Figure 1.
Time- and dose-dependent activation of Akt by AGEs in 3T3-L1 cells.
(A) Serum-starved quiescent 3T3-L1 cells were exposed to AGEs (100 µg/ml) for the indicated times. (B) Serum-starved quiescent 3T3-L1 cells were exposed to various concentrations of AGEs for 15 min. (C) Serum-starved quiescent 3T3-L1 cells were pretreated with 15 µM LY294002 for 30 min, and then exposed to 100 µg/ml AGEs for 15 min. Total cell lysates were immunoblotted with antibodies recognizing phospho-Akt (Aktser473), Akt or actin. (D) Serum-starved quiescent 3T3-L1 cells were pretreated with 15 µM LY294002 for 30 min, and then exposed to 100 µg/ml AGEs for 15 min. Total cell lysates were immunoblotted with antibodies recognizing phospho-Akt (AktThr308), Akt or actin. (E) Serum-starved quiescent 3T3-L1 cells were pretreated with 15 µM LY294002 for 30 min, and then exposed to 100 µg/ml AGEs for 15 min. Total cell lysates were immunoblotted with antibodies recognizing phospho-PDK1 (PDK1Ser241), PDK1 or actin. Data are representative of three independent experiments yielding similar results. *, statistically significant differences (*, P <0.05 versus control).
Figure 2.
The effect of NAC, Tiron, DPI and apocynin on AGEs-stimulated Akt activation in 3T3-L1 cells.
(A) Cells were exposed to 100 µg/ml AGEs for 15 min in the serum-free medium. After treatment, cells were incubated with 20 µM CM-H2DCFDA for 30 min at 37°C. The ROS production was determined by a fluorescence reader (excitation/emission: 485/520 nm). The data represent mean ± the standard error (SE) of results from three independent experiments. Increases in the AGEs-induced ROS were statistically significant at this time point. (*: P<0.05) (B) After 18 hr serum starvation, 3T3-L1 cells were treated with 2 mM NAC, 2 mM Tiron, 50 µM DPI, 25 µM apocynin, or RAGE antibodies (1 µg/ml) for 60 min, and then ROS production was determined by a fluorescence reader (excitation/emission: 485/520 nm). After 18 hr serum starvation, 3T3-L1 cells were treated with 2 mM NAC (C), 2 mM Tiron (D), 50 µ M DPI (E), or 25 µM apocynin (F) for 60 min, and then challenged with 100 µg/ml AGEs for 15 min. Cell lysates were immunoblotted with antibodies specific for phospho-Akt (Aktser473), Akt or actin. Data are representative of three independent experiments yielding similar results.
Figure 3.
Involvement of Src and RAGE in AGEs-stimulated Akt activation in 3T3-L1 cells.
(A): Serum-starved quiescent 3T3-L1 cells were pretreated with and without 10 µM PP2 for 30 min, and then challenged with 100 µg/ml AGEs for 15 min. Total cell lysates were immunoblotted with antibodies specific for phospho-Akt (Aktser473), Akt or actin. Serum-depleted cells were pretreated with and without 10 µM PP2 for 30 min (B), 2 mM NAC (C) or RAGE antibodies (1 µg/ml) (D) for 60 min, and then challenged with 100 µg/ml AGEs for 15 min. Total cell lysates were immunoblotted with antibodies specific for phospho-Src (SrcTyr416), total Src (Src), Akt, phospho-Akt (Aktser473) or actin. Data are representative of three independent experiments yielding similar results.
Figure 4.
Transactivation of IGF-1R by AGEs in 3T3-L1 cells.
Serum-depleted 3T3-L1 cells were stimulated with 100 µg/ml AGEs for 15 min, and cell lysates were immunoprecipitated with IGF-1R (A) or IRS-1 (B) antibodies followed by Western blotting with phospho-tyrosine, phospho-IGF-1Rβ(IGF-1RβpY1135/1136), phospho-IGF-1R (IGF-1RβpY1131), total IGF-1Rβ (IGF-1Rβ), IRS-1 or actin antibodies. Serum-depleted 3T3-L1 cells were pretreated with and without 2 µM AG1024 for 30 min and then challenged with 100 µg/ml AGEs for 15 min. Cell lysates were immunoprecipitated with IGF-1Rβsubunit (IGF-1β) antibody followed by immunoblotting with phospho-IGF-1Rβ (IGF-1RβpY1135/1136) and IGF-1Rβ(C). Total cell lysates were also immunoblotted with antibodies specific for phospho-Akt (Aktser473) or Akt (D). Data are representative of three independent experiments yielding similar results.
Figure 5.
Transactivation of IGF-1R by AGEs is mediated by Src in 3T3-L1 cells.
(A) Serum-depleted 3T3-L1 cells were pretreated with and without 10 µM PP2 for 30 min and then challenged with 100 µg/ml AGEs for 15 min. Cell lysates were immunoprecipitated with IGF-1Rβantibody followed by immunoblotting with phospho-tyrosine, IGF-1Rβ or phospho-IGF-1Rβ (IGF-1RβpY1135/1136) antibodies. (B) Total lysates from cells treated with and without 2 µM AG1024 and then challenged with 100 µg/ml AGEs for 15 min were subjected to Western blotting with antibodies specific for phospho-Src (SrcTyr416), Src or actin antibodies. (C) Total lysates from cells treated with and without 100 µg/ml AGEs were immunoprecipitated with the IGF-1Rβantibody followed by immunoblotting with IGF-1Rβ, Src and p-Src antibodies. (D) Serum-starved 3T3-L1 cells were pretreated with and without 2 mM NAC for 60 min and then challenged with 100 µg/ml AGEs for 15 min. Cell lysates were immunoprecipitated with IGF-1Rβantibody followed by immunoblotting with phospho-tyrosine or phospho-IGF-1Rβ (IGF-1RβpY1135/1136) antibodies. The data represent mean ± the standard error (SE) of results from three independent experiments. The densitometrical data were shown as the means ± SEM of three independent experiments. *P<0.05 compared with control group and **P<0.05 compared with AGEs group.
