ovarian estrogens play a major role in the regulation of energy homeostasis ( gao and horvath , 2008 ; hill et  al . , 2013 ; mauvais - jarvis et  al . , decreased levels of estradiol ( e2 ) after menopause or ovariectomy ( ovx ) are also associated with hyperphagia , reduced energy expenditure , and weight gain ( rogers et  al . , 2009 ; mauvais - jarvis et  al . , , e2 replacement therapy prevents ovx - induced obesity by decreasing feeding and increasing energy expenditure ( gao and horvath , 2008 ; finan et  al . , likewise , hormone replacement therapy reverses the development of obesity and metabolic dysfunctions in postmenopausal women ( wren , 2009 ; mauvais - jarvis et  al . , 2013 ) . studies have also suggested variations in meal size and body weight in rats depending on the stage of the estrous cycle ( tritos et  al . , 2004 ) , as well as during pregnancy and lactation ( garca et  al . , 2003 ) . genetic models of loss of function of estrogen receptors ( ers ) , which are widely expressed in the hypothalamus ( shughrue et  al . , 1997 ) , have demonstrated that mice with global or brain - specific targeted disruption of er alpha ( er ) are obese , as a consequence of hyperphagia and hypometabolism ( heine et  al . , 2000 ; similarly , mice and patients deficient for the aromatase enzyme , which mediates the conversion of androgens to estrogens , develop obesity ( grumbach and auchus , 1999 ; jones et  al . interestingly , estrogens display a nucleus - specific action within the hypothalamus to modulate energy balance , particularly within the arcuate ( arc ) and ventromedial ( vmh ) nuclei . vmh - specific delivery of adeno - associated viral vectors silencing er in mice and rats leads to marked obesity , impaired glucose tolerance , and reduced energy expenditure ( musatov et  al . , 2007 ) . of note , these genetic manipulations did not alter food intake , indicating that estrogens actions in the vmh modulate specifically energy expenditure . in keeping with this , female mice lacking er in hypothalamic steroidogenic factor-1 ( sf1 ) neurons of  the vmh exhibit reduced energy expenditure and brown adipose tissue ( bat)-mediated thermogenesis , leading to obesity , despite normal feeding ( xu et  al . , 2011 ) . in contrast , deletion of er in proopiomelanocortin ( pomc ) neurons of the arc leads to hyperphagia without changes in energy expenditure ( xu et  al . , 2011 ) . finally , concomitant deletion of er from both sf1 and pomc neurons recapitulates both phenotypes , causing hypometabolism , hyperphagia , and severe obesity ( xu et  al . , 2011 ) . despite this evidence , the molecular and cellular events mediating e2-induced negative energy balance and bat thermogenesis remain elusive . thus , the aim of this study was to investigate the hypothalamic mechanism mediating e2-induced thermogenesis . we show that central e2 regulates bat thermogenesis through er and activation of the sympathetic nervous system ( sns ) by modulating hypothalamic amp - activated protein kinase ( ampk ) , specifically in the vmh . ovx rats gained significantly more weight and developed a marked hyperphagia ( figures 1a and 1b ) . subcutaneous ( sc ) treatment of ovx rats with e2 induced a marked reduction in body weight and food intake ( figures 1a and 1b ) leading to a state of negative energy balance ( figure  s1a available online ) . ovx rats showed the expected increase in serum luteinizing hormone ( lh ) and decrease in circulating e2 levels ( confirming the efficiency of the ovx procedure ) , while sc e2 treatment recover both parameters to physiological levels ( figures s1b and s1c ) . for this reason , and in order to avoid a possible interference of fluctuations of ovarian e2 production during the estrous cycle , we used ovx rats in all the experiments ( with the exception of figures 7 and s7a  s7c , where intact rats were used ) . the anorectic effect of e2 was found to be associated with decreased npy mrna and increased pomc mrna levels in the arc of ovx rats treated with e2 ( figures 1c and 1d ) . e2-treated rats showed decreased phosphorylated levels of ampk ( pampk ) and acc ( pacc , which is inactive when phosphorylated ) in the whole hypothalamus ( figure  1e ) . consistent with acc activation , we found that e2 increased the hypothalamic malonyl - coa levels ( figure  1f ) . to establish the functional dependence of e2 s actions on hypothalamic ampk and malonyl - coa levels , we investigated whether pharmacological activation of central ampk could reverse the anorectic effects of e2 . intracerebroventricular ( icv ) administration of the