normalized 18F-FDG uptake was derived from the area (mm3) within the inguinal bed that showed an SUV value greater than 13; this cut off was derived by visually defining the increased area of activity using a contour tool and then normalizing that minimum value across all the scans to the injected dose (SUV) and applying that cut off to all mice. The injected dose was decay-corrected to the actual start time of the PET scan.

Telemetric transponder implantation and ingWAT temperature measurements Enriori et al., 2011 Enriori P.J.

Sinnayah P.

Simonds S.E.

Garcia Rudaz C.

Cowley M.A. Leptin action in the dorsomedial hypothalamus increases sympathetic tone to brown adipose tissue in spite of systemic leptin resistance. Remote biotelemetry was performed using pre-calibrated sensitive transmitters (PDT-4000 G2 E-Mitter sensors, Mini Mitter Company, Starr Life Science, Holliston, MA). IngWAT temperature was measured as described previously (). E-Mitters were implanted under isoflurane anesthesia beneath the ingWAT pad and secured in place by suture. Mice were allowed one-week recovery before studies were commenced. Signals emitted by the E-Mitter telemetric transponders were detected by a receiver positioned underneath the animal’s home cage and analyzed using VitalView software (Mini Mitter Company, Starr Life Science, Holliston, MA). 10-week-old C57BL/6 male mice were allowed access to food (fed) for at least 72 h and the same mice subsequently food-restricted (from 7 pm to 7 am). IngWAT temperature measurements were taken every min and averaged over the indicated times.