THE INTEGRATION OF HORMONAL AND CENTRAL SIGNALS AND THE IMPACTS OF ENDOCRINE DISRUPTING COMPOUNDS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0228769
• Project No.: NJ06107
• Project Start Date: Jul 1, 2012
• Project End Date: Jun 30, 2017

Project Director
Roepke, TR.

Recipient Organization
RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY
3 RUTGERS PLZA
NEW BRUNSWICK,NJ 08901-8559

Performing Department
Animal Sciences

Non Technical Summary
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.

Animal Health Component

20%

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
711	3840	1010	15%
711	3840	1020	20%
711	3840	1150	15%
702	3840	1010	15%
702	3840	1020	20%
702	3840	1150	15%

Knowledge Area
711 - Ensure Food Products Free of Harmful Chemicals, Including Residues from Agricultural and Other Sources; 702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
3840 - Laboratory animals;

Field Of Science
1010 - Nutrition and metabolism; 1020 - Physiology; 1150 - Toxicology;

Keywords

neuroendocrinology

toxicology

physiology

hypothalamus

energy homeostasis

estradiol

environmental estrogens

electrophysiology

gene expression

body weight

food intake

energy expenditure

estrogen receptors

transgenic mice

Goals / Objectives
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. 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. 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. 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. 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.Due to disinterest in allocating funding to examine the effect of bisphenol A on energy homeostasis, we would like to reallocate our own efforts away from any further investigation into bisphenol A and to focus our endeavors on Zeranol and flame retardants (PDBE-47 and organophosphate flame retardants (OPFR)).

Project Methods
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.

Progress 07/01/12 to 06/30/17

Outputs
Target Audience:The target audiences for this research are food producers and food handlers (both agribusiness and factory producers), scientists, and other groups that are interested in the negative effects of contaminants, chemicals, and natural toxins in food and the home/work environment on child development, behavior, and normal physiological functions that control energy balance. These effects may cause health problems like obesity, diabetes, metabolic syndrome, in adults Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This research project has provided research training opportunities in for over 40 undergraduate students, seven graduate students (Jennifer Yang, Kyle Mamounis, Elizabeth Krumm, Gwyndolin Vail, Ali Yasrebi, Sabrina Walley and Kristine Conde), and two postdoctoral researchers (Drs. Jason Magby and Pu Hu). Jennifer, Kyle, Ali, Liz, and Gwyn all attended international scientific meetings to present their research. Undergraduate student Vipa Patel presented her project on the effects of adult exposures to flame retardants on energy balance, glucose homeostasis, hormone production, and hypothalamic gene expression at aninternational symposium in Brazil. Other undergraduates involved in this research leading to authorship in a publication include Anna Hsieh, Marisa Snyder, Michelle Hernandez, Alejandra Villalobos, Taylor Tillery, Janelle Rivera, Victoria Wright, Sarah O'Leary, Nick Margolis, and Naomi Shvedov. This research project serves as the basis for training graduate and undergraduate students in hypothesis generation and testing, experimental design, research methodologies and data analysis. These studies also involve additional informal teaching about energy balance, neuroscience, neuroendocrinology, reproduction, maternal programming, and endocrine disruption. Students have gained the opportunity for experiential learning in rodent physiology, handling, and surgery. How have the results been disseminated to communities of interest?We have published 12 original manuscripts this past year with 1 more resubmitted and two invited reviews accepted in the past month. We currently have another 2 in preparation for publication in 2017 and 3-4 in preparation for 2018. I alone have presented this data at 8 international and national conferences in addition to the presentations of my undergraduate and graduate students and postdoctoral researchers. Furthermore, I have used the data collected from these studies to submitted 3 R01 applications and 3 R21 applications to the NIH. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The goal of these studies has been to improve our understanding of the effect of endocrine disruption on hypothalamic functions especially energy balance, glucose metabolism, reproduction and thermoregulation through either adult or developmental exposures. We have collected baseline data for experiments involving exposure to endocrine disrupting compounds during adulthood and during perinatal development. In our first publication, we reported the effects of ovariectomy and estradiol replacement on body weight gain, feeding and energy balance in wild-type, ERalpha knockouts and ERalpha knockin/knockout (that lack a functional DNA binding domain in the ERalpha gene). We continued these studies in the transgenic mice with the added variable of a low-fat vs. high-fat diet and testing for glucose tolerance and insulin sensitivity and gene expression in the liver. This project was published in 2017. Another study published in 2016 used these same transgenic mouse models to examine the expression of selected genes using quantitative real-time PCR in the hypothalamus of wildtype, ERalpha knockouts and ERalpha knockin/knockouts after treatment with estradiol. We have also expanded this project by employing cutting-edge RNA Sequencing technology to examine the entire expressed genome (the transcriptome) in the arcuate nucleus from these transgenic mouse models after estradiol treatment. This project was also published in 2017. An additional study was conducted utilizing the same mouse models to examine the effects of maternal high fat diets on adult female offspring energy homeostasis and glucose homeostasis. These data demonstrate, for the first time, that ERalpha is involved in the response of adult female offspring to maternal high fat diets. The first half of this data was published in 2017 and the second half is in preparation for submission in December 2017. An R01 NIH application was submitted in 2015 based on these studies and will be resubmitted in 2018. We completed studies on the effects of maternal exposures to nonylphenol and alpha-zearanol on the energy balance and glucose homeostasis and hypothalamic gene expression in adult male and female offspring in mice. This study will be submitted for publication in the coming year. We have also completed sample collection and animal experimentation in a project examining the effects of maternal exposure to flame retardants, a mixture of organophosphate flame retardants (TPP, TCP, TDCPP), on offspring energy balance and glucose homeostasis. This project is nearing completion and will be submitted for publication in 2018 and for submission of an NIH R01 in February 2018. We are also conducting experiments examining the effects of flame retardants on gene expression and neuronal activity in adult male and female mice. We found significant effects on arcuate gene expression of cation channels and hormone receptors in males and females. These studies are complete and under second review for publication. Data from this project was used for an NIH R21 application focusing on the neurophysiological effects of these exposures on arcuate neurons, which was funded in March 2017. Another area of research is the examination of the effects of high fat diets differing in fatty acid composition (saturated vs polyunsaturated) on adult energy homeostasis either through adult diets or maternal diets. Individual studies on male and female mice were both accepted for publication in 2017. The maternal experiment is nearing completion and will be submitted in 2018 for publication and potentially for another NIH R01 application. Collectively, these data have sought to characterize how estradiol, high-fat diets, and endocrine disrupting compounds impact energy balance, especially during pregnancy, early infant, and childhood development. We have successfully highlighted the impact of these influences on obesity and associated syndromes in both sexes. This research has also impacted the field by using techniques and tools not employed in the past in addressing these concerns and may provide clues as to the impacts these chemicals and diets have on the developing brain

