Experiments were performed in accordance with ethics guidelines approved by The University of Melbourne, Monash University and St Vincents Research Institutes Animal Ethics Committee (10323, 10324, 10352, 10385, 10427, 21712, 22282, 22404, 25349 and 28097). Mice were maintained on a 12 h light-dark cycle in a temperature-controlled high-barrier facility with free access to food and water per NHMRC Australian Code of Practice for the Care and Use of Animals. C57BL/6J mice were sourced from the Animal Resources Centre, Australia, whereas Agrp-IRES-Cre (strain 012899), db/db (strain 000697), Npy-GFP (strain 006417), Pomc-GFP (strain 009593), LSL-Cas9 (strain 028551), NZO (strain 002105) mice were sourced from Jackson Laboratories. To generate Agrp-IRES-Cre;;LSL-Cas9-GFP (AgRP-Cas9) mice, hemizygous Agrp-IRES-Cre mice were bred with homozygous LSL-Cas9-GFP mice. All experimental interventions were performed in male rodents aged 8–10 weeks old, unless stated otherwise. Male Sprague–Dawley rats (Animal Resources Centre, Australia) were housed individually with nesting and enrichment material at a room temperature of 23 ± 2 °C, room humidity 40–70%, on a reverse 12 h light/dark cycle (lights off at 09:00). Animals were fed a standard chow (Barastoc, Ridley AgriProducts), a high-fat high-sugar diet (mice: 43% and 20% of total energy from fat and carbohydrate respectively, SF04-001, Specialty Feeds; rats: 30% fats of total energy SF17-204, Specialty Feeds) or a high-fat high-cholesterol diet (40% of total energy from fat and 2% cholesterol, SF16-033, Specialty Feeds). To induce late-stage type 2 diabetes in mice, male C57Bl/6J mice were fed a HFHS diet for 4 weeks before being receiving up to 6 intraperitoneal injections of streptozotocin (40 mg kg−1 (Sigma) in 50 mM sodium citrate buffer pH 4.5) over the following 2 weeks. Blood glucose levels were monitored and mice exhibiting stable blood glucose levels of >15 mM were used for downstream experiments. For all experiments, random allocation was used for assignment of individual mice to experimental groups, and sample sizes were chosen on the basis of prior work and according to standards in the field.
DNA was extracted from tail biopsies using Tissue Extract-PCR Buffers (MDX004, Meridian Bioscience) and DNA was amplified by PCR using MyTaq HS Red Mix (BIO-25048, Meridian Bioscience) with the following primers to detect cre (forward: 5′-GCGGTCTGGCAGTAAAAACTATC-3′, reverse: 5′-GTGAAACAGCATTGCTGTCACTT-3′), LSL-Cas9 (wt forward: 5′-AAGGGAGCTGCAGTGGAGTA-3′, wt reverse: 5′-mCAGGACAACGCCCACACA-3′, mt forward: 5′-TCCCCATCAAGCTGATCC -3′, mt reverse: 5′-CTTCTTCTTTGGGGCCATCT-3′), Npy-GFP (common forward: 5′-TATGTGGACGGGGCAGAAGATCCAGG-3′, wt reverse: 5′-CCCAGCTCACATATTTATCTAGAG-3′, mt reverse: 5′-GGTGCGGTTGCCGTACTGGA-3′), Pomc-GFP (forward 5′-AAGTTCATCTGCACCACCG-3′, reverse 5′-TGCTCAGGTAGTGGTTGTCG-3′) alleles. The following primers were used to monitor the CRISPR-mediated deletion of the mouse insulin receptor gene (ΔInsrCRISPR): forward 5′-GAGATGGTCCACCTGAAGGA-3′, reverse 5′-GTGAAGGTCTTGGCAGAAGC-3′.
For immunohistochemistry on brain, mice were anaesthetized and perfused transcardially with heparinized saline (10,000 units l−1 porcine heparin) followed by 10% neutral buffered formalin. Brains were post-fixed for 16 h and kept for three days at 4 °C in 30% sucrose in PBS to cryoprotect the tissue, before freezing on dry ice. Thirty-micrometre sections (120 mm apart) were cut in the coronal plane throughout the entire rostral–caudal extent of the hypothalamus. Sections were stored in cryoprotectant (30% ethylene glycol, 20% glycerol in PBS) at −20 °C for long term storage. For the detection of hyaluronic acid and versican only, sections were subjected to heat-induced epitope retrieval using citrate acid buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) at 95 °C for 20 min.
