Journal of Steroid Biochemistry & Molecular Biology 139 (2014) 25–32 Contents lists available at ScienceDirect Journal of Steroid Biochemistry and Molecular Biology jo ur nal home page: www.elsevier.com/locate/jsbmb Estrogen reduces endoplasmic reticulum stress to protect against glucotoxicity induced-pancreatic -cell death Suwattanee Kooptiwut a,∗ , Pitchnischa Mahawong a , Wanthanee Hanchang a , Namoiy Semprasert a , Suchada Kaewin a , Thawornchai Limjindaporn b , Pa-thai Yenchitsomanus c a Department of Physiology, Faculty of Medicine Siriraj Hospital, Mahidol University, 2 Prannok Road, Bangkok 10700, Thailand b Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, 2 Prannok Road, Bangkok 10700, Thailand c Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, 2 Prannok Road, Bangkok 10700, Thailand a r t i c l e i n f o Article history: Received 15 February 2013 Received in revised form 27 September 2013 Accepted 30 September 2013 Keywords: Glucotoxicity Estrogen Pancreatic -cell Apoptosis Endoplasmic reticulum stress a b s t r a c t Estrogen can improve glucose homeostasis not only in diabetic rodents but also in humans. However, the molecular mechanism by which estrogen prevents pancreatic -cell death remains unclear. To investigate this issue, INS-1 cells, a rat insulinoma cell line, were cultured in medium with either 11.1 mM or 40 mM glucose in the presence or the absence of estrogen. Estrogen significantly reduced apoptotic -cell death by decreasing nitrogen-induced oxidative stress and the expression of the ER stress markers GRP 78, ATF6, P-PERK, PERK, uXBP1, sXBP1, and CHOP in INS-1 cells after prolonged culture in medium with 40 mM glucose. In contrast, estrogen increased the expression of survival proteins, including sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA-2), Bcl-2, and P-p38, in INS-1 cells after prolonged culture in medium with 40 mM glucose. The cytoprotective effect of estrogen was attenuated by addition of the estrogen receptor (ER and ER) antagonist ICI 182,780 and the estrogen membrane receptor inhibitor G15. We showed that estrogen decreases not only oxidative stress but also ER stress to protect against 40 mM glucose-induced pancreatic -cell death. © 2013 Elsevier Ltd. All rights reserved. Introduction Type 2 diabetes mellitus (DM) is a major health problem with an increasing prevalence worldwide [1]. Hyperglycemia simu- lates -cell dysfunction and eventually causes -cell death [2,3]. This condition is known as glucotoxicity [4]. The major mecha- nism of high glucose-induced cell death is possibly attributable to oxidative stress [5], which results from several pathways, Abbreviations: ER, endoplasmic reticulum; UPR, unfolded protein response; GRP-78, glucose-regulated protein 78; CHOP, CCAAT/enhancer-binding protein homologous protein; PI, propidium iodide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide thiazolyl blue; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; cDNA, complementary DNA; RIPA, radioim- munoprecipitation assay; BCA, bicinchoninic acid; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; Ero 1, endoplasmic reticulum oxi- doreductin I; SERCA, sarco-/endoplasmic reticulum Ca 2+ ATPase; GPER, G protein-coupled estrogen receptor; CREB, cyclic AMP response element–binding protein. ∗ Corresponding author. Tel.: +66 2 419720; fax: +66 2 4115009. E-mail addresses: siskw@mahidol.ac.th, S kooptiwut@hotmail.com (S. Kooptiwut), pornma79@gmail.com (P. Mahawong), sinmn@mahidol.ac.th (N. Semprasert), thawornchai.lim@mahidol.ac.th (T. Limjindaporn), ptyench@gmail.com (P.-t. Yenchitsomanus). including substrate-driven overproduction of mitochondrial reac- tive oxygen species, advanced glycation end product formation and glucose auto-oxidation [6]. Several lines of evidence suggest that endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of pancreatic -cell dysfunction and cell death [7–9]. The ER is a cellular organelle that has an important function in folding, assembling and delivering newly synthesized proteins to their target locations. Pancreatic -cells contain a well-developed ER because they normally synthesize and secrete a large quantity of insulin [10]. During insulin resistance or hyperglycemic conditions, ER function is increased as a result of enhanced insulin produc- tion. However, exceeding the functional capacity of ER leads to a large quantity of unfolded proteins, thereby generating ER stress [11,12]. The accumulation of unfolded proteins stimulates the unfolded protein response (UPR), which is an adaptive mechanism to decrease the amount of unfolded proteins [13]. UPR up-regulates glucose-regulated protein 78 (GRP78) to facilitate unfolded pro- tein breakdown and reduces ER stress [14]. In case of chronic or unresolved ER stress, apoptosis is triggered [15]. UPR stimulates apoptosis by activating CCAAT/enhancer-binding protein homolo- gous protein (CHOP), which is a pro-apoptotic transcription factor [16]. Then, activated CHOP alters gene expression [17]. 0960-0760/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jsbmb.2013.09.018