《人体的社交网络——共生微生物》课外文献阅读：Gut Microbiota Orchestrates Energy Homeostasis during Cold.pdf_P6-P10

Cell Verrucomicrobia, and Tenericutes were less abundant in the in the cold-transplanted mice(Figures 21-2K) and had decreased cold samples, while RT samples were less abundant in Deferri- ingSAT and pgVAT volumes and weights(Figures 3A-3F and bacteres(Figure 1K. When looking at the most significantly $4B). Hounsfield unit(HU) analysis of the microCT scans re- changed OTUs using analysis of variance, Akkermansia mucin- vealed that cold microbiota-transplanted mice had higher in- 7 family were among the top nine most shifted gSAT and pgvat density compared to the controls(Figures bacteria(Figures S3G and S3H). Verrucomicrobia phylum was and 3H). Together, these data suggest that the cold microbiota presented by eight different OTUs, all part of the same species: contributes to the increased insulin sensitivity observed during kkermansia muciniphila, which we found highly decreased with cold exposure and leads to decreased total fat coupled with old exposure(Figures S3E and S3F). The changes in the major increased fat density. bacterial phyla were confirmed by qPCR in the sequenced, as ell as in independent sets of SPF and conventional animals Cold Microbiota Promotes Browning, Energy (Figures S3l-S3L). Together, these results demonstrate a major Expenditure, and Cold Tolerance shift in lower biota in response to cold exposure. To investigate whether the higher density and the decreased fat amount(Figures 3A-3H) are originating from the differences in Cold Microbiota Transplantation Increases Insulin the adipocyte volume, we measured the adipocyte size distribu- Sensitivity tion using high content imaging. Cold-transplanted mice had To investigate the importance of the microbiota changes during increased number of small and decreased number of large adi- cold, we transplanted the microbiota from 30 days cold-exposed pocytes in the ingSAT and pgVAT depots(Figures 31-3L.The or control RT mice to germ-free(GF) mice by co-habitation and adipose depots excised from the cold-transplanted animals again confirmed the shifts in the donors and the recipient mice were darker in appearance. All these phenotypic events are (Figures S3K and S3L) As expected, cold exposure of donor characteristic features of mature beige adipocytes. Therefore, mice led to a marked increase in the insulin sensitivity(Figure 2A). we investigated whether cold microbiota could affect the brown- Strikingly, cold microbiota transplanted mice also showed ing of the white fat depots and found that cold -transplanted mice increased sensitivity to insulin ( Figure 2B), suggesting that cold had marked increase in the brown fat-specific markers in the in- microbiota alone is sufficient to transfer part of this phenotype. gSAT, and surprisingly, also in the pgVAT depots(Figures 3M The increased insulin sensitivity was further investigated using and 30). The increased browning of ingSAT was consistent hyperinsulinemic-euglycemic clamp in awake and unrestrained with the increased Ucp1-positive cells in the cold-transplanted mice. Cold mice showed a marked increase in the glucose infu- mice( Figure 3N). There was a tendency bordering significance sion rates( GIr)needed to maintain the clamped glucose levels toward increased brown fat marker expression in the interscap- nd an increase in the stimulated glucose disappearance(Rd) ular BAT (iBAT) depots of the col planted mice, albeit at levels(Figures 2C, 2D, and S4A). To investigate the peripheral smaller scale compared to ingSAT and pgVAT(Figure 3P) glucose uptake, we co-administered 2[C]deoxyglucose(2 Together, these data suggest that cold microbiota alone can [CDG)during the clamp. While no changes were observed in be sufficient to induce beige/brown fat formation primarily in the glucose uptake from interscapular BAT (iBAT), brain, soleus, the ing SAT and pgVAT, and to a smaller magnitude, in the or quadriceps muscle, there was a large increase in the uptake iBAT depots. The increased browning was consistent with the from inguinal subcutaneous and perigonadal (epididymal in enhanced resting EE(REE)of the cold-transplanted mice(Fig ales)visceral depots of the WAT (ngSAT and pgVAT, respec- ure 3Q), suggesting increased energy dissipation. To further tively)(Figure 2E. These observations were further corroborated investigate its functional relevance, we exposed the cold-trans in glucose-stimulated