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

Figures

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  Fig. 1. Adipose-derived neurotrophic factor NT-3 correlates with UCP1 expression and sympathetic innervation in adipose tissue. aUcp1 mRNA expression in iWAT of 20-day-old postnatal pups, and 3- and 6-month-old mice (n = 5/group, one-way ANOVA, F (2,12) = 18.04, P = 0.0002, *indicates statistical significance vs. 20D with Turkey’s multiple comparisons test). b UCP1 and TH protein levels in iWAT of 20-day-old postnatal pups and 3-month-old adult mice (n = 5/group, * indicates statistical significance vs. 20D using unpaired two-tailed t test). c Representative images of TH-positive sympathetic nerve innervation in iWAT (left panel, from three replicates/group, scale bar = 2000 µm) and quantitation of mean nerve fiber density, mean nerve fiber length, and mean branching points normalized to total adipose tissue area (right panel) in iWAT of 20-days-old postnatal pups and 3-month-old adult mice (n = 3/group, * indicates statistical significance vs. 20D using unpaired two-tailed t test). d Tissue distribution of Nt-3 expression (iBAT n = 7; iWAT n = 5; others n = 4) (*P < 0.0001 iWAT vs iBAT with unpaired two-tailed t test). e NT-3 protein levels measured by ELISA in iBAT and iWAT of…

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  Fig. 2. Administration of NT-3 protein promotes beiging in postnatal mice and protects adult mice from diet-induced obesity. a–c Ucp1 and other BAT-specific gene expression (a, n = 5/group, * indicates statistical significance between saline and NT-3 treatments with unpaired two-tailed t test), UCP1 and TH protein levels (b, n = 5/group, * indicates statistical significance between saline and NT-3 treatments with unpaired two-tailed t-test) and representative UCP1-immunostaining images showing UCP1-positive beige adipocytes in iWAT (c, from three replicate animals/group, scale bar = 150 µm) of 20-day-old postnatal pups with daily intraperitoneal (i.p.) NT-3 (50 μg/kg) injection for 10 days. d–g Body weight (d, saline n = 9; NT-3 n = 8. * Indicates statistical significance with unpaired two-tailed t test, saline vs NT-3 treatments: D19, P = 0.04; D20, P = 0.038; D21, P = 0.022; D22, P = 0.04; D24, P = 0.03; D27, P = 0.019; D29, P = 0.013; D31, P = 0.013; D34, P = 0.006; D36, P = 0.009; D38, P = 0.009; D40, P = 0.008; D43, P = 0.006; D44, P = 0.006; D45, P = 0.004; D48, P = 0.019; D50, P = 0.027; D52, P = 0.0496; D55, P = 0.026; D57, P = 0.025; D59, P = 0.024; D62, P = 0.018; D64, P =…

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  Fig. 3. Overexpression of NT-3 in adipocytes promotes cold-induced thermogenesis and protects mice from diet-induced obesity. a–c Nt-3 mRNA (a, WT n = 3, AdNT3-Tg n = 4, * indicates statistical significance between WT and AdNT3-Tg with unpaired two-tailed t test), NT-3 protein levels measured by immunoblotting (b, WT n = 7, AdNT3-Tg n = 5, * indicates statistical significance between WT and AdNT3-Tg with unpaired two-tailed t test) and ELISA (c, n = 4/group, * indicates statistical significance between WT and AdNT3-Tg with unpaired two-tailed t test) in iWAT of WT and AdNT3-Tg mice. d–h Core body temperature (d, n = 5/group, * indicates statistical significance between WT and AdNT3-Tg with unpaired two-tailed t test), fat pad mass (e, n = 4/group, * indicates statistical significance between WT and AdNT3-Tg with unpaired two-tailed t test), Ucp1 and other thermogenic gene expression in iWAT (f, WT n = 4, AdNTF3-Tg n = 6, * indicates statistical significance between WT and AdNT3-Tg with unpaired two-tailed t test), UCP1 protein levels in iWAT (g, n = 3, *indicates statistical significance between WT and AdNT3-Tg with unpaired two-tailed t test), and representative UCP1 immunostaining…

