Autologous fecal transplantation from a lean state potentiates caloric restriction effects on body weight and adiposity in obese mice

Autologous fecal transplantation (FT-A) emerges as a promising strategy to modulate gut microbiota with minimal side effects since individual´s own feces are transplanted. With the premise of improving obesity and its associated disorders, we investigated if fecal microbiota transplantation (FMT), heterologous and autologous, potentiates the effects of a moderate caloric restriction (CR) in high-fat diet (HFD)-induced obese mice. Mice were randomized into control, HFD, CR (12 weeks on HFD and 6 weeks under CR), FT-H (similar to CR and FMT carried out with feces from controls, weeks 17 & 18), and FT-A (administration of their own feces before developing obesity at weeks 17 & 18). Our study demonstrated that FMT, and, especially, FT-A potentiates the effects of a moderate CR on weight loss and adiposity in the short term, by decreasing feed efficiency and increasing adipose tissue lipolysis. Although FT-A produced a significant increase in bacterial richness/diversity, FMT did not significantly modify gut microbiota composition compared to the CR at phyla and bacteria genera levels, and only significant increases in Bifidobacterium and Blautia genera were observed. These results could suggest that other mechanisms different from bacterial microbiota engraftment participates in these beneficial effects. Thus, FT-A represents a very positive synergetic approach for obese patients that do not respond well to moderate restrictive diets.


Results
Effects of fecal transplantation (heterologous and autologous) on body weight, feed efficiency, adipose tissue and liver weight. The increased body weight gain induced by a HFD (p < 0.0001 vs. control) was lower in those animals under a moderate caloric restriction for 6 weeks (p < 0.01 vs. control and vs. HFD). This lower body weight gain observed in animals under CR was more evident in the fecal-transplanted mice and especially in the FT-A group as significant differences were observed among the FT-A mice and the CR group (p < 0.0001 FT-A vs. HFD and p < 0.05 vs. CR). No differences were observed in body weight gain among the FT-A group and the controls at the end of the experiment (Fig. 1B). The feed efficiency ratio, representing the body weight gain relative to energy intake in calories, was significantly higher in the HFD group (p < 0.001 vs. control). A moderate caloric restriction was able to partially impair such increase (p < 0.05 vs. control). FT-A mice showed a lower feed efficiency ratio in comparison with the HFD and also compared to the CR group (p < 0.05 vs. HFD and CR), although no statistical differences were observed among FT-A and FT-H animals (Fig. 1C). No differences were observed in food intake (expressed as grams ingested per day or calories ingested per day per animal) among the three groups under CR (Supplementary table 1). A similar pattern was observed in adipose tissue; thus, the ingestion of a HFD for 18 weeks was accompanied by a significant increase in subcutaneous fat and also in total visceral fat and in each of the fat pads collected (p < 0.0001 vs. control) (Fig. 1D) whereas fecal transplantation, especially the FT-A group, showed a significant lower size of total visceral fat compared to the HFD group (p < 0.01 vs. HFD), being more evident in the mesenteric and retroperitoneal fat depots (p < 0.0001 and p < 0.05 vs. HFD respectively). In addition, no significant differences were observed in the weight of these fat depots in the FT-A group in comparison with the controls and also compared to the FT-H animals (Fig. 1D). Histological examination of mesenteric and retroperitoneal adipose tissues showed fewer and bigger adipocytes in HFD-fed mice ( Fig. 1E-I). FT-A mice showed a significant increase in the number of mesenteric adipocytes (p < 0.05 vs. HFD and vs. CR) with no statistical differences when compared to the controls. Thus, the size of the mesenteric adipocytes in FT-A mice were significantly smaller than those observed in the HFD group, and also in comparison with the CR mice (p < 0.05) and the FT-H (p < 0.01) with no differences when compared to the controls ( Fig. 1F-H). A similar pattern was observed in retroperitoneal adipose tissue although less significant than in