Effects of endocrine disruptors on obesity

The Developmental Exposed DES Animal Model to Study Obesity, 2008

Summary

Environmental chemicals with hormone-like activity can disrupt the programming of endocrine signalling pathways that are established during perinatal life and result in adverse consequences that may not be apparent until much later in life. Increasing evidence implicates developmental exposure to environmental hormone mimics with a growing list of adverse health consequences in both males and females. Most recently, obesity has been proposed to be yet another adverse health effect of exposure to endocrine disrupting chemicals (EDCs) during critical stages of development. Obesity is quickly becoming a significant human health crisis because it is reaching epidemic proportions worldwide, and is associated with chronic illnesses such as diabetes and cardiovascular disease. In this review, we summarize the literature reporting an association of EDCs and the development of obesity, and further describe an animal model of exposure to diethylstilbestrol that has proven useful in studying mechanisms involved in abnormal programming of various oestrogen target tissues during differentiation. Together, these data suggest new targets (i.e. adipocyte differentiation and mechanisms involved in weight homeostasis) of abnormal programming by EDCs, and provide evidence that support the scientific term ‘the developmental origins of adult disease’. The emerging idea of an association of EDCs and obesity expands the focus on obesity from intervention and treatment to include prevention and avoidance of these chemical modifiers.

The developmental exposed DES animal model to study obesity

The role of environmental chemicals in the development of obesity is an emerging area of research which is focusing on the identification of obesogens, their possible molecular targets and potential cellular mechanisms through which they might act. Thus, to determine if environmental chemicals with hormone‐like activity are playing a role in the development of obesity and, further, study potential mechanisms involved, we used an experimental mouse model of perinatal DES exposure which was developed and characterized in our laboratory to study altered developmental programming of the reproductive tract which is well known to result in disease and dysfunction.

To study the mechanisms involved in DES toxicity, we developed an animal model using outbred CD‐1 mice treated with DES by subcutaneous injections on days 9–16 of gestation (the period of major organogenesis in the mouse) or days 1–5 of neonatal life (a period of cellular differentiation of the reproductive tract and a critical period of immune, behavioral and adipocyte differentiation). The developmental DES animal model has been used to successfully duplicate, and in some cases, predict, many of the alterations (structural, function, cellular and molecular) observed in similarly DES‐exposed humans.

Although our major focus has been on reproductive tract abnormalities and subfertility/infertility, we also examined the effects of DES on body weight. Treatment of female mice with DES on days 1–5 of neonatal life using a low dose of 0.001 mg/day did not affect body weight during treatment but was associated with a significant increase in body weight as adults. Further, data indicated that the increase in body weight in these DES‐exposed mice was associated with an increase in the percent of body fat as determined by mouse densitometry. Featured image schematically shows the weight patterns of low dosed DES‐treated mice and controls.

Unlike the low dose of DES (0.001 mg/kg), the high neonatal DES dose of 1 mg/day caused a significant decrease in body weight of female mice during treatment on days 1–5 but this was followed by a ‘catch up’ period lasting until about 2 months of age and then finally resulting in a significant increase in body weight of DES‐treated mice as compared with controls. The high body weight in all DES‐treated mice, both low and high doses, was maintained throughout adulthood. These data suggest that numerous pathways are probably involved in programming for obesity because DES at different doses resulted in obesity whether or not pups were under weight during treatment.

As the densitometry images suggested DES‐treated mice had excessive abdominal fat which has been previously reported to be associated with cardiovascular disease and diabetes, weights of various fat pads were measured to determine if specific fat pads were affected by DES treatment or whether it was a generalized effect throughout the mouse. At 6–8 months of age, fat pad weights were compared in DES‐treated mice (1 mg/kg) and controls; inguinal, parametrial, gonadal and retroperitoneal fat pads were all increased in DES‐treated mice as compared with controls; however, brown fat weights were not significantly different between DES and controls.