For detection of aggrecan, GFP, hyaluronic acid, mCherry, versican, tenascin C, HAPLN1, neurocan, phosphacan, brevican, WFA, WFA–FITC, PGP9.5 and AgRP, sections were incubated at room temperature for 1 h in blocking buffer (0.3% Triton X-100, 5% normal goat serum, Gibco, Thermo Fisher, 0.02% sodium azide) and then overnight at 4 °C in 1% blocking buffer containing either rabbit anti-aggrecan (1:1,000, AB1031, Millipore), chicken anti-GFP (1:2,000; ab13970, Abcam), biotinylated hyaluronic acid binding protein (1:100, 385911, Millipore), rabbit anti-dsRed (1:2,000, 600-401-379, Rockland), rabbit anti-versican (1:1,000, AB1033, Millipore), tenascin C (1:500, M1-B4, Developmental Studies Hybridoma Bank), HAPLN1 (1:500, 9/30/8-A-4, Developmental Studies Hybridoma Bank), neurocan (1:300, 1F6-S, Developmental Studies Hybridoma Bank), phosphacan (1:300, 3F8, Developmental Studies Hybridoma Bank), brevican (1:500, 610895, BD Transduction Laboratories), biotinylated WFA (1:2,000, L1516; Sigma-Aldrich), WFA–FITC (1:2,000, FL-1351-2, Vector Laboratories), rabbit anti PGP9.5 (1:1,000, 14730-1-AP, Proteintech), or guinea pig anti-AgRP (1:500, AS506, Antibodies Australia). After washing with PBS-T (0.3% Triton X-100 in PBS + 0.02% sodium azide), sections were incubated with goat anti-chicken–Alexa Fluor 488 (ab150169, Abcam), goat anti-rabbit–Alexa Fluor 488, goat anti-rabbit–Alexa Fluor 595 or goat anti-rabbit–Alexa Fluor 647 (ab150077, ab150080 or ab150083, respectively, Abcam), Streptavidin–Alexa Fluor 594 or Streptavidin–Alexa Fluor 647 Streptavidin (405240, BioLegend) in 5% blocking buffer for 2 h at room temperature. Sections were mounted with Mowiol 4–88 mounting media and visualized with an Olympus BX61 microscope. Images were captured with an Olympus BX61 camera, acquired using Olympus cellSens Dimension software v2.1 and processed using ImageJ software v1.53 s (NIH). Images for cell internalization were captured using a Zeiss LSM880 Airyscan Fast confocal microscope, acquired using Zeiss ZEN software v2.1 and processed using ImageJ v1.53 s (NIH). Brightness and contrast have been adjusted to aid in the analysis and visualization.
For ingWAT, eWAT, liver and BAT immunohistochemistry, tissue was immediately dissected and fixed in buffered formalin solution on a rocking platform for 48 h at room temperature. Tissues were embedded in paraffin, and 5-µm sections 100 µm apart were prepared. For haematoxylin and eosin histology, sections were incubated in haematoxylin for 3 min followed by 30 s in eosin. For detection of UCP1, ingWAT sections were subjected to antigen retrieval in citrate acid buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) at 95 °C for 20 min. Sections were incubated at room temperature for 1 h in 5% blocking buffer and then overnight at 4 °C in rabbit anti-UCP1 (1:1,000; ab10983, Abcam), in 1% blocking buffer. Following washing in PBS-T, sections were incubated with goat anti-rabbit Alexa Fluor 488 (ab150077, Abcam) secondary antibody in 5% blocking buffer for 2 h at room temperature. Sections were incubated in DAPI (20 ng ml−1 in PBS) for 10 min then mounted with Mowiol 4–88 mounting media and visualized with an Olympus BX61 microscope. Images were captured with an Olympus BX61 camera, acquired using Olympus cellSens Dimension software v2.1 and processed using ImageJ software v1.53 s (NIH). Brightness and contrast have been adjusted to aid in the analysis and visualization.
Mice were injected intraperitoneally with vehicle (PBS) or insulin (3 mU g−1, Actrapid, Nova Nordisk) and mice were transcardially perfused (as described above) after 15 min with 10% neutral buffered formalin. For p-STAT3 signalling, mice were intravenously injected with vehicle (PBS) or leptin (20 μg per mouse in a volume of 100 μl) and transcardially perfused after 30 min with 10% neutral buffered formalin. The brains were post-fixed for 16 h on a rocking platform at room temperature and then kept for two days in 30% sucrose in PBS to cryoprotect the tissue, before freezing on dry ice. 30 µm sections were cut in the coronal plane throughout the entire rostral–caudal extent of the hypothalamus. Sections were pre-treated for 20 min in freshly prepared 1% NaOH, 1% H2O2 in PBS, washed in PBS, incubated for 10 min in 0.3% glycine, washed in PBS and incubated for 10 min in 0.03% SDS. Sections were then blocked in 5% blocking buffer for 1 h at room temperature and incubated for 48 h with rabbit anti-p-AKT (Ser473) (1:300; 4060, Cell Signaling Technology), rabbit anti p-STAT3 (Tyr705) (1:500, number 9131S, Cell Signaling Technology), or rabbit anti fluor in 1% blocking buffer. Sections were then incubated in 5% blocking buffer containing either goat anti-rabbit Alexa Fluor 647 (ab150083, Abcam), goat anti-rabbit Alexa Fluor 594 (ab150080, Abcam) or biotinylated goat anti-rabbit (BA-1000, Vector Laboratories, no sodium azide in 5% blocking buffer). Florescence sections were mounted with Mowiol 4–88 mounting media and visualized using Olympus BX61 microscope. Images were captured with an Olympus BX61 camera, acquired using Olympus cellSens Dimension software v2.1 and processed using ImageJ software v1.53 s (NIH). For chromogenic detection, p-AKT signal was amplified using Vectastain ABC-HRP Kit (1;500, PK-4000, Vector Laboratories) and visualized using 0.1% H2O2 DAB solution (3,30-diaminobenzidine, ICN980681, Thermo Fisher) Peroxidase Substrate Kits (Vector Laboratories). p-STAT3 and p-AKT immunopositive cells were visualized with a Leica DM2000 LED bright field microscope using a Leica DMC6200 camera and Leica Application Suite X software. Brightness and contrast have been adjusted to aid in the analysis and visualization.
The ARC PNN was stereologically assessed throughout the entire rostro-caudal ARC. The ARC was divided into three regions, including the rostral ARC (−1.22/−1.58 mm anterior–posterior), medial ARC (−1.58/−1.94 mm anterior–posterior) and caudal ARC (−1.94/−2.18 mm anterior–posterior). PNN was quantified in the VMH and RSG cortex (−1.58/−1.94 mm anterior–posterior).