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Mamounis, K.J., Yasrebi, A., and Roepke, T.A. Linoleic acid causes greater weight gain than saturated fat without hypothalamic inflammation in the male mouse. Journal of Nutritional Biochemistry 40:122-131.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Yang, J.A., Stires, H., Belden, W., and Roepke, T.A. The arcuate estrogen-regulated transcriptome: estrogen response element-dependent and -independent signaling of ERalpha in female mice. Endocrinology 158(3):612-626
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Yasrebi, A., Rivera, J.A., Krumm, E.A., Yang, J.A., and Roepke, T.A. Activation of estrogen response element-independent ERalpha signaling protects female mice from diet-induced obesity. Endocrinology 158(2):319-334.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Roepke, T.A, Yasrebi, A., Villalobos, A., Krumm, E.A., Yang, J.A., and Mamounis, K.J. Loss of ERalpha partially reverses the effects of maternal high-fat diet on energy homeostasis in female mice. Scientific Reports. 7(1):6381.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2017 Citation: Mamounis, K.J., Hernandez, M., Margolies, N., Yasrebi, A., and Roepke, T.A. Interaction of 17?-estradiol and fatty acids on energy and glucose homeostasis in females. Nutritional Neuroscience. July 7:1-14. (Epub ahead of print)
• Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Vail, G., and Roepke, T.A. Membrane-initiated estrogen signaling via Gq-coupled GPCR in the central nervous system. Steroids  Review (refereed) accepted.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Convergence of Membrane-Initiated and Nuclear-Initiated Estrogen Signaling in Regulating the Expression and Activity of a Potassium Current. 2017 International Workshop in Neuroendocrinology, Coc�n, Chile. August 6-9.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Yasrebi, A., and Roepke, T.A. The role of ERalpha-mediated ERE-dependent and ERE-independent signaling in feeding and exploratory behaviors in male and female mice. Society for Neuroscience, Washington, D.C., November 11-15.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Vail, G., and Roepke, T.A. The effects of organophosphate flame retardants on the M-current in NPY neurons in adult mice. Society for Neuroscience, Washington, D.C., November 11-15.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Conde, K., and Roepke, T.A. Ghrelin inhibition of the M-current in KNDy neurons and the impact of 17beta-estradiol. Society for Neuroscience, Washington, D.C., November 11-15.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Walley, S.N., Yasrebi, A., and Roepke, T.A. The effects of maternal exposure to organophosphate flame-retardants on offspring feeding and exploratory behaviors. Society for Neuroscience, Washington, D.C., November 11-15.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The target audiences for this research are food producers and food handlers (both agribusiness and factory producers), scientists and other groups that are interested in the negative effects of contaminants, chemicals, and natural toxins in food and the home/work environment on child development, behavior, and normal physiological functions that control energy balance. These effects may cause health problems like obesity, diabetes, metabolic syndrome, in adults Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This research project has provided research training opportunities in 2016 for the following graduate students: Kyle Mamounis, Jennifer Yang, Gwyndolin Vail, Sabrina Walley and Ali Yasrebi. Jennifer attended international scientific meetings to present their research. Undergraduate Vipa Patel presented her project on the effects of adult exposures to flame retardants on energy balance, glucose homeostasis, hormone production, and hypothalamic gene expression at an international symposium in Brazil. Other undergraduates involved in this research include Taylor Tillery, Janelle Rivera, Victoria Wright, Nick Margolis, Jessica Medina, Steven Rabel, and Daniel Regan. This research project serves as the basis for training graduate and undergraduate students in hypothesis generation and testing, experimental design, research methodologies and data analysis. These studies also involve additional informal teaching about energy balance, neuroscience, neuroendocrinology, reproduction, maternal programming, and endocrine disruption. Students also gain the opportunity for experiential learning in rodent physiology, handling, and surgery. How have the results been disseminated to communities of interest?We have published 6 manuscripts this past year with 2 more recently accepted in the past month. We currently have another 2 in revision and another 2 in preparation for publication in 2017. Furthermore, I have used the data collected from these studies to submitted 1 R01 applications and 3 R21 applications to the NIH. What do you plan to do during the next reporting period to accomplish the goals?We will submit for publication the studies discussed above including 1) the effects of maternal high fat diet on female offspring using the wild-type, ERKO, and KIKO mouse models; 2) the arcuate transcriptome project in the wild-type, ERKO, and KIKO mouse models; 3) The effects of high-fat diets containing either high PUFA or high SFA on energy balance in males; and 4) females; and 6) offspring; 7) the effects of flame retardant exposures on adult energy homeostasis and gene expression. Finally, we will complete the studies examining the effects of maternal exposures to flame retardants (OPFR) on adult offspring energy and glucose homeostasis and prepare for publication. I also have a new graduate student, Sabrina Walley, who will conduct the maternal OPFR exposure studies as a part of her thesis.

