Investigating the Response of Obesogens to Adipogenesis Transcription Factors in Adipocytes

Obesity in the United States is estimated to occur in 35.7% of American adults, and 17% of American children as reported by the CDC. This is a 22% increase from the numbers in 1962. In an effort to combat this disease, an increasing number of studies have been conducted recently that aim to correlate the environmental chemical exposure of humans to obesity. This has led to the classification of certain environmental contaminants as “obesogens” [1]. Specifically, two chemicals have generated significant interest as obesogens - Bisphenol-A (BPA) (Figure 1) and tributyltin (TBT).  These chemicals are found to disrupt the development of lipid metabolism and are observed to alter adipocyte metabolism by causing increased lipid deposits and storage; hence, adipogenesis is observed in fat cells.

BPA is a compound commonly used in polycarbonate plastics, in addition to “epoxy resin” [1] and mimics estrogenic activity. It is typically ingested and can affect humans through its presence in plastics and the lining of food cans [2]. Throughout many countries, including the United States and Canada, BPA has been speculated to cause many disorders such as obesity and diabetes. Canada was the first country to declare BPA as a toxin in 2010, followed by the European Union and the USA based on studies suggesting that exposure to BPA irreversibly affects lipid metabolism of fetuses, infants, and young children [9].

TBT, an organotin molecule, is typically found in paint and inhibits the growth of barnacles, bacteria and other marine animals [3].  In addition to its use in paint, TBT is also used in plastics, and as an anti-yellowing agent to preserve wood, paper, and similar compounds. It is also an irritant for human skin and eyes and can have harmful effects if inhaled. More importantly, this compound is of interest because it has been linked to obesity and is found in water plastic bottles.

In-vitro studies showed that BPA increases differentiation of adipocytes, and in combination with insulin, it accelerates the preadipocyte stem cell differentiation by up-regulating adipogenic genes such as PPARγ [5,7]. In an in vivo study, mice exposed to BPA either by injection or through water experienced a postnatal increase in weight [5]. Offspring of these BPA-exposed mice experienced the same metabolically altered adipose tissue effects (i.e., increase in postnatal weight). This study showed that pregnant mice displayed faster maturation of fat cells and larger increase in the number of adipocytes in adipose tissue.The same study also found that TBT disrupts the body’s controls over adipogenesis and energy balance, through disruption of the activity of the transcription factors RXR and PPARγ. Thus, exposure to TBT is believed to cause an increase in body fat and results in obesity.

We propose to investigate the effect of each BPA and TBT individually on transcription factors that regulate adipogenesis in adipocytes. We hypothesize that the addition of obesogens, such as TBT and BPA, will cause an upregulation in the transcription factors responsible for adipogenesis - PPARγ, CEBP, CREB, and SREBP - because it has been observed that those obesogens increase the number and size of fat droplets in adipose tissue cells. Adipogenesis is defined as the process in which the cell differentiates from a preadipocyte stem cell into an adipocyte. Adipogenesis plays an important role in energy homeostasis. A better understanding of the action of these obesogens through the intermediate macromolecules involved in adipogenesis can help us more effectively combat obesity linked to environmental factors. The expression levels of the mentioned key transcription factors for adipogenesis upon exposure of adipocytes to obesogens will be investigated.

The mouse 3T3-L1 preadipocyte cell line will be used for these experiments. These cells can be differentiated into functional adipocytes by exposing them to a standard adipogenic hormone cocktail. Pre-adipocytes with a secreted Gaussialuciferase (Gluc) reporter gene will be cultured and differentiated into adipocytes. These reporter cells have a binding region that is recognized by each transcription factor. When a transcription factor protein binds to the binding domain, it initiates transcription and expression of the Gluc gene (i.e., like an On switch). Therefore, by monitoring the level of Gluc production, we can infer the activation of the different transcription factors.

The cells will be cultured and seeded in preparation for treatment. The pre-adipocytes will be exposed to BPA or TBT at various concentrations on the day of differentiation. We expect to gain insight into the effects of these chemicals on adipocyte differentiation through monitoring the transcription factors.

Since Gluc is secreted into the culture medium, we will assay for Gluc activity in these samples to determine the extent of transcription factor activation. Reporter cells for 4 transcription factors - PPAR-gamma, CEBP, CREB, and SREBP – are available in the Jayaraman laboratory. Luciferase activity will be determined using a BioLux® Gaussia Luciferase Assay Kit and measured by a luminometer. The effect of obesogens on the transcription factors will be used as an indirect measure of adipogenesis, as these molecules are well established to control and regulate adipogenesis. In other words, an increase in activity of transcription factors will lead to an increase in luciferase activity or vice-versa.

We will also visually investigate lipid accumulation throughout the experiment by staining cells with the AdipoRed stain. Following the hypothesis, cells exposed to obesogens would show more luciferase activity and have larger lipid droplets than the control cells not exposed to obesogens. One of the functions of adipocytes is to store fat, so by comparing lipid accumulation in adipocytes that have been treated with BPA or TBT with the control group, we expect to determine how each obesogen impacts adipogenesis and adipocyte lipid accumulation.

In conclusion, our objective is to determine the qualitative and quantitative effects of two obesogens, BPA and TBT, on lipid accumulation and transcription factor activation in adipocytes. Previous studies have shown a correlation between obesity and BPA and TBT, however no study has focused on the activation of transcription factors in adipocytes by these obesogens and its effect on lipid accumulation. Through these various experimental procedures, we will determine whether these chemicals, which are found in everyday materials, negatively alter the adipocyte function and pose a public health risk to obesity.