All image quantification was performed in ImageJ v1.53 s (NIH). Raw images underwent background subtraction using a rolling ball algorithm to minimize background and any potential variance in tissue autofluorescence. To quantify area and intensity of the PNN within each brain region (ARC, VMH or RSG cortex) images were thresholded and binarized to create a region of interest (ROI) mask of only the PNN. For each brain area, PNN ROI area (µm2) and intensity was calculated. This process was automated to minimize bias and to account for differences in brain nuclei size across multiple images. Brain nuclei were defined in accordance with the Paxinos and Franklin Mouse Brain Atlas (http://labs.gaidi.ca/mouse-brain-atlas/). The area and intensity of PNN within each region was normalized to the respective control.
To determine the co-localization of ECM components (hyaluronic acid, HAPLN1, tenascin C, aggrecan, versican, phosphacan, brevican, neurocan) within the PNN (WFA-positive staining), 2 masks were generated per image: one for the total PNN staining and another for component staining within the ARC. The overall area and intensity were calculated for the total PNN structure. The area and intensity for components within the PNN was determined by quantifying the expression within the total PNN mask only. This allowed for the characterization of ECM components expressed specifically within the ARC PNN. The area and intensity of PNN within each region was normalized to the respective control. To determine the co-localization of the WFA-labelled ARC PNN within the ARC PNN components, two masks were generated per image: one for total PNN staining and another for component staining within the ARC. The overall area and intensity were calculated for the total component structure. The area and intensity for the PNN comprising the components was determined by quantifying the WFA expression within the total component mask only. The area and intensity of PNN within each region was normalized to the respective control. This combined approach further characterizes the specificity of the components to the PNN region. Brightness and contrast have been adjusted to aid in the analysis and visualization.
To determine which metabolically relevant ARC neurons are encased within the PNN during the development of metabolic disease we analysed brains taken from 0, 4- and 12-week HFHS-fed Npy-GFP (to visualize AgRP/NPY neurons) and Pomc-GFP (to visualize POMC neurons) mice. ARC sections were stained for GFP and WFA as described in ‘Immunohistochemistry’ and analysed using ImageJ v1.53 s (NIH) software. To determine the number of GFP positive neurons encased within the PNN we generated two masks. To define the PNN structure in the ARC, images were thresholded and binarized to create a PNN mask. To identify individual GFP positive neurons, images were thresholded and binarized to create a GFP mask. To define individual GFP neurons, the GFP masks were segmented using a watershed separation algorithm. The total number of GFP positive cells were counted within the whole ARC area and within the PNN mask. This quantified the percent of GFP cells encompassed by the PNN in the ARC.
To determine the intensity of the PNN that specifically surrounds individual GFP cells in the ARC, GFP images were thresholded and binarized. An ROI of 1.29 µm (average size of ECM surrounding cortical neurons47) was generated around each GFP cell using dilate, distance map and Voronoi processes in ImageJ v1.53 s software. This generated a mask capable of specifically analysing PNN bordering individual GFP cells. Using this mask, PNN staining intensity surrounding GFP cells present within the ARC PNN was determined.
Mice were fasted overnight and housed individually in transparent cages with ad libitum access to water. Two hours after the beginning of the light cycle (at 09:00) pre-weighed food was presented to the mice and mice were undisturbed and discreetly observed for 90 min. Momentary behaviour was scored every 30 s over a 90-min observation. Behaviour at each 30 s interval was recorded according to the following classifications: feeding (animal at hopper trying to obtain food, chewing, or gnawing), drinking (animal licking at the water spout), grooming (animal scratching, biting or licking any part of its anatomy), resting (animal curled up, resting head with eyes closed), active (animal showing activity, including locomotion, sniffing, rearing), or inactive (animal immobile when aware, or signs of sickness behaviour). Data were collated into 5-min bins, and several variables were assessed including the average percentage of time the mice spent engaging in each recorded behaviour (percentage of total behaviour), food intake, the transition from eating to resting and the time to satiety (the time when the frequency of eating behaviour intersects with the frequency of resting behaviour).
All stereotaxic injections were undertaken under isoflurane anaesthetic using an Ultra Precise Stereotaxic Instrument (963 Kopf) or Ultra Precise Rotational Stereotaxic Instrument (69100, RWD Life Sciences) alongside stereotaxic nanoinjectors (788130, KD Scientific) with Neurosyringes (Hamilton). To induce hypothalamic inflammation, mice were bilaterally injected with a 1:1:1 cocktail containing AAVs expressing GFP (AAV-CMV-eGFP, Addgene) and ligands for TNF (AAV-CMV-TNF) and TGFβ (AAV-CMV-TGFβ) or control AAV alone (AAV-CMV-eGFP, Addgene). To inhibit hypothalamic inflammation mice received bilateral injections of a 1:1:1:1 AAV cocktail containing expressing soluble TNF Receptor Superfamily 1 A (AAV-CMV-sTNFR1A), soluble TGFβR2 (AAV-TRE-sTGFβR2 and AAV-CMV-TetOFF) and AAV-CMV-eGFP vector, or control AAV alone (AAV-CMV-eGFP, Addgene). All inflammatory AAVs we delivered at ~1012 GU ml−1. To disassemble the PNN within the ARC, mice received bilateral (unless stated otherwise) administration of 15 mU per side of active chABC (C3667, Sigma; dissolved in 1 M trehalose) or heat-inactivated chABC protein as a vehicle (chABC in 1 M trehalose48 was heat-inactivated at 85 °C for 45 min, as previously described49) in a total volume of 150 nl per side. To pulse the PNN within the ARC or RSG, mice received bilateral (unless stated otherwise) administration of biotinylated WFA (0.3 µg per side, in a volume of 150 nl). To disrupt the insulin receptor in AgRP neurons, 12-week HFHS-fed AgRP-Cas9 mice were stereotaxically injected with AAV vectors expressing U6-driven guide RNA’s targeting the Insr gene or a scrambled sequence (5′-GTGTAGTTCGACCATTCGTG-3′) alongside a CAG driven mCherry FLEX switch. Unless otherwise stated injections were delivered bilaterally into the ARC (coordinates, bregma: anterior–posterior, −1.70 mm; dorsal–ventral, −5.85 mm; lateral, ±0.18 mm, 200 nl per side) or into the RSG (coordinates, bregma: anterior–posterior, −1.70 mm; dorsal–ventral, −1.00 mm; lateral, ±0.20 mm, 200 nl per side). WFA–biotin was injected unilaterally into the cc (coordinates, bregma: anterior–posterior, −1.70 mm; dorsal–ventral, −1.50 mm; lateral, ±0.20 mm, 200 nl per side).
