《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

Cell Cold-Exposed and Cold Microbiota-Transplanted Mice observed a marked increase in the lengths and weights of the Have Increased Intestinal Absorptive Surface small intestine in the cold-exposed mice as early as 9 days after Next, we monitored short-chain fatty acids( SCFAs), volatile com- initiation of cold exposure(Figures S5A-S5C), persisting up to pounds, and organic acids associated with gut flora activity using 30 days of cold(Figures 5G and S5D). Microbiota depletion mass spectrometry (Tables S1 and S2). In lipid cecal extracts, also led to increased intestinal length and weight, however, the butyrate, the primary energy source in colon and the most abun- changes in this case were more pronounced after 30 days of dant SCFA(Ferreyra et al., 2014), was markedly decreased in Abx treatment and were consistent with the increased intestinal ntibiotic-treated mice and, accordingly, increased upon gut length in the GF mice(Figures 5G-5l and S5D). Cold exposure of flora transplantation(Table S2). Similarly, succinate, a frequent the Abx-treated and GF mice led to dramatic increases of their eria Wichmann et al., 2013), was decreased in the absence of over 150% compared to the RT controls, demonstrating remark gut flora. We observed increase of propionate, butyrate, lactate, able plasticity of the small intestinal absorptive tissue in and succinate in cold-transplanted mice (Table S2). These results response to the increased energy demand( Figures 5G-5l could indicate increased fermentation activity of cold over RT mi- S5D, and S5E). The rest of the tissues, such as the colon, stom- crobiota, associated with increased energy harvest. ach, iBAT, or quadriceps muscle did not show obvious morpho- 6. As mentioned, during long-term cold exposure and after the logical changes(Figures S5D-S5G), except the decreased WAT al weight loss, the Bw stabilizes despite the constantly levels described above. The increased intestinal lengt increased EE rates and heat production, suggesting increased present 3 weeks after the end of the cold exposure in the donor nutrient absorption from the relatively stable food intake. Oral mice that were used to transplant the GF mice(Figure 5J). Strik glucose tolerance tests (OGTn in cold-exposed mice with or ingly, cold microbiota-transplanted mice also showed a marked without microbiota depletion showed an elevated glucose increase in the intestinal lengths and weights compared to the ak following glucose gavage compared to Rt controls(Fig- RT-transplanted controls, suggesting that the microbiota es 5A and S4C-S4G)after 15 min, but also faster clearance, contributed to this phenotype(Figures 5L, 5M, and S5K). To consistent with the increased insulin sensitivity(Figure S4H). further investigate the changes in the intestinal morphology terestingly, no differences were observed in the initial glucose we measured the intestinal perimeter and villus length and found peak when glucose was administered intraperitoneally (Fig- that both were increased in the cold mice and were further ure S4D). This suggests that orally administered glucose is rapidly enlarged in the cold-exposed Abx-treated mice(Figures 5N- taken up in cold-exposed mice and in microbiota-depleted mice. 5P). This characteristic, however, was not transferred by the The rapid glucose uptake was observed also in the cold-trans- microbiota transplantation, consistent with the proportional in- planted mice, which showed increased glucose peaks 7.5 and crease in the intestinal lengths and weights in the transplanted 15 min after glucose gavage(Figure 5B)and no changes in the mice, compared to the donors in which the ratio we eight versus insulin release compared to the RT-transplanted(Figure S4J). length increased by 1.8-fold. This was consistent with increased triglyceride uptake and To investigate the intestinal morphological changes, we quan non-esterified fatty acid levels in the cold-transplanted mic the relative contribution erent cell types (Figures 5C and 5D), suggesting increased total energy harvest composing it-stem cells and Paneth cells in the bottom of the levels following oral gavage in the cold-transplanted mice. To crypt and enterocytes, goblet cells, and enteroendocrine cells onfirm that cold exposure leads to increase in the calorie up- along the villi. In our models, the number of the enteroendocrine take, we measured the fecal caloric content using bomb calorim- cells was increased in the cold-exposed and cold-exposed etry and calculated the total energy uptake Cold-exposed mice Abx-treated mice, but also in the cold-transplanted mice,pro- showed increased caloric uptake, and this was phenocopied in portional to the overall increase in the average cell number(Fig the cold-transplanted mice(Figures 5E and 5F). These data sug- ures S6A-S6E. There was an antibiotics-dependent effect in gested increased intestinal absorptive capacity following cold the number of goblet cells, which were increased upon xposure, which is transferable by the microbiota transplanta- microbiota depletion, but no changes were observed in the tion. We therefore looked at the intestine in more detail and cold-transplanted mice(Figures S6F-S6H, S6L, and S6M (C and D) Plasma triglycerides(C) and free fatty acids(D)during oral fat tolerance test in RT-or cold microbiota-transplanted mice as in( B)(n= 6 per group). F)Total caloric uptake during 24 hr of cold-or RT-exposed(E, or RT-transplanted (F)mice(n= 8 per group). Mice were kept two per cage. Each cage