Synbiotic Supplementation Modulates Gut Microbiota, Regulates Beta-catenin Expression and Prevents Weight Gain in Ob/ob Mice

Background: Obesity is one of the main health problems in the world today and dysbiosis seem to be one of the factors involved. The aim of this study was to examine the impact of synbiotic supplementation in obesity and microbiota in ob/ob mice. 20 animals were divided into four groups: Obese Treated (OT) and Control (OC), Lean Treated (LT) and Control (LC). All animals received standard diet for 8 weeks. Treated groups received a synbiotic in water while nontreated groups received water. After 8 weeks, all animals were sacriced and gut tissue mRNA isolation and stool samples by microbiota analysis were collected. Beta-catenin, occludin, cadherin and zonulin were analyzed in gut tissue by RT-qPCR. Microbiome DNA was extracted from stool samples and sequenced using the Ion PGM Torrent platform. Results: The synbiotic supplementation reduced body weight gain in OT group comparing with OC (p=0.0398), increase of Enterobacteriaceae (p=0.005) and decrease of Cyanobacteria (p=0.047), Clostridiaceae (p=0.026), Turicibacterales (p=0.005) and Coprococcus (p=0.047). In the other hand, a signicant reduction of Sutterella bacteria (p=0.009) and Turicibacter (p=0.005) was observed in LT group compared to LC. Alpha and beta diversities were differ between all treated groups. Beta-catenin gene expression was signicantly decreased in the gut tissue of OT group (p ≤ 0.0001) when compared to other groups. No changes were observed in occludin, cadherin and zonulin gene expression in the gut tissue. Conclusion: The synbiotics supplementation prevents excessive weight gain, modulates the gut microbiota, and reduces beta-catenin expression in ob/ob mice.

The term symbiotic refers to food ingredients or dietary supplements that combine probiotics and prebiotics in a form of synergism, for example, a mixtures of fructooligosaccharides (FOS) with lactisol Bi dobacteriase with Lactobacillus [4]. These promote the survival and implantation of probiotics in the large intestine. Some studies have shown that the consumption of symbiotics prevents bacterial translocation, epithelial invasion and inhibits bacterial adhesion of the mucosa and the production of antimicrobial peptides, reducing in ammation and stimulating host immunity [5,6]. Intestinal colonization of synbiotic bacteria seems to be a good strategy to reduce the damage caused by SIBO and gut permeability. Probiotics are live microbial food supplements that promote: the maintenance of mucosal integrity [7]; the GM balance; the increment of humoral and cellular immunity and the reduction of cholesterol and triglycerides (TG) [8,9]. Prebiotics are derived from naturally occurring carbohydrates in some vegetables, which are not hydrolyzed by digestive enzymes and reach the large intestine to be digested by GM. This type of supplement works as an energy source to the growth of bene cial bacteria [7].
The leptin de ciency causing hyperphagia, excessive nutrient intake and reduced energy expenditure leading to the development of MetS, with visceral obesity, IR, mimetizing obesity as well in humans [10]. Due to this phenotype that easily facilitates the development of IR, T2DM and in ammatory response, these animals are used in several experimental studies [11,12]. Due to paucity and the lack of standardization in studies evaluating the supplementation of prebiotics, probiotics and synbiotics in obesity, we proposed a study using an experimental model of leptin de cient obese mice (ob/ ob mice) to test the effects of synbiotic supplementation in this experimental model to evaluate the possible modulation of obesity, gut microbiota, gut integrity, and permeability.

Animals
Twenty adults male ob/ ob mice were housed in a temperature, humidity-, and ventilation-controlled vivarium, with a 12-h light/ dark cycle. All procedures for animal experimentation followed the ethical guidelines of the Helsinki Declaration of 1975 (NIH Publication No. 85 − 23, revised 1996)  The animal's weight was measured using a digital balance (Gehaka, Model BK4001, Brazil) and the weight gain was calculated as the difference between body weight measured at the beginning and at the end of the protocol.
After the period of treatment, the animals were anesthetized with ketamine hydrochloride (0.1 mL/ kg) intraperitoneally and sacri ced. Hepatic tissue samples were collected for histological analysis. Gut tissue samples were collected for analysis of the mRNAs of genes related to gut integrity [beta-catenin (5'

