Nonylphenol Promotes the Differentiation of 3T3-L1 Preadipocytes

Background Nonylphenol (NP) induces obesity, we elucidated the inuence of NP on the differentiation of 3T3-L1 preadipocytes and characterize the key stages in the underlying mechanism. Methods 3T3-L1 preadipocytes were cultured until contact inhibition occurred and subsequently induced to differentiate using the MDI (methylisobutylxanthine, dexamethasone, and insulin) induction protocol. The cells were exposed to NP during the middle and late stages of the MDI-induced differentiation. After 24 h of exposure to NP, cell growth and differentiation were evaluated under an inverted phase-contrast microscope, lipid deposition was assessed by Oil Red O (ORO) staining, and the levels of the lipid-metabolism–related proteins CCAAT/enhancer-binding protein α (C/EBPα), fatty acid synthesis (FAS), peroxisome proliferator-activated receptor γ (PPARγ), and sterol regulatory element binding protein 1 (SREBP1) were determined by western blotting.


Abstract
Background Nonylphenol (NP) induces obesity, we elucidated the in uence of NP on the differentiation of 3T3-L1 preadipocytes and characterize the key stages in the underlying mechanism.
Methods 3T3-L1 preadipocytes were cultured until contact inhibition occurred and subsequently induced to differentiate using the MDI (methylisobutylxanthine, dexamethasone, and insulin) induction protocol. The cells were exposed to NP during the middle and late stages of the MDI-induced differentiation. After 24 h of exposure to NP, cell growth and differentiation were evaluated under an inverted phase-contrast microscope, lipid deposition was assessed by Oil Red O (ORO) staining, and the levels of the lipidmetabolism-related proteins CCAAT/enhancer-binding protein α (C/EBPα), fatty acid synthesis (FAS), peroxisome proliferator-activated receptor γ (PPARγ), and sterol regulatory element binding protein 1 (SREBP1) were determined by western blotting.
Results 1) Compared with the control group, the lipid droplets in the NP-treated cells were signi cantly more abundant and bigger,and the levels of C/EBPα, FAS, PPARγ and SREBP1 were signi cantly higher (P < .001).
2) The intensity of ORO staining was stronger and there were more intensely stained lipid droplets in the NP-treated cells during the middle stages than the late stage the levels of the C/EBPα, FAS, PPARγ and SREBP1 during the middle stages were approximately higher than those during the late stage (P < .001).
Conclusion NP promotes proliferation, differentiation, and lipid accumulation in 3T3-L1 preadipocytes, and increases the expression of the C/EBPα, FAS, PPARγ and SREBP1 and NP mainly promotes the proliferation and differentiation of 3T3-L1 preadipocytes during the middle stages of the MDI-induced differentiation.