wa (G and H) Small intestine and colon lengths of cold-exposed mice with or without Abx treatment (n= 8 per group)(g)or cold-exposed and RT-kept GF mice(n= 6 er group)(H) Representative images of cecum, small intestine, and colon of mice as in(EH). W Small intestine and colon lengths of 30 days cold-exposed or RT-kept donor mice used for microbiota-transplantation, 23 days after start of cohabitation at RT ths of RT-or cold microbiota-transplanted mice as in(B)(n=8 per group), 21 days after transplantation, and GF controls(n= 4). (M) Representative images of cecum, small intes colon of mice as in( (N-P) H&E staining of duodenum of cold-exposed mice with or without Abx treatment () and morphometric quantifications of duodenal perimeter (o)and villi length(P)(n=8 per group in triplicates, data show mean SEM). ee also Figures s5 and S6. 368 Cell 163. 1360-1374 December 3. 2015 2015 Elsevier Inc

Cold-Exposed and Cold Microbiota-Transplanted Mice Have Increased Intestinal Absorptive Surface Next, we monitored short-chain fatty acids (SCFAs), volatile com￾pounds, and organic acids associated with gut flora activity using mass spectrometry (Tables S1 and S2). In lipid cecal extracts, butyrate, the primary energy source in colon and the most abun￾dant SCFA (Ferreyra et al., 2014), was markedly decreased in antibiotic-treated mice and, accordingly, increased upon gut flora transplantation (Table S2). Similarly, succinate, a frequent product of primary fermenters that is utilized by butyrogenic bac￾teria (Wichmann et al., 2013), was decreased in the absence of gut flora. We observed increase of propionate, butyrate, lactate, and succinate in cold-transplanted mice (Table S2). These results could indicate increased fermentation activity of cold over RT mi￾crobiota, associated with increased energy harvest. As mentioned, during long-term cold exposure and after the initial weight loss, the BW stabilizes despite the constantly increased EE rates and heat production, suggesting increased nutrient absorption from the relatively stable food intake. Oral glucose tolerance tests (OGTT) in cold-exposed mice with or without microbiota depletion showed an elevated glucose peak following glucose gavage compared to RT controls (Fig￾ures 5A and S4C–S4G) after 15 min, but also faster clearance, consistent with the increased insulin sensitivity (Figure S4H). Interestingly, no differences were observed in the initial glucose peak when glucose was administered intraperitoneally (Fig￾ure S4I). This suggests that orally administered glucose is rapidly taken up in cold-exposed mice and in microbiota-depleted mice. The rapid glucose uptake was observed also in the cold-trans￾planted mice, which showed increased glucose peaks 7.5 and 15 min after glucose gavage (Figure 5B) and no changes in the insulin release compared to the RT-transplanted (Figure S4J). This was consistent with increased triglyceride uptake and non-esterified fatty acid levels in the cold-transplanted mice (Figures 5C and 5D), suggesting increased total energy harvest levels following oral gavage in the cold-transplanted mice. To confirm that cold exposure leads to increase in the calorie up￾take, we measured the fecal caloric content using bomb calorim￾etry and calculated the total energy uptake. Cold-exposed mice showed increased caloric uptake, and this was phenocopied in the cold-transplanted mice (Figures 5E and 5F). These data sug￾gested increased intestinal absorptive capacity following cold exposure, which is transferable by the microbiota transplanta￾tion. We therefore looked at the intestine in more detail and observed a marked increase in the lengths and weights of the small intestine in the cold-exposed mice as early as 9 days after initiation of cold exposure (Figures S5A–S5C), persisting up to 30 days of cold (Figures 5G and S5D). Microbiota depletion also led to increased intestinal length and weight, however, the changes in this case were more pronounced after 30 days of Abx treatment and were consistent with the increased intestinal length in the GF mice (Figures 5G–5I and S5D). Cold exposure of the Abx-treated and GF mice led to dramatic increases of their intestinal lengths amounting to almost 35% and weights of over 150% compared to the RT controls, demonstrating remark￾able plasticity of the small intestinal absorptive tissue in response to the increased energy demand (Figures 5G–5I, S5D, and S5E). The rest of the tissues, such as the colon, stom￾ach, iBAT, or quadriceps muscle did not show obvious morpho￾logical changes (Figures S5D–S5G), except the decreased WAT levels described above. The increased intestinal length was still present 3 weeks after the end of the cold exposure in the donor mice that were used to transplant the GF mice (Figure 5J). Strik￾ingly, cold microbiota-transplanted mice also showed a marked increase in the intestinal lengths and weights compared to the RT-transplanted controls, suggesting that the microbiota contributed to this phenotype (Figures 5L, 5M, and S5K). To further investigate the changes in the intestinal morphology, we measured the intestinal perimeter and villus length and found that both were increased in the cold mice and were further enlarged in the cold-exposed Abx-treated mice (Figures 5N– 5P). This characteristic, however, was not transferred by the microbiota transplantation, consistent with the proportional in￾crease in the intestinal lengths and weights in the transplanted mice, compared to the donors in which the ratio weight versus length increased by 1.8-fold. To