Adipose tissue-derived neurotrophic factor 3 regulates sympathetic innervation and thermogenesis in adipose tissue

Activation of brown fat thermogenesis increases energy expenditure and alleviates obesity. Sympathetic nervous system (SNS) is important in brown/beige adipocyte thermogenesis. Here we discover a fat-derived “adipokine” neurotrophic factor neurotrophin 3 (NT-3) and its receptor Tropomyosin receptor kinase C (TRKC) as key regulators of SNS growth and innervation in adipose tissue. NT-3 is highly expressed in brown/beige adipocytes, and potently stimulates sympathetic neuron neurite growth. NT-3/TRKC regulates a plethora of pathways in neuronal axonal growth and elongation. Adipose tissue sympathetic innervation is significantly increased in mice with adipocyte-specific NT-3 overexpression, but profoundly reduced in mice with TRKC haploinsufficiency (TRKC +/−). Increasing NT-3 via pharmacological or genetic approach promotes beige adipocyte development, enhances cold-induced thermogenesis and protects against diet-induced obesity (DIO); whereas TRKC + /− or SNS TRKC deficient mice are cold intolerant and prone to DIO. Thus, NT-3 is a fat-derived neurotrophic factor that regulates SNS innervation, energy metabolism and obesity.

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Policy information about availability of computer code Data collection 1. The expression of genes of interest was measured by a one-step quantitative RT-PCR with TaqMan Universal PCR Master Mix reagents (ThermoFisher Scientific, Waltham, MA) using an Applied Biosystems QuantStudio 3 real-time PCR system (ThermoFisher Scientific). 2. Histological and IHC images were captured using an Olympus DP73 photomicroscope or Zeiss 710 NLO Laser Scanning Confocal Microscope. 3. Immunoblotting was visualized using a Li-COR Imager System (Li-COR Biosciences, Lincoln, NE). 4. Tissue NE content was measured by HPLC. 5.For bioinformatics analysis, raw reads were filtered to remove adaptor sequences and low-quality data using SOAPnuke (v1.5.2, https:// github.com/BGI-flexlab/SOAPnuke) and mapped to reference sequences (University of California Santa Cruz Mouse Genome Browser mm9 Assembly) using Hierarchical Indexing for Spliced Alignment of Transcripts (HISAT2, v2.0.4, http://www.ccb.jhu.edu/software/hisat/ index.shtml).
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April 2020
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability The RNAseq data have been deposited to Gene Expression Omnibus (GEO) database with the accession code GSE173503. All data generated in this study are available and reported. A source data file is included with the paper.

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Sample size
The experimental sample sizes were estimated based on our previous published data that are sufficient to detect statistical differences among the groups ( Data exclusions Most of the data were included for analysis. Only exception is, in real-time RT-PCR measurement, CT value for some samples were not detected due to experimental error and these samples were excluded from analysis. This includes one sample for Tgm1 and one sample for Pgm1 in Figure 7B, one sample for Pgc1b and one sample for Fgf21 in Suppl. Figure 10H, and one sample for Pparg, one sample for Pgc1b and two samples for Pgc1a in Suppl. Figure 20C.
Replication A replication of 3 to 14 animals each group were used for animal studies, gene expression, immunobloting and histology analysis. For primary sympathetic neuronal culture, between 40 and 168 neurons each group were analyzed. The results were the same.
Randomization For all experiments, animals with the same genotypes were randomly assigned into control and experimental groups.

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We applied blinding to immunohistochemistry sample analysis. For other experiments, blinding is not necessary because all groups including animals and cell experiments were treated the same way.
Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.  Validation These antibodies were used as recommended by the manufacturer and as cited extensively in previous publications. We have also tested different concentrations of each antibody and used the optimized concentration for each antibody. We further ensured that signals in Western Blots were present at the expected size for each target The relevant information for each primary antibody, from the manufacturer and online databases, is as follows: UCP1 for western ABCAM ab23841, Rabbit polyclonal to UCP1. Tested by the manufacturer for detecting UCP1 in mouse, rat and dog by western blotting (WB) and immunohistochemistryPolyclonal Goat IgG (IHC), using mouse and rat brown adipose tissue as positive controls in WB. Ninety-nine Citations from the CiteAb database (https://www.citeab.com/antibodies/758073-ab23841-anti-ucp1antibody?des=641734b5b4769e80 α-tubulin Advanced BioChemicals ABCENT4777, rabbit polyclonal antibody, detects α-tubulin from human, mouse and rat by WB, IHC, IF and ELISA. cFos Millipore ABE457, rabbit polyclonal antibody. Evaluated by manfacturer by Western Blot in PMA(TPA) treated HeLa cell lysate.
GFP aves labs GFP1010, Chicken polyclonal antibody. Recommended by the manufacturer to detect GFP by ELISA, ICC, IHC, WB. From manufacturer's notes: Antibodies were analyzed by western blot analysis (1:5000 dilution) and immunohistochemistry (1:500 dilution) using transgenic mice expressing the GFP gene product. One hundred and sixteen citations from CiteAb database (https:// www.citeab.com/antibodies/575207-gfp-1010-green-fluorescent-protein-1-0-mg?des=708459386bbbe89e). Alexa Fluor 680 Goat Anti-Rabbit IgG (H ⁄ L), highly cross-adsorbed, Invitrogene A21109. According to manufacturer, to minimize cross-reactivity, these goat anti-rabbit IgG (H+L) whole secondary antibodies have been affinity purified and cross-adsorbed against bovine IgG, goat IgG, mouse IgG, rat IgG, and human IgG. Cross-adsorption or pre-adsorption is a purification step to increase specificity of the antibody resulting in higher sensitivity and less background staining. The secondary antibody solution is passed through a column matrix containing immobilized serum proteins from potentially cross-reactive species. Only the nonspecific-binding secondary antibodies are captured in the column, and the highly specific secondaries flow through. The benefits of this extra step are apparent in multiplexing/multicolor-staining experiments (e.g., flow cytometry) where there is potential cross-reactivity with other primary antibodies or in tissue/cell fluorescent staining experiments where there are may be the presence of endogenous immunoglobulins. 201 citations from CiteAb: https://www.citeab.com/antibodies/2401223-a-21109-goat-anti-rabbit-igg-h-l-highlycross-adso?des=cb3719bfcb178f19.
Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 680, Invitrogen A21084. This secondary antibody is designed for fluorescent Western blot detection on various near-infrared fluorescence instruments. This antibody can be used for multi-color and multiplexing detection when using other antibodies conjugated to compatible Alexa Fluor dyes and wavelengths. Other applications of this antibody include immunofluorescent and fluorescent imaging applications when using instrumentation with appropriate excitation and detection capabilities. 56 citations from CiteAb: https://www.citeab.com/antibodies/2401206-