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Placental endoplasmic reticulum stress in gestational diabetes: the potential for therapeutic intervention with chemical chaperones and antioxidants

View Article: PubMed Central - PubMed

ABSTRACT

Aims/hypothesis: The aim of this work was to determine whether placental endoplasmic reticulum (ER) stress may contribute to the pathophysiology of gestational diabetes mellitus (GDM) and to test the efficacy of chemical chaperones and antioxidant vitamins in ameliorating that stress in a trophoblast-like cell line in vitro.

Methods: Placental samples were obtained from women suffering from GDM and from normoglycaemic controls and were frozen immediately. Women with GDM had 2 h serum glucose levels > 9.0 mmol/l following a 75 g oral glucose tolerance test and were treated with diet and insulin when necessary. Western blotting was used to assess markers of ER stress. To test the effects of hyperglycaemia on the generation of ER stress, a new trophoblast-like cell line, BeWo-NG, was generated by culturing in a physiological glucose concentration of 5.5 mmol/l (over 20 passages) before challenging with 10 or 20 mmol/l glucose.

Results: All GDM patients were well-controlled (HbA1c 5.86 ± 0.55% or 40.64 ± 5.85 mmol/mol, n = 11). Low-grade ER stress was observed in the placental samples, with dilation of ER cisternae and increased phosphorylation of eukaryotic initiation factor 2 subunit α. Challenge of BeWo-NG with high glucose activated the same pathways, but this was as a result of acidosis of the culture medium rather than the glucose concentration per se. Addition of chemical chaperones 4-phenylbutyrate and tauroursodeoxycholic acid and vitamins C and E ameliorated the ER stress.

Conclusions/interpretation: This is the first report of placental ER stress in GDM patients. Chemical chaperones and antioxidant vitamins represent potential therapeutic interventions for GDM.

Electronic supplementary material: The online version of this article (doi:10.1007/s00125-016-4040-2) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

No MeSH data available.


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Both chemical chaperones and antioxidants effectively suppress high glucose-induced ER stress, but only antioxidants reduce the degree of metabolic acidosis. BeWo-NG cells were treated with different concentrations of glucose in the presence of either chemical chaperones, 4-PBA (500 μmol/l) and TUDCA (250 μmol/l), or vitamin C (Vit C; 500 μmol/l) or vitamin E (Vit E; 500 μmol/l) for 24 h. (a) Levels of ER stress markers were determined by western blotting. β-actin was used as loading control. (b) Phosphorylation status is presented as the ratio between phosphorylated and total protein. Data are presented as mean ± SEM, n = 3. The y-axis shows the relative level of p-eIF2α eIF2α or the ratio of p-eIF2α/eIF2α. (c) pH of the culture media after the experiment. (d) Effect of vitamins on high glucose-induced lactate production. Data are presented as median, n = 3–6. †p < 0.05 and ††p < 0.01 vs 10 mmol/l glucose in the glucose-only control group; ‡p < 0.05 and ‡‡p < 0.01 vs 20 mmol/l glucose in the glucose-only control group
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Fig3: Both chemical chaperones and antioxidants effectively suppress high glucose-induced ER stress, but only antioxidants reduce the degree of metabolic acidosis. BeWo-NG cells were treated with different concentrations of glucose in the presence of either chemical chaperones, 4-PBA (500 μmol/l) and TUDCA (250 μmol/l), or vitamin C (Vit C; 500 μmol/l) or vitamin E (Vit E; 500 μmol/l) for 24 h. (a) Levels of ER stress markers were determined by western blotting. β-actin was used as loading control. (b) Phosphorylation status is presented as the ratio between phosphorylated and total protein. Data are presented as mean ± SEM, n = 3. The y-axis shows the relative level of p-eIF2α eIF2α or the ratio of p-eIF2α/eIF2α. (c) pH of the culture media after the experiment. (d) Effect of vitamins on high glucose-induced lactate production. Data are presented as median, n = 3–6. †p < 0.05 and ††p < 0.01 vs 10 mmol/l glucose in the glucose-only control group; ‡p < 0.05 and ‡‡p < 0.01 vs 20 mmol/l glucose in the glucose-only control group

Mentions: Chemical chaperones, including 4-phenylbutyrate (4-PBA) and tauroursodeoxycholic acid (TUDCA), have been widely demonstrated to be effective in reducing ER stress [23], while antioxidants, such as vitamins C and E, suppress oxidative stress in placental explants [24]. Therefore, we investigated the effects of chemical chaperones and antioxidants in the suppression of high glucose-induced ER stress. Both chemical chaperones and antioxidants were effective in reducing phosphorylation of eIF2α (Fig. 3a, b), while all other ER stress markers and residential proteins remained unchanged (Fig. 3a). Surprisingly, vitamins, but not chemical chaperones, significantly restored the pH of the normal media to ∼6.7 and reduced lactate accumulation by over 50% in the presence of 10 and 20 mmol/l glucose (Fig. 3c, d). Vitamin C or vitamin E alone suppressed high glucose-induced ER stress and restored the pH, but were not as effective when combined (data not shown). These results reveal potential beneficial effects of both chemical chaperones and vitamins C and E in reducing ER stress. Additionally, not only can the vitamins suppress the ER stress induced by high glucose but they may also be used to prevent hyperglycaemia-induced acidosis.Fig. 3


