Oscillating glucose induces microRNA-185 and impairs an efficient antioxidant response in human endothelial cells

Lucia La Sala, Monica Cattaneo, Valeria De Nigris, Gemma Pujadas, Roberto Testa, Anna Rita Bonfigli, Stefano Genovese, Antonio Ceriello

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

Background: Intracellular antioxidant response to high glucose is mediated by Cu/Mn-superoxide dismutases (SOD-1/SOD-2), catalase (CAT) and glutathione peroxidases (GPx), particularly glutathione peroxidase-1 (GPx-1). Although oscillating glucose can induce a more deleterious effect than high glucose on endothelial cells, the mechanism by which oscillating glucose exerts its dangerous effects is incompletely understood; however, the involvement of oxidative damage has been generally accepted. In this study we sought to determine whether oscillating glucose differentially modulates antioxidant response, and to elucidate the potential regulatory mechanisms exerted by the microRNA-185 (miR-185). Methods: Human endothelial cells were exposed for 1 week to constant and oscillating high glucose. SOD-1, SOD-2, CAT and GPx-1, as well as two markers of oxidative stress [8-hydroxy-2'-deoxyguanosine (8-OHdG) and the phosphorylated form of H2AX (γ-H2AX)] were measured at the end of the experiment. Intracellular miR-185 was measured and loss-of function assays were performed in HUVEC. Bioinformatic tool was used to predict the link between miR-185 on 3'UTR of GPx-1 gene. Luciferase assay was performed to confirm the binding on HUVEC. Results: After exposure to constant high glucose SOD-1 and GPx-1 increased, while in oscillating glucose SOD-1 increased and GPx-1 did not. SOD-2 and CAT remained unchanged under both conditions. A critical involvement of oscillating glucose-induced miR-185 in the dysregulation of endogenous GPx-1 was found. Computational analyses predict GPx-1 as miR-185's target. HUVEC cultures were used to confirm glucose's causal role on the expression of miR-185, its target mRNA and protein and finally the activation of antioxidant response. In vitro luciferase assays confirmed computational predictions targeting of miR-185 on 3'-UTR of GPx-1 mRNA. Knockdown of miR-185, using anti-miR-185 inhibitor, was accompanied by a significant upregulation of GPx-1 in oscillating glucose. 8-OHdG and γ-H2AX increased more in oscillating glucose than in constant high glucose. Conclusions: Glucose oscillations may exert more deleterious effects on the endothelium than high glucose, likely due to an impaired response of GPx-1, coupled by the upregulation of miR-185.

Original languageEnglish
Article number71
JournalCardiovascular Diabetology
Volume15
Issue number1
DOIs
Publication statusPublished - 2016

Fingerprint

MicroRNAs
Endothelial Cells
Antioxidants
Glucose
Catalase
3' Untranslated Regions
Luciferases
Superoxide Dismutase
Up-Regulation
glutathione peroxidase GPX1
Messenger RNA
Glutathione Peroxidase
Computational Biology
Endothelium
Oxidative Stress

Keywords

  • Antioxidant defense
  • GPx-1
  • MiR-185
  • Oscillating glucose
  • Oxidative stress

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Cardiology and Cardiovascular Medicine

Cite this

Oscillating glucose induces microRNA-185 and impairs an efficient antioxidant response in human endothelial cells. / La Sala, Lucia; Cattaneo, Monica; De Nigris, Valeria; Pujadas, Gemma; Testa, Roberto; Bonfigli, Anna Rita; Genovese, Stefano; Ceriello, Antonio.

In: Cardiovascular Diabetology, Vol. 15, No. 1, 71, 2016.

Research output: Contribution to journalArticle

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keywords = "Antioxidant defense, GPx-1, MiR-185, Oscillating glucose, Oxidative stress",
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T1 - Oscillating glucose induces microRNA-185 and impairs an efficient antioxidant response in human endothelial cells

