adipose tissue is an important metabolic regulator of energy balance ( langin , 2010 ) . in mammals , three types of adipose tissues white adipose tissue ( wat ) is essentially composed of white adipocytes containing one single large lipid vesicle . wat is mainly located in the abdominal and subcutaneous areas of the body and is highly adapted to store excess energy in the form of triacylglycerides . conversely , beige and brown adipocytes are highly energy - expending ( lee et  al . , 2014a ) . they are able to dissipate energy in the form of heat via the action of uncoupling protein-1 ( ucp1 ) ( tiraby and langin , 2003 ) . brown adipocytes reside in discrete brown adipose tissue ( bat ) depots , such as the interscapular area in rodents , whereas beige adipocytes are intermingled with white adipocytes in wat . both brown and beige adipocytes have a multilocular morphology and large numbers of mitochondria and express a common set of brown / beige fat - selective genes , including ucp1 ( lee et  al . , 2014b ) . in rodents , heat generated by brown or beige adipocytes via ucp1 is essential to survive the cold season ( cohen and spiegelman , 2015 ) . in both cell types , the cold response is mainly regulated by the sympathetic nervous system , in particular by the 3-adrenergic signaling pathway ( cannon and nedergaard , 2004 ) . in addition to its role in thermogenesis and fatty acid oxidation , it has been reported that bat activated by the 3-adrenergic signaling pathway can improve glucose homeostasis ( peirce and vidal - puig , 2013 ) . because recent studies have shown that brown / beige adipocytes exist in quantifiable amounts in humans ( betz and enerbck , 2015 ) , these fat types have been rediscovered as potential therapeutic targets for weight loss . therefore , understanding brown / beige adipocyte function and finding new ways to modulate their activity has become a major scientific point of interest ( betz and enerbck , 2015 ) . among other factors ( , 2012 , puigserver et  al . , 1998 , vernochet et  al . , 2012 ) , the epigenetic regulator lysine - specific demethylase 1 ( lsd1 ) ( cai et  al . , 2014 , metzger et  al . , 2005 , metzger et  al . , 2016 ) , a flavin  adenine dinucleotide ( fad)-dependent amine oxidase , has been shown to regulate the differentiation and function of white and beige adipocytes ( duteil et  al . , 2014 , in wat , lsd1 is induced by cold and involved in mediating the effects of 3-adrenergic signaling . notably , we recently demonstrated that increased expression of lsd1 in mouse wat promotes the development of thermogenically active beige adipocytes and suppresses metabolic disorders such as obesity and type 2 diabetes ( duteil et  al . , 2014 ) . moreover , in mature white adipocytes , elevation of lsd1 levels is sufficient to activate  mitochondria biogenesis , oxidative phosphorylation , and thermogenesis ( duteil et  al . , 2014 ) . in contrast , the role of lsd1 in bat has not been investigated in detail . brown fat cells exhibit a gene expression profile distinct from either beige or white adipocytes ( wu et  al . , 2012 ) . because lsd1 is a key regulator of metabolism in mature white or beige adipocytes , we asked whether lsd1 might also play a role in controlling brown adipocyte function . in this study , we show that selective ablation of lsd1 or inactivation of its catalytic activity in brown adipocytes triggers a profound whitening of bat . lsd1 loss and genetic or chemical inhibition elicits a dramatic rise in expression of wat - selective genes while impairing the expression of bat - selective genes . our data demonstrate that lsd1 acts via a dual mechanism by positively regulating the expression of bat - selective genes in  cooperation with nrf1 and actively repressing the expression of wat - selective genes in concert with the corest complex . the whitening of bat observed upon depletion or inhibition of lsd1 is accompanied by a shift from oxidative to glycolytic metabolism associated with an accumulation of di- and triacylglycerides . taken together , our results demonstrate that lsd1 is essential to maintain the metabolic function of brown adipose tissue . to investigate the role of lsd1 in bat , we crossed mice harboring conditional lsd1 alleles ( zhu et  al . , 2014 ) with the ucp1-cre deleter strain ( turpin et  al . , 2014 ) , which resulted in cre - mediated loss of lsd1 selectively in brown and beige adipocytes ( lsd1 , hereafter named lsd1 ) ( figures s1a and s1b ) . at 10  weeks of age , lsd1 mice exhibited a profound morphological whitening of their bat ( figure  1a ) . this whitening was accompanied by an 40% increase in tissue mass ( figure  1b ) , a switch from multilocular to unilocular morphology , and