Impacts
What was accomplished under these goals? The goal of these studies is to improve our understanding of the effect of endocrine disruption on hypothalamic functions especially energy balance, glucose metabolism, reproduction and thermoregulation through either adult or developmental exposures. We are currently in the process of collecting baseline date for experiments involving exposure to endocrine disrupting compounds during adulthood and during perinatal development. In our first publication, we reported the effects of ovariectomy and estradiol replacement on body weight gain, feeding and energy balance in wild-type, ERalpha knockouts and ERalpha knockin/knockout (that lack a functional DNA binding domain in the ERalpha gene). We have continued these studies in the transgenic mice with the added variable of a low-fat vs. high-fat diet and testing for glucose tolerance and insulin sensitivity and gene expression in the liver. This project is complete and was recently accepted for publication. Another study using these same transgenic mouse models has been completed and is being resubmitted for publication. This study examined the expression of selected genes in the hypothalamus of wildtype, ERalpha knockouts and ERalpha knockin/knockouts after treatment with estradiol. These data were published in 2016. We have also expanded this project by employing cutting-edge RNA Sequencing technology to examine the entire expressed genome (the transcriptome) in the arcuate nucleus from these transgenic mouse models after estradiol treatment. This project is currently under revision for publication. Another study using a maternal high fat diet paradigm examines the effects of maternal high fat diets on adult female offspring energy homeostasis and glucose homeostasis using wild type, ERalpha knockout, and ERalpha knockin/knockout transgenic strains. These data demonstrate, for the first time, that ERalpha is involved in the response of adult offspring to maternal high fat diets. These data are also being prepared for publication and will be submitted shortly. An R01 NIH application was submitted in 2015 based on these studies and will be resubmitted in 2017. We completed studies on the effects of maternal exposures to nonylphenol and alpha-zearanol on the energy balance and glucose homeostasis and hypothalamic gene expression in adult male and female offspring in mice. This study will be submitted for publication in the coming year. We have also completed sample collection and animal experimentation in a project examining the effects of maternal exposure to flame retardants, a mixture of organophosphate flame retardants (TPP, TCP, TDCPP), on offspring energy balance and glucosue homeostasis. This project is being repeated to increase sample size of litters for a resubmission of an NIH R01 in June 2017 and publication later in 2017. We are also conducting experiments examining the effects of flame retardants on gene expression and neuronal activity in adult male and female mice. We found significant effects on arcuate gene expression of cation channels and hormone receptors in males and females. These studies are complete and will be submitted for publication in 2017. Data from this project was used for an NIH R21 application focusing on the neurophysiological effects of these exposures on arcuate neurons. The application was scored high and we are awaiting the funding decision. Another area of research is the examination of the effects of high fat diets differing in fatty acid composition (saturated vs polyunsaturated) on adult energy homeostasis either through adult diets or maternal diets. The first aim of this project was recently accepted for publication. Data from the second and third aim of this project will be submitted for publication in 2017. Collectively, these data seek to characterize how estradiol impacts energy balance and to characterize the potential impacts that exposures to environmental contaminants and/or high fat diet during pregnancy and early infant and childhood development have on the obesity epidemic in both sexes. This research will also impact the field by using techniques and tools not employed in the past in addressing these concerns and may provide clues as to the impacts these chemicals and diets have on the developing brain

