regulation of body weight and adiposity relies on a homeostatic system balancing energy intake with energy expenditure .
well - documented evidence gathered during the past 35 years has established that induction of thermogenesis in brown adipocytes of mice and rats can reduce obesity ( rothwell and stock , 1979 ) . the maintenance of body temperature by brown adipose tissue ( bat ) thermoregulation utilizes energy stores in proportion to ambient temperature ( kozak , 2010 ) .
importantly , bat - based non - shivering thermogenesis may be important in humans as well ( yoneshiro and saito , 2015 ) .
therefore , adiposity may be regulated by the capacity of the individual to manipulate brown adipocyte phenotypes at reduced temperature , thereby constituting a strategy to reduce metabolic efficiency ( jaroslawska et  al . , 2015 ) .
during the past decade , accumulating data have demonstrated that the gut microbiota impacts body weight and energy homeostasis ( tremaroli and bckhed , 2012 ) .
microbial diversity as well as the relative proportions between the members of main phyla firmicutes and bacteroidetes have been associated with regulation of obesity in both mice and humans ( ley et  al . , 2005 ,
the gut microbiota is important for energy harvest on a polysaccharide - rich diet ( bckhed et  al . , 2004 ) and also contributes to diet - induced obesity ( dio ) by modulating different pathways involving triglyceride storage and fatty acid oxidation ( bckhed et  al . , 2007 ) .
interestingly , microbiota from lean twins can protect against obesity induced by microbiota from obese twins , when transferred to mice ( ridaura et  al .
bile acids ( bas ) produced by hepatocytes , stored in the gall bladder and released into the gut after a meal , are metabolized by the gut microbiota to generate an array of ba species such as secondary bas , e.g. , lithocholic and deoxycholic acid ( sayin et  al . , 2013 ) .
these can activate nuclear receptor farnesoid x receptor ( fxr ) and g - coupled receptor tgr5 , located on ibat , to regulate thermogenesis ( prawitt et  al .
recent studies have linked changes in the gut microbiota in a cold environment to the thermogenic potential of brite cells through enhanced type 2 cytokine signaling by the innate immune system ( lee et  al . , 2015 , chevalier et  al . , 2015 ) .
here we show , in contrast to the role of brite cells in determining resistance to dio in c57bl/6j mice at reduced temperature ( surez - zamorano et  al . , 2015 ) , that acute changes in energy balance in mice fed a high - fat diet at 12c is associated with changes in the gut microbiota , increased ba metabolism , and induction of ibat thermogenesis .
we fed mice with high - fat diet ( hfd ) and chow diet ( chd ) at thermoneutrality and at reduced ambient temperatures to assess the effects of reduced temperature on cold - induced energy expenditure and development of dio . at 12c and 17c ,
the reduced fat mass and adiposity ( figures 1a , 1b , s1a , and s1b ) together with increased food intake in mice fed either hfd or chd resulted in increased energy expenditure ( figures 1c and 1d ) , demonstrating that reduced temperature blocks dio .
reduction of ambient temperature from 29c to 17c and 12c induced ibat and inguinal fat ( ing ) uncoupling protein 1 ( ucp1 ) mrna and protein expression in mice fed either chd or hfd ( figures 1e1h and s1c ) .
however , ucp1 expression in ing was only 6% of that observed in ibat . in addition , pgc1a ( peroxisome proliferator - activated receptor coactivator 1-alpha ) and adrb1 ( beta3-adrenergic receptor ) expression , which are associated with improved metabolism , was significantly induced in ibat and ing by cold ( figures s1d and s1e ) . reduced cellular energy levels lead to increased phosphorylation of ampk and was associated with increased phosphorylation of acc and increased expression of cpt1a , indicating increased hepatic fatty acid oxidation ( figures 1i1k ) . at 12c mice had decreased expression of biomarkers of hepatic lipogenesis ( figure  s1f ) , a phenotype associated with reduced plasma cholesteryl esters ( ces ) and triacylglycerols ( tags ) in mice fed hfd at 12c compared to 29c ( ces , 12c 6,102.5   446 versus 29c 8,421.1   490.7 , p  < 0.05 ; tags , 12c 155.1   50.4 versus 29c 409.4   43.4 , p  < 0.05 ) .