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  Fig. 4. NT-3 regulates sympathetic neuronal growth and innervation in adipose tissue. a Representative βIII-tubulin immunofluorescent images of sympathetic ganglia neurons treated with NT-3 (100 ng/ml) (left panel, from three independent treatments/group, scale bar = 100 µm) and quantitation of the number of neurites per neuron, total neurite length per neuron, maximal neurite length per neuron, and average neurite length per neurite per neuron (right panel, control n = 80, NT-3 n = 60, * indicates statistical significance between control and NT-3 treatments with unpaired two-tailed t test). Neurons from sympathetic ganglia at thoracic T1–T4 levels from 6- to 8-week-old mice were isolated and used in the experiment. b, c Representative images of TH-positive sympathetic nerve innervation in eWAT (b, left panel, from four replicate animals/group, scale bar = 2000 µm) and iWAT (c, left panel, from four replicate animals/group, scale bar = 2000 µm) and quantitation of mean nerve fiber length and mean nerve fiber branching points normalized to total adipose tissue area in eWAT (b, right panel, n = 4/group, * indicates statistical significance between WT and AdNT3-Tg with unpaired two-tailed t…

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  Fig. 5. TRKC is required for the neurotrophic effects of NT-3. a Representative βIII-tubulin immunofluorescent images of sympathetic ganglia neurons isolated from WT and TRKC + /− mice (left panel, from three independent treatments/group, scale bar = 100 µm) and quantitation of the number of neurites per neuron, total neurite length per neuron, maximal neurite length per neuron, and average neurite length per neurite per neuron (right panel, control n = 158, TRKC + /− n = 162, * indicates statistical significance between WT and TRKC + /− with unpaired two-tailed t test). b Representative βIII-tubulin immunofluorescent images of sympathetic ganglia neurons isolated from fl/fl and STRKCKO mice treated with phosphate-buffered saline (PBS) or NT-3 (100 ng/ml) (left panel, from three independent treatments/group, scale bar = 100 µm) and quantitation of total neurite length per neuron (right panel, fl/fl n = 135, fl/fl + NT-3 n = 49, STRKCKO n = 168, STRKCKO + NT-3 n = 42. Statistical significance was analyzed with two-way ANOVA. fl/fl vs STRKCKO F (1, 390) = 13.25 P = 0.0003; PBS vs NT-3 F (1, 390) = 7.680 P = 0.0059. * Indicates statistical significance with Turkey’s multiple comparisons…

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  Fig. 6. NT-3/TRKC regulates sympathetic ganglia neuronal activity. a, b Representative images of TH, Fast Blue (FB) and TRKC-EYFP labeling (a, from five replicate animals for RT, and 7 replicate animals for cold, scale bar = 75 µm), and percentage of single-, double-, and triple-labeled neurons (b, RT = 5, cold = 7, * indicates statistical significance between RT and cold treatments with unpaired two-tailed t test) in sympathetic ganglia at thoracic T2 level in TRKC-Cre::R26EYFP mice at room temperature (RT) or subjected to a 7-day cold challenge. In panel b, the calculation of single-, double-, and triple-labeled neurons is mutually exclusive, i.e., the number of single-labeled neurons does not include double- or triple-labeled neuron numbers, and the number of double-labeled neurons does not include triple-labeled neuron numbers. c Percentage of FB-positive (FB + ) and FB-negative (FB-) neurons in TH/TRKC-positive neurons in sympathetic ganglia at thoracic T2 level in TRKC-Cre::R26EYFP mice at room temperature (RT) or subjected to a 7-day cold challenge (RT = 5, COLD = 7, a vs b: FB + in RT vs FB + in cold, P = 0.010 with unpaired two-tailed t test). d, e Representative images (d, from…