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Yasrebi, A., Hsieh, A., Mamounis, K.J., Krumm, E.A., Yang, J.A., Magby, J., Hu, P., and Roepke, T.A. Regulation of GHSR signaling pathway in the arcuate nucleus and NPY neurons by fasting, diet-induced obesity and 17beta-estradiol. Molecular and Cellular Endocrinology 422:42-56
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Gotthardt, J.D., Verpeut, J.L., Yeomans, B.L., Yang, J.A., Yasrebi, A., Roepke, T.A., and Bello, N.T. Intermittent Fasting Promotes Fat Loss with Lean Mass Retention, Increased Hypothalamic Norepinephrine Content, and Increased Neuropeptide Y Gene Expression in Diet-Induced Obese Male Mice. Endocrinology 157:679-691
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Yang, J.A., Yasrebi, A., Mamounis, K. and Roepke, T.A. ERE-dependent and ERE-independent gene regulation by 17beta-estradiol in the arcuate nucleus of female mice. Steroids 107:128-138
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Roepke, T.A., Yang, J.A., Yasrebi, A, Mamounis, K., Oruc, E., Zama, A., and Uzumcu, M. Endocrine disrupting compounds regulate genes involved in energy homeostasis in female rats exposed during development. Reproductive Toxicology 62:18-26
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Hu, P., Liu, J., Yasrebi, A., Gotthard, J.D., Bello, N.T., Pang, Z., and Roepke, T.A. G-protein-coupled membrane Estrogen Receptor signaling mediates rapidly excites corticotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus in female mice. Endocrinology 157:3604-3620
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Yang, J.A., Snyder, M, Yasrebi, A., and Roepke, T.A. The interactions of fasting, caloric restriction, and diet-induced obesity with 17beta-estradiol on KNDy neuropeptides and their receptors in the female mouse. Molecular and Cellular Endocrinology 437:35-50.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Adult Exposure to Flame Retardants Alters Energy Homeostasis, Peptide Hormone Levels and Hypothalalmic and Liver Gene Expression. Patel, V., Krumm, E.A., Tillery, T., Yasrebi, A., Shen, J., Guo, G., and Roepke, T.A. National Institute for Environmental Health Sciences (NIEHS) 25 Years of Endocrine Disruption Research: Past Lessons and Future Directions, Bethesda, MA. September 21-22, 2016

