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Insulin Increases Sestrin 2 Content by Reducing Its Degradation through the PI 3 K/mTOR Signaling Pathway.

Chai D, Wang G, Zhou Z, Yang H, Yu Z - Int J Endocrinol (2015)

Bottom Line: Sestrin (SESN) is known as a cysteine sulfinic acid reductase.While mechanisms underlying the expression of SESN are not fully understood.Following SESN2 knockdown insulin-stimulated PKB phosphorylation was enhanced and accompanied by reduced PTEN content.

View Article: PubMed Central - PubMed

Affiliation: Fujian Key Laboratory of Chinese Materia Medica, Biomedical Drug Research and Development Center, Fujian University of Traditional Chinese Medicine, Fuzhou 350108, China.

ABSTRACT
Sestrin (SESN) is known as a cysteine sulfinic acid reductase. Recently, nonredox functions of SESN in metabolic regulation and antitumor property have been recognized. While mechanisms underlying the expression of SESN are not fully understood. Here we report that insulin markedly increased SESN2 level in HepG2 cells through mTOR activation. To determine whether insulin affects SESN2 degradation, we assessed SESN2 turnover by applying the protein synthesis inhibitor, cycloheximide (CHX), and found that following insulin treatment SESN2 protein levels were reduced significantly slower than non-insulin-treated cells. Furthermore, the proteasomal inhibitor, MG132, dramatically increased SESN2 protein and its ubiquitination level while in the presence of MG132 insulin did not further increase SESN2 content, suggesting that insulin increases SESN2 content mainly via inhibiting its proteasomal degradation. We then explored the potential feedback role of SESN2 in insulin signaling by SESN2 siRNA knockdown in HepG2 cells. Following SESN2 knockdown insulin-stimulated PKB phosphorylation was enhanced and accompanied by reduced PTEN content. Taken together, our study suggests that insulin upregulates SESN2 content via the PI3K/mTOR signaling pathway and this effect is attributed to decreased SESN2 degradation. Furthermore, SESN2 via modulating PTEN plays a negative feedback role in insulin signaling.

No MeSH data available.


Related in: MedlinePlus

Insulin or the proteasomal inhibitor MG132 blocks SESN2 degradation. (a) HepG2 cells were pretreated with CHX for 30 min followed by a treatment with or without 100 nM of insulin (INS) for the indicated times. The cells were then lysed for SESN2 western blotting. Right panel is the plot of densitometric analysis. *P < 0.05 compared with noninsulin treatment. (b) and (c) HepG2 cells were treated with the indicated reagents for 30 min, followed by a treatment with or without 100 nM insulin for 8 h. The cells were then lysed for SESN2 western blotting. (d) HepG2 cells were incubated with or without insulin or MG132 for 18 h and the cells were lysed. Western blotting of SESN2 and β-actin (loading control) of total cellular proteins (left panel) or SESN2 immunoprecipitation followed by polyubiquitin antibody western blotting (right panel). IP indicates the immunoprecipitation antibody; IB indicates the immunoblotting antibody. Bottom panel of left panel: western blotting of the input samples. A representative blot of three independent experiments.
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fig3: Insulin or the proteasomal inhibitor MG132 blocks SESN2 degradation. (a) HepG2 cells were pretreated with CHX for 30 min followed by a treatment with or without 100 nM of insulin (INS) for the indicated times. The cells were then lysed for SESN2 western blotting. Right panel is the plot of densitometric analysis. *P < 0.05 compared with noninsulin treatment. (b) and (c) HepG2 cells were treated with the indicated reagents for 30 min, followed by a treatment with or without 100 nM insulin for 8 h. The cells were then lysed for SESN2 western blotting. (d) HepG2 cells were incubated with or without insulin or MG132 for 18 h and the cells were lysed. Western blotting of SESN2 and β-actin (loading control) of total cellular proteins (left panel) or SESN2 immunoprecipitation followed by polyubiquitin antibody western blotting (right panel). IP indicates the immunoprecipitation antibody; IB indicates the immunoblotting antibody. Bottom panel of left panel: western blotting of the input samples. A representative blot of three independent experiments.

