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AICAR and Metformin Exert AMPK-dependent Effects on INS-1E Pancreatic β-cell Apoptosis via Differential Downstream Mechanisms.

Dai YL, Huang SL, Leng Y - Int. J. Biol. Sci. (2015)

Bottom Line: Both AICAR and metformin protected INS-1E cells from palmitate-induced apoptosis, as reflected by decreases in both cleaved caspase 3 protein expression and caspase 3/7 activity, and these protective effects were abrogated by AMPK inhibitor compound C.The protective action of AICAR was probably mediated by the suppression of triacylglycerol accumulation, increase in Akt phosphorylation and decrease in p38 MAPK phosphorylation, while metformin might exert its protective effect on INS-1E cells by decreases in both JNK and p38 MAPK phosphorylation.Our results provided new and informative clues for better understanding of the role of AMPK in β-cell apoptosis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zu Chong Zhi Road 555, Shanghai 201203, China.

ABSTRACT
The role of AMP-activated protein kinase (AMPK) in pancreatic β-cell apoptosis is still controversial, and the reasons for the discrepancies have not been clarified. In the current study, we observed the effects of two well-known AMPK activators 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and metformin, on apoptosis in rat insulinoma INS-1E cells, and further explored their possible mechanisms. Both AICAR and metformin protected INS-1E cells from palmitate-induced apoptosis, as reflected by decreases in both cleaved caspase 3 protein expression and caspase 3/7 activity, and these protective effects were abrogated by AMPK inhibitor compound C. The protective action of AICAR was probably mediated by the suppression of triacylglycerol accumulation, increase in Akt phosphorylation and decrease in p38 MAPK phosphorylation, while metformin might exert its protective effect on INS-1E cells by decreases in both JNK and p38 MAPK phosphorylation. All these regulations were dependent on AMPK activation. However, under standard culture condition, AICAR increased JNK phosphorylation and promoted INS-1E cell apoptosis in an AMPK-dependent manner, whereas metformin showed no effect on apoptosis. Our study revealed that AMPK activators AICAR and metformin exhibited different effects on INS-1E cell apoptosis under different culture conditions, which might be largely attributed to different downstream mediators. Our results provided new and informative clues for better understanding of the role of AMPK in β-cell apoptosis.

No MeSH data available.


Related in: MedlinePlus

Effects of AICAR and metformin on INS-1E cell apoptosis under standard culture condition. (A, B) Under standard culture condition, INS-1E cells were stimulated with 1 mM AICAR or 2 mM metformin for 16 h, followed by immunoblotting of JNK phosphorylation (A) and cleaved caspase 3 (B). 0.25 mM palmitate was set as positive control. (C) INS-1E cells were preincubated with or without JNK-IN-8 (0.3 μM) for 30 min, and then exposed to 1 mM AICAR for 16 h, followed by immunoblotting of JNK phosphorylation and cleaved caspase 3. (D) INS-1E cells were preincubated with or without compound C (10 μM) for 30 min, and then exposed to 1 mM AICAR for 16 h, followed by immunoblotting of AMPK, ACC and JNK phosphorylation and cleaved caspase 3. Representative immunoblots were shown and protein contents of cleaved caspase 3 and P-JNK were quantified. Three or four independent experiments were performed. *P<0.05 and **P<0.01 vs control (cells not exposed to palmitate) or corresponding columns as indicated; ns, not significant. Palm, palmitate; Met, metformin.
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Figure 5: Effects of AICAR and metformin on INS-1E cell apoptosis under standard culture condition. (A, B) Under standard culture condition, INS-1E cells were stimulated with 1 mM AICAR or 2 mM metformin for 16 h, followed by immunoblotting of JNK phosphorylation (A) and cleaved caspase 3 (B). 0.25 mM palmitate was set as positive control. (C) INS-1E cells were preincubated with or without JNK-IN-8 (0.3 μM) for 30 min, and then exposed to 1 mM AICAR for 16 h, followed by immunoblotting of JNK phosphorylation and cleaved caspase 3. (D) INS-1E cells were preincubated with or without compound C (10 μM) for 30 min, and then exposed to 1 mM AICAR for 16 h, followed by immunoblotting of AMPK, ACC and JNK phosphorylation and cleaved caspase 3. Representative immunoblots were shown and protein contents of cleaved caspase 3 and P-JNK were quantified. Three or four independent experiments were performed. *P<0.05 and **P<0.01 vs control (cells not exposed to palmitate) or corresponding columns as indicated; ns, not significant. Palm, palmitate; Met, metformin.

Mentions: Considering the possibility that activation of JNK by AICAR might cause cell apoptosis under standard culture condition, INS-1E cells were incubated with 1 mM AICAR or 2 mM metformin for 16 h. Assessment of JNK phosphorylation and cleaved caspase 3 protein expression was performed. Under this condition, chronic treatment of AICAR indeed increased JNK phosphorylation, whereas metformin decreased JNK phosphorylation (P<0.01 vs control) (Fig. 5A). Simultaneously, we observed a moderate increase in the cleaved form of caspase 3 (P<0.05 vs control) after treatment with AICAR, but metformin had no effect on it (Fig. 5B). This demonstrated that AICAR, but not metformin, would trigger cell apoptosis under standard culture condition.