the mesenteric depot ( Fig. 1G-I). Antibiotic (neomycin + ampicillin) treatment for 6 days prior fecal transplants largely abolishes the effects of FT (both heterologous and autologous) on body weight gain and adipose tissue size (Supplementary figure 1A-B). A significant increase was observed in liver size and transaminases plasma levels after the ingestion of a HFD. Six weeks under CR was able to counteract such increase independently of the fecal transplantations carried out (Supplementary figure 2A-B). Table 1, the ingestion of a HFD for 18 weeks induced a significant increase in glucose and insulin serum levels as well as in the insulin resistance index, the HOMA index, compared to the control animals (p < 0.05). A moderate CR diet for 6 weeks did not result in significant improvements in these parameters. However, FT-mice (both heterologous and autologous FT-mice) showed a significant decrease in all parameters (p < 0.05 vs. HFD and p < 0.05 vs. CR). No differences were observed on insulin and the HOMA index when comparing the FT-H or FT-A groups with the controls ( Table 1). The ingestion of a HFD resulted in a significant reduction of the mRNA levels of GLUT-4 in mesenteric fat (Fig. 1J), however, this decrease was not counteracted by the CR nor FT-H, whereas a significant increase was observed in FT-A (p = 0.05 vs. HFD and CR). TG serum levels were significantly increased after the ingestion of a HFD for 18 weeks (p < 0.05) and FT were able to significantly impair such increase, being especially evident in the FT-A group (p < 0.05 vs. HFD and vs. CR) ( Table 1). Our results showed that HFD mice presented a significant increase in LBP plasma levels and CR was able to reduce such increase, independently of the fecal transplantations (Supplementary figure 5). Effects of CR and FMT on mesenteric GLUT4 mRNA levels. Data are expressed as mean ± SEM of at least 8 animals per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs Control; # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs HFD, a p < 0.05 vs CR , $ p < 0.05, $$ p < 0.01 vs FT-H.

Fecal Transplantation (Heterologous and Autologous) induces lipolysis in white adipose tissue (WAT).
To understand the molecular mechanisms underlying the antiadiposity effects of FT on white adipose tissue, the expression of the main lipolysis-related enzymes in mesenteric and retroperitoneal adipose tissues was evaluated (Fig. 2). A significant increase in total ATGL protein expression was observed in both transplanted-mice in comparison with the HFD group (p < 0.05) (Figs. 2B,H). Significant higher CGI58 mRNA levels were also observed in the FT-A group in mesenteric adipose tissue (p < 0.05 vs. CR) ( Fig. 2A). HSL activity is regulated by reversible phosphorylation in serine residues. PKA phosphorylates HSL at Ser 563 and Ser 660, which stimulates HSL activity. In contrast, phosphorylation of HSL at Ser 565 by AMPK prevents activation by PKA, inhibiting lipolysis. Thus, to better elucidate the mechanisms underlying the lipolytic actions of FT, we investigated the effects of FT (both heterologous and autologous) on HSL phosphorylation in Ser 563, Ser 660 and Ser 565 in both mesenteric and retroperitoneal fat pads from four animals per group (Figs. 2F,L). No significant differences were observed on mesenteric HSL protein expression ( Fig. 2C-F) whereas a significant increase was observed on HSL phosphorylation in Ser 660 in FT-A mice in mesenteric fat compared to HFD (p < 0.05 vs. HFD and p = 0.08 vs. CR) ( Fig. 2D-F). No significant differences were observed on HSL phosphorylation in Ser 563 (Fig. 2E,F). Concerning retroperitoneal adipose tissue, the significant decrease observed on ATGL after the ingestion of a HFD was counteracted by FT, especially by FT-A at the protein level (p < 0.05 vs. HFD for both FT-H and FT-A) ( Fig. 2H-L). FT was also able to counteract the lower total HSL protein levels induced by the HFD (Fig. 2I-L), as well as the HFD-induced reduction in HSL phosphorylation in ser660 (it did not reach statistical significance) and ser563 (p < 0.05 FT-A vs. HFD and vs. CR) (Fig. 2J-L). No statistical differences were observed in HSL phosphorylation in Ser 565 in either mesenteric or retroperitoneal adipose tissue (data not showed).

Fecal Transplantation (Heterologous and Autologous) increases fatty acid oxidation in liver.