investigate the intestinal morphological changes, we quan￾tified the relative contribution of the different cell types composing it—stem cells and Paneth cells in the bottom of the crypt and enterocytes, goblet cells, and enteroendocrine cells along the villi. In our models, the number of the enteroendocrine cells was increased in the cold-exposed and cold-exposed Abx-treated mice, but also in the cold-transplanted mice, pro￾portional to the overall increase in the average cell number (Fig￾ures S6A–S6E). There was an antibiotics-dependent effect in the number of goblet cells, which were increased upon microbiota depletion, but no changes were observed in the cold-transplanted mice (Figures S6F–S6H, S6L, and S6M). (C and D) Plasma triglycerides (C) and free fatty acids (D) during oral fat tolerance test in RT- or cold microbiota-transplanted mice as in (B) (n = 6 per group). (E and F) Total caloric uptake during 24 hr of cold- or RT-exposed (E), or RT-transplanted (F) mice (n = 8 per group). Mice were kept two per cage. Each cage was considered as one pooled sample (n = 4). Data in (E) and (F) show mean ± SEM. (G and H) Small intestine and colon lengths of cold-exposed mice with or without Abx treatment (n = 8 per group) (G) or cold-exposed and RT-kept GF mice (n = 6 per group) (H). (I) Representative images of cecum, small intestine, and colon of mice as in (E)–(H). (J) Small intestine and colon lengths of 30 days cold-exposed or RT-kept donor mice used for microbiota-transplantation, 23 days after start of cohabitation at RT (n = 6 per group). (K) Stomach, small intestine, cecum, and colon weights of donor mice as in (J). (L) Small intestine and colon lengths of RT- or cold microbiota-transplanted mice as in (B) (n = 8 per group), 21 days after transplantation, and GF controls (n = 4). (M) Representative images of cecum, small intestine, and colon of mice as in (L). (N–P) H&E staining of duodenum of cold-exposed mice with or without Abx treatment (N) and morphometric quantifications of duodenal perimeter (O) and villi length (P) (n = 8 per group in triplicates, data show mean ± SEM). See also Figures S5 and S6. 1368 Cell 163, 1360–1374, December 3, 2015 ª2015 Elsevier Inc

Cell B Cold e ZaRT+Abx/cold+Abx E RT transplanted Cold transplanted old transplanted m Cold Vs RT RT+Abx Apoptotic」 心φ小小A國少yy分 Cold+Abx Anti-apoptotic Tissue remodelling Glucose upte DAPI N RT transplanted Cold transplanted Cleaved caspe- RT transplanted Cold transplanted 最 RT RT+Abx Cleaved casp 28 kDa Legend on next page) ce163,1360-1374, December3,2015@2015 Elsevier Inc.1369

RT RT+Abx Cold Cold+Abx 500 1000 1500 2000 2500 3000 3500 4000 -10 0 10 20 30 40 50 Microvilli length (nm) Microvilli distribution (%) 1000 1500 2000 2500 [***] Average microvilli length (nm) RT RT+Abx Cold Cold+Abx RT transplanted 0 300 600 900 1200 1500 1800 2100 2400 2700 3000 3300 3600 3900 0 10 20 30 40 50 Microvilli length (nm) Microvilli distribution (%) *** ** *** * B C D E GF Cold transplanted RT transplanted Average microvilli length (nm) * 1000 1500 2000 2500 F Cold transplanted GF RT Cold Cold + Abx RT+ Abx 0 2 4 6 8 10 12 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. -log(p-value) Cold vs RT RT+Abx vs RT Cold+Abx vs RT A H I Germ-free RT transplanted Cold transplanted * * * 0 1 2 3 Glucose uptake * * * 0 1 2 3 4 Anti-apoptotic *** **** * 0.0 0.5 1.0 1.5 2.0 2.5 Tissue remodelling **** ** ** ** * ** *** * Cleaved casp3 Pcna RT transplanted Cold transplanted 17 kDa 28 kDa 0.0 0.5 1.0 1.5 2.0 2.5 * 0.0 0.5 1.0 1.5 2.0 2.5 2.5 4.5 6.5 Normalized RNASeq counts (RT=1) RT Cold RT+Abx Cold+Abx * * * ** *** * * * * *** * *** *** * *** *** ** *** *** **** * *** *** * *** ***** *** *** ***** ***** * *** *** * **** *** Pro-apoptotic Anti￾apoptotic Remodelling, oncogenic Glucose uptake * *** *** *** *** **** Relative mRNA expression (RT=1) * * * γ-tubulin β-actin 48 kDa 40 kDa RT transplanted Cold transplanted 0 1 2 3 4 * Cleaved casp3 / Pcna RT transplanted Cold transplanted β-actin / γ-tubulin M N O G * 5 RT RT+Abx Cold 17 kDa 28 kDa Cleaved casp3 Pcna RT Cold RT+Abx 0.0 0.5 1.0 1.5 Cleaved casp3 / Pcna [*] RT RT+Abx Cold Cold+Abx log fold change (2) -1.0 -0.5 0.0 0.5 1.0 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 PCA log fold change (1) J K DAPI EdUTP RT RT+Abx Cold Cold+Abx RT transplanted Cold transplanted EdUTP DAPI ◄ ▼ ▼ ▼ L RT transplanted Cold transplanted ▼ Click-IT TUNEL OsO4 / Toluidine blue Slc2a2 Slc5a1 Slc2a1 Slc2a5 Il15 Bcl2l1 Mcl1 Casp1 Casp3 Casp4 Casp8 Casp12 Apaf1 Gzmb Tnfsf10 Fas Fasl Bid Thbs1 Vegfa Mmp7 Frat1 Sgk1 Slc5a1 Slc2a2 Il15 Bcl21 Mcl1 Bcl3 Wnt2b Thbs1 Vegfa Actb Sgk1 (legend on next page) Cell 163, 1360–1374, December 3, 2015 ª2015 Elsevier Inc. 1369

Cell stme is a highly specific and robust marker for Lgr5 positive 2009), were strongly suppressed in all cells. Quantification of the Olfm4+ cells showed increment mice(Figure S6N) Indeed, apoptosis and anti-apoptotic inter only in the intestine of cold-exposed Abx-treated mice, consis- leukin-15 signaling(Obermeier et al., 2006) were among the tent with their most pronouncedly enlarged intestine(Figures top-regulated pathways in the mice with increased intestinal sur- S61-S6K). These data suggest that cold exposure leads to a face( Figures 6H, 6l, and S6N). Using the TUNEL assay, we number of changes in the intestinal composition, which in the observed that compared to the RTmice, the apoptosis was mark case of the enteroendocrine cells, are in part transferable by edly reduced in the villi of all other groups(Figure 6J). This