Metabolism toxicity and susceptibility of decabromodiphenyl ether (BDE-209) exposure on BRL cells with insulin resistance

doi:10.21203/rs.3.rs-1552684/v2

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Metabolism toxicity and susceptibility of decabromodiphenyl ether (BDE-209) exposure on BRL cells with insulin resistance

https://doi.org/10.21203/rs.3.rs-1552684/v2

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Type 2 diabetes mellitus (T2DM) is a metabolic disease characterized by insulin resistance (IR) and has attracted worldwide attention due to its high prevalence. As a typical persistent organic pollutant, decabromodiphenyl ether (BDE-209) has been detected in food and human samples and the concentration trends to increase year by year. In addition, it has been proved to have the potential to increase the risk of IR, but it is rarely reported whether it could aggravate IR in T2DM. Therefore, in this study, the IR-BRL (Buffalo Rat Liver cells with IR) model was applied to study the metabolism toxicity and susceptibility of BDE-209. Results showed that BDE-209 could inhibit the glucose absorption and increase the levels of TC and TG, ultimately lead to the disorder of glucolipid metabolism in IR-BRL cells. Besides, it also could cause cell damage by increasing the levels of AST, ALT and MDA in cells. Moreover, its potential mechanisms were to: (1) affect the transport of glucose, synthesis of glycogen and fatty acid via IRS-1/GLUT4 and IRS-1/PI3K/AKT/GSK-3β pathways; (2) impact the proliferation and differentiation by regulating the expression of Mek1/2, Erk1/2 and mTOR proteins and genes. Furthermore, susceptibility analysis showed that there was a significant synergism interaction between IR and BDE-209, which suggested that IR-BRL cells were more susceptible to the metabolism toxicity induced by BDE-209.

Decabromodiphenyl ether

insulin resistance

Susceptibility；Integrated biomarker responses

Diabetes is a widespread metabolic disease with the global number of patients having been predicted to reach 700 million by 2045 (IDF, 2019). It could be roughly divided into type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). In view of the predominance of T2DM patients in the general diabetic population (accounting for over 90%), T2DM has been the focus of diabetes research. Previous studies found that exposure to adverse environmental factors such as PM10, SO₂ and NO₂ was closely related with T2DM, suggesting that environmental problem has been the one of the main causes of T2DM (Shan et al., 2020). What’s more, an in vitro study on rat liver BRL cells showed that the risks of DEHP-induced metabolic toxicity and insulin resistance in cells with insulin resistance were higher than that of normal cells, demonstrating that T2DM patients were more susceptible to DEHP (Ding et al., 2019).

Decabromodiphenyl ether (BDE-209) is known as the most widely (accounting for almost 82%) used one in commercial polybrominated diphenyl ethers (PBDEs) which have been commonly applied in construction, plastics, textile and electronic products industries as flame retardants (Cai et al., 2020). In addition, BDE-209 is a typical persistent organic pollutant and could enter into environment and human body through many ways (Cai et al., 2020). Because of its bioaccumulation, high lipophilicity, and hard degradable, BDE-209 has been found in a variety of environmental media, food and human samples, and the potential health risks of BDE-209 is worthy of attention (Abdallah & Harrad, 2014; Chen et al., 2020; Fernandes et al., 2016; Li et al., 2010; Śmiełowska & Zabiegała, 2020; Berg et al., 2017; Vizcaino et al., 2014). Evidences have shown that BDE-209 was involved in various diseases such as immune injury, thyroid and liver dysfunction (Feng et al., 2015; Sun et al., 2020; Wang et al., 2019). Besides, recently researches indicated that BDE-209 was associated with the disorder of glycolipid metabolism for it could induce high fasting glucose in rats even at the lowest dosage (0.05 mg/kg) of 8 weeks treatment (Zhang et al., 2013). Zhu et al. (2019) found that an increase of the serum levels of glucose, insulin, and triglyceride were observed in mice after 28 days of BDE-209 exposure, so was the decrease of high-density lipoprotein level and the damaged of hepatic structures and adipose tissue. Moreover, Yanagisawa et al. (2019) found that BDE-209 could contribute to the enhancement of diet-induced hyperglycemia through disruption of glucose homeostasis in the obese mouse, demonstrating that BDE-209 may be a risk factor for obesity-related metabolic disorders. Although those studies shed light on the metabolism toxicity of BDE-209, there was a lacking of comprehensive analysis and evaluation of indicators. Additionally, to the best of our knowledge, whether the T2DM patients are more susceptible to BDE-209 remains unknown.

