Environmental Toxicology and Pharmacology 43 (2016) 7–12
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Bisphenol A disrupts glucose transport and neurophysiological role of IR/IRS/AKT/GSK3 axis in the brain of male mice Jing Li a,1 , Yixin Wang a,1 , Fangfang Fang a , Donglong Chen a , Yue Gao a , Jingli Liu a,b , Rong Gao a , Jun Wang a,∗ , Hang Xiao a,∗ a Key Lab of Modern Toxicology (NJMU), Ministry of Education. Department of Toxicology, School of Public Health, Nanjing Medical University, Nanjing 211166, China b Department of Laboratory Medicine, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing 210000, China
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Article history: Received 25 July 2015 Received in revised form 20 November 2015 Accepted 22 November 2015 Keywords: BPA Insulin signaling Glucose transporters Neurodegenerative diseases Mice
a b s t r a c t Bisphenol A (BPA), one of the most prevalent chemicals for daily use, was recently reported to disturb the homeostasis of energy metabolism and insulin signaling pathways, which might contribute to the increasing prevalence rate of mild cognitive impairment (MCI). However, the underlying mechanisms are remained poorly understood. Here we studied the effects of low dose BPA on glucose transport and the IR/IRS/AKT/GSK3 axis in adult male mice to delineate the association between insulin signaling disruption and neurotoxicity mediated by BPA. Mice were treated with subcutaneous injection of 100 g/kg/d BPA or vehicle for 30 days, then the insulin signaling and glucose transporters in the hippocampus and prefrontal cortex were detected by western blot. Our results showed that mice treated with BPA displayed significant decrease of insulin sensitivity, and in glucose transporter 1, 3 (GLUT1, 3) protein levels in mouse brain. Meanwhile, hyperactivation of IR/IRS/AKT/GSK3 axis was detected in the brain of BPA treated mice. Noteworthily, significant increases of phosphorylated tau and -APP were observed in BPA treated mice. These results strongly suggest that BPA exposure significantly disrupts brain insulin signaling and might be considered as a potential risk factor for neurodegenerative diseases. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Bisphenol A (BPA), a kind of environmental endocrine disrupting chemicals (EDCs), is present ubiquitously in our lives. It is used commercially in products containing polycarbonate plastics such as food and drink packaging materials and infusion bags which sometimes may be boiled, microwave heated, autoclaved and brushed (Vandenberg et al., 2009). It has been reported that more than 90 percent of the population aged above six in the USA has detectable levels of BPA in urine and blood (Taylor et al., 2011). Since BPA is rapidly metabolized, it suggests that human exposure to BPA might be continuous and via multiple sources, such as inhalation and contact, not only limited for ingestion (Stahlhut et al., 2009). The detection of adverse effects in numerous of animal models upon exposure to environmentally relevant doses of BPA that correspond to those observed in humans, strongly supports that the endocrine
∗ Corresponding authors. E-mail addresses:
[email protected] (J. Wang),
[email protected] (H. Xiao). 1 These authors have contributed equally to this study. http://dx.doi.org/10.1016/j.etap.2015.11.025 1382-6689/© 2016 Elsevier B.V. All rights reserved.
disrupting activities of BPA contribute to adverse effects on human health. Recently, the adverse effects of BPA on glucose homeostasis have been demonstrated in numerous studies. Glucose-stimulated insulin secretion (GSIS) and serum insulin were found to be elevated upon exposure to 100 g/kg/day BPA, suggesting the direct effect of BPA on pancreatic -cell function (Nadal et al., 2009). Furthermore, BPA affects hormone signaling and causes endocrine dysfunction by binding to estrogen receptors and promotes both agonist and antagonist activity (Pan et al., 2013). Also it has been shown to cause persistent aberrations in spontaneous behavior and in learning and memory in rodents (Yang et al., 2014). Several investigations have been undertaken to explore the neurotoxic effects of BPA, especially in fetal brain development and its promotion of neurodegenerative diseases. In vitro studies support that BPA causes adverse neurological effects as it induces impairments in dendritic and synaptic development in cultured fetal rat hypothalamic cells (Leranth et al., 2008) and inhibits neurite extension in rat pheochromocytoma (PC12) cells (Seki et al., 2011). Furthermore, BPA may act as a DNA methylation agent to alter gene expression in the rodent brain, which may be a plausible reason for the developmental neurotoxic effects (Wolstenholme et al., 2011).
