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The goal of this research is to elucidate the multiple receptor-mediate mechanisms through which estradiol and environmental estrogens from food production and food storage (BPA, Zeronal, etc.) can impact energy homoeostasis and other hypothalamic functions. This research will address basic neurological effects of these compounds and further enhance our knowledge of the impacts that environmental estrogens have on human health by using novel approaches (transgenic mouse models and electrophysiological techniques) and integration with whole animal studies. <P>First, I will examine the effects of ERE-independent estrogen signaling (ERalpha and Gq-mER) in the control of gene expression and energy homeostasis and determine if BPA and Zeranol activate these same pathways. I will determine if E2, BPA and Zeronal alter energy homeostasis and gene expression in adults through other ERalpha-mediated, ERE-independent signaling mechanisms using the recently developed ERalpha KI/KO transgenic mouse model, which does not have ERE-dependent signaling, compared to alphaERKO and wild-type mouse strains. <P>Secondly, I will examine the effects of maternal exposure to environmental estrogens (BPA, Zeronal) and whether these effects function via ERE-dependent or independent estrogen signaling to control of gene expression and energy homeostasis using the same transgenic mouse strains. <P>Thirdly, I will examine the effects of acute E2 treatment (within mice) on the neuronal excitability of hypothalamic arcuate neurons to determine if environmental estrogens will mimic or inhibit E2's effects, using both intact males and ovariectomized females. <P>Finally, I will examine the effects of in vivo exposure to environmental estrogens during development or adulthood on the expression of cation channel subunits i.e., KATP, KCNQ, T-type Cav3.1, TRPC, etc., in POMC and NPY neurons and alter the activity of the respective currents in both males and females. Electrophysiological studies using whole-cell patch clamp techniques coupled with single-cell quantitative real-time PCR will determine if treatment with environmental estrogens will alter cation channel expression and activity in POMC and NPY neurons. Data from these experiments will be analyzed and published in peer-reviewed journals and presented at national and international conferences. Data from these studies will advance the field of neurotoxicology by utilizing techniques (electrophysiology) to address issues surrounding the effects of environmental estrogens on brain functions. Data from these studies may impact how food safety is regulated in regards to environmental estrogens.
Non-Technical Summary:<br/>
The impacts of chemicals that can mimic the actions of estrogens on physiological systems like energy balance (weight gain, food intake, glucose, etc.), and thereby contribute to the obesity epidemic in both juvenile and adult populations, are of increasing interest to the scientific community, regulatory organizations and the general population. The research outlined in the project will determine the underlying cellular mechanisms behind these effects using unique transgenic strains of mice exposed both during development and in adulthood to environmental estrogens delivered to humans through food production, storage and distribution. Because many of these environmental estrogens are found in food containers and used in food production, the data from this research project may inform governmental decision-making in regards to the safety of food both at the state and federal level.
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Approach:<br/>
This research will have 3 different experimental methods. The first methods involve conducting whole animal experiments on males and females that will assess the effects of estradiol and enviromental estrogens on physiological functions related to energy balance (weight gain, food intake, energy expenditure, fat accumulation, etc.) in unique transgenic mouse models. This will be done using lab animal monitoring equipment and MRI. Secondly, those same animals will be sacrificed at the completion of the whole animal experiments and hypothalamic brain tissue will be collected, as well as peripheral tissues like the liver, muscle and fat tissue. This tissues will be prepared for and analyzed by gene microarray and quantitative real-time PCR for alterations of genes in the various tissues known to be involved in energy balance and cellular physiology. The last experimental method will examine the cellular physiology of neurons involved in energy balance in the hypothalamus, using whole-cell patch clamp electrophysiology. This technique has not been used when examining the effects of environmental estrogens in the brain and will yield new data and new approaches to the study of neurotoxicology. Data from all three methods and experimental approaches will be prepared for publication in peer-reviewed journals as well as conferences and symposiums. Milestones for this research will be monitored and assessed by the presentation of the data as stated above. The manuscripts generated from this research will inform public policy decisions on the health and safety of environmental estrogens in our food. I will also be mentoring undergraduate and graduate students and instructing them on the various aspects of this research plan, the techniques, data collection and analysis and preparation of manuscripts.
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Progress:<br/>
2011/10 TO 2012/09<br/>
OUTPUTS: The aim of this project is to understand the effects of endocrine disrupting compounds (environmental estrogens) from food production and food storage (bisphenol-A, phthalates, Zeronal, etc.) on energy homoeostasis and other hypothalamic functions and to determine which steroid signaling pathways are involved including those functioning through membrane-mediated actions. In the past year, I have focused on setting up my laboratory, training technicians and graduate students in common experimental techniques, and initiating experiments. Currently, my lab is conducting studies determining the effect of 17beta-estradiol replacement post-ovariectomy on energy balance in female transgenic mice to establish a baseline. These studies examine the effects of 17beta-estradiol replacement on body weight gain, oxygen consumption, food intake, fat accumulation and glucose/triglyceride regulation when fed normal chow and hi-fat chow in adult females. Also, hypothalamic genes involved in energy balance will be examined in these treated females for changes in gene expression. Furthermore, the effects of endocrine disrupting compounds on the electrophysiological characteristics of hypothalamic neurons are just now being conducted. Outreach activities included presentation of data generated from this project at the 5th Annual Pioneers in Endocrinology Symposium at Rutgers University on September 24, 2012, by the graduate students. In addition, the research conducted this year will serve as the basis for an NIH R01 proposal to be submitted in February 2013 and focusing on the effects of endocrine disruption from maternal exposures on adult energy homeostasis using transgenic mouse models.
<br/>PARTICIPANTS: Troy A. Roepke, PI. Dr. Roepke designs, and oversees or conducts all experiments, analyzes and interprets data and prepares written reports and manuscripts. Training and Professional Development: This research project has provided research training opportunities in 2012 for the following graduate students: Jennifer Yang and Kyle Mamounis. In addition, Michelle Hernandez, Pauline Chen, and Anna Hsieh, undergraduate students in the laboratory, gained valuable hands-on research experience in conducting these studies.
<br/>TARGET AUDIENCES: The target audiences for this research are food producers and food handlers (both agribusiness and factory producers) and includes those that are interested in the deleterious effects of contaminants and natural toxins on child development, behavior and normal physiological functions that control obesity. This research project serves as the basis for training graduate and undergraduate students in hypothesis testing, experimental design, research methodologies and data analysis. These studies also involve additional informal teaching about energy balance, neuroscience, neuroendocrinology, reproduction, maternal programming of development and endocrine disruption. Students also gain the opportunity for experiential learning in rodent reproductive physiology, handling and surgery.
<br/>PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
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IMPACT: The goal of these studies is to improve our understanding of the effect of endocrine disruption on energy balance and obesity through either development exposures. We are currently in the process of collecting baseline date for experiments involving exposure to endocrine disrupting compounds during adulthood and during pre-natal and post-natal development. We have also collaborated with Dr. Mehmet Uzumcu on experiments in rats exposed developmentally to endocrine disrupting compounds, in particular, examining the effects of these compounds on hypothalamic gene expression using highly sensitive gene expression assays. Early results suggest that there are several genes involved in energy balance that are altered in the hypothalamus from adults exposed to bisphenol A and other compounds. These experiments will be completed in the coming year and submitted for publication.