Lack of insulin response in human umbilical vein endothelial cells from pregestational maternal obesity may result from endoplasmic reticulum stress

摘要

Pregestational maternal obesity (PGMO) is associated with adverse cardio-metabolic newborn outcome. PGMO also causes insulin-desensitization in human umbilical vein endothelial cells (HUVECs). The endoplasmic reticulum stress (ERS) has been related to the development of obesity-associated insulin resistance. However, whether ERS is present and involved in insulin-desensitization in HUVECs from PGMO is unknown. Objective: (1) To assay whether HUVECs from women with PGMO show increased ERS markers, and (2) to evaluate the involvement of ERS in insulin-induced nitric oxide (NO) synthesis in HUVECs. Methods: HUVECs were isolated from normal or PGMO pregnancies from the Hospital Clínico UC-CHRISTUS (Chile). Cells were incubated (8 hours) in absence or presence of tunicamycin (5 μmol/L, ERS inducer), tauroursodeoxy-cholic acid (TUDCA, 100 μmol/L, ERS reducer), or both. We evaluated the protein level of CCAAT-enhancer-binding protein homologous protein (CHOP), tribbles-like protein 3 (TRB3), and phosphorylation and total protein level of protein kinase RNA-like endoplasmic reticulum kinase (PERK), eukaryotic translation initiator factor 2-alpha (eIF2α), inositol-requiring enzyme 1-alpha (IRE1α), and c-jun N-terminal kinase 1 (JNK1) by western blot. X-box binding protein 1 (XBP1) mRNA processing was evaluated by PCR. Synthesis of NO was induced by incubation of cells with insulin (1 nmol/L, 20 min) and measured by 4-amino-5-methylamino-2', 7'-difluorofluorescein diacetate (DAF-FM) fluorescence. Results: Activator phosphorylation of PERK (1.7 ±0.3 fold) and eIF2α (1.6 ±0.3 fold), and protein abundance of CHOP (2.5 ±0.5 fold) and TRB3 (1.5 ±0.2 fold) were increased (P< 0.05) in HUVECs from PGMO compared with normal pregnancies. Tunicamycin increased the levels of all ERS markers in cells from normal but not from PGMO pregnancies. Conversely, TUDCA reversed PGMO-increased levels of ERS markers, but did not alter the levels of all ERS markers in HUVECs from normal. Activator phosphorylation of IRE1α and JNK1 were unaltered, and there was not splicing of XBP1 mRNA. Finally, NO synthesis was increased (2.9 ±0.2 fold, P< 0.05) in response to insulin by HUVECs from normal, but not from PGMO pregnancies. Tunicamiycin reduced insulin-increased NO synthesis in cells from normal, but not from PGMO pregnancies. TUDCA did not alter insulin-induced NO synthesis neither in normal nor in PGMO pregnancies. Conclusions: HUVECs from women with PGMO show ERS by activation of PERK branch, suggesting that PERK branch-associated ERS could results in PGMO reduced insulin sensi-tization. The increase of TRB3 protein level suggests a potential role for this protein as inductor of insulin desensitization in this type of foetoplacental endothelium. Finally, ERS is a PGMO-associated condition that induces insulin desensitization in HUVECs.