Figure 6.
Involvement of phospho-Cav-1 in AGEs-mediated Akt activation in 3T3-L1 cells.
(A) Serum-depleted 3T3-L1 cells were pretreated with and without 50 µM β-MCD for 60 min and then challenged with 100 µg/ml AGEs for 15 min. Total cell lysates were immunoblotted with antibodies specific for phospho-Akt (Aktser473), Akt or actin. Data are representative of three independent experiments yielding similar results. (B) Serum-depleted 3T3-L1 cells were pretreated with and without 20 µM β-MCD for 60 min and then challenged with 100 µg/ml AGEs for 15 min. Cell lysates were subjected to Western blotting with antibodies specific for phospho-Src (SrcTyr416), total Src (Src) or actin antibodies. (C) Serum-depleted 3T3-L1 cells were pretreated with and without 50 µM β-MCD for 60 min and then challenged with 100 µg/ml AGEs for 15 min. After treatment, cells were incubated with 20 µM CM-H2DCFDA for 30 min at 37°C. The ROS production was determined by a fluorescence reader (excitation/emission: 485/520 nm). The data represent mean ± the standard error (SE) of results from three independent experiments. (D) Serum-depleted 3T3-L1 cells were stimulated with 100 µg/ml AGEs for 15 min, and cell lysates were immunoprecipitated with IGF-1Rβ antibody followed by immunoblotting with phospho-Cav-1 (Cav-1 tyr14), Cav-1 or IGF-1Rβ. (E) Cell lysates from vector control and cells expressing Cav-1 Y14F were subjected to Western blotting with phospho-Cav-1 (Cav-1 tyr14), Cav-1, phospho-Akt or Akt. (F) Cell lysates from vector control and cells expressing Cav-1 Y14F were subjected to Western blotting with phospho-Src (SrcTyr416), Src or actin antibodies. Cell lysates were also immunoprecipitated with IGF-1Rβantibody followed by immunoblotting with phospho-IGF-1Rβ (IGF-1RβpY1135/1136) or IGF-1Rβ. (G) Serum-depleted cells were pretreated with and without 10 µM PP2 for 30 min and then challenged with 100 µg/ml AGEs for 15 min. Total cell lysates were immunoblotted with antibodies specific for phospho-caveolin-1 (Cav-1 tyr14), Cav-1 or actin. Data are representative of three independent experiments yielding similar results. *, statistically significant differences (* and **, P<0.05 versus control).
Figure 7.
Promotion of adipogenesis of 3T3-L1 cells by AGEs-treatment.
(A) 3T3-L1 cells were treated with and without 100 µg/ml AGEs during differentiation.The adipogenic induction medium contains 0.5 mM isobutylmethylxanthine, 1 mM dexamethasone, and 1.7 mM insulin. At day 12, cells were subjected to the Oil Red O staining. The oil droplet contents were quantified by measuring the OD510 nm. (*, P<0.05 versus control) (B) 3T3-L1 cells were treated with and without 100 µg/ml AGEs during differentiation. At day 5, cell lysates were subjected to Western blot analysis using the PPARγ, C/EBPα, aP2 and the loading control actin antibodies. (C) Cells from 3T3-L1 and 100 µg/ml AGEs-treated cells were harvested on day 8 after adipogenic induction and were subjected to GPDH activity assay by reading the absorbance of NADH at 340 nm. The results are the means ± SEM of three independent experiments. (*, P<0.05 vs. control) (D) 3T3-L1 cells were treated with and without 100 µg/ml AGEs in the presence of and absence of 10 µM AG1024, 15 µM LY294002, or 5 µM Akt inhibitor during differentiation. Four days after adipogenic induction, cells were subjected to the Oil Red O staining and the oil droplet contents were quantified by measuring the OD510 nm. (E) 3T3-L1 cells were treated with and without 100 µg/ml AGEs in the presence of and absence of 10 µM AG1024, 15 µM LY294002, or 5 µM Akt inhibitor during differentiation. The concentration of DMSO is 0.05% for control and inhibitor-treated groups. At day 5, cell lysates were subjected to Western blot analysis using PPARγ, aP2 and actin antibodies. (*, P<0.05 DMSO versus inhibitors; **, P<0.05 DMSO vs. DMSO+AGEs, ***, P<0.05 DMSO+AGEs vs. inhibitors+AGEs).
Figure 8.
A proposed model for the activation of Akt by AGEs in 3T3-L1 cells.
AGEs, via RAGE, activate NAD(P)H oxidase and produce reactive oxygen species (ROS) which then stimulate Src kinase activity. Src subsequently phosphorylates and activates IGF-1 receptor which acts as a stimulator for PI3-kinase, PDK-1 and Akt. Src also phosphorylates caveolin-1 and enhances IGF-1 receptor-mediated signaling.