Liver plays an important role in the regulation of plasma glucose levels. In addition, liver is a modulator of peripheral insulin and its secretion can control insulin sensitivity (Guerra & Gastaldelli, 2020). Principal component analysis (PCA) is a multivariable technique for analyzing tables of data, which can be used to overview complex data and extract the important information from the data (Abdi & Williams, 2010). A method for assessing the overall effects of multiple biomarkers in the entire system termed Integrated biomarker responses (IBR) was generally used to evaluate the toxic effects of aquatic organisms, which exposed to contaminants at different detection sites. This method contribute to research the difference of sensitivity of different biomarkers to pollutants (Damiens et al., 2007). Factorial analysis is an efficient method to investigate the influence of some factors on the target variables and to explore the interaction between factors (Ehlen et al., 2005). With the possible risk of BDE-209 exposure to T2DM, the differences between the effect of BDE-209 on normal and IR should be investigated. However, there have been few reports on whether IR were more sensitive to exposure to BDE-209.Therefore, in this study, Buffalo Rat Liver (BRL) cells accompanied by PCA, IBR and factorial analysis were applied to investigate the metabolism toxicity, potential molecular mechanisms and susceptibility of BDE-209.

2.1 Materials

BDE-209 (CAS NO 1163-19-5) was purchased from Zhenhuai Biotechnology Co. Ltd (Shanghai, China); Insulin was obtained from Hefei Bomei Biotechnology Co. Ltd (Hefei, China); 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Dimethyl sulfoxide (DMSO) were obtained from Sigma Chemical Co. (St. Louis, MO, USA); the Glucose oxidase-peroxidase, TC, TG, AST, ALT, MDA (malondialdehyde) and BCA Assay Kit were obtained from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China); Dulbecco’s modified Eagle’s minimum essential medium (DMEM), fetal bovine serum (FBS) and Polyvinylidene fluoride (PVDF) membranes was purchased from Bio-Rad (America); GAPDH #5174, IRS-1 #2390, Phospho-IRS-1 #3203, PI3 Kinase p85 #4257, GLUT4 #2213, AKT #4685, Phospho-AKT #13038, GSK-3β #9315, Phospho-GSK-3β #5558, mTOR #2983, Mek1/2 #8727, Erk1/2 #4695, Phospho-Erk1/2 #4370, Peroxidase-AffiniPure Goat Anti-Rabbit IgG (H + L) and Peroxidase-AffiniPure Goat Anti-Mouse IgG (H + L) were purchased from Cell Signaling Technology, Inc (America).

2.2 Cell culture

BRL cells were obtained from the Institute of Cell Biology, Chinese Academy Sciences (Shanghai, China). Cells were cultured in DMEM medium supplemented with 10% FBS at 37 ℃ in a humidified incubator containing 5% CO₂.

2.3 Establishment of IR-BRL model

BRL cells (2×10⁴ cells/mL, 200 µL per well) were cultured overnight in flat-bottomed 96-well plates, and then incubated with insulin at different concentrations (1×10^− 7, 5×10^− 7, 1×10^− 6, 5×10^− 6 and 1×10^− 5 mol/L), while the wells without insulin were taken as control. After 24 h incubation, cells were washed by pre-cooling phosphate buffer saline (PBS) and cultured in DMEM medium supplemented with 10% FBS for further 12, 24, 36, 48, 60 and 72 h. Finally, glucose oxidase-peroxidase kit was adopted to evaluate the glucose level in the supernatant of each well.

2.4 Effect of BDE-209 on IR-BRL cells

IR-BRL model was established at the optimum condition in section 2.3, then the IR-BRL cells were incubated in different concentrations (0, 6.25, 12.5, 25, 50 and 75 µmol/L) of BDE-209 for 24, 48 and 72 h. For each well, MTT (20 µL, 1 mg/mL) was added and incubated at 37 ℃ for 4 h. After that, the formed formazan was dissolved in DMSO (100 µL) and read at 570 nm.

2.5 Effect of BDE-209 on glycose and lipid metabolism of IR-BRL cells

2.5.1 Glucose absorption capacity

According to results of section 2.3 and 2.4, the optimal modeling method and exposure dose of BDE-209 were selected for subsequent experiments. Cells were divided into five groups: (1) Control group; (2) IR group (5×10^− 6 mol/L insulin);(3) IR + BDE-209 group (5×10^− 6 mol/L insulin + 10.5, 21 or 42 µmol/L BDE-209). After 24, 48 or 72 h treatment with BDE-209, the glucose level in the supernatant was detected using the Glucose oxidase-peroxidase kit following the manufacturer’s specifications.

