www.nature.com/scientificreports OPEN Glycation of paraoxonase 1 by high glucose instigates endoplasmic reticulum stress to induce Received: 21 October 2016 Accepted: 03 March 2017 endothelial dysfunction in vivo Published: 04 April 2017 Wei Yu1,2,*, Xiaoli Liu2,*, Liru Feng2, Hui Yang2, Weiye Yu2, Tiejian Feng2, Shuangxi Wang3, Jun Wang2 & Ning Liu1 High-density lipoprotein (HDL) modulates low-density lipoprotein and cell membrane oxidation through the action of paraoxonase-1 (PON1). Endoplasmic reticulum (ER) stress has been linked to a wide range of human pathologies including diabetes, obesity, and atherosclerosis. Previous studies have reported that PON1 is glycated in diabetes. The aim of this study is to investigate whether and how PON1 glycation contributes to endothelial dysfunction in diabetes. ER stress markers were monitored by western blot. Endothelial function was determined by organ bath. Incubation of recombinant PON1 proteins with high glucose increased PON1 glycation and reduced PON1 activity. Exposure of HUVECs to glycated PON1 induced prolonged ER stress and reduced SERCA activity, which were abolished by tempol, apocynin, BAPTA, and p67 and p22 siRNAs. Chronic administration of amino guanidine or 4-PBA prevented endothelial dysfunction in STZ-injected rats. Importantly, injection of glycated PON1 but not native PON1 induced aberrant ER stress and endothelial dysfunction in rats, which were attenuated by tempol, BAPTA, and 4-PBA. In conclusion, glycation of PON1 by hyperglycemia induces endothelial dysfunction through ER stress. In perspectives, PON1 glycation is a novel risk factor of hyperglycemia-induced endothelial dysfunction. Therefore, inhibition of oxidative stress, chelating intracellular Ca2+, and ER chaperone would be considered to reduce vascular complications in diabetes. Diabetes mellitus is usually associated with the development of atherosclerosis and nephropathy, which is char- acterized by endothelial dysfunction1,2. Advanced glycation end-products (AGEs) are a heterogeneous group of products which protein and lipids are covalently bound to sugar residues under hyperglycemic and oxidative stress situations, which is proposed to play a major role in the pathogenesis of diabetic complications3. High-density lipoprotein (HDL) associated paraoxonase-1 (PON1) is primarily responsible for the anti-oxidative properties of HDL in retarding the oxidation of low-density lipoprotein (LDL) and cell mem- branes4–6. By modulating the oxidation of LDL, PON1 abolishes the ox-LDL-stimulated induction of monocyte-chemotactic protein-1 (MCP1) produced by endothelial cells, thereby preventing monocyte/endothe- lial cell interaction in one of the earliest processes of atherosclerosis7,8. PON1 is low in subjects with diabetes, leading to dysfunctional HDL with impaired antioxidant capacity9–11. In diabetes, there is an inverse relationship between PON1 activity and circulating oxidized LDL levels, indicative of the major role of PON1 in retarding LDL oxidation12,13. Glycation of paraoxonase-1 inhibits its activity and impairs the ability of HDL to metabolize membrane lipid hydroperoxides14. It has been reported that dysfunction of PON is related to vascular oxidative stress15 and vascular damage5,16,17. However, the mechanism needs to be defined. The normal endoplasmic reticulum (ER) is the principal site of protein synthesis, folding, and maturation. ER stress has been linked to a wide range of human pathologies including diabetes, obesity, atherosclerosis, cancer, neurodegenerative disorders, and inflammatory conditions18,19. ER stress may be triggered by hyperglycemia, oxi- dative stress, Ca2+ overload, ischemia, and hypoxia. In normal condition, unfolded or misfolded proteins in ER 1Central Laboratory, Second Hospital, Jilin University, Changchun 130041, China. 2Shenzhen Center for Chronic Disease Control, Shenzhen 518020, China. 3Department of Pharmacology, College of Pharmacy, Xinxiang Medical University, Xinxiang, 453003, China. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to J.W. (email: [email protected]) or N.L. (email: [email protected]) SCIENTIFIC REPORTS | 7:45827 | DOI: 10.1038/srep45827 1 www.nature.com/scientificreports/ are sent to the cytoplasm by a “retro-translocation mechanism” to be degraded by the ubiquitin proteasome sys- tem20. However, ER stress causes the accumulation of unfolded and misfolded proteins, leading to an “unfolded protein response (UPR)”, resulting in cellular dysfunctions21. Previous studies have shown that glycation of LDL triggered ER Stress and induced endothelial dysfunction22,23. Based on the literature evidence, we hypothesized that PON1 glycation may promote endothelial dysfunction via ER stress. In this study, we reported that recombinant PON1 protein was glycated by high glucose in vitro. Glycated PON1 (Gly-PON1) instigated ER stress via the oxidation and inhibition of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) in endothelial cells and induced endothelial dysfunction in rats. In perspectives, PON1 glycation is a risk factor of endothelial dysfunction in diabetes. Materials and Methods Materials. Antibodies against phospho-eukaryotic translation initiation factor 2α (eIF2α ), and 3-nitrotry- osine (3-NT) were obtained from Cell Signaling Biotechnology (Danvers, MA). The antibodies against phos- pho-PKR (protein kinase R)-like ER kinase (PERK), CHOP, ATF6, BIP, SERCA, scrambled small interfering RNA (siRNA), and the specific siRNA for p67 and p22 were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Amino guanidine (AG), streptozotocin (STZ), tempol, 1,2-bis (2-aminophenoxy) ethane-N4-tetraacetic acid (BAPTA), 4-phenyl butyric acid (4-PBA), tunicamycin, D-glucose, acetylcholine (ACh), sodium nitroprus- side (SNP), phenylephrine (PE) and dihydroethidium (DHE) were purchased from Sigma-Aldrich Company or Caymen chemical Company. Fluo-4 NW kits were obtained from Invitrogen Inc. (Carlsbad, CA). All other chemicals, if not indicated, were purchased from Sigma-Aldrich (St. Louis, MO). Preparation of glycated of PON1. To prepare glycated PON1, recombinant PON1 protein (10 μ g) from Abcam Company was incubated with 0.3 mmol/l EDTA at 37 °C, pH 7.4 for 3 days in freshly prepared D-glucose. For normal and glycated PON1, 1 mmol/l DTPA was also added and incubations were under nitrogen. Modification was terminated by repeat extensive dialysis as described above24. Highly glycated PON1 was gener- ated by buffer exchange of native PON1 into PBS, pH 7.4, dilution to 3 mg/ml protein, and addition of CuCl2 to a final concentration of 10 μmol/l for 24 hours, under air at 37 °C. PON1 preparations were sterile filtered (0.22 μm), stored in the dark under nitrogen at 4 °C, and used within 1 month of preparation. The PON1 pools were tested for endotoxin contamination by the Limulus Amebocyte Lysate (Bio-Whittaker, Walkersville, MD) according to the manufacturer’s suggestion. Determination of PON1 activity. As described previously14, PON1 activity was measured by adding 20 μL of sample to Tris buffer (100 mmol/L, pH 8.0) containing 2 mmol/l CaCl2 and 1 mmol/L paraoxon (Sigma). PON1 activity was measured using phenylacetate as a substrate and the reaction mixture contained 750 μ L of 0.1 mol/L Tris- HCl (pH 8.5), 1 mmol/L CaCl2, 125 μ L of 12 mmol/L phenylacetate and 125 μ L of diluted serum with water (1:10). Initial rates of hydrolysis were determined by following the increase of phenol concentration at 270 nm at 37 °C. Enzyme activities were expressed in international units per 1 liter of serum (U/L). An international unit is the amount of hydrolyzed substrate in mmol/minute. Cell cultures. Human umbilical vein endothelial cells (HUVECs) were grown in EBM (Clonetics Inc. Walkersville, MD) supplemented with 2% fetal bovine serum, penicillin (100 u/ml), and streptomycin (100 μ g/ml). In all experiments, cells were between passages 3 and 8. All cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Cells were grown to 70–80% confluency before being treated with different agents. Transfection of siRNA into cells. Transient transfection of siRNA was carried out according to Santa Cruz’s protocol25. Briefly, the siRNAs were dissolved in siRNA buffer (20 mM KCl; 6 mM HEPES, pH 7.5; 0.2 mM MgCl2) to prepare a 10 μ M stock solution. Cells grown in 6-well plates were transfected with siRNA in transfec- tion medium containing liposomal transfection reagent (Lipofectamine RNAiMax, Invitrogen, Shanghai branch, China). For each transfection, 100 μl transfection medium containing 4 μl siRNA stock solution was gently mixed with 100 μ l transfection medium containing 4 μ l transfection reagent. After 30-min incubation at room tempera- ture, siRNA-lipid complexes were added to the cells in 1.0 ml transfection medium, and cells were incubated with this mixture for 6 h at 37 °C. The transfection medium was then replaced with normal medium, and cells were cultured for 48 h. Western blotting. As described previously26, cells or aortic tissues were homogenized on ice in cell-lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 μ g/ml leupeptin, and 1 mM PMSF). Cell was lysated with cell-lysis buffer. The protein content was assayed by BCA protein assay reagent (Pierce, USA). 20 μ g proteins were loaded to SDS-PAGE and then transferred to membrane. Membrane was incubated with