Effect of SR141716A on diet intake, body weight and body composition

Consistent with previous studies, treatment with SR141716A of mice with Diet-induced Obesity (DIO) (HFD + SR) transiently reduced calorie intake and induced weight loss when compared with vehicle-treated mice with Diet-Induced Obesity (DIO) (HFD + Vehicle) (Fig. 1b,c)20. To assess the effect of SR141716A beyond its effect on weight loss and calorie intake, pair-feeding was conducted in diet intake-matched control (PFSR), and food intake was adjusted in body weight-matched (BWM) controls (Fig. 1a). The temporary reduction of calorie intake in HFD + SR mice during the first week was diminished by day 9 of treatment, reaching similar intake as vehicle-treated Diet-Induced Obesity (DIO) group (HFD + Vehicle) (Fig. 1b). However, we noted consecutive weight loss in SR141716A-treated Diet-Induced Obesity (DIO) group (HFD + SR) through the end of treatment (Fig. 1c). To maintain the same body weight in body weight-matched (BWM) group as SR141716A-treated Diet-Induced Obesity (DIO) group (HFD + SR), their food was restricted to even lower intake level than SR141716A-treated Diet-Induced Obesity (DIO) group (HFD + SR) (Fig. 1b).

Figure 1 SR141716A causes transient reduction in diet intake and persistent weight loss when compared to vehicle-treated HFD fed control. (a) Diet-Induced Obesity (DIO) model was generated by feeding C57BL/6 J male mice with high-fat diet (HFD + Vehicle) whereas their lean, age-matched controls were fed low-fat diet (LFD + Vehicle). High Fat Diet-fed mice were treated with either SR141716A (10 mg/kg/day) (HFD + SR) or vehicle (0.1% Tween 80) (HFD + Vehicle) by daily oral gavage for 4 weeks starting at week 12. In order to assess the anti-inflammatory effect of SR141716A beyond its effect on calorie intake inhibition and weight loss in Diet-Induced Obesity (DIO) phenotype, pair-feeding was conducted in diet-intake matched controls (PFSR) and diet intake was adjusted in bodyweight-matched controls (BWM; n = 8–10 mice/group). (b) Daily energy intake during 4 week treatment with SR141716A in Diet-Induced Obesity (DIO) mice was recorded, Area Under the Curve (AUC) was calculated from the 5 replicated experiments. (c) Daily body weight of each group of mice is shown during the whole period of treatment; AUC was calculated from the 5 replicated experiments which were identical to the replicates in Fig. 1b. Area Under Curve (AUC) was calculated with Trapezoidal rule in R software. Generalized Estimating Equation (GEE) was performed to fit a repeated measurement logistic regression in SPSS. Data are shown as mean ± SD. Data with different superscript letters are significantly different (P < 0.05). (d) Pearson correlation between changes in body weight and caloric intake within different groups was assessed using R software. (e) Total fat mass gain and changes in lean mass was assessed at the baseline and after 4 weeks of treatment with Dual Energy X-ray absorptiometry (DEXA). Data are shown as mean ± SD. Data with different superscript letters are significantly different (P < 0.05). (f) The surface area of 100 adipocytes was determined and then averaged to represent mean adipocyte size for each mouse using ImageJ software (National Institutes of Health). Data are shown as mean \(\pm \) SD. Data with different superscript letters are significantly different (P < 0.05) according to post hoc ANOVA one-way statistical analysis. (n = 10). (g) Weights of fat pads and livers were assessed at the end of the treatment. Full size image

To examine the effect of SR141716A beyond its effect on calorie intake, the pair-fed control (PFSR) mice were fed with the same amount of high fat diet as consumed by the SR141716A-treated Diet-Induced Obesity (DIO) mice (HFD + SR). The weight loss pattern in pair-fed control (PFSR) group was similar to HFD + SR during the first two weeks of treatment, but then pair-fed control (PFSR) group started to gain weight, reaching body weight close to vehicle-treated Diet-Induced Obesity (DIO) mice (HFD + Vehicle), by the end of the treatment (Fig. 1c). Correlation between changes in body weight and caloric intake within different groups demonstrated consistent weight loss in SR141716A-treated Diet-Induced Obesity (DIO) group (HFD + SR) regardless of its high level of calorie intake, close to vehicle-treated Diet-Induced Obesity (DIO) group (HFD + Vehicle) (Fig. 1d).

