Fig. 3: Vaspin inhibition of adrenergic signaling involves LRP1 binding and modulation of PDE activities.

A Regulatory mechanisms of adrenergic signaling and levels of intervention (created in BioRender, https://BioRender.com/sxodmnd). B Cell culture supernatant of human vaspin (SERPINA12) after 3 h starvation (control) and parallel blocking of vaspin secretion using BFA (BFA–after 3 h prior medium change, final–post stimulation with CL) before signal transduction and lipolysis assays in differentiated primary brown adipocytes from VasTg measured by ELISA (n = 3 per condition). C–E Western blot analysis and (D) quantification of basal and CL-induced PKA-activation (n = 3 (basal) and 4 (CL)) as well as (E) free fatty acid release in differentiated primary brown adipocytes from VasTg and WT mice after blocking vaspin secretion (NEFA, n = 5–12 per condition). F BAT Lrp1 expression in WT mice housed at 30 °C, 23 °C or 8 °C (n = 6 per temperature). G–J Knockdown of LRP1: (G) Western blot analysis and (H) quantification of LRP1 expression, (I) basal and CL-induced PKA-activation as well as (J) free fatty acid release (NEFA, n = 10–12 per condition, please see source data file) in vaspin-treated differentiated imBA, with or without siRNA-mediated Lrp1 knockdown. K, L Non-heparin-binding (NHB) vaspin variant: (K) Western blot analysis and (L) quantification basal and CL-induced PKA-activation in vaspin and NHB-treated and control differentiated imBA (n = 3/3). M LRP1 ligand RAP: Western blot analysis of basal and CL-induced PKA-activation in RAP or vaspin treated differentiated imBA. N Blocking of vaspin binding to LRP1 (RAP preincubation) or LRP1 internalization by clathrin-mediated endocytosis (using CPZ): Quantification of basal and CL-induced free fatty acid release with or without vaspin treatment in differentiated imBA (n = 19–20 per condition, please see source data file). O, P Inhibition of Gas signaling using PTX: (O) Western blot analysis and (P) quantification of basal and NE-induced PKA-activation in vaspin-treated differentiated imBA with PTX pretreatment (n = 3/3). Q, R Inhibition of PDE activities using IMBX: (Q) Western blot analysis and (R) quantification of basal and CL-induced PKA-activation in differentiated primary brown adipocytes from VasTg and WT mice (n = 3/3). S–U Inhibition of PDE3 and PDE4 using Cilo and Ro: S) Western blot analysis and (T) quantification of basal and CL-induced PKA-activation in differentiated primary brown adipocytes from VasTg and WT mice with Cilo or Ro pretreatment and controls (n = 3/3/3). U Inhibition of total PDE activities using IBMX, Cilo or Ro: Quantification of basal and CL-induced free fatty acid release with or without vaspin treatment in differentiated imBA (n = 10–20 per condition, please see source data file). V-X ELISA-based analysis of TAMRA-vaspin binding to LDL receptors (V) - LDLR in black, vLDLR in red; LRP receptors (W) - LRP2 in black, LRP4 in red, LRP5 in orange, LRP6 in yellow, LRP10 in green); and class B scavenger receptors (X) - SR-BI in black, CD36 in red. Nonlinear regression analysis was performed to determine EC₅₀ presented in Table 1. Data are presented as mean ± SEM of at least two (B–E, J–L, M–U) or three (G–I) independent experiments. WT or control samples are indicated as black circles, VasTg samples as orange circles, Vaspin-treated samples as green circles, Vaspin NHB-treated samples as blue circles. Statistical significance was evaluated by one-way ANOVA with Šídák’s (B, D, E, L) or uncorrected Fischer’s LSD (N, R, U), or two-way ANOVA with Dunett’s (I) or Tukey’s (F, J, P) post-hoc or uncorrected Fischer’s LSD (T), or unpaired two-sided t-test (H). *p value < 0.05, **p value < 0.01, ***p value < 0.001.