AICAR and Metformin Exert AMPK-dependent Effects on INS-1E Pancreatic β-cell Apoptosis via Differential Downstream Mechanisms.

Dai YL, Huang SL, Leng Y - Int. J. Biol. Sci. (2015)

Effects of AICAR and metformin on INS-1E cell apoptosis under standard culture condition. (A, B) Under standard culture condition, INS-1E cells were stimulated with 1 mM AICAR or 2 mM metformin for 16 h, followed by immunoblotting of JNK phosphorylation (A) and cleaved caspase 3 (B). 0.25 mM palmitate was set as positive control. (C) INS-1E cells were preincubated with or without JNK-IN-8 (0.3 μM) for 30 min, and then exposed to 1 mM AICAR for 16 h, followed by immunoblotting of JNK phosphorylation and cleaved caspase 3. (D) INS-1E cells were preincubated with or without compound C (10 μM) for 30 min, and then exposed to 1 mM AICAR for 16 h, followed by immunoblotting of AMPK, ACC and JNK phosphorylation and cleaved caspase 3. Representative immunoblots were shown and protein contents of cleaved caspase 3 and P-JNK were quantified. Three or four independent experiments were performed. *P<0.05 and **P<0.01 vs control (cells not exposed to palmitate) or corresponding columns as indicated; ns, not significant. Palm, palmitate; Met, metformin.
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Figure 5: Effects of AICAR and metformin on INS-1E cell apoptosis under standard culture condition. (A, B) Under standard culture condition, INS-1E cells were stimulated with 1 mM AICAR or 2 mM metformin for 16 h, followed by immunoblotting of JNK phosphorylation (A) and cleaved caspase 3 (B). 0.25 mM palmitate was set as positive control. (C) INS-1E cells were preincubated with or without JNK-IN-8 (0.3 μM) for 30 min, and then exposed to 1 mM AICAR for 16 h, followed by immunoblotting of JNK phosphorylation and cleaved caspase 3. (D) INS-1E cells were preincubated with or without compound C (10 μM) for 30 min, and then exposed to 1 mM AICAR for 16 h, followed by immunoblotting of AMPK, ACC and JNK phosphorylation and cleaved caspase 3. Representative immunoblots were shown and protein contents of cleaved caspase 3 and P-JNK were quantified. Three or four independent experiments were performed. *P<0.05 and **P<0.01 vs control (cells not exposed to palmitate) or corresponding columns as indicated; ns, not significant. Palm, palmitate; Met, metformin.
Mentions: Considering the possibility that activation of JNK by AICAR might cause cell apoptosis under standard culture condition, INS-1E cells were incubated with 1 mM AICAR or 2 mM metformin for 16 h. Assessment of JNK phosphorylation and cleaved caspase 3 protein expression was performed. Under this condition, chronic treatment of AICAR indeed increased JNK phosphorylation, whereas metformin decreased JNK phosphorylation (P<0.01 vs control) (Fig. 5A). Simultaneously, we observed a moderate increase in the cleaved form of caspase 3 (P<0.05 vs control) after treatment with AICAR, but metformin had no effect on it (Fig. 5B). This demonstrated that AICAR, but not metformin, would trigger cell apoptosis under standard culture condition.

Bottom Line: Both AICAR and metformin protected INS-1E cells from palmitate-induced apoptosis, as reflected by decreases in both cleaved caspase 3 protein expression and caspase 3/7 activity, and these protective effects were abrogated by AMPK inhibitor compound C.The protective action of AICAR was probably mediated by the suppression of triacylglycerol accumulation, increase in Akt phosphorylation and decrease in p38 MAPK phosphorylation, while metformin might exert its protective effect on INS-1E cells by decreases in both JNK and p38 MAPK phosphorylation.Our results provided new and informative clues for better understanding of the role of AMPK in β-cell apoptosis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zu Chong Zhi Road 555, Shanghai 201203, China.

ABSTRACT
The role of AMP-activated protein kinase (AMPK) in pancreatic β-cell apoptosis is still controversial, and the reasons for the discrepancies have not been clarified. In the current study, we observed the effects of two well-known AMPK activators 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and metformin, on apoptosis in rat insulinoma INS-1E cells, and further explored their possible mechanisms. Both AICAR and metformin protected INS-1E cells from palmitate-induced apoptosis, as reflected by decreases in both cleaved caspase 3 protein expression and caspase 3/7 activity, and these protective effects were abrogated by AMPK inhibitor compound C. The protective action of AICAR was probably mediated by the suppression of triacylglycerol accumulation, increase in Akt phosphorylation and decrease in p38 MAPK phosphorylation, while metformin might exert its protective effect on INS-1E cells by decreases in both JNK and p38 MAPK phosphorylation. All these regulations were dependent on AMPK activation. However, under standard culture condition, AICAR increased JNK phosphorylation and promoted INS-1E cell apoptosis in an AMPK-dependent manner, whereas metformin showed no effect on apoptosis. Our study revealed that AMPK activators AICAR and metformin exhibited different effects on INS-1E cell apoptosis under different culture conditions, which might be largely attributed to different downstream mediators. Our results provided new and informative clues for better understanding of the role of AMPK in β-cell apoptosis.

No MeSH data available.


Related in: MedlinePlus