Background
In recent years, the prevalence of obesity has rapidly increased worldwide, especially in young individuals [1]. According to the obesity and overweight data reported by the World Health Organization (WHO) [2], approximately 2 billion adults were overweight in 2016 and among these, 650 million individuals were obese. It is estimated that 2.7 billion adults will be overweight by 2025, with 1 billion of them considered obese. Although it is commonly believed that obesity is mainly related to lifestyle, dietary habits, and genetic factors [3], studies have suggested that the obesity epidemic is correlated with the increased production of chemicals after World War II [4]. This has led to the proposal of the "environmental obesogen" hypothesis in recent years. Endocrine-disrupting chemicals (EDCs) are a key class of presumptive environmental obesogens; in particular, certain EDCs such as nonylphenol (NP) [5], bisphenol A [6], and phthalates [7] at various exposure levels have been proven to promote weight increase and obesity in animal models or human.
NP is a typical lipophilic EDC capable of in uencing secretion of hormones [8], development of the reproductive system [9], airway in ammation [10],and functions of the nervous system [11]. NP may enter the human body through the use of household products and consumption of contaminated foods and drinks, such as tap water [12]or even infant formula powders [13]. Zhang [14] and Chang [15] studies have found that perinatal exposure to NP increases the body weight, fat mass, and total cholesterol level in the serum. Furthermore, in utero or neonatal exposure to NP causes hyperadrenalism and metabolic syndrome in the F1 generation . We have also shown that NP can induce diabetes mellitus [16]and nonalcoholic fatty liver disease [17], thereby resulting in metabolic syndrome [18]. The ndings described above indicate that NP may be a potential chemical stressor for obesity and obesity-related diseases.
However, the key period and mechanism underlying the effect of NP are currently unclear, and investigation of adipose tissue expansion, especially adipocyte differentiation, is critical to the understanding of obesity.
Previous studies [19] have reported that 3T3-L1 preadipocytes have the potential to differentiate from broblasts into adipocytes, and mature adipocytes differentiated from 3T3-L1 cells by using the MDI (methylisobutylxanthine, dexamethasone, insulin) induction protocol are ideal for the establishment of adipocyte models. The commonly used standard procedure for the MDI-induced differentiation of 3T3-L1 preadipocytes is as follows: First, 3T3-L1 preadipocytes with proliferative ability are cultured until contact inhibition occurs (i.e., the cells enter the G0 phase of the cell cycle and stops dividing). These contactinhibited preadipocytes are then subjected to MDI (methylis obutylxanthine, dexamethasone and insulin) induction using the hormones methylisobutylxanthine, dexamethasone, and insulin. Under the stimulation of MDI, preadipocytes enter a speci c cell division phase known as clonal expansion and ultimately differentiate into mature adipocytes. Therefore, 3T3-L1 preadipocytes were selected for this study. After 24 h of exposure, lipid deposition in the preadipocytes was examined by Oil Red O (ORO) staining, and the levels of lipid-metabolism-related proteins C/EBPα, FAS, PPARγ and SREBP1 were determined by western blotting to determine the key period and underlying mechanism of the pro-obesity effect of NP. The results of this study can provide a theoretical basis for early and precise intervention of obesity and obesity-related diseases. (1) Middle stage: NP working solution was prepared using the MDI induction medium described in Figure  1, and cells were concurrently exposed to NP for 24 h while induced to differentiate in the MDI induction medium. Subsequently, all other steps of the MDI induction protocol in Figure 1 were followed, and routine sampling was performed.
(2) Late stage: After inducing differentiation using the procedure described in Figure 1, the cells were exposed to the NP working solution for 48 h on Day 10 of induced differentiation, and routine sampling was performed.

Assessment of lipid deposition by Oil Red O staining
Oil Red O (ORO) staining was performed on successfully differentiated preadipocytes. The lipid droplets in the cells on each stained slide were observed and photographed under an optical microscope at 50× and 200× magni cation [22].

Statistical analysi
Statistical analysis of all the data was performed using SPSS 18.0 with a signi cance level of α = 0.05. One-way analysis of variance (ANOVA) was used to compare the differences among groups, and the least signi cant difference (LSD) test was adopted for pairwise comparisons when intragroup differences were observed. All the data are expressed as mean ± SD.

In uence of DMSO on the viability of 3T3-L1 preadipocytes and basis for the selection of DMSO concentration
To determine if DMSO exposure affected the viability of 3T3-L1 cells, the MTT assay was performed ( Figure. 4). The OD (wavelength: 490 nm) of the cells cultured with 0.1% or 0.01% DMSO was not signi cantly different from that of the control group, which indicates that DMSO at these concentrations did not in uence cell viability. However, as the difference in cell viability between the 0.1% DMSO-treated cells and control group was somewhat bigger than that caused by 0.01% DMSO, the latter DMSO concentration was selected for further experiments (Figure 4).

In uence of NP exposure on the differentiation of 3T3-L1 preadipocytes
The intensity of ORO staining in 3T3-L1 preadipocytes increased in correlation with the NP dose during all the two stages of the induced differentiation. Compared with the cells during the late stage, mid-stage cells had a denser distribution, higher staining intensity, and higher degree of differentiation ( Figure 5