Mentions: Since insulin treatment rapidly upregulated hepatic SESN2 protein within 1 h, it is unlikely that the stimulation is via the increase of SESN2 protein synthesis. We hence explored whether insulin-stimulated SESN2 expression is due to the blockage of its degradation. For this purpose, HepG2 cells were pretreated with the protein synthesis inhibitor cycloheximide (CHX) to prevent the production of newly synthesized SESN2, before insulin treatment. We observed that, in the presence of insulin, SESN2 disappeared slower than non-insulin-treated cells (Figure 3(a)), indicating that insulin may block or attenuate SESN2 degradation. To further identify which degradation pathway is involved in SESN2 degradation, we examined the potential role of two major cellular protein-degrading systems, that is, proteasome and lysosome on SESN2 turnover. HepG2 cells were pretreated with either the proteasomal inhibitor, MG132, or the lysosomal inhibitor, chloroquine (CQ), for 30 min. The cells were then further treated with or without 100 nM insulin for additional 8 h. We observed that MG132 treatment alone significantly elevated SESN2 level, while insulin did not further increase SESN2 content in MG132 pretreated cells (Figure 3(b)). In contrast, the lysosomal inhibitor CQ did not exert a notable effect on SESN2 level (Figure 3(b)). Additionally, in the presence of the protein synthesis inhibitor cycloheximide, insulin and MG132 were still able to increase SESN2 content, further supporting our notion that insulin blocks proteasome-mediated SESN2 degradation (Figure 3(c)). If SESN2 is ubiquitinated, we could be able to detect upper bands with higher molecular weights of SESN2 due to its covalent binding to ubiquitins. And, indeed, we observed that both insulin and MG132 treatment enhanced the density of upper bands of SESN2 (Figure 3(d), left panel). Finally, we immunoprecipitated SESN2 protein followed with the polyubiquitin antibody detection and observed that SESN2 protein was polyubiquitinated which was enhanced by both insulin and MG132 (Figure 3(d), right panel).


Insulin Increases Sestrin 2 Content by Reducing Its Degradation through the PI 3 K/mTOR Signaling Pathway.

Chai D, Wang G, Zhou Z, Yang H, Yu Z - Int J Endocrinol (2015)

