Supplemental Data

Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Effect of aldosterone on protein amount of Glut1 and Glut4.
  3T3-L1 adipocytes were treated with 10^-8 M to 10^-5 M aldosterone for 16 h and then stimulated with 17 nM insulin for 5 min. Cell lysates were subjected to SDS-PAGE, and immunoblotted with anti-Glut1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) or anti-Glut4 (Abcam Japan, Tokyo, Japan) antibody.
• Supplemental Figure 2 - Effect of aldosterone on Ser³⁰⁷ phosphorylations of IRS1.
  (A) 3T3-L1 adipocytes were treated with 10^-8 M to 10^-5 M aldosterone for 16 h and then stimulated with 17 nM insulin for 5 min. Cell lysates were subjected to SDS-PAGE, and immunoblotted with anti-Ser³⁰⁷ phospho-specific IRS1 antibody. (B) Cells were preincubated with 10 μ M BMS345541 (BMS, IKKβ inhibitor) or 20 nM rapamycin (Rapa, mTORC1 inhibitor) for 30 min, and treated with 10^-5 M aldosterone for 2 h. Cell lysates were subjected to SDS-PAGE, and immunoblotted with anti-Ser³⁰⁷ phospho-specific IRS1 antibody. Results are means ± SE of three separate experiments. *p<0.05 versus untreated cells by one-way ANOVA with Bonferroni test.
• Supplemental Figure 3 - Aldosterone phosphorylates NFκ B and S6 kinase.
  3T3-L1 adipocytes were preincubated with 20 μ M SP600125 (SP, JNK inhibitor), 100 nM Calphostin C (CalC, PKC inhibitor), 10 μ M BMS345541 (BMS, IKKβ inhibitor), or 20 nM rapamycin (Rapa, mTORC1 inhibitor) for 30 min. Cells were then treated with 10^-5 M aldosterone for 2 h. Cell lysates were subjected to SDS-PAGE, and immunoblotted with (A) anti-Thr¹⁸³/Tyr¹⁸³ phospho-specific SAPK/JNK antibody, (B) anti-phospho-specific pan PKC (β II Ser⁶⁶⁰) antibody, (C) anti-Ser⁵³⁶ phospho-specific NFκ B antibody, and (D) anti-Ser^235/236 phospho-specific S6 kinase antibody. These antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA).
• Supplemental Figure 4 - Effect of IKKβ or S6 kinase knockdown on aldosterone- induced degradation of IRS proteins.
  3T3-L1 adipocytes were transfected with siRNA for IKKβ or S6 kinase knockdown by Nucleofector system (Amaxa Inc, MD). The electroporation was conducted according to the manufacture’s instruction. In brief, differentiated 3T3-L1 adipocytes were scraped with 0.25% trypsin and 0.5 mg/ml collagenase. After washing with PBS, cells were mixed with Nucreofector solution and 1 μ g siRNA. Then electroporation was conducted. The siRNA sense sequences for IKKβ was 5’-GCCAAGGAGGAGAUCUCCGAAGAUA-3’. The siRNA sense sequences for S6 kinase was 5’-GGACAUGGCAGGAGUGUUU-3’. Stealth RNAi negative control medium GC duplex (Invitrogen) was used as control scramble (Scr) siRNA. After incubation for 24 h, cells were treated with 10^-5 M aldosterone for 16 h, and subjected to SDS-PAGE and immunoblotted with indicated antibodies.
• Supplemental Figure 5 - Effect of proteasomal inhibitors on aldosterone-induced degradation of IRS1 and IRS2.
  3T3-L1 adipocytes were pretreated with 10 μ M lactacystin or 4 nM MG132 (Merck Ltd., Tokyo, Japan) for 30 min, treated with 10^-8 M to 10^-5 M aldosterone for 16 h, and then stimulated with 17 nM insulin for 5 min. Cell lysates were subjected to SDS-PAGE, and immunoblotted with (A, C) anti-IRS1 antibody or (B, D) anti-IRS2 antibody. (A, B) Results are means ± SE of three separate experiments. *p<0.05 versus untreated cells by one-way ANOVA with Bonferroni test.
• Supplemental Figure 6 - Putative mechanisms underlying the aldosterone-induced insulin resistance in 3T3-L1 adipocytes.
  Aldosterone stimulates production of ROS through GR but not MR. ROS activates IKKβ and TORC1, both of which phosphorylate serine residues on IRS1 in LDM fraction. Serine-phosphorylated IRS1 is translocated to proteasome in cytosol, and degraded subsequently. Since IRS1 located in LDM fraction is essential for transmitting insulin’s metabolic signals leading to Glut4 translocation to the plasma membrane, the reduction of IRS1 at LDM should be crucial for the pathogenesis of aldosterone-induced insulin resistance in 3T3-L1 adipocytes.