increased lipid content ( figures 1d and 1e ) . in lsd1 mice , the morphology of mitochondria was severely altered with substantially augmented size and less densely packed cristae ( figure  s1c ) . in accordance , the dna content of mitochondria was decreased by around 60% in lsd1 mice ( figure  s1d ) , indicating degradation and dysfunction of this organelle . these observations suggested that loss of lsd1 in brown adipocytes shifted bat toward a white fat - like phenotype . accordingly , we expected to see changes in the transcriptional program of brown adipocytes of lsd1 mice that  resemble the gene expression profile observed in white adipocytes . we therefore determined the transcriptome of lsd1-deleted bat and compared it with the transcriptome of wat of control mice , which was described previously ( fitzgibbons et  al . , 2011 ; figure  1f ; table s1 ) . the vast majority of wat - selective genes ( 95% ) was upregulated in bat of lsd1 mice , whereas 67% of bat - selective genes showed reduced expression ( figure  1f ; table s1 ) . qrt - pcr analysis confirmed that the white fat transcriptome was enhanced to the detriment of the brown fat program , as illustrated by significantly increased expression of representative wat - selective genes such as bmp4 , egfr , pdk3 , fads1 , apoe , klhl13 , rtn1 , bmp1 , and wls ( figure  1 g ) and decreased expression of representative bat - selective genes ( ucp1 , slc27a2 , cox8b , acadl , cox6a1 , acadm , oplah , prdm16 , and cox17 ) ( figure  1h ) in lsd1 mice . in line with these rna expression data , western blot analyses showed that bat of lsd1 mice expressed higher levels of apoe and reduced levels of ucp1 protein ( figure  s1e ) . these data suggest that , in bat , lsd1 is required to repress the expression of white fat - selective genes while supporting the brown fat - selective gene expression program . to determine whether lsd1 demethylase activity is required to maintain the brown fat program , we engineered mice homozygous for a conditional , enzymatically inactive mutant lsd1 knockin allele ( lsd1 , hereafter named lsd1 ) ( figures s1f  s1i ) . in bat of these mice , the endogenous lsd1 gene is replaced by an enzymatically inactive lsd1 mutant allele following ucp1-cre - mediated recombination . as observed in lsd1-depleted mice , we found that bat of lsd1 mice was increased in size and contained a large proportion of unilocular adipocytes ( figures 1a and 1c1e ) . although lsd1 mrna levels remained unchanged ( figure  s1h ) , the expression levels of wat - selective genes were increased , whereas the levels of bat - selective genes were decreased ( figures 1 g and 1h ; figure  s1i ) . in conclusion , alterations in bat morphology and transcription pattern in lsd1 mice resemble those of lsd1 mice , demonstrating that lsd1 demethylase activity contributes to the phenotype observed in genetically modified mice . because the expression of ucp1 is initiated in utero between embryonic day ( e ) 18.5 and e19.5 , lsd1 ablation by ucp1-cre - mediated recombination is expected to occur around birth ( giralt et  al . , 1990 ) . to investigate the role of lsd1 in adult bat , 10-week - old wild - type mice were treated for 5  days with the lsd1-specific nanomolar affinity inhibitor qc6688 ( lsd1(i ) ) or vehicle . bat of mice treated with lsd1(i ) showed a profoundly pale color and increased size compared with that of vehicle - treated mice ( figure  2a ; figure  s2a ) . h&e staining of bat sections revealed that lsd1(i)-treated adipocytes had larger lipid droplets compared with those of vehicle - treated mice ( figure  2b ) . the expression levels of representative wat - selective genes were increased at both mrna and protein levels ( figures 2c and 2e ) , whereas expression of representative bat - selective genes was decreased in qc6688-treated mice ( figures 2d and 2e ) . these data corroborate our findings in lsd1 mice and suggest that catalytically active lsd1 is required to maintain the metabolic properties of bat . to investigate the cell autonomy of lsd1(i ) in bat , we treated differentiated primary brown adipocytes ( hereafter named brown adipocytes ) isolated from the stromal vascular fraction with lsd1(i ) or vehicle for 3  days . as shown by oil red o staining , lsd1(i)-treated brown adipocytes accumulated more fat than vehicle - treated cells ( figure  2f ) and shifted their transcription pattern to a white adipocyte - like program ( figures s2b  s2d ) . together , these data demonstrate that the gene expression program of brown adipocytes is controlled by the catalytic activity of lsd1 in a cell - autonomous manner . next we determined genome - wide lsd1 chromatin occupancy in brown adipocytes by chromatin immunoprecipitation