For hyperinsulinaemic–euglycaemic clamps, mice were anaesthetized under isoflurane and the right jugular vein was catheterized for infusions, as previously described4. Catheters were attached to an implant button (BMSW25, RWD Life Sciences). Implant buttons were capped allowing for group mousing of mice and catheters were kept patent by flushing daily with 40 µl heparinized saline. On the day of the experiment, food was removed at 07:00. After 3.5 h fasting, a primed (1 min, 1.25 μCi min−1) continuous infusion (0.05 μCi min−1) of [3-3H]glucose (NET331A001MC, PerkinElmer) was administered to measure whole-body glucose turnover, as described4. Ninety minutes later, mice received a 40 mU kg−1 insulin bolus over 10 min which was followed by continuous insulin infusion (4 mU kg−1 min−1 in gelofusine). Euglycaemia (~8–10 mM blood glucose) was maintained by a variable infusion of a 30% glucose solution.
Tail blood samples were collected during steady-state conditions (rate of appearance (Ra) = rate of disappearance (Rd)) and at 80, 90, 100, 110, and 120 min for determination of Rd and Ra, as described above. At 120 min, a 13 μCi bolus of [14C]-2-deoxy-d-glucose (NEC495A250UC, PerkinElmer) was injected into the jugular vein, and blood was sampled at 122, 125, 135, 145 and 155 min. At the end of the experiment tissues were extracted for glucose uptake determinations.
HFHS-fed C57BL/6J mice were bilaterally injected with vehicle or chABC into the ARC. 24 h food intake was determined for intra-ARC chABC-treated mice and a cohort of intra-ARC treated vehicle-treated mice were pair-fed, whereby food availability was restricted to the average food consumed by intra-ARC chABC-treated mice.
Metabolic measurements were undertaken in the Melbourne Mouse Metabolic Phenotyping Platform (The University of Melbourne, Australia). Glucose tolerance tests were performed on 6 h fasted conscious mice respectively by injecting d-glucose (2 mg per g of lean body mass and 1 mg per g lean mass for db/db and HFHS + streptozotocin mice) into the peritoneal cavity and measuring glucose in tail blood immediately before and at 0, 15, 30, 45, 60, 90 and 120 min after injection using an Accu-Check glucometer (Roche). The areas under glucose excursion curves were determined and expressed as mM × min. Fasted (12 h fast) plasma insulin or glucose levels were determined using a Rat/Mouse Insulin ELISA (EZRMI-13K, Merck Millipore) or an Accu-Check glucometer respectively. The HOMA-IR was calculated using the equation [(glucose × insulin)/405]. Adiposity was measured using TD-NMR minispec with OPUS 7.0 spectroscopy software (Bruker Optics).
Mice were acclimated for 24 h and then monitored for 48 h in an environmentally controlled Promethion Metabolic Screening System (Sable Systems International) fitted with indirect open circuit calorimetry, food consumption and activity monitors to measure activity, caloric intake and energy expenditure. Data were recorded and extracted using MetaScreen v2.3.15.13 and Macro Interpreter v23.6.0 (Sable Systems International). Respiratory quotients were calculated as the ratio of CO2 production over O2 consumption respiratory exchange ratio and energy expenditure was calculated using the Weir equation (energy expenditure (kcal h−1) = 60 × (0.003941 × VO2 + 0.001106 × VCO2). To account for difference in body mass/composition energy expenditure was analysed and adjusted using ANCOVA using scripts available at the National Mouse Metabolic Phenotyping Centers (MMPC) energy expenditure analysis page (https://www.mmpc.org/shared/regression.aspx).
To provide an index of ingWAT and BAT thermogenesis, infrared thermography was used to measure temperature changes in the inguinal and interscapular regions as described previously50. The FLIR T1010 thermal imaging camera (FLIR Systems Australia) was mounted onto a tripod and animals were positioned at a standardized distance of 70 cm from the camera. Animals were anaesthetized, shaved in the regions of interest and whole-body images were collected in both the prone and supine positions. Temperatures were analysed using the FLIR ResearchIT Max 4 program (FLIR Systems). The peak temperatures within the ingWAT and BAT was determined.