A recent study describes a role for developmental genes in the origins of obesity and body fat distribution in mice and humans. Therefore, exposure to environmental chemicals with hormonal activity may be altering gene expression involved in programming adipocytes. Several genes have been implicated in altering adipocyte distribution and function such as Hoxa5, Gpc4 and Tbx15 and fat cell distribution such as Thbd, Nr2f1 and Sfrp2. We investigated changes in gene expression by microarray analysis in uterine samples from DES‐treated mice (1 mg/kg) compared with controls at 19 days of age. In these samples, genes involved in adipocyte distribution were not altered in the uterus following neonatal DES exposure, however, genes involved in fat distribution were. Thbd and Nr2f1 were significantly downregulated and Sfrp2 was significantly upregulated in DES‐treated uteri compared with controls. These findings support the idea that environmental oestrogens may play a role in regulating the expression of obesity‐related genes in development.

Although DES‐treated mice were similar in weight to controls at 2 months, high dose DES (1 mg/kg) mice exhibited elevated serum levels of leptin, adiponectin, IL‐6 and triglycerides before overweight and obesity developed suggesting these endpoints may be important early markers of subsequent adult disease. The elevated levels of leptin and adiponectin may indicate insensitivity to these hormones and/or a loss of the negative feedback mechanisms that regulate adipogenesis. Further, at 6 months of age, insulin and all of the serum markers except triglycerides were found to be significantly elevated as compared with controls.

As, the balance of activity levels and food intake are known contributors to obesity, activity was measured in DES (1 mg/day) and control mice at 2 months of age before a difference in body weight could be detected. Individual mice were placed in an Opto‐Max motor activity chamber (Columbus Instruments, Columbus, OH, USA) and their ambulatory activity measured. Overall, there was no statistical difference in this parameter between the two groups although the DES group showed less movement as compared with controls as the experiment progressed. This difference, however, was not sufficient to explain the enhanced weight gain in DES mice as they aged. Additional measures of activity will be necessary before decrease in activity can be ruled out as a contributing factor to the development of obesity in these mice.

Feed consumption was also measured over a 2‐week period for control and DES‐treated mice (1 mg/kg). Although DES‐treated mice ate more than controls over the course of the experiment (approximately 3 g more), the amounts were not statistically different from controls. Taken into account, both the marginal decrease in activity and the increase in food intake in DES‐treated mice as compared with controls, it is unlikely these two measurements can solely explain the development of obesity in DES‐treated mice.

Glucose levels were also measured in DES (1mg/kg) and control female mice at 2 months of age prior to the development of obesity (Newbold et al., 2007a,b). Twenty‐five percent of the DES‐treated mice had significantly higher glucose levels than controls; these mice also showed a slower clearance rate of glucose from the blood because higher levels were seen throughout the experiment. It is important to note that altered glucose levels were observed in these mice before they developed excessive weight. Perhaps additional glucose measurements in older mice may help determine if a higher percentage of mice are affected with age, and if higher and sustained levels of glucose can be demonstrated. To date, however, our data suggest that overweight and obesity observed in perinatal DES‐treated mice will be associated with the development of diabetes, similar to the association of obesity with diabetes in humans. Interestingly, earlier studies from our laboratory have shown a high prevalence of islet cell hyperplasia in the pancreas of DES‐treated mice supporting the idea that these mice have abnormal glucose metabolism.

Although female mice exposed to DES at low or high doses developed obesity as adults, these phenomena were not apparent in similarly exposed males. In fact, DES‐treated males were smaller than corresponding controls and the decrease weight was dose dependent. This sex‐specific effect is not surprising because organizational effects of sex steroids during critical developmental periods have been well known for years to cause changes in the long‐term anatomy and function of the hypothalamic nuclei which regulates reproduction as well as body weight. Certainly, additional studies are necessary to investigate the effects of EDCs on male weight homeostasis and adipocytes because it is not clear whether this is a compound‐specific effect, an oestrogenic effect or typical of EDCs with other hormonal activities. Again, our findings point out the complexities of the mechanisms associated with the development of obesity.

References

DES DIETHYLSTILBESTROL RESOURCES

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