Background: Childhood obesity is commonly associated with signs of endothelial dysfunction, characterized by impairment of insulin signaling and vascular NO availability. Recently both these features have been associated with endoplasmic reticulum (ER) stress, however the role of ER stress in the mechanism/s leading to vascular dysfunction in childhood obesity remains still to be established.
Objective and Hypotheses: To evaluate ER stress and insulin-stimulated NO availability in Human Umbilical Vein Endothelial Cells HUVECs cultured with plasma obtained from severely obese (OB) and normal-weight (C) prepubertal children.
Method: Plasma were obtained from OB- (n=15, age: 9.3±2.00; SDS BMI: 2.38±0.27) and C-children (n=14, age: 9.3±1.7; SDS BMI: 0.16±0.026). Fasting insulin and glycaemic levels were measured. HUVECs were cultured with 10% C- and OB-plasma for 24 h and their effects on Grp-78/Bip, eLF2α/phospho-eLF2α, IKBα/phospho-IKBα, NF-kB and CHOP, respectively ER stress and inflammatory markers, were quantified by flow cytometry analysis. Moreover, cells were cultivated for 72 h in the same experimental conditions and insulin (100 nM) was added during the last 24 h to determine: HUVECs viability (MTT analysis); eNOS activity (conversion of L-[3H]-arginine into L-[3H]-citrulline) and NO bioavailability (intracellular cGMP levels by EIA).
Results: OB-children presented higher fasting insulin levels (19.7±8.5 vs 6.25±1.56 mU/ml, P=0.0004) when compared to C-children. No difference was found between two groups in terms of fasting glycaemia (86.5±5.85 vs 83.3±5.85 mg/dl; P=0.829). ER stress induction was assessed by increased expression of Grp78/Bip, NF-kB, CHOP (P<0.05) and enhanced phosphorylation of eIF2α and IKBα. Moreover, insulin significantly increased eNOS activity (0.38±0.07 vs 0.19±0.02 pmoles/NO per mg prot tot, P<0.05) and cGMP levels (1.2 folds) in HUVECs treated with C-plasma, whereas it was uneffective in OB-plasma treated cells.
Conclusions: Taken together our in vitro data demonstrate that plasma from obese children are able to increased endothelial ER stress. This is associated to increased inflammation and reduced insulin-stimulated NO bioavailability.
20 - 22 Sep 2014
European Society for Paediatric Endocrinology