Cell tion remain poorly understood. Here, we show that the micro- controls. Profiling of the microbiota composition by 16S rRNA biota remodeling is an important contributor of the beige fat gene sequencing followed by principal coordinates analysis induction during cold and a key factor that promotes energy up-(PCoA) based on weighted UniFrac distance, showed major al- ke by increasing the intestinal absorptive area, thus orches- terations of the microbiota content both in cecum and feces trating the overall energy homeostasis during increased energy samples of cold-exposed animals(Figures 1F, S2A, and S2B) As expected, Firmicutes was the richest phylum in all samples n average 69.10%)(Figures 1I and 1J). Bacteroidetes was RESULTS the most abundant phylum(on average 63. 50%)in all samples except the cold-exposed day 31 samples(Figures S2C-S2E) Cold Exposure Changes the Gut Microbiota We observed differences in operational taxonomic units(OTUs) Composition abundance at phylum level in Bacteroidetes, Firmicutes, Verru Short-term cold exposure for up to 10 days leads to increased comicrobia, Deferribacteres, Cyanobacteria, and Actinobacte- EE relative to the energy uptake and suppresses BW and white ria, and differences in oTU numbers at phylum-level in all the fat mass gain(Figures S1A-S1F)(u et al., 2012, 2013). To above plus Deferribacteres based on factor summary bar chart investigate the importance of the acutely consumed food and Individual species, or family-based hierarchical clustering using caloric harvest during cold exposure, we restricted the food ac- the average-neighbor method, confirmed the major shift of the ess during the initial 8 hr(hr)of cold exposure or depleted the microbiota composition and showed clustering of the samples intestinal microbiota using broad range antibiotics(Abx) admin- from the cold-exposed versus the room temperature(RT)groups istered in the drinking water. The higher fecal caloric content af- in both feces and cecum samples (Figures 1G, S2D, S2F, S3A, ter complete microbiota depletion was confirmed using bomb and S3B). Comparison of phylum level proportional abundance calorimetry(Figure S1G)and was consistent with previous re- in feces showed shifts in proportions(Figures 1H and S2C) ports(El Kaoutari et al., 2013), suggesting lower energy harvest especially in the ratio Firmicutes/Bacteroidetes where Firmi- from the food. Restricting the food access during acute cold cutes abundance(from 18.6% in RT up to 60.5% under cold exposure led to decreased body temperature(Figures 1A and increased over Bacteroidetes(from 72.6% in RT to 35.2% under 1B) compared to ad libitum-fed control mice and to a marked cold). The verrucomicrobia phylum was almost absent from both drop in the blood glucose and BW at cold(Figures S1H-S1J). feces and cecum after the cold exposure(from 12.5% for the Rt The decreased tolerance to cold and lowered blood glucose to 0.003% for the cold in cecum)(Figures 1H, S2C, and S2E) levels were also evident in the Abx-treated mice and the changes Interestingly, similar shifts, although less pronounced were relatively stable during short and long-term microbiota ciated with genetic and high fat diet-induced obesity (Turnbaugh depletion up to 4 weeks of treatment (Figures 1C-1E and S1K- et al., 2006). The shifts in phylum abundance correlated v S1R), despite the stable food intake and slightly increased water richness of the species present in them. Firmicutes phylum consumption (Figures S1S and S1T). These data suggest that the increased its richness in feces up to 78. 1% under cold exposure energy harvest during acute cold contributes to maintaining the (compared to 65% in RT) and Bacteroidetes decreased it to body temperature, and the intestinal microbiota is supporting 18.8%(compared to 29.7%in RT)(Figures 1I and 1J), without We observed that over time the overall fat loss was attenuated versity index(Figures S3C and S3D). From the 3. 