Assessing body composition after four weeks of SR141716A intervention in Diet-Induced Obesity (DIO) mice (HFD + SR) showed a significant reduction in fat gain when compared to vehicle-treated Diet-Induced Obesity (DIO) mice (HFD + Vehicle), while there was no difference in lean mass (Fig. 1e). Because SR141716A-treated Diet-Induced Obesity (DIO) mice (HFD + SR) demonstrated less fat mass when compared to body weight-matched control (BWM), the data suggested that other factors are associated with the use of SR141716A besides its effect on calorie intake and weight loss (Fig. 1e). Lower fat mass within the SR141716A-treated group (HFD + SR) has been characterized with less adiposity. Assessing the area of the adipocytes demonstrated significant shrinkage in adipocytes of the SR141716A-treated Diet-Induced obesity (DIO) mice (HFD + SR) when compared to vehicle-treated Diet-Induced Obesity (DIO) mice (HFD + Vehicle), pair-fed controls (PFSR), and body weight-matched controls (BWM) (Fig. 1f). Furthermore, vehicle-treated Diet-Induced obesity mice (HFD + Vehicle) group demonstrated the presence of crown-like structures of macrophages surrounding the adipocytes, which were absent in other groups.

Adipose tissue fibrosis, in obese phenotype is associated with an increase in local inflammation. The Picrosirius red fibrillar collagens were interspersed among the adipocytes in vehicle-treated Diet-Induced obesity (HFD + Vehicle) group. SR141716A treatment in Diet-Induced Obesity (DIO) mice (HFD + SR) resulted in a significant suppression of adipose tissue fibrosis and consequently further reduction in local adipose tissue inflammation and dysfunction (Supplementary Figure 1a,b).

Lighter fat pad (mainly in the epididymal fat pad) in SR141716A-treated Diet-Induced Obesity (DIO) mice (HFD + SR) was associated with smaller liver weight when compared with vehicle-treated Diet-Induced obesity (DIO) (HFD + Vehicle), pair-fed Diet –induced Obesity (DIO) control (PFSR), and body weight-matched control (BWM) mice (Fig. 1g).

Effect of SR141716A on systemic and local inflammation

Based on the active role of macrophages in the initiation of inflammation in adipose tissue, we examined the changes in macrophage population in adipose tissue. Intervention treatment of Diet-Induced Obesity (DIO) mice with SR141716A (HFD + SR) demonstrated significant reduction in the ratio of macrophages/adipocytes as compared with vehicle-treated Diet-Induced Obesity (DIO) (HFD + Vehicle) mice, pair-fed control(PFSR), and body weight matched-control (BWM) (Fig. 2a). Flow cytometric analysis of the subset of macrophages showed a significant reduction in both frequency and the absolute number of pro-inflammatory M1 macrophages with SR141716A treatment (HFD + SR) when compared to vehicle-treated Diet-Induced Obesity (DIO) controls (HFD + Vehicle) (Fig. 2b,c). The inflammatory profile was also assessed by examining chemokines and cytokines in the serum for systemic inflammation. Treatment of Diet-Induced obesity (DIO) mice with SR141716A (HFD + SR) led to lower level of IL-17, monocyte chemoattractant protein-1 (MCP-1), eotaxin, and macrophage inflammatory protein-1α (MIP-1α) when compared to vehicle-treated Diet-Induced Obesity (DIO) mice (HFD + Vehicle) (Fig. 2d–g). The same trend was seen with TNF-α, IL-6, RANTES, MIP-1β and MIP-2 but the differences were not significant. Changes in lipopolysaccharide (LPS) as a primary stimulator of macrophages has been demonstrated21.

Figure 2 SR141716A attenuates local and systemic inflammation in diet-induced obesity. Experiments Diet-Induced Obesity (DIO) were set up as described in Fig. 1 legend. (a) Adipose Tissue Macrophages (ATMs) were quantified per 100 adipocytes by Spot Studio v1.0 Analysis Software. (b,c) Kidney fat was isolated from 10 mice in each group. The ratio (b) and total cell number (c) of kidney fat F4/80 and CD11c+ cells was studied. (d–g) Effect of SR141716A on plasma cytokine levels, (d) IL-17 levels (e) Monocyte chemoattractant protein-1(MCP-1) levels, (f) Eotaxin levels, and (g) Macrophage inflammatory protien-1α (MIP-1α) levels in plasma were quantified with multiplex immunoassays. (h,k) Effect of SR141716A treatment on the mRNA level of (h) RORγ, (i) TNF-𝛼, (j) iNOS, and (k) IL-6, in the epididymal adipose tissue was examined. Data shown as mean \(\pm \) SD. Data with different superscript letters are significantly different (P < 0.05) according to post hoc ANOVA one-way statistical analysis. (n = 5 except LFD + Vehicle; n = 4). Full size image

We also investigated the inflammation profile of adipose tissue and colon locally, and to that end, the mRNA level of RORγ, TNF-α, iNOS, and IL-6 was quantified in adipose tissue (Fig. 2h,k). Overall, intervention treatment of Diet-Induced Obesity (DIO) mice with SR141716A (HFD + SR) led to the improvement of the inflammatory state of adipose tissue beyond its effect on diet restriction.