pheno- cold microbiota transplantation. type was transferred in the cold-transplanted animals, which Microvilli form the brush border on the apical epithelial surface retained the anti-apoptotic phenotype of the GF and Abx-treated of the small intestine, and a single enterocyte can have as many mice (Figures 6K-60). Conversely, RT-transplanted as 1, 000 microvilli, each one formed by cross-linked actin bun- acquired increased apoptosis, exhibited reduction of the anti- dles. They increase the surface area of the absorptive cell apoptotic //15, Bcl2/1(coding isoform Bcl2 -XL), and Mcl1 ex- 125-fold. Using quantitative electron microscopy(EM), we pression(Pelletier et al., 2002) and showed increased caspase found that the microvilli length is substantially increased in the 3 activation(Figures 6M-6O). Concomitantly, the mice with cold-exposed, as well as in microbiota-depleted mice(Figures increased intestinal surface had augmented vascularization and 6A-6C), thus further largely increasing the intestinal surface tissue remodeling gene expression and showed marked increase rea. Strikingly, these differences were also transferred in the in the main apical (Sglt1, gene Slc5a1)and basolateral(Glut2, cold microbiota-transplanted mice, which showed increased Slc2a2) glucose transporters(Figures 6l and 6M). Together, these microvilli lengths (Figures 6D-6F). Together, these results data suggest a mechanistic explanation of the increased intesti- demonstrate that during increased energy demand, specifically nal surface area and glucose permeability, which can be trans- cold exposure, there is a dramatic increase in the intestinal ferred by the cold microbiota transplantation absorptive surface area due to the increased intestinal, villi and microvilli lengths, and cold microbiota transfer alone can Cold Microbiota Increases Intestinal Absorption in an be sufficient to induce these changes. ohila-Sensitive Manner To finally demonstrate that the increased intestinal surface corre- Reduced Apoptosis Underlies the Increased Intestinal sponds to enhanced absorptive capacity of the intestine, we did ex vivo experiments in isolated segments from the middle to To uncover the mechanisms of the microbiota-epithelium cross- proximal jejunum of the microbiota-transplanted mice Mucosal talk responsible for the observed gut phenotype, we deep to serosal D[1-c glucose(D[C]G)apparent diffusion coeffi- equenced the transcriptome from proximaL jejunum of RT, cient was higher in cold-transplanted mice(Figure 7A), suggest RT+Abx, Cold, and Cold+Abx mice. The expression profiles ing increased intestinal glucose absorption. This was consistent markedly differed between the groups( Figure 6G), and unbiased with the increased D[C]G present in intestinal tissue after 1 hr of pathway enrichment analysis revealed changes in pathways transport and lower residual D[CG levels in the lumen(Figures involved in cytoskeletal remodeling, tissue growth, WNT 7B and 7C). Cold microbiota mice also had prolonged intestinal signaling, apoptosis, and immune response common for mice transit time, proportional to the increase in the intestinal length with increased intestinal surface(Cold, RT+Abx, and Cold+Abx), of the corresponding animals(Figure 7D). Since the increased i when compared to RT mice(Figures 6H, 61, and S6N) Anti-micro- testinal surface area was also present in the microbiota-deplete bial response and TNF signaling, which promote apoptosis and mice, we assumed that absence of certain bacterial strains cell shedding, and are activated by bacteria through NF-KB and rather than increased abundance, could be responsible for the TLR pathways(Hausmann, 2010: Spehlmann and Eckmann, observed intestinal phenotype following the cold microbiota Figure 6. Presence and Composition of Microbiota Determine Length of Microvilli on Brush Border of Small Intestine (A and D)Electron micrographs of jejunal enterocyte microvilli of cold-exposed mice with or without Abx treatment (A), or GF, RT, and cold microbiota- ansplanted mice 19 days after transplantation(D) Scale bars, 2 and E Morphometric quantification of microvilli length distribution in( B)as in (A) and(E as in(D) (C and F) Average microvilli lengths of mice as in (A)and(D), respectively. (G) Principal component analysis(PCoA) of gene expression data in proximal jejunum of mice as in(A) (H) Top commonly regulated pathways(Meta Core pathway enrichment) in RT, RT+Abx, Cold, and Cold+ Abx differential gene expression and survival; 6: GM-CSF signaling: 7: TGF, WNT, and cytoskeletal remodeling: 8: signal transduction, AKT signaling: 9: cell adhesion, chemokines and adhesion; and 10: IL-15 signaling via JAK-STAT cascade. and M) Relative m RNA expression in proximal jejunum of mice as in (A), or GF, or RT-and cold microbiota-transplanted GF (M)quantified by RNa seq(or real- time PCR (M)and normalized to the average expression of the housekeeping Rplpo (36b4)and Rps16(GF are n=4; rest are n=8 per group). Significance in (was alculated using general linear model with negative binomial distribution. d k Terminal deoxynucleotidyl transferase(duTP)nick end labeling (TUNEL assay for apoptotic cells double labeled with DAPl of proximal jejunum paraffin ections of mice as in (A)or(D) Scale bars, 200 um. ( Semi-fine um thick EM sections of proximal jejunum stained with toluidine blue displaying apoptotic cells in dark blue(marked with arrowheads)of mice as in (D) Round, goblet cells. Scale bar, 20 um. (N and o)Western blotting of lysates from proximal jejunum of mice as in(D)and (A) and respective quantifications (o)normalized to loading controls See also Figure S6. 1370 Cell 163. 1360-1374 December 3. 2015 2015 Elsevier Inc

