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Insulin requires A1 adenosine receptors expression to reverse gestational diabetes-increased L-arginine transport in human umbilical vein endothelium

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Abstract

Gestational diabetes mellitus (GDM) associates with increased L-arginine transport and extracellular concentration of adenosine in human umbilical vein endothelial cells (HUVECs). In this study we aim to determine whether insulin reverses GDM-increased L-arginine transport requiring adenosine receptors expression in HUVECs. Primary cultured HUVECs from full-term normal (n = 38) and diet-treated GDM (n = 38) pregnancies were used. Insulin effect was assayed on human cationic amino acid transporter 1 (hCAT1) expression (protein, mRNA, SLC7A1 promoter activity) and activity (initial rates of L-arginine transport) in the absence or presence of adenosine receptors agonists or antagonists. A1 adenosine receptors (A1AR) and A2AAR expression (Western blot, quantitative PCR) was determined. Experiments were done in cells expressing or siRNA-suppressed expression of A1AR or A2AAR. HUVECs from GDM exhibit higher maximal transport capacity (maximal velocity (V max)/apparent Michaelis Menten constant (K m), V max/K m), which is blocked by insulin by reducing the V max to values in cells from normal pregnancies. Insulin also reversed the GDM-associated increase in hCAT-1 protein abundance and mRNA expression, and SLC7A1 promoter activity for the fragment −606 bp from the transcription start point. Insulin effects required A1AR, but not A2AAR expression and activity in this cell type. In the absence of insulin, GDM-increased hCAT-1 expression and activity required A2AAR expression and activity. HUVECs from GDM pregnancies exhibit a differential requirement of A1AR or A2AAR depending on the level of insulin, a phenomenon that represent a condition where adenosine or analogues of this nucleoside could be acting as helpers of insulin biological effects in GDM.

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Abbreviations

GDM:

Gestational diabetes mellitus

NO:

Nitric oxide

eNOS:

Endothelial NO synthase

HUVECs:

Human umbilical vein endothelial cells

hPMECs:

Human placental microvascular endothelial cells

ARs:

Adenosine receptors

A1AR:

A1 adenosine receptors

A2AAR:

A2A adenosine receptors

A2BAR:

A2B adenosine receptors

A3AR:

A3 adenosine receptors

hCAT-1:

Human cationic amino acid transporter 1

hENT1:

Human equilibrative nucleoside transporters 1

siRNA:

Short interference RNAs

KDA1AR:

A1AR knockdown cells

KDA2AAR:

A2AAR knockdown cells

CHOP:

C/EBP homologous protein 10.

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Acknowledgments

Authors thank Mrs Amparo Pacheco and Mrs Ninoska Muñoz from CMPL, Pontificia Universidad Católica de Chile, for excellent technical and secretarial assistance, respectively.

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Authors and Affiliations

  1. Cellular and Molecular Physiology Laboratory (CMPL), Division of Obstetrics and Gynaecology, School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, P.O. Box 114-D, Santiago, 8330024, Chile

    Enrique Guzmán-Gutiérrez, Fabián Pardo, Andrea Leiva & Luis Sobrevia

  2. Faculty of Health Sciences, Universidad San Sebastián, Concepción, 4080871, Chile

    Enrique Guzmán-Gutiérrez & Axel Armella

  3. Department of Basic Sciences, Faculty of Sciences, Universidad del Bío-Bío, Chillán, 3780000, Chile

    Fernando Toledo

  4. Department of Physiology, Faculty of Pharmacy, Universidad de Sevilla, Seville, E-41012, Spain

    Luis Sobrevia

  5. University of Queensland Centre for Clinical Research (UQCCR), Faculty of Medicine and Biomedical Sciences, University of Queensland, Herston, QLD, 4029, Australia

    Luis Sobrevia

Authors
  1. Enrique Guzmán-Gutiérrez
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  2. Axel Armella
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  3. Fernando Toledo
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  4. Fabián Pardo
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  5. Andrea Leiva
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  6. Luis Sobrevia
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Corresponding author

Correspondence to Luis Sobrevia.

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Funding

This works was supported by Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT 1150377, 1150344, and 11150083), Chile.

Conflict of interest

The authors declare that they have no competing interests.

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Guzmán-Gutiérrez, E., Armella, A., Toledo, F. et al. Insulin requires A1 adenosine receptors expression to reverse gestational diabetes-increased L-arginine transport in human umbilical vein endothelium. Purinergic Signalling 12, 175–190 (2016). https://doi.org/10.1007/s11302-015-9491-2

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