We also observed a significant increase in both the percentage and numbers of CD4 + GATA3 + Th2 cells (anti-inflammatory T cell subset) following treatment with SR141716A of Diet-Induced Obesity (DIO) mice (HFD + SR) in adipose tissue (Supplementary Figure 2a,b).

Myeloid Derived-Suppressor Cells (MDSC) that are GR-1 + CD11b + have been identified as potent anti-inflammatory cells. In the current study, we noted that Myeloid Derived-Suppressor Cells (MDSCs) were increased with SR141716A treatment in Diet-Induced Obesity (DIO) mice (HFD + SR) when compared to vehicle-treated Diet-Induced Obesity (DIO) (HFD + Vehicle) in adipose tissue (Supplementary Figure 2c,d). We also assessed the changes in blood Myeloid Derived-Suppressor Cells (MDSCs) and found a significant decrease in Myeloid Derived-Suppressor Cells (MDSCs) following SR141716A treatment (Supplementary Figure 2e), thereby suggesting that there may be increased migration of such cells into the adipose tissue following SR141716A treatment.

Differential analysis of complete blood count (CBC) revealed significant leukocytosis in Diet-Induced Obesity (DIO) mice (HFD + Vehicle) when compared to SR141716A-treated Diet-Induced Obesity (DIO) (HFD + SR) and lean (LFD + Vehicle) mice. Leukocytosis in Diet-Induced Obesity (DIO) mice was more pronounced in the neutrophil subpopulation, which is the first responder to an inflammatory signal. Our data suggested that treating Diet-Induced Obesity (DIO) mice with SR141716A reduces neutrophilic leukocytosis (Supplementary Table 1). Furthermore, SR141716A treatment balanced the increased level of hemoglobin and hematocrit (HCT%) in Diet-Induced Obesity (DIO) mice. (Supplementary Table 1). Collectively, our data suggested that intervention treatment of Diet-Induced Obesity (DIO) mice with SR141716A (HFD + SR) attenuates systemic and local inflammation.

Effect of SR141716A on metabolic parameters

Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT) showed remarkable improvement in metabolic parameters in Diet-Induced Obesity (DIO) mice when treated with SR141716A (HFD + SR) as compared with vehicle-treated Diet-Induced Obesity (DIO) (HFD + Vehicle) and pair-fed (PFSR) control mice (Fig. 3a,b). The intervention treatment of SR141716A in Diet-Induced Obesity (DIO) mice (HFD + SR) improved the serum metabolic parameters such as Fasting Blood Glucose (FBG), triglycerides (TGs), high-density lipoprotein (HDL), low-density lipoprotein (LDL), homeostatic model assessment (HOMA) index) when compared to vehicle-treated Diet-Induced Obesity (DIO) (HFD + Vehicle) and pair-fed to SR141716A (PFSR). (Supplementary Table 2). Additionally, SR141716A treatment reversed the increase in Free Fatty Acids in diet-induced obesity (Supplementary Figure 3).

Figure 3 SR141716A ameliorates metabolic dysfunction in diet-induced obesity. Experiments Diet-Induced Obesity (DIO)were set up as described in Fig. 1 legend. (a) Glucose tolerance test (GTT) and (b) Insulin tolerance test (ITT) of mice fed LFD + Vehicle (n = 10), HFD + Vehicle (n = 10), HFD + SR (n = 9) and Pair-fed to SR141716A (PFSR) (n = 10). Each animal received by oral gavage 1.5 g/kg body mass of glucose (25% D-glucose). Blood glucose levels were determined after 15, 30, 60 and 120 minutes. Insulin-tolerance tests were carried out on un-fasted animals by i.p injection of 1.5 U/kg body mass of insulin. Blood glucose levels were detected after 15, 30, 60 and 120 minutes. Generalized Linear Mixed Model (GLMM) was performed to calculate p values for the repeated measures in SPSS. Mean Area Under the Curve (AUC) from triplicate experiments measured between 0–120 minutes after glucose (GTT) and insulin (ITT) load. AUC was assessed with Trapezoidal rule in R software. Data with different superscript letters are significantly different. GTT (P < 0.01), ITT (P < 0.05). Full size image

