Fig. 8: Neuritin1-deficient brown adipose cells show a reduced metabolic response to isoproterenol, diminished maximal respiration and mouse recombinant NRN1 protein promotes PKA activation and induction of CI-NDFUB8 respiratory subunit.
From: Identification of Neuritin 1 as a local metabolic regulator of brown adipose tissue
A Relative Nrn1 mRNA expression in Scr and siNRN1 preadipocytes. Data are from 3 separate differentiation experiments. B Representative imatge of PPARγ1 and γ2, UCP1, HSL and β-tubulin expression in Scr and siNRN1 brown adipocytes at day 10 of differentiation. p-HSL and phosphorylated by PKA substrates (p-PKA) expression (C) and quantification (D and E) in Scr and siNRN1 adipocytes (day 10 of differentiation), in basal and treated with Isoproterenol (10 μM) for 2 h (p-HSL n = 6 from 2 experiments performed in triplicate (p-HSL) and n = 3 from 1 representative experiment performed in triplicate (p-PKA)). Ppargc1a mRNA (F) and Ucp1 mRNA (G) expression in Scr and siNRN1 adipocytes cells, in basal and treated with isoproterenol (10 μM) for 2 h. Data are mean of 2 experiments performed in triplicate. H Seahorse representative experiments for Scr and siNRN1 brown adipocytes at day 10 of differentiation (Scr n = 4 and siNRN1 n = 5). I Mitochondrial respiration values referred to basal WT. Data are the mean of 5 independent experiments. J, K Expression (J) and quantification (K) of β3 Adrenergic Receptors in Scr and siNRN1 brown adipocytes at day 10 of differentiation. Data are mean of 4 independent experiments. L NRN1 SEC fractions run on 10% Bis-Tris NuPAGE Midi SDS-PAGE gels with MES running buffer. BenchMark protein markers (Invitrogen) were included for reference. M Abundance of PKA phosphorylated substrates (p-PKA) in brown adipocytes treated with 0.1, 0.5 and 1 μg/ml of recombinant NRN1 protein. N, O Ppargc1a (N) and Ucp1(O) mRNA expression in adipocytes treated with NRN1 (100 ng/ml) during 2 hours (n = 6 from 3 independent experiments). P Mitochondrial respiration values referred to basal WT respiration in untreated brown adipocytes non-treated (white bars) and cells treated with NRN1 at a concentration of 100 ng/ml (orange bars) from day 4 of differentiation (Representative experiment, Basal n = 7 and NRN1 treated adipocytes n = 8). Q, R Expression (Q) and quantification (R) of CI-NDUFB8 subunit in Scr adipocytes cells treated chronically with NRN1 (100 ng/ml) from day 4 of differentiation until day 10. Data are the mean of 3 independent experiments. Data are MEAN+/− SEM. Statistical differences according to a two-sided Student´s t test (A, I, K, N, O, P and Q) and two-way ANOVA followed by Sidak’s multiple comparison test (D–F and G). Source data are provided as a Source Data file.