Although no significant differences were observed in the mRNA levels of PPARα, ACOX or CPT1α in adipose tissue due to the fecal transplantations (data not showed), a significant increase was observed in the hepatic expression of all of these genes in the FT-A group compared to the HFD animals (Fig. 3). Six weeks under a moderate caloric restriction and the heterologous fecal transplantation were also accompanied by significant increases in the mRNA levels of ACOX, CPT1α and PPARα in liver, however, the induction of these beta-oxidation genes was more potent in those animals treated with autologous fecal transplants (p < 0.001 vs. HFD and vs. CR) (Fig. 3). In addition, significant differences were observed in the mRNA levels of ACOX when comparing both types of transplants (p < 0.05 FT-H vs. FT-A).

Effects of Fecal Transplantation (Heterologous and Autologous) on bacterial diversity/richness.
Two indexes of bacterial richness (Observed species and Chao-1) and one of dominance (Simpson index) have been evaluated. At the end of the experimental period, a significant decrease in bacterial richness was observed after the ingestion of a HFD. This was not counteracted by 6 weeks on CR (Fig. 4). A tendency to improve such decrease was observed in the FT-A group, being especially significant with the Observed species index (p = 0.05 vs. CR and p < 0.05 vs. FT-H) and the Chao-Index (p < 0.05 vs. CR and FT-H). Interestingly, when α-diversity was compared among T1 (24 hours before the first transplant was carried out) and the end of the experimental period (T4), no differences were observed in the CR group or in the FT-H groups (Fig. 5A,B). However, a significant increase was observed in α-diversity in the FT-A group (p < 0.05-p = 0.01 for Observed species and the Chao-1 indexes respectively), while no differences were found in the Simpson index (Fig. 5C).

Effects of Fecal Transplantation (Heterologous and Autologous) on microbial communities'
composition. Concerning GM composition (differential abundance among groups of animals), the analysis carried out at the end of the experimental period (T4) revealed that the most abundant phyla in all groups were Bacteroidetes and Firmicutes, representing around of the 90% of phyla in stools (Fig. 6A). It is interesting to note that the increase observed in Firmicutes and, on the contrary, the decrease observed in Bacteroidetes in the HFD group was partially restored by the CR, and independently of the type of transplant carried out (Fig. 6A).
When comparing the five groups using a classical univariate analyses, five phyla resulted statistical different: Cyanobacteria, Firmicutes, Tenericutes, TM7 and Actinobacteria (Table 2). However, the differences observed in TM7 and Actinobacteria abundance could be due to the slight increase observed in FT-A in comparison with the other groups (data not showed), although it did not reach statistical significances when compared to the CR group. Temporal dynamics at phylum level were also analyzed. The ingestion of a HFD induced a significant decrease in the presence of Actinobacteria, Bacteroidetes, Cyanobacteria and Tenericutes along the experimental period (p = 0.0001-p.003) (Fig. 6B). When studying the temporal dynamics among the FT-A group against their   Figures 6-10). To achieve the number of samples needed for statistical analyses (n = 4), two gels/blots were used and were processed in parallel. Loading control (Ponceau S) has been run in the same blot to normalize the results. Data are expressed as mean ± SEM of 4 animals per group in the western-blot analyses while all animals were included for the quantification of CGI58 mRNA levels (at least 8 animals per group). Data are presented in comparison to the controls considered as 1. *p < 0.05, **p < 0.01, vs Control; # p < 0.05, ## p < 0.01, vs HFD, a p < 0.05, aa p < 0.01 vs CR, $$ p < 0.01 vs FT-H. (2020) 10:9388 | https://doi.org/10.1038/s41598-020-64961-x www.nature.com/scientificreports www.nature.com/scientificreports/ placebo (CR), significant increases were observed on TM7, Tenericutes and Verrucomicrobia in FT-A animals (p = 0.0006-p = 0.45) (Fig. 6C). Of interest, the increased abundance of TM7 in FT-A resulted significant only after applying the first transplant. No statistical differences were observed among the FT-H and FT-A groups in the longitudinal study at phyla level. Only slight differences were observed in Actinobacteria (p = 0.047) and Verrucomicrobia (p = 0.00), although these differences disappeared at the end of the experimental period, T4 (Fig. 6D).