To generate the AAV-gScrambled (pAAV-U6>mScramble-GTGTAGTTCGACCATTCGTG)-CAG > LL:rev(mCherry):rev(LL):WPRE) and AAV-gIR (pAAV[-U6>mInsr[gRNA-TATCGACTGGTCCCGTATCC]-U6>mInsr[gRNA-GTCTGTCCAGGCACCGCCAA]-CAG > LL:rev(mCherry):rev(LL):WPRE) viral vectors, sgRNAs were first designed using online CRISPR tools (http://crispr.mit.edu and http://chopchop.cbu.uib.no/). Potential off-target gRNA binding was assessed in silico using Off-Spotter (https://cm.jefferson.edu/Off-Spotter/) and guides exhibiting ≥3 mismatch with non-specific genomic regions were considered. For AAV-gScrambled a pUp-U6>Scrambled gRNA vector was generated using the Gibson assembly of a pDONR P4-P1R backbone and primers 5′-GGGGACAACTTTGTATAGAAAAGTTGGAGGGCCTATTTCCCATGATTC-3′ and 5′-GGGGACTGCTTTTTTGTACAAACTTGAAAAAAGCACCGACTCGGTGCC-3′. For AAV-gIR a pUp-U6>mInsr[gRNA-TATCGACTGGTCCCGTATCC]-U6>mInsr[gRNA-GTCTGTCCAGGCACCGCCAA] gRNA vector was generated using the Gibson assembly of a AarI digested pUp-U6-gRNA-AarI-Stuffer-AarI backbone and primers 5′-ATATCTTGTGGAAAGGACGAAACACCGTATCGACTGGTCCCGTATCCG-3′ and 5′-AACTTGCTATTTCTAGCTCTAAAACTTGGCGGTGCCTGGACAGAC-3′. For both AAV-gScrambled and AAV-gIR the p-Up vectors were cloned alongside pDown-CAG and pTail-LL:rev(mCherry):rev(LL) to generate the final vectors by LR reaction using the Gateway method. The AAV plasmids were used to generate recombinant viral vectors packaged into the AAV-DJ/8 pseudotype supplied at a titre of >2 × 1013 GC ml−1). All vector cloning and AAV packaging was carried out by VectorBuilder (Chicago, IL). The recombinant AAV vectors expressing inflammatory factors TNF (AAV-CMV-TNF) and TGFβ (AAV-CMV-TGFβ), or soluble TNF receptor superfamily 1A (AAV-CMV-sTNFRA1) or TGFβ receptor 2 (AAV-TRE-sTGFβR2 and AAV-CMV-TetOFF), were manufactured in-house as described previously51. In brief, cDNA constructs carrying the relevant gene expression cassettes flanked by AV2 terminal repeats in an AAV expression plasmid were transfected with the pDGM6 packaging plasmid into HEK293T cells (Sigma; authenticated by Sigma and not tested for mycoplasma contamination) by means of the calcium phosphate precipitate method to produce AAV6 vectors. At 72 h after transfection, the media and cells were harvested for purification via heparin affinity column (HiTrap, GE Healthcare) chromatography and overnight ultracentrifugation before re-suspension in sterile physiological Ringer’s solution and titre determination via quantitative PCR-based reaction (Applied Biosystems) as described previously52. Purified vectors were stored frozen until the day of use, at which time they were rapidly thawed at room temperature and diluted in sterile PBS for administration via stereotaxic injection as described herein.
12-week HFHS-fed C57BL/6J or aged-matched chow-fed controls received bilateral injections of vehicle or chABC into the ARC. 3 days post-injection (before differences in body weights were seen), mice were fasted for 6 h. To assess insulin extravasation into the ARC mice were administered insulin–FITC (50 µg per mouse in a volume of 100 µl, intravenous injection, I3661, Sigma) or FITC (64.3 µmol per mouse in a volume of 100 µl, intravenous injection, F3651, Sigma). To assess leptin extravasation into the ARC, mice were administered leptin-647 (20 μg per mouse in a volume of 100 μl). Mice were perfused (as described above) 30 min post-injection. To assess insulin extravasation into the ARC irrespective of the BBB, mice were administered insulin–FITC (1 µg per mouse in a volume of 2 µl) directly into the lateral ventricles. To do this, mice were anaesthetized and stereotaxically injected (as described above) insulin–FITC at a rate of 200 nl min−1 into the lateral ventricles (coordinates, bregma: anterior–posterior, −0.20 mm; dorsal–ventral, −2.4 mm; lateral, +0.10 mm). Mice were perfused (as described above) 20 min from the start of injection. To assess insulin–FITC brains were post-fixed overnight and cryoprotected in 30% sucrose in PBS. To retain spontaneous fluorescence signal, brains and sections were kept in the dark and were mounted and imaged immediately after sectioning.
Under isoflurane anaesthetic 12-week HFHS-fed C57BL/6J or AgRP-Cas9 mice were implanted stereotaxically with guide cannulas into the right lateral ventricle (0.2 mm posterior, 1.0 mm lateral from bregma). Guide cannula was positioned 1.3 mm above the injection site (1 mm ventral to the surface of the skull). AgRP-Cas9 mice were treated with either AAV-gScrambled or AAV-gIR and underwent guide cannula placement 7 days post AAV administration. Mice were administered intracerebroventricular vehicle (ddH2O), fluorosamine (100 µg per day or 250 µg per day) in a volume of 2 µl and all compounds were delivered approximately 1 h before lights off (19:00).
Conscious mice were restrained by scruffing and inverted parallel to the floor with the chin at ~180-degree angle with the neck. Using a 10 µl tip, a pipettor was loaded with 5 µl of vehicle (ddH2O) or fluorosamine (1 mg per mouse in 20 µl or 5 mg per mouse in 20 µl). The tip of the filled pipettor was placed near the left nostril at a 45-degree angle, and the drug was ejected to form a small 5 µl droplet at tip for the mouse to inhale. Immediately after the mouse inhaled the first droplet the remaining solution was ejected to form another small droplet for the mouse to inhale through the same nostril. The mouse was held in this position for 15 s before the procedure was repeated in the right nostril. The mouse was returned to the cage for 2 min and the process was repeated so that each mouse received four droplets of 5 µl each, delivering a total of 20 µl of solution. All drugs were administered delivered approximately 1 h before lights off (19:00).