Insulin or the proteasomal inhibitor MG132 blocks SESN2 degradation. (a) HepG2 cells were pretreated with CHX for 30 min followed by a treatment with or without 100 nM of insulin (INS) for the indicated times. The cells were then lysed for SESN2 western blotting. Right panel is the plot of densitometric analysis. *P < 0.05 compared with noninsulin treatment. (b) and (c) HepG2 cells were treated with the indicated reagents for 30 min, followed by a treatment with or without 100 nM insulin for 8 h. The cells were then lysed for SESN2 western blotting. (d) HepG2 cells were incubated with or without insulin or MG132 for 18 h and the cells were lysed. Western blotting of SESN2 and β-actin (loading control) of total cellular proteins (left panel) or SESN2 immunoprecipitation followed by polyubiquitin antibody western blotting (right panel). IP indicates the immunoprecipitation antibody; IB indicates the immunoblotting antibody. Bottom panel of left panel: western blotting of the input samples. A representative blot of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: Insulin or the proteasomal inhibitor MG132 blocks SESN2 degradation. (a) HepG2 cells were pretreated with CHX for 30 min followed by a treatment with or without 100 nM of insulin (INS) for the indicated times. The cells were then lysed for SESN2 western blotting. Right panel is the plot of densitometric analysis. *P < 0.05 compared with noninsulin treatment. (b) and (c) HepG2 cells were treated with the indicated reagents for 30 min, followed by a treatment with or without 100 nM insulin for 8 h. The cells were then lysed for SESN2 western blotting. (d) HepG2 cells were incubated with or without insulin or MG132 for 18 h and the cells were lysed. Western blotting of SESN2 and β-actin (loading control) of total cellular proteins (left panel) or SESN2 immunoprecipitation followed by polyubiquitin antibody western blotting (right panel). IP indicates the immunoprecipitation antibody; IB indicates the immunoblotting antibody. Bottom panel of left panel: western blotting of the input samples. A representative blot of three independent experiments.
Mentions: Since insulin treatment rapidly upregulated hepatic SESN2 protein within 1 h, it is unlikely that the stimulation is via the increase of SESN2 protein synthesis. We hence explored whether insulin-stimulated SESN2 expression is due to the blockage of its degradation. For this purpose, HepG2 cells were pretreated with the protein synthesis inhibitor cycloheximide (CHX) to prevent the production of newly synthesized SESN2, before insulin treatment. We observed that, in the presence of insulin, SESN2 disappeared slower than non-insulin-treated cells (Figure 3(a)), indicating that insulin may block or attenuate SESN2 degradation. To further identify which degradation pathway is involved in SESN2 degradation, we examined the potential role of two major cellular protein-degrading systems, that is, proteasome and lysosome on SESN2 turnover. HepG2 cells were pretreated with either the proteasomal inhibitor, MG132, or the lysosomal inhibitor, chloroquine (CQ), for 30 min. The cells were then further treated with or without 100 nM insulin for additional 8 h. We observed that MG132 treatment alone significantly elevated SESN2 level, while insulin did not further increase SESN2 content in MG132 pretreated cells (Figure 3(b)). In contrast, the lysosomal inhibitor CQ did not exert a notable effect on SESN2 level (Figure 3(b)). Additionally, in the presence of the protein synthesis inhibitor cycloheximide, insulin and MG132 were still able to increase SESN2 content, further supporting our notion that insulin blocks proteasome-mediated SESN2 degradation (Figure 3(c)). If SESN2 is ubiquitinated, we could be able to detect upper bands with higher molecular weights of SESN2 due to its covalent binding to ubiquitins. And, indeed, we observed that both insulin and MG132 treatment enhanced the density of upper bands of SESN2 (Figure 3(d), left panel). Finally, we immunoprecipitated SESN2 protein followed with the polyubiquitin antibody detection and observed that SESN2 protein was polyubiquitinated which was enhanced by both insulin and MG132 (Figure 3(d), right panel).

Bottom Line: Sestrin (SESN) is known as a cysteine sulfinic acid reductase.While mechanisms underlying the expression of SESN are not fully understood.Following SESN2 knockdown insulin-stimulated PKB phosphorylation was enhanced and accompanied by reduced PTEN content.

View Article: PubMed Central - PubMed

Affiliation: Fujian Key Laboratory of Chinese Materia Medica, Biomedical Drug Research and Development Center, Fujian University of Traditional Chinese Medicine, Fuzhou 350108, China.

ABSTRACT
Sestrin (SESN) is known as a cysteine sulfinic acid reductase. Recently, nonredox functions of SESN in metabolic regulation and antitumor property have been recognized. While mechanisms underlying the expression of SESN are not fully understood. Here we report that insulin markedly increased SESN2 level in HepG2 cells through mTOR activation. To determine whether insulin affects SESN2 degradation, we assessed SESN2 turnover by applying the protein synthesis inhibitor, cycloheximide (CHX), and found that following insulin treatment SESN2 protein levels were reduced significantly slower than non-insulin-treated cells. Furthermore, the proteasomal inhibitor, MG132, dramatically increased SESN2 protein and its ubiquitination level while in the presence of MG132 insulin did not further increase SESN2 content, suggesting that insulin increases SESN2 content mainly via inhibiting its proteasomal degradation. We then explored the potential feedback role of SESN2 in insulin signaling by SESN2 siRNA knockdown in HepG2 cells. Following SESN2 knockdown insulin-stimulated PKB phosphorylation was enhanced and accompanied by reduced PTEN content. Taken together, our study suggests that insulin upregulates SESN2 content via the PI3K/mTOR signaling pathway and this effect is attributed to decreased SESN2 degradation. Furthermore, SESN2 via modulating PTEN plays a negative feedback role in insulin signaling.

No MeSH data available.


Related in: MedlinePlus