2.5.2 TC and TG levels

The experiment design and the exposure duration of BDE-209 was the same as section 2.5.1. After the exposure, cells were washed by pre-cooling PBS and lysed by RIPA Lysis Buffer with protease and phosphatase inhibitor (Beyotime Biotechnology, China). Then, TC and TG levels of cell lysates were detected by TC and TG assay kit, respectively.

2.6 Effect of BDE-209 on hepatocyte function of IR-BRL cells

AST and ALT activities were applied to reflect the hepatocyte function. In briefly, IR-BRL cells were treated with BDE209 at the concentrations of 10.5, 21 and 42 µmol/L, and the subsequent AST and ALT activities in the supernatant were recorded by the commercial kit.

2.7 Effect of BDE-209 on cell oxidative stress of IR-BRL cells

Cells were treated and lysed as described in section 2.5.2, and then the MDA level in the lysates were tested by MDA assay kit.

2.8 Western blot analysis

Western blot was applied to assay the expressions of GAPDH, IRS-1, p-IRS-1, GLUT4, PI3K, AKT, p-AKT, GSK-3β, p-GSK-3β, mTOR, MEK1/2, ERK1/2 and p-ERK1/2 in the IR-BRL cells after treatment with BDE209. The total proteins were separated by RIPA Lysis Buffer with protease and phosphatase inhibitor, and the protein concentration of lysates were determined using BCA assay kit. The proteins (20 µg) were isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes. Then, the PVDF membranes were incubated by TBST (Tris-buffered saline-Tween20 (0.1%)), 5% skimmed milk powder and 1.5% BSA (Albumin from bovine serum) at 37°C for 2 h. After being washed by pre-cooling PBS for three times, the PVDF membranes were incubated with specific primary antibodies and oscillated at low speed overnight at 4°C. The incubated membranes were washed six times with TBST, followed by incubation in horseradish peroxidase conjugated anti-rabbit or anti-mouse antibodies (1:5000) (CST, America) at 37°C for 1 h. Finally, the membranes were washed with TBST for six times and photographed by the Vilber FUSION SOLO gel imaging system (Vilber, France). Densitometry analysis of specific bands was analyzed with the Quantity One software4.6.9 (Bio-Rad, America).

2.9 RT-PCR analysis

The total RNA was extracted from IR-BRL cells treated with or without BDE209 using Trizol reagent (Takara Bio, Japan), and then reverse-transcribed by the Prime Script TM RT-PCR Kit (Takara Bio, Japan). Q-PCR (Quantitative real time polymerase chain reaction) was performed with a Light Cycler® 96 Instrument (Roche Applied Science) using SYBR® Premix Ex TaqTM II Kit (Takara) according to the instructions. The primers sequences are listed in Table 1. Relative RNA concentrations were calculated using the 2^−△△CT, where CT was the mean threshold cycle value and GAPDH was used as normalize.

Table 1

Fluorescent quantitative PCR primers
Genes	Primer Sequences
Gapdh	F: 5'-GGCACAGTCAAGGCTGAGAATG-3' R: 5'-ATGGTGGTGAAGACGCCAGTA-3'
Irs-1	F: 5'-TGTGCCAAGCAACAAGAAAG-3' R: 5'-AAGGAGACGAGACTTTGGCA-3'
Pi3k	F: 5'-GATGTCTGCGTTAGGGCTTACC-3' R: 5'-TCAGCATCATGGAGAACAGGAT-3'
Akt	F: 5'-GGCAGGAGGAGGAGACGATGG-3' R: 5'-TTCATGGTCACACGGTGCTTGG-3'
Glut4	F: 5'-GGGCTGTGAGTGAGTGCTTTC-3' R: 5'-CAGCGAGGCAAGGCTAGA-3'
Gsk-3β	F: 5'-TACCCATACGATGTTCCAGAT-3' R: 5'-CACCGTTGAGGACCCGTCCCA-3'
Mtor	F: 5'-GGCTTCTGAAGATGCTGTCC-3' R: 5'-GAGTTCGAAGGGCAAGAGTG-3'
Mek1	F: 5'-CTGGCAATGGTGGAGTGGTGTTC-3' R: 5'-TGATCTCGCCGTCACTGTAGAAGG-3'
Mek2	F: 5'-GCTGTCCGCAACCAGATCATCC-3' R: 5'-TCTCGCCGTCGCTGTAGAAGG-3'
Erk1/2	F: 5'-CTCAAGCCTTCCAACCTC-3' R: 5'-TTCCACGGCACCTTATTT-3'

