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et al. Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity.

Figure 7 Cold Microbiota Increases Intestinal Absorption Due to Absence of A. muciniphila Show full caption (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 μm. (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 μm. (O) Semi-fine 1-μm 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 μm. (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

Figure S7 A. muciniphila Supplementation over Cold Microbiota Reduces Intestinal Length but Does Not Affect the Browning, Related to Figure 7 Show full caption (A) Representative images of cecum, small and large intestine of mice transplanted with cold microbiota with or without A. muciniphila co-transplantation. (B) Insulin levels during OGTT as in Figure 7 F of mice transplanted with cold microbiota with or without A. muciniphila co-transplantation (n = 6 per group, day 23). (C) Insulin tolerance test normalized to initial glycemia of mice as in (A) (n = 6 per group, day 16). (D) Relative mRNA expression in ingSAT tissues 5 weeks after transplantation of mice as in (A) quantified by real-time PCR and normalized to the house keeping beta-2-microglobulin (B2m) Rplp0 (36b4) and Rps16 (n = 6 per group). (E–G) Infrared temperature readings of eye (E), ventral (F) or dorsal (G) temperature after 4 hr cold exposure (day 18). (H–J) Infrared temperature readings of eye (H), ventral (I) or dorsal (J) temperature after 12 hr cold exposure (day 18). (K and L) Relative abundance of Bacteroidetes (K) and Firmicutes (L) phylum in mice transplanted with RT or cold microbiota with or without A. muciniphila 21 days after transplantation, quantified by qPCR and normalized to bacterial universal 16SrRNA (V4-V5 region). In (E-L) n = 10 cold transplanted, n = 6 cold co-transplanted with A. muciniphila. (M–P) (M) Bacterial abundance per g of feces (day 10), (N) intestinal length, (O) duodenum perimeter, and (P) distribution of microvilli lengths from EM images from GF mice monocolonized with A. muciniphila at 7 weeks of age and kept at RT for 12 days (n = 4 per group). (Q and R) (Q) Body weight, and (R) fat pad weight of cold exposed 7 weeks old C57BL6J mice, exposed to cold for 7 days and gavaged daily with fresh A. muciniphila monoculture resuspended in anaerobic PBS or by vehicle (PBS). (S) Food consumption of mice as in (P). Values show the food intake of 2 mice per 24 hr. (T) Relative bacterial abundance of mice as in (Q) 7 days after the start of treatment, quantified by qPCR and normalized to bacteria universal 16SrRNA (V4-V5 region). (U) Area under the curve during the first 30 min of OGTT as in Figure 7 I of mice as in (Q) day 6 days after the start of treatment. In (Q)–(R) n = 5 per group. All values show mean ± sd. Significance was calculated using non-paired two tailed Student’s t test.∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001.

To finally demonstrate that the increased intestinal surface corresponds 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-C] glucose (D[C]G) apparent diffusion coefficient was higher in cold-transplanted mice ( Figure 7 A), suggesting increased intestinal glucose absorption. This was consistent with the increased D[C]G present in intestinal tissue after 1 hr of transport and lower residual D[C]G levels in the lumen ( Figures 7 B and 7C). Cold microbiota mice also had prolonged intestinal transit time, proportional to the increase in the intestinal length of the corresponding animals ( Figure 7 D). Since the increased intestinal 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 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 (), which improves gut barrier function and is linked to obesity (). 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 7 A–7C) and decreased the intestinal transit time ( Figure 7 D). Moreover, the increased intestinal length caused by cold microbiota transplantation was fully reverted in the cold microbiota + A. muciniphila-transplanted animals ( Figures 7 E and S7 A). These results were consistent with the OGTT, which showed a limited increase in the glucose peak 15 min after the gavage ( Figure 7 F) and no differences in the insulin levels between the groups ( Figure S7 B). Neither differences were observed in the tolerance to insulin and cold, nor in the expression of the beige fat markers ( Figures S7 C–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 Bacteroidetes/Firmicutes ratio in the cold-transplanted mice ( Figures S7 K 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 S7 M–S7P), suggesting 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 abundance was not yet affected by the cold exposure at this time interval, showing that changes in their ratio is not a prerequisite for the intestinal remodeling, and change in A. muciniphila precedes the remodeling of these major phyla ( Figures 7 G–7J and S7 Q–S7T). A. muciniphila re-colonization during the cold exposure decreased the OGTT peak and prevented the cold-induced increase in the intestinal absorptive capacity ( Figures 7 K–7M and S7 U). Accordingly, re-colonizing A. muciniphila reverted 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 7 N–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 sufficient to prevent the adaptive increase in the intestinal absorptive functions that maximize the caloric uptake during cold.