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:The target audiences for this research are food producers and food handlers (both agribusiness and factory producers), scientists and other groupsthat are interested in the negative effects of contaminants, chemicals, and natural toxins in food and the home/work environmenton child development, behavior,and normal physiological functions that control energy balance. These effects may cause health problems like obesity, diabetes, metabolic syndrome, in adults. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This research project has provided research training opportunities in 2015for the following graduate students: ElizabethKrumm, Kyle Mamounis, Jennifer Yang, and Grahma Vail. Elizabeth, Kyle, and Jennifer have each attended international scientific meetings to present their research. Elizabeth presented at Society for Neuroscience. Kyle presented at Experimental Biology. Jennifer presented at Society for Neuroscience and Society for Behavioral Neuroendocrinology.UndergraduatesWendy Flores and Taylor Tillery received training in molecular biology while completing a projectexaming the effects of maternalexposures nonylphenol and Zeranol on adult hypothalamic geneexpression, whichpertain directly to the aims of the grant. Vipa Patel, another undergraduate student, is completing studies initiated by Elizabeth Krumm for her thesis. This project examines the effects of flame retardants on gene expression, energy balance, and control of glucose in male and female mice. All three of these undergraduate students have had the opportunity to present their research at local student research symposiums.Other undergraduates involved in thisresearch include Michelle Hernandez, Janelle Rivera, Ruleena Barreto, Alejandra Villalobos, Victoria Wright, Nick Margolis,Steven Rabel, Christina Kaye, Gina Giunta, Daniel Regan, and Lubna Begum. This research project serves as the basis for training graduate andundergraduate students in hypothesis generation and testing, experimental design, research methodologies and dataanalysis. These studies also involve additional informal teaching about energy balance, neuroscience,neuroendocrinology, reproduction, maternal programming, and endocrine disruption. Students also gain the opportunity forexperiential learning in rodent physiology, handling, and surgery. How have the results been disseminated to communities of interest?We currentlyhave 4 manuscripts at some stage of the review process with 5-6 planned in the next 6 months.Furthermore, I have used the data collected from these studies to submitted 4 R01 applications and 2 R21 applications to the NIH. What do you plan to do during the next reporting period to accomplish the goals?We will submit for publication many of the studies discussed above including 1)the effects of maternal high fat diet on female offspring using the wild-type, ERKO, and KIKOmouse models; 2) diet-induced obesity and estradiol replacement in the wild-type, ERKO, and KIKOmouse models; 3) The effects of high-fat diets containing either high PUFA or high SFA on energy balance in males; and 4) females; 5) the intereactions of estradiol, high-fat diet, and caloric restriction on arcuate gene expression; and 6) the arcuate transcriptome project in the wild-type, ERKO, and KIKOmouse models.Finally,will completed the studiess examining the effects of flame retardants (PBDE-47 and OPFR) on adulthypothalamic gene expression in males and females wild-type mice and the effects of maternal exposure to these compoundson adult offspring energy and glucose homeostasis and prepare for publication. I also have a new graduate student, Graham Vail, who will conduct studies on the effects of estrogenic endocrine-disrupting compounds on arcuate neuronal activity for their thesis project.