Quanti cation and Analysis of Total RNA Integrity
The concentration of total RNAs extracted was determined by spectrophotometry [NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA)]. RNA preparation was considered protein free when the A260/280 ratio was between 1.8 and 2.0. For samples that did not reach these values, puri cations were performed using the RNeasy™ Mini Kit (Qiagen, Hilden, Germany). The integrity and purity of the RNAs were analyzed by 1% agarose gel electrophoresis. Only samples whose bands corresponding to ribosomal RNA (RNA) 18 and 28S were shown to be intact for analysis under ultraviolet light. The RNAs were maintained at -80°C until use.

Real-time PCR
One hundred nanograms were used for Real-time PCR analysis. PCR was performed in a 15 µL reaction mixture containing 7.5 µL 2 × SYBR Green Reaction Mix (Invitrogen Life Technologies, Carlsbad, CA, USA), 0.3 µL each primer (10 pmol), 0.3 µL Super Script III RT/Platinum Taq Mix (10 pmol/µL), 0.15 µL ROX Reference Dye, and 5 µL sample in water. Gene-speci c primers were used. All the primer sequences are listed in Table 1. Reactions were performed using StepOne™ Real-Time PCR System (Applied Biosystem -Foster City, CA -EUA).

Analysis of results
Data analysis were processed through the Quantitative Insights Into Microbial Ecology (QIIME) software package v1.8 [14]. Reads were ltered by length (> 200 pb), quality (Phred Score = 30) and minimum expected error (0.1) utilizing the USEARCH tool [15]. The remaining sequences were grouped into Operational Taxonomic Units (OTUs), based on 97% similarity, using the UCLUST algorithm. Singletons were removed. OTUs were classi ed taxonomically, using the Greengenes 16S reference database v. 13.8 [16]. Alpha-diversity index (Shannon, Simpson, Chao1, Observed_OTUs, Faith's Phylogenetic diversity and Pielou's Evenness) and Beta diversity index (Weighted Unifrac, Unweighted Unifrac, Bray Curtis and Jaccard) were calculated based on the rare ed OTU table using 54789 sequences per sample. The Principal Coordinates Analysis (PCoA) plot for each of the beta diversity index was generated using Emperor [17]. Compositions of microbiota communities were summarized by proportion at different taxonomy levels, including genus, family, order, class, and phylum ranks. The Kruskal-Wallis test was performed to explore differences in alpha diversity index. Differences in community composition (beta diversity) were assessed using Permutational Multivariate Analysis of Variance (PERMANOVA). Kruskal-Wallis and PERMANOVA analysis were corrected by Benjamini & Hochberg method.
Microorganism features distinguishing fecal microbiota were identi ed using the linear discriminant analysis (LDA) effect size (LEfSe) method for biomarker discovery, which emphasizes both statistical signi cance and biological relevance (metagenomic biomarker discovery and explanation). LEfSe uses the Kruskal-Wallis rank-sum test with a normalized relative abundance matrix to detect features with signi cantly different abundances between assigned taxa and performs LDA to estimate the effect size of each feature.

Statistical Analysis
All data were expressed as mean or median (depending on the distribution pattern of the variables). Minimum (Min), Maximum (Max) and Standard Deviation (DP) values were set. For Gaussian distribution variables we used t-test, one-way ANOVA and the Newman-Keuls post-test of multiple comparisons. For non-Gaussian distribution variables, the Mann-Whitney, Kruskal-Wallis and Dunn's multiple-comparison tests were used. Chi-square tests and Fisher's exact test were used to compare the histological scores between the groups. A p value < 0.05 was considered signi cant. All calculations and graphs were performed with SPSS 17.0 software (SPSS Inc., Chicago, IL, USA).

Weighing of animals
The body weight gain after synbiotic supplementation in the OT group was signi cantly lower when compared with OC group (p = 0.0398). No relevant modi cations were observed in the body weight gain when it was compared LT and LC groups (Fig. 1).