To determine PNN turnover in the ARC, RSG, or CC, mice received stereotaxic injections of WFA–biotin as described in ‘Stereotaxic surgery’. At experimental endpoints mice were transcardially perfused and assessment of pulse labelled ARC PNN was identified by immunofluorescent detection of WFA–biotin (PNN at the time of pulse) and WFA–FITC (total PNN) as described in ‘Immunohistochemistry’.
To chase the pulsed WFA–biotin in the ARC, sections were imaged and analysed using ImageJ v1.53 s (NIH) software. Raw images underwent background subtraction using a rolling ball algorithm to minimize background and tissue autofluorescence. To quantify staining area within the ARC, images were thresholded and binarized to create ROI masks for WFA–biotin and WFA–FITC. For each image, staining ROI area (µm2) and intensity was calculated.
To validate the PNN tracker technique, C57BL/6J mice were stereotaxically injected unilaterally with WFA (0.3 µg per side, in a volume of 150 nl) to pulse the PNN into one side of the ARC and saline injected into the other side. One day later mice were transcardially perfused and ARC brain sections were stained and analysed for PNN tracker analysis. To determine how faithfully the pulsed WFA–biotin represents the current PNN we quantified the percentage area to which WFA–biotin (pulse labelled) co-localizes with WFA–FITC (total present PNN).
To validate that the chased WFA–biotin signal represents bona fide PNN staining we stereotaxically injected WFA (0.3 µg per side, in a volume of 150 nl) bilaterally into the ARC of 8-week-old C57BL/6J mice. 3 days later mice received unilateral ARC injections of chABC (15 mU per side in a volume of 150 nl) or vehicle to disassemble the WFA–biotin bound PNN. To determine the specificity of pulsed WFA–biotin we quantified and compared the area and intensity of WFA–biotin staining in the chABC and vehicle-treated sides of the ARC.
To determine PNN turnover in lean and obese mice, we stereotaxically injected WFA–biotin (0.3 µg per side, in a volume of 150 nl) bilaterally into the ARC of 12-week HFHS-fed C57BL/6J mice or aged-matched controls. Brains were extracted either the day after surgery (day 0) or following 1, 3, 5 and 10 weeks post-injection. Brain sections were stained for the presence of WFA–biotin and WFA–FITC, and we quantified the area of WFA–biotin staining as described above. To determine PNN turnover we compared WFA-labelled PNN present at the start of the experiment (day 0) to that which remained at weeks 1, 3, 5 and 10. WFA–FITC labelling of the PNN was performed at each time point to validate the presence of the ARC PNN and ensure changes in WFA–biotin labelling were not due to loss of the PNN over time. The same process was used to assess turnover in the RSG and blood vessels of the CC.
Microdissected ARC tissues were incubated in the extraction buffer, containing 8 M urea, 0.5% Triton X-100, 5 mM Tris 2-carboxyethylphosphine and cOmplete mini ETDA-free protease inhibitor cocktail (Merck) for 30 min with gentle mixing and then homogenized. Samples were centrifuged for 30 min at 5,000 rpm and the supernatant was collected and buffer exchanged using Amicon Ultracell-10k MWCO centrifugal tubes into PBS. Protein concentration of each sample was estimated using Bradford assay. Thirty μg of each protein extract was reduced using 5 mM dithiothreitol for 30 min at 50 °C and alkylated with 10 mM iodoacetamide for an hour at room temperature before blotting onto 0.45 μm PVDF membrane (Millipore, IPVH20200) and dried. Each sample spot was transferred into a 96-well plate and blocked using 1% (v/v) polyvinylpyrrolidone solution in water.
The disaccharide analysis procedure was adapted from53 with the following modifications. GAG disaccharides were released from the PVDF sample spots using an enzyme mix containing 5 mU chABC (Sigma, Cat# C3667), 50 ng each of heparinase I/II/III (R&D Systems) in 100 mM ammonium acetate pH 7 with 5 mM calcium chloride and incubated at 30 °C overnight. An additional mixture of purified GAG polysaccharides containing 1 μg each of bovine kidney heparan sulfate (Sigma-Aldrich, H7640), 10 μg shark chondroitin sulfate (Signma-Aldrich, C4382) and 1 μg of Streptococcus equi hyaluronic acid (Sigma-Aldrich, 53747) were digested alongside samples as enzyme reaction controls, and as retention time standards. Digested disaccharides were collected and dried under low pressure for labelling using 2-AB (2-aminobenzamide), according to a commercially available protocol (Ludger LT-KAB-VP24-Guide-v2.0). Samples, alongside a standard mix of 8 common HS (Iduron, HS mix) and 8 common chondroitin sulfate disaccharides (Iduron, chondroitin sulfate mix), were labelled with 2-AB and washed with octanal twice to remove excess labelling agent. Cleaned samples in the aqueous layer were dried and resuspended in 75% (v/v) acetonitrile with 10 mM ammonium acetate, pH 6.8.