Olfm4 is a highly specific and robust marker for Lgr5 positive stem cells. Quantification of the Olfm4+ cells showed increment only in the intestine of cold-exposed Abx-treated mice, consis￾tent with their most pronouncedly enlarged intestine (Figures S6I–S6K). These data suggest that cold exposure leads to a number of changes in the intestinal composition, which in the case of the enteroendocrine cells, are in part transferable by cold microbiota transplantation. Microvilli form the brush border on the apical epithelial surface of the small intestine, and a single enterocyte can have as many as 1,000 microvilli, each one formed by cross-linked actin bun￾dles. They increase the surface area of the absorptive cell !25-fold. Using quantitative electron microscopy (EM), we found that the microvilli length is substantially increased in the cold-exposed, as well as in microbiota-depleted mice (Figures 6A–6C), thus further largely increasing the intestinal surface area. Strikingly, these differences were also transferred in the cold microbiota-transplanted mice, which showed increased microvilli lengths (Figures 6D–6F). Together, these results demonstrate that during increased energy demand, specifically cold exposure, there is a dramatic increase in the intestinal absorptive surface area due to the increased intestinal, villi, and microvilli lengths, and cold microbiota transfer alone can be sufficient to induce these changes. Reduced Apoptosis Underlies the Increased Intestinal Surface To uncover the mechanisms of the microbiota-epithelium cross￾talk responsible for the observed gut phenotype, we deep sequenced the transcriptome from proximal jejunum of RT, RT+Abx, Cold, and Cold+Abx mice. The expression profiles markedly differed between the groups (Figure 6G), and unbiased pathway enrichment analysis revealed changes in pathways involved in cytoskeletal remodeling, tissue growth, WNT signaling, apoptosis, and immune response common for mice with increased intestinal surface (Cold, RT+Abx, and Cold+Abx), when compared to RT mice (Figures 6H, 6I, and S6N). Anti-micro￾bial response and TNF signaling, which promote apoptosis and cell shedding, and are activated by bacteria through NF-kB and TLR pathways (Hausmann, 2010; Spehlmann and Eckmann, 2009), were strongly suppressed in all microbiota-depleted mice (Figure S6N). Indeed, apoptosis and anti-apoptotic inter￾leukin-15 signaling (Obermeier et al., 2006) were among the top-regulated pathways in the mice with increased intestinal sur￾face (Figures 6H, 6I, and S6N). Using the TUNEL assay, we observed that compared to the RT mice, the apoptosis was mark￾edly reduced in the villi of all other groups (Figure 6J). This pheno￾type was transferred in the cold-transplanted animals, which retained the anti-apoptotic phenotype of the GF and Abx-treated mice (Figures 6K–6O). Conversely, RT-transplanted mice acquired increased apoptosis, exhibited reduction of the anti￾apoptotic Il15, Bcl2l1 (coding isoform Bcl2-XL), and Mcl1 ex￾pression (Pelletier et al., 2002) and showed increased caspase 3 activation (Figures 6M–6O). Concomitantly, the mice with increased intestinal surface had augmented vascularization and tissue remodeling gene expression and showed marked increase in the main apical (Sglt1, gene Slc5a1) and basolateral (Glut2, Slc2a2) glucose transporters (Figures 6I and 6M). Together, these data suggest a mechanistic explanation of the increased intesti￾nal surface area and glucose permeability, which can be trans￾ferred by the cold microbiota transplantation. Cold Microbiota Increases Intestinal Absorption in an Akkermansia-muciniphila-Sensitive Manner To finally demonstrate that the increased intestinal surface corre￾sponds to enhanced absorptive capacity of the intestine, we did ex vivo experiments in isolated segments from the middle to proximal jejunum of the microbiota-transplanted mice. Mucosal to serosal D[1-14C] glucose (D[14C]G) apparent diffusion coeffi- cient was higher in cold-transplanted mice (Figure 7A), suggest￾ing increased intestinal glucose absorption. This was consistent with the increased D[14C]G present in intestinal tissue after 1 hr of transport and lower residual D[14C]G levels in the lumen (Figures 7B and 7C). Cold microbiota mice also had prolonged intestinal transit time, proportional to the increase in the intestinal length of the corresponding animals (Figure 7D). Since the increased in￾testinal surface area was also present in the microbiota-depleted mice, we assumed that absence of certain bacterial strains, rather than increased abundance, could be responsible for the observed intestinal phenotype following the cold microbiota Figure 6. Presence and Composition of Microbiota Determine Length of Microvilli on Brush Border of Small Intestine (A and D) Electron micrographs of jejunal enterocyte microvilli of cold-exposed mice with or without Abx treatment (A), or GF, RT-, and cold microbiota￾transplanted mice 19 days after transplantation (D). Scale bars, 2 mm. (B and E) Morphometric quantification of microvilli length distribution in (B) as in (A) and (E) as in (D). (C and F) Average microvilli lengths of mice as in (A) and (D), respectively. (G) Principal component analysis (PCoA) of gene expression data in proximal jejunum of mice as in (A). (H) Top commonly regulated pathways (MetaCore pathway enrichment) in RT, RT+Abx, Cold, and Cold+Abx differential gene expression