Yong Xu, M.D., Ph.D. - US grants

DESCRIPTION (provided by applicant): The increasing incidence of obesity is a major health issue facing the world and increased understanding of body weight regulation may lead to effective strategies to combat obesity and related disorders, such as diabetes. The sex hormone, estrogen, plays a beneficial role in maintaining normal body weight as women show dramatically increased risks for developing obesity and diabetes when they enter menopause. Hormone replacement therapy may be a way to reduce these risks, but actions of estrogen via its receptors in the peripheral tissues cause unwanted effects, such as cancer and heart diseases. Thus, one objective of the proposed study is to determine whether the anti-obesity effects of estrogen are mediated by one estrogen receptor isoform, ER(, expressed by specific populations of brain cells, namely POMC neurons and SF1 neurons. To this end, we will generate mouse models with ER( deleted selectively in POMC neurons and/or SF1 neurons, and assess whether the metabolic benefits induced by estrogen are abrogated in these animals. We will also generate mice whose POMC or SF1 neurons lack activity of PI3 kinase, which is an intracellular molecule activated by estrogen. By assessing effects of estrogen in these mice, we will determine if the PI3 kinase is required for estrogenic actions on energy balance. Thus, the proposed study will not only advance our understanding about the mechanisms by which sex hormone regulates brain functions to provide a coordinated regulation of body weight, but also help identify rationale targets for developing more specific estrogen therapies that provide metabolic benefits with no or fewer side effects.

DESCRIPTION (provided by applicant): Obesity is a major risk factor for type II diabetes and cardiovascular disease and increased understanding of body weight regulation may lead to effective strategies to combat obesity and diabetes. The sex hormone, estrogen, plays a beneficial role in maintaining normal body weight and glucose balance as women show dramatically increased risks for developing obesity and diabetes when they enter menopause. Hormone replacement therapy may be a way to reduce these risks, but actions of estrogen via its receptors in the peripheral tissues cause unwanted effects, such as cancer and heart disease. Evidence indicates that estrogen acts in the brain to reduce body weight and improve glucose profile, but the mechanisms underlying these beneficial effects are not fully understood. To this end, three objectives will be pursued in the current grant. (1) It has been shown that estrogen suppresses food intake and improves glucose balance by acting upon one estrogen receptor isoforms, ER1, present in a subset of brain cells, namely POMC neurons. However, the downstream neural circuits recruited by these POMC neurons to mediate effects of estrogen remain unknown. Mouse models will be generated in which melanocortin 4 receptor (MC4R), the receptor for the POMC product, will be re-expressed in two distinct site of the brain at the null background. These models will be used to determine if MC4R in these sites is sufficient to mediate anorexigenic and anti-diabetic effects of estrogen. (2) Actions of ER1 in another population of brain cells (SF1 neurons) are shown to increase energy expenditure, but the intracellular signaling initiated by ER1 to achieve this regulation are unclear. Mice with FoxO1 deleted only in SF1 neurons will be used to determine if FoxO1 in SF1 neurons is required to mediate estrogenic effects on energy expenditure. (3) Finally, the functions of ER1 in other brain sites will be examined. Mice will be generated with ER1 deleted only in a forebrain structure, amygdala. These mice will be used to determine if ER1 in the amygdala provides redundant mechanisms to regulate energy and glucose balance. Thus, the proposed study will not only advance our understanding about the mechanisms by which sex hormone regulates brain functions to provide a coordinated regulation of body weight and glucose, but also help identify rational targets for developing more specific estrogen therapies that provide metabolic benefits with no or fewer side effects.