2.10 Susceptibility analysis

In this part, data on the effect of BDE-209 on BRL and IR-BRL cells were submitted as supplementary documentation. PCA was applied to reduce the dimension of these indexes. IBR method was applied to comprehensively evaluate the cytotoxicity, and it was conducted according to our previous research (Liao et al., 2021). In the factorial analysis, values of p < 0.05 were considered interaction, and the calculation of IBR values was in accordance with our previous research

2.11 Statistical analysis

Statistical analyses were performed using SPSS (version 16.0; SPSS Inc., Chicago, America). All the data were expressed as mean ± standard deviation. The differences between groups were assessed by one-way analysis (ANOVA). In the ANOVA, post hoc multiple comparisons were made simultaneously using Turkey test. Values of p < 0.05 were considered statistically significant difference.

3.1 Establishment of IR-BRL model

IR usually refers to an impaired biological response to insulin, and it is known to underlie the deterioration of glucose homeostasis that leads to the typical forms of T2DM (Bailey, 1999). As shown in Table 2, after the treatment with 5×10^− 6 mol/L or 1×10^− 5 mol/L insulin for 24 h, the glucose concentration in supernatant of BRL cells was significantly higher than that in the control group (p < 0.05). In addition, this significance was maintained until 72 h, suggesting that the IR-BRL model was established successfully and it could be available at least during 72 h. Therefore, 5×10^− 6 mol/L insulin incubated for 24 h were selected to build IR-BRL cell model.

Table 2

Effect of insulin treatment on glucose concentration in supernatant of BRL cells
Insulin (mol·L^− 1)	Glucose(mg/L)
Insulin (mol·L^− 1)	12 h	24 h	36 h	48 h	60 h	72 h
0	2501.92 ± 194.14	2563.81 ± 99.53	2554.96 ± 55.21	3439.59 ± 47.88	3532.33 ± 62.38	3115.03 ± 114.04
1×10^− 7	2798.08 ± 127.89	2890.91 ± 73.83	2674.31 ± 179.07	3641.92 ± 106.05	3544.97 ± 154.53	3224.62 ± 143.81
5×10^− 7	3067.73 ± 50.21^a	2674.31 ± 100.41	2550.54 ± 203.00	3536.54 ± 55.12	3692.51 ± 126.67	3380.58 ± 14.60
1×10^− 6	3076.57 ± 53.04^a	2692.00 ± 195.35	2780.40 ± 149.05	3570.27 ± 193.58	3671.43 ± 63.65	3426.95 ± 117.50
5×10^− 6	2859.97 ± 138.03^a	2939.54 ± 134.15^a	2988.16 ± 146.67^a	3865.33 ± 120.63^a	3852.68 ± 98.77^a	3582.91 ± 64.89^a
1×10^− 5	2979.32 ± 46.57 ^a	2952.80 ± 97.75^a	3019.10 ± 141.80^a	3915.91 ± 157.94^a	3918.96 ± 64.89^a	3540.76 ± 38.63^a
Values are expressed as mean ± standard deviation (n = 3)
^ap<0.05, compared with the control group

3.2 Effect of BDE-209 on IR-BRL cell viability

Viability of IR-BRL cells were evaluated by MTT assay after treatment with BDE-209 for 24, 48 and 72 h, and a dose and time dependent inhibited manner was observed as BDE-209 concentration increasing (Fig. <link rid="fig8">1</link>–1). Therefore, BDE-209 caused concentration dependent cytotoxic effects on IR-BRL cells. When treated with BDE-209 for 48h, a significant variation was shown and the IC₅₀ of BDE-209 (48 h) on IR-BRL cells was 41.61µmol/L.

3.2 Effect of BDE-209 on glycose and lipid metabolism of IR-BRL cells

3.2.1 Glucose absorption capacity

As shown in Fig. 1–2, the glucose levels in the supernatant of cells in IR group significantly (p < 0.05) increased compared with the control group, indicating that the IR-BRL model was successfully established. Additionally, obvious dose-dependent manner was exhibited in IR + BDE-209 group as compared with that in the IR group, and a significant signal (p < 0.05) was observed when the exposure concentration of BDE-209 was 42 µmol/L at each experiment time point (24, 48 and 72 h). The results revealed that the BDE-209 could further exacerbate reduction in glucose uptake in IR-BRL cells, and this effect was dose-dependently.