Analysis of intestinal microbiota
Microorganisms belonging to the bacteria kingdom were searched and quanti ed following the taxonomic classi cation of phyla, class, order, family and genus (species subdivision was not contemplated in the analysis).
The metagenome analysis LEfSe approach was applied to identify the key phylotypes responsible for the difference between the groups. Bacteroides and Bacteroidaceae, which were most abundant in the treated groups (lean and obese) where compared to controls, were the dominant phylotypes that contributed to the difference between the intestinal microbiota of treated groups ( Fig. 4a and b).
Regarding the phyla the proportion of Bacteroidetes and Firmicutes observed among samples in the OT group compared to the OC was 71.02% and 18.19%, respectively (Fig. 5a). Figure 5b shows the heatmap representation calculated at the genus level of the classi ed reads obtained. The Cyanobacteria phylum and the Turicibacterales order were reduced in OT group (p = 0.047, p = 0.005 respectively) and among bacteria classes there was an increase in the Gammaproteobacteria (p = 0.005), in the bacteria order Enterobacteriales (p = 0.005) and in the bacteria family Enterobacteriaceae (p = 0.005). In addition, the Clostridiaceae family (p = 0.026) and the bacteria genus Turicibacter (p = 0.005) and Coprococcus (p = 0.047) were decreased in OT group (Fig. 6).

Gene expression analysis
A signi cantly decrease in gene expression of beta-catenin in the gut tissue was observed in OT group when compared to OC group (p = 0.0479) and in LT group when compared to LC group (p = 0.0030) (Fig.   8). On the other hand, no signi cant changes were veri ed among the groups in gene expression of cadherin (p = 0.4048), occludin (p = 0.2063) and zonulin (ZO-1) (p = 0.171) in gut tissue (see Additional le 2).

