According to the latest report of International Diabetes Federation in 2009, there was 1 in 11 adults (20–79 years) have diabetes (463 million people). In addition, the number of T2DM population accounts for over 90% of the total number of diabetic patients, which leads to the widespread concern about the health of this population. T2DM could cause a variety of complications, even death. Thus, the predisposing factors and related mechanisms of T2DM deserve further study. Previous researches showed that exposure to POPs including BDE-209 could induce disorders of glycose and lipid metabolism, which might lead to T2DM (Starling & Hoppin, 2015). What’s more, it is reported that the insulin resistance cells in metabolism toxicity were more sensitive than normal cells, suggesting that T2DM population is more susceptible to pollutants. In this study, IR-BRL cells were applied to evaluated the metabolism toxicity, susceptibility and underlying mechanism of BDE-209.
It is well known that abnormal metabolism of glucose and lipid is the main clinical feature of diabetes mellitus. Most persistent organic toxic substances can interfere with glucose and lipid metabolism. It has been reported that hexafluoropropylene dimeric acid interferes with glycolipid metabolism in young mice by altering glucose-related genes (Conley et al., 2021). Moreover, 3, 3', 4, 4', 5-PENTachlorobenzene has been reported to disrupt glycose and lipid metabolism by interfering with peroxisome fatty acid oxidation and ketogenesis (Gadupudi et al., 2018). Meanwhile, exposure to low doses of PCB126 may reduce glycose and lipid metabolism in organisms by interfering with physiological oscillations of circadian genes (Shen et al., 2019), illustrating that exposure to BDE-209 possibly interfere with glucose and lipid metabolism.
In addition, liver is an important organ of glycose and lipid metabolism, therefore, liver injury can cause glycose and lipid metabolism disorder. It was found that two persistent organic pollutants affect glycose and lipid metabolism by down-regulating multiple genes involved in lipid and glucose metabolism in human hepatocytes through interaction (Ambolet-Camoit et al., 2015). The levels of TC and TG were usually detected to reflect the condition of glycose and lipid metabolism, the results were consistent with previous report that BDE-209 could increase TC and TG activities in BRL cells. (Alimu et al., 2021; Zhu et al., 2019; Zhang et al., 2013). Moreover, higher activities of TC and TG in the IR groups compared to the same dose groups without IR were observed, suggesting glycose and lipid metabolism were interfered both in IR cells and normal cells, and BDE-209 caused more serious damage to IR cells, which demonstrated that IR cells were more sensitive than normal cells.
In addition, studies have shown that BDE-209 can cause liver injury, and the degree of liver tissue and function injury in T2DM patients can be reflected by detecting the activity of ALT and AST (Ali et al., 2018). MDA is one of the products of lipid peroxidation in cells, which levels can reflect the state of oxidative stress and oxidative damage of the body (Del Rio et al., 2005). It has been reported that exposure to BDE-209 could elevate the activities of AST and ALT in hippocampal neurons, which was harmful to liver (Chen et al., 2022). The level of MDA in digestive gland was found increased in a dependent manner with the time and concentration of exposure to BDE-209 (Zhu et al., 2019). Our results showed that BDE-209 cause elevated AST, ALT and MDA levels in normal BRL cells and cause liver damage, which was consistent with the studies described above. Meanwhile, the activities of above indexes were more significant in IR-BRL cells than that in normal cells, which could be speculated that IR-BRL cells were more sensitive to BDE-209 than normal cells.
Insulin receptor signaling pathway concerned glucose transport and regulated glucose homeostasis through its effects on the liver and other tissue(Boucher et al., 2014). IRS-1 is an insulin receptor substrate, where tyrosine residues were phosphorylated by activated IR to further exert the biological effects of insulin and promote glucose metabolism (Tanti & Jager, 2009). Glucose transport is one of the most important steps in the process of glucose uptake in cells (Illsley & Baumann, 2020), which is responsible by the Glut family with 13 well known glucose transporters, among the rest, GLUT4 is a subtype of the 12-transmembrane domain sugar transporter family and the most important transporter in the process (Huang & Czech, 2007). Our previous research has found that DEHP inhibited glucose transport by interfering with the expression of IRS-1/GLUT4 signaling pathway (Ding et al., 2019). It also has been reported that some pollutants could reduce insulin sensitivity and inhibit glucose transport by down regulating GLUT4 expression on cell membrane (Robinson et al., 2018). In our research, the decreased trends of IRS-1 and GLUT4 mRNA and protein expression were observed in BRL cells, which is treated by BDE-209, and the results are similar to others and our previous findings. Moreover, the extend of decreasing is more significant in IR BRL cells than that in normal BRL cells, suggesting IR BRL cells is more sensitive to BDE-209.
The IRS-1/PI3K/AKT/GSK-3β signaling pathway is one of the most important pathways which controlled the synthesis of glycogen and fatty acid (Xiong et al., 2020). PI3K and AKT are signal elements downstream of IRS in insulin signaling pathway, and their expression plays an important role in insulin signaling (Franke et al., 2003). GSK-3β is an important serine-threonine protein kinase in the downstream of insulin signaling pathway, which is controlled by insulin and mainly regulates glycogen and fatty acid synthesis (Zhang et al., 2018). Researchers investigated that the AKT could be activated by persistent organic pollutants and decreased the expression of PI3K, thus reduce the secretion of insulin (Karandrea et al., 2017). It is reported that mostly POPs can decrease the expression of GSK-3β and interfere with glucolipid metabolism, which cause the enhancing of insulin resistance (Wohua & Weiming, 2019). In our study, the secretions and expressions of PI3K, AKT and GSK-3β were significantly decreasing in the treatment groups, furthermore, the decreased trends of expressions were more outstanding in IR-BRL cells, which is consistent with our previous researches.
Mek1/2 and Erk1/2 signaling pathways are located downstream of insulin signaling pathway and involving in regulating the growth, proliferation and differentiation of cells (Chang et al., 2003). mTOR is an atypical serine-threonine protein kinase, the member of Pi3k related kinase family, which plays an important regulatory role in cells proliferation and differentiation (Laplante & Sabatini, 2012). Research has reported that the downward trend of Mek1/2 and Erk1/2 could cause cells apoptosis (McCubrey et al., 2007). The previous study showed that the decrease of mTOR cause the apoptosis of cells(Feng et al., 2005), which is similar to our research that the expressions of Mek1/2, Erk1/2 and mTOR were mostly down-regulation in cells, and the extend were more intensity in IR-BRL cells.
To comprehensively evaluate the damage caused by BDE-209 to cells, the IBR analysis method was used. IBR was an important method for evaluating the effects of exposure to chemical pollutants and detecting responses to environmental stress (Beliaeff & Burgeot, 2002), which could comprehensively consider a variety of biomarkers. In our research, six biomarkers were selected including TG, AST, p-IRS-1/IRS-1, PI3K, p-GSK-3β/GSK-3β and Erk1/2 mRNA, which accurately reflect glycose and lipid metabolism, hepatic function injury, glucose transport and regulated, and cells damage. Our study found that various biomarkers showed different sensitivities to BDE-209 exposure and the IBR value of IR-BRL cells was always larger than that of BRL cells at the same dose, indicating that the IBR conversion values of all indicators of IR-BRL cells after exposure to BDE-209 were higher than those of normal BRL cells, suggesting they were subjected to stronger metabolic toxicity, which is corresponding to above research. It was speculated that people with type 2 diabetes may face greater metabolic stress than the normal population. In conclusion, exposure to BDE-209 may cause metabolic toxicity in people with type 2 diabetes, and the toxic effects may be more severe than in the normal population.