consistent with reduced adiposity , glucose tolerance was improved in mice fed hfd at 12c compared to 17c or 29c and was similar in both hfd- and chd - fed mice at 12c ( figure  1l ) .
reduced ambient temperature also improved insulin tolerance of mice on either hfd or chd ( figures s1 g and s1h ) .
the improved glucose tolerance was associated with increased glut4 ( glucose transporter type 4 ) mrna and protein levels in ibat and skeletal muscle ( figures s1i  s1k ) .
induction of irs1 ( insulin receptor substrate 1 ) mrna at 17c reflected the improved insulin tolerance at reduced ambient temperature ( figure  s1l ) .
furthermore , expression of pepck ( phosphoenolpyruvate carboxykinase ) and g6pc ( glucose-6-phosphatase catalytic subunit ) mrna levels was increased in liver of mice housed at 17c and 12c ( figures s1 m and s1n ) , indicating increased hepatic gluconeogenesis , which provides energy for thermogenesis in the cold ( himms - hagen , 1995 ) . to directly investigate the impact of diet and ambient temperature on mouse gut microbiota , the variable region 4 ( v4 ) of bacterial 16s rrna genes was amplified by pcr and sequenced using the illumina miseq platform .
we observed 4,090 distinct otus ( from 9,442,775 reads ; ranging from 75,344 to 159,323 reads per sample ) . to investigate how diet and ambient temperature affected the microbiota phylogenetic richness in each caecum sample
, we analyzed the -diversity , as assessed by  rarefication and phylogenetic diversity ( figure  2a ) .
similar to previous reports , we found that diet is a key factor in shaping phylogenetic diversity ( hildebrandt et  al . , 2009 , turnbaugh et  al . , 2009 ) and observed that the caecal microbiota of mice on chd had higher phylogenetic diversity compared with mice on hfd .
we next performed principal coordinate analysis ( pcoa ) of unweighted unifrac distances between the caecum samples from the different groups to determine the effects of diet and ambient temperature on the microbiota . a clear separation between the communities , driven by diet ,
was observed at the first principal coordinate ( x axis ) , which explained 27% of the variance ( figure  2b ) .
the second principal coordinate ( y  axis ) accounted for 13% of the variance and separated the communities by ambient temperature within the diets ( figure  2b ) .
moreover , the communities of mice maintained at 12c separated from those at 17c and 29c independently of the diets with the strongest effect in the hfd groups ( figure  2b ) .
analysis at the phylum level indicated that the caecal microbiota was dominated by five major phyla : actinobacteria , bacteroidetes , firmicutes , proteobacteria , and verrucomicrobia ( figure  2c ) .
mice fed hfd at 12c and 17c were associated with a bloom of proteobacteria and a reduction in bacteroidetes and an increase in firmicutes ( figure  2c ) .
shifts in bacteroidaceae , rikenellaceae , and s24 - 7 ( all bacteroidetes ) , particularly in s24 - 7 on chd and bacteroidaceae on hfd , were observed ( figure  2d ) .
the increase in firmicutes under all conditions was due to increases in clostridiaceae , lachnospiraceae , and ruminococcaceae ( figure  2d ) .
interestingly , a bloom in erysipelotrichaceae was observed in both diets , but only at 29c ( figure  2d ) .
the bloom in proteobacteria at reduced temperature was largely accounted for  by desulfovibrionaceae ( figure  2d ) , which has been previously associated with metabolic health ( caesar et  al . , 2015 ) . to identify taxonomic differences in the microbiota composition of mice fed hfd or chd and
challenged by differences in the ambient temperature and to identify specific bacterial taxa or species - level phylotypes that contribute to the adiposity phenotype , we applied linear discriminant analysis ( lda ) effect size ( lefse ) with lda score > 2 ( segata et  al . , 2011 ) .
