Elsevier

Chemosphere

Volume 253, August 2020, 126772
Chemosphere

The livestock growth-promoter zeranol facilitates GLUT4 translocation in 3T3 L1 adipocytes

https://doi.org/10.1016/j.chemosphere.2020.126772Get rights and content

Highlights

  • The mycotoxin derivative zeranol used to promote growth in livestock facilitates glucose uptake in adipocytes.

  • GLUT4 expression and translocation, and Akt phosphorylation was increased by the growth promoter.

  • Replacing the mycotoxin by a generally accepted alternative is possible.

Abstract

Zeranol is an approved but controversial growth-promoting agent for livestock in North America. It is a mycotoxin metabolite secreted by the Fusarium family fungi. The regulatory bodies in this region have established the acceptable daily intake and exposure below the level would not significantly increase the health risk for humans. However, their European counterparts have yet to establish an acceptable level and do not permit the use of this agent in farm animals. Given the growth-promoting ability of zeranol, its effect on energy metabolism was investigated in the current study. Our results indicated that zeranol could induce glucose transporter type 4 (GLUT4) expression in 3T3 L1 cells at 10 μM and initiate the translocation of the glucose transporter to the membrane as assayed by confocal microscopy. The translocation was likely triggered by the increase of GLUT4 and p-Akt. The insulin signal transduction pathway of glucose translocation was analyzed by Western blot analysis. Since no increase in the phosphorylated insulin receptor substrate in zeranol-treated cells was evidenced, the increased p-Akt and GLUT4 amount should be the mechanism dictating the GLUT4 translocation. In summary, this study showed that zeranol could perturb glucose metabolism in differentiated 3T3 L1 adipocytes. Determining the growth-promoting mechanism is crucial to uncover an accepted alternative to the general public.

Introduction

Mycotoxins are fungal metabolites, and those isolated from food and food products are mostly generated by fungi grown in the pre-/post-harvesting or storage stage (Richard, 2007). Aflatoxins which are potent toxins to the liver (Bullerman and Caballero, 2003) caused an outbreak of mycotoxicosis in Kenya in 2004, and 125 lives were lost from contaminated maize (Azziz-Baumgartner et al., 2005). Zeranol or α-zearalanol is a mycoestrogen derived from the mycotoxins zearalenone and α-zearalenol (Daeseleire et al., 2017), and the metabolism, toxicology and detection methodology of the chemical have been reviewed (Baldwin et al., 1983). The acceptable daily intake (ADI) of zeranol residue was evaluated and established by the Joint Food and Agriculture Organization of the United Nations (FAO)/World Health Organization Expert (WHO) Committee on Food Additives (1997). The U.S. Food and Drug Administration has allowed zeranol to be used as a growth-promoter (brand name ‘Ralgro’) in livestock under Code of Federal Regulations Title 21. The European Food Safety Authority, however, has yet to establish the ADI (EFSA, 2007). The European Union countries prohibit the use of zeranol in farm animals or import meat and meat products with zeranol residue from non-EU countries under the Directive 96/22/EC and Directive 2003/74/EC. In the other parts of the world, such as Singapore and China, this agent is also prohibited to use in farm animals.

Zeranol is produced by fungi of the genus Fusarium (Mirocha et al., 1979; Bennett and Klich, 2003). It is an estrogen receptor (ER) agonist and can generate estrogen collaboratively with leptin by increasing the expression of aromatase in breast pre-adipocytes (Zhong et al., 2010). Because of its estrogenicity, the undesirable effect on breast carcinogenesis similar to that of estrogen could be a concern. Zeranol facilitates the growth and transformation of MCF-10A breast cells (Ye et al., 2011b). Its catechol metabolites could also introduce oxidative DNA damage (Fleck et al., 2012). It suppresses the tumor suppressor gene p53 expression in breast cancer cells (Ye et al., 2011a) and induces the proto-oncogene c-fos and the growth factor epidermal growth factor receptor (EGFR) expression in breast xenograft (Deng et al., 2009). Unlike estrogen, zeranol administration starting from the prepubertal stage does not promote chemical-induced mammary tumorigenesis; however, it may disrupt the normal ovarian functioning (Yuri et al., 2004).

Contrasting to the adverse effects, some favorable actions of zeranol have also been documented, like improving the vascular function in ovariectomized rats (Zhen et al., 2011) and reversing bone loss in rats without ovarian hormones (Zong et al., 2012).

The expression and translocation of glucose transporter type 4 (GLUT4) in adipose tissue are important for regulating glucose uptake from peripheral circulation (Moraes-Vieira et al., 2016). Besides, the gene expression of GLUT4 is known to be regulated by various physiological and pathological circumstances. The expression is reduced in obesity, type 2 diabetes and after long term fasting (Weems and Olson, 2011). GLUT4 activation and translocation can be facilitated by the P13 K/Akt/mTOR axis (Cong et al., 1997; Poloz and Stambolic, 2015).

Given its growth-promoting effect, we hypothesized that zeranol might perturb glucose metabolism in fat cells. Since GLUT4 is only expressed in mature adipocytes but not in preadipocytes (Taher et al., 2015), the effect of zeranol on lipid and glucose metabolic status was investigated in a differentiated 3T3 L1 cell model in the present study.

Section snippets

Chemicals

Zeranol (Catalog no. 0292, 98% purity, the stock solution was stored in −20 °C, insulin (Catalog no. I6634), 3-isobutyl-1-methylxanthine, dexamethasone (Catalog no. D4902), isopropanol (Catalog no. I9516), aprotinin (Catalog no. A1153), leupeptin (Catalog no. L2284), ethylenediaminetetraacetic acid (EDTA) (Catalog no. E9884), pepstatin (Catalog no. P4265), sodium deoxycholate (Catalog no. D6750), and sodium dodecyl sulfate (SDS) (Catalog no. L3771) were obtained from Sigma-Aldrich, St Louis,

Zeranol induced glucose uptake in 3T3 L1 cells

The effect of zeranol on glucose uptake was determined by a commercial kit as described. The result indicated that zeranol at 10 μM significantly increased 2-NBDG uptake in 3T3 L1 adipocytes (Fig. 1).

Reduced lipolysis in zeranol-treated adipocytes

Since the glucose uptake was found increased by zeranol treatment, we subsequently investigated the mycotoxin’s effect on lipolysis. The absorbance data suggested that zeranol treatment at 10 μM suppressed the breakdown of fat in the adipocytes (Fig. 2).

Zeranol did not promote adipogenesis

Adipogenesis describes the differentiation

Discussion

In the present study, we demonstrated that zeranol increased GLUT4 translocation. The mechanism could be attributed to the increased expression and activated Akt. Because of the reduced amounts of p-IRS, the insulin signaling upstream to Akt was unlikely involved in the translocation. A summary of findings is presented in Fig. 7.

Insulin may translocate GLUT4 vesicles to the cell surface in two ways, i.e. by activating the IRS/PI3K/Akt pathway or initiating the cleavage process of the tether

Credit author statement

YQ Tan was responsible for designing, carrying out the experiments, and manuscript preparation. Q Li and L Wang assisted the experiments and the data preparation. LC Chiu-Leung helped prepare and edit the manuscript, and performed the statistical analysis. LK Leung planned and oversaw the execution of the project, and prepared the manuscript.

Declaration of competing interest

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.

Acknowledgment

This research was funded by Chinese University of Hong Kong Grant no. SLS7105454.

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