AU - La Sala, Lucia

AU - Cattaneo, Monica

AU - De Nigris, Valeria

AU - Pujadas, Gemma

AU - Testa, Roberto

AU - Bonfigli, Anna Rita

AU - Genovese, Stefano

AU - Ceriello, Antonio

PY - 2016

Y1 - 2016

N2 - Background: Intracellular antioxidant response to high glucose is mediated by Cu/Mn-superoxide dismutases (SOD-1/SOD-2), catalase (CAT) and glutathione peroxidases (GPx), particularly glutathione peroxidase-1 (GPx-1). Although oscillating glucose can induce a more deleterious effect than high glucose on endothelial cells, the mechanism by which oscillating glucose exerts its dangerous effects is incompletely understood; however, the involvement of oxidative damage has been generally accepted. In this study we sought to determine whether oscillating glucose differentially modulates antioxidant response, and to elucidate the potential regulatory mechanisms exerted by the microRNA-185 (miR-185). Methods: Human endothelial cells were exposed for 1 week to constant and oscillating high glucose. SOD-1, SOD-2, CAT and GPx-1, as well as two markers of oxidative stress [8-hydroxy-2'-deoxyguanosine (8-OHdG) and the phosphorylated form of H2AX (γ-H2AX)] were measured at the end of the experiment. Intracellular miR-185 was measured and loss-of function assays were performed in HUVEC. Bioinformatic tool was used to predict the link between miR-185 on 3'UTR of GPx-1 gene. Luciferase assay was performed to confirm the binding on HUVEC. Results: After exposure to constant high glucose SOD-1 and GPx-1 increased, while in oscillating glucose SOD-1 increased and GPx-1 did not. SOD-2 and CAT remained unchanged under both conditions. A critical involvement of oscillating glucose-induced miR-185 in the dysregulation of endogenous GPx-1 was found. Computational analyses predict GPx-1 as miR-185's target. HUVEC cultures were used to confirm glucose's causal role on the expression of miR-185, its target mRNA and protein and finally the activation of antioxidant response. In vitro luciferase assays confirmed computational predictions targeting of miR-185 on 3'-UTR of GPx-1 mRNA. Knockdown of miR-185, using anti-miR-185 inhibitor, was accompanied by a significant upregulation of GPx-1 in oscillating glucose. 8-OHdG and γ-H2AX increased more in oscillating glucose than in constant high glucose. Conclusions: Glucose oscillations may exert more deleterious effects on the endothelium than high glucose, likely due to an impaired response of GPx-1, coupled by the upregulation of miR-185.

AB - Background: Intracellular antioxidant response to high glucose is mediated by Cu/Mn-superoxide dismutases (SOD-1/SOD-2), catalase (CAT) and glutathione peroxidases (GPx), particularly glutathione peroxidase-1 (GPx-1). Although oscillating glucose can induce a more deleterious effect than high glucose on endothelial cells, the mechanism by which oscillating glucose exerts its dangerous effects is incompletely understood; however, the involvement of oxidative damage has been generally accepted. In this study we sought to determine whether oscillating glucose differentially modulates antioxidant response, and to elucidate the potential regulatory mechanisms exerted by the microRNA-185 (miR-185). Methods: Human endothelial cells were exposed for 1 week to constant and oscillating high glucose. SOD-1, SOD-2, CAT and GPx-1, as well as two markers of oxidative stress [8-hydroxy-2'-deoxyguanosine (8-OHdG) and the phosphorylated form of H2AX (γ-H2AX)] were measured at the end of the experiment. Intracellular miR-185 was measured and loss-of function assays were performed in HUVEC. Bioinformatic tool was used to predict the link between miR-185 on 3'UTR of GPx-1 gene. Luciferase assay was performed to confirm the binding on HUVEC. Results: After exposure to constant high glucose SOD-1 and GPx-1 increased, while in oscillating glucose SOD-1 increased and GPx-1 did not. SOD-2 and CAT remained unchanged under both conditions. A critical involvement of oscillating glucose-induced miR-185 in the dysregulation of endogenous GPx-1 was found. Computational analyses predict GPx-1 as miR-185's target. HUVEC cultures were used to confirm glucose's causal role on the expression of miR-185, its target mRNA and protein and finally the activation of antioxidant response. In vitro luciferase assays confirmed computational predictions targeting of miR-185 on 3'-UTR of GPx-1 mRNA. Knockdown of miR-185, using anti-miR-185 inhibitor, was accompanied by a significant upregulation of GPx-1 in oscillating glucose. 8-OHdG and γ-H2AX increased more in oscillating glucose than in constant high glucose. Conclusions: Glucose oscillations may exert more deleterious effects on the endothelium than high glucose, likely due to an impaired response of GPx-1, coupled by the upregulation of miR-185.

KW - Antioxidant defense

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KW - Oscillating glucose

KW - Oxidative stress

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