3.2.2 TC and TG levels

TC and TG levels were the commonly used indicators that could evaluate the condition of lipid metabolism. As shown in Fig. 2 − 1, an increase signal was shown in IR group for both of the TC and TG levels as compared with that of the control group, suggesting that IR could interfere the lipid metabolism of BRL cells. In addition, the TC and TG levels in IR + BDE-209 groups (21 and 42 µmol/L exposure for 48 and 72 h, p < 0.05) were remarkably higher than that of the IR group, however, no difference was observed at 24 h. Collectively, these findings showed that BDE-209 could induce the disorder of lipid metabolism, and this effect would be more significant with higher dose and longer exposure period. In general, these data indicated that BDE-209 could aggravate the disorder of lipid metabolism in IR-BRL cells.

3.3 Effect of BDE-209 on hepatocyte function of IR-BRL cells

AST and ALT are the main indexes to detect liver cell injury. Results showed that AST and ALT levels were enhanced in the IR group as compared with that of the control groups, nevertheless, no distinct difference was observed except for the ALT level at 48 h (p < 0.05). Moreover, this enhancement was intensified by the exposure of BDE-209 in a dose-dependent manner except for the ALT level at 72 h with BDE-209 concentration of 10.5 µmol/L. Furthermore, AST level in IR + BDE-209 group (42 µmol/L) displayed a significant change (exposure for 48 and 72 h) in comparison to that in the IR group. Similar to AST, significance of ALT level only appeared at concentration of 42 µmol/L for 48 h, however, no difference was observed at 24 h and all exposure concentration exhibited noteworthy variation at 72 h. The results indicated that BDE-209 could exacerbate IR-induced hepatocyte damage.

3.4 Effect of BDE-209 on cell oxidative stress

MDA levels were detected to evaluate the oxidative stress in IR-BRL cells induced by BDE-209, and the results were shown in Fig. 2–3. Compared with the control group, MDA levels in IR group were dramatically (p < 0.05) increased, suggesting IR could strengthen the oxidative stress. Moreover, BDE-209 could up-regulate MDA levels in a dose-dependent manner, and significant differences (p < 0.05) were observed in all BDE-209 exposure groups except for 10.5 µmol/L exposure at 24 h as compared with that of IR group. In short, these detections demonstrated that BDE-209 could worsen the oxidative stress of IR-BRL cells.

3.5 Effect of BDE-209 on expressions of insulin receptor signaling pathway proteins

3.5.1 Expressions of glucose transport related proteins

Western Blot was applied to evaluate the expression of related proteins in insulin resistance signaling pathway, and the results of glucose transport related proteins were shown in Fig. 3 − 1. As shown in Fig. 3 − 1 (A) and (B), the p-IRS-1/IRS-1 ratio and the expression level of GLUT4 appeared to have an arresting decline trend (p < 0.05) in IR-BRL cells as compared with that of control group. In addition, these indicators displayed a tendency of decreasing in IR + BDE-209 groups, but no significant difference was observed as compared with that of IR group. These results proved that IR could disrupt the expressions of glucose transport related proteins, and this effect was strengthened with the presence of BDE-209.

3.5.2 Expressions of glycogen and fatty acid synthesis related proteins

Analyses on expressions of PI3K, AKT, GSK-3β and related phosphorylated proteins were shown in Fig. 3 − 1. It can be seen from Fig. 3 − 1 (C), (D) and (E) that a remarkably reduction of the PI3K expression level and p-GSK-3β/GSK-3β ratio was exhibited in IR group in comparison with that of the control group (p < 0.05). Besides, these indicators continued to decrease after the addition of BDE-209, and significant differences (p < 0.05) were observed in each BDE-209 exposure group as compared with that of IR group. However, no obviously difference was detected in the ratio of p-AKT/AKT between the IR group and the control group. Whereas, a further reduction tendency was observed at the presence of BDE-209 and a significantly change (p < 0.05) was shown when the concentration of BDE209 was 42 µmol/L. Hence, the results provided evidence that the treatment of BDE-209 could interfere with the expressions of glycogen and fatty acid synthesis related proteins in IR-BRL cells.

