Fig 2: SWELL1 protein expression regulates insulin-stimulated PI3K-AKT2-AS160 signaling.a, b Western blots detecting SWELL1, pAKT2Ser474, AKT2, pAS160Thr642, AS160, and β-actin in wildtype (WT, black), SWELL1 knockout (KO, red), and adenoviral reexpression of SWELL1 in KO (KO + SWELL1 O/E, blue) 3T3-F442A adipocytes stimulated with 0 and 10 nM insulin for 15 min (a) and densitometric quantification for pAKT2/β-actin and pAS160/β-actin (b) (n = 3 independent experiments for each condition). c Mean inward and outward current densities recorded at −100 and +100 mV from WT (black, n = 5 cells), KO (red, n = 4 cells), and KO + SWELL1 O/E (blue, n = 4 cells) 3T3-F442A preadipocytes. d SN-401/DCPIB chemical structure. e ICl,SWELL inward and outward current over time upon hypotonic (210 mOsm) stimulation and subsequent inhibition by 10 μM SN-401 in a HEK-293 cell. f, g Western blots detecting SWELL1, pAKT2Ser474, and β-actin in WT 3T3-F442A preadipocytes treated with either vehicle or SN-401 (10 µM) for 96 h (f) (n = 2 each) and the corresponding densitometric ratio (g). h Western blots detecting SWELL1 and β-actin in WT 3T3-F442A adipocytes treated with either vehicle or SN-401 (10 µM) for 96 h (n = 6 each) and the corresponding densitometric ratio below. i, j Western blots detecting pAKT2Ser474, AKT2, pAS160Thr642, AS160, and β-actin in WT (n = 9 each) and SWELL1 KO (n = 3 each) 3T3-F442A adipocytes treated with either vehicle or SN-401 (500 nM) for 96 h and stimulated with 0 and 10 nM insulin for 15 min (i) and the corresponding densitometric ratio for pAKT2/β-actin and pAS160/β-actin respectively (j). Data were represented as mean ± SEM. Two-tailed unpaired t-test was used in b, c, g, h, and j where *, **, and *** represent p values of <0.05, <0.01, and <0.001, respectively. ns difference did not exceed the threshold for significance.

Fig 3: ApoA-I phosphorylates IRβ, IRS1, Akt and AS160 in HSKMCs. HSKMCs were pre-incubated at 37 °C for 16 h in serum-free MEM-α with or without lipid-free apoA-I then incubated with insulin as described in the legend to Fig. 1. The cells were lysed, subjected to SDS-PAGE and immunoblotted for (A) total and phosphorylated IRβ, (B) total and phosphorylated IRS-1, (C) total and phosphorylated Akt, (D) total and phosphorylated AS160 using β-actin as the loading control. Values represent the mean ± SD of three independent experiments.*p < 0.05, **p < 0.01, ***p < 0.001 vs. control (open bars).

Fig 4: CDN1163 showed no changes in TBC1D4Thr642 phosphorylation. Human myotubes were pretreated with 0.1 ​μM CDN1163 for 5 days. Prior to harvesting, half of the samples were treated with 100 ​nM insulin. Proteins were isolated and expressions of total TBC1D4 and TBC1D4 phosphorylated at Thr642 were assessed by immunoblotting. Expression of pTBC1D4 was normalised to total TBC1D4 expression. Data are presented as mean ​± ​SEM from eight individual experiments (n ​= ​8). ∗Statistically significant compared to basal (unstimulated) cells (p ​< ​0.05, unpaired t-test).

Fig 5: BMP signaling in adipocytes upregulates total levels of GLUT4, but does not interfere with insulin-stimulated GLUT4 translocation to the plasma membrane. (a) 3T3-L1 adipocytes were stimulated with PBS, BMP2 or BMP6 and analyzed after 4 h, 10 h and 24 h of stimulation for Glut4 expression via qRT-PCR. Data are means + SEM of 4 independent experiments with triplicate measurements each. Statistical significance was calculated using 1-way ANOVA and Bonferroni post-hoc test. (b,c) Protein level of GLUT4 as determined via intracellular immunostaining and flow cytometric analysis gated on mature adipocytes. Representative histograms. (d,e) GLUT4myc7-GFP overexpressing 3T3-L1 adipocytes36 were used to monitor GLUT4 translocation to the cell surface. Cells were stimulated with control or insulin in combination with BMP2 or BMP6 for 30 minutes as indicated. (e) Data are means of multiple flow cytometric measurements (MFI) as fold induction of control + SEM, n = 3. (f) Analysis of AS160 phosphorylation as a critical step upstream of GLUT4-storage vesicle (GSV) fusion with the plasma membrane. Mature 3T3-L1 cells were stimulated with PBS (−), BMP2 (B2), BMP6 (B6), Insulin (I) or Rosiglitazone (R) for 30 minutes. Lysates were analyzed by immunoblotting with indicated antibodies, β-Actin served as a loading control. MFI = Mean Fluorescence Intensity, MNE = Mean Normalized Expression.