comparisons. Legend: 1: immune response, TNF-R2 signaling; 2: main growth factor signaling cascades; 3: IGF family signaling in colorectal cancer; 4: c-Kit ligand signaling during hemopoiesis; 5: apoptosis and survival; 6: GM-CSF signaling; 7: TGF, WNT, and cytoskeletal remodeling; 8: signal transduction, AKT signaling; 9: cell adhesion, chemokines and adhesion; and 10: IL-15 signaling via JAK-STAT cascade. (I and M) Relative mRNA expression in proximal jejunum of mice as in (A), or GF, or RT- and cold microbiota-transplanted GF (M) quantified by RNA seq (I) or real￾time PCR (M) and normalized to the average expression of the housekeeping Rplp0 (36b4) and Rps16 (GF are n = 4; rest are n = 8 per group). Significance in (I) was calculated using general linear model with negative binomial distribution. (J and K) Terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) assay for apoptotic cells double labeled with DAPI of proximal jejunum paraffin sections of mice as in (A) or (D). Scale bars, 200 mm. (L) Semi-fine 1-mm thick EM sections of proximal jejunum stained with toluidine blue displaying apoptotic cells in dark blue (marked with arrowheads) of mice as in (D). Round, goblet cells. Scale bar, 20 mm. (N and O) Western blotting of lysates from proximal jejunum of mice as in (D) and (A) and respective quantifications (O) normalized to loading controls. See also Figure S6. 1370 Cell 163, 1360–1374, December 3, 2015 ª2015 Elsevier Inc

Cell Cold transplanted 一03 0 Cold tra h!_■ G Days of cold exposure H -- Cold ◆cold+ A. muciniphila 3 求40、◆cod+ A.muciniphila K 1500200025 Microvilli length (um) Click-IT TUNE 5201◆cAmm曲 Time(min) Oso /Toluidine blue P 5n日,Ame Cold transplanted Legend on next page) ce163,1360-1374, December3,2015@2015 Elsevier Inc.1371

Intestinal length (cm) 5+7.5 weeks 0 5 10 35 36 37 38 39 RT transplanted Cold transplanted Cold transplanted +A.muciniphila ** * ns F 0 1 2 3 Cold transplanted OGTT (normalized) Cold+A.muciniphila transplanted * Time (min) 0 5 10 15 * 0.0 0.1 0.2 0.3 0.4 Tissue glucose after 1h transport (pM Gluc/mg of wet tissue/cm) * ns 0 10 20 30 40 50 60 70 0 1 2 3 Time (min) Mucosal to serosal glucose permeability (pM Glucose/mg wet tissue/cm) Cold transplanted RT transplanted Cold transplanted +A.muciniphila * * 0 100 200 300 400 Transit time (min) * A BC D E 13 6 -1 0 1 2 3 Days of cold exposure ∆ weight (g) compared to day 1 Cold Cold+A.muciniphila ** * Cold transplanted RT transplanted Cold transplanted +A.muciniphila Length (cm) 0 20 40 60 0.0 0.5 1.0 1.5 2.0 Time (min) Mucosal to serosal glucose permeability (pM glucose/mg wet tissue/cm) * Cold Cold+A.muciniphila 0 30 60 90 15 5 10 15 20 25 Time (min) ** Cold Cold+A.muciniphila Lu men glucose after 1h transport (%) Il15 Bcl2l1 Mcl1 Thbs1 Actb Wnt2b Slc5a1 Slc2a2 0.0 0.5 1.0 1.5 Relative mRNA expresssion Cold * ** *** Cold+A.muciniphila * * ** ** Il15 Slc5a1 Slc2a2 0.0 0.5 1.0 1.5 Cold transplanted Cold transplanted +A.muciniphila * ** * anti-apoptotic remodelling glucose uptake G L M K H P Q EdUTP N Cold DAPI Cold+A.muciniphila Click-IT TUNEL ▼ ▼ ◄◄ ◄ ◄ O Cold Cold+A.muciniphila OsO4 / Toluidine blue Small intestine Colon 0 7.5 15 30 60 90 120 0 500 1000 1500 2000 2500 3000 3500 0 10 20 30 40 Microvilli length (µm) Microvilli distribution (%) * Cold Cold+A.muciniphila I J EM Cold Cold+A.muciniphila OGTT - Glycemia (mM) Small intestine Colon 6 8 10 33 35 37 39 * Cold Cold+A.muciniphila Cold Cold +A.muciniphila 0 5 10 15 20 * Lumen glucose after 1h transport (%) * (legend on next page) Cell 163, 1360–1374, December 3, 2015 ª2015 Elsevier Inc. 1371

Cell muciniphila) is a S7Q-S7T) A. muciniphila re-colonization during the cold expo- Gram-negative bacterium that commonly constitutes 3%-5% sure decreased the oGTT peak and prevented the cold of the gut microbial community. A. muciniphila within the mucus induced increase in the intestinal absorptive capacity(Figures layer is implicated in the control of host mucus turnover(Belzer 7K-7M and S7U). Accordingly, re-colonizing A. muciniphila re- and de Vos, 2012), which improves gut barrier function and is verted the cold-induced decrease in the apoptosis levels ar linked to obesity(Everard et aL., 2013). Since A. muciniphila is reduced the expression of the key tissue remodeling, anti- the most abundant species of the Verrucomicrobia, the most apoptotic, and glucose uptake genes during cold(Figures negatively affected phylum in response to cold exposure, we 7N-7Q). Combined, these results underscore that the cold investigated whether this strain alone could revert part of the exposure-induced decrease of A. muci enables transplanted phenotype. Co-transplantation of A. muciniphila increasing the intestinal absorptive surface by altering several fully prevented the cold microbiota transferable increase of the key regulatory pathways, and co-transfer of this strain together intestinal glucose absorption(Figures 7A-7C) and decreased with the cold microbiota, or during the cold exposure, is suffi- the intestinal transit time( Figure 7D). Moreover, the increased in- cient to prevent the adaptive increase in the intestinal absorp- stinal length caused by cold microbiota transplantation was tive functions that maximize the caloric uptake during cold fully reverted in the cold microbiota +A. muciniphila-transplanted animals(Figures 7E and S7A). These results were consistent with DISCUSSION the OGTT, which showed a limited increase in the glucose peak 15 min after the gavage(Figure 7F)and no differences in the insu- During evolution, mammals developed