3.5.3 Expressions of proliferation and differentiation related proteins

In order to determine the effect BDE-209 exposure on the proliferation and differentiation of IR-BRL cells, Mek1/2, p-Erk1/2, Erk1/2 and mTOR were selected as target proteins in this research. As the results shown in Fig. 3 − 1 (F), (G) and (H), the expression of Mek1/2 and mTOR were significantly (p < 0.05) inhibited in IR-BRL cells as compared with the that of the control group. Moreover, this inhibition was strengthened by the exposure of BDE-209, and significant difference (p < 0.05) was observed in Mek1/2 expression compared with that of IR group. Although there was no significant difference in the ration of p-Erk1/2/Erk1/2 between the control and IR group, an obvious (p < 0.05) tendency of decreasing was shown in IR-BRL cells after the addition of BDE-209. The results revealed that BDE-209 could further down regulate the expressions of proliferation and differentiation related proteins in IR-BRL cells.

3.6 Effect of BDE-209 on expressions of insulin receptor signaling pathway genes

3.6.1 Expressions of glucose transport-related genes

Effects of BDE-209 on gene expressions of Irs-1 and Glut4 were shown in Fig. 3 − 2 (A) and (B). A significance falling (p < 0.05) of Irs-1 gene expression was observed in IR group as compared with the that of the control group, and this significant was maintained when BDE-209 appeared. However, there was no distinct differences of Irs-1 mRNA expression in BDE-209 + IR groups as compared with that of IR group. A slight reduction of Glut4 gene expression was discovered in IR-BRL cells as compared to that in BRL cells. Whereas, significant reductions of Glut4 gene expression in IR + BDE-209 exposure groups were disclosed as compared with that of IR group. These results were consistent with the expressions of glucose transport related proteins, suggesting that BDE-209 could inhibit the transport of glucose by affecting IRS-1/GLUT4 pathway.

3.6.2 Expressions of glycogen and fatty acid synthesis related genes

As shown in Fig. 3 − 2 (C), (D) and (E), the mRNA expression of Pi3k in IR group was not obviously altered as compared with that of the control group. Nevertheless, the mRNA expression of Pi3k in all IR + BDE-209 groups were significantly lower that of IR group (p < 0.05). For Akt, a negatively correlated with the concentration of BDE-209 was exhibited. Specifically, compared to the control group, IR could cause obvious decrease (p < 0.05) of mRNA expression of Akt, and this decrease displayed a tendency to even worsen when administration of BDE-209 in IR-BRL cells. Differently, no obvious statistical difference was observed in Gsk-3β mRNA expression between control and IR group. What’s more, although the expression of Gsk-3β mRNA was reduced by the effect of 10.5 and 21 µmol/L BDE-209, there was no significant difference in this indicator compared with that of IR group. These results indicated that BDE-209 could influence the synthesis of glycogen and fatty acid by affecting IRS-1/PI3K/AKT/GSK-3β pathway.

3.6.3 Expressions of proliferation and differentiation related genes

As shown in Fig. 3 − 2 (F), (G), (H) and (I), there was a downward trend in Mek1, Mek2, Erk1/2 and Mtor mRNA expressions of IR-BRL cells compared with that of the control group. Additionally, these gene expressions continued to decline overall after the addition of BDE-209. In particular, Mek1, Mek2 and Erk1/2 mRNA expressions was significantly (p < 0.05) inhibited as compared with that of the IR group. These observations were consistent with previous studies of proteins, suggesting that BDE-209 could inhibit the proliferation and differentiation of IR-BRL cells by affecting the expression of related proteins and genes.

3.7 Susceptibility analysis

3.7.1 Principal component analysis

The results of PCA were shown in Table 3 − 1 and Table 3 − 2. As shown in Table 3 − 1, there were two components with eigenvalues greater than 1, which together explain 82.798% of the total variance. These results indicated that the first principal components (PC1) accounted for 65.714% of the total variance, and the parameters of PI3K, mTOR, Mek1 mRNA, etc. had the larger load. The second principal components (PC2) accounted for 17.084% of the total variance and the parameter AKT, p-AKT/AKT, p-Erk1/2/Erk1/2, etc. had the larger load. In summary, two principal factors were selected from the original 22 variables employing PCA.