using lsd1 antibody followed by massive parallel sequencing ( chip - seq ) . the antibody was validated previously and specifically recognizes lsd1 ( duteil et  al . , 2014 ) . we found that lsd1 occupied the promoter of 75% of the bat - selective genes ( 103 genes ) and 68% of the wat - selective genes ( 158 genes ) differentially regulated in lsd1 mice ( figure  2 g ) , suggesting a dual mechanism by which lsd1 represses the expression of wat - selective genes while activating that of bat - selective genes . hypergeometric optimization of motif enrichment ( homer ) motif searches revealed that promoter occupancy of lsd1 significantly correlated with the presence of binding sites for the transcription factors nrf1 and rest ( figure  2h ) . thus , we hypothesized that lsd1 might positively regulate bat - selective genes in  cooperation with nrf1 while repressing wat - selective gene expression in concert with the corest complex . chip with nrf1 antibody followed by qpcr analysis in brown adipocytes showed that lsd1 and nrf1 co - localized at promoters of representative bat - selective genes such as acadm , oplah , and cox6a1 ( figure  3a ) . ucp1 was not part of the analysis because our genome - wide cistrome analysis by chip - seq showed no lsd1 occupancy . importantly , nrf1 was not present at the promoter of wat - selective genes , as illustrated by figure  s3a . because lsd1 is known to activate gene expression by erasing dimethyl groups at lysine 9 of histone 3 ( h3k9me2 ) , we hypothesized that inhibition of lsd1 would modify methylation levels of h3k9 at bat - selective genes . chip - qpcr analysis for h3k9me2 revealed that levels of h3k9me2 were increased upon lsd1(i ) treatment at promoters of bat - selective genes ( figure  3b ) . in parallel , levels of h3k4me2 were reduced in lsd1(i)-treated cells ( figure  s3b ) , indicating a repressive transcriptional context . to investigate whether lsd1 and nrf1 are present in the same protein complex , nuclear extracts from brown adipocytes western blot analysis showed that lsd1 and nrf1 co - fractionate , suggesting the existence of an nrf1/lsd1complex ( figure  s3c ) . furthermore , lsd1 and nrf1 co - immunoprecipitated , confirming interaction between these two endogenously expressed proteins in bat ( figure  s3d ) . accordingly , small interfering rna ( sirna)-mediated knockdown of nrf1 ( figure  s3e ) significantly decreased transcript levels of bat - selective genes but did not impair wat - selective gene expression ( figure  3c ) . together , these data demonstrate that lsd1 controls the expression of bat - selective genes in cooperation with nrf1 by targeting h3k9 methylation levels . next we analyzed whether an lsd1-associated repressor complex might regulate the wat - selective gene program in bat . for this purpose , nuclear protein extracts from bat were used for immunoprecipitation with three distinct anti - lsd1 antibodies directed against various lsd1 domains followed by liquid chromatography - tandem mass spectrometry ( lc - ms / ms ) . the intersection of the three  interactomes uncovered 163 proteins commonly interacting with lsd1 ( figure  s3f ) and revealed the repressive corest complex as a significantly enriched protein group ( table s2 ) . interaction between lsd1 and corest has already been well established ( lee et  al . , 2005 , metzger et  al . , 2005 , shi et  al . , 2004 ) . accordingly , co - immunoprecipitation experiments and size exclusion chromatography demonstrated that , also in bat , endogenous lsd1 interacts with members of the corest complex ( figures s3 g and s3h ) . to test whether the  corest complex indeed represses the expression of wat - selective genes , we knocked down the complex members rcor1 and rcor3 in brown adipocytes ( figure  s3i ) . cells treated with rcor1 or rcor3 sirna upregulated wat - selective genes ( figure  3d ) without affecting bat - selective genes , as exemplified by oplah and acadl ( figure  3d ) , and accumulated more lipids than cells treated with control sirna ( figure  s3j ) . in addition , chip - qpcr analyses confirmed the colocalization of rcor1 and rcor3 with lsd1 specifically at promoters of wat - selective genes ( figure  3e ) but not at promoters of bat - selective genes ( figure  s3k ) . because lsd1 is known to repress gene expression by erasing dimethylation of lysine 4 of histone 3 ( h3k4me2 ) , we analyzed whether inhibition of lsd1 is sufficient to alter methylation levels of h3k4 at wat - selective genes . chip - qpcr analysis showed that levels of h3k4me2 were increased upon lsd1(i ) treatment at promoters of wat - selective genes ( figure  3f ) . concomitantly , levels of h3k9me2 were decreased at these promoters ( figure  s3l ) , indicating an active transcriptional context . together , our data demonstrate that lsd1 represses wat - selective gene expression in cooperation with the corest complex in bat by reciprocal modulation of h3k4 and h3k9 methylation levels . lsd1 mice exhibit a profound morphological whitening of bat associated with large lipid accumulation . to identify further changes in global gene expression in lsd1 mice that might account for the observed whitening of lsd1-depleted brown fat the analysis ( figure  4a ) uncovered pathways regulating energy production and fatty acid catabolism such as  the tricarboxylic acid ( tca ) cycle , electron transport chain ( etc ) , and -oxidation . the expression of genes representing these pathways was downregulated ( figure  4b ; figure  s4a ) , suggesting that oxidative metabolism is lowered in bat of lsd1 mice , confirming our data in wat ( duteil et  al . , 2014 ) . interestingly , the second most enriched pathway was  glycolysis and gluconeogenesis ,  which we did not observe in wat . in line with these findings , we observed increased expression of glucose transporters and the majority of glycolytic enzymes catalyzing glucose - to - pyruvate conversion in lsd1 mice ( figure  4b ; figure  s4b ) . we thus hypothesized that lsd1 deficiency in bat not only leads to reduction of oxidative functions but also  increases glycolytic capacities . to evaluate bioenergetic changes upon inhibition of lsd1 , we employed a seahorse xf analyzer ( agilent technologies ) to measure the extracellular acidification rate ( ecar ) as an index of glycolytic activity ( using a glycolysis stress kit ) and the oxygen consumption rate ( ocr ) as an index of respiratory capacity ( using the cell mito stress test kit ) in brown adipocytes treated with lsd1(i ) or vehicle . the rate of glycolysis following glucose administration was higher upon lsd1(i ) treatment compared with vehicle ( figure  4c ; figure  s4c ) . after addition of oligomycin , an inhibitor of mitochondrial atp synthesis , the maximum glycolytic capacity of lsd1(i)-treated cells remained higher than in control cells , demonstrating that not only the rate of glycolysis but also the glycolytic reserve capacity were enhanced in lsd1-inactivated cells ( figure  4c ; figure  s4c ) . in parallel , the ocr was lower  in  lsd(i)-treated cells compared with vehicle ( figures s4d and s4e ) , and , subsequently , the ocr / ecar ratio was decreased , demonstrating that lsd1 inhibition shifted mitochondrial metabolism toward the glycolytic pathway in brown adipocytes ( figure  4d ) . furthermore , oxygen consumption resulting from mitochondrial respiration measured in bat of lsd1 mice was reduced compared with control mice , confirming lower  oxidative phosphorylation ( figure  s4f ) . taken together , our data  show that glycolytic activity is enhanced in the absence of lsd1 , whereas oxidative phosphorylation is reduced . in addition , glucose uptake and activity of key glycolytic enzymes such as hexokinase ( hk ) , phosphofructokinase ( pfk ) , enolase ( eno ) , and lactate dehydrogenase ( ldh ) were higher in bat of lsd1 mice compared with control mice ( figure  4e ) , promoting increased glycolytic capacity . of note , the expression levels of genes involved in pyruvate metabolism , including the rate - limiting mitochondrial pyruvate transporters ( mpc1 and mpc2 ) and enzymes catalyzing the conversion from pyruvate to acetyl - coenzyme a ( coa ) ( e.g. , dlat and pdhx ) , were decreased in lsd1 mice ( figure  4b ; figure  s4 our genome - wide analysis also revealed that expression of glutamic - oxaloacetic transaminase 1 ( got1 ) and 2 ( got2 ) as well as slc25a12 and phosphoenolpyruvate carboxykinase 2 ( pck2 ) was upregulated in lsd1 mice compared with control ( figure  4b ; figure  s4 g ) . got1 and 2 are responsible for conversion of downstream pyruvate intermediate oxaloacetate into aspartate and phosphoenolpyruvate , respectively ( birsoy et  al . , 2015 ) . in particular , got1 was shown to be essential for aspartate regeneration to restore etc dysfunction ( birsoy et  al . , 2015 ) . etc dysfunction is associated with a drop in nicotinamide adenine dinucleotide ( nad)/nadh ratio because nad can not be oxidized by complex i. however , despite decreased oxidative  phosphorylation ( oxphos ) , the nad / nadh ratio was increased in bat of lsd1 mice ( figure  s4h ) . this might be , at least in part , mediated by higher got1 activity ( figure  4e ) . an increased nad / nadh ratio is also in favor of increased glycolysis ( zhang et  al . , 2007 ) . similarly , expression profiling of metabolic genes in lsd1 mice and mice treated with lsd1(i ) revealed that lsd1 demethylase activity strongly contributes to