AMP-activated protein kinase inhibits IGF-I signaling and protein synthesis in vascular smooth muscle cells via stimulation of insulin receptor substrate 1 S794 and tuberous sclerosis 2 S1345 phosphorylation

Abstract

AMP-activated protein kinase (AMPK) inhibits IGF-I actions, but the mechanism by which AMPK functions is undefined. This study identified signaling events that were induced by AMPK that mediated inhibition of IGF-I-stimulated phosphoinosotide-3-kinase (PI3K) pathway activation. The AMPK activator metformin stimulated AMPK Thr172 phosphorylation and inhibited IGF-I-stimulated phosphorylation of Akt/tuberous sclerosis 2 (TSC2)/mammalian target of rapamycin (mTOR)/p70S6 kinase (p70S6K). Expression of constitutively active forms of AMPK suppressed IGF-I-stimulated activation of Akt/TSC2/mTOR/p70S6K and protein synthesis, whereas AMPK knockdown resulted in enhanced responses to IGF-I. To determine the mechanism by which AMPK inhibited IGF-I signaling, the role of insulin receptor substrate-1 (IRS-1) was examined. Both metformin and constitutively activated AMPK enhanced phosphorylation of IRS-1 Ser794, which led to decreased IRS-1 tyrosine phosphorylation and recruitment of the p85 subunit of PI3K. Overexpression of IRS-1 S794A was associated with increased IGF-I-stimulated IRS-1 tyrosine phosphorylation, p85 association, and protein synthesis. To determine whether other signaling molecules mediated the effect of AMPK, TSC2 function was examined. Cells overexpressing TSC2/S1345A (the site of AMPK phosphorylation) were less responsive to metformin-induced inhibition of p70S6 kinase. These findings are relevant to whole animal physiology because administration of metformin to mice resulted in inhibition of IGF-I-stimulated phosphorylation of Akt/mTOR/p70S6K. In conclusion, AMPK functions to inhibit IGF-I-stimulated PI3K pathway activation through stimulation of IRS-1 serine 794 phosphorylation. Because IGF-I is an important stimulant of the anabolic response, this effect of AMPK could account for part of its inhibitory effect on protein synthesis, thus allowing more efficient energy use by other cellular processes.

Figure 1

AMPK suppresses IGF-I-stimulated Akt/TSC2/p70S6K phosphorylation. pSMC were grown to confluency in DMEM-NG growth medium. A and B, The cells were incubated in serum-free medium for 40 h. Metformin (Met) was added for the last 18 h at the concentration indicated followed by IGF-I (10 ng/ml) for 10 min. The cell lysates were immunoblotted with anti-phospho- (p-)AMPK (T172), anti-AMPK, anti-p-Akt(S473), anti-Akt, anti-p-TSC2(S939), anti-TSC2, anti-p-mTOR(S2448), anti-mTOR, anti-p-p70S6K(T389), and anti-p70S6K antibodies, respectively. The bar graphs show the relative differences in protein phosphorylation wherein the scanning densitometry value for each phosphoprotein is divided by the corresponding value for total protein. Each experiment was repeated at least three times, and the bars represent mean ± sem. P < 0.05 indicates a significant difference.

Figure 2

Constitutively active AMPK suppresses IGF-I signaling. pSMC were transduced with pLenti-LacZ (control), pLenti-AMPK (1-312), or pLenti-AMPK(1-312)T172D, respectively (both are constitutively active forms of AMPK). A and B, Cells were serum deprived for 18 h and stimulated with IGF-I (10 ng/ml) for 10 min. The cell lysates were immunoblotted with anti-phospho- (p-)Akt(S473), anti-Akt, anti-p-TSC2(S939), anti-TSC2, anti-p-mTOR(S2448), anti-mTOR, anti-p-p70S6K(T389), anti-p70S6K, anti-HA, and anti-β-actin antibodies, respectively. The bar graphs show the relative differences in protein phosphorylation wherein the band intensity of each phosphoprotein is divided by the corresponding total protein band intensity. The error bars represent mean ± sem. P < 0.05 indicates a significant difference.

Figure 3

Knockdown of AMPK enhances IGF-I signaling. pSMC were transduced with empty vector (Si-EVT) or an AMPK shRNA template. A and B, The cell line with highest efficiency of AMPK knockdown (Si-AMPK3) was selected (Supplemental Fig. 3, B and C) to analyze the effect of metformin on IGF-I stimulation of Akt and downstream signaling. Si-AMPK3-expressing cells were serum deprived for 40 h and metformin (5 mm) added for the last 18 h before IGF-I (10 ng/ml) was added for 10 min. The cell lysates were immunoblotted with anti-phospho- (p-)AMPK(T172), anti-AMPK, anti-p-Akt(S473), anti-Akt, anti-p-TSC2(S939), anti-TSC2, anti-p-mTOR(S2448), anti-mTOR, anti-p-p70S6K(T389), and anti-p70S6K antibodies, respectively. The bar graphs show the relative differences in protein phosphorylation wherein the band intensity of each phosphoprotein is divided by the corresponding total protein band intensity. Error bars represent mean ± sem. P < 0.05 indicates a significant difference.