86e n di- changing the overall bacterial diversity based on the Shan despite the stable food intake and EE(Figures S1A-S1F), sug- detected, using Welcht test done across the two groups ofsa US gesting compensatory mechanisms that enable increased ples using the abundance metrics, 252 OTUs(within 44 familie caloric harvest from the consumed food. To investigate whether were significantly different(p 0.05). Of the selected families, this prolonged cold exposure causes changes in the intestinal there were mixed responses in Firmicutes, Proteobacteria, microbiota, we collected feces at days 0, 11, and 31 and cecum and Bacteroidetes, however, those within Actinobacteria, Rectal body temperature(BT) of food restricted or ad libitum-fed C57BI6J mice after 4 and 8 hr(hr) of cold exposure(n 8 per group) 3) change in BT compared to initial as in(A) (C) Rectal BT after 3 hr of cold exposure of male mice treated or not treated with antibiotics (n =8 per group) (E Change in BT compared to initial as in(D) (F) Principal coordinates analysis(PCoA) based on weighted UniFrac analysis of OTUs. Each symbol represents a single sample of feces after 31 days of cold- exposed (n =8)or RT controls(n= 6 per group) Hierarchical clustering diagram using the average-neighbor(HC-AN) method comparing feces of 31 sed mice (n= 6). Associated heatmap shows the relative abundance of representative oTUs selected for p< 0.05, obtained with a Welch t test comparison groups and then grouped into families. One representative oTu with the greatest difference between the two group means from each family is se inclusion in the heatmap diagram OTUs are shown as: Phylum, Class, Order, Family, Genus, and Species. R, RT; C, cold-exposed and J)Richness represented as the pr of oTUs classified at the phylum rank. Feces. (J) Cecum. In (HJ)n=5+6(cecum) or 6 +8(feces). (K Heatmap tree comparing selected oTUs abundance from feces of T controls (n=6, inner rings) and 31 days cold-exposed mice(n=8, outer rings)and their phylogenic relationships. The OTUs representative of differentially abundant families are selected as described in(H) 1362cel163,1360-1374, December3,2015@2015 Elsevier Inc.homeostasis of the new host following microbiota transplanta￾tion remain poorly understood. Here, we show that the micro￾biota remodeling is an important contributor of the beige fat induction during cold and a key factor that promotes energy up￾take by increasing the intestinal absorptive area, thus orches￾trating the overall energy homeostasis during increased energy demand. RESULTS Cold Exposure Changes the Gut Microbiota Composition Short-term cold exposure for up to 10 days leads to increased EE relative to the energy uptake and suppresses BW and white fat mass gain (Figures S1A–S1F) (Wu et al., 2012, 2013). To investigate the importance of the acutely consumed food and caloric harvest during cold exposure, we restricted the food ac￾cess during the initial 8 hr (hr) of cold exposure or depleted the intestinal microbiota using broad range antibiotics (Abx) admin￾istered in the drinking water. The higher fecal caloric content af￾ter complete microbiota depletion was confirmed using bomb calorimetry (Figure S1G) and was consistent with previous re￾ports (El Kaoutari et al., 2013), suggesting lower energy harvest from the food. Restricting the food access during acute cold exposure led to decreased body temperature (Figures 1A and 1B) compared to ad libitum-fed control mice and to a marked drop in the blood glucose and BW at cold (Figures S1H–S1J). The decreased tolerance to cold and lowered blood glucose levels were also evident in the Abx-treated mice and the changes were relatively stable during short and long-term microbiota depletion up to 4 weeks of treatment (Figures 1C–1E and S1K– S1R), despite the stable food intake and slightly increased water consumption (Figures S1S and S1T). These data suggest that the energy harvest during acute cold contributes to maintaining the body temperature, and the intestinal microbiota is supporting this process. We observed that over time, the overall fat loss was attenuated despite the stable food intake and EE (Figures S1A–S1F), sug￾gesting compensatory mechanisms that enable increased caloric harvest from the consumed food. To investigate whether this prolonged cold exposure causes changes in the intestinal microbiota, we collected feces at days 0, 11, and 31 and cecum post-mortem of cold-exposed mice and room temperature (RT) controls. Profiling of the microbiota composition by 16S rRNA gene sequencing, followed by principal coordinates analysis (PCoA) based on weighted UniFrac distance, showed major