At genus level, when comparing the five groups of animals at the end of the experimental period (T4), 19 genera resulted statistical significant among the groups (Table 3). Six weeks under caloric restriction was able to impair the HFD effects on the abundance of Lactococcus, Anaerotruncus, Oscillospira, Streptococcus, Bilophila and Ruminococcus (Table 3). Fecal transplants did not translate into substantial changes at genus level in comparison with the effects of caloric restriction and only significant increases were observed in Bifidobacterium and Blautia genera (Table 3). In fact, when comparing the three groups under CR, only significant differences were observed on these two bacterial genera: Blautia and Bifidobacterium (FDR: 0.0438). Thus, higher abundance of Blautia was observed in both FT animals in comparison to the placebo group (CR) and also higher presence of Bifidobacterium was observed in both FT compared to CR, being more evident in the FT-A mice (Supplementary figure 3). Temporal dynamics at genera level were also analyzed. The increase observed in Bifidobacterium and Blautia was observed after the first transplant, as can be observed in Fig. 6E. No significant differences were observed among FT-H and FT-A at bacterial genus level.
Taking into account Bray-Curtis index, all groups clustered together at the beginning of the experimental period (Supplementary figure 4). The ingestion of a HFD was crucial. In fact, the controls and the other groups  www.nature.com/scientificreports www.nature.com/scientificreports/ (HFD, CR and both transplanted mice) were represented in two different clusters (PERMANOVA r 2 : 0.59426, p < 0.001) at T1. At the end of the experimental period (T4), the same pattern was observed, although the clusters seem to be more homogenous (PERMANOVA r 2 : 0.53974, p < 0.001) and the FT-A was represented inside the others (Supplementary figure 4). These results were plotted according to the first two principle components and the clustering of samples was represented accounting for 54.5% of total variation (Component 1 = 41.5% and Component 2 = 13%).

Discussion
Autologous fecal transplantation emerges as a promising strategy to modulate gut microbiota with minimal long-term side effects since individual´s own feces are transplanted avoiding the risks of donor resistance genes and donor-recipient mismatches. Several recent studies have confirmed the potential for autologous fecal transplantation for remediation of gut microbiota after antibiotic treatments [30][31][32] . Here, we have developed a mouse model of autologous transplantation in which each animal received their own feces but collected before the mice   www.nature.com/scientificreports www.nature.com/scientificreports/ were obese. The extrapolation of this model into humans will require the creation of fecal banks, along with the proper legislation and safety controls.
Our main finding is that only two autologous fecal transplantations were able to potentiate the effects of a moderate energy restriction on weight loss and adiposity in the short term, possibly by decreasing feed efficiency and by increasing adipose tissue lipolysis and hepatic fatty acid oxidation. Although heterologous fecal transplantation also showed positive effects, the majority of the results suggested that autotransplant is apparently more potent than CR alone and also than the FT-H group as these animals better mimic the physiology of mice fed with a standard diet (control animals).
The lower body weight gain observed in the FT-A mice seems to be related to the ability of the autologous fecal transplantation to reduce the amount of energy absorbed from food (feed efficiency), similarly to the results obtained by Lai et al., (2018) that demonstrated that HFD-fed mice receiving FT from control-exercised donors showed remarkably reduced food efficacy 33 . In this context, a vast majority of studies have demonstrated that a high Firmicutes/Bacteroidetes ratio is associated to increased capacity for harvesting energy from diet, as observed in obese subjects/animals 15,34 . Similarly, our HFD animals exhibited a higher proportion of Firmicutes and lower abundance of Bacteroidetes in comparison with those fed with a control/standard diet, which could explain the increased feed efficiency observed in these animals. Energy restriction was able to significantly counteract the increase observed in Firmicutes with no significant effects on Bacteroidetes and feed efficiency. Fecal transplantations, both heterologous and autologous, did not exert any significant effects on the abundance of these phyla suggesting that the lower feed efficiency observed in these animals are independent of the abundance of Firmicutes and Bacteroidetes phyla, which contrasts with other studies that observed that FT intervention mitigated the HFD-disrupted gut microbiota at phylum level 35 . These differences could be explained by the different methodology used in both studies (FT once a day for 8 weeks vs. once per week for only two weeks in our study). It seems that longer and more frequent transplants are needed in order to induce changes in gut microbiota at phylum level. However, since only two FT are able to induce in the short term a significant decrease in feed efficiency and a lower body weight and adiposity; this is a more interesting approach to extrapolate into humans.
