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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

SESN2 siRNA knockdown enhances insulin signaling by repressing the PTEN level. HepG2 cells were transfected with either the control or the SESN2 siRNA for 48–72 h as indicated in Methods. (a) The cells were harvested for western blotting with indicated SESN antibodies. (b) and (c), HepG2 cells were transfected with either the control or the SESN2 siRNA for 48–72 h. The cells were then serum-starved for 4 h, followed by 100 nM insulin treatment for 15 min. Western blotting was performed with indicated antibodies. Right panels show the results of densitometric analysis. *P < 0.05 compared with noninsulin treatment.
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fig4: SESN2 siRNA knockdown enhances insulin signaling by repressing the PTEN level. HepG2 cells were transfected with either the control or the SESN2 siRNA for 48–72 h as indicated in Methods. (a) The cells were harvested for western blotting with indicated SESN antibodies. (b) and (c), HepG2 cells were transfected with either the control or the SESN2 siRNA for 48–72 h. The cells were then serum-starved for 4 h, followed by 100 nM insulin treatment for 15 min. Western blotting was performed with indicated antibodies. Right panels show the results of densitometric analysis. *P < 0.05 compared with noninsulin treatment.

Mentions: Long-term activation of insulin signaling-evoked negative feedback regulation is one of the major mechanisms for downregulating its signaling and this can be achieved by certain downstream signaling components, such as mTOR [18]. To explore a potential contribution of SESN2 in insulin signaling, HepG2 cells were transfected with the SESN2 siRNA or the control siRNA. We observed that SESN2 siRNA concentration dependently reduced SESN2 protein levels (Figure 4(a), upper panel) while other family members of SESN were not affected (Figure 4(a), lower panel). We then examined whether insulin-stimulated PKB phosphorylation could be altered by SESN2 siRNA knockdown. As shown in Figure 4(b), insulin-stimulated PKB phosphorylation at both sites (i.e., Ser473 and Thr308) was enhanced after SESN2 knockdown (Figure 4(b)). In line with this finding, the downstream PKB signaling, S6K phosphorylation was also increased after SESN2 knockdown (Figure 4(b)). Since PKB phosphorylation is regulated by the upstream PI3K, we examined the tumor suppressor PTEN, a phosphatase that degrades the PI3K product and therefore negatively regulates insulin signaling [18]. As shown in Figure 4(c), SESN2 siRNA knockdown significantly reduced PTEN level to ~50%. These results implicate that insulin-stimulated SESN2 expression will in turn affect its signaling transduction by PTEN-mediated PI3K downregulation.


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)

SESN2 siRNA knockdown enhances insulin signaling by repressing the PTEN level. HepG2 cells were transfected with either the control or the SESN2 siRNA for 48–72 h as indicated in Methods. (a) The cells were harvested for western blotting with indicated SESN antibodies. (b) and (c), HepG2 cells were transfected with either the control or the SESN2 siRNA for 48–72 h. The cells were then serum-starved for 4 h, followed by 100 nM insulin treatment for 15 min. Western blotting was performed with indicated antibodies. Right panels show the results of densitometric analysis. *P < 0.05 compared with noninsulin treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: SESN2 siRNA knockdown enhances insulin signaling by repressing the PTEN level. HepG2 cells were transfected with either the control or the SESN2 siRNA for 48–72 h as indicated in Methods. (a) The cells were harvested for western blotting with indicated SESN antibodies. (b) and (c), HepG2 cells were transfected with either the control or the SESN2 siRNA for 48–72 h. The cells were then serum-starved for 4 h, followed by 100 nM insulin treatment for 15 min. Western blotting was performed with indicated antibodies. Right panels show the results of densitometric analysis. *P < 0.05 compared with noninsulin treatment.
Mentions: Long-term activation of insulin signaling-evoked negative feedback regulation is one of the major mechanisms for downregulating its signaling and this can be achieved by certain downstream signaling components, such as mTOR [18]. To explore a potential contribution of SESN2 in insulin signaling, HepG2 cells were transfected with the SESN2 siRNA or the control siRNA. We observed that SESN2 siRNA concentration dependently reduced SESN2 protein levels (Figure 4(a), upper panel) while other family members of SESN were not affected (Figure 4(a), lower panel). We then examined whether insulin-stimulated PKB phosphorylation could be altered by SESN2 siRNA knockdown. As shown in Figure 4(b), insulin-stimulated PKB phosphorylation at both sites (i.e., Ser473 and Thr308) was enhanced after SESN2 knockdown (Figure 4(b)). In line with this finding, the downstream PKB signaling, S6K phosphorylation was also increased after SESN2 knockdown (Figure 4(b)). Since PKB phosphorylation is regulated by the upstream PI3K, we examined the tumor suppressor PTEN, a phosphatase that degrades the PI3K product and therefore negatively regulates insulin signaling [18]. As shown in Figure 4(c), SESN2 siRNA knockdown significantly reduced PTEN level to ~50%. These results implicate that insulin-stimulated SESN2 expression will in turn affect its signaling transduction by PTEN-mediated PI3K downregulation.

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