ampk activator aicar decreased hypothalamic malonyl - coa concentration ( figure  1 g ) and reversed the hypophagia observed in rats treated with e2 ( figure  1h ) . to dissect the relative contribution of the anorectic effect of e2 to its impact on energy homeostasis , a group of vehicle - treated pair - fed rats ( i.e. , received the average amount of food eaten by the e2-treated rats the day before ) was studied . sc administration of e2 to ovx rats decreased body weight largely exceeding that of pair - fed ovx rats , indicating a food - independent weight loss ( figures 2a and 2b ) . sc administration of e2  increased the mrna expression or protein content of thermogenic markers , such as uncoupling protein-1 ( ucp1 ) , peroxisome - proliferator - activated receptor - gamma coactivator 1 alpha ( pgc1 ) and beta ( pgc1 ) in the bat ( figures 2c and 2d ) , while no changes in browning markers ( waldn et  al . , 2012 ; ohno et  al . 2012 ) were detected in white adipose tissue ( wat ) ( figure  s2a ) . peripheral administration of e2 induced a slight upregulation of the thermogenic program in liver but not in skeletal muscle ( figures s2b and s2c ) . no changes in inflammatory markers were detected in muscle either ( figure  s2c ) . we found that icv administration of e2 induced weight loss that was significantly larger than that observed in ovx pair - fed animals ( figures 2e and 2f ) . consistently , the mrna expression or protein content of ucp1 , pgc1 , and pgc1 was markedly increased in bat of icv e2-treated rats when compared to both vehicle - treated and pair - fed animals ( figures 2 g and 2h ) , whereas no changes in browning markers were found in wat ( figure  s2d ) . no increase in the expression of thermogenic or inflammatory markers was detected in the liver or muscle after icv e2 administration ( figures s2e and s2f ) . icv e2-treated ( 5  nmol ) rats showed decreased weight ( figure  3a ) , anorexia ( figure  3b ) , as well as a significantly higher body temperature ( figure  3c ) and energy expenditure ( ee ) ( figure  3d ) associated with reduced respiratory quotient ( rq ) ( which indicates increased lipid mobilization ; figure  3e ) and locomotor activity ( la ) ( figure  3f ) , which overall promote a negative energy balance ( figure  s3a ) . notably , a single icv administration of e2 caused a marked increase in bat sympathetic nerve traffic recorded directly by microneurography ( figure  3 g ) . icv e2 also decreased ampk activation in the mediobasal hypothalamus ( mbh ) ( figure  3h ) . in order to further investigate whether icv e2-induced increase in thermogenic markers correlated with changes in bat thermogenesis , we used thermographic imaging analysis ( martnez de morentin et  al . , 2012 ; whittle et  al . , 2012 ) . a total of 1  nmol of icv e2 induced a marked weight loss , anorexia , and an increase in core temperature ( figure  4a ) . the increase in body temperature was associated with elevated ucp1 protein levels in bat ( figure  4b ) and a significant rise in the temperature of the skin surrounding interscapular bat ( figure  4c ) , indicating elevated thermogenesis . no changes were detected in wat browning program after icv treatment with e2 ( figure  s4a ) . the effects evoked by icv e2 are not related to leakage of e2 from the cerebrospinal fluid , because circulating levels of e2 remain unchanged after icv treatment ( figure  s4b ) . ampk in vmh plays a major role in the modulation of bat thermogenesis ( lpez et  al . , 2010 ; martnez de morentin et  al . , 2012 ; whittle et  al . , 2012 ) . thus , we aimed to investigate whether icv e2 elicits changes in vmh ampk signaling with arc samples used as control . the specificity of the vmh and arc dissections was confirmed by analyzing the mrna of specific markers , namely sf1 and pomc , respectively ( figure  s4c ) . we found that icv administration of 1  nmol of e2 elicited similar changes to those observed in the mbh after 5  nmol of the hormone , namely decreased vmh ampk activity ( figure  4d ) . icv administration of 0.5  nmol of e2 also induced weight loss , anorexia , increased ucp1 in bat , and decreased hypothalamic ampk signaling ( data not shown ) . no changes were found in the arc ampk pathway after icv e2 injections ( figure  s4d ) . of note , the effect of icv e2 administration was not related to increased inflammatory responses in the vmh ( where , in fact , inflammatory markers showed reduced expression ) ( figure  s4e ) or muscle ( figure  s4f ) . given that icv e2 induced a marked increase in the activity of the sns subserving bat , we aimed to