Effect of SR141716A on gut barrier integrity

SR141716A counteracted diet-induced colonic mucosal barrier dysfunction during high-fat diet feeding by modulating the mucosal thickness (Fig. 4a,b). The expression of mucus-related genes, Mucin2 (Muc2) and Kruppel-Like Factor 4 (KLF4) were increased with SR141716A treatment in Diet-Induced Obesity (DIO) mice (HFD + SR) when compared with vehicle-treated Diet-Induced Obesity (DIO) mice (HFD + Vehicle), pair-fed to SR141716A (PFSR) controls, and body weight-matched (BWM) controls (Fig. 4c,d). SR141716A did not show any effect on the Trefoil Factor 3 (Tff3) gene expression (Fig. 4e). Trefoil Factor family of secretory proteins are expressed by the gastrointestinal mucus layer. Although their functions are not clear, they are expected to be protective by stabilizing mucus layer and healing disrupted epithelium. To visualize histological changes better in the colon of different groups, the image of the whole colon is included. (Supplementary Figure 4)

Figure 4 SR141716A restores gut barrier function in diet-induced obesity. Experiments Diet-Induced Obesity (DIO) were set up as described in Fig. 1 legend. (a) Representative Periodic Acid Schiff images were used for in situ mucus layer staining, scale bar,100 μm. (b) Thickness of the mucus layer measured by histological image analysis software MetaMorph (LFD + Vehicle, n = 5; HFD + Vehicle, n = 5; SR, n = 6; PFSR, n = 5; and BWM, n = 6). (c–e) mRNA expression analysis by qRT-PCR of mucus-related genes in the colonic mucosa. (f) Intestinal permeability was measured by quantitation of levels of serum FITC-Dextran (4 kDa) following oral gavage (n = 5 except LFD + Vehicle, n = 4). (g) Plasma lipopolysaccharide (LPS) level in Diet-Induced Obesity (DIO) mice treated with SR141716A for four weeks and controls was quantified (n = 5). (h) Myeloperoxidase (MPO) levels in colonic tissue were measured (n = 5). Data are shown as mean \(\pm \) SD. Data with different superscript letters are significantly different (P < 0.05) according to post hoc ANOVA one-way statistical analysis. Full size image

The hallmark of colonic inflammation due to fat-dense diet is gut leakage and subsequently elevated levels of lipopolysaccharide (LPS) and the leukocyte enzyme myeloperoxidase. To examine the effect of the SR141716A treatment on gut integrity, we performed in vivo intestinal permeability assay using an FITC-labelled dextran method. Less leakage in the gut of SR141716A-treated Diet-Induced Obesity (DIO) mice (HFD + SR) was observed when compared to vehicle-treated Diet-Induced Obesity (DIO) (HFD + Vehicle) and pair-fed to SR14716A (PFSR) controls (Fig. 4f). Taken together, these data indicated that SR141716A intervention treatment in Diet-Induced Obesity (DIO) mice (HFD + SR) ameliorates the compromised mucosal layer and gut leakage in Diet-Induced Obesity (DIO) phenotype.

Additionally, our data showed that there was a significant reduction in lipopolysaccharide (LPS) levels in the serum of SR141716A-treated Diet-Induced Obesity (DIO) mice (HFD + SR) when compared to vehicle-treated Diet-Induced Obesity (DIO) (HFD + Vehicle), pair-fed Diet-Induced Obesity (DIO) control (PFSR), and body weight matched-control (BWM) mice (Fig. 4g). Local inflammation in colonic tissue was also determined by assessing the level of myeloperoxidase. SR141716A-treated Diet-Induced Obesity (DIO) mice (HFD + SR) showed significant improvement in colonic inflammation, independent of its effect on weight loss and diet intake (Fig. 4h). Based on such criteria, we propose that improvement in colon morphology following blockade of cannabinoid CB1 receptor in obese phenotype is associated with amelioration of gut inflammation.