In the same line, a lower richness of microbiome gene content and taxa has been tightly linked to a higher feed efficiency 36 . Fecal microbiota transplantation in mice was associated with increased bacterial richness 37 Table 3. Statistical comparisons of abundance of the major genera present in gut when control mice were compared with those fed with a HFD, submitted or not to a moderate caloric restriction and under fecal transplants (heterologous and autologous). A false discovery rate (FDR) < 0.05 was considered significant. FDR was obtained comparing the five groups using Kruskal Wallist test and the Benjamini-Hochberg approach.
our study has demonstrated that autologous fecal transplantation exerted a significant increase in α-diversity in the longitudinal analysis, which could explain, at least in part, the lower feed efficiency observed in these animals at the end of the experimental period and, therefore, the lower body weight gain. These results could suggest that the anti-obesity effects of autologous fecal transplantation are more related to changes in bacterial diversity rather than in wide phylum levels. In addition, FT and especially autologous fecal transplantation significantly improved insulin resistance despite a HFD, as previously demonstrated in individuals with metabolic syndrome 26 and also in mice 35 , which reinforces the benefits of FT on obesity and associated disorders. These positive results observed in glucose metabolism could be secondary, at least in part, to the increased expression of the facilitated glucose transporter member 4 (GLUT4) in adipose tissue 38 , although more studies are needed to deeply investigate the underlying mechanisms. The lower body weight gain observed in FT-A mice was also accompanied by a lower adiposity, being especially evident in total visceral fat and specifically in the mesenteric and retroperitoneal fat pads. These are the fat depots strongly linked to insulin resistance, type 2 diabetes, hypertension, and dyslipidemia, suggesting that the reduction of these adipose tissues could also contribute to the protective actions observed on insulin resistance and other metabolic alterations. Elucidations of molecular mechanisms that favor adipose tissue decline such as increased lipolysis and/or β-oxidation are of interest. In fact, the significant increase observed in lipolysis in both fat depots could explain the lower weight of adipose tissue in general and also the lower size of adipocytes. Lipolysis is a complex process that is highly regulated and involves the coordinated participation of several lipid droplet proteins and also several lipases such as adipose triglyceride lipase (ATGL/desnutrin), hormone sensitive lipase (HSL), and monoacylglycerol lipase 39 . A significant increase in total ATGL protein expression was observed in both fat depots in FT-mice in comparison with the HFD group. Lipase activity of ATGL largely depends on its coactivation by comparative gene identification 58 (CGI-58) and a significant increase was also observed in the mRNA levels of this gene especially in mesenteric fat. The activity of HSL is also well known to be regulated posttranscriptionally by reversible phosphorylation and our study suggested a significant increase in HSL protein levels and activity (via phosphorylation) in both fat depots. Since a very recent study suggested that adipose tissue from mice exposed to a HFD present "obesity memory", which means that present a tissue-autonomous lipolytic defect allowing increased efficiency of lipid storage 40 , synergistic approaches are needed to counteract such defects. Thus, the use of FMT emerges as a very positive and necessary approach along with a caloric restriction to reduce adiposity and obesity despite the "obesity memory" of the mesenteric and retroperitoneal adipocytes induced by the ingestion of a HFD.
Increased rates of lipolysis in mesenteric adipose tissue could be associated with fat liver accumulation 41 and insulin resistance 42 . However, increasing lipolysis in adipose tissue does not necessarily increase serum free fatty acids (FFAs) levels because it could cause a shift within adipocytes or liver towards increased fatty acid utilization and, thus, protect against obesity. In line with this, our study has demonstrated that FMT in rodents reduces weight loss and fat mass through increased lipolysis but without developing fatty liver or increasing circulating FFA. Moreover, FMT also improves insulin resistance and this could be associated with FMT-induced fatty acid oxidation in liver, as suggested by our mRNA results.