a number of adaptive re- lin levels between the groups(Figure S7B). Neither differences sponses to energy scarcity. Microbial diversity of the human gut ere observed in the tolerance to insulin and cold nor in the is the result of co-evolution between microbial communities and expression of the beige fat markers(Figures S7C-S7J), together their hosts. We assumed that this co-evolution favored maxi- suggesting that A. muciniphila does not negatively affect the mizing uptake of calories from the consumed food during pe- browning A. muciniphila colonization reverted the changes in the Bacteroi- Indeed, cold exposure led to dramatic changes of the microbiota detes/Firmicutes ratio in the cold-transplanted mice( Figures S7K composition, increasing Firmicutes versus Bacteroidetes ratios nd S7L) Therefore, we investigated the importance of the rest of and almost completely depleting the verrucomicrobia phylum the bacterial consortium by mono-colonizing GF mice with We found that these changes favored enhanced energy extrac A muciniphila and observed no differences in the intestinal length tion during cold. Interestingly, in part this is rendered possible by and duodenum perimeter, while there was a small decrease of the an adaptive mechanism of the host that increases the overall in- microvilli length bordering significance(Figures S7M-S7P), sug- testinal absorptive surface, due to a marked elongation of the to- gesting that A. muciniphila is necessary, but not sufficient to tal intestinal, villi, and microvilli lengths. When transplanted to revert the intestinal lengthening. In contrast, daily gavage of GF-recipient mice, the cold microbiota alone was sufficient to A muciniphila to cold-exposed mice decreased their BW and promote this increased intestinal absorptive surface area by fat mass gain and shortened their intestine and microvilli after lengthening the gut and the epithelial microvilli. Similar changes 7 days of cold exposure. The Bacteroidetes/Firmicutes abun- in the gut morphology were observed in microbiota-depleted dance was not yet affected by the cold exposure at this time in- mice, which is also a condition of negative energy balance, sug- erval, showing that changes in their ratio is not a prerequisite gesting that the increase in the intestinal absorptive surface is a for the intestinal remodeling, and change in A. muciniphila pre- general adaptive mechanism promoting caloric uptake when cedes the remodeling of these major phyla (Figures 7G-7J and food is available Figure 7. Cold Microbiota Increases Intestinal Absorption Due to Absence of A. muciniphila e in jejunal segments excised from RT, cold -, and cold+ A. muciniphila-transplanted mice(n= 5 per group); with mucosal to serosal glucose permeability (A), radioactive glucose tracer in tissue(B, and in the lumen (c )of jejunum segment after 1 hr of transport (D)Intestinal transit time of RT-, cold, and cold+ A. muciniphila-transplanted mice as in(a(n= 6 per group). (E Intestinal length in mice transplanted with RT(n= 9), cold(n= 10), and cold+ A muciniphila (n 6)microbiota 6 weeks after transplantation. (F)OGTT in cold-(n= 10) and cold+ A muciniphia (n= 6-transplanted male mice as in (G)Body weight change compared to day O of 7-week-old mice, exposed to cold for 7 days and gavaged daily with fresh A. muciniphila or vehicle(PBS)(n=5 per estinal length of mice as in(G) microvilli of mice as in(G)Scale bar, 2 um. Morphometric quantification of microvilli length distribution of the EM images as shown in((n=5 per group). OGTT of mice as in(G)6 days after start of treatment. (L and M) Ex vivo measurements of glucose transport in jejunal segments excised from mice as in(G)(n 5 per group) with mucosal to serosal glucose Bability(L, radioactive glucose tracer in tissue after 1 hr of transport(M) TUNEL assay for apoptotic cells double-labeled with daPl of proximal jejunum paraffin sections of mice as in bar, 200 um. (O)Semi-fine sections of proximal jejunum stained with toluidine blue showing apoptotic cells in (marked with arrowheads). Round goblet cells. Scale bar, 20 um. (P and Q)Relative mRNA expression in proximal jejunum of mice as in(G)or (A), (P)or(Q), respectively, quantified by real-time PCR and normalized to the average expression of the house keeping rplpo (36b4)and Rps 16. See also Figure S7. 1372cel163,1360-1374, December3,2015@2015 Elsevier Inc

transplantation. Akkermansia muciniphila (A. muciniphila) is a Gram-negative bacterium that commonly constitutes 3%–5% of the gut microbial community. A. muciniphila within the mucus layer is implicated in the control of host mucus turnover (Belzer and de Vos, 2012), which improves gut barrier function and is linked to obesity (Everard et al., 2013). Since A. muciniphila is the most abundant species of the Verrucomicrobia, the most negatively affected phylum in response to cold exposure, we investigated whether this strain alone could revert part of the transplanted phenotype. Co-transplantation of A. muciniphila fully prevented the cold microbiota transferable increase of the intestinal glucose absorption (Figures 7A–7C) and decreased the intestinal transit time (Figure 7D). Moreover, the increased in￾testinal length caused by cold microbiota transplantation was fully reverted in the cold microbiota +A. muciniphila-transplanted animals (Figures 7E and S7A). These results were consistent with the OGTT, which showed a limited increase in the glucose peak 15 min after the gavage (Figure 7F) and no differences in the insu￾lin levels between the groups (Figure