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With global aging, the prevalence of obesity, type 2 diabetes and neurodegenerative diseases have been astonishingly increasing. These diverse pathologies shared the same characteristic, insulin resistance. Therefore, the role of insulin in human health has become more and more critical and may be relevant to the age-related disorders, which are associated with disturbances in glucose metabolism. Noteworthily, the brain was once considered to be an insulin insensitive tissue. However, Havrankova et al. indicated the widespread presence of insulin receptors (IRs) and higher levels of insulin in the central nervous system (CNS) than periphery (Havrankova et al., 1978), where it plays a critical physiological role. In the past decade, growing evidence from in vivo and in vitro studies confirms that insulin and its receptor, can affect nervous physiology, including energy homeostasis and cognitive processes (de la Monte, 2012). As in peripheral tissues, insulin signaling starts with binding to the IR, which is activated by auto tyrosine phosphorylation. IR then phosphorylates insulin receptor substrate (IRS) proteins, and the activated IRSs serve as docking sites to activate the downstream signals such as (protein kinase B) AKT/PKB and glycogen synthase kinase 3 beta (GSK-3), which play crucial roles in the development of neuronal structure (Chiu and Cline, 2010). Therefore, understanding of the link between insulin signaling axis disruption and neurotoxicity could provide potential therapeutic strategies targeting BPA-mediated neurotoxicity. Our previous work suggested that BPA contributed to endocrine dysfunction by interfering insulin biosynthesis and secretion (Liu et al., 2013). Based on the work, the present study was undertaken to assess its deregulation of the IR/IRS/AKT/GSK3 axis, which might represent a key-contributing factor to the neurodegenerative process that culminates in Alzheimer-like dementia.
2. Materials and methods
of 0, 15, 30, 60 and 120 min after insulin administration using an Accu-Check compact glucometer (Roche, Madrid, Spain). 2.3. Western blot analysis Tissues were homogenized with the help of ultrasonic cell disrupter in 200 L of cold RIPA lysis buffer (Sigma–Aldrich) with protease inhibitor. The homogenates were centrifuged at 12,000×g for 10 min at 4 ◦ C and supernatants were measured using a BCA Protein Assay Kit. For western blots, 50 g of proteins were resolved with 10% SDS-PAGE and transferred to PVDF membranes (Millipore). The membranes were blocked in 5% nonfat dried milk. Blots were then incubated with the primary antibodies at 4 ◦ C overnight. After washing, membranes were incubated at room temperature with anti-mouse or anti-rabbit horseradish peroxidase-conjugated IgG (1:50,000). Immunoreactivity was detected using an enhanced chemiluminescence reaction (Millipore). Densitometrica nalysis employed Image J (NIH, USA). The following primary antibodies (Cell Signaling Technology or proteintech, China) and dilutions were used: antiinsulin (15848-1-AP, 1:2000), anti-p-IR Tyr1355 (#3024, 1:1000), anti-IR (1:2000, #3025), anti-p-IRS1 (1:500, BS4835), anti-IRS1 (1:500, #2382), anti-p-AKT Ser473 (1:2000, #4060), anti-AKT (1:2000, #4691), anti-p-GSK3 (1:1000, #D85E12), anti-GSK3 (1:2000, #27C10), anti-GLUT1 (1:1000, 21829-1-AP), anti-GLUT3 (1:1000, 20403-1-AP), anti-GLUT4 (1:2000, ab18831), anti-GAPDH (1:10,000 dilution; Sigma–Aldrich). 2.4. Statistical analysis All data were normalized and expressed as mean ± SEM. The unpaired Student’s t test and repeated-measures one-way ANOVA were used as appropriate for comparison between groups of mice. Statistical significance was assumed at p < 0.05.
2.1. Animals and treatment 3. Results All experiments involving animals and tissue samples were conducted in accordance with the guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH) (USA), and all procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University (China). The C57BL6 male mice, 7–8 weeks old and 25–30 g weight, were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). The mice were housed throughout the experiments under specific pathogen free (SPF) conditions, with controlled illumination (12 h light/12 h dark cycles), humidity (30–50%), and temperature (18–22 ◦ C). Adult mice were randomly assigned to receive 50ul corn oil (vehicle) or BPA (100 g/kg/day; Sigma–Aldrich) for 30 days. Subcutaneous injection was chosen for the present study to mimic the nonfood sources of BPA exposure. Then, 24 h after the last dosing, the animals were sacrificed by cervical dislocation. The brain was rapidly removed, and the hippocampus and frontal cortex were immediately dissected and frozen at −80 ◦ C for further experiment.