Placental endoplasmic reticulum stress in gestational diabetes: the potential for therapeutic intervention with chemical chaperones and antioxidants
Both chemical chaperones and antioxidants effectively suppress high glucose-induced ER stress, but only antioxidants reduce the degree of metabolic acidosis. BeWo-NG cells were treated with different concentrations of glucose in the presence of either chemical chaperones, 4-PBA (500 μmol/l) and TUDCA (250 μmol/l), or vitamin C (Vit C; 500 μmol/l) or vitamin E (Vit E; 500 μmol/l) for 24 h. (a) Levels of ER stress markers were determined by western blotting. β-actin was used as loading control. (b) Phosphorylation status is presented as the ratio between phosphorylated and total protein. Data are presented as mean ± SEM, n = 3. The y-axis shows the relative level of p-eIF2α eIF2α or the ratio of p-eIF2α/eIF2α. (c) pH of the culture media after the experiment. (d) Effect of vitamins on high glucose-induced lactate production. Data are presented as median, n = 3–6. †p < 0.05 and ††p < 0.01 vs 10 mmol/l glucose in the glucose-only control group; ‡p < 0.05 and ‡‡p < 0.01 vs 20 mmol/l glucose in the glucose-only control group
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Fig3: Both chemical chaperones and antioxidants effectively suppress high glucose-induced ER stress, but only antioxidants reduce the degree of metabolic acidosis. BeWo-NG cells were treated with different concentrations of glucose in the presence of either chemical chaperones, 4-PBA (500 μmol/l) and TUDCA (250 μmol/l), or vitamin C (Vit C; 500 μmol/l) or vitamin E (Vit E; 500 μmol/l) for 24 h. (a) Levels of ER stress markers were determined by western blotting. β-actin was used as loading control. (b) Phosphorylation status is presented as the ratio between phosphorylated and total protein. Data are presented as mean ± SEM, n = 3. The y-axis shows the relative level of p-eIF2α eIF2α or the ratio of p-eIF2α/eIF2α. (c) pH of the culture media after the experiment. (d) Effect of vitamins on high glucose-induced lactate production. Data are presented as median, n = 3–6. †p < 0.05 and ††p < 0.01 vs 10 mmol/l glucose in the glucose-only control group; ‡p < 0.05 and ‡‡p < 0.01 vs 20 mmol/l glucose in the glucose-only control group
Mentions: Chemical chaperones, including 4-phenylbutyrate (4-PBA) and tauroursodeoxycholic acid (TUDCA), have been widely demonstrated to be effective in reducing ER stress [23], while antioxidants, such as vitamins C and E, suppress oxidative stress in placental explants [24]. Therefore, we investigated the effects of chemical chaperones and antioxidants in the suppression of high glucose-induced ER stress. Both chemical chaperones and antioxidants were effective in reducing phosphorylation of eIF2α (Fig. 3a, b), while all other ER stress markers and residential proteins remained unchanged (Fig. 3a). Surprisingly, vitamins, but not chemical chaperones, significantly restored the pH of the normal media to ∼6.7 and reduced lactate accumulation by over 50% in the presence of 10 and 20 mmol/l glucose (Fig. 3c, d). Vitamin C or vitamin E alone suppressed high glucose-induced ER stress and restored the pH, but were not as effective when combined (data not shown). These results reveal potential beneficial effects of both chemical chaperones and vitamins C and E in reducing ER stress. Additionally, not only can the vitamins suppress the ER stress induced by high glucose but they may also be used to prevent hyperglycaemia-induced acidosis.Fig. 3

View Article: PubMed Central - PubMed

ABSTRACT

Aims/hypothesis: The aim of this work was to determine whether placental endoplasmic reticulum (ER) stress may contribute to the pathophysiology of gestational diabetes mellitus (GDM) and to test the efficacy of chemical chaperones and antioxidant vitamins in ameliorating that stress in a trophoblast-like cell line in vitro.

Methods: Placental samples were obtained from women suffering from GDM and from normoglycaemic controls and were frozen immediately. Women with GDM had 2&nbsp;h serum glucose levels &gt; 9.0&nbsp;mmol/l following a 75&nbsp;g oral glucose tolerance test and were treated with diet and insulin when necessary. Western blotting was used to assess markers of ER stress. To test the effects of hyperglycaemia on the generation of ER stress, a new trophoblast-like cell line, BeWo-NG, was generated by culturing in a physiological glucose concentration of 5.5&nbsp;mmol/l (over 20 passages) before challenging with 10 or 20&nbsp;mmol/l glucose.

Results: All GDM patients were well-controlled (HbA1c 5.86&thinsp;&plusmn;&thinsp;0.55% or 40.64&thinsp;&plusmn;&thinsp;5.85&nbsp;mmol/mol, n&thinsp;=&thinsp;11). Low-grade ER stress was observed in the placental samples, with dilation of ER cisternae and increased phosphorylation of eukaryotic initiation factor 2 subunit &alpha;. Challenge of BeWo-NG with high glucose activated the same pathways, but this was as a result of acidosis of the culture medium rather than the glucose concentration per se. Addition of chemical chaperones 4-phenylbutyrate and tauroursodeoxycholic acid and vitamins C and E ameliorated the ER stress.

Conclusions/interpretation: This is the first report of placental ER stress in GDM patients. Chemical chaperones and antioxidant vitamins represent potential therapeutic interventions for GDM.

Electronic supplementary material: The online version of this article (doi:10.1007/s00125-016-4040-2) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

No MeSH data available.


Related in: MedlinePlus