DESCRIPTION (provided by applicant): Obesity is a major risk factor for type II diabetes and cardiovascular disease. Increased understanding of body weight regulation may lead to effective strategies to combat obesity. Hypothalamic neurons, including pro-opiomelanocortin (POMC) neurons and steroidogenic factor-1 (SF1) neurons, integrate multiple metabolic cues to provide a coordinated control of energy homeostasis. In our pilot studies, we found that a nuclear receptor co-activator, namely steroid receptor co-activator-1 (SRC1), is expressed in majority of POMC and SF1 neurons. We observed that hypothalamic SRC1 interacts with pSTAT3 and SF1. In particular, the hypothalamic SRC1-pSTAT3 interaction can be enhanced by leptin, but is disrupted in mice with diet-induced obesity (DIO). Importantly, we observed the similar SRC1-pSTAT3 dissociation in the hypothalami from obese humans. These raise the hypotheses that (1) SRC1 in POMC and/or SF1 neurons mediate leptin actions through its interactions with pSTAT3 or SF1; (2) the dysfunction of hypothalamic SRC1 contributes to the development of DIO; (3) interventions enhancing hypothalamic SRC1 functions interaction can be used to prevent or treat obesity. Consistent with this, we found that SRC1lox/lox/POMC-Cre mice, which lack SRC1 in both mature and developing POMC neurons, are less sensitive to leptin-induced anorexia and more susceptible to DIO. Importantly, we identified a small chemical that enhances the hypothalamic SRC1-pSTAT3 interaction and partially prevents DIO. Objectives of the current application are to generate mice lacking or overexpressing SRC1 only in mature POMC neurons (Aim 1) or SF1 neurons (Aim 2), and systemically examine the effects of such deletion/overexpression on energy homeostasis and leptin sensitivity. In addition, a series of in vivo and in vitro experiments are designed to explore the molecular mechanisms by which hypothalamic SRC1 interacts with pSTAT3 and SF1, and regulates their transcriptional activities. Finally, we will continue to test the anti-obesity efficacy of the small chemical in a number of rodent obese models and to explore molecular mechanisms and action targets of the chemical. Thus, these experiments may reveal novel mechanisms underlying the development of obesity, identify hypothalamic SRC1 as a rational target for the treatment of obesity, and lead to discovery of an obesity drug candidate suitable for clinical trials.

Sexual dimorphism exists in body weight balance, but underlying mechanisms remain elusive. We screened a number of neural populations that are known to play key roles in the regulation of energy homeostasis, and found that hypothalamic neurons expressing pro-opiomelanocortin (POMC) display sex differences in two fundamental functions: (1) expressing the Pomc gene and (2) firing action potentials. In particular, female POMC neurons express more Pomc gene and firing more frequently than male POMC neurons. Mechanisms for this sexual dimorphism are not clear and little is known about whether these sex differences contribute to sexually dimorphic regulation of energy balance. Through POMC neuron-specific transcriptome analyses, we identified a number of genes that are expressed in a sexually dimorphic fashion. These include TAp63 (a transcription factor) which is dominant in female POMC neurons, as well as Gabra5 (a GABAA receptor subunit) and SK3 (a small-conductance Ca2+-activated K+ channel) which are both dominant in male POMC neurons. Three mutant mice will be generated to have each of these three genes deleted specifically in POMC neurons, respectively. These mouse strains (both males and females) will be used to determine the physiological roles of these three genes in regulating POMC neuron firing and/or Pomc gene expression, and in maintaining energy homeostasis in different sexes. The functional interactions between these sexually dimorphic genes with the sex hormones will also be examined. Results from these studies will test a hypothesis that multiple sexually dimorphic genes in POMC neurons contribute to the sex differences in POMC neuron functions and body weight balance.

Project 1 -­ Project Summary    Numerous nuclear receptors (NRs) or transcription factors (TFs) have been identified as important regulators of  body weight. However, anti-­obesity regimens targeting these individual molecules alone are far from satisfying.  Coactivators  interact  with  a  broad  range  of  NRs/TFs  and  may  serve  as  master  regulators  that  coordinate  and  synergize actions of multiple metabolic signals. High levels of Steroid Receptor Coactivator-­1 and -­2 (SRC-­1 and  SRC-­2) are expressed in the hypothalamus, the key brain region controlling feeding and body weight balance.  The pilot observations led to a hypothesis that hypothalamic SRC-­1 and SRC-­2 coactivate STAT3 and FoxO1,  repectively,  to  provide  coordinated  control  of  energy  metabolism.  Aim  1  will  determine  whether  hypothalamic  SRC-­1 fine-­tunes STAT3 transcription activity to mediate the anti-­obesity effects of leptin. Mouse models lacking  or  overexpressing  SRC-­1  only  in  leptin-­responsive  neurons  have  been  generated.  Metabolic  parameters  in  response  to  different  diets  or  to  leptin  treatment  will  be  assessed  in  these  mice.  Importantly,  the  molecular  mechanisms  by  which  the  SRC1-­pSTAT3  complex  regulates  leptin  signaling  will  be  delineated.  Aim  2  will  determine  whether  human  SRC-­1  mutations  impair  leptin-­STAT3  pathway  in  the  hypothalamus  and  cause  obesity.  Using  the  CRISPR  technology,  a  knockin  mouse  line  has  been  generated  to  mimic  a  SRC-­1  genetic  mutation associated with human obesity. Metabolic phenotypes of these mice will be characterized, and leptin-­ STAT3  actions  and  STAT3  transcription  activity  will  be  evaluated.  Aim  3  will  determine  whether  hypothalamic  SRC-­2 coativates FoxO1 transcriptional activity to facilitate energy reservations. Mice lacking or overexpressing  SRC-­2  in  mature  POMC  neurons  have  been  generated,  with/without  FoxO1  overexpression.  Metabolic  phenotypes will be characterized in all these models and FoxO1 transcriptional activity will also be evaluated.   

