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Figure 3 Abx-Mediated Depletion of the Intestinal Microbiota Reduces Body Fat and Caloric Uptake from the Diet Show full caption (A) Weight of untreated (n = 10), AE-treated (n = 8), and VA-treated (n = 10) mice after BMT, relative to day 0. (B) Weight of periovarian fat in untreated (n = 15), AE-treated (n = 14), and VA-treated (n = 10) mice 28 days after BMT. (C) Representative photographs of intestines and cecum in untreated, AE-treated, and VA-treated mice 28 days after BMT. (D) Quantification of weight of intestines and cecum including contents (from duodenum to rectum) of untreated (n = 5), AE-treated (n = 5), and VA-treated (n = 5) mice 28 days after BMT. (E–J) Energy intake during 24 hr (E), energy excreted as feces during 24 hr (F), fraction of energy intake absorbed (absorbed energy [ingested calories after subtraction of excreted calories] divided by ingested calories) (G), energy expenditure rate and total energy expenditure during 24 hr (H), cumulative and total distance traveled (I), and respiratory exchange ratio (RER) for untreated (n = 10) and AE-treated (n = 10) mice 13 days after BMT (J). Light and dark cycle indicated by white and gray background, respectively. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; n.s., not significant. Data are presented as mean ± SEM. See also Results except (C) and (D) represent at least two independent experiments.p < 0.05,p < 0.01,p < 0.001; n.s., not significant. Data are presented as mean ± SEM. See also Figure S4

In addition to showing impaired hematopoietic recovery, we noticed that the mice treated with abx lost around 20% of their body weight during the first weeks after transplant while untreated mice lost only about 3% ( Figure 3 A). Although all treatment groups regained their baseline weight at the end of the 28-day experiment, abx-treated mice had less visceral adipose tissue (VAT) compared to untreated mice as assessed by the amount of periovarian fat ( Figure 3 B), which is one of the largest VAT depots in female mice (). Furthermore, the reduced weight of periovarian fat in AE-treated mice was abolished in mice harboring the abx-resistant flora ( Figure S4 A), indicating that the reduction in VAT deposits was mediated by depletion of the flora. As previously observed in abx-treated () and germ-free (GF) mice (), the weights of the cecum and intestines (small and large bowel) in abx-treated mice were nearly double that of untreated mice ( Figures 3 C and 3D). The increase in cecal and intestinal weight accounted for about 10% of the total body weight and likely contributed to the weight re-gain of abx-treated mice, despite the loss of VAT. The cecal content also showed a darkened color in abx-treated mice compared to untreated mice, which was not the result of hematochezia as tests for occult hemoglobin in cecum and large intestine were negative (data not shown). It is known that VAT is preferentially lost when dietary restriction is implemented (), and since the intestinal flora supports the host by breaking down complex dietary fibers that are otherwise not digestible (), we hypothesized that depletion of the flora decreased the amount of energy harvested from carbohydrates in the diet. In support of this, AE-treated mice had higher fecal output ( Figure S4 B) despite consuming a comparable mass of food ( Figure S4 C) compared to mice with an intact flora. To further determine the metabolic phenotype, AE-treated and untreated mice were singly housed in metabolic cages. The fraction of energy absorbed from the diet was 8% lower in AE-treated mice based on energy intake and energy excreted in feces ( Figures 3 E–3G). Abx-treated mice also had a lower energy expenditure compared to untreated mice ( Figures 3 H and S4 D), which was partly due to less movement ( Figure 3 I). Reduced nutritional absorption could be a result of disrupted epithelial function in the intestine. However, intestinal epithelial integrity, as measured by leakage of FITC-dextran into the systemic circulation, was not altered in AE-treated mice compared to untreated mice ( Figure S4 E). Furthermore, pathology scores for intestinal apoptosis, inflammation, and erosion were not increased in AE-treated mice compared to untreated mice ( Figure S4 F). Metabolic profiling also showed that AE-treated transplanted mice had a higher dependence on fat metabolism compared with transplanted mice with an intact flora ( Figure 3 J). This was inferred from the respiratory exchange ratio (RER), which varies depending on what fuel is metabolized, with a value of 1 expected when purely carbohydrates are metabolized and a value of 0.7 expected when purely fats are metabolized (). The lower RER in AE-treated mice is thus consistent with the notion that abx-treated mice compensated for reduced caloric uptake from carbohydrates by utilizing endogenous fat, resulting in reduced VAT.