Olanzapine-induced endoplasmic reticulum stress and inflammation in the hypothalamus were inhibited by an ER stress inhibitor 4-phenylbutyrate
Introduction
Antipsychotics (APs) are widely used in the treatment of schizophrenia. However, APs particularly olanzapine and clozapine, are associated with weight gain and other metabolic side-effects. Previous studies have suggested that multiple factors are involved in AP-induced weight gain, including the histamine H1 receptor and its associated 5′ AMP-activated protein kinase (AMPK) signaling (He et al., 2014), H3 receptor (Lian et al., 2014; Poyurovsky et al., 2013, 2005), serotonin receptor 2C (Lord et al., 2017), ghrelin-mediated growth hormone secretagog receptor (GHS-R) signaling (Tagami et al., 2016; Zhang et al., 2014b), and neuropeptides (Huang et al., 2006; Zhang et al., 2014b). It has long been established that the cannabinoid receptor 1 (CB1 receptor) is involved in the metabolic mechanisms of food intake (Lazzari et al., 2011; Manca et al., 2013; Mastinu et al., 2012, 2013). Recently, a study in female rats reported that olanzapine-induced food intake and weight gain was markedly inhibited by the CB1 receptor inverse agonists rimonabant or NESS06SM (Lazzari et al., 2017), suggesting the importance of the CB1 receptor in AP-induced weight gain. Moreover, interesting research demonstrated that the microbial fermentation of prebiotics, B-GOS® attenuated olanzapine-induced weight gain in female rats (Kao et al., 2018). However, in the clinic, there is still a lack of medication for preventing and treating AP-induced weight gain. To find out the interaction of APs with other signaling pathways that mediate energy balance is very important for treating AP-induced weight gain and for development of novel non-obesogenic APs.
The endoplasmic reticulum (ER) is a continuous membrane system in the cells of eukaryotic organisms. ER stress refers to a pathophysiological process in which ER function is disordered under conditions of hypoxia, oxidative stress, undernutrition or imbalance of calcium ion homeostasis (Cnop et al., 2012). ER stress is mediated by three proteins: protein kinase R-like ER kinase (PERK), inositol requiring enzyme 1(IRE1) and activating transcription factor 6 (ATF6) (Cnop et al., 2012). Under non-stress conditions, the three proteins are inactive in combination with glucose-regulated protein 78 (GRP78). GRP78 is also referred to as the immunoglobulin heavy chain-binding protein (BiP) (Lee, 2005). When ER stress occurs, PERK, IRE1, and ATF6 dissociate from GRP78, and PERK is then activated by phosphorylation. pPERK thereby phosphorylates the downstream eukaryotic initiation factor 2α (eIF2α) to inhibit the translation and synthesis of transcription factor 4 (ATF4). ATF4 up-regulates GRP78/BiP expression (PERK-eIF2α signaling), and this effect contributes to the restoration of endoplasmic reticulum homeostasis (Cnop et al., 2012). Therefore, up-regulation of GRP78/BiP expression is a widely used marker of ER stress (Cnop et al., 2012). It is noteworthy that activation of hypothalamic ER stress PERK- eIF2α signaling leads to obesity (Cnop et al., 2012; Ozcan et al., 2009; Ramirez and Claret, 2015; Zhang et al., 2008). In high-fat diet (HFD) induced obese rodents (both males and females), the expression of pPERK, peIF2α and GRP78/BiP in the hypothalamus was significantly increased (Cakir et al., 2013; Melo et al., 2014). In male mice model, activation of hypothalamic ER stress leads to hyperphagia and inhibition of intra-capsular brown adipose tissue (IBAT) decomposition and heat production, resulting in severe obesity (Ozcan et al., 2009; Qiang et al., 2017). Conversely, inhibition of the hypothalamic ER stress by 4-phenylbutyrate (4-PBA) and tauroursodeoxycholic acid (TUDCA) reduces food intake and increases heat production in IBAT in male obese mice, thereby inhibiting obesity (Melo et al., 2014; Ozcan et al., 2009; Ramirez and Claret, 2015)
In the pathological process of obesity, hypothalamic ER stress is related to inflammation (Zhang et al., 2008). It has been demonstrated that activation of hypothalamic PERK- eIF2α signaling activates nuclear factor kappa B (NFκB), one of the most important transcriptional regulators in inflammatory response (Zhang et al., 2008). HFD feeding significantly activated hypothalamic ER stress and IKKβ-NFκB signaling, whereas intraventricular injection of an ER stress inhibitor TUDCA markedly inhibited hypothalamic IKKβ-NFκB signaling, accompanied by decreased food intake and weight gain in mice (gender not shown) (Zhang et al., 2008). Activation of hypothalamic IKKβ-NFκB signaling by constitutively active IKKβ cloned lentiviral vector induced increased food intake and weight gain in mice. On the contrary, inhibition of hypothalamic IKKβ-NFκB signaling by dominant-negative IKKβ cloned lentiviral vector significantly protected mice from obesity (Zhang et al., 2008). These results suggested that overnutrition activates hypothalamic IKKβ-NFκB signaling at least partly via elevating hypothalamic ER stress. Moreover, it has been found in male mice that NFκB activation increased the transcriptional activity of pro-opiomelanocortin (POMC) in the hypothalamus, suggesting involvement of NFκB in feeding control (Jang et al., 2010). Furthermore, the involvement of NFκB in a male murine model of neurodevelopment disorder has been reported recently (Bonini et al., 2016).