2.2. Glucose and insulin tolerance tests For intraperitoneal glucose tolerance tests (IPGTT), after 30 days of treatment with BPA, animals were fasted overnight for 16 h, and then intraperitoneally injected with glucose at 2 g/kg body weight. Blood samples were collected from the angular vein and measured for blood glucose levels at 0, 15, 30, 60 and 120 min. For the intraperitoneal insulin tolerance tests (IPITT), 6-hour-fasted animals were injected intraperitoneally with 1 IU/kg body weight soluble insulin. Blood glucose was measured at the time points
3.1. BPA decreases insulin sensitivity and plasma insulin level in adult mice The IPGTT and IPITT were performed to investigate the effect of BPA on glucose homeostasis and insulin sensitivity. As depicted in Fig. 1A, BPA exerted no obvious effect on blood glucose levels, and the AUC of glucose in each group displayed no significant difference (Fig. 1A). However, BPA treated mice showed decreased insulin sensitivity in comparison to control mice when insulin tolerance tests were performed (p < 0.05; Fig. 1B), and the corresponding AUC was significantly increased in BPA-treated mice (p < 0.05). 3.2. BPA impairs IR/IRS/AKT/GSK3ˇ axis in the hippocampus and prefrontal cortex Since peripheral insulin homeostasis is closely associated with the insulin signaling IR/IRS/AKT/GSK3 axis, the next step was to ascertain whether BPA disrupts this signaling axis in the brain. Our data indicated that insulin levels in both hippocampus (p < 0.05) and prefrontal cortex (p < 0.01) were obviously increased in BPAtreated mice. Similarly, the phosphorylated IR (Tyr1355) as well as the downstream signal protein phosphorylated IRS1 (Tyr896 and Ser307) were significantly increased in both hippocampus (p < 0.05) and prefrontal cortex (p < 0.01) in BPA-treated mice (Fig. 2). Since AKT plays a crucial role in insulin signaling, we then assessed the expression of this signal protein. As shown in Fig. 3, the phosphorylated AKT (Ser473) was obviously increased in hippocampus (p < 0.01) and prefrontal cortex (p < 0.05) in BPA-treated mice. In parallel with the effect of BPA on AKT phosphorylation, BPA
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Fig. 1. Effect of BPA on glucose homeostasis and insulin sensitivity shown by IPGTT and IPITT. (A) Plasma glucose concentrations (mmol/L) following administration of glucose (2 g/kg body weight) in fasted adult male mice. The mean total glucose AUC was shown at the same time points (n = 5–7). (B) Plasma glucose concentrations (mmol/L) following injection of insulin in fasted adult male mice (1 IU/kg body weight). The mean total glucose AUC was shown at the same time points (n = 6–7). Statistical differences were determined by Student’s t test. * p < 0.05 compared with vehicle control. Data are expressed as mean ± S.E.M.
treatment also increased the inactivation of GSK3 (Ser9) in the hippocampus (p < 0.05) and prefrontal cortex (p < 0.01) (Fig. 3).
prefrontal cortex (p < 0.01) were obviously increased in BPA-treated mice (Fig. 4).
3.3. BPA disturbs glucose transport in the hippocampus and prefrontal cortex
3.4. BPA increases APP and tau phosphorylation in the hippocampus and prefrontal cortex
Since treatment with BPA contributed to the disturbance of insulin function in the peripheral plasma as mentioned above, we next sought to investigate whether BPA could affect glucose transport in CNS. As shown in Fig. 4, the expression levels of GLUT1 (p < 0.05) and GLUT3 (p < 0.01) in the hippocampus were significantly decreased, while GLUT4 in the hippocampus (p < 0.05) and
The effect of BPA on the tau phosphorylation and -APP expression relevant to brain insulin signaling disruption was further determined. It indicated that the levels of APP and tau phosphorylation at Ser199 and Ser396 in both hippocampus and prefrontal cortex were increased in BPA-treated mice (p < 0.05, Fig. 5).
Fig. 2. Effect of BPA on insulin signals in the hippocampus and prefrontal cortex. The expression of Insulin, IR, phosphorylated IR (Tyr1355), IRS1 and phosphorylated IRS1 (Tyr896, Ser307) in both hippocampus and prefrontal cortex in vehicle and BPA treated animals. All phosphorylated protein expression levels were normalized relative to their total protein and other protein expression levels were normalized relative to a GAPDH loading control. Data are expressed as mean ± S.E.M of duplicates from three independent experiments with protein of seven mice. Statistical differences were determined by Student’s t test. * p < 0.05, ** p < 0.01 compared with vehicle control.