maintaining proper energy metabolism in bat ( figures s4b , s4 g , and s4i ) . taken together , these data show that glycolysis is enhanced in lsd1-inactivated bat , whereas oxidative metabolism is lowered . the analysis of genome - wide lsd1 chromatin occupancy in bat by chip - seq revealed that glucose transporters and genes involved in glycolysis are direct targets of lsd1 ( figure  s4j ) . chip - qpcr of brown adipocytes showed promoter occupancy of representative genes by rcor1 , rcor3 , and lsd1 ( figure  4f ) . in parallel , the levels of h3k4me2 were enriched , whereas the levels of h3k9me2 were lowered upon lsd1(i ) treatment ( figure  4 g ; figures s4k and s4l ) . these results demonstrate that lsd1 is directly repressing glycolytic genes to ensure physiological metabolic processes in brown adipocytes . to further investigate our phenotype , we performed metabolomic and lipidomic analyses of bat of 10-week - old control and lsd1 mice . these analyses revealed an accumulation of intermediates of glycolysis , including glyceraldehyde-3-phosphate and glycerol , in bat of lsd1 mice ( figure  5a ) accompanied by augmentation of long - chain diacyl- and triacylglycerides ( dags and tags , respectively ) ( figure  5b ) . in accordance with an increase in long - chain fatty acids , the transcript levels of fatty acid elongases ( elovl1 , elovl4 , and elovl7 ) and fatty acid desaturases ( fads1 and fads2 ) were upregulated in the absence of lsd1 ( figures 4b and 5c ) or lsd1 demethylase activity ( figures s5a and s5b ) . the analysis of genome - wide lsd1 chromatin occupancy in bat by chip - seq revealed that genes encoding enzymes involved in fatty acid elongation are direct targets of lsd1 ( figures s5c  s5f ) . dag and tag are glycerol esters derived from dihydroxyacetone phosphate ( dhap ) and fatty acids . allocation of fatty acids necessary for glyceride synthesis might be due to decreased lipolysis , increased de novo lipogenesis , or increased fatty acid uptake in bat of lsd1 mice . increased fatty acid synthase ( fasn ) activity ( figure  5d ) and low acetyl - coa carboxylase phosphorylation ( figure  s5 g ) are in favor of enhanced de novo lipogenesis in lsd1 mice . to assess lipolysis efficiency , we measured lipase activity . the activity of lipases present in brown adipocytes was reduced by 50% in lsd1 mice , suggesting that hydrolysis of lipids into fatty acids is severely impaired ( figure  5e ) . in addition , the activity of lipoprotein lipase ( lpl ) , which hydrolyzes triglycerides in lipoproteins such as those found in chylomicrons and very low - density lipoprotein ( vldl ) to facilitate fatty acid uptake from the bloodstream , was increased upon loss of lsd1 ( figure  5f ) . finally , to test whether fatty acid oxidation was altered , we measured mitochondrial respiration in the presence of palmitoyl - l - carnitine as substrate in saponine - permeabilized bat . the data revealed that lsd1-deficient bat failed to properly use fatty acids ( figure  5 g ) . increased fatty acid synthesis and lower lipolysis are in agreement with the observation of an increased nad / nadh ratio ( figure  s4h ) . altogether , these findings show that lsd1 regulates lipid accumulation in bat . in summary , our data show that lsd1 positively regulates the expression of oxidative metabolism - related genes with concomitant repression of the gene program responsible for glycolysis and fatty acid biosynthesis . accordingly , loss of lsd1 in brown adipose tissue drives a shift from oxidative to glycolytic metabolism accompanied by aberrant triglyceride accumulation . lsd1 male mice gained significantly more weight ( 40% ) than their control littermates between 10 and 30  weeks of age ( figure  6a ) . the weight gain of lsd1 mice was not due to increased food intake or decreased mobility ( figures s6b  s6d ) . body composition analysis assessed with a whole - body magnetic resonance analyzer ( echomri ) revealed that 30-week - old lsd1 mice accumulated more fat than their control littermates , resulting in a lower lean / fat mass ratio compared with control mice ( figure  6b ) . these data were corroborated by a dramatically increased bat mass in 30-week - old lsd1 mice as well as in lsd1 mice ( figures s6e and s6f ) , associated with 45% augmentation in fatty acid content of bat ( figure  6c ) . in accordance , histological analyses revealed a robust phenotypic conversion of bat in lsd1 and lsd1 mice to a wat - like  appearance ( figure  s6 g ) . in addition , we noticed robustly reduced ucp1 protein levels , as shown by immunocytochemistry ( figure  s6 g ) . observed morphological changes in bat are accompanied by increased levels of intermediates of glycolysis , glycerol , dag , and