Table 3

− 1 Total Variance Explained
Component	Rotation Sums of Squared Loadings
Component	Total	% of Variance	Cumulative %
1	14.457	65.714	65.714
2	3.759	17.084	82.798

Table 3

− 2 Rotated Component Matrix
Parameter	Component
Parameter	PC1	PC2
glucose	-0.691	-0.559
TC	-0.447	-0.702
TG	-0.577	-0.734
AST	-0.221	-0.888
ALT	-0.729	-0.532
MDA	-0.555	-0.800
p-IRS-1/IRS-1	0.878	0.292
PI3K	0.958	0.135
p-AKT/AKT	-0.012	0.873
p-GSK-3β/GSK-3β	0.766	0.292
GLUT4	0.446	0.668
Mek1/2	0.846	0.380
p-Erk1/2/Erk1/2	0.131	0.959
mTOR	0.974	-0.078
Irs-1 mRNA	0.887	-0.229
Pi3k mRNA	0.850	0.355
Akt mRNA	0.884	0.343
Gsk-3β mRNA	0.034	-0.790
Mek1 mRNA	0.908	0.366
Mek2 mRNA	0.422	0.766
Erk1/2 mRNA	0.843	0.386
Mtor mRNA	0.841	0.368

3.7.2 Integrated biomarker response

According to the results of PCA, six biomarkers (TG, AST, p-IRS-1/IRS-1, PI3K, p-GSK-3β/GSK-3β and Erk1/2 mRNA) were selected for subsequent IBR analysis. Star charts drawn with standardized biomarker responses make it possible to visualize the comprehensive condition of BRL cells with or without IR after different dose of BDE-209 exposure. It is observed that the area of star plot of IR-BRL cells was larger than that of BRL cells at the same exposure dose of BDE-209 (Figure. 4-1A-D). In addition, the area of the star plot was positively correlated with the exposure dose in general. A stacked bar chart was drawn to display the contribution of selected biomarkers to IBR value. As shown in Fig. 4 − 2, for BRL cells, TG and AST are the most affected indicators in the 42 µmol/L exposure group. While for IR-BRL cells, p-IRS-1/IRS-1, PI3K, p-GSK-3β/GSK-3β and Erk1/2 mRNA all contributed greatly in each group, and the proportion of TG increased gradually. The IBR value represents the area of the star chart. Considering that the higher IBR value means the stronger biological responses and poorer health condition, these results indicated that the harmful effect of BDE-209 on test cells increased with dose, and this effect was more pronounced on IR-BRL cells.

3.7.3 Factorial analysis

According to the results of factorial analysis, there were interactive influences between IR and BDE-209 exposure on p-IRS-1/IRS-1 (F = 2.059, p < 0.01), PI3K (F = 0.505, p < 0.01), p-GSK-3β/GSK-3β (F = 2.278, p < 0.01), GLUT4 (F = 2.348, p < 0.01),

p-Erk1/2/Erk1/2 (F = 2.275, p < 0.01), mTOR (F = 0.759, p < 0.05), Pi3k mRNA (F = 1.594, p < 0.01), Akt mRNA (F = 2.066, p < 0.01), Gsk-3β mRNA (F = 1.297, p < 0.01),

Mek1 mRNA (F = 1.186, p < 0.01), Mek2 mRNA (F = 2.231, p < 0.01), Mtor mRNA (F = 2.498, p < 0.01) (Table 4). The results suggested that the effect of BDE-209 on BRL cells with or without IR was dose-dependent in general, and this effect was more significant on IR-BRL cells.

Table 4

The results of Factorial analysis
Parameter	F	p
Parameter	F	IR×BDE-209 exposure
glucose	1.158	0.749
TC	0.763	0.496
TG	0.898	0.654
AST	2.163	0.628
ALT	0.420	0.945
MDA	1.138	0.634
p-IRS-1/IRS-1	2.059	< 0.01
PI3K	0.505	< 0.01
p-AKT/AKT	0.213	0.073
p-GSK-3β/GSK-3β	2.278	< 0.01
GLUT4	2.348	< 0.01
Mek1/2	0.863	0.761
p-Erk1/2/Erk1/2	2.275	< 0.01
mTOR	0.759	< 0.05
Irs-1 mRNA	2.474	0.882
Pi3k mRNA	1.594	< 0.01
Akt mRNA	2.066	< 0.01
Gsk-3β mRNA	1.297	< 0.01
Mek1 mRNA	1.186	< 0.01
Mek2 mRNA	2.231	< 0.01
Erk1/2 mRNA	1.725	0.132
Mtor mRNA	2.498	< 0.01

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.

