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Serum glucocorticoid inducible kinase (SGK)-1 protects endothelial cells against oxidative stress and apoptosis induced by hyperglycaemia

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Abstract

Diabetic hyperglycaemia causes endothelial dysfunction mainly by impairing endothelial nitric oxide (NO) production. Moreover, hyperglycaemia activates several noxious cellular pathways including apoptosis, increase in reactive oxygen species (ROS) levels and diminishing Na+–K+ ATPase activity which exacerbate vascular damage. Serum glucocorticoid kinase (SGK)-1, a member of the serine/threonine kinases, plays a pivotal role in regulating NO production through inducible NO synthase activation and other cellular mechanisms. Therefore, in this study, we aimed to investigate the protective role of SGK-1 against hyperglycaemia in human umbilical endothelial cells (HUVECs). We used retrovirus to infect HUVECs with either SGK-1, SGK-1Δ60 (lacking of the N-60 amino acids—increase SGK-1 activity) or SGK-1Δ60KD (kinase-dead constructs). We tested our hypothesis in vitro after high glucose and glucosamine incubation. Increase in SGK-1 expression and activity (SGK-1Δ60) resulted in higher production of NO, inhibition of ROS synthesis and lower apoptosis in endothelial cell after either hyperglycaemia or glucosamine treatments. Moreover, in this study, we showed increased GLUT-1 membrane translocation and Na+−K+ ATPase activity in cell infected with SGK-1Δ60 construct. These results suggest that as in endothelial cells, an increased SGK-1 activity and expression reduces oxidative stress, improves cell survival and restores insulin-mediated NO production after different noxae stimuli. Therefore, SGK-1 may represent a specific target to further develop novel therapeutic options against diabetic vascular disease.

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Acknowledgments

We thank Dr Suzanne Conzen, Department of Medicine section of Haematology/Oncology, University of Chicago, USA. This work has been supported by the following grants: the Italian Ministry of Health–IRCSS Monzino RF2007; Research Project 2009, Fondazione Roma, Italy; 2008 and 2010–2011 PRIN, Italian Department of Research and University; Fondazione Umberto Di Mario, Rome.

Conflict of interest

Francesca Ferrelli, Donatella Pastore, Barbara Capuani, Marco F Lombardo, Marcel Blot-Chabaud, Andrea Coppola, Katia Basello, Angelica Galli, Giulia Donadel, Maria Romano, Sara Caratelli, Francesca Pacifici, Roberto Arriga, Nicola Di Daniele, Paolo Sbraccia, Giuseppe Sconocchia, Alfonso Bellia, Manfredi Tesauro, Massimo Federici, David Della-Morte, Davide Lauro declare that they have no conflict of interest.

Ethical standard

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Human and animal rights

This article does not contain any studies with human or animal subjects performed by the any of the authors.

Informed consent

The informed consent disclosure statements is not applicable for this study since the human cells used were purchased from a biocompany.

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Authors and Affiliations

  1. Laboratory of Molecular Medicine, Department of Systems Medicine, University of Rome Tor Vergata, Montpellier, 1 Street, 00133, Rome, Italy

    Francesca Ferrelli, Donatella Pastore, Barbara Capuani, Marco F. Lombardo, Andrea Coppola, Katia Basello, Giulia Donadel, Sara Caratelli, Francesca Pacifici, Roberto Arriga, Nicola Di Daniele, Paolo Sbraccia, Alfonso Bellia, Manfredi Tesauro, Massimo Federici, David Della-Morte & Davide Lauro

  2. Faculté de Pharmacie, UMR-S608, INSERM, Université de la Méditerranée, Marseille, France

    Marcel Blot-Chabaud, Nicola Di Daniele, Alfonso Bellia & Davide Lauro

  3. Department of Medicine, Fondazione Policlinico di Tor Vergata, Rome, Italy

    Angelica Galli & Maria Romano

  4. Institute of Translational Pharmacology, National Research Council, Rome, Italy

    Sara Caratelli & Giuseppe Sconocchia

  5. IRCCS San Raffaele Pisana - Research Centre, Rome, Italy

    David Della-Morte

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Correspondence to Davide Lauro.

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592_2014_600_MOESM1_ESM.tif

Fig. S1. L-NAME inhibits SGK-1-mediated increase in NO production. NO production assayed in basal state (a) and after glucose (25mM glucose, HG), (b) or glucosamine (10mM Gluc-N ) () (c) treatments for 72 hours with insulin stimulation 10-7 M (1 hour) and with L-NAME (100 μM) 30 minutes before insulin. Supplementary material 1 (TIFF 765 kb)

592_2014_600_MOESM2_ESM.tif

Fig. S2. Mannitol treatment of infected HUVEC. Infected HUVEC were treated with mannitol 20 mM for 72 hours as osmotic control. After treatments with glucose and glucosamine, NO production (a), ROS (b) and apoptosis (c) were measured using cytofluorimetric analysis. No differences were observed among different infected cell groups. Supplementary material 2 (TIFF 673 kb)

592_2014_600_MOESM3_ESM.tif

Fig. S3. SGK-1 reduces apoptosis through Fox3a inhibition. Sub-cellular localization of FoxO3a in cytoplasm and membrane fraction, before and after insulin stimulation (10-7 M, 30 minutes after 16–18 hours of serum starvation) and/or GSK650394 treatments. In basal condition, FoxO3a distribution was predominantly cytoplasmatic in pLPCX (a), SGK-1wt (e) and SGK-1Δ60 (i) infected HUVECs cells, whereas it was predominantly nuclear in SGK-1Δ60KD (o). SGK-1 inhibitor GSK650394 induced a significant decrease of FoxO3a in the cytoplasm of SGK-1wt (g) while did not affect the FoxO3a cytoplasm levels of SGK-1Δ60 (m)-infected cells. Similar effect was present after insulin stimulus (f, l). In cells infected with SGK-1Δ60KD, FoxO3a distributions were mainly nuclear also after insulin and/or inhibitor treatments (p, r). Supplementary material 3 (TIFF 641 kb)

592_2014_600_MOESM4_ESM.tif

Fig. S4. Schematic diagram of mechanisms of endothelial cells protection mediated by overactivation of SGK1 (SGK-1Δ60). Infected HUVECs by SGK-1Δ60 respond to hyperglycaemic and insulin stimuli by the following: 1. increasing NO production and eNOS phosphorylation; 2. reducing ROS levels, 3. decreasing apoptosis, 4. increasing of FOXOa3 phosphorylation and its translocation in the cytoplasm, 5. enhancing GLUT-1 translocation; and 6. increasing in Na+-K+ ATPase activity. Supplementary material 4 (TIFF 685 kb)

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Ferrelli, F., Pastore, D., Capuani, B. et al. Serum glucocorticoid inducible kinase (SGK)-1 protects endothelial cells against oxidative stress and apoptosis induced by hyperglycaemia. Acta Diabetol 52, 55–64 (2015). https://doi.org/10.1007/s00592-014-0600-4

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