investigate whether adrenergic receptors blockade impact the central effect of icv e2 on bat thermogenic program . central administration of 1  nmol of e2 was reversed ( in a feeding - independent manner ) by pharmacological blockade of beta 3 adrenergic receptor ( 3-ar ) with the specific antagonist sr59230a ( lpez et  al . , 2010 ) the increase in body weight induced by sr59230a was associated with appropriate reversal of icv e2-induced activation of temperature ( figure  4 g ) and bat ucp1 expression ( figure  4h ) . we performed stereotaxic administration of e2 into the vmh or the arc . to assess the effects of e2 in both hypothalamic nuclei , we performed anatomical analysis of the injection routes , as well as a combined injection of vehicle or e2 with fluorescein - isothiocyanate ( fitc ) , which allowed us to control the diffusion of the treatment . both vmh and arc injections were specific , and the treatment did not spread to other hypothalamic areas ( figure  5a ) ; moreover , no damage in vmh or arc structures was found when the sections were stained with toluidine blue ( figure  s5a ) . we found that administration of e2 in the vmh , but not in the arc , induced a significant rise in temperature ( figure  5b ) associated with an upregulation of bat ucp1 at both mrna and protein levels ( figure  5c ) . in order to confirm these data using an alternative approach , we used microdialysis , which recapitulated the e2-induced increase in bat ucp1 ( figure  s5c ) . to better control for the specificity of vmh administration ( as indicated by the absence of fitc in this nucleus ) , no changes in ucp1 expression in the bat were detected ( figure  s5e ) . this evidence indicates that the effect of e2 on bat thermogenic program is specific of the vmh and it is not related to e2 spreading to adjacent areas . in line with the temperature and anatomical data , administration of e2 into the arc did not affect the expression of thermogenic markers in the bat ( figure  5d ) . in addition , these effects are not related to leakage or diffusion of central e2 from the brain , because circulating levels of e2 remained unchanged after vmh and arc injections ( figure  s5f ) . we then assessed the effect of vmh and arc e2 injection on the central mechanism modulating bat thermogenic program . rats receiving e2 in the vmh ( but not arc ) displayed a significant increase in neuronal activation in the inferior olive ( io ) and the raphe pallidus ( rpa ) , as defined by c - fos staining ( figure  5e ) . the rpa and io have been involved in the sympathetic activation of bat in response to cold ( shibata et  al . , 1999 ; uno and shibata , 2001 ; cano et  al . , 2003 ; cannon and nedergaard , 2004 ) . we also analyzed the effect of specific stereotaxic injection of e2 on the ampk pathway in the vmh versus arc . the administration of e2 into the vmh decreased the protein levels of pampk and pacc in this nucleus ( figure  5f ; left images and graph ) . in contrast , no effect was detected on ampk pathway when e2 was microinjected into the arc ( figure  5f ; right images and graph ) . the main upstream ampk kinases are the tumor suppressor lkb1 and ca / calmodulin - dependent protein kinase kinase alpha and beta ( camkk and camkk ) , which phosphorylate ampk at thr172 ; ampk phosphorylation levels are also regulated by protein phosphatase-2c ( pp2c ) ( carling et  al . , 2011 ; we found that 2  hr after specific vmh e2 injection , the protein levels of camkk and plkb1 were decreased , whereas the levels of pp2c were increased in the vmh ( figure  5 g ) . consistent with these data , ampk phosphorylation status was also reduced 2  hr after e2 treatment in the vmh ( figure  s5 g ) . finally , e2 caused no changes in the activity of pi3k and akt signaling in the vmh ( just an increase in total akt levels ; figure  5h ) , and as previously described ( gao et  al . , 2007 ) , e2 induced a marked increase in the pstat3 levels in the vmh ( figure  5h ) . to elucidate the contribution of ampk activity in the vmh to the  thermogenic program in the bat after e2 central treatment ,  adenoviruses encoding constitutively active ampk-ca , together with gfp or control adenovirus expressing gfp alone ( minokoshi et  al . , 2004 ; lpez et  al . , 2008 , 2010 ; martnez de morentin et  al . , 2012 ; whittle et  al . , 2012 ) , were injected stereotaxically into the vmh or the arc of ovx rats treated with vehicle or e2 . infection efficiency was assessed by ( a ) expression of gfp in the vmh ( figure  6a ) or in the arc ( figure  6e ) and ( b ) increased phosphorylation of acc ( figure  s6a ) . quantification analyses showed that when the injections were directed to the vmh , the vast majority of gfp - positive cells ( 98.4% ) reside in the vmh ( figure  s6b ) . on the other hand , when the arc was targeted , we achieved a similar level of specificity with 95% of gfp - positive cells in this nucleus ( figure  s6c ) . administration of ampk-ca adenoviruses into the vmh reversed the weight loss in ovx rats treated with e2 ( figure  6b ) with no alteration in food intake ( figure  6c ) . overexpression of ampk-ca in the vmh was also associated with a reduction in expression of ucp1 , pgc1 , and pgc1 in the bat , as well as reduced core temperature , of ovx rats treated with e2 ( figure  6d ) . this effect was vmh specific , since ampk-ca adenoviruses injected into the arc of ovx rats treated with e2 caused a slight increase in body weight in comparison to e2 gfp - treated rats ( figure  6f ) , which was associated with increased feeding ( figure  6 g ) but unaltered expression of thermogenic markers in the bat ( figure  6h ) . a particular feature of adenoviral experiments is that administration of ampk-ca had no effect on body weight per se in ovx animals treated with vehicle . a possible explanation for this is that ampk was already maximally activated in the ovx state ( data not shown ) . thus , we determined whether chronic inactivation of ampk in the vmh or the arc can alter markers of bat activation of ovx rats . stereotaxic delivery of a dominant - negative isoform of ampk ( ampk-dn ) ( figure  s6a ) into the vmh , but not into the arc , caused feeding - independent weight  loss that began on the day following the injection ( figures s6d , s6e , s6 g and s6h ) . administration of ampk-dn into the  vmh , but not into the arc , of ovx rats increased the expression of ucp-1 , pgc1 , and pgc1 in the bat ( figures s6f and s6i ) . to gain further insight into the physiological effects of endogenous e2 on the aforementioned vmh ampk - sns - bat axis , we studied female rats at diestrus that display low - to - mid circulating levels of e2 , in contrast to preovulatory animals with peak e2 concentrations ( figures s1b and 7a ) . diestrus rats had higher core temperature ( figure  7a ) and bat ucp1 protein levels ( figure  7b ) than ovx rats , as well as decreased ampk signaling in  the vmh ( figure  7c ) . consistent with these data , icv administration of the combined er- antagonist ici 182.780 ( movrare - skrtic et  al . , 2014 ) to intact female rats induced feeding - independent weight gain ( figure  s7a ) in association with decreased ucp1 expression in the bat ( figure  s7b ) and increased ampk signaling in the vmh ( figure  s7c ) . 2002 ) to intact female rats induced weight gain and a tendency for food intake to increase ( figure  7d ) in association with decreased bat ucp1 expression ( figure  7e ) and increased ampk signaling in the vmh ( figure  7f ) . in keeping with this , icv administration of the specific er agonist ppt ( snchez - criado et  al . , 2004 , 2006 ; roa et  al . , 2008 ) to ovx rats recapitulated the effects of e2 by eliciting weight loss and anorexia ( figure  s7d ) , associated with increased ucp1 expression in bat ( figure  s7e ) and reduced ampk signaling in the vmh ( figure  7f ) . vmh administration of a lentivirus encoding an shrna that silence er to intact female rats induced weight gain without changes in feeding behavior ( figure  7 g ) and simultaneous decrease in ucp1 expression in bat ( figure  7h ) and an increase in ampk signaling in the vmh ( figure  7i ) . conversely , overexpression of a wild - type isoform of er in the vmh of ovx rats , using adenoviral vector , induced feeding - independent weight loss ( figure  s7 g ) in association with increased bat ucp1 expression ( figure  s7h ) and decreased ampk signaling in the vmh ( figure  s7i ) . this study identifies a link between the effects of e2 on hypothalamic ampk and bat thermogenesis . specifically , we show that activation of bat thermogenic program and subsequent energy expenditure depends on er-mediated inactivation of vmh ampk . overall , these results offer a physiological mechanism , via the energy sensor ampk , for the central actions of e2 on er that is of relevance in linking the changes in energy balance and obesity - associated alterations in the gonadal axis . conditions in which ovarian estrogens are lacking , such as ovx or menopause , are associated with hyperphagia and decreased caloric expenditure that results in a positive energy balance leading to obesity . estrogen replacement reverts this phenotype both in women and female