Cell ORT transplanted ORT o Cold trans 日cok Time(hrs) ORT transplanted a cold transplanted 害 e(hrs) Time(hrs) Time(hrs) Time(hrs) Time(hrs) Figure 4. Cold Microbiota Prevents Hypothermia (A and B)Rectal temperature (A)or temperature change(B)of RT- or cold-transplanted mice before or after 4 hr of cold exposure(n =8 per group) (C)Infrared images of representative RT- or cold-transplanted mice after 4 hr cold exposure. f Infrared temperature readings from eye(d (E, or dorsal()region of mice (G-I)Infrared temperature readings from eye(G), ventral (H), or dorsal ( region of mice as in(C)before or after 12 hr of cold exposure (Figure 4c)demonstrated that cold microbiota-transplanted tween the groups(Figures 4E and 4H), the maximal ventral mice are fully resistant to cold stress as shown by the eye tem- heat differences remained constant also after 12 hr of cold expo- perature measurements, representative of the internal body tem- sure(Figures 4F and 4l). These data suggest a mechanistic perature(Figures 4D and 4G). Analysis of the dorsal and ventral explanation for the increased insulin sensitivity and demonstrate infrared images showed that the inguinal and the interscapular that the cold microbiota alone is sufficient to induce tolerance to regions contribute to the overall tolerance to cold. Specifically, cold, increased EE and lower fat content, and this effect is while the differences in dorsal temperatures were transient be- partially mediated by the browning of the white fat depots gSAT and pgvAT of cold- and RT-transplanted mice 21 days after transplantation using the Ct scans. Scale bar, 5 mm (B)Weight of fat pads of cold-or RT-transplanted mice after 5.5 weeks(n= 6 per group). C-H)IngSAT or pgVAT volumes(C and E, or densities(G and H)of mice as in(A. Change in each fat pad volume(D and F)(n= 12 per group, except [E]and [F where n=6 per group) of same mice scanned at day 3 and day 21 after transplantation. and )Cell size profiling of adipocytes from ingSAT (), or pgVAT ()of RT-or cold-transplanted mice 21 days after transplantation. The values show from the sponding fractions from each animal sEM(n= 6 for each panel and L) H&E staining on paraffin sections from ingSAT (K or pgVAT (L of RT- or cold-transplanted mice (M, O, and P)Relative mRNA expression in ingSAT (M), pgVAT (O), or iBAT(P)of RT- or cold-transplanted mice(n= 6 per group), quantified by real-time PCR and normalized house keeping beta-2-microglobulin(B2M). Immunohistochemistry of Ucpl and DAPl on paraffin sections from ingSAT in RT-or cold-transplanted mice as in(k). Resting energy expenditure(REE in Ri-or cold-transplanted mice, measured between day 3 and day 21 after bacterial colonization (n= 6 per group). Scale bars in(K). (L, and(N), 100 um. 366 Cell 163. 1360-1374 December 3. 2015 2015 Elsevier Inc.(Figure 4C) demonstrated that cold microbiota-transplanted mice are fully resistant to cold stress as shown by the eye tem￾perature measurements, representative of the internal body tem￾perature (Figures 4D and 4G). Analysis of the dorsal and ventral infrared images showed that the inguinal and the interscapular regions contribute to the overall tolerance to cold. Specifically, while the differences in dorsal temperatures were transient be￾tween the groups (Figures 4E and 4H), the maximal ventral heat differences remained constant also after 12 hr of cold expo￾sure (Figures 4F and 4I). These data suggest a mechanistic explanation for the increased insulin sensitivity and demonstrate that the cold microbiota alone is sufficient to induce tolerance to cold, increased EE, and lower fat content, and this effect is partially mediated by the browning of the white fat depots. Figure 3. Cold Microbiota Promotes Browning of WAT (A) 3D reconstitution of the ingSAT and pgVAT of cold- and RT-transplanted mice 21 days after transplantation using the CT scans. Scale bar, 5 mm. (B) Weight of fat pads of cold- or RT-transplanted mice after 5.5 weeks (n = 6 per group). (C–H) IngSAT or pgVAT volumes (C and E), or densities (G and H) of mice as in (A). Change in each fat pad volume (D and F) (n = 12 per group, except [E] and [F] where n = 6 per group) of same mice scanned at day 3 and day 21 after transplantation. (I and J) Cell size profiling of adipocytes from ingSAT (I), or pgVAT (J) of RT- or cold-transplanted mice 21 days after transplantation. The values show % from the total number of analyzed cells. Bars show mean of the pooled corresponding fractions from each animal ± SEM (n = 6 for each panel). (K and L) H&E staining on paraffin sections from ingSAT (K) or pgVAT (L) of RT- or cold-transplanted mice. (M, O, and P) Relative mRNA expression in ingSAT (M), pgVAT (O), or iBAT (P) of RT- or cold-transplanted mice (n = 6 per group), quantified by real-time PCR and normalized to the house keeping beta-2-microglobulin (B2M). (N) Immunohistochemistry of Ucp1 and DAPI on paraffin sections from ingSAT in RT- or cold-transplanted mice as in (K). (Q) Resting energy expenditure (REE) in RT- or cold-transplanted mice, measured between day 3 and day 21 after bacterial colonization (n = 6 per group). Scale bars in (K), (L), and (N), 100 mm. –3 –2 –1 0 ** ∆°C after 4 h cold 34 35 36 37 38 Rectal T (°C) ** * n.s. 0 4 32 33 34 35 36 37 38 0 12 ** Eye T (°C) 32 33 34 35 36 37 38 Inguinal T (°C) ** 0 12 34 35 36 37 38 39 40 Interscapular T (°C) 0 12 p = 0.1 25.0 38.0 °C eye / right lateral ventral dorsal RT transplanted Cold transplanted 32 34 36 38 Eye T (°C) ** 0 4 34 36 38 40 Interscapular T (°C) * 0 4 31 33 35 37 39 Inguinal T (°C) ** 0 4 A B RT transplanted Cold transplanted C DE F GH I Time (hrs) Time (hrs) Time (hrs) Time (hrs) Time (hrs) Time (hrs) Time (hrs) RT transplanted Cold transplanted RT transplanted Cold transplanted Figure 4. Cold Microbiota Prevents Hypothermia (A and B) Rectal temperature (A) or temperature change (B) of RT- or cold-transplanted mice before or after 4 hr of cold exposure (n = 8 per group). (C) Infrared images of representative RT- or cold-transplanted mice after 4 hr cold exposure. (D–F) Infrared temperature readings from eye (D), ventral (E), or dorsal (F) region of mice as in (C) before or after 4 hr of cold exposure. (G–I) Infrared temperature readings from eye (G), ventral (H), or dorsal (I) region of mice as in (C) before or after 12 hr of cold exposure. 1366 Cell 163, 1360–1374, December 3, 2015 ª2015 Elsevier Inc