Discussion
The results of ORO staining indicate that NP exposure promoted lipid deposition in 3T3-L1 preadipocytes.
ORO staining, which involves the use of Oil Red O, a liposoluble dye that readily dissolves in fat and speci cally stains lipids within tissues, is the most commonly used method for the detection of abnormalities in lipid droplets [26,27]. Wada et al. [28] have found that NP exposure signi cantly promotes the accumulation of triglycerides after the differentiation of 3T3-L1 preadipocytes into mature adipocytes, and thus their results are consistent with the results of the present study. Another study by Masuno et al. [29] showed that NP could promote the proliferation of fully differentiated 3T3-L1 cells. We [16,17]have also identi ed NP exposure as a risk factor for the onset of type II diabetes mellitus (DM) and non-alcoholic fatty liver disease (NAFLD). Furthermore, studies have proven that obesity is also a risk factor for the onset of type II DM and NAFLD [30,31]. In view of these ndings, it can be deduced that NP exposure can promote the proliferation and differentiation of 3T3-L1 preadipocytes and lipid accumulation in cells, thereby leading to obesity. This may be the mechanism by which NP causes the onset of type II DM and NAFLD. Results of the measurement of lipid metabolism-related protein levels revealed that NP exposure promoted the expression of the lipid-metabolism-related proteins C/EBPα, FAS, PPARγ, and SREBP1 in 3T3-L1 preadipocytes [32,33]. These proteins play key roles in the proliferation, differentiation, maturation, and lipid metabolism of adipocytes [34]. Speci cally, PPARγ is pivotal in lipid utilisation and storage, lipoprotein metabolism, and adipokine expression; C/EBPα is essential for the survival of mature adipocytes as high C/EBPα expression can trigger and accelerate adipocyte differentiation; SREBP1 is the main transcriptional mediator of lipid homeostasis as it can activate genes related to fatty acid synthesis, including the FAS and acetyl-CoA carboxylase genes. FAS is a key regulatory enzyme that guides the de novo synthesis of fats from fatty acids and plays an important role in the synthesis and deposition of lipids in animal cells and participates in PPARγ activation in mouse embryonic broblasts [35]. Therefore, a large number of studies have regarded the increase in the expression levels of PPARγ, C/EBPα, SREBP1, and FAS as an indicator of lipid formation. For instance, the differentiation process of 3T3-L1 mouse preadipocytes is accompanied by upregulation of C/EBPα, PPARγ, and SREBP1 levels [6,32]. In contrast, hypaphorine (an indole alkanoid) blocks the differentiation and lipid accumulation of 3T3-L1 adipocytes by inhibiting the expression of the PPARγ, C/EBPα, SREBP1, and FAS genes during the differentiation process [36]. Hao [5]and Zhang [14] have found that perinatal NP exposure leads to a signi cant increase in the mRNA levels of PPARγ, SREBP1, FAS, and lipoprotein lipase (LPL) in the adipose tissues of F1 and F2 rats, and these observations are consistent with the results of the present study. In view of the results described above, we deduce that NP may promote proliferation, differentiation, and lipid deposition in 3T3-L1 preadipocytes by increasing the expression of the lipid-metabolism-related proteins C/EBPα, FAS, PPARγ, and SREBP1. It is possible that NP shows the same effect in vivo, ultimately resulting in the onset of obesity. Although a large number of studies have proven that EDCs can in uence the proliferation and differentiation of preadipocytes, the key periods of in uence of EDCs on adipocytes during the differentiation of preadipocytes into mature adipocytes have rarely been reported. Therefore, the investigation of the key period of in uence of NP exposure on preadipocyte proliferation and differentiation is of great signi cance. If the proliferation and differentiation of preadipocytes during the key period of in uence can be inhibited by chemical or physical methods, the incidence of obesity and obesity-related diseases caused by EDCs, such as NP, can be effectively reduced. In the present study, preadipocytes exposed to NP had a higher distribution density and intensity of staining during the middle stages of induced differentiation compared with the levels during the late stage. In addition, the levels of the lipid-metabolism-related proteins C/EBPα, FAS, PPARγ, and SREBP1 in the medium-and high-NP dose groups were also signi cantly higher during the middle stages of the differentiation than during the late stage. Therefore, it can be deduced that the key periods of the NP in uence on 3T3-L1 preadipocyte differentiation may be the middle stages of MDI-induced differentiation .

Conclusion
NP increases the expression of lipid-metabolism-related proteins, thereby promoting the proliferation, differentiation, and intracellular lipid accumulation of 3T3-L1 preadipocytes, with the possible key periods of in uence being the middle stages of MDI-induced differentiation. The results of this study can provide a theoretical basis for early and precise intervention of obesity and obesity-related diseases.   In uence of DMSO on the viability of 3T3-L1 cells In uence of NP exposure on 3T3-L1 preadipocytes proliferation during the middle and late stages of the induced differentiation. In uence of NP exposure on the lipid-metabolism-related proteins C/EBPα, FAS, PPARγ, and SREBP1 in 3T3-L1 preadipocytes during the middle and late stages of differentiation. avs Control group, P 0.05. bvs 40 pM group, P 0.05.cvs 40 nM group, P 0.05.

Figure 7
The levels of lipid-metabolism-related proteins in 3T3-L1 preadipocytes During the middle and late stages of induced differentiation. *vs late stage, P < .05.

Supplementary Files
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