Fig 2: TRKC is required for the neurotrophic effects of NT-3.a Representative ßIII-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 ßIII-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 test). Neurons from sympathetic ganglia at thoracic T1–T4 levels from 6- to 8-week-old mice were isolated and used in these experiments. c, d Representative images of TH-positive sympathetic nerve innervation in eWAT (c, left panel, from three replicate animals/group, scale bar = 2000 µm) and iWAT (d, 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 (c, right panel, n = 3/group, * indicates statistical significance between WT and TRKC + /- with unpaired two-tailed t test) and iWAT (d, right panel, n = 4/group, * indicates statistical significance between WT and TRKC + /- with unpaired two-tailed t test) in TRKC + /- mice and their littermate control mice after a 7-day cold challenge. e, f Representative images of TH-positive sympathetic nerve innervation in eWAT (e, left panel, from three replicate animals/group, scale bar = 2000 µm) and iWAT (f, 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 (e, right panel, n = 3/group, * indicates statistical significance between fl/fl and STRKCKO with unpaired two-tailed t test) and iWAT (f, right panel, n = 4/group, * indicates statistical significance between fl/fl and STRKCKO with unpaired two-tailed t test) in fl/fl and STRKCKO mice after a 7-day cold challenge. All data are expressed as mean ± SEM.

Fig 3: 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 = 0.02; D66, P = 0.014; D69, P = 0.008), body composition (e, saline n = 9; NT-3 n = 6, * indicates statistical significance between saline and NT-3 treatments with unpaired two-tailed t test), fat pad morphology (f) and fat pad mass (g, n = 8/group, * indicates statistical significance between saline and NT-3 treatments with unpaired two-tailed t test) in 6-week-old mice with NT-3 (50 µg/kg) i.p. injection every day for the first 2 weeks, then three times a week for 8 weeks while fed HFD. h, i Oxygen consumption (h) and heat production (i) in 6-week-old mice with NT-3 (50 µg/kg) i.p. injection every day for the first 2 weeks, then three times a week for 8 weeks while fed HFD (n = 7/group, * indicates statistical significance between saline and NT-3 treatments with unpaired two-tailed t test). j, k UCP1 and TH protein levels in iBAT (j, saline n = 9, NT-3 n = 8. * Indicates statistical significance between saline and NT-3 treatments with unpaired two-tailed t test) and representative H&E and UCP1 immunostaining in iBAT and iWAT (k, from three replicate animals/group, scale bar for iBAT = 75 µm, and for iWAT=150 µm) of 6-week-old mice with NT-3 (50 µg/kg) i.p. injection every day for the first 2 weeks, then three times a week for 8 weeks while fed HFD. l Correlation between serum NT-3 levels and body weight in mice fed a HFD (n = 14, P = 0.034 with simple linear regression and Pearson’s correlation test, F (1, 12) = 5.748). All data are expressed as mean ± SEM.

Fig 4: 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 20-day-old postnatal pups and 3-month-old adult mice (iBAT 20D n = 6, 3 M n = 5; iWAT 20D n = 10, 3 M n = 6) (two-way ANOVA, 20D vs 3 M, F (1, 24) = 62.2, P < 0.0001; iBAT vs iWAT, F (1, 24) = 21.95, P < 0.0001. * indicates statistical significance as shown in (e) with Turkey’s multiple comparisons test). f The expression of Nt-3 mRNA in adipocytes and stromal vascular fraction (SVF) in iBAT and iWAT (iBAT n = 6; iWAT adipocyte n = 8, SVF n = 5, two-way ANOVA, cell type (adipocyte vs SVF), F (1, 21) = 4.118, P = 0.055; tissue (iBAT vs iWAT), F (1, 21) = 85.65, P < 0.0001. * indicates statistical significance as shown in (f) with Turkey’s multiple comparisons test). g, h The expression of Nt-3 (g) and Ucp1 (h) in iWAT during postnatal development in mice (n = 4/group). i, j The expression of Nt-3 (i) and Ucp1 (j) in iBAT during postnatal development in mice (n = 4/group). k, l NT-3 protein levels measured by ELISA in iWAT (k, time 0 n = 4; 6 h, 1d and 7d n = 5) and eWAT (l, time 0 n = 4; 6 h, 1d and 7d n = 5) during cold exposure. k One-way ANOVA with Turkey’s multiple comparisons test, F (3, 15) = 39.79, P < 0.0001. a vs c: time 0 vs 1 day, P = 0.018; a vs d: time 0 vs 7 days, P < 0.0001; b vs c: 6 h vs 1 day, P = 0.035; b vs d: 6 h vs 7 day, P < 0.0001; c vs d: 1 day vs 7 days, P < 0.0001. l One-way ANOVA with Turkey’s multiple comparisons test, F (3, 15) = 64.81, P < 0.0001. a vs c: time 0 vs 1 day, P = 0.0047; a vs d: time 0 vs 7 days, P = 0.0037; b vs c: 6 h vs 1 day, P = 0.012; b vs d: 6 h vs 7 days, P = 0.0039; c vs d: 1 day vs 7 days, P = 0.0069. All data are expressed as mean ± SEM.

Fig 5: Co-immunoprecipitation assay to demonstrate interactions between neurotrophin-3 (NT-3) and the neurotrophin receptors TrkA and TrkB in the retinas of diabetic rats.The NT-3 band was immunoprecipitated within a complex between ligand and receptors. The TrkA and TrkB bands, present in the immunoprecipitates with an NT-33-reactive antibody, represent the interaction between NT-3 and the receptors TrkA and TrkB in diabetic (D) and control (C) rats.