al￾terations of the microbiota content both in cecum and feces samples of cold-exposed animals (Figures 1F, S2A, and S2B). As expected, Firmicutes was the richest phylum in all samples (on average 69.10%) (Figures 1I and 1J). Bacteroidetes was the most abundant phylum (on average 63.50%) in all samples except the cold-exposed day 31 samples (Figures S2C–S2E). We observed differences in operational taxonomic units (OTUs) abundance at phylum level in Bacteroidetes, Firmicutes, Verru￾comicrobia, Deferribacteres, Cyanobacteria, and Actinobacte￾ria, and differences in OTU numbers at phylum-level in all the above plus Deferribacteres based on factor summary bar chart. Individual species, or family-based hierarchical clustering using the average-neighbor method, confirmed the major shift of the microbiota composition and showed clustering of the samples from the cold-exposed versus the room temperature (RT) groups in both feces and cecum samples (Figures 1G, S2D, S2F, S3A, and S3B). Comparison of phylum level proportional abundance in feces showed shifts in proportions (Figures 1H and S2C), especially in the ratio Firmicutes/Bacteroidetes where Firmi￾cutes abundance (from 18.6% in RT up to 60.5% under cold) increased over Bacteroidetes (from 72.6% in RT to 35.2% under cold). The Verrucomicrobia phylum was almost absent from both feces and cecum after the cold exposure (from 12.5% for the RT to 0.003% for the cold in cecum) (Figures 1H, S2C, and S2E). Interestingly, similar shifts, although less pronounced, are asso￾ciated with genetic and high fat diet-induced obesity (Turnbaugh et al., 2006). The shifts in phylum abundance correlated with the richness of the species present in them. Firmicutes phylum increased its richness in feces up to 78.1% under cold exposure (compared to 65% in RT) and Bacteroidetes decreased it to 18.8% (compared to 29.7% in RT) (Figures 1I and 1J), without changing the overall bacterial diversity based on the Shannon di￾versity index (Figures S3C and S3D). From the 3,864 OTUs detected, using Welch t test done across the two groups of sam￾ples using the abundance metrics, 252 OTUs (within 44 families) were significantly different (p < 0.05). Of the selected families, there were mixed responses in Firmicutes, Proteobacteria, and Bacteroidetes, however, those within Actinobacteria, Figure 1. Cold Exposure Changes the Gut Microbiota Composition (A) Rectal body temperature (BT) of food restricted or ad libitum-fed C57Bl6J mice after 4 and 8 hr (hr) of cold exposure (n = 8 per group). (B) Change in BT compared to initial as in (A). (C) Rectal BT after 3 hr of cold exposure of male mice treated or not treated with antibiotics (n = 8 per group). (D) Rectal BT after 4 hr and 24 hr of cold exposure in antibiotics-treated or control female mice (n = 6 per group). (E) Change in BT compared to initial as in (D). (F) Principal coordinates analysis (PCoA) based on weighted UniFrac analysis of OTUs. Each symbol represents a single sample of feces after 31 days of cold￾exposed (n = 8) or RT controls (n = 6 per group). (G) Hierarchical clustering diagram using the average-neighbor (HC-AN) method comparing feces of 31 days cold-exposed mice (n = 8) and their RT controls (n = 6). Associated heatmap shows the relative abundance of representative OTUs selected for p < 0.05, obtained with a Welch t test comparison of the two groups and then grouped into families. One representative OTU with the greatest difference between the two group means from each family is selected for inclusion in the heatmap diagram. OTUs are shown as: Phylum, Class, Order, Family, Genus, and Species. R, RT; C, cold-exposed. (H) Comparison of phylum-level proportional abundance of cecum and feces of up to 31 days cold-exposed or RT control mice. (I and J) Richness represented as the proportions of OTUs classified at the phylum rank. (I) Feces. (J) Cecum. In (H)–(J) n = 5 + 6 (cecum) or 6 + 8 (feces). (K) Heatmap tree comparing selected OTUs abundance from feces of RT controls (n = 6, inner rings) and 31 days cold-exposed mice (n = 8, outer rings) and their phylogenic relationships. The OTUs representative of differentially abundant families are selected as described in (H). See also Figures S1, S2, and S3. 1362 Cell 163, 1360–1374, December 3, 2015 ª2015 Elsevier Inc