Previous studies have illustrated that the most obesogenic APs, including olanzapine, clozapine and risperidone, significantly activated ER stress PERK- eIF2α signaling in β-pancreatic cells (Ozasa et al., 2013) and the male mouse liver (Lauressergues et al., 2012), therefore leading to glucose and lipid metabolism dysfunction. A recent study in a female rat model reported that acute clozapine treatment (treated once and rats were sacrificed 1 h after the treatment) increased the protein expression of eIF2α and IRE1 in the liver (Weston-Green et al., 2018). This evidence suggested that AP treatment did activate ER stress PERK-eIF2α signaling. However, the effect of olanzapine on hypothalamic ER stress PERK-eIF2α signaling and IKKβ-NFκB signaling is unclear. The present study aimed to examine the role of hypothalamic ER stress PERK- elF2α signaling and its related IKKβ-NFκB signaling in olanzapine-induced weight gain.
Section snippets
Cell culture and olanzapine treatment
The human neuroblastoma SH-SY5Y cell line was purchased from American Type Culture Collection (Rockville, MD, USA). The cells were cultured in Dulbecco`s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, Wuhan, Hubei, China, #31,966,021) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Thermo Fisher Scientific, #10,270,098 and #15070063, respectively) at 37 °C in a humidified 5% CO2 incubator. The cells were regularly subcultured to maintain them in the logarithmic
Olanzapine dose-dependently activated PERK- eIF2α and IKKβ-NFκB signaling in human neuroblastoma SH-SY5Y cells
The protein expression of key molecules of PERK- eIF2α and IKKβ-NFκB signaling was examined by Western blot. As shown in Fig. 1A-B, compared with the control group (treated with DMSO), olanzapine 100 μM treated for 24 h markedly increased the protein expression of pPERK (increased by 107.3 ± 26.9%, p < 0.05), peIF2α (by 70.7 ± 9.3%, p < 0.01), ATF4 (by 57.7 ±14.8%, p < 0.01) and GRP78/BiP (by 44.6 ± 2.0%, p < 0.01). 50 μM olanzapine did not significantly increase the protein expression of
Discussion
The present study firstly demonstrated that olanzapine induced aberrant activation of ER stress PERK-eIF2α signaling in human neuroblastoma SH-SY5Y cells, suggesting that olanzapine might directly act on the neurons to induce ER stress. The animal study provides the first evidence that sub-chronic olanzapine treatment activated the hypothalamic PERK-eIF2α signaling. The protein expression of pPERK positively correlated with food intake, weight gain and feeding efficiency. Previous studies
Conclusions
In conclusion, our study revealed that olanzapine treatment markedly activated the hypothalamic ER stress PERK-eIF2α signaling and inflammatory IKKβ-NFκB signaling, and these effects were related to olanzapine-induced weight gain. Co-treatment with an ER stress inhibitor, 4-PBA, inhibited olanzapine-induced weight gain via suppressing PERK-eIF2α signaling and the related inflammatory IKKβ-NFκB signaling in the hypothalamus. This study may shed light on future possibilities for development of
List of author contributions
Prof T Sun and X-F Huang designed this study. Drs M He, J Li, G Gao, Ms T Zhou, W Li, J Hu and J Chen performed and managed this project study. M He and J Li undertook the statistical analysis. M He wrote the draft of the manuscript. Drs Sun, Huang and He contributed in the discussion of interpretation of the results. All authors have contributed to and have approved the final manuscript.
Conflicts of interest
None.
Acknowledgements
The work was supported by the National Natural Science Foundation of China (81803515, 21805218 and 21703163), the Wuhan University of Technology fund for First-class University and First-class Discipline Construction Projects (472-20162008), the Natural Science Foundation of Hubei Province (2018CFB342 and 2018CFB348) and the Fundamental Research Funds for the Central Universities (173120003). We thank Ms Hongqin Wang and Prof. Chao Deng (University of Wollongong) for their kind help with the
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