rodents ( carr , 2003 ; gao and horvath , 2008 ; rogers et  al . , 2009 ; wren , 2009 ; finan et  al . recent evidence indicates that hypothalamic kisspeptin / neurokinin b / dynorphin neurons promote cutaneous vasodilatation and participate in the e2 modulation of body temperature ( mittelman - smith et  al . , 2012 ; rance et  al . , also , genetic models have shown that some of the actions of estrogens on energy homeostasis are centrally mediated through activation of er in key hypothalamic nuclei , such as the arc and the vmh ( musatov et  al . regard , compelling data have demonstrated that e2 , the most important ovarian estrogen , inhibits feeding in a leptin - independent ( but stat3-dependent ) manner , acting on pomc , npy , and melanin - concentrating hormone ( mch ) neurons in the hypothalamus ( mystkowski et  al . , 2000 ; furthermore , it has been proposed that e2 modulates ghrelin signaling and that this endocrine interaction may account for important sex differences in the regulation of food intake ( clegg et  al . , 2007 ) . however , e2 action on feeding does not explain the entire change in body weight , as shown here in experiments assessing the effect of estrogens in pair - fed animals . recent literature indicates that hypothalamic ampk pathway is a central regulator of energy homeostasis that acts by controlling both feeding and energy dissipation ( minokoshi et  al . , 2004 ; lpez et  al . , 2008 , 2010 ; andrews et  al . , 2008 ; martnez de morentin et  al . , 2012 ; therefore , we hypothesized that e2-induced actions on energy balance could be mediated by hypothalamic ampk . our data showed that peripheral administration of e2 induced a negative energy balance in obese and hyperphagic ovx rats , associated with inactivation of ampk signaling and increased malonyl - coa levels in the hypothalamus . notably , central pharmacological activation of ampk , using aicar , was able to restore hypothalamic malonyl - coa concentration and reversed the anorectic effect evoked by e2 administration . overall , these data indicate that hypothalamic ampk mediated the actions of e2 on energy balance . these results are in line with previous evidence suggesting an anabolic role for ampk in the hypothalamus , which is in contrast with its peripheral catabolic function ( minokoshi et  al . , 2002 ; icv administration of e2 promoted a state of negative energy balance , characterized by hypophagia , increased energy expenditure , and bat thermogenesis , leading to marked weight loss . this effect was associated with a robust decrease in hypothalamic ampk signaling , neuronal activation in rpa and io ( two nuclei which have been functionally linked to the regulation of bat thermogenesis ) ( morrison , 1999 ; shibata et  al . , 1999 ; uno and shibata , 2001 ; cano et  al . , 2003 ; morrison et  al . , 2014 ) , and increased sns tone to bat . while based on the literature we do believe that our activation ( c - fos ) data in io and caudal rpa are related to bat activity , we can not rule out the possibility that at least part of the responses observed might be related to other functions , such as motor control , as previously described ( welsh and llins , 1997 ; apps , 1999 ) . importantly , the above effects are reversed by pharmacological blockage of 3-ar and appear to derive from vmh - specific actions of e2 , so that the impact of e2 on bat thermogenesis derives from its ability to modulate ampk activity in the vmh . interestingly , when e2 was given in the neighboring arc , which has been primarily involved in the regulation of feeding rather than energy expenditure ( williams and elmquist , 2012 ; yeo and heisler , 2012 ) , no effect was detected on bat thermogenic program . likewise , genetic activation of ampk within the vmh , but not in the arc , abolished the effect of central e2 on bat program markers . in contrast , genetic activation of ampk in the arc reversed e2 anorectic action but had no effects on bat function . overall , these data confirm that e2 modulates energy homeostasis in a brain site - specific fashion ( xu et  al . , 2011 ) and uncover a molecular mechanism ( i.e. modulation of ampk , by which e2 specifically modulates bat metabolism ) . also , in agreement with elegant studies from clegg and colleagues ( heine et  al . , 2000 ; , 2007 ; xu et  al . , 2011 ) , our data using pharmacological and viral approaches also suggest that the effect of e2 on the vmh ampk - sns - bat axis is mediated by er. remarkably , physiological fluctuations in e2 levels during estrous cycle and blockade of e2 actions also modulate the