tag ( figures s6h and s6i ) . in 30-week - old lsd1 mice , we observed lower energy expenditure , determined by decreased whole - body oxygen consumption ( vo2 ) ( figure  6d ; figures s6j and s6k ) and co2 production ( vco2 ) ( figure  6e ; figures s6l and s6 m ) , resulting in an increased respiratory quotient ( rq , vco2/vo2 ratio ) ( figure  6f ) . in addition , calorimetric parameters deduced from these analyses indicated that energy expenditure was decreased in lsd1 mice ( figure  6 g ; figures s6n and s6o ) . we observed that circulating glucose levels in 10-week - old lsd1 mice were lower compared with control littermates ( figure  6i ) . nevertheless , we did not notice any difference in glucose tolerance of lsd1 mice relative to control mice at this age ( figure  6j ) . importantly , with aging , control mice developed progressive glucose intolerance , contrary to lsd1 mice , which conserved efficient glucose uptake ( figure  6j ) . age - developed glucose intolerance is generally associated with loss of insulin sensitivity and contributes to type 2 diabetes . to unravel whether lsd1 mice are resistant to age - induced type 2 diabetes , we performed an intraperitoneal insulin sensitivity test ( ipist ) and determined serum insulin levels . despite dramatic alterations in glucose metabolism , we did not observe any difference in insulin sensitivity ( figure  s6p ) or in total insulin levels ( figure  s6q ) , showing that only glucose but not insulin metabolism is altered in lsd1 mice . in summary , these data show that lsd1 is required in bat to maintain a proper energy balance with aging . white and brown adipocytes have distinct transcriptional programs that facilitate long - term lipid storage or thermogenesis , respectively . understanding these programs is of the highest interest for developing new strategies to counteract obesity . the epigenetic determinants that regulate white versus brown fat - selective gene expression have not yet been completely elucidated . in this study , we show that the histone demethylase lsd1 plays a key role in the maintenance of brown fat metabolic properties . lsd1 accomplishes this mission via a dual mechanism by inhibiting the expression of wat - selective genes and simultaneously promoting the expression of bat - selective genes . these opposing functions of lsd1 are orchestrated by the assembly in different protein complexes : a repressive corest complex to inhibit the expression of wat - selective genes and an nrf1-containing complex to promote the expression of bat - selective genes ( figure  7 ) . our data point out the importance of lsd1 demethylase activity in controlling bat metabolism in  vivo . indeed , our engineered mouse model , lsd1 mice , as well as wild - type mice treated with an lsd1-specific inhibitor , basically recapitulate the phenotype observed in lsd1 mice . alterations in bat morphology and transcriptome in lsd1 and lsd(i)-treated mice resemble that of lsd1 mice and suggest that catalytically active lsd1 significantly contributes to the maintenance of the bat gene expression program and function . however , additional scaffolding functions of lsd1 , which might be independent of the enzymatic activity ( i.e. , the recruitment of proteins and/or the assembly in different protein complexes ) might be impaired upon loss of lsd1 in lsd1 mice . in contrast , in knockin mice , the enzymatically inactive lsd1 mutant protein might be able to perform , at least in part , these functions , which might explain a less severe phenotype . coordinated action of lsd1 with  members of the corest complex , in particular rcor1 and rcor3 , represses the expression of wat - selective genes . although lsd1 has been shown to interact with corest in various tissues ( lee et  al . , 2005 , metzger et  al . , 2005 , shi et  al . , 2004 , yang et  al . , 2011 here we demonstrate for the time that the corest complex plays a critical role in the repression of the wat - selective genes in bat , thereby strongly contributing to the maintenance of brown fat characteristics . in bat , similar to lsd1 , rcor1 is ubiquitously expressed in somatic cells , whereas the expression of rcor2 and rcor3 seems to be more restricted ( yang et  al . , 2011 ) . this suggests that the lsd1 corepressor complex might assemble with different rcor family members to carry out specific functions in different cell types . furthermore , lsd1 interacts with nrf1 to activate the expression of bat - selective genes . we have shown previously that , in wat , lsd1 acts together with nrf1 to positively regulate oxphos and thermogenesis ( duteil et  al . , 2014 ) . elevated lsd1 levels favor beigening of wat by increasing oxidative metabolism , uncoupling , thermogenesis , and the number and size of mitochondria . our data provide evidence that the absence of lsd1 in bat causes dramatic mitochondrial dysfunction associated with decreased transcript levels of oxphos - related genes . it is interesting that lsd1 in cooperation with nrf1 shares comparable functions in beige and brown adipocytes . whether the mechanism of regulation of oxidative metabolism is conserved in all metabolically active tissues remains unclear . to clarify this hypothesis , it would be necessary to analyze the metabolism of skeletal muscle and liver in lsd1 knockout mice . our analyses uncovered that lsd1 is necessary to regulate metabolism , particularly fatty acid oxidation , de novo lipogenesis , and lipolysis , leading to increased dag and tag accumulation and whitening of lsd1 bat . in addition , lsd1 depletion / inhibition leads to elevated glucose uptake and increased glycolytic capacities in mice . this may contribute to dag and tag accumulation because the glycerol backbone for triglyceride synthesis can be derived from the glycolytic intermediate dihydroxyacetone phosphate . furthermore , pyruvate is converted into citrate in the tca cycle and utilized to provide acetyl - coa to initiate de novo lipogenesis . however , further experiments would be necessary to provide a direct link between these processes . nevertheless , the role of lsd1 in lipid metabolism has  been confirmed in this study because lsd1 mice show increased activity of fatty acid synthase and decreased activity of lipases . altogether , alterations in glycolysis and fatty acid oxidation might contribute to aberrant production of dag and tag , which leads to increased fat accumulation . transcriptional and metabolic changes induced by lsd1 ablation have secondary effects on body weight gain and glucose tolerance with aging . these findings are of interest , considering that many mouse models possessing defects in adipose tissue function and metabolism , such as loss of mitochondrial uncoupling ( liu et  al . , 2003 ) and fatty acid or ceramide metabolism ( ellis et  al . , 2015 , turpin et  al . , 2014 ) , do not develop obesity . a hypothesis might be that lsd1-deficient brown adipocytes secrete signaling molecules that exert a potential inhibitory function on energy expenditure in other metabolic organs such as white adipose tissue , liver , or muscle . consequently , we carefully inspected our transcriptome analysis for differential expression of endocrine molecules , cytokines , or other signaling molecules . this analysis failed to identify any known candidates that might explain the phenotype in our mice . this , however , does not exclude the possibility that lsd1 regulates the expression of yet undescribed endocrine molecules in brown adipocytes . previous studies showed that the ablation of the brown fat - determining transcriptional regulator prdm16 is required to suppress the expression of wat - selective genes in bat ( harms et  al . , 2014 ) however , the expression of the most typical bat - selective genes was only slightly reduced upon prdm16 ablation , which could explain why prdm16 knockout mice are not prone to obesity ( harms et  al . , 2014 ) . in line with this publication , zeng et  al . ( 2016 ) showed that lsd1 associates with prdm16 to repress expression of wat - selective genes . of note , our chip - seq data identified prdm16 as a direct lsd1 target , and prdm16 expression was reduced in lsd1-deleted bat . however , immunoprecipitation , size exclusion chromatography , and mass spectrometry analyses did not provide any evidence for association of lsd1 and prdm16 in bat , suggesting that lsd1 acts upstream of prdm16 . ( 2016 ) also claim that lsd1 represses the expression of hydroxysteroid 11--dehydrogenase isozyme 1 ( hsd11b1 ) independently from prdm16 . even though hsd11b1 is 1.5-fold upregulated in our lsd1 mice , our cistrome analysis did not provide any evidence for binding of lsd1 to the hsd11b1 promoter . even though the phenotypes of our lsd1 mice and the mice engineered by zeng et  al . ( 2016 ) are close , the differences observed in the follow - up cistrome and transcriptome analyses and the interpretation of the molecular mechanism could be explained by the fact that they did not use a brown fat - specific cre deleter strain . ( 2016 ) proposed that zfp516 associates with lsd1 to promote ucp1 promoter occupancy and gene expression . in clear contrast , our mass spectrometry analysis does not confirm interaction of endogenously expressed lsd1 and zfp516 in brown adipocytes . in addition , our chip - seq showed that the ucp1 promoter is not occupied by lsd1 in brown adipocytes . our findings indicate that the use of lsd1 inhibitors would lead to a shift