The labelled disaccharides were separated by liquid chromatography using a SeQuant ZIC-HILIC column (200 Å pore size, 3.5 µm particle size, 1 mm × 150 mm) at 35 °C using an Agilent 1260 Infinity II with fluorescence detection. The mobile phase solvent A (10 mM NH4Ac, pH 6.8) and solvent B (90% acetonitrile in 10 mM NH4Ac pH 6.8) were run at a constant flow rate of 60 μl min−1 in microflow mode with gradient parameters as follows: 0–3 min, 100% B; 4–14 min, 94% B; 34 min, 86% B; 47 min, 75% B; 51 min, 60% B; 52–57 min, 60% B; 58– 65 min, 100% B. Fluorescence detection was carried out with excitation and emission wavelengths set at 320 nm and 420 nm, respectively. Peaks were identified using the standard panel and polysaccharide digest control as retention time standards and the abundances were quantified manually by peak area.
Npy-GFP male mice were placed on a HFHS diet for 12 weeks before being stereotaxically injected with either vehicle or chABC in the ARC 3 days before electrophysiological characterization. Mice were anaesthetized with isoflurane prior to brain extraction, and brains were incubated in ice-cold artificial cerebrospinal fluid (aCSF) of the following composition: 127 mM NaCl, 1.2 mM KH2PO4, 1.9 mM KCl, 26 mM NaHCO3, 3 mM D-glucose, 7 mM mannitol, 2.4 mM CaCl2, 1.3 mM MgCl2 (saturated with 95% O2 and 5% CO2, pH 7.4). Coronal sections (250 μm) of the ARC were cut using a vibratome (Leica VTS1000S). Slices were heated for 30 min at 34 °C and then allowed to cool to room temperature prior to recording. Slices were placed in a recording chamber and continuously perfused with room temperature aCSF.
Npy-GFP neurons in the ARC were visualized using fluorescence and differential interference contrast optics with infrared video microscopy (AxioCam MRm, Zeiss) and an upright microscope (BX51WI, Olympus). For current clamp recordings, patch pipettes (8–11 MΩ) were pulled from thin-walled borosilicate glass (Sutter Instruments, BF150-86-10) using a horizontal puller (Sutter Instruments) and filled with intracellular solution containing 140 mM potassium gluconate, 10 mM HEPES, 10 mM KCl, 1 mM EGTA, 4 mM Na-ATP, 0.3 mM Na-GTP and 10 mM Biocytin (300 mOsm and pH 7.3, with osmolality and pH adjusted with sucrose and KOH accordingly). In voltage-clamp recordings to examine K+ currents, patch pipettes (3–6 MΩ) were filled with intracellular solution containing 130 mM potassium gluconate, 6 mM NaCl, 4 mM NaOH, 11 mM EGTA, 1 mM CaCl2, 10 mM HEPES, 1 mM MgCl2, 2 mM Na-ATP, 0.2 mM Na-GTP, 0.1% biocytin (295 mOsm and pH 7.3, with osmolality and pH adjusted with sucrose and KOH accordingly). Cells with a series resistance of >20 MΩ were not included in the analysis. Recordings were made in the presence of tetrodotoxin, where 11 depolarizing pulses applied from −40 to +60 mV for 500 ms in 10 mV increments from a holding potential of −80 mV. A 50 ms prepulse to 0 mV was used to inactivate any residual voltage-dependent Na+ current. Whole-cell recordings were made using a Double IPA Integrated Patch amplifier controlled with SutterPatch software (Sutter Instruments) with all current clamp data filtered at 5 kHz. Data were analysed using Sutterpatch (Sutter Instruments) and Clampfit 10.7 (Axon Instruments).
The mediobasal hypothalamus was microdissected and snap frozen in liquid nitrogen. Tissues were mechanically homogenized in 100 μl ice-cold RIPA lysis buffer (ab156034, Abcam, UK, containing PhosStop Phosphatase Inhibitor, 1 tablet per 10 ml; Roche PHOSS-RO) and clarified by centrifugation (13,000 rpm for 20 min at 4 °C). Tissue lysates were resolved by SDS–PAGE and immunoblotted as described previously (PMID: 31509751). Antibodies used are rabbit phospho-IR (Tyr1162, Tyr1163) polyclonal antibody (1:1,000, 44–804 G, Invitrogen, MA), rabbit monoclonal anti-IR (1:1,000, 3025x, Cell Signaling), rabbit-β-actin polyclonal antibody (1:2,000, 4967, Cell Signaling Technology), mouse GAPDH monoclonal antibody (1:5,000, 60004-1-Ig, Proteintech), mouse monoclonal anti-tubulin (1:2,000, T5168, Sigma).
