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p21(WAF1/CIP1) Expression is Differentially Regulated by Metformin and Rapamycin.

Molnar Z, Millward AB, Tse W, Demaine AG - Int J Chronic Dis (2014)

Bottom Line: Here we investigated the effect of metformin and rapamycin on mTOR-related phenotypes in cell lines of epithelial origin.These effects were associated with reduced AMPK activation, affecting downstream mTOR signalling.However, the inhibition of the mTOR pathway by rapamycin did not have a negative effect on p21 expression, suggesting that metformin regulates p21 upstream of mTOR.

View Article: PubMed Central - PubMed

Affiliation: Renal Unit and Diabetes Clinical Research Unit, Derriford Hospital, Plymouth, PL6 8DH, UK.

ABSTRACT
The mammalian target of rapamycin (mTOR) pathway plays an important role in the development of diabetic nephropathy and other age-related diseases. One of the features of DN is the elevated expression of p21(WAF1/CIP1). However, the importance of the mTOR signalling pathway in p21 regulation is poorly understood. Here we investigated the effect of metformin and rapamycin on mTOR-related phenotypes in cell lines of epithelial origin. This study reports that metformin inhibits high glucose-induced p21 expression. High glucose opposed metformin in regulating cell size, proliferation, and protein synthesis. These effects were associated with reduced AMPK activation, affecting downstream mTOR signalling. However, the inhibition of the mTOR pathway by rapamycin did not have a negative effect on p21 expression, suggesting that metformin regulates p21 upstream of mTOR. These findings provide support for the hypothesis that AMPK activation may regulate p21 expression, which may have implications for diabetic nephropathy and other age-related pathologies.

No MeSH data available.


Related in: MedlinePlus

Western blot results indicate that AMPK inhibition is associated with the reversal of metformin-induced p21 downregulation in HEK293 cells. In these experiments, the protein level of P-S6KThr389, cyclin D1, P-AMPKα12Thr172, and P-mTORSer2448 was measured in order to confirm the expected effects of treatments. mTOR and β-actin were used to control equal protein loading. (a) The cells were treated with culture medium containing 5.5 mM D-glucose. Mannitol (Man) and D-glucose (HG) were added for three days at the indicated concentrations (mM). The cells were treated with metformin (Met) for 24 h at the indicated concentrations (mM). (b) Compound C (Cc) reverses metformin-induced p21 downregulation. The cells were treated with Met and Cc for 24 h at the indicated concentrations (mM and μM, resp.). (c) HEK293 cells stably transfected with shRNA expression plasmids targeting AMPKα2 (KD) or nonsilencing (NS) were cultured with or without 8 mM metformin for 18 h in whole cell culture medium. (d) The proteasome inhibitor carbobenzoxy-Leu-Leu-leucinal (MG132) prevents metformin-induced (Met) downregulation of p21. HEK293 cells stably transfected with shRNA expression plasmids targeting AMPKα2 (KD) or nonsilencing (NS) were cultured with or without 8 mM metformin and 10 μM MG132 overnight in whole cell culture medium. Vehicle effects were controlled by adding 0.05% DMSO to the conditions.
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fig6: Western blot results indicate that AMPK inhibition is associated with the reversal of metformin-induced p21 downregulation in HEK293 cells. In these experiments, the protein level of P-S6KThr389, cyclin D1, P-AMPKα12Thr172, and P-mTORSer2448 was measured in order to confirm the expected effects of treatments. mTOR and β-actin were used to control equal protein loading. (a) The cells were treated with culture medium containing 5.5 mM D-glucose. Mannitol (Man) and D-glucose (HG) were added for three days at the indicated concentrations (mM). The cells were treated with metformin (Met) for 24 h at the indicated concentrations (mM). (b) Compound C (Cc) reverses metformin-induced p21 downregulation. The cells were treated with Met and Cc for 24 h at the indicated concentrations (mM and μM, resp.). (c) HEK293 cells stably transfected with shRNA expression plasmids targeting AMPKα2 (KD) or nonsilencing (NS) were cultured with or without 8 mM metformin for 18 h in whole cell culture medium. (d) The proteasome inhibitor carbobenzoxy-Leu-Leu-leucinal (MG132) prevents metformin-induced (Met) downregulation of p21. HEK293 cells stably transfected with shRNA expression plasmids targeting AMPKα2 (KD) or nonsilencing (NS) were cultured with or without 8 mM metformin and 10 μM MG132 overnight in whole cell culture medium. Vehicle effects were controlled by adding 0.05% DMSO to the conditions.

