Fig. 6. sFNDC4 insulin-sensitizing effects in 3T3L1 adipocytes require interaction with GPR116 and involve Gs-cAMP signaling.

a WB of the indicated proteins: overnight incubation (O/N—16 h) of 3T3L1 adipocytes with FcsFNDC4 (FcsF4) or Fc with 10 nM of insulin or without insulin (w/o). Following O/N incubations, the cells were serum starved for 3 h. After that, cells were stimulated with insulin at the indicated concentrations (0, 0.5, 1 nM) for 5 min. b WB of the indicated proteins: 3T3L1 adipocytes were treated overnight with insulin (10 nM) or w/o and FcsFNDC4 or Fc in the presence of antiGPR116 (ab111169) (0.4 µg/ml) or isotype control (0.4 µg/ml). Prior acute insulin stimulation for 5 min, cells were incubated in serum-free media (SFM) for 3 h. For a, b, this experiment was performed at least two times and experiment repeats are shown in Supplementary Fig. 5 and quantification of blots in Supplementary Fig. 6. c Tritium-labeled glucose uptake at the indicated conditions. Cells were treated as in b and, after serum starvation, were stimulated with 1 nM insulin for 20 min and glucose transport was initiated by the addition of  ³H-2DG (PerkinElmer Life Sciences) (0.25 μCi/well, 50 μM unlabeled 2-deoxyglucose) for 5 min, when the experiment was terminated. n = 3 replicate wells of a representative experiment out of two experiments is shown. d, e Stimulation of 3T3L1 adipocytes luciferase reporter stable cells lines with the indicated concentrations of stimuli. Stimulation was 3–4 h for the CRE-, SRE-, and SRF-luc2P cell lines and 16 h for the NFAT-RE luc2P cell line. For e, antiGPR116 (ab111169) 0.4 μg/ml was added 30 min prior the addition of the rec. proteins then media was removed and replaced with media containing the indicated concentrations of rec. proteins. In d, n = 3 replicate wells and NFAT control n = 6 replicate wells of a representative experiment out of at least 3 independent experiments is shown. In e, n = 4 independent experiments. f WB: 3T3L1 adipocytes and g WB: adipocytes derived from mouse primary SVF cells were incubated in SFM for 3 h and then stimulated in SMF with the indicated dose of rec. protein or antiGPR116 antibody 0.4 μg/ml for g and for the indicated duration of incubation (min). For d, f, g, at least three independent experiments were performed. In d, e, phosphodiesterase (PDE) inhibitor, IBMX (0,5 mM), was present in the media during the treatment of Cre2LucP adipocytes, whereas in f, g no phosphodiesterase (PDE) inhibitors were present. In c–e, bars are mean ± SEM and statistics represents Student’s unpaired two-tailed t test. p p-value, ns non-significant, NT non-treated. Phospho-antibodies: pAKT^Ser473, pCREB^Ser133, pAS160^Thr642. Source data are provided as a Source data file.