RNA was extracted using TRIzol reagent (Invitrogen) and total RNA quality and quantity determined using a NanoDrop 3300 v2.8.1 (Thermo Scientific). mRNA was reverse-transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and processed for quantitative real-time PCR using SYBR Green PCR Master Mix (4309155, Applied Biosystems). The following primers were used for SYBR green expression assays: Adamst4 (forward-GAACGGTGGCAAGTATTGTGAGG, reverse-TTCGGTGGTTGTAGGCAGCACA), Adamst5 (forward-CTGCCTTCAAGGCAAATGTGTGG, reverse-CAATGGCGGTAGGCAAACTGCA), Ikkb (forward-GCAGACTGACATTGTGGACCTG, reverse-ATCTCCTGGCTGTCACCTTCTG), Il6 (forward-GGTGCCCTGCCAGTATTCTC, reverse-GGCTCCCAACACAGGATGA), Kcna4 (forward-GCAGATTGCTGAATGACACCTCG, reverse-GGACAAGCAAAGCATCGAACCAC), Kcnb1 (forward-GAGGAGTTCGACAACACGTGCT, reverse-TGAGTGACAGGGCAATGGTGGA), Kcnb2 (forward-GCTGGAGAAACCTAACTCGTCC, reverse-CTCGTCGTTTTCTTGCAGCTCTG), Kcnc3 (forward-GAAGAGGTGATTGAAACCAACAGG, reverse-TGGGCTCTTGTCTTCTGGAGAC), Kcnc4 (forward-CCAGCTCGAATCGCCCATTTAC, reverse-AGCACCGCATTAGCATCGCCAT), Kcnd2 (forward-CCTACATGCAGAGCAAGCGGAA, reverse-GTGGTTTTCTCCAGGCAGTGAAG), Kcnd3 (forward-AGAAGAGGAGCAGATGGGCAAG, reverse-CTTGATGGTGGAGGTTCGTACAG), Kcnj11 (forward-TGCGTCACAAGCATCCACTCCT, reverse-GGACATTCCTCTGTCACCATGC), Kcnj3 (forward-CAGTTCGAGGTTGTCGTCATCC, reverse-CCCAAAGCACTTCGTCCTCTGT), Kcnj6 (forward-GGAACTGGAGATTGTGGTCATCC, reverse-TCTTCCAGCGTTAGGACAGGTG), Kcnj9 (forward-TCTCACCTCTCGTCATCAGCCA, reverse-GCTTCGAGCTTGGCACGTCATT), Kcnma1 (forward-CCTGAAGGACTTTCTGCACAAGG, reverse-ACTCCACCTGAGTGAAATGCCG), Kcnn3 (forward-TCCACCGTCATCCTGCTTGGTT, reverse-CAGGCTGATGTAGAGGATACGC), Kcnq3 (forward-AAGCCTACGCTTTCTGGCAGAG, reverse-ACAGCTCGGATGGCAGCCTTTA), Mmp13 (forward-AGCAGTTCCAAAGGCTACAACT, reverse-GGATGCTTAGGGTTGGGGTC), Mmp14 (forward-AGCACTGGGTGTTTGACGAA, reverse-CCGGTAGTACTTATTGCCCCG), Mmp2 (forward-GTCGCCCCTAAAACAGACAA, reverse-GGTCTCGATGGTGTTCTGGT), Mmp9 (forward-GCTGACTACGATAAGGACGGCA, reverse-TAGTGGTGCAGGCAGAGTAGGA), Nfkb1 (forward-GCTGCCAAAGAAGGACACGACA, reverse-GGCAGGCTATTGCTCATCACAG), Rn18s (forward-CAGCTCCAAGCGTTCCTGG, reverse-GGCCTTCAATTACAGTCGTCTTC), sTgfβr2 (forward-AAGGGTTCAGCCTACACCTT, reverse-GTCGGGACTGCTGGTGGTGT), sTnfr1α (forward-GGTTATCTTGCTAGGTCTTTG, reverse-GATCCCTACAAATGATGGAG), Tgfb1 (forward-GGATACCAACTATTGCTTCAG, reverse-TGTCCAGGCTCCAAATATAG), Tgfb2 (forward-CTAATGTTGTTGCCCTCCTACAG, reverse-GCACAGAAGTTAGCATTGTACCC), Tgfbr1 (forward-GGACCATTGTGTTACAAGAAAGC, reverse-CATGGCGTAACATTACAGTCTGA), Tgfbr2 (forward-TCCTAGTGAAGAACGACTTGACC, reverse-TACCAGAGCCATGGAGTAGACAT), Timp1 (forward-TCTTGGTTCCCTGGCGTACTCT, reverse-GTGAGTGTCACTCTCCAGTTTGC), Timp3 (forward-GCTAGAAGTCAACAAATACCAG, reverse-TAGTAGCAGGACTTGATCTTG) and Tnf (forward-CTGTGAAGGGAATGGGTGTT, reverse-GGTCACTGTCCCAGCATCTT).
Gene expression was normalized to Rn18s and relative quantification was achieved using the ΔΔCT method. Reactions were performed using a Bio-Rad CFX 384 touch (Bio-Rad).
To determine the interaction of insulin with PNN components in vitro, flat-bottom 96-well plates were first coated with 10 μg ml−1 poly-l-lysine overnight, followed by a rinsing with water. A purified CSPGs mix containing neurocan, phosphacan, versican and aggrecan (CC117, Merk Millipore), purified aggrecan (A1960, Merk Millipore) or purified C4S (S9004, Selleck Chemicals), were coated onto the 96-well plates at a concentration of 10 μg ml−1 for 4 h at room temperature, followed by a rinse with water. Insulin–FITC was incubated on plates containing ECM at concentration ranging from 5–1 mg ml−1 for 2 h at room temperature and protected from light. Control wells contained either no ECM, bovine serum albumin (10 μg ml−1) or poly-l-lysine alone. Wells were washed 3 times with water and imaged using a SPECTROstar Nano Microplate Reader (BMG Labtech, Germany). To digest PNN or to negate PNN negative charge, wells were incubated with either chABC (0.5 U ml−1) or poly-l-arginine (10 μg ml−1, P7762, Merk Millipore) for 1 h at 37° C after the ECM coating, washed 3 times with water and then incubated with insulin–FITC.
Statistical analyses were performed using GraphPad Prism version 10 (GraphPad Software). Statistical significance was determined by a one-way or two-way ANOVA with multiple comparisons or repeated-measures, one or two-tailed paired or unpaired Student’s t-tests, ANCOVA, or simple linear regression as appropriate. P < 0.05 was considered significant: *P < 0.05, **P < 0.01 and ***P < 0.001. Statistical details of individual experiments such as exact values of n and exact statistical tests can be found in figures and legends.
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
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