S7B). Neither differences were observed in the tolerance to insulin and cold, nor in the expression of the beige fat markers (Figures S7C–S7J), together suggesting that A. muciniphila does not negatively affect the browning or the sensitivity to insulin. Interestingly, A. muciniphila colonization reverted the changes in the Bacteroi￾detes/Firmicutes ratio in the cold-transplanted mice (Figures S7K and S7L). Therefore, we investigated the importance of the rest of the bacterial consortium by mono-colonizing GF mice with A. muciniphila and observed no differences in the intestinal length and duodenum perimeter, while there was a small decrease of the microvilli length bordering significance (Figures S7M–S7P), sug￾gesting that A. muciniphila is necessary, but not sufficient to revert the intestinal lengthening. In contrast, daily gavage of A. muciniphila to cold-exposed mice decreased their BW and fat mass gain and shortened their intestine and microvilli after 7 days of cold exposure. The Bacteroidetes/Firmicutes abun￾dance was not yet affected by the cold exposure at this time in￾terval, showing that changes in their ratio is not a prerequisite for the intestinal remodeling, and change in A. muciniphila pre￾cedes the remodeling of these major phyla (Figures 7G–7J and S7Q–S7T). A. muciniphila re-colonization during the cold expo￾sure decreased the OGTT peak and prevented the cold￾induced increase in the intestinal absorptive capacity (Figures 7K–7M and S7U). Accordingly, re-colonizing A. muciniphila re￾verted the cold-induced decrease in the apoptosis levels and reduced the expression of the key tissue remodeling, anti￾apoptotic, and glucose uptake genes during cold (Figures 7N–7Q). Combined, these results underscore that the cold exposure-induced decrease of A. muciniphila enables increasing the intestinal absorptive surface by altering several key regulatory pathways, and co-transfer of this strain together with the cold microbiota, or during the cold exposure, is suffi- cient to prevent the adaptive increase in the intestinal absorp￾tive functions that maximize the caloric uptake during cold. DISCUSSION During evolution, mammals developed a number of adaptive re￾sponses to energy scarcity. Microbial diversity of the human gut is the result of co-evolution between microbial communities and their hosts. We assumed that this co-evolution favored maxi￾mizing uptake of calories from the consumed food during pe￾riods of increased energy demand, such as cold exposure. Indeed, cold exposure led to dramatic changes of the microbiota composition, increasing Firmicutes versus Bacteroidetes ratios and almost completely depleting the Verrucomicrobia phylum. We found that these changes favored enhanced energy extrac￾tion during cold. Interestingly, in part this is rendered possible by an adaptive mechanism of the host that increases the overall in￾testinal absorptive surface, due to a marked elongation of the to￾tal intestinal, villi, and microvilli lengths. When transplanted to GF-recipient mice, the cold microbiota alone was sufficient to promote this increased intestinal absorptive surface area by lengthening the gut and the epithelial microvilli. Similar changes in the gut morphology were observed in microbiota-depleted mice, which is also a condition of negative energy balance, sug￾gesting that the increase in the intestinal absorptive surface is a general adaptive mechanism promoting caloric uptake when food is available. Figure 7. Cold Microbiota Increases Intestinal Absorption Due to Absence of A. muciniphila (A–C) Ex vivo measurements of glucose transport in jejunal segments excised from RT-, cold-, and cold+ A. muciniphila-transplanted mice (n = 5 per group); with mucosal to serosal glucose permeability (A), radioactive glucose tracer in tissue (B), and in the lumen (C) of jejunum segment after 1 hr of transport. (D) Intestinal transit time of RT-, cold-, and cold+ A. muciniphila-transplanted mice as in (A) (n = 6 per group). (E) Intestinal length in mice transplanted with RT (n = 9), cold (n = 10), and cold+ A. muciniphila (n = 6) microbiota 6 weeks after transplantation. (F) OGTT in cold- (n = 10) and cold+ A. muciniphila (n = 6)-transplanted male mice as in (A). (G) Body weight change compared to day 0 of 7-week-old mice, exposed to cold for 7 days and gavaged daily with fresh A. muciniphila or vehicle (PBS) (n = 5 per group). (H) Intestinal length of mice as in (G). (I) Electron micrographs of jejunal enterocyte microvilli of mice as in (G) Scale bar, 2 mm. (J) Morphometric quantification of microvilli length distribution of the EM images as shown in (I) (n = 5 per group). (K) OGTT of mice as in (G) 6 days after start of treatment. (L and M) Ex vivo measurements of glucose transport in jejunal segments excised from mice as in (G) (n = 5 per group); with mucosal to serosal glucose permeability (L), radioactive glucose tracer in tissue after 1 hr of transport (M). (N) TUNEL assay for apoptotic cells double-labeled with DAPI of proximal jejunum paraffin sections of mice as in (G). Scale bar, 200 mm. (O) Semi-fine 1-mm thick EM sections of proximal jejunum stained with toluidine blue showing apoptotic cells in dark blue (marked with arrowheads). Round, goblet cells. Scale bar, 20 mm. (P and Q) Relative mRNA expression in proximal jejunum of mice as in (G) or (A), (P) or (Q), respectively, quantified by real-time PCR and normalized to the average expression of the house keeping Rplp0 (36b4) and Rps16. See also Figure S7. 1372 Cell 163, 1360–1374, December 3, 2015 ª2015 Elsevier Inc