Concerning gut microbiota composition at genera level, the ingestion of a HFD was accompanied by changes in several bacterial genera, whereas a moderate caloric restriction was able to counteract the changes induced by the HFD in the abundance of Lactococcus, Anaerotruncus, Oscillospira, Streptococcus, Bilophila and Ruminococcus. These results are in line with the study from Wang et al., (2018) that demonstrates that gut microbiota mediates the antiobesity effects of a caloric restriction 37 . However, FMT did not significantly modify gut microbiota composition compared to the CR and only significant increases in Bifidobacterium and Blautia genera were observed. Interestingly, the increase in both bacterial genera was only observed after the transplantations. Bifidobacterium, that belongs to Actinomycetes phylum, is one of the most numerous commensal bacteria present in mammalian gut. It helps Bacteroides degrade polysaccharides 43 and inhibits exogenous cholesterol absorption from the small intestine 44 , although the beneficial effects on lipid metabolism and body weight could depend on the strain 45 . In general terms, Bifidobacterium has been suggested as a potential therapeutic candidate for management of obesity 45 ; thus, the increase observed after FMT could mediate, at least in part, the lower body weight, adiposity and insulin resistance observed in these mice. Blautia is also a bacterial genera significantly and inversely associated with visceral fat accumulation in adults, regardless of gender 46 . In fact, different approaches that improve obesity and associated disorders such as laparoscopic sleeve gastrectomy or silybin, a naturally occurring hepatoprotective agent, have also demonstrated to increase the abundance of this genus 47,48 . More studies are needed to deeply investigate the mechanisms involved in the potential association among Bifidobacterium and Blautia abundance and improvements in obesity. Bifidobacteria and Bacteroides spp are known to reinforce intestinal integrity 49 . Although we have not directly measured gut permeability, we quantified one marker of bacterial translocation in plasma (LBP, supplementary figure 5). Our results showed that HFD mice presented a significant increase in LBP plasma levels as expected, and CR was able to reduce such increase independently of the fecal transplantations. These results suggested that the beneficial effects observed after fecal transplantations are not secondary to improvements in bacterial translocation and, therefore, the health-promoting effects of Bifidobacterium seem to be independent to their actions on intestinal barrier function. In addition, the slight changes observed in gut microbiota composition after FMT could suggest that engraftment of bacterial microbiota is not needed to achieve such beneficial effects on obesity and adiposity in the short term and other components within the feces such as bacteriophages, or even nonliving components such as metabolites, could influence host energy homeostasis 16 . In fact, sterile fecal filtrates from resveratrol-fed mice have been demonstrated to be sufficient to improve glucose homeostasis in obese mice 50,51 . This issue deserves further investigation.
Antibiotic-treated animals are commonly used for FMT studies but problems with reproducibility, baseline values and antibiotic resistance genes should be considered 52 . Our study shows that all the positive effects observed after FMT disappeared if animals were previously treated with antibiotics for 6 days, which is in line with Scientific RepoRtS | (2020) 10:9388 | https://doi.org/10.1038/s41598-020-64961-x www.nature.com/scientificreports www.nature.com/scientificreports/ a previous study that demonstrated that antibiotic administration largely abolishes the metabolism-regulatory functions of gut microbiota and abrogated the health-beneficial effect of gut microbiota 37 . Thus, our results should be taken into account in the design of strategies to modify gut microbiota in the context of antibiotic resistance era.
To sum up, we have demonstrated that fecal transplantation and, especially, autologous fecal transplantation, potentiates the effects of a moderate energy restriction on weight loss and adiposity in the short term, possibly by decreasing feed efficiency and increasing adipose tissue lipolysis and, possibly, via increased hepatic fatty acid oxidation. Although autologous fecal transplantation produced a significant increase in bacterial richness/diversity, no significant changes were observed on gut microbiota composition at phyla and/or bacteria genera level and only slight changes were observed in the abundance of Bifidobacterium and Blautia genera, suggesting that other mechanisms different from changes in gut microbiota composition or bacterial microbiota engraftment participates in such beneficial effects. The additive effects of fecal transplantation and, especially, autologous procedure and energy restriction in obesity and its associated metabolic disorders could be very positive for those obese patients that do not respond well to moderate restrictive diets. In fact, these synergic effects could improve dietary adherence and could help to achieve the loss of weight needed to improve health with no need to follow more restrictive diets or to start other therapeutic/surgical options. These results could also reinforce the need for stool banking to deposit "lean" feces for later use.

