Fig 2: ERα 1-280 domain increases IRS1 protein stability and enhances insulin signaling.a Diagram of truncated ERα protein domains. b Effect of ERα on insulin sensitivity in HepG2 cells, n = 3 independent cells; for IRS1, Vehicle versus ERα-Vehicle, P < 0.0001, Insulin versus ERα-Insulin, P = 0.0005; for pAKT-S473, Vehicle versus ERα-Vehicle, P = 0.0025, Vehicle versus Insulin, P < 0.0001, ERα-Vehicle versus ERα-Insulin, P < 0.0001, Insulin versus ERα-Insulin, P = 0.0001; for pAKT-T308, Vehicle versus Insulin, P < 0.0001, ERα-Vehicle versus ERα-Insulin, P < 0.0001, Insulin versus ERα-Insulin, P < 0.0001. c Effect of ERα 1-280 on insulin sensitivity in HepG2 cells, n = 3 independent cells; for IRS1, Vehicle versus ERα 1-280-Vehicle, P < 0.0001, Insulin versus ERα 1-280-Insulin, P = 0.0034; for pAKT-S473, Vehicle versus ERα 1-280-Vehicle, P = 0.0457, Vehicle versus Insulin, P < 0.0001, ERα 1-280-Vehicle versus ERα 1-280-Insulin, P < 0.0001, Insulin versus ERα 1-280-Insulin, P = 0.0007; for pAKT-T308, Vehicle versus Insulin, P = 0.0040, ERα 1-280-Vehicle versus ERα 1-280-Insulin, P = 0.0003, Insulin versus ERα 1-280-Insulin, P = 0.0397. d Effect of ERα DBD + AF2 on insulin sensitivity in HepG2 cells, n = 3 independent cells; for pAKT-S473, Vehicle versus Insulin, P < 0.0001, ERα DBD + AF2-Vehicle versus ERα DBD + AF2-Insulin, P < 0.0001; for pAKT-T308, Vehicle versus Insulin, P = 0.0002, ERα DBD + AF2-Vehicle versus ERα DBD + AF2-Insulin, P = 0.0003. e Interaction between ERα 1-280 and IRS1 protein domains. The experiments were repeated independently twice. Representative blots were shown. f Effect of ERα 1-280 on IRS1 phosphorylation at S302 in HepG2 cells, n = 3 independent cells; for pIRS1-S302, P = 0.0227; for IRS1, P = 0.0187. g Effect of ERα on IRS1 phosphorylation at S302 in HepG2 cells, n = 3 independent cells; for pIRS1-S302, P = 0.0207; for IRS1, P = 0.0154. h Effect of ERα 1-280 on IRS1 ubiquitination in HEK293T cells. The experiments were repeated independently twice. Representative blots were shown. i Effect of ERα 1-280 on IRS2 ubiquitination in HEK293T cells. The experiments were repeated independently twice. Representative blots were shown. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unpaired Two-tailed Student’s t test (f, g), Two-way ANOVA with Tukey’s multiple comparisons test (b, c, d). CNTR: Control. Source data are provided as a Source Data file.

Fig 3: H19 inhibition regulates IRS1 levels in HDAC6 dependent manner and impairs insulin signaling in C2C12 cells. A Differentiated C2C12 cells were transfected with the scramble or H19 siRNA (5 nM) and treated with wither DMSO or the HDAC inhibitor, SAHA (10 µM). On termination of incubation (24 h), cells were lysed and 30 µg protein was resolved on SDS-PAGE and subjected to Western Blot analysis using anti-IRS1 antibody. HSC70 was used as the loading control. The blot presented is a representative blot and given below is the densitometric analysis of three independent experiments. B Differentiated C2C12 cells were transfected with either scramble or HDAC6 siRNA (5–25 nM) and after 48 h of transfection, cells were lysed and 1 μg of total RNA was reverse transcribed and the transcript levels of Hdac6 and Irs1 were detected by qRT-PCR. 18S rRNA was taken as the endogenous control. C Differentiated C2C12 cells were transfected as in “B” and after 48 h of transfection, 20 µg of protein was resolved on SDS-PAGE and probed using anti-HDAC6 and anti-IRS1 antibodies. HSC70 was used as the loading control. The blot presented is a representative figure and given below is the densitometric analysis of three independent experiments. D Differentiated C2C12 cells were transfected with the scramble or H19 siRNA (5 nM) alone or together with HDAC6 siRNA (25 nM). After 48 h of transfection, cells were lysed and 1 μg of total RNA was reverse transcribed and the transcript levels of Irs1 were determined by qRT-PCR. 18S rRNA was taken as the endogenous control. Also, after a similar transfection, 20 µg of protein from scramble or H19 siRNA alone or together with HDAC6 siRNA transfected cells was resolved on SDS-PAGE and probed using anti-IRS1 antibody. HSC70 was used as the loading control. The blot presented is a representative figure and given below is the densitometric analysis of at least five independent experiments. C2C12 cells were transfected with either control vector or HDAC6 cloned vector (0.5 µg and 1 μg) and 48 h post-transfection, cells were lysed and the transcript levels of Hdac6 (E) or Irs1 (F) were determined by qRT-PCR. 18S rRNA was used as the endogenous control. G Differentiated C2C12 cells were transfected with either control vector or HDAC6 overexpression vector (0.5 µg and 1 μg) and 48 h post-transfection, cells were lysed and 20 µg lysate was resolved on SDS-PAGE and probed using anti-HDAC6 and anti-IRS1 antibody. HSC70 was used as the loading control. The blot presented is a representative figure and given below is the densitometric analysis of four independent biological replicates. Differentiated C2C12 cells were transfected with either the scramble or H19 siRNAs and after 48 h of transfection, cells were treated with insulin (100 nM, 20 min). On termination of incubation, cells were lysed and probed for IRS1, pIRS1 (H) and Akt, pAkt (I) protein levels by Western Blot analysis using specific antibodies. Vinculin was taken as the loading control. All experiments were done at least thrice and densitometric analyses of the same are provided. Values are means ± SD. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001

Fig 4: 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 5: (Continued)(D,E) The expression levels of INSR, IRS1, total Akt and insulin-stimulated pAkt (Ser473/Thr308), and plasma membrane Glut4 content, were measured by RT-qPCR and/or WB in both organ culture tissue and isolated adipocytes from VAT samples of normal-weight subjects (n = 4 independent samples per group), under the same normoxic/hypoxic conditions as in (B,C). Representative WBs and mean densitometric analyses of WBs are shown. *P < 0.05 vs normoxia [Student's t-test].