Figure 1.
Reduction of phosphorylated Akt by angiotensin II.
(A) Representative Western blot analysis of phosphorylated Akt (p-Akt), total Akt, phosphorylated ERK1,2 (p-ERK) and total ERK1,2 in extracts from cells incubated 8 h in adipogenic medium without (-) or with (+) 1.5×10−5 M angiotensin II (AII) during the last 2 h. (B) p-Akt/Akt ratios determined from Western blots in the conditions indicated in A and expressed relative to the value in the corresponding controls maintained in adipogenic medium without angiotensin II. The mean pAkt/Akt for the controls in these experiments was 1.56±0.27 (n = 5 donors). (C) The Western blots illustrate phosphorylated Akt (p-Akt), total Akt, phosphorylated ERK1,2 (p-ERK) and total ERK1,2 in extracts from cells maintained in basal culture conditions and treated without (−) or with (+) angiotensin II during 2 h. (D) The p-Akt/Akt ratios were determined from Western blots and normalized with respect to the corresponding controls in the absence of angiotensin II. The mean pAkt/Akt value for the control condition in these experiments was 0.43±0.11 (n = 8 donors). Blots in part A were overexposed to enhance low chemoluminescent signals for phosphoproteins at this time point. The symbol * denotes p<0.05.
Figure 2.
Increased phosphorylated Akt by inhibition of ERK1,2 activation.
(A) Representative Western blots of phosphorylated Akt (p-Akt), total Akt, phosphorylated ERK1,2 (p-ERK), total ERK1,2 and β-actin in extracts from cells incubated for 60 min with 1×10−5 M U0126, 1×10−5 M PD98059 or vehicle. (B) The p-Akt/Akt ratios were determined from Western blots and values were normalized with respect to those determined in control cultures maintained in the basal medium supplemented with vehicle. The mean value for pAkt/Akt and p-ERK1,2/ERK1,2 in the controls were 0.41±0.08 and 1.53±0.30, respectively. The p-Akt/Akt (black bars) increase and the p-ERK1,2/ERK1,2 (white bars) decrease after incubation of cell cultures with the inhibitors of ERK1,2 activation (n = 5 donors). Symbols * and ** denote significant differences with respect to the control condition without inhibitors, at p<0.05 and p<0.01, respectively.
Figure 3.
Increased phosphorylated Akt by ERK1 knockdown.
(A) The Western blots illustrate ERK1, ERK2 and β-actin levels in cell lysates obtained 9 days after infection with lentiviral vectors carrying shRNA specific for ERK1 (shRNA ERK1) or a scrambled sequence (shRNA scr). (B) Representative Western blots of phosphorylated Akt (p-Akt), total Akt, phosphorylated ERK1,2 (p-ERK), total ERK1,2 and β-actin in cell cultures transduced either with ERK1 or scrambled shRNA constructs. When indicated (+), nine days after transduction with shRNA scr, cell cultures were exposed for 2 h to 1×10−5 M U0126 in basal medium. (C) The p-Akt/Akt ratios were determined from Western blots of cells transduced with shRNA scr (white bars) or shRNA ERK1 (black bar), and treated under the experimental conditions specified in part B, (n = 3 donors). The mean pAkt/Akt ratio in the control condition was 0.39±0.14. The symbols * and ** denote significantly different mean values, at p<0.05 and p<0.01, respectively (One-way ANOVA followed by Tukey’s post-hoc test).
Figure 4.
The inhibitor U0126 abolishes angiotensin II-dependent reduction of phosphorylated Akt.
(A) Representative Western blot analysis of phosphorylated and total Akt in extracts from cells treated with 1×10−5 M U0126 for 60 min before exposure to angiotensin II (AII) for additional 60 min. Immunodetection of phosphorylated and total ERK1,2 illustrates p-ERK1,2 decrease after treatment with U0126. β-actin abundance was included as reference. The symbols+and – respectively denote presence or absence of AII or U0126 in the culture medium. (B) The p-Akt/Akt ratios were normalized with respect to the value measured in control cells without AII or U0126 (U). The mean pAkt/Akt value in the control condition was 0.36±0.10 (white bar). Different letters denote significantly different values, p<0.01; one-way ANOVA followed by Tukey’s post-hoc test; n = 4 donors).
Figure 5.
The inhibitor U0126 abolishes angiotensin II-dependent reduction of phosphorylated FoxO1 and FoxO4.
SGBS cells were treated with 1×10−5 M U0126 (U) for 30 min before addition of angiotensin II (AII) and incubated in culture medium with insulin for additional 60 min. The phosphorylated level of ERK1,2 (A), Akt (B), FoxO1 (C) and FoxO4 (D) in cells treated with U plus AII were compared with those determined in cells treated solely with AII or U. The p-ERK1,2/ERK1,2 ratio and the p-Akt/Akt ratio in each experimental condition were expressed relative to the value measured in control cells without AII or U. Mean p-ERK1,2/ERK1,2, p-Akt/Akt values in the control condition were 0.87±0.17 and 0.40±0.12, respectively (white bars in A and B). Due to technical limitations, the p-FoxO1 and p-FoxO4 levels were expressed relative to β-tubulin abundance and normalized versus the corresponding value in the control condition specified above. Mean p-FoxO1/β-tubulin and p-FoxO4/β-tubulin ratios in the control condition were 0.14±0.04 and 0.37±0.07, respectively (white bars in C and D). Different letters denote significantly different values, p<0.05, n = 4 (One-way ANOVA followed by Tukey’s post-hoc test).