this analysis revealed 46 discriminative features in the chd - fed mice ( figure  s2a ) and 34 in the microbiota of hfd - fed mice ( figure  s2b ) .
similar otus belonging to class bacilli in particular orders turicibacterales and lactobacillales , the genus allobaculum in the family erysipelotrichaceae , as well as the genus rc4 - 4 in the family peptococcaceae ( figures s2a and s2b ) were increased in mice maintained at 29c independent of diet .
the microbiota of mice fed hfd maintained at 12c was enriched in a corriobacteria such as adlercreutzia , number of clostridia from the mogibacteriaceae and ruminococcaceae families , and desulfovibrionales such as desulfovibrio ( figure  s2b ) .
similar changes were observed in mice fed chd maintained at 17c ( figure  s2a ) , all of which were lean and glucose tolerant .
reduced ambient temperature caused a robust induction of hepatic enzymes engaged in the conversion of cholesterol to primary bas , including cyp7a1 ( cholesterol 7alpha - hydroxylase ) , cyp8b1 ( sterol 12alpha - hydroxylase ) , and cyp27a1 ( sterol 27-hydroxylase ) , as well as in taurine production and taurine conjugation to bas ( figures 2e , s2c , and s2d ) .
accordingly , the ba profile was drastically altered and dominated by unconjugated ba ( ca , and - , mca ) at 29c in mice fed a hfd , whereas primary taurine - conjugated bas , i.e. , tca , tmca , and tmca , were elevated in plasma of mice maintained at 12c on both diets ( figures 2f2h ) .
fgf21 was 7- and 2.5-fold upregulated in ibat at 12c on a hfd and chd , respectively ( figure  s2e ) .
furthermore , type 2 iodothyronine deiodinase expression , which is associated with increased thermogenesis , was induced at reduced temperature ( figure  s2e ) , as previously observed ( de jesus et  al . , 2001 ) .
the tgr5 receptor , which binds to microbially produced secondary bas ( kawamata et  al . , 2003 ) , was induced at 12c while thyroid hormone receptor beta was not ( figures s2e  s2 g ) . to investigate whether the gut microbiota and bas were altered in response to reduced temperature or reduction in obesity
, we performed a kinetic experiment and analyzed the microbiota in mice that were fed hfd for 4  weeks at 29c and then transferred to 12c for 6  days . increased fat mass and adiposity after 4  weeks of a hfd at 29c was not affected by reduced ambient temperature ( figures 3a and 3b ) , and the increased demand for fuel was satisfied by a 20% increase in food intake within 2  days of cold exposure ( figure  3c ) .
cold exposure induced ucp1 expression 5-fold in ibat after 1  day at 12c ( figure  3d ) , similar to the increase observed after 4  weeks at 12c ( compare with figure  1e ) .
the induction of ucp1 in ing was very low and therefore not associated with  the initial response required for survival in the cold ( figure  3d ) .
the gut microbiota and ba composition were shifted to the cold - adapted state within 1  day , demonstrating that these changes were independent of reduced adiposity ( figures 3e3h ) .
the bas profile , already modified 1  day after cold exposure , was dominated by conjugated bas ( tca , tmca , tmca , and tmca ) , whereas the levels of unconjugated bas ( mca , mca , and mca ) were reduced compared to 29c ( figure  3e ) .
analysis on caecal microbiota composition showed a significant decrease in the phylogenetic diversity already 2  days after cold exposure compared to 29c ( figure  3f ) . at the phylum level , deferribacteres increased and verrucomicrobia decreased within 1  day of cold ( figure  3 g ) .