Mentions: The expression of p21 was investigated in the context of AMPK/mTOR/S6K signalling by western blotting. Metformin treatment caused a decrease in p21 expression in a dose-dependent manner (Figure 6(a)). The expression level of phosphorylated S6K and cyclin D1 was also investigated. As expected, metformin caused downregulation of both P-S6K and cyclin D1 with more pronounced effects in the 5–8 mM concentration range. High glucose treatments (15–30 mM) increased the expression of p21, cyclin D1, and P-S6K and at 25 mM concentration blunted the inhibitory effects of metformin. Similar to high glucose, compound C had an opposing effect of metformin-induced AMPK activation, S6K dephosphorylation, and p21 downregulation (Figure 6(b)). The inhibitory effect of metformin on p21 expression was also confirmed in HEK293 cells stably expressing ShRNA against AMPKα2 (Figure 6(c)). In AMPKα2-deficient cells, metformin-induced AMPK activation was reduced. Correspondingly, reciprocal changes in mTOR phosphorylation could also be observed. Metformin treatment reduced p21 expression in the nonsilencing control condition. In contrast, in the AMPKα2 knockdown condition the inhibitory effect of metformin on p21 expression was less pronounced. The expression of p21 can be regulated by the proteasome and recently it has been suggested that AMPK activation may inhibit the function of the proteasome [46–48]. To investigate whether metformin-induced downregulation of p21 is proteasome-dependent, the proteasome inhibitor, carbobenzoxy-Leu-Leu-leucinal (MG132), was used in control and AMPKα2-deficient cells. In both control and knockdown cells, the downregulation of p21 was prevented by MG132 treatments (Figure 6(d)). In order to confirm the above western blot results and obtain information about changes in p21 localisation, immunofluorescence microscopy was performed on metformin and high glucose treated HEK293 cells. As expected, metformin treatment decreased the expression of p21 and high glucose treatment reversed metformin-induced p21 downregulation (Figure 7). In addition, high glucose treatment enhanced the nuclear compartmentalisation of p21.


p21(WAF1/CIP1) Expression is Differentially Regulated by Metformin and Rapamycin.

Molnar Z, Millward AB, Tse W, Demaine AG - Int J Chronic Dis (2014)