Figure 4

AMPK phosphorylates IRS-1 Ser794, leading to inhibition of IGF-I-stimulated IRS-1/PI3K/Akt activity. Constitutively active AMPK transduced cells and control cells were serum deprived for 18 h before the addition of IGF-I (10 ng/ml) for 10 min (A and B, lanes 1–8). pSMC were serum deprived for 40 h and exposed to metformin for the last 18 h before IGF-I was added (A and B, lanes 9–12). A, The cell lysates were immunoprecipitated with anti-IGF-IR antibody and immunoblotted with anti-phospho- (p-)Tyr (PY99). To control the loading, the blot was stripped and immunoblotted with an anti-IGF-IR antibody. The association of IRS-1 with the P85α subunit of PI3K was determined by immunoprecipitating cell lysates with an anti-IRS-1 antibody and immunoblotting with an anti-P85α antibody. B, The phosphorylation of IRS-1 at Ser794 and tyrosine phosphorylation of IRS-1 were determined by immunoprecipitating cell lysates with anti-IRS-1 antibody and immunoblotting with anti-p-IRS-1(S794) or anti-p-Tyr (PY99) antibodies, respectively. The bar graphs show the differences in protein phosphorylation wherein scanning densitometry value of each phosphoprotein band is divided by the value for the corresponding control protein, e.g. IGF-IR or IRS-1. The error bars represent mean ± se. P > 0.05 indicates that the difference was not statistically significant. P < 0.05 indicates a significant difference.

Figure 5

Ser794 phosphorylation of IRS-1 inhibits IGF-I-stimulated Akt/TSC2/p70S6K signaling pathway activity. pSMC were transduced with pLenti-IRS-1 WT (control), pLenti-IRS-1 S794A, or pLenti-IRS-1 S794D, respectively. A–C, To analyze the role of IRS-1 S794 in IGF-I signaling, transduced cells were serum deprived for 18 h and stimulated with IGF-I for 10 min. The tyrosine phosphorylation of IRS-1 and the association of IRS-1 with the P85α subunit were determined by immunoprecipitating cell lysates with an anti-IRS-1 antibody and immunoblotting with anti-phospho- (p-)Tyr (PY99) and anti-P85α antibodies, respectively. The cell lysates were immunoblotted with anti-p-Akt(S473), anti-Akt, anti-p-TSC2(S939), anti-TSC2, anti-p-mTOR(S2448), anti-mTOR, anti-p-p70S6K(T389), or anti-p70S6K. D and E, IRS-1 WT or IRS-1 S794A cells were serum starved for 40 h, and metformin (Met) added for the last 18 h before IGF-I was added for 10 min. The cell lysates were immunoblotted with anti-p-AMPK(T172), anti-AMPK, anti-p-Akt(S473), anti-Akt, anti-p-TSC2(S939), anti-p-TSC2, anti-p-mTOR(S2448), anti-mTOR, anti-p-p70S6K(T389), and anti-p70S6K antibodies, respectively. F, Measurement of protein synthesis was conducted in cells transduced with WT IRS-1 and both IRS-1 mutants. The cell protein synthesis responses to IGF-I were compared between IRS-1 WT cells and the IRS-1 mutant cells. Each bar indicates the fold increase over basal and represents the pool of at least three independent experiments. P < 0.01, indicates significant difference when cells expressing IRS-1 S794A or IRS-1 S794D were compared with IRS-1/WT control cells. The bar graphs show the differences in phosphorylated proteins wherein the phosphoprotein band intensity was divided by the corresponding total protein. For the IRS-1 immunoprecipitates, the IRS-1 phosphoprotein or p85α band intensities were divided by the intensity of the IRS-1 band. The error bars represent mean ± se. P < 0.05 indicates a significant difference.

Figure 6

AMPK-dependent phosphorylation site of TSC2 is not involved in AMPK inhibition of Akt activation, but it does modify p70S6K phosphorylation. pSMC were transduced with pLenti-TSC2 WT (control) or pLenti-TSC2 S1345A. A, Transduced cells were serum starved for 40 h, and 3 mm metformin (Met) was added for the last 18 h before IGF-I was added for 10 min. The cell lysates were immunoblotted with anti-phospho- (p-)AMPK(T172), anti-AMPK, anti-p-Acc(S79), anti-p-Akt(S473), anti-Akt, anti-p-mTOR(S2448), anti-mTOR, anti-p-p70S6K(T389), anti-p70S6K, and anti-β-actin antibodies, respectively. B, Transduced cells were serum starved for 18 h, and rapamycin (Rapa, 5 nm) was added for 1 h before IGF-I was added for 10min. The cell lysates were immunoblotted with anti-p-Akt(S473), anti-Akt, anti-p-mTOR(S2448), anti-mTOR, anti-p-p70S6K(T389), or anti-p70S6K antibodies, respectively. The bar graphs show the difference in protein phosphorylation wherein the intensity of each phosphoprotein band was divided by the corresponding total protein band intensity. The error bars represent mean ± sem. ns, Not statistically significant. P < 0.05 indicates a significant difference. P > 0.05 indicates not statistically significant.

Figure 7

AMPK suppresses IGF-I signaling in vivo. The 6- to 8-wk-old male C57/B6 mice were injected ip with metformin (250 mg/kg body weight, diluted in 1× PBS) or 1× PBS (control) daily for 3 consecutive days. Before they were killed, the mice were injected ip with IGF-I (1 mg/kg body weight, diluted in PBS) or PBS (control). After 15 min, they were euthanized. A and B, Aortic extracts were prepared as in Materials and Methods and immunoblotted using anti-phospho- (p-)AMPK(T172), anti-AMPK, anti-p-Akt (S473), anti-Akt, anti-p-mTOR (S2448), anti-mTOR, anti-p-p70S6K(T389), or anti-p70S6K antibody. The bar graph shows the difference in protein phosphorylation in aortic extracts from groups of mice that received different treatments. The phosphoprotein band intensity was divided by the band intensity of the corresponding total protein. Error bars represent mean ± sem. P < 0.05 indicates a significant difference.