Our current study demonstrated that BDE-209 induced glycose and lipid metabolism and cell damage. BDE-209 could inhibit the transport of glucose combined with the synthesis of glycogen and fatty acid by affecting IRS-1/GLUT4 and IRS-1/PI3K/AKT/GSK-3β pathways. In addition, BDE-209 could interfered with the proliferation and differentiation of cells through affecting the expression of Mek1/2, Erk1/2 and mTOR proteins and genes. Furthermore, the susceptibility analysis showed that IR-BRL cells were more susceptible to the metabolism toxicity induced by BDE-209.

Authors Contributions

Conceptualization: Guanghua Mao, Methodology: Guanghua Mao, Data Curation: Guanghua Mao, Junjie Tang, Taotao Liao, Xiaoxiang Shi, FangYuan Dong, Formal analysis: Guanghua Mao, Junjie Tang, Writing - Original Draft: Guanghua Mao, Junjie Tang, Taotao Liao, Writing - Review & Editing: Guanghua Mao, Funding acquisition: Guanghua Mao, Project administration: Guanghua Mao, Visualization: Guanghua Mao, Junjie Tang, Investigation: Junjie Tang, Taotao Liao, Software: Xiaoxiang Shi, Weiwei Feng, Yao Chen, Validation: FangYuan Dong, Ting Zhao, Liuqing Yang, Project administration: Xiangyang Wu, Supervision: Xiangyang Wu, Liuqing Yang

Ethical Approval

There is no ethical issue in this article.

Consent To Participate

All co-authors consent to participate in this research.

Consent To Publish

The Author confirms:

That the work described has not been published before (except in the form of an abstract or as part of a published lecture, review, or thesis),

That it is not under consideration for publication elsewhere,

That its publication has been approved by all co-authors, if any,

That its publication has been approved (tacitly or explicitly) by the responsible authorities at the institution where the work is carried out.

The Author agrees to publication in the Journal indicated below and also to publication of the article in English by Springer in Springer's corresponding English- language journal.

Funding

This work was supported by State Key Laboratory of Environmental Chemistry and Ecotoxicology Open Fund [KF 2020-11], Natural Science Foundation of Jiangsu province [BK20160497], Natural Science Research of Jiangsu Higher Education Institutions of China [16KJB610005], Senior Talent Foundation of Jiangsu University [15JDG031], Priority Academic Program Development of Jiangsu Higher Education Institutions and Collaborative Innovation Center for Water Treatment Technology and Materials.

Competing Interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Availability of data and materials

The datasets generated or analyzed during this study are available from the corresponding author

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Metabolism toxicity and susceptibility of decabromodiphenyl ether (BDE-209) exposure on BRL cells with insulin resistance

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Version 2

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Materials

2.2 Cell culture

2.3 Establishment of IR-BRL model

2.4 Effect of BDE-209 on IR-BRL cells

2.5 Effect of BDE-209 on glycose and lipid metabolism of IR-BRL cells

2.5.1 Glucose absorption capacity

2.5.2 TC and TG levels

2.6 Effect of BDE-209 on hepatocyte function of IR-BRL cells

2.7 Effect of BDE-209 on cell oxidative stress of IR-BRL cells

2.8 Western blot analysis

2.9 RT-PCR analysis

2.10 Susceptibility analysis

2.11 Statistical analysis

3. Results

3.1 Establishment of IR-BRL model

3.2 Effect of BDE-209 on IR-BRL cell viability

3.2 Effect of BDE-209 on glycose and lipid metabolism of IR-BRL cells

3.2.1 Glucose absorption capacity

3.2.2 TC and TG levels

3.3 Effect of BDE-209 on hepatocyte function of IR-BRL cells

3.4 Effect of BDE-209 on cell oxidative stress

3.5 Effect of BDE-209 on expressions of insulin receptor signaling pathway proteins

3.5.1 Expressions of glucose transport related proteins

3.5.2 Expressions of glycogen and fatty acid synthesis related proteins

3.5.3 Expressions of proliferation and differentiation related proteins

3.6 Effect of BDE-209 on expressions of insulin receptor signaling pathway genes

3.6.1 Expressions of glucose transport-related genes

3.6.2 Expressions of glycogen and fatty acid synthesis related genes

3.6.3 Expressions of proliferation and differentiation related genes

3.7 Susceptibility analysis

3.7.1 Principal component analysis

3.7.2 Integrated biomarker response

3.7.3 Factorial analysis

4. Discussion

5. Conclusion

Declarations

References

Supplementary Files

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