Discussion
The present study demonstrated that dietary supplementation of synbiotics prevented excessive weigh gain in obese mice when compared to controls, modulated the GM and reduced the gene expression of beta-catenin expression. The last mentioned is an important gene involved in tight junction signaling, in ammation and obesity [18].
The mechanism by which synbiotic supplementation could in uence the loss of weight has not been fully elucidated. However, corroborating our results, studies have demonstrated that dietary supplementation with probiotics and prebiotics combination is able to modulate the GM of mice and obesity humans, which leads to signi cant changes in the prevalence of speci c intestinal bacteria. These population with dietary supplementation may bene t from the decrease of energy harvest capacity from diet and thus reduce weight gain [19,20].
In the present study the intragroups analyses of alpha and beta diversities were differ between all treated groups and control group. Addicionaly, the intergroups analyses also showed a signi cant difference between lean and obese treated and control groups. Our data demosntrate that the richness and evenness of the GM in lean and obese animals were different before treatment and this difference became more evident after synbiotic supplementation. Corroboring our ndings, studies with probiotics supplementation in human and mice may change GM richness and diversity for conditions such as obesity and metabolic disorders [21,22]. Lower diversity in the GM has been linked to obesity, higher IR, higher visceral fat and numerous in ammatory conditions [23]. Thus, GM diversity could be linked to body weight.
The gut microbiota is mainly composed by bacteria from the Bacteroidetes and Firmicutes phylum. Proteobacteria, Verrucomicrobia, Actinobacteria, Fusobacteria, and Cyanobacteria are present in minor proportions [24]. Adequate amounts of Cyanobacteria are considered bene cial for the host to diminish in ammation through NF-κB inhibition, and, consequently, reduce proin ammatory cytokines, protecting the host against oxidative stress [25]. However, increased Cyanobacteria have been associated with obesity [26] and recent study published by Shao et al. showed that after weight loss the abundance of these bacteria decreases [27]. Our ndings showed Cyanobacteria phylum reduction in the OT group after supplementation associated with weight loss, similarly to Shao et al [27].
Turicibacter is a genus in the Firmicutes phylum of bacteria that has been found most commonly in the gut [28]. Although this bacteria has been related to greater energy extraction from the diet and that would be related to obesity [1], the data on the literature are con icting. There are studies that also show a negative correlation between the amount of Turicibacter and NF-κB and associate a lower amount of this bacteria with the most in amed individuals with obesity and steatosis [3]. In our study, the analyzes of fecal microbiota of ob/ob mice treated (obese and lean) after synbiotics supplementation demonstrated a decrease of Turicibacter genus when compared to control groups, which could be a interesting nding, responsible for reducing the energy extraction from the diet. Besides, we observed an increase of Enterobacteriaceae family in relation to the control groups. This family is a group of bacteria considered bene cial either by promoting protection of the intestinal barrier or regulating the growth of other bacteria that promote gastrointestinal disorders [29].
The other relevant nding of our study was the decrease in Clostridiaceae family, and Coprococcus genus in the OT group and a reduction in the abundance of Sutterella genus in LT group. Clostridiaceae family is the group of bacteria present mainly in obese and in T2DM animals [30] and is associated with dysbiosis in adults and children [31]) and in ammatory bowel disease in adults [32]. In consonance with our ndings, a recent study demonstrated that probiotic supplementation (Lactobacillus paracasei) is able to reduce the abundance of Clostridiaceae [33]. On the other hand, Coprococcus, a genus in the Firmicutes phylum, when increased has been associated with a high-fat diet in mice [34]. Bacteria of the genus Sutterella have been often associated with in ammatory bowel disease and disrupt of the intestinal epithelial homeostasis [35]. It is evident that the consumption of high-protein and high-sugar diets increase Sutterella in the gut [36] and that probiotics supplementation reduces their abundance [37], which corroborates our ndings in the LT group.
In our study, a signi cant increase in the following groups of gut bacteria was observed in the LT group: Bacteroides and Lactococcus genus, Enterobacteriales order, Bacteroidaceae, Prevotelaceae and Enterobacteriaceae family. The abundance of some of these bacteria is linked to the improvement of the integrity of the intestinal barrier [38].
The main role of the intestinal barrier is to separate the internal environment from the luminal content, and the complex system of intercellular junctions, including tight junctions, seals together the epithelial cells to form a continuous layer [39]. In our study we observed a lower expression of intestinal betacatenin in OT and LT groups compared to control groups. There was no difference in cadherin, occludin and ZO-1. The beta-catenin is one of the proteins that compose the tight junctions which are primarily responsible for maintenance of the intestinal permeability barrier, regulating the passage of ions and solutes between cells by the paracellular pathway [40]. However, when beta-catenin expression is increased it would enter the cell nucleus and would induce the activation of NF-kB, pro-in ammatory genes and the expression of others oncogenes, which are important for development of some intestinal diseases [41,42] and hepatocellular carcinoma (HCC) [43]. Evidence about the effect of synbiotic supplementation on beta-catenin modulation is scarce. Our results are consistent with Kuugbee et al. that has shown an inhibition of beta-catenin signaling pathway after probiotic [44] and synbiotic supplementation [18]. Based on these results we can infer that the reduction in beta-catenin expression improves the permeability of the intestinal barrier, preventing the passage of endotoxins from the intestinal lumen through the intestinal barrier and consequently not triggering in ammatory cytokines, which are important for the development of obesity. On the other hand, there are studies showing increased expression of tight junction after synbiotic administration, highlighting ZO-1, occludin and claudin, but not evaluating beta-catenin [19,45]. Apparently, modulation of intestinal tight junctions happens with prolonged use of symbiotic, which perhaps justi es no difference in gene expression of cadherin, occludin and ZO-1 in our study.
There were strengths and limitations in our study that should be considered. The strengths of our study were the combination of four different probiotics strains, the choice of isogenic mice, the microbial sequencing techniques and the rigorous evaluation of gene expression and liver histology performed by a specialist. However, our study also had some limitations. First, we only chose one probiotic ber to include in the synbiotic supplementation and the treatment period has lasted only 8 weeks. Perhaps for these reasons, we have not observed more consistent results in gene expression in these animals. Despite these limitations, we believe that our results are encouraging and support the consideration of larger, welldesigned studies to evaluate synbiotics supplementation as obesity prevention.
In conclusion, our experimental study with animal model shows that synbiotics supplementation is effective to prevent excessive weigh gain, positively modulates the gut microbiota, reduces beta-catenin expression, but was not able to improve other tight junctions gene expressions. Our data support the evidence of bene cial effects of synbiotics supplementation on prevention of obesity. Nonetheless, more randomized controlled trials are needed.     Analysis of the distribution of bacteria in lean control (LC) and lean treated (LT) groups according to phyla, order, family and genus.