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Fig. 3. Effect of BPA on AKT, GSK3 signal pathways in the hippocampus and prefrontal cortex. The protein expression levels of AKT, phosphorylated AKT (Ser473), GSK3 and phosphorylated GSK3 (Ser9) in vehicle and BPA treated animals. All phosphorylated protein expression levels were normalized relative to their total protein and other protein expression levels were normalized relative to a GAPDH loading control. Data are expressed as mean ± S.E.M of duplicates from three independent experiments with protein of seven mice. Statistical differences were determined by Student’s t test. * p < 0.05, ** p < 0.01 compared with vehicle control.
4. Discussion Over the last several decades, numerous researches have suggested that exposure to low doses of BPA has been reported to possess weak estrogenic, anti-estrogenic properties and promote insulin secretion, which exerts adverse effects on cognitive behaviors, learning and memory in adult rodents (Neel and Sargis, 2011). Since estrogens play essential roles in insulin secretion, synaptic plasticity and cognitive function of adult brain (Barha and Galea, 2010), it is reasonable to explore whether BPA affects these processes. Therefore, in the present study, we sought to assess the association between BPA exposure and brain insulin signaling axis. The dose of BPA (100 g/kg/day) we used in this study was approximate to the predicted safe reference dose of 50 g/kg/day and below the lowest observed effect level (LOAEL) of 50 mg/kg/day, which was established by the U.S. Environmental
Protection Agency (EPA). Besides, it is recommended in several papers and is considered as the low dose in vivo studies (Liu et al., 2013). Our data suggested that mice exposed to BPA for 30 days displayed a tendency of dysregulation of insulin sensibility, while the glucose tolerance was normal compared with vehicle (Fig. 1). As Nadal reported, glucose-stimulated insulin secretion (GSIS) and serum insulin level elevated upon exposure to 100 g/kg/day BPA (Nadal et al., 2009), so the cause of this symptom may be the response of the pancreas to produce sufficient insulin to compensate for insulin resistance. Glucose transporters (GLUT), which are responsible for glucose transport, play important roles in transferring the glucose across capillaries and plasma membranes in astrocytes and neurons. GLUT4 is insulin regulated glucose transporter in the CNS, where they maintain hippocampus dependent cognitive functions. GLUT1 and GLUT3 are independent of insulin,
Fig. 4. Effect of BPA on glucose transport in the hippocampus and prefrontal cortex. The protein expression levels of GLUT1, GLUT3 and GLUT4 in vehicle and BPA treated animals. All protein expression levels were normalized relative to a GAPDH loading control. Data are expressed as mean ± S.E.M of duplicates from three independent experiments with protein of seven mice. Statistical differences were determined by Student’s t test. * p < 0.05, ** p < 0.01 compared with vehicle control.
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Fig. 5. Effect of BPA on the expression of APP and phosphorylation TAU (Ser199, Ser396) in the hippocampus and profrontal cortex. The protein expression levels of APP and phosphorylation TAU (Ser199, Ser396) in vehicle and BPA treated animals. All phosphorylated protein expression levels were normalized relative to their total protein and other protein expression levels were normalized relative to a GAPDH loading control. Data are expressed as mean ± S.E.M of duplicates from three independent experiments with protein of seven mice. Statistical differences were determined by Student’s t test. *p < 0.05, ** p < 0.01 compared with vehicle control.
which are low-Km (Michaelis constant) transporters expressed in many tissues and are responsible for the glucose uptake in the unfed state. GLUT1 facilitates a continual basal level of glucose uptake and helps glucose transport across the blood brain barrier (BBB), and subsequent uptake from the mesenchymal cells into neurons which occurs via GLUT3 (Apelt et al., 1999). In this work, we demonstrated that BPA exposure resulted in GLUT4 up-regulation. The reason for this observation is unknown, we speculated that the acatastatic up-regulation of GLUT4 expression in CNS may ascribe to the stimulatory effects of insulin (Ngo et al., 2009). Therefore, further detailed studies with different doses of BPA or exposure times may confirm and provide insight into this phenomenon. For the GLUT1 and 3, the expression levels were obviously decreased in the brain, it may due to the impairment of persistent low doses of BPA (D’Cruz et al., 2012). In parallel with the disturbance of glucose transport, we also observed the expression of neuronal IR/IRS/AKT/GSK3 signaling, APP and hyperphosphorylation of Tau (Ser199, Ser396). Our data demonstrated that the key insulin signaling, such as IR and IRS were aberrantly phosphorylated in BPA-treated brain when compared to vehicle. In addition, AKT phosphorylation (Ser473), GSK3 phosphorylation (Ser9), -APP and hyperphosphorylation of Tau in the brain were also significantly increased in BPA-treated group. These results were in line with those observed in APP/SP1 mice that p-AKT and p-GSK3 were aberrant expressed in this AD model (Sonoda et al., 2010). Recently, Chen et al. showed an aberrant and sustained activation of neuronal IR/IRS/AKT/GSK3 axis in the early stages of AD (Chen and Zhong, 2013), which further supported our results that BPA exerts its neurotoxic effects partially through insulin signaling axis disruption. Moreover, aberrant high level of insulin in brain, known as hyperinsulinaemia, has been proposed to negatively regulate extracellular A degradation by modulating insulin degrading enzyme (IDE) activity (van der Heide et al., 2006). Thus, we speculated that the up-regulation of
insulin in BPA-treated mice in the present study may contribute to increase A levels in the brain. In addition, up-regulation of AKT phosphorylation has been also described in hippocampal and cortical neurons of AD brain (Sonoda et al., 2010), in the current study, it revealed that in BPA-treated mice, p-AKT was obviously increased, which further elucidated the increment of -APP in the brain. The PI3K/AKT/GSK3 axis is directly regulated by IRS1, the major insulin receptor substrate protein, which is activated by Tyrphosphorylation. This signaling also has negative feedback that acts through IRS1 serine phosphorylation (Ser307), the best studied inhibitory residue that causes IRS1 inactivation by disrupting IRS1 binding to IR and inhibiting its tyrosine phosphorylation. The irregular insulin signaling and AKT/GSK3 axis regulated by IRS1 inactivation represents one of the major causes of insulin resistance (White, 2006), it indicates that IRS1 is phosphorylated at inhibitory serine residues (Ser307) by increasing AKT/GSK3 activity in AD neurons which leading to resistance to both insulin and IGF-1 (Moloney et al., 2010). In the present research, we found that 30-day BPA exposure led to significant high phosphorylated level of IRS1 serine phosphorylation (Ser307), however, we could not fully clarify why Tyr-phosphorylation was also increased. Therefore, further research is still needed. Although increased inactive GSK3 has been proved to be associated with the decrease hyperphosphorylation of tau (Bhat et al., 2004), the role of GSK3 in tau pathologies has not yet been clearly defined. Interestingly, both levels of active and inactive GSK3 were increased in AD patients, suggesting the dysregulation of both inhibitory and stimulatory forms of GSK3 (Griffin et al., 2005). Some other groups also provided evidence that the inactive GSK3 in AD was inconsistent with its proposed role as a tau kinase (Sheppard et al., 2012). These results, might be in part, explained the mechanism of increased phosphorylated GSK3 (Ser9) in the present study.
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5. Conclusions The important contribution of the present work is that we provide evidence for the adverse effects of BPA on insulin secretion, insulin sensibility, especially in the brain on -APP, p-tau and glucose transport dysregulation. Of note, insulin signaling in the brain might be involved in this process. Therefore, insights into the role of insulin signaling could be responsible for BPA-mediated neurotoxicity. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgements This work was supported by the National Natural Science Foundation of China (81273115, 81473012 and 81072329), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Science and Technology Plan Project of Jiangsu Province (SBL2014020070) and the Training and Innovation Program of Jiangsu Graduate (CXZZ13 0598), and the Key Laboratory of Modern Toxicology (Nanjing Medical University), Ministry of Education (NMUMY201402). The founders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. References Apelt, J., Mehlhorn, G., Schliebs, R., 1999. Insulin-sensitive GLUT4 glucose transporters are colocalized with GLUT3-expressing cells and demonstrate a chemically distinct neuron-specific localization in rat brain. J. Neurosci. Res. 57, 693–705. Barha, C.K., Galea, L.A.M., 2010. Influence of different estrogens on neuroplasticity and cognition in the hippocampus. Biochim. Biophys. Acta 1800, 1056–1067. Bhat, R.V., Haeberlein, S.L.B., Avila, J., 2004. Glycogen synthase kinase 3: a drug target for CNS therapies. J. Neurochem. 89, 1313–1317. Chen, Z.C., Zhong, C.J., 2013. Decoding Alzheimer’s disease from perturbed cerebral glucose metabolism: implications for diagnostic and therapeutic strategies. Prog. Neurobiol. 108, 21–43. Chiu, S.L., Cline, H.T., 2010. Insulin receptor signaling in the development of neuronal structure and function. Neural Dev. 5, 7. D’Cruz, S.C., Jubendradass, R., Jayakanthan, M., Rani, S.J.A., Mathur, P.P., 2012. Bisphenol A impairs insulin signaling and glucose homeostasis and decreases steroidogenesis in rat testis: an in vivo and in silico study. Food Chem. Toxicol. 50, 1124–1133.
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