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Increased O-Linked N-Acetylglucosamine Modification of NF-ΚB and Augmented Cytokine Production in the Placentas from Hyperglycemic Rats

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Abstract

Inflammation as a result of NF-κB activation may result from the classical (canonical) pathway, with disconnection of the IκB inhibitor and subsequent nuclear translocation or, alternatively, by post-translational modifications of modulatory proteins or NF-κB subunits (non-canonical pathway). We hypothesized that hyperglycemia-induced increased glycosylation with O-linked N-acetylglucosamine (O-GlcNAc) of NF-κB in placental tissue leads to augmented production of pro-inflammatory cytokines, culminating in placental dysfunction and fetal restriction growth. Single injections of streptozotocin (40 mg/kg) or vehicle were used to induce hyperglycemia or normoglycemia, respectively, in female Wistar rats. After 3 days, rats were mated and pregnancy confirmed. Placental tissue was collected at 21 days of pregnancy. Placental expression of p65 subunit was similar between groups. However, nuclear translocation of p65 subunit, showing greater activation of NF-κB, was increased in the hyperglycemic group. Reduced expression of IκB and increased expression of phosphorylated IκBSer32 were observed in the placenta from hyperglycemic rats, demonstrating increased classical NF-κB activation. Augmented modification of O-GlcNAc-modified proteins was found in the placenta from hyperglycemic rats and p65 subunit was a key O-GlcNAc target, as demonstrated by immunoprecipitation. Tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) expressions were increased in the placenta from hyperglycemic rats. Furthermore, placental weight was increased, whereas fetal weight was decreased under hyperglycemic conditions. TNF-α and IL-6 demonstrated positive correlations with placental weight and negative correlations with fetal weight and placental efficiency. Therefore, under hyperglycemic conditions, a modulatory role of O-GlcNAc in NF-κB activity was demonstrated in the placenta, contributing to fetal and placental dysfunction due to inflammatory cytokine exacerbation.

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Acknowledgments

This study was supported by the Fundação de Amparo à Pesquisa do Estado de Mato Grosso (FAPEMAT, 151371/2014 to F.R.G.; 211997/2015 to V.V.L.), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, 23038009165/2013-48 to V.V.L.), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, 471675/2013-0 and 305823/2015-9 to F.R.G; 445777/2014-1 to V.V.L.).

Author Contribution Statement

VDJ, JSG, and RAF performed the molecular experiments. VDJ and ADF conducted cytokine measurements. VDJ, FRG, and VVL performed the statistical analysis. FRG designed the hypothesis. RCT, FSC, VVL, and FRG provided the financial support, supervised the study, and continuously contributed with ideas and expertise for the project and revisions for the paper.

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Authors and Affiliations

  1. Institute of Biological Sciences and Health, Federal University of Mato Grosso, Av. Valdon Varjão, Barra do Garças, MT, 78600-000, Brazil

    Vanessa Dela Justina, Jéssica S. Gonçalves, Raiany Alves de Freitas, Adriany Dias Fonseca, Gustavo T. Volpato, Victor Vitorino Lima & Fernanda R. Giachini

  2. Ribeirão Preto Medical School, University of Sao Paulo, Ribeirão Preto, SP, Brazil

    Rita C. Tostes & Fernando S. Carneiro

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  1. Vanessa Dela Justina
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  8. Victor Vitorino Lima
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Correspondence to Victor Vitorino Lima.

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All procedures were performed in accordance with the Guiding Principles in the Care and Use of Animals, approved by the Ethics Committee on Animal Research (CEUA) of the Federal University of Mato Grosso (23108.120946/2015-83).

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The authors declare that they have no conflicts of interest.

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Dela Justina, V., Gonçalves, J.S., de Freitas, R.A. et al. Increased O-Linked N-Acetylglucosamine Modification of NF-ΚB and Augmented Cytokine Production in the Placentas from Hyperglycemic Rats. Inflammation 40, 1773–1781 (2017). https://doi.org/10.1007/s10753-017-0620-7

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