from oxidative to glycolytic metabolism in bat ,  resulting in weight gain but improved glucose tolerance . these findings need to be considered because lsd1 inhibitors entered clinical trials , for example in the treatment of mixed - lineage leukemia ( feng et  al . , 2016 ) . in conclusion , all mice were housed in the pathogen - free barrier facility of the university medical center freiburg in accordance with institutional guidelines approved by the regional board . mice were maintained in a temperature- and humidity - controlled animal facility with a 12-hr light / dark cycle , free access to water , and a standard rodent chow ( kliba , breeding , 3807 ) . animals were killed by cervical dislocation , and tissues were immediately collected , weighed , frozen in liquid nitrogen , or processed for further analyses . in  vivo experiments , including food consumption , serum analysis , glucose tolerance tests , temperature measurements , and energy expenditure , were described previously ( duteil et  al . , 2014 ) . the lsd1 inhibitor qc6688 was administrated per os at 5  mg / kg in 0.5% methylcellulose ( sigma ; product m0512 - 110 g ; viscosity , 4,000 centipoise [ cp ] ) . immediately before the measurement , cells were washed with unbuffered dmem as described previously ( wu et  al . , 2007 ) . plates were placed into the xf24 instrument for measurement of ocr and ecar with the xfp glycolysis stress kit ( 103017 - 100 , seahorse ) for ecar measurements ( glucose , 10  mm ; oligomycin , 1  m ; and 2 deoxiglucose ( 2-dg ) , 50  mm ) or the cell mito stress kit ( 103010 - 100 , seahorse ) for ocr measurements ( oligomycin , 1  m ; carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone ( fccp ) , 0.5  m ; and rotenone / antimycin a , 0.25  m ) . total lipids were extracted from 50  mg of bat using a lipid extraction kit ( sta-612 , cell biolabs ) and quantified using a lipid quantification kit ( sta-613 , cell biolabs ) according to the manufacturer s recommendations . bat was digested overnight with proteinase k , and dna was extracted with phenol - chloroform . mitochondria and nuclear dna were amplified by qpcr using cox2 and fasn primers ( table s4 ) , respectively . tissue samples were grinded with a retsch mm440 instrument and further extracted as described in giavalisco et  al . lc - ms measurements were performed using a waters acquity ultra performance liquid chromatography ( uplc ) system coupled to a thermo fisher scientific qexactive mass spectrometer . significance was calculated by(1)two - tailed student s t test for figures 1 g and 1h , 2c and 2d , 4d and 4e , 5c5 g , and 6c6i and figures s1a , s1d , s2a , s2c , s2d , s4b , s4c , s4e s6e , s6j , s6l , s6n , and s6q;(2)one - way anova for figures 3a and 3c3e and 4f and figures s3a , s3e , s3k , and s5d;(3)two - way anova for figures 3b and 3f , 4 g , and 6a , 6b , and 6j and figures s3b , s3l , s4k , s4l , s5e , s5f , s6a , and s6p;(4)wilcoxon rank - sum test for figures 5a and 5b and figures s6h and s6i ; and(5)analysis of covariance ( ancova ) for figures s6k , s6 m , and s6o . two - tailed student s t test for figures 1 g and 1h , 2c and 2d , 4d and 4e , 5c5 g , and 6c6i and figures s1a , s1d , s2a , s2c , s2d , s4b , s4c , s4e s6e , s6j , s6l , s6n , and s6q ; one - way anova for figures 3a and 3c3e and 4f and figures s3a , s3e , s3k , and s5d ; two - way anova for figures 3b and 3f , 4 g , and 6a , 6b , and 6j and figures s3b , s3l , s4k , s4l , s5e , s5f , s6a , and s6p ; wilcoxon rank - sum test for figures 5a and 5b and figures s6h and s6i ; and analysis of covariance ( ancova ) for figures s6k , s6 m , and s6o . heatmaps were generated by centering and normalizing expression values with cluster 3.0 and importing them to mev viewer .
summaryprevious work indicated that lysine - specific demethylase 1 ( lsd1 ) can positively regulate the oxidative and thermogenic capacities of white and beige adipocytes . here 
 we investigate the role of lsd1 in brown adipose tissue ( bat ) and find that bat - selective lsd1 ablation induces a shift from oxidative to glycolytic metabolism . 
 this shift is associated with downregulation of bat - specific and upregulation of white adipose tissue ( wat)-selective gene expression . 
 this results in the accumulation of di- and triacylglycerides and culminates in a profound whitening of bat in aged lsd1-deficient mice . 
 further studies show that lsd1 maintains bat properties via a dual role . 
 it activates bat - selective gene expression in concert with the transcription factor nrf1 and represses wat - selective genes through recruitment of the corest complex . in conclusion , 
 our data uncover lsd1 as a key regulator of gene expression and metabolic function in bat .