Obesity is a major risk factor for type II diabetes and metabolic syndromes. Increased understanding of food intake and body weight regulation may lead to effective strategies to combat obesity and diabetes. Serotonin (5-HT) neurons in the dorsal Raphe nucleus (DRN) play an essential role in regulating feeding behavior. Enhanced brain 5-HT actions robustly inhibit food intake and body weight but little is known about how firing activity of 5-HTDRN neurons is regulated. Based on pilot observations, the Xu and Tong labs put forward a general hypothesis that 5-HTDRN neurons integrate dopamine (DA) and GABA inputs to promote food intake. The first objective is to determine whether DA released by neurons in the ventral tegmental area inhibits 5-HTDRN neurons to promote food intake. In vivo optogenetic studies will be carried out to determine whether photostimulation of the DAÆDRN circuit increases food intake while photoinhibition of the same circuit inhibits eating; a DA receptor (DRD2) will be deleted in 5-HT neurons to test whether this deletion decreases food intake and body weight and block effects of DAÆDRN activation in mice. The second objective is to determine whether GABA released by neurons in the lateral hypothalamus inhibits 5-HTDRN neurons to promote food intake. Similar optogenetic studies will be carried out to determine whether photostimulation of the GABAÆDRN circuit increases food intake while photoinhibition of the same circuit inhibits eating; a GABA receptor (?2) will be deleted in 5-HT neurons to test whether this deletion decreases food intake and body weight and block effects of GABAÆDRN activation in mice. The third objective is to determine whether DA and GABA signals converge on the same or distinct subsets of 5-HTDRN neurons and whether stimulation of both DA and GABA-originated circuits produce redundant or synergistic effects on food intake. Thus, accomplishment of these experiments may advance our understanding about the physiological roles of brain 5-HT system in the regulation of feeding and energy balance, we may also identify novel targets (e.g. DRD2 and ?) for therapeutic development of human diseases, e.g. obesity.

PROJECT SUMMARY Dramatic decline in circulating 17?-estradiol (E2) in post-menopausal women has been associated with development of obesity and glucose dysregulations. While E2 administration in post-menopausal women may correct these issues, the estrogen therapy is often associated with side effects, including reproductive endocrine toxicity and breast cancer. Targeting specific estrogen receptors (ERs) and ER-expressing populations may produce anti-obesity and anti-diabetes benefits with fewer side effects. We demonstrated that estrogen receptor-? (ER?) in the ventrolateral subdivision of the ventromedial hypothalamus (vlVMH) is essential to maintain body weight and glucose balance. Here we seek to unravel the molecular and neurocircuitry mechanisms for these ER? neurons by testing a general hypothesis that E2-sensitive ER?vlVMH neurons detect nutritional/glycemic fluctuations, and recruit multiple downstream neural circuits to maintain energy and glucose homeostasis. The first objective is to determine the glucose and energy-regulatory effects of the ER?vlVMH-originated projections to a few brain regions, including the dorsal Raphe nuclei (DRN) and medial posterior arcuate nucleus of the hypothalamus (mpARH). The second objective is to determine whether two ionic channel genes, namely, Abcc8 and Ano4, regulate the firing responses of ER?vlVMH neurons to various alterations in blood glucose and/or feeding states; we will also examine the physiological functions of these channels on whole-body energy/glucose balance. The third objective is to establish Clic1 as a novel ER? target gene, and to determine whether Clic1 in ER?vlVMH neurons mediates actions of E2 to maintain energy and glucose balance. Accomplishment of these studies will unravel ionic mechanisms by which ER?vlVMH neurons detect dynamic changes in energy and glucose balance, and reveal the ER?vlVMH-originated neural networks that respond to these changes and therefore restore energy/glucose homeostasis. We will also delineate molecular mechanisms by which E2 regulates ER?vlVMH neuron functions and energy/glucose balance, and may identify potential targets for treatment of metabolic disorders associated with menopause.