and basal conditions( Figures 2F and 2G), planted mice to acute cold and monitored the internal body which in addition showed increased glucose uptake in iBAT. temperature, as well as ventrally or dorsally, indicative of the Interestingly, the cold microbiota transferred the fat-specific temperature emitted from ingSAT or iBAT depots. The rectal glucose disposal phenotype to the transplanted mice as temperature measurements showed that the RT-transplanted measured by 2[C DG uptake(Figure 2H)and by positron emis- mice had decreased body temperature following 4 hr of cold sion tomography-computed tomography(microPET-CT). Spe- exposure, but only a mild temperature drop was detected in cifically, both ingSAT and pgVAT, but not quadriceps muscle, the cold-transplanted mice(Figures 4A and 4B). Accordingly, showed increased [F]fluorodeoxyglucose([FFDG)uptake the infrared imaging and quantification of the different regions nd B) Intraperitoneal insulin tolerance test (m)in RT and 25 days cold-exposed mice(A), or RT-and cold microbiota- planted mice(B)relative to initial blood glucose, ( n 8 per group (C-E Euglycemic-hyperinsulinemic clamp of awake mice as in(A. Rate of disappearance ofH-D-glucose( C). GIR time course during the hyperinsulinar mp(D). 2[ C]DG uptake in various tissues(B(n= 6+6) 2 'C]DG tracer uptake in tissues 45 min after iP tracer and glucose(2 g/kg BW) administration in mice as in (a)(n =6 per group). and H)2[CDG uptake in tissues 30 min after administration under basal conditions in anesthetized rt (n 9)and cold (n= 10)(G); or RT- and cold- ( J, and L) Positron emission tomography-computer t hy (microPET-CT) measurement of[F]FDG uptake in ingSAT(O), pgVAT (), or quadriceps muscle (Lin basal conditions of RT-and cold-transplanted mice as in(B)(n= 6 per group). Transversal[F]FDG PET-CT images of ing SAT and pgVAT of mice as in 0) and (). See also Figure S4 1364cel163,1360-1374, December3,2015@2015 Elsevier Inc

Verrucomicrobia, and Tenericutes were less abundant in the cold samples, while RT samples were less abundant in Deferribacteres (Figure 1K). When looking at the most significantly changed OTUs using analysis of variance, Akkermansia muciniphila and S24-7 family were among the top nine most shifted bacteria (Figures S3G and S3H). Verrucomicrobia phylum was represented by eight different OTUs, all part of the same species: Akkermansia muciniphila, which we found highly decreased with cold exposure (Figures S3E and S3F). The changes in the major bacterial phyla were confirmed by qPCR in the sequenced, as well as in independent sets of SPF and conventional animals (Figures S3I–S3L). Together, these results demonstrate a major shift in lower gut microbiota in response to cold exposure. Cold Microbiota Transplantation Increases Insulin Sensitivity To investigate the importance of the microbiota changes during cold, we transplanted the microbiota from 30 days cold-exposed or control RT mice to germ-free (GF) mice by co-habitation and again confirmed the shifts in the donors and the recipient mice (Figures S3K and S3L). As expected, cold exposure of donor mice led to a marked increase in the insulin sensitivity (Figure 2A). Strikingly, cold microbiota transplanted mice also showed increased sensitivity to insulin (Figure 2B), suggesting that cold microbiota alone is sufficient to transfer part of this phenotype. The increased insulin sensitivity was further investigated using hyperinsulinemic-euglycemic clamp in awake and unrestrained mice. Cold mice showed a marked increase in the glucose infusion rates (GIR) needed to maintain the clamped glucose levels and an increase in the stimulated glucose disappearance (Rd) levels (Figures 2C, 2D, and S4A). To investigate the peripheral glucose uptake, we co-administered 2-[14C]deoxyglucose (2 [ 14C]DG) during the clamp. While no changes were observed in the glucose uptake from interscapular BAT (iBAT), brain, soleus, or quadriceps muscle, there was a large increase in the uptake from inguinal subcutaneous and perigonadal (epididymal in males) visceral depots of the WAT (ingSAT and pgVAT, respectively) (Figure 2E). These observations were further corroborated in glucose-stimulated and basal conditions (Figures 2F and 2G), which in addition showed increased glucose uptake in iBAT. Interestingly, the cold microbiota transferred the fat-specific glucose disposal phenotype to the transplanted mice as measured by 2[14C]DG uptake (Figure 2H) and by positron emission tomography-computed tomography (microPET-CT). Specifically, both ingSAT and pgVAT, but not quadriceps muscle, showed increased [18F]fluorodeoxyglucose ([18F]FDG) uptake in the cold-transplanted mice (Figures 2I–2K) and had decreased ingSAT and pgVAT volumes and weights (Figures 3A–3F and S4B). Hounsfield unit (HU) analysis of the microCT scans revealed that cold microbiota-transplanted mice had higher ingSAT and pgVAT density compared to the controls (Figures 3G and 3H). Together, these data suggest that the cold microbiota contributes to the increased insulin sensitivity observed during cold exposure and leads to decreased total fat coupled with increased fat density. Cold Microbiota Promotes Browning, Energy Expenditure, and Cold Tolerance To investigate whether the higher density and the decreased fat amount (Figures 3A–3H) are originating from the differences in the adipocyte volume, we measured the adipocyte size distribution using high content imaging. Cold-transplanted mice had increased number of small and decreased number of large adipocytes in the ingSAT and pgVAT depots (Figures 3I–3L). The adipose depots excised from the cold-transplanted animals were darker in appearance. All these phenotypic events are characteristic features of mature beige adipocytes. Therefore, we investigated whether cold microbiota could affect the browning of the white fat depots and found that cold-transplanted mice had marked increase in the brown fat-specific markers in the ingSAT, and surprisingly, also in the pgVAT depots (Figures 3M and 3O). The increased browning of ingSAT was consistent with the increased Ucp1-positive cells in the cold-transplanted mice (Figure 3N). There was a tendency bordering significance toward increased brown fat marker expression in the interscapular BAT (iBAT) depots of the cold-transplanted mice, albeit at smaller scale compared to ingSAT and pgVAT (Figure 3P). Together, these data suggest that cold microbiota alone can be sufficient to induce beige/brown fat formation primarily in the ingSAT and pgVAT, and to a smaller magnitude, in the iBAT depots. The increased browning was consistent with the enhanced resting EE (REE) of the cold-transplanted mice (Figure 3Q), suggesting increased energy dissipation. To further investigate its functional relevance, we exposed the cold-transplanted mice to acute cold and monitored the internal body temperature, as well as ventrally or dorsally, indicative of the temperature emitted from ingSAT or iBAT depots. The rectal temperature measurements showed that the RT-transplanted mice had decreased body temperature following 4 hr of cold exposure, but only a mild temperature drop was detected in the cold-transplanted mice (Figures 4A and 4B). Accordingly, the infrared imaging and quantification of the different regions Figure 2. Cold Microbiota Transplantation Increases Insulin Sensitivity and WAT Glucose Uptake (A and B) Intraperitoneal insulin tolerance test (ITT) in RT and 25 days cold-exposed mice (A), or RT- and cold microbiota-transplanted mice (B) relative to initial blood glucose, (n = 8 per group). (C–E) Euglycemic-hyperinsulinemic clamp of awake mice as in (A). Rate of disappearance of 3 H-D-glucose (C). GIR time course during the hyperinsulinemic clamp (D). 2[14C]DG uptake in various tissues (E) (n = 6 + 6). (F) 2[14C]DG tracer uptake in tissues 45 min after IP tracer and glucose (2 g/kg BW) administration in mice as in (A) (n = 6 per group). (G and H) 2[14C]DG uptake in tissues 30 min after administration under basal conditions in anesthetized RT (n = 9) and cold (n = 10) (G); or RT- and coldtransplanted mice (n = 3) (H). (I, J, and L) Positron emission tomography-computer tomography (microPET-CT) measurement of [18F]FDG uptake in ingSAT (I), pgVAT (J), or quadriceps muscle (L) in basal conditions of RT- and cold-transplanted mice as in (B) (n = 6 per group). (K) Transversal [18F]FDG PET-CT images of ingSAT and pgVAT of mice as in (I) and (J). See also Figure S4. 1364 Cell 163, 1360–1374, December 3, 2015 ª2015 Elsevier Inc

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 temperature measurements, representative of the internal body temperature (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 between the groups (Figures 4E and 4H), the maximal ventral heat differences remained constant also after 12 hr of cold exposure (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