Impacts
What was accomplished under these goals? The goal of these studies is to improve our understanding of the effect of endocrine disruption on hypothalamic functionsespecially energy balance, glucose metabolism, reproduction and thermoregulation through either adult or developmentalexposures. We are currently in the process of collecting baseline date for experiments involving exposure to endocrinedisrupting compounds during adulthood and during perinatal development. In our first publication, we reportedthe effects of ovariectomy and estradiol replacement on body weight gain, feeding and energy balance in wild-type, ERalphaknockouts and ERalpha knockin/knockout (that lack a functional DNA binding domain in the ERalpha gene).We have continued these studies in the transgenic mice with the added variable of a low-fat vs. high-fat diet and testing for glucose tolerance and insulin sensitivity and gene expression in the liver.This project is complete and will be submitted in the next year for publication. Another study using these same transgenic mouse models has beencompleted and is being resubmitted for publication. Thisstudy examinedthe expression of selected genesin the hypothalamus ofwildtype,ERalpha knockouts and ERalpha knockin/knockouts after treatment with estradiol. We have also expanded this project by employing cutting-edge RNA Sequencing technology to examine the entire expressed genome (the transcriptome) in the arcuate nucleus from these transgenic mouse models after estradiol treatment. These data are currently being analyzed and will be prepared for publication in the coming year. We completed studies on the effects of maternal exposures to nonylphenol and alpha-zearanol on the energy balanceand glucose homeostasis of adult male and female offspring in mice. These preliminary experiments have been used an NIHapplication for federal extramural funding. Undergraduate students are currently investigating the effects of these exposureson hypothalamic gene expression. This study will be submitted forpublication in the coming year. We have also completed sample collection and animal experimentation in a project examining the effects of maternal exposure to flame retardants (PBDE-47 and a mixture of organophosphate flame retardants (TPP, TCP, TDCPP)) on offspring energy balance and glucosue homeostasis. We are currently analyzing tissues for gene expression prior to submission for publication in coming year. We have also submitted an NIH R01 based on this project focusing on OPFR. That application has been reviewed and will be resubmitted in Decemeber 2015. We are also conducting experiments examining the effects of flame retardants on gene expression andneuronal activity in adult male and female mice.We found significant effects on arcuate gene expression in males and females and will be completing these studies in the coming year and submitting for publication. Data from this project was used for an NIH R21 application submitted in October 2015. Another study using a maternal exposure paradigm examines the effects of maternal high fat diets on adult offspring energyhomeostasis and glucose homeostasis in females using wild type, ERalpha knockout and ERalpha knockin/knockouttransgenic strains. These data demonstrate, for the first time, that ERalpha is involved in the response of adult offspring tomaternal high fat diets. These data are also being prepared for publication. An R01 NIH application was submitted in 2015 based on these studies and will be resubmitted in March 2016. Another area of research is the examination of the effects of high fat diets differing in fatty acid composition (saturated vspolyunsaturated) on adult energy homeostasis either through adult diets or maternal diets. Sample collection and animal experimentation has been completed. Data from this project will be submitted for publication within the next year. Collectively, these data seek tocharacterize how estradiol impacts energy balance and to characterize the potential impacts that exposures to environmentalcontaminants and/or high fat diet during pregnancy and early infant and childhood development have on the obesity epidemicin both sexes.This research will also impact the field by using techniques and tools not employed in the past in addressing these concernsand may provide clues as to the impacts these chemicals and diets have on the developing brain.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: Mamounis K.J. and Roepke, T.A. Obesity and Hypothalamic Inflammation from High Fat Diets of Different Fatty Acid Profile. Experimental Biology, Boston, MA. March 28-April 1, 2015.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: Regulation of hypothalamic functions by ERE-dependent and EREindependent mechanisms. The Yin/Yang of Estrogen Signaling in the Control of Energy Homeostasis. Experimental Biology, Boston, MA. March 28-April 1, 2015.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: Yasrebi, A., Hsieh, A., Mamounis, K.J., Krumm, E.A., J. A. Yang, J.A., and Roepke, T.A. Regulation of GHSR signaling pathway in the arcuate nucleus and NPY neurons by fasting, diet-induced obesity and 17beta-estradiol in mice. Society for Neuroscience, Washington, D.C., November 15-19.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: Krumm, E.A., Yasrebi, A., Yang, J.A., Mamounis, K.J., and Roepke, T.A. The role of ERalpha signaling in the effects of maternal obesity on adult offspring energy homeostasis, Society for Neuroscience, Washington, D.C., November 15-19
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: Yang, J.A., Mamounis, K.J., Yasrebi, A., and Roepke, T.A. Regulation of gene expression by 17beta-estradiol in the arcuate nucleus of the mouse through ERE-dependent and ERE-independent mechanisms, Society for Neuroscience, Washington, D.C., November 15-19

