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Metformin attenuates palmitate-induced endoplasmic reticulum stress, serine phosphorylation of IRS-1 and apoptosis in rat insulinoma cells.

Simon-Szabó L, Kokas M, Mandl J, Kéri G, Csala M - PLoS ONE (2014)

Bottom Line: The underlying endoplasmic reticulum (ER) stress response can lead to even β-cell death (lipoapoptosis).Assessment of palmitate-induced lipoapoptosis by fluorescent microscopy and by detection of caspase-3 showed a significant decrease in metformin treated cells.Our results indicate that the β-cell protective activity of metformin in lipotoxicity can be at least partly attributed to suppression of ER stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary; MTA-SE Pathobiochemistry Research Group, Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary.

ABSTRACT
Lipotoxicity refers to cellular dysfunctions caused by elevated free fatty acid levels playing a central role in the development and progression of obesity related diseases. Saturated fatty acids cause insulin resistance and reduce insulin production in the pancreatic islets, thereby generating a vicious cycle, which potentially culminates in type 2 diabetes. The underlying endoplasmic reticulum (ER) stress response can lead to even β-cell death (lipoapoptosis). Since improvement of β-cell viability is a promising anti-diabetic strategy, the protective effect of metformin, a known insulin sensitizer was studied in rat insulinoma cells. Assessment of palmitate-induced lipoapoptosis by fluorescent microscopy and by detection of caspase-3 showed a significant decrease in metformin treated cells. Attenuation of β-cell lipotoxicity was also revealed by lower induction/activation of various ER stress markers, e.g. phosphorylation of eukaryotic initiation factor 2α (eIF2α), c-Jun N-terminal kinase (JNK), insulin receptor substrate-1 (IRS-1) and induction of CCAAT/enhancer binding protein homologous protein (CHOP). Our results indicate that the β-cell protective activity of metformin in lipotoxicity can be at least partly attributed to suppression of ER stress.

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Related in: MedlinePlus

Phosphorylation of eIF2α.Insulinoma cells were treated with palmitate (500 µM) alone or together with metformin (10 µM, 100 µM) at 70–80% confluence. Cell lysates were prepared after 8 h and the phosphorylation and expression level of eIF2α were assessed by Western blot analysis using antibodies specific to phosphorylated (upper panel) and total (lower panel) eIF2α, respectively. Typical results of three independent experiments are shown. The results were quantified by densitometry and are shown as normalized relative band densities. Data are presented as mean values ± S.D. of three experiments in arbitrary units (palmitate-treated = 100%); aP<0.05, bP<0.01 v.s. untreated control; **P<0.01 v.s. palmitate-treated.
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pone-0097868-g004: Phosphorylation of eIF2α.Insulinoma cells were treated with palmitate (500 µM) alone or together with metformin (10 µM, 100 µM) at 70–80% confluence. Cell lysates were prepared after 8 h and the phosphorylation and expression level of eIF2α were assessed by Western blot analysis using antibodies specific to phosphorylated (upper panel) and total (lower panel) eIF2α, respectively. Typical results of three independent experiments are shown. The results were quantified by densitometry and are shown as normalized relative band densities. Data are presented as mean values ± S.D. of three experiments in arbitrary units (palmitate-treated = 100%); aP<0.05, bP<0.01 v.s. untreated control; **P<0.01 v.s. palmitate-treated.

Mentions: PERK is responsible for the attenuation of general translation through phosphorylation of eIF2α. This phenomenon was well detectable in palmitate-treated RINm5F cells by Western blot using a P-eIF2α specific primary antibody (Fig. 4). The amount of phosphorylated eIF2α was approximately 3-times higher in treated v.s. untreated cells, strongly indicating the activation of PERK-initiated events of the UPR. A partial inhibition of eIF2α phosphorylation was observed when palmitate was administered together with metformin. The antidiabetic agent was only effective at higher (100 µM) concentration, and P-eIF2α was still increased to about twice the control level (Fig. 4).