Materials and Methods
Animal experiments. Forty-two male mice (C57BL/6 J) (5 weeks old) purchased from Charles River (Barcelona, Spain) were randomly assigned to the following groups: i) Control: fed with a normal chow diet (Standard diet, 801010 RM1A (P), SDS, Essex, UK) for 18 weeks; ii) HFD: animals fed with a HFD (60% of kcal from fat: D12492) (Research Diets Inc., New Brunswick, NJ, USA) for 18 weeks; iii) CR group: mice fed with a HFD for 12 weeks and 6 weeks under CR (−25% of daily calories). A 25% degree of energy restriction was chosen following the majority of studies carried out (ranging from 20 to 40% of energy restriction) 53,54 . These mice received water by oral gavage at the latter two weeks (weeks 17 and 18, once per week); iv) FT-H: similar to the previous group. These animals received feces from control mice at the latter two weeks (weeks 17 & 18, once per week); v) FT-A: similar to the previous group but with administration of their own feces before developing obesity (at the beginning of the experimental period) (Fig. 1A). The amount of diet provided to restricted animals (groups: CR, FT-H and FT-A) was calculated on the basis of spontaneous food intake (calories) quantified in HFD-fed animals. The CR group was also submitted to oral gavage (water-placebo); therefore, these mice were under the same stressful conditions than the FT-H and FT-A animals.
The first day of the experimental period (day 0) stools from control mice and FT-A animals were collected and frozen at −80 °C. At week 17 and week 18, 70 mg of the stools were hydrated in 250 µl of distilled water. Transplant into recipient mice was achieved by oral gavage of 152 µl of the supernatant obtained after centrifugation. Pooled stools from controls were used for transplantation in FT-H mice, whereas each mouse from the FT-A group received their own feces collected at day 0 following this procedure (Fig. 1A).
Antibiotic pretreatment is commonly used in clinical practice in FMT transplantation for C. difficile infection 19 and also in studies of FMT in rodents 52 . Thus, in order to check if antibiotic pretreatment potentially increase the response of transplants on body weight gain and adiposity, another three groups of mice were included in the design of the experiment. These groups were similar to CR, FT-H and FT-A mice but treated with antibiotics for 6 days before performing the first fecal transplantation (1 mg/ml ampicillin and 1 mg/ml neomycin in the drinking water) (ampicillin provided by Normon, Madrid, Spain and neomycin provided by Sigma, St. Louis, MO, USA).
All mice were euthanized after 18 weeks of treatment and samples were collected after a 5 h fasting period. White adipose tissue (WAT) from different anatomical locations (mesenteric, epidydimal, retroperitoneal and subcutaneous), liver, muscle and other organs were dissected, weighed and immediately frozen in liquid nitrogen or fixed in 10% formalin and paraffin embedded for subsequent histological studies. Stools were collected at the beginning of the project (time point 0; T0), 24  Biochemical parameters and Bacterial Translocation. Serum samples were collected from cardiac puncture after 5 hours of fasting. Levels of glucose, triglycerides, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using an automatic biochemical analyzer (Cobas C711, Madrid, Spain). Insulin was quantified by a commercial ELISA Kit following manufacturer's instructions (EMD Millipore, MO, USA). Insulin resistance was calculated using the homeostasis model assessment of insulin resistance (HOMA-IR) as previously described 55 . Plasma levels of lipopolysaccharide binding protein (LBP) were measured using an ELISA from Hycult Biotech (Uden, The Netherlands) 56 .
Histological analyses. Following formalin fixation, adipose tissue from different fat depots (mesenteric, retroperitoneal and epididymal) were dehydrated and paraffin embedded. Tissue sections (5 μm-thick) were rehydrated and stained with hematoxylin-eosin (H&E) according to standard protocols. The fields were evaluated with the final magnification of 40×. Digital photographs were taken from each histological section and the number of adipocytes and their size was quantified using automatized software (Image-J Software).