Effect of SR141716A on Endocannabinoid (eCB) System

Obesity has been characterized by over-activation of eCB system13. In the current study, we found that CB1 receptor expression was down-regulated with the SR141716A treatment of Diet-Induced Obesity (DIO) mice (HFD + SR) when compared to vehicle-treated Diet-Induced Obesity (DIO) mice (HFD + Vehicle) (Fig. 5a). The level of endogenous ligands of cannabionoid receptors in adipocytes and serum was assessed by Liquid Chromatograph/Mass Spectrometry/Mass Spectrometry (LC/MS/MS). We observed a significant reduction in adipose tissue anandamide (AEA) in Diet-Induced Obesity (DIO) mice treated with SR141716A (HFD + SR) when compared to the control Diet-Induced Obesity (DIO) mice (HFD + Vehicle) (Fig. 5b,c). We were unable to detect significant levels of 2-arachidonyl glycerol (2-AG) in all samples. It is well established that Cannabinoid CB1 receptor agonists increase cannabinoid CB1 receptor activity whereas cannabinoid CB1 receptor antagonists decrease its expression22. While the reasons for this effect are not clear, it is believed that the use of the antagonist leads to increase in endocannabinoids that cannot act on the receptors to activate them.

Figure 5 SR141716A attenuates overactivity of endocannabinoid system in diet-induced obesity. Experiments Diet-Induced Obesity (DIO) were set up as described in Fig. 1 legend. (a) Adipose tissue CB1 mRNA levels in SR141716A-treated Diet-Induced Obesity (DIO) (HFD + SR), vehicle-treated Diet-Induced Obesity (DIO) (HFD + Vehicle), lean mice (LFD + Vehicle) and Pair-fed to SR141716A (PFSR) control mice was assessed by RT-PCR. (b) White adipose tissue AEA levels from the same mice (percent of control values) were measured with Liquid Chromatograph/Mass Spectrometry/Mass Spectrometry (LC/MS/MS) (n = 3). N-archidonoylethanolamine (AEA) levels (percent of LFD + Vehicle) were calculated in the epididymal adipose tissue of HFD + SR, HFD + Vehicle, and PFSR (n = 3). Data are shown as mean \(\pm \) SD. Data with different superscript letters are significantly different (P < 0.05) according to post hoc ANOVA one-way statistical analysis. Full size image

Effect of SR141716A on adipogenic related-genes

Next, we investigated the effect of SR141716A on adipose tissue metabolism, which was assessed by RT-PCR for lipogenesis, oxidation and differentiation genes. We observed that SR141716A treatment of Diet-Induced Obesity (DIO) mice (HFD + SR) increased the mRNA expression of markers of lipid oxidation such as carnitine palmitoyltransferase-1 (CPT1), acyl-CoAoxidase (ACOX1), peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), and peroxisome proliferator-activated receptor alpha (PPARα) (Fig. 6a), as well as adipocyte differentiation including CCAAT/enhancer–binding protein-α (C/EBPα)) and peroxisome proliferator-activated receptor γ (PPARγ) (Fig. 6b). Changes in lipogenic properties of adipose tissue were examined by acetyl-CoA carboxylase (ACC1) and fatty acid synthase (FASN) quantification (Fig. 6c). Together, our data suggested that the shrinkage in fat mass in SR141716-treated Diet-Induced Obesity (DIO) mice (HFD + SR) was associated with an increase in lipid oxidation differentiation and lipogenesis.

Figure 6 SR141716A improves adipose tissue metabolism in diet-induced obesity. Experiments Diet-Induced Obesity (DIO) were set up as described in Fig. 1 legend. mRNA expression of markers of (a) lipid oxidation (CPT1; ACOX1; PGC-1α; and PPARα), (b) adipocyte differentiation (C/EBPα, PPARγ), and (c) lipogenesis (ACC1; FASN) was measured in epididymal fat depots (n = 5). Data are shown as mean ± SD. Data with different superscript letters are significantly different (P < 0.05) according to post hoc ANOVA one-way statistical analysis. Full size image

Effect of SR141716A on dysbiosis of gut microbiota in diet-induced obesity

To test the role of gut microbiota, we performed 16S rRNA metagenomic sequencing of both variable regions (V3 + V4) of fecal samples in different groups of our study (n = 5 per group), and rarefied to a depth of 10,000 reads per sample. We arranged microorganisms in Operational Taxonomic Units (OTUs) to standardize grouping based on 97% similarities in DNA sequence (Supplementary Table 3). The data obtained demonstrated that overall, microbial communities were strongly structured by diet. Alpha diversity was calculated based on the Chao1 index to estimate the diversity of microorganisms in regards to their numbers and their similarities in abundance, (Fig. 7a). Also, beta diversity was studied by principal coