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Intermittent high glucose implements stress-induced senescence in human vascular endothelial cells: role of superoxide production by NADPH oxidase.

Maeda M, Hayashi T, Mizuno N, Hattori Y, Kuzuya M - PLoS ONE (2015)

Bottom Line: Impaired glucose tolerance characterized by postprandial hyperglycemia, which occurs frequently in elderly persons and represents an important preliminary step in diabetes mellitus, poses an independent risk factor for the development of atherosclerosis.Interestingly, in intermittent high glucose, this effect was more pronounced as well as increase of p21 and p16INK4a , senescence related proteins with DNA damage.However, telomerase was not activated and telomere length was not shortened, thus stress-induced senescence was shown.

View Article: PubMed Central - PubMed

Affiliation: Department of Geriatrics, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.

ABSTRACT
Impaired glucose tolerance characterized by postprandial hyperglycemia, which occurs frequently in elderly persons and represents an important preliminary step in diabetes mellitus, poses an independent risk factor for the development of atherosclerosis. Endothelial cellular senescence is reported to precede atherosclerosis. We reported that continuous high glucose stimulus causes endothelial senescence more markedly than hypertension or dyslipidemia stimulus. In the present study, we evaluated the effect of fluctuating glucose levels on human endothelial senescence. Constant high glucose increased senescence-associated-β-galactosidase (SA-β-gal) activity, a widely used marker for cellular senescence. Interestingly, in intermittent high glucose, this effect was more pronounced as well as increase of p21 and p16INK4a , senescence related proteins with DNA damage. However, telomerase was not activated and telomere length was not shortened, thus stress-induced senescence was shown. However, constant high glucose activated telomerase and shortened telomere length, which suggested replicative senescence. Intermittent but not constant high glucose strikingly up-regulated the expression of p22phox, an NADPH oxidase component, increasing superoxide. The small interfering RNA of p22phox undermined the increase in SA-β-gal activity induced by intermittent high glucose. Conclusively, intermittent high glucose can promote vascular endothelial senescence more than constant high glucose, which is in partially dependent on superoxide overproduction.

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Related in: MedlinePlus

eNOS activity in HUVECs exposed to high glucose.NG, constant normal glucose (5 mM); HG, constant high glucose (22 mM); and N/HG, 5 mM alternating with 22 mM glucose. (A) Typical Western blots for total eNOS, Ser-1177 phosphorylated eNOS, and Thr-495 phosphorylated eNOS are shown. GAPDH served as the loading control. (B-D) Summary of quantification of densitometric measurement of the immunoblot data. Total eNOS expression and eNOS phosphorylation levels were normalized to GAPDH and total eNOS, respectively. (E) NO production by HUVECs, using the fluorescent dye DAF-2-DA. (F) The data of NOx (NO2- and NO3-), NO metabolites measured by HPLC.
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pone.0123169.g005: eNOS activity in HUVECs exposed to high glucose.NG, constant normal glucose (5 mM); HG, constant high glucose (22 mM); and N/HG, 5 mM alternating with 22 mM glucose. (A) Typical Western blots for total eNOS, Ser-1177 phosphorylated eNOS, and Thr-495 phosphorylated eNOS are shown. GAPDH served as the loading control. (B-D) Summary of quantification of densitometric measurement of the immunoblot data. Total eNOS expression and eNOS phosphorylation levels were normalized to GAPDH and total eNOS, respectively. (E) NO production by HUVECs, using the fluorescent dye DAF-2-DA. (F) The data of NOx (NO2- and NO3-), NO metabolites measured by HPLC.

Mentions: We have previously shown that eNOS plays a pivotal role in the regulation of the senescence program in vascular endothelial cells [2,6,7,8]. Under constant or intermittent high glucose, however, eNOS total expression, Ser-1177 eNOS phosphorylation, and Thr-495 eNOS dephosphorylation levels were substantially similar to those under normal glucose (Fig 5A–5D). When we examined the basal NO levels from HUVECs using the fluorescent dye DAF-2 DA, no significant difference was found among the three groups (Fig 5E). Concentration of NOx; metabolites of NO, NO2- + NO3-, in culture medium also supports those data (Fig 5F).


Intermittent high glucose implements stress-induced senescence in human vascular endothelial cells: role of superoxide production by NADPH oxidase.

Maeda M, Hayashi T, Mizuno N, Hattori Y, Kuzuya M - PLoS ONE (2015)

eNOS activity in HUVECs exposed to high glucose.NG, constant normal glucose (5 mM); HG, constant high glucose (22 mM); and N/HG, 5 mM alternating with 22 mM glucose. (A) Typical Western blots for total eNOS, Ser-1177 phosphorylated eNOS, and Thr-495 phosphorylated eNOS are shown. GAPDH served as the loading control. (B-D) Summary of quantification of densitometric measurement of the immunoblot data. Total eNOS expression and eNOS phosphorylation levels were normalized to GAPDH and total eNOS, respectively. (E) NO production by HUVECs, using the fluorescent dye DAF-2-DA. (F) The data of NOx (NO2- and NO3-), NO metabolites measured by HPLC.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400006&req=5

pone.0123169.g005: eNOS activity in HUVECs exposed to high glucose.NG, constant normal glucose (5 mM); HG, constant high glucose (22 mM); and N/HG, 5 mM alternating with 22 mM glucose. (A) Typical Western blots for total eNOS, Ser-1177 phosphorylated eNOS, and Thr-495 phosphorylated eNOS are shown. GAPDH served as the loading control. (B-D) Summary of quantification of densitometric measurement of the immunoblot data. Total eNOS expression and eNOS phosphorylation levels were normalized to GAPDH and total eNOS, respectively. (E) NO production by HUVECs, using the fluorescent dye DAF-2-DA. (F) The data of NOx (NO2- and NO3-), NO metabolites measured by HPLC.
Mentions: We have previously shown that eNOS plays a pivotal role in the regulation of the senescence program in vascular endothelial cells [2,6,7,8]. Under constant or intermittent high glucose, however, eNOS total expression, Ser-1177 eNOS phosphorylation, and Thr-495 eNOS dephosphorylation levels were substantially similar to those under normal glucose (Fig 5A–5D). When we examined the basal NO levels from HUVECs using the fluorescent dye DAF-2 DA, no significant difference was found among the three groups (Fig 5E). Concentration of NOx; metabolites of NO, NO2- + NO3-, in culture medium also supports those data (Fig 5F).

Bottom Line: Impaired glucose tolerance characterized by postprandial hyperglycemia, which occurs frequently in elderly persons and represents an important preliminary step in diabetes mellitus, poses an independent risk factor for the development of atherosclerosis.Interestingly, in intermittent high glucose, this effect was more pronounced as well as increase of p21 and p16INK4a , senescence related proteins with DNA damage.However, telomerase was not activated and telomere length was not shortened, thus stress-induced senescence was shown.

View Article: PubMed Central - PubMed

Affiliation: Department of Geriatrics, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.

ABSTRACT
Impaired glucose tolerance characterized by postprandial hyperglycemia, which occurs frequently in elderly persons and represents an important preliminary step in diabetes mellitus, poses an independent risk factor for the development of atherosclerosis. Endothelial cellular senescence is reported to precede atherosclerosis. We reported that continuous high glucose stimulus causes endothelial senescence more markedly than hypertension or dyslipidemia stimulus. In the present study, we evaluated the effect of fluctuating glucose levels on human endothelial senescence. Constant high glucose increased senescence-associated-β-galactosidase (SA-β-gal) activity, a widely used marker for cellular senescence. Interestingly, in intermittent high glucose, this effect was more pronounced as well as increase of p21 and p16INK4a , senescence related proteins with DNA damage. However, telomerase was not activated and telomere length was not shortened, thus stress-induced senescence was shown. However, constant high glucose activated telomerase and shortened telomere length, which suggested replicative senescence. Intermittent but not constant high glucose strikingly up-regulated the expression of p22phox, an NADPH oxidase component, increasing superoxide. The small interfering RNA of p22phox undermined the increase in SA-β-gal activity induced by intermittent high glucose. Conclusively, intermittent high glucose can promote vascular endothelial senescence more than constant high glucose, which is in partially dependent on superoxide overproduction.

Show MeSH
Related in: MedlinePlus