Baicalein protects rat insulinoma INS-1 cells from palmitate-induced lipotoxicity by inducing HO-1

RESEARCH ARTICLE Baicalein protects rat insulinoma INS-1 cells from palmitate-induced lipotoxicity by inducing HO-1 Hyun Jeong Kwak1, Dongki Yang2, Yongha Hwang3, Hee-Sook Jun3, Hyae Gyeong Cheon1,4* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Pharmacology, Gachon University College of Medicine, Incheon, Republic of Korea, 2 Department of Physiology, Gachon University College of Medicine, Incheon, Republic of Korea, 3 College of Pharmacy and Gachon Institute of Pharmaceutical Science, Gachon University, Incheon, Republic of Korea, 4 Gachon Medical Research Institute, Gil Medical Center, Incheon, Republic of Korea * Abstract OPEN ACCESS Citation: Kwak HJ, Yang D, Hwang Y, Jun H-S, Cheon HG (2017) Baicalein protects rat insulinoma INS-1 cells from palmitate-induced lipotoxicity by inducing HO-1. PLoS ONE 12(4): e0176432. https://doi.org/10.1371/journal.pone.0176432 Editor: Salvatore V. Pizzo, Duke University School of Medicine, UNITED STATES Received: December 6, 2016 Objective β-Cell dysfunction plays a central role in the pathogenesis of type 2 diabetes (T2D), and the identification of novel approaches to improve β-cell function is essential to treat this disease. Baicalein, a flavonoid originally isolated from the root of Scutellaria Baicalensis, has been shown to have beneficial effects on β-cell function. Here, the authors investigated the molecular mechanism responsible for the protective effects of baicalein against palmitate (PA)-induced impaired β-cell function, and placed focus on the role of heme oxygenase (HO)-1. Accepted: April 11, 2017 Published: April 26, 2017 Methods Copyright: © 2017 Kwak et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Rat pancreatic β-cell line INS-1 cells or mouse pancreatic islets were cultured with PA (500 μM) to induce lipotoxicity in the presence or absence of baicalein (50 μM), and the expressions of the ER stress markers, ATF-3, CHOP and GRP78 were detected by Western blotting and/or qPCR. The involvement of HO-1 was evaluated by HO-1 siRNA transfection and using the HO-1 inhibitor ZnPP. Data Availability Statement: All relevant data are within the paper. Funding: This research was supported by a grant from the Korean Health Technology R&D Project through the Korean Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI14C1135), and by Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (Grant no. 2015R1D1A1A01058235). The funders had no role in study design, data Results Baicalein reduced PA-induced ER stress and inflammation and enhanced insulin secretion, and these effects were associated with the induction of HO-1. Furthermore, these protective effects were attenuated by ZnPP and by HO-1 siRNA. Pretreatment of PD98059 (an ERK inhibitor) significantly inhibited the protective effects of baicalein and blocked HO-1 induction. On the other hand, CO production by RuCO (a CO donor) ameliorated PA-induced ER stress, suggesting that CO production followed by HO-1 induction may contribute to the protective effects of baicalein against PA-induced β-cell dysfunction. PLOS ONE | https://doi.org/10.1371/journal.pone.0176432 April 26, 2017 1 / 15 Beta cell protection by baicalein-mediated HO-1 upregulation collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Conclusion Baicalein protects pancreatic β-cells from PA-induced ER stress and inflammation via an ERK-HO-1 dependent pathway. The authors suggest HO-1 induction in pancreatic β-cells appears to be a promising therapeutic strategy for T2D. Introduction Pancreatic β-cell dysfunction plays a central role in the development of diabetes mellitus, and it has been documented that chronic exposure to elevated free fatty acid (FFA) levels causes β-cell dysfunction and apoptosis [1]. Although the mechanism of lipotoxicity has not been fully clarified, accumulating evidence suggests the induction of endoplasmic reticulum (ER) stress might contribute to the detrimental effects of FFA on β-cells [2]. ER is a specialized organelle responsible for the synthesis, initial posttranslational modification, folding, and export of secretion of membrane proteins. When ER homeostasis is disrupted by biochemical, physiological, or pathological stimuli, unfolded or misfolded proteins accumulate in ER lumen, overwhelm ER chaperons, such as, glucose-regulated protein 78 (GRP78), and cause ER stress. Moderate ER stress may represent a defense mechanism against external stimuli, but excessive or persistent ER stress eventually triggers programmed cell death or apoptosis by activating the caspase-12, C/EBP homology protein (CHOP), or the c-Jun-N-terminal kinase (JNK)-dependent pathway [3]. Recently, considerable evidence has been presented that suggests ER stress contributes to FFAinduced β-cell dysfunction by upregulating pro-apoptotic effectors, such as, CHOP [4]. Consequently, pharmacological agents that alleviate ER stress may provide novel means of protecting β-cells from lipotoxicity. Baicalein is a flavonoid that exerts broad pharmacological effects, including anti-inflammatory and antioxidant effects, and was originally isolated from the roots of Scutellaria Baicalensis [5]. Recently, much interest has been focused on the antidiabetic effects of baicalein. In particular, it has been demonstrated that baicalein improves hyperglycemia and glucose intolerance, and promotes insulin secretion by inhibiting islet cell apoptosis in streptozotocin-induced diabetic mice [6]. Heme oxygenase-1 (HO-1) is an anti-inflammatory molecule that catalyzes heme breakdown to carbon monoxide, biliverdin, and iron, and is induced by numerous stressors including endotoxin, cytokines, and oxidants [7]. In humans and mice, loss of HO-1 function in Kupffer cells and PBMCs leads to early death due to increased susceptibility to inflammation [8–10]. Interestingly, several studies have shown that chemical inducers of HO-1 ameliorate obesity and diabetes in different models [11]. Furthermore, efforts to identify pharmacological or natural agents with HO-1-inducing ability were initiated after it was reported HO-1 induction in islets afforded protection from the detrimental effects of pro-inflammatory cytokines [12]. The iron produced by the catalytic breakdown of HO-1 produces ROS, and bilirubin has been reported to have both cytotoxic and cytoprotective effects [13, 14]. However, CO has vasodilatory effects, and inhalation of CO has been reported to protect tissues against hyperoxia and to reduce obesity, hyperglycemia, and insulin resistance [15 (...truncated)