Metformin attenuates palmitate-induced endoplasmic reticulum stress, serine phosphorylation of IRS-1 and apoptosis in rat insulinoma cells.

Simon-Szabó L, Kokas M, Mandl J, Kéri G, Csala M - PLoS ONE (2014)

Phosphorylation of eIF2α.Insulinoma cells were treated with palmitate (500 µM) alone or together with metformin (10 µM, 100 µM) at 70–80% confluence. Cell lysates were prepared after 8 h and the phosphorylation and expression level of eIF2α were assessed by Western blot analysis using antibodies specific to phosphorylated (upper panel) and total (lower panel) eIF2α, respectively. Typical results of three independent experiments are shown. The results were quantified by densitometry and are shown as normalized relative band densities. Data are presented as mean values ± S.D. of three experiments in arbitrary units (palmitate-treated = 100%); aP<0.05, bP<0.01 v.s. untreated control; **P<0.01 v.s. palmitate-treated.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4045581&req=5

pone-0097868-g004: Phosphorylation of eIF2α.Insulinoma cells were treated with palmitate (500 µM) alone or together with metformin (10 µM, 100 µM) at 70–80% confluence. Cell lysates were prepared after 8 h and the phosphorylation and expression level of eIF2α were assessed by Western blot analysis using antibodies specific to phosphorylated (upper panel) and total (lower panel) eIF2α, respectively. Typical results of three independent experiments are shown. The results were quantified by densitometry and are shown as normalized relative band densities. Data are presented as mean values ± S.D. of three experiments in arbitrary units (palmitate-treated = 100%); aP<0.05, bP<0.01 v.s. untreated control; **P<0.01 v.s. palmitate-treated.
Mentions: PERK is responsible for the attenuation of general translation through phosphorylation of eIF2α. This phenomenon was well detectable in palmitate-treated RINm5F cells by Western blot using a P-eIF2α specific primary antibody (Fig. 4). The amount of phosphorylated eIF2α was approximately 3-times higher in treated v.s. untreated cells, strongly indicating the activation of PERK-initiated events of the UPR. A partial inhibition of eIF2α phosphorylation was observed when palmitate was administered together with metformin. The antidiabetic agent was only effective at higher (100 µM) concentration, and P-eIF2α was still increased to about twice the control level (Fig. 4).

Bottom Line: The underlying endoplasmic reticulum (ER) stress response can lead to even β-cell death (lipoapoptosis).Assessment of palmitate-induced lipoapoptosis by fluorescent microscopy and by detection of caspase-3 showed a significant decrease in metformin treated cells.Our results indicate that the β-cell protective activity of metformin in lipotoxicity can be at least partly attributed to suppression of ER stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary; MTA-SE Pathobiochemistry Research Group, Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary.

ABSTRACT
Lipotoxicity refers to cellular dysfunctions caused by elevated free fatty acid levels playing a central role in the development and progression of obesity related diseases. Saturated fatty acids cause insulin resistance and reduce insulin production in the pancreatic islets, thereby generating a vicious cycle, which potentially culminates in type 2 diabetes. The underlying endoplasmic reticulum (ER) stress response can lead to even β-cell death (lipoapoptosis). Since improvement of β-cell viability is a promising anti-diabetic strategy, the protective effect of metformin, a known insulin sensitizer was studied in rat insulinoma cells. Assessment of palmitate-induced lipoapoptosis by fluorescent microscopy and by detection of caspase-3 showed a significant decrease in metformin treated cells. Attenuation of β-cell lipotoxicity was also revealed by lower induction/activation of various ER stress markers, e.g. phosphorylation of eukaryotic initiation factor 2α (eIF2α), c-Jun N-terminal kinase (JNK), insulin receptor substrate-1 (IRS-1) and induction of CCAAT/enhancer binding protein homologous protein (CHOP). Our results indicate that the β-cell protective activity of metformin in lipotoxicity can be at least partly attributed to suppression of ER stress.

Show MeSH
Related in: MedlinePlus