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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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Treatment with metformin only.Insulinoma cells were treated with 500 µM palmitate (positive control) or metformin (10 µM, 100 µM) at 70–80% confluence. PDI, CHOP, cleaved caspase-3, Total and phosphorylated eIF2α, total and phosphorylated JNK (two isoforms), total and phosphorylated c-Jun and β-actin as a constitutive reference protein were detected by Western blot analysis using cell lysates prepared after 8 h. Total RNA was also prepared after 8 h and unspliced (uXBP-1) and spliced (sXBP-1) XBP-1 mRNA sequences were amplified by RT-PCR. PstI restriction endonuclease cleavage yields two fragments (153 and 294 bp) from uXBP-1 while leaves sXBP-1 uncut (421 bp). The products were separated by 2% agarose gel electrophoresis. Typical results of three independent experiments are shown.
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pone-0097868-g008: Treatment with metformin only.Insulinoma cells were treated with 500 µM palmitate (positive control) or metformin (10 µM, 100 µM) at 70–80% confluence. PDI, CHOP, cleaved caspase-3, Total and phosphorylated eIF2α, total and phosphorylated JNK (two isoforms), total and phosphorylated c-Jun and β-actin as a constitutive reference protein were detected by Western blot analysis using cell lysates prepared after 8 h. Total RNA was also prepared after 8 h and unspliced (uXBP-1) and spliced (sXBP-1) XBP-1 mRNA sequences were amplified by RT-PCR. PstI restriction endonuclease cleavage yields two fragments (153 and 294 bp) from uXBP-1 while leaves sXBP-1 uncut (421 bp). The products were separated by 2% agarose gel electrophoresis. Typical results of three independent experiments are shown.

Mentions: As it was shown in Fig. 2, metformin treatment in the absence of palmitate did not affect the intensity of apoptosis or necrosis in RINm5F cells. The possible effect of metformin on the investigated parameters of the UPR including caspase-3 activation and c-Jun phosphorylation was also tested in our experimental conditions. Palmitate treatment was applied as a positive control. Metformin (10 or 100 µM) did not cause any statistically significant change in the expression level of PDI, CHOP, eIF2α, c-Jun or JNK; in the phosphorylation of the latter three proteins or in the activation of caspase-3 (Fig. 8).


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)

Treatment with metformin only.Insulinoma cells were treated with 500 µM palmitate (positive control) or metformin (10 µM, 100 µM) at 70–80% confluence. PDI, CHOP, cleaved caspase-3, Total and phosphorylated eIF2α, total and phosphorylated JNK (two isoforms), total and phosphorylated c-Jun and β-actin as a constitutive reference protein were detected by Western blot analysis using cell lysates prepared after 8 h. Total RNA was also prepared after 8 h and unspliced (uXBP-1) and spliced (sXBP-1) XBP-1 mRNA sequences were amplified by RT-PCR. PstI restriction endonuclease cleavage yields two fragments (153 and 294 bp) from uXBP-1 while leaves sXBP-1 uncut (421 bp). The products were separated by 2% agarose gel electrophoresis. Typical results of three independent experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0097868-g008: Treatment with metformin only.Insulinoma cells were treated with 500 µM palmitate (positive control) or metformin (10 µM, 100 µM) at 70–80% confluence. PDI, CHOP, cleaved caspase-3, Total and phosphorylated eIF2α, total and phosphorylated JNK (two isoforms), total and phosphorylated c-Jun and β-actin as a constitutive reference protein were detected by Western blot analysis using cell lysates prepared after 8 h. Total RNA was also prepared after 8 h and unspliced (uXBP-1) and spliced (sXBP-1) XBP-1 mRNA sequences were amplified by RT-PCR. PstI restriction endonuclease cleavage yields two fragments (153 and 294 bp) from uXBP-1 while leaves sXBP-1 uncut (421 bp). The products were separated by 2% agarose gel electrophoresis. Typical results of three independent experiments are shown.
Mentions: As it was shown in Fig. 2, metformin treatment in the absence of palmitate did not affect the intensity of apoptosis or necrosis in RINm5F cells. The possible effect of metformin on the investigated parameters of the UPR including caspase-3 activation and c-Jun phosphorylation was also tested in our experimental conditions. Palmitate treatment was applied as a positive control. Metformin (10 or 100 µM) did not cause any statistically significant change in the expression level of PDI, CHOP, eIF2α, c-Jun or JNK; in the phosphorylation of the latter three proteins or in the activation of caspase-3 (Fig. 8).

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