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: The target audieces for this research are food producers and food handlers (both agribusiness and factory producers) and includes those that are interested in the deleterious effect of contaminants and natural toxins on child development, behavior and normal physiological functions that control obesity. Changes/Problems: The only change to this grant is that we have dropped the investigation into bisphenol A due to the disinterest in the NIH persuant to PA-12-185/184 in funding grants examining BPA's effects on energy homeostasis. We are now focusing our endeavors on Zeranol and flame retardants (PDBE-47 and organophosphate flame retardants (OPFR)). What opportunities for training and professional development has the project provided? This research project has provided research training opportunities in 2014 for the following graduate students: Elizabeth Krumm, Kyle Mamounis, Jennifer Yang. In addition, Josh Gilens, an undergraduate in Animal Sciences, conducting his senior honors thesis project on the effects of perinatal exposures to Zeranol (and nonylphenol) on adult offspring energy and glucose homeostasis. Wendy Flores and Taylor Tillery are examing the effects of these exposures on adult hypothalamic gene expression. These undergraduate projects pertain directly to the aims of the grant. Other undergraduates involved in this research include Michelle Hernandez, Janelle Rivera, Ruleena Barreto, Alejandra Villalobos, Victoria Wright, Nick Margolis, Steven Rabel, Christina Kaye, and Gina Giunta. This research project serves as the basis for training graduate and undergraduate students in hypothesis generation and testing, experimental design, research methodologies and data analysis. These studies also involve additional informal teaching about energy homeostasis, neuroscience, neuroendocrinology, reproduction, maternal programming and endocrine disruption. Students also gain the opportunity for experiential learning in rodent physiology, handling and surgery. How have the results been disseminated to communities of interest? One peer-reviewed article has been published and 2 have been submitted and are in revision. Three more articles are in preparation. Furthermore, I have used the data collected from the perinatal Zeranol study for submission of an NIH R21 application to NIEHS. That data was also recently presented at an international conference (Prenatal Programming and Toxicology IV, Boston, MA, Oct 26-29, 2014). I have also been invited to present my findings on estradiol's control of energy homeostasis at Experimental Biology meeting in Boston, MA, March 28-April 1. What do you plan to do during the next reporting period to accomplish the goals? We have completed studies on the effects of maternal high fat diet on female offspring using the wild-type, ERKP and KIKO mouse models. Experiments are completed and analysis of tissue samples will be completed by March 2015 for publication. We are currently conducting studies on the effects of oral estradiol replacement on energy and glucose homeostasis in ovariectomized females from wild type, ERKO and KIKO genotypes when fed a normal fat diet or a high fat (45%) diet. Experiments will be completed by December 2014 and samples analyzed by May 2015 for submission for publication. Finally, my graduate student, Elizabeth Krumm, is examing the effects of flame retardants (PBDE-47 and OPFR) on adult hypothalamic gene expression in males and females wild-type mice and the effects of maternal exposure to these compounds on adult offspring energy and glucose homeostasis. These studies will be combined with the Zeranol studies for future grant applications. I have recently hired a new electrophysiogist/post doctoral researcher who is examining the membrane-mediated and nuclear-mediated effects of environmental estrogens on hypothalamic neuronal activity and cation current expression and activity.

Impacts
What was accomplished under these goals? The goal of these studies is to improve our understanding of the effect of endocrine disruption on hypothalamic functions especially energy balance, glucose metabolism, reproduction and thermoregulation through either adult or developmental exposures. We are currently in the process of collecting baseline date for experiments involving exposure to endocrine disrupting compounds during adulthood and during perinatal development. We have completed our first project characterizing the effects of ovariectomy and estradiol replacement on body weight gain, feeding and energy balance in wild-type, ERalpha knockouts and ERalpha knockin/knockout (that lack a functional DNA binding domain in the ERalpha gene). This study shows that estradiol, a reproductive steroid, controls energy balance (feeding, energy expenditure, etc.) through mechanisms that involve gene regulation controlled by one of its nuclear receptors (ERalpha) acting on a specific promotor in the genome. However, the data also shows that other ERalpha-mediated mechanisms are involved in the control of energy balance during development. We have also completed studies on the control of gene expression in the hypothalamus by estradiol in wild-type, ERalpha knockouts and ERalpha knockin/knockouts, which suggest that estradiol controls gene expression through mechanism that are independent of classical estrogen-response-elementmediated DNA transcription. These data have been submitted for publication. We have completed studies on the effects of maternal exposures to nonylphenol and alpha-zearanol on the energy balance and glucose homeostasis of adult male and female offspring in mice. These preliminary experiments have been used an NIH application for federal extramural funding. Undergraduate students are currently investigating the effects of these exposures on hypothalamic gene expression. Studies will be repeated prior to publication and if funding is awarded. We are also conducting experiments examining the potential effects of endocrine disruptor compounds on neuronal activity and gene expression through both nuclear-initiated and membrane-initiated mechanisms. We are currently collecting samples for gene expression analysis in support of this project. Another study using a maternal exposure paradigm examines the effects of maternal high fat diets on adult offspring energy homeostasis and glucose homeostasis in females using wild type, ERalpha knockout and ERalpha knockin/knockout transgenic strains. These data demonstrate, for the first time, that ERalpha is involved in the response of adult offspring to maternal high fat diets. These data are also being prepared for publication and for an application for extramural funding. Another area of research is the examination of the effects of high fat diets differing in fatty acid composition (saturated vs polyunsaturated) on adult energy homeostasis either through adult diets or maternal diets. Collectively, these data seek to characterize how estradiol impacts energy balance and to characterize the potential impacts that exposures to environmental contaminants and/or high fat diet during pregnancy and early infant and childhood development have on the obesity epidemic in both sexes. This research will also impact the field by using techniques and tools not employed in the past in addressing these concerns and may provide clues as to the impacts these chemicals and diets have on the developing brain.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Mamounis KJ, Yang JA, Yasrebi A, Roepke TA. 2014. Estrogen response element-independent signaling partially restores post-ovariectomy body weight gain but is not sufficient for 17beta-estradiol's control of energy homeostasis. Steroids 81:88-98