aforementioned axis , indicating that this integrative response is not a particular phenomenon restricted to conditions of estrogen deficiency , such as ovx , but also a mechanism of relevance in the physiological regulation of energy homeostasis by ovarian estrogens . in recent years , hypothalamic metabolic energy sensors , such as ampk , several metabolic hormones , such as leptin , ghrelin , insulin , and thyroid hormones ( minokoshi et  al . , 2004 ; andrews et  al . , 2008 ; lpez et  al . , 2008 , 2010 ) , and more recently bone morphogenetic protein 8b ( bmp8b ) ( whittle et  al . , 2012 ) , have been shown to regulate energy balance acting through those energy sensors . here , we provide evidence that e2 , a hormone that markedly influences energy and metabolic homeostasis , acts on the same sensors to control both feeding and energy expenditure . notably , the mechanisms described here for the thermogenic actions of e2 display considerable commonalities with those previously unveiled by our group for thyroid hormone and bmp8b ( lpez et  al . , 2010 ; whittle et  al . , 2012 ) . thus , the vmh ampk - sns - bat axis appears to act as a canonical central mechanism integrating central and peripheral regulation of energy stores . in this regard , our data points to ampk in the vmh as a potential target for the treatment of obesity and other metabolic disorders associated to conditions of endocrine deregulation . the relevance of such a pharmacological target is emphasized by recent reports showing that nicotine - induced anorexia and weight loss is in part mediated by actions on ampk - expressing neurons in the vmh to increase energy expenditure ( martnez de morentin et  al . , 2012 ) , which could be exploited as a potential therapeutic strategy . in addition , dimarchi , tschop , and colleagues have recently reported an elegant approach based on the development of a combinatorial peptide of a glucagon - like peptide-1 ( glp-1)-estrogen conjugate for the treatment of obesity and metabolic syndrome ( finan et  al . , 2012 ) . they demonstrated that selective activation of ers in glp-1-targeted tissues enhances the benefits of both e2 and glp-1 agonism on body weight , glucose homeostasis , and lipid metabolism . considering that both glp-1 receptor and er are highly expressed in the vmh ( shughrue et  al . , 1997 ; gu et  al . , 2013 ) raises the possibility that the metabolic benefits of e2 and glp-1 agonism may be mediated by selective modulation of ampk in the vmh . , we show that central e2 inactivates ampk in the vmh through er , which increases sns tone , upregulates bat thermogenesis , and increases energy expenditure , leading to weight loss . thus , our data add more evidence to the fact that ampk , specifically in the vmh , is a canonical mechanism modulating energy dissipation via sns activation of bat . this observation provides insights into the physiological regulation of energy balance and its perturbation in estrogen - deficient states and suggests that the vmh ampk - sns - bat axis may be a potential therapeutic target for the treatment of obesity . female sprague - dawley and wistar unilever ( for the microdialysis experiments ) rats ( 250300 g ; animalario general usc , santiago de compostela , spain ; harlan , horst , the netherlands ; and charles river , wilmington ) were used for the experiments . all animals were housed on a 12  hr light ( 8:00 to 20:00 ) 12  hr dark cycle , in a temperature- and humidity - controlled room . the animals were allowed free access to standard laboratory pellets of rat chow and tap water . the experiments were performed in agreement with the international law on animal experimentation and were approved by the usc local ethical committee and the ministry of science and innovation of spain ( project i d pi12/01814 ) , animal experimental committee of the amc , and the university of iowa institutional animal care and use committee . sprague - dawley rats were bilaterally ovariectomized ( ovx ) or sham operated , as described previously , under ketamine / xylacine anesthesia ( 50  mg / kg , intraperitoneally ) ( vigo et  al . , 2007 ; roa et  al . , 2009 ; liu et  al . , all treatments ( central or peripheral ) on ovx rats were carried out 2  weeks after surgery to ensure a total washout of endogenous ovarian hormones . for the experiments of e2 replacement , ovx rats received a daily sc injection of e2 ( estradiol benzoate ; eb , 2  g dissolved in 100  l of sesame oil ; both from sigma ; saint louis ) or vehicle ( 100  l of sesame oil ; control rats ) during 7  days . eb was chosen for sc administration , as this synthetic estrogen is endowed with higher bio - potency and stability and it has been often used in various protocols of peripheral estrogenization ( clegg et  al . , 2007 ; roa et  al . , 2008 ) . we used 17-estradiol ( 17-e2 , as the major endogenous estrogen ) in our central ( icv , vmh , or arc ) experiments in a mix dmso : saline ( 1:10 ) . intact female ( see below ) or ovx rats were subjected to a protocol of daily icv injections of 17-e2 ( 0.5  nmol , 1  nmol , or 5  nmol dissolved in 5  l of saline containing 10% of dmso ; sigma ; saint louis ) or vehicle ( 5  l of saline containing 10% of dmso ; control rats ) for 37  days . for the experiments of pharmacological ampk activation , rats were icv treated with aicar ( 5  g dissolved in 5  l of dmso ; sigma , saint louis ) or vehicle ( 5  l of dmso ) , for 2 and 4  hr . for the experiments of er stimulation of blockage , we used icv administration of the following : ( a ) the selective estrogen receptor alpha ( er ) agonist ppt ( 1  nmol dissolved in 5  l of saline containing 10% of dmso ; tocris bioscience , bristol ) ( roa et  al . , 2008 ) , ( b ) the combined er- antagonist named ici 182.780 ( 2.5  nmol dissolved in 5  l of saline containing 10% of dmso ; tocris bioscience , bristol ) ( movrare - skrtic et  al . , 2014 ) , ( c ) and the specific er antagonist mpp ( 2.5  nmol dissolved in 5  l of saline containing 10% of dmso ; tocris bioscience , bristol ) ( harrington et  al . , 2003 ) . the 3-ar specific antagonist sr59230a ( 3  mg / kg / day ; tocris bioscience , bristol ) ( lpez et  al . , 2010 ) was administrated subcutaneously , starting during 2  days before the icv e2 injections . for more details of implantation of microdialysis probes and central treatments , see supplemental experimental procedures . freely moving rats were treated within the vmh or arc with 17-e2 ( sigma , saint louis ; 0.1  nmol in 1  l of saline containing 10% of dmso ) or vehicle ( 1  l of saline containing 10% of dmso ) during 2 or 12  hr . fitc ( sigma , saint louis ) was used to control the diffusion of the treatment . adenoviral ( gfp , ampk-ca , or ampk-dn : viraquest , north liberty ; er wildtype : signagen ; rockville ) or lentiviral ( gfp , er shrna ; santa cruz biotechnology ; santa cruz ) were stereotaxically delivered as previously reported ( lpez et  al . , 2008 , 2010 ;  martnez de morentin et  al . , 2012 ; whittle et  al . , animals were treated for 57  days in the case of adenoviruses and 16  days in the case of lentiviruses , due to the longer infective period needed . sample processing , analysis of ee , la , and rq ; nuclear magnetic resonance analysis ; sympathetic nerve activity ( sna ) recording ; temperature measurements ; e2 , lh , and malonyl - coa assays ; and real - time quantitative pcr , in  situ hybridization , western blotting , and immunohistochemistry were performed as previously shown ( lpez et  al . , 2008 , 2010 ; mrna and protein data were expressed in relation ( % ) to control ( vehicle - treated ) rats . statistical significance was determined by student s t test when two groups were compared or anova and post hoc two - tailed bonferroni test when more than two groups were compared . f.r .- p . , and l.p . performed the in  vivo experiments ( hormonal , drug , and viral treatments ) , the analytical methods ( in situ hybridization , western blotting , real - time quantitative pcr , and hormone measurements ) , the analysis of temperature , ee , rq , la , and nmr . m.l . developed the hypothesis , coordinated and directed the project , and wrote the manuscript .
summaryestrogens play a major role in the modulation of energy balance through central and peripheral actions . here , we demonstrate that central action of estradiol ( e2 ) inhibits amp - activated protein kinase ( ampk ) through estrogen receptor alpha ( er ) selectively in the ventromedial nucleus of the hypothalamus ( vmh ) , leading to activation of thermogenesis in brown adipose tissue ( bat ) through the sympathetic nervous system ( sns ) in a feeding - independent manner . genetic activation of ampk in the vmh prevented e2-induced increase in bat - mediated thermogenesis and weight loss . 
 notably , fluctuations in e2 levels during estrous cycle also modulate this integrated physiological network . 
 together , these findings demonstrate that e2 regulation of the vmh ampk - sns - bat axis is an important determinant of energy balance and suggest that dysregulation in this axis may account for the common changes in energy homeostasis and obesity linked to dysfunction of the female gonadal axis .