Western blot results indicate that AMPK inhibition is associated with the reversal of metformin-induced p21 downregulation in HEK293 cells. In these experiments, the protein level of P-S6KThr389, cyclin D1, P-AMPKα12Thr172, and P-mTORSer2448 was measured in order to confirm the expected effects of treatments. mTOR and β-actin were used to control equal protein loading. (a) The cells were treated with culture medium containing 5.5 mM D-glucose. Mannitol (Man) and D-glucose (HG) were added for three days at the indicated concentrations (mM). The cells were treated with metformin (Met) for 24 h at the indicated concentrations (mM). (b) Compound C (Cc) reverses metformin-induced p21 downregulation. The cells were treated with Met and Cc for 24 h at the indicated concentrations (mM and μM, resp.). (c) HEK293 cells stably transfected with shRNA expression plasmids targeting AMPKα2 (KD) or nonsilencing (NS) were cultured with or without 8 mM metformin for 18 h in whole cell culture medium. (d) The proteasome inhibitor carbobenzoxy-Leu-Leu-leucinal (MG132) prevents metformin-induced (Met) downregulation of p21. HEK293 cells stably transfected with shRNA expression plasmids targeting AMPKα2 (KD) or nonsilencing (NS) were cultured with or without 8 mM metformin and 10 μM MG132 overnight in whole cell culture medium. Vehicle effects were controlled by adding 0.05% DMSO to the conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig6: Western blot results indicate that AMPK inhibition is associated with the reversal of metformin-induced p21 downregulation in HEK293 cells. In these experiments, the protein level of P-S6KThr389, cyclin D1, P-AMPKα12Thr172, and P-mTORSer2448 was measured in order to confirm the expected effects of treatments. mTOR and β-actin were used to control equal protein loading. (a) The cells were treated with culture medium containing 5.5 mM D-glucose. Mannitol (Man) and D-glucose (HG) were added for three days at the indicated concentrations (mM). The cells were treated with metformin (Met) for 24 h at the indicated concentrations (mM). (b) Compound C (Cc) reverses metformin-induced p21 downregulation. The cells were treated with Met and Cc for 24 h at the indicated concentrations (mM and μM, resp.). (c) HEK293 cells stably transfected with shRNA expression plasmids targeting AMPKα2 (KD) or nonsilencing (NS) were cultured with or without 8 mM metformin for 18 h in whole cell culture medium. (d) The proteasome inhibitor carbobenzoxy-Leu-Leu-leucinal (MG132) prevents metformin-induced (Met) downregulation of p21. HEK293 cells stably transfected with shRNA expression plasmids targeting AMPKα2 (KD) or nonsilencing (NS) were cultured with or without 8 mM metformin and 10 μM MG132 overnight in whole cell culture medium. Vehicle effects were controlled by adding 0.05% DMSO to the conditions.
Mentions: The expression of p21 was investigated in the context of AMPK/mTOR/S6K signalling by western blotting. Metformin treatment caused a decrease in p21 expression in a dose-dependent manner (Figure 6(a)). The expression level of phosphorylated S6K and cyclin D1 was also investigated. As expected, metformin caused downregulation of both P-S6K and cyclin D1 with more pronounced effects in the 5–8 mM concentration range. High glucose treatments (15–30 mM) increased the expression of p21, cyclin D1, and P-S6K and at 25 mM concentration blunted the inhibitory effects of metformin. Similar to high glucose, compound C had an opposing effect of metformin-induced AMPK activation, S6K dephosphorylation, and p21 downregulation (Figure 6(b)). The inhibitory effect of metformin on p21 expression was also confirmed in HEK293 cells stably expressing ShRNA against AMPKα2 (Figure 6(c)). In AMPKα2-deficient cells, metformin-induced AMPK activation was reduced. Correspondingly, reciprocal changes in mTOR phosphorylation could also be observed. Metformin treatment reduced p21 expression in the nonsilencing control condition. In contrast, in the AMPKα2 knockdown condition the inhibitory effect of metformin on p21 expression was less pronounced. The expression of p21 can be regulated by the proteasome and recently it has been suggested that AMPK activation may inhibit the function of the proteasome [46–48]. To investigate whether metformin-induced downregulation of p21 is proteasome-dependent, the proteasome inhibitor, carbobenzoxy-Leu-Leu-leucinal (MG132), was used in control and AMPKα2-deficient cells. In both control and knockdown cells, the downregulation of p21 was prevented by MG132 treatments (Figure 6(d)). In order to confirm the above western blot results and obtain information about changes in p21 localisation, immunofluorescence microscopy was performed on metformin and high glucose treated HEK293 cells. As expected, metformin treatment decreased the expression of p21 and high glucose treatment reversed metformin-induced p21 downregulation (Figure 7). In addition, high glucose treatment enhanced the nuclear compartmentalisation of p21.

Bottom Line: Here we investigated the effect of metformin and rapamycin on mTOR-related phenotypes in cell lines of epithelial origin.These effects were associated with reduced AMPK activation, affecting downstream mTOR signalling.However, the inhibition of the mTOR pathway by rapamycin did not have a negative effect on p21 expression, suggesting that metformin regulates p21 upstream of mTOR.

View Article: PubMed Central - PubMed

Affiliation: Renal Unit and Diabetes Clinical Research Unit, Derriford Hospital, Plymouth, PL6 8DH, UK.

ABSTRACT
The mammalian target of rapamycin (mTOR) pathway plays an important role in the development of diabetic nephropathy and other age-related diseases. One of the features of DN is the elevated expression of p21(WAF1/CIP1). However, the importance of the mTOR signalling pathway in p21 regulation is poorly understood. Here we investigated the effect of metformin and rapamycin on mTOR-related phenotypes in cell lines of epithelial origin. This study reports that metformin inhibits high glucose-induced p21 expression. High glucose opposed metformin in regulating cell size, proliferation, and protein synthesis. These effects were associated with reduced AMPK activation, affecting downstream mTOR signalling. However, the inhibition of the mTOR pathway by rapamycin did not have a negative effect on p21 expression, suggesting that metformin regulates p21 upstream of mTOR. These findings provide support for the hypothesis that AMPK activation may regulate p21 expression, which may have implications for diabetic nephropathy and other age-related pathologies.

No MeSH data available.


Related in: MedlinePlus