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: 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. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This research project has provided research training opportunities in 2013 for the following graduate students: Jennifer Yang and Kyle Mamounis. In addition, Michelle Hernandez, Pauline Chen, Josh Gilens, Marisa Snyder, Anthony Fruges, Ruleena Barreto, Janelle Rivera, saima Usmani, and Anna Hsieh, undergraduate students in the laboratory, gained valuable hands-on research experience in conducting these studies.. 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 dditional 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. How have the results been disseminated to communities of interest? One peer-reviewed article has been published and three more are in preparation. I have also given seminars at two Rutgers University programs/department - EOHSI and Department of Nutritional Sciences. What do you plan to do during the next reporting period to accomplish the goals? In the past year, I have moved my laboratory to a new location and restarted our experiments. We have completed and published an initial study on estradiol's actions through the ERalpha on energy balance in ovariectomized females. We are repeating this studies using multiple diets incudling high fat diets with different compositions of saturated and polyunsaturated fatty acids. Furthermore, the effects of endocrine disrupting compounds on the electrophysiological characteristics of hypothalamic neurons are just now being conducted. We also examining the effects of diet and caloric restriction on gene expression in the hypothalamus and in specific neurons like NPY and POMC

Impacts
What was accomplished under these goals? The goal of these studies is to improve our understanding of the effect of endocrine disruption on hypothalamic functions, especially energy balance, after adult or maternal/development exposures. We have completed our first project characterizing the effects of ovariectomy and estradiol replacement on body weight gain, feeding and energy balance in wild-type, ERalpha knockouts and ERalpha knockin/knockout (that lack a functional DNA binding domain in the ERalpha gene). This study shows that estradiol, a reproductive steroid, controls energy balance (feeding, energy expenditure, etc.) through mechanisms that involve gene regulation controlled by one of its nuclear receptors (ERalpha) acting on a specific promotor in the genome. However, the data also shows that other ERalpha-mediated mechanisms are involved in the control of energy balance during development. These data have been accepted for publication. We have also completed studies on the control of gene expression in the hypothalamus by estradiol in wild-type, ERalpha knockouts and ERalpha knockin/knockouts, which suggest that estradiol controls gene expression through mechanism that are independent of classical estrogen-response-element-mediated DNA transcription. These data are being prepared for publications. We are also currently conducting studies on the effects of maternal exposures to nonylphenol and alpha-zearanol on the energy balance and glucose homeostasis of adult male and female offspring in mice. These preliminary experiments will be utilized for an application for federal extramural funding. We are also conducting experiments showing that these compounds can alter neuronal activity of POMC and NPY neurons through both nuclear-initiated and membrane-initiated mechanisms. We are currently collecting samples for gene expression analysis in support of this project. Another study using a maternal exposure paradigm examines the effects of maternal high fat diets on adult offspring energy homeostasis and glucose homeostasis in females using wild type, ERalpha knockout and ERalpha knockin/knockout transgenic strains. These data demonstrate, for the first time, that ERalpha is involved in the response of adult offspring to maternal high fat diets. These data are also being prepared for publication and for an application for extramural funding. Collectively, these data seek to characterize how estradiol impacts energy balance and to characterize the potential impacts that exposures to environmental contaminants and/or high fat diet during pregnancy and early infant and childhood development have on the obesity epidemic in both sexes. This research will also impact the field by using techniques and tools not employed in the past in addressing these concerns and may provide clues as to the impacts these chemicals and diets have on the developing brain.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Mamounis KJ, Yang JA, Yasrebi A, Roepke TA. Steroids. 2013 Estrogen response element-independent signaling partially restores post-ovariectomy body weight gain but is not sufficient for 17?-estradiols control of energy homeostasis. Steroids Nov 16. [Epub ahead of print]

Progress 10/01/11 to 09/30/12

Outputs
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. 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. 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. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
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.

Publications

• No publications reported this period