bacteroidetes increased and firmicutes decreased , but not until after 6  days at 12c ( figure  3 g ) , to increase the bacteroidetes / firmicutes ratio ( figure  3h ) . at the family level , the most abundant phylotypes affected by cold exposure were bacteroidaceae , rikenellaceae , porphyromonadaceae , deferribacteraceae , lactobacillaceae , clostridiaceae , and peptostreptococcaceae ( figure  s3a ) .
the fraction of others , such as s24 - 7 , clostridiales , lachnospiraceae , ruminococcaceae , erysipelotrichaceae , and desulfovibrionaceae ( figure  s3a ) did not change when mice were transferred to cold for 6  days , suggesting that these microbial phylotypes are associated with hfd feeding .
similar to the initial cold - exposure experiment for 4  weeks , we observed that adlercreutzia and bacteroides were increased after 6  days of cold exposure ( figure  s3b ) .
the firmicutes , lactobacillus , clostridiaceae , genus smb53 , dehalobacterium , peptostreptococcaceae , and mogibacteriaceae decreased at 12c ( figure  s3b ) .
long - term cold exposure induced a bloom in proteobacteria , particularly in desulfovibrionaceae ( figure  s2b ) , but it did not change during the short - term exposure at 12c ( figure  s3a ) .
these data suggest that in parallel with ucp1 induction , changes in microbiota and bas occur when ambient temperature is reduced , but independent of changes in adiposity . to assess the capacity of the gut microbiota from mice maintained at reduced ambient temperature to modulate the development of dio
mice colonized with microbiota from donors kept at 12c had reduced fat mass and adiposity as well as significantly higher expression of ucp1 and dio2 mrna and protein in ibat compared with mice colonized with microbiota from 29c ( figures 4a4e ) .
expression of adrb3 , pgc1a , and fgf21was not altered in ibat ( figure  4c ) ; no changes in gene expression were detected in ing of recipient mice ( figure  4f ) .
the glucose response was improved in the recipient mice colonized with microbiota from donor kept at 12c , but no differences occurred in glut4 protein levels in muscle and ibat ( figures 4 g , s4a , and s4b ) .
recipient mice , colonized with microbiota from donors kept at 12c , had increased hepatic expression of cyp8b1 and cyp7b1 ( 25-hydroxycholesterol 7alpha - hydroxylase ) and csd ( cysteine sulfinic acid decarboxylase ) , leading to an altered ba profile ( figures s4c and 4h ) .
these mice had an increased proportion of conjugated , and a reduced proportion of primary unconjugated , bas ( ca , cdca , and mca ) and secondary bas ( dca and udca ) in the plasma ( figure  4h ) .
gut microbiota transferred from donors housed at 12c influenced hepatic lipid metabolism , inducing ampk phosphorylation augmenting lipid -oxidation in liver compared with microbiota from 29c ( figures 4i4k and s4d ) .
nonetheless , lipid analysis showed a modest increase in total tag in plasma in mice colonized with microbiota from  donor kept at 12c ( figure  s4e ) .
these results suggest that functional differences between microbiota of mice kept  at 12c and 29c can be transferred to recipient mice kept at 23c .
a primary and essential task of all endothermic animals is to maintain a normal body temperature by inducing thermogenesis in response to cold . here , we demonstrate that reducing ambient temperature from 29c to 12c or even 17c also protects the host from dio , an effect that is associated with increased ucp1 expression in ibat .
however , recent studies have shown that the pathway for activating thermogenesis may be more complex than the traditional model based on ibat thermogenesis .
thermogenesis occurs not only in ibat , but also by the induction of brite cells in wat at 29c by administration of interleukin 4 ( qiu et  al . ,
similarly , the thermogenic program in ibat may be activated by bas at 29c ( zietak and kozak , 2016 ) .
recently , it was shown that the depletion of the microbiota by antibiotic treatment or gf conditions stimulates the induction of brite cells by enhanced type 2 cytokine signaling from eosinophil infiltration ( surez - zamorano et  al . , 2015 ) . here , we show that both the acute and chronic exposure to reduced ambient temperature leads to induction of ucp1 in ibat and rapid changes in the composition of the gut microbiota and ba metabolism .