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  Fig. 7. RNA-seq analysis of sympathetic ganglia from AdNT3-Tg and TRKC + /− mice after 1-day cold challenge. a Heatmap of the expression of genes regulated in the opposite direction in sympathetic ganglia from thoracic T1 to T4 levels of AdNT3-Tg and TRKC + /− mice after 1-day cold challenge. T1–T4 sympathetic ganglia from six mice each group was pooled for RNA-seq analysis. b, c Real-time RT-PCR validation of the genes that are downregulated in sympathetic ganglia of AdNT3-Tg mice (b, n = 5 for WT (except for Tgm1 and Pgm1, where n = 4); n = 5 for AdNT3-Tg. * Indicates statistical significance between WT and AdNT3-Tg with unpaired two-tailed t test), but are upregulated in sympathetic ganglia of TRKC + /− mice (c, n = 5/group, * indicates statistical significance between WT and TRKC + /− with unpaired two-tailed t test) after 1-day cold exposure. d Measurement of neurite growth in sympathetic neurons treated with NT-3 (100 ng/ml) in the presence or absence of CXCL10 (100 ng/ml) (PBS n = 50, CXCL10 n = 43, NT-3 n = 47, CXCL10 + NT-3 n = 40. Statistical significance was analyzed by two-way ANOVA; with or without CXCL10 treatment, F (1, 210) = 95.47 P < 0.0001; with or without NT-3…

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  Fig. 8. Mice with haploinsufficiency of TRKC exhibit impaired cold-induced thermogenesis and are prone to diet-induced obesity. a–d Body temperature (a, WT n = 8, TRKC + /− n = 6, * indicates statistical significance between WT and TRKC + /− with unpaired two-tailed t test), and Ucp1 and other thermogenic gene expression in iWAT (b, WT n = 4, TRKC + /− n = 5), rWAT (c, WT n = 5, TRKC + /− n = 6) and iBAT(d, WT n = 4, TRKC + /− n = 5) of WT and TRKC + /− mice during a 7-day cold exposure (* indicates statistical significance between WT and TRKC + /− with unpaired two-tailed t test). e–g UCP1 and TH protein levels in iWAT (e, WT n = 5, TRKC + /− n = 3, * indicates statistical significance between WT and TRKC + /− with unpaired two-tailed t test) and rWAT (f, WT n = 3, TRKC + /− n = 4, * indicates statistical significance between WT and TRKC + /− with unpaired two-tailed t test), and representative UCP1 immunostaining images showing UCP1-positive beige adipocytes in iWAT and rWAT (g, from three replicate animals/group, scale bar = 150 µm) of WT and TRKC + /− mice during a 7-day cold exposure. h–l Body weight (h, WT n = 7, TRKC + /− n = 8, * indicates statistical significance between WT and…

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  Fig. 9. Mice with specific deletion of TRKC in sympathetic neurons exhibit impaired cold-induced thermogenesis and are prone to diet-induced obesity. a–f Core body temperature (a, n = 4/group, * indicates statistical significance between fl/fl and STRKCKO with unpaired two-tailed t test), fat pad mass (b, n = 4/group, * indicates statistical significance between fl/fl and STRKCKO with unpaired two-tailed t test), Ucp1 and other thermogenic gene expression in iBAT (c, for Ucp1, fl/fl n = 9, STRKCKO n = 7; for other genes, fl/fl n = 5, STRKCKO n = 4, * indicates statistical significance between fl/fl and STRKCKO with unpaired two-tailed t test), Representative H&E and UCP1 immunostaining in iBAT (d, from three replicate animals/group, scale bar = 75 µm), Ucp1 and other thermogenic gene expression in iWAT (e, fl/fl n = 10, STRKCKO n = 6, * indicates statistical significance between fl/fl and STRKCKO with unpaired two-tailed t test), and representative UCP1 immunostaining images showing UCP1-positive beige adipocytes in iWAT (f, from three replicate animals/group, scale bar=150 µm) of fl/fl and STRKCKO mice during a 7-day cold challenge. g–l Body composition (g, n = 5/group, *indicates…

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  Fig. 10. Schematic illustration of the role of fat-derived NT-3 and its receptor TRKC at sympathetic ganglia in the regulation of SNS innervation in adipose tissue. Briefly, the neurotrophic factor NT-3 secreted from adipose tissue acts through its receptor TRKC in sympathetic ganglia to promote SNS innervation in brown and white adipose tissues, which in turn stimulates BAT thermogenesis and WAT beiging, leading to increased energy expenditure and protection from diet-induced obesity.