the results suggest that the induction of cold - mediated thermogenesis involves the activation of ibat thermogenesis by a mechanism involving modulation of ba metabolism and ampk phosphorylation by the gut microbiota .
a key question is whether the improved obesity / diabetic phenotype of mice with dio reared in the cold is dependent on the effects of the gut microbiota .
we observed that cold exposure is linked to altered caecal microbiota and that mice on both diets shared microbial phylotypes associated with changes in ambient temperature .
bacteria belonging to bacilli and erysipelotrichaceae , usually associated with obesity ( turnbaugh et  al . , 2009 ) , were enriched in mice at 29c , whereas adlercreutzia and desulfovibrio , associated with leanness ( caesar et  al .
, 2015 , goodrich et  al . , 2014 ) , were increased at 12c .
changes in the composition of gut microbiota occurring within 1  day of exposure at 12c and in the absence of changes in adiposity suggest that the changes in microbiota are driven by changes in temperature rather than merely reflecting obesity .
the major microbiota changes occurring in parallel with induction of ucp1 were reduced firmicutes apparent after 1  day and an increase in bacteroidetes after 6  days , changes previously linked with protection against obesity ( ley et  al . , 2005 , turnbaugh et  al . , 2008 ) .
( 2015 ) , we identified a decreased abundance of verrucomicrobia and an increased abundance of deferribacteres when mice were exposed to cold , suggesting that altered abundance of members in these phyla contribute to the cold - induced phenotype .
changes in other bacteria after longer cold exposure may be in response to altered adiposity of the host .
the mechanism by which the gut microbiota influences adiposity may include modulation of ba metabolism ( sayin et  al . , 2013 ) .
bas are microbial - derived metabolic regulators , which can suppress dio through increased energy expenditure ( watanabe et  al . , 2012 ) . here
we show that lower ambient temperature increased production of bas and expression of genes related to ba synthesis .
the increased levels of conjugated bas may be attributed to reduced levels of lactobacillus , which has high deconjugation capacity upon cold exposure .
these observations were confirmed in the kinetics study where conjugated bas were increased and lactobacillus was decreased after 1  day at 12c .
the increased prevalence of taurine species in the cold might antagonize fxr receptor ( sayin et  al . , 2013 ) and protect against dio as fxr - deficient mice are resistant to dio ( prawitt et  al .
, 2011a ) . in agreement with inhibition of fxr signaling in the cold - exposed animals , we observed increased expression of enzymes involved in ba synthesis and increased ba pool size .
the cold - exposed mice had a ba profile that resembled that of gf mice , which also are resistant to dio and have increased expression of phosphorylated ampk in the liver ( bckhed et  al . , 2007 ) .
ampk activation inhibits fxr activity ( lien et  al . , 2014 ) and also promotes energy expenditure .
accordingly , we observed increased expression of phosphorylated acc as well as downstream cpt1a expression , which is associated with increased fatty acid oxidation and energy expenditure .
importantly , we observed this phenotype both following cold - exposure as well as after microbiota transfer , suggesting that the phenotype is linked to the altered microbiota . in the current study ,
cold exposure at 12c causes a strong and rapid induction of the brown adipose phenotype in ibat , but not in ing , an increased whole - animal energy expenditure , and a complete suppression of dio .
we demonstrated that transplantation of caecal material from mice reared at 12c to gf recipients improved their metabolic phenotype .
( 2015 ) demonstrated that transplantation of caecal material from cold - exposed mice reduced obesity and improved insulin sensitivity .
however , the brown adipocyte phenotype in ing wat in our study is minor ( 1%5% ) compared to the robust ibat thermogenic phenotype .
our investigations suggest that changes in the gut microbiota in response to the cold exposure mediate ba metabolism , possibly through changes in ampk and fxr signaling , to complement sympathetic signaling in the regulation of thermogenesis in ibat and resistance to dio .
protocol i. breeding pairs of c57bl6/j ( b6 ) mice were obtained from the jackson laboratory .
adult b6 mice at 1220  weeks of age were divided into 6 groups ( n  = 810 mice / group ) .
three groups were ad libitum fed hfd ( 58% energy in kcal from fat , ain-76a 9g03 research diets ) ; another three groups received chd ( 11% energy in kcal from fat , 5053 , rodent diet 20 , labdiet ) .
mice were single - housed with a 12  hr light/12  hr dark cycle at environmental temperatures of 12c , 17c , and 29c for 4  weeks .
b6 mice at 8  weeks of age were individually housed at 29c and ad libitum fed a hfd for 4  weeks ( n  = 6 mice ) .
thereafter , mice were transferred to 12c for 1 , 2 , 4 , and 6  days and received ad libitum a hfd ( n  = 6 mice / time point ) . for both protocols , mice were anaesthetized with an overdose of cocktail composed of ketamine / xylazine / chlorpromazine administered subcutaneously to collect blood samples by cardiac puncture .
blood samples were centrifuged for 10  min at 2,400  rpm and stored at 20c .
mice were sacrificed by cervical dislocation , and tissues and caecal content were quickly removed and stored at 80c for further analysis .
animal experiments performed at  different temperatures were approved by the local committee for the ethical treatment of experimental animals of warmia - mazury university ( nr 38/2011 ) , olsztyn , poland .
fat mass and lean mass were measured by nuclear magnetic resonance ( nmr , bruker ) .
food intake was calculated on a weekly basis and expressed as the amount of energy in kj . at the end of the experiment
bacterial gdna was extracted using the genematrix stool dna purification kit ( eurx ) from the caecum of mice fed hfd and maintained at 17c or 29c .
genomic dna from caecum of mice fed hfd at 12c and chd at 12c , 17c , and 29c was isolated using the repeated bead beating ( rbb ) method ( salonen et  al . , 2010 ) .
details about 16s rrna amplification , sequencing , microbiota data analysis , and ba analyses are provided in supplemental experimental procedures .
the data are expressed as mean   sem and analyzed using graphpad prism 6.0 .
statistical differences for single variables were analyzed by mann - whitney test or one - way anova and tukey s multiple comparison post hoc test for  three groups ( 29c , 17c , and 12c ) the level of significance was set at p  < 0.05 ; p  <
0.05 ; p  < 0.01 ; p  < 0.001 ; p  < 0.0001 .
m.z . was involved in design of the experiment and performed physiological and molecular phenotypes and participated in writing the manuscript ; p.k .- d . was involved in design and performed experiments concerning microbial profiling and transplant experiments and participated in writing the manuscript ; l.h.m . and m.s .
designed the initial hypothesis and participated in the design and the writing of the manuscript ; f.b . was involved in design and interpretation of experiments concerning microbial profiling and transplant experiments and participated in the design and the writing of the manuscript .
 summarymaintenance of body temperature in cold - exposed animals requires induction of thermogenesis and management of fuel . here 
 , we demonstrated that reducing ambient temperature attenuated diet - induced obesity ( dio ) , which was associated with increased ibat thermogenesis and a plasma bile acid profile similar to that of germ - free mice . 
 we observed a marked shift in the microbiome composition at the phylum and family levels within 1  day of acute cold exposure and after 4  weeks at 12c . 
 gut microbiota was characterized by increased levels of  adlercreutzia , mogibacteriaceae , ruminococcaceae , and desulfovibrio and reduced levels of bacilli , erysipelotrichaceae , and the genus rc4 - 4 . 
 these genera have been associated with leanness and obesity , respectively . 
 germ - free mice fed a high - fat diet at room temperature gained less adiposity and improved glucose tolerance when transplanted with caecal microbiota of mice housed at 12c compared to mice transplanted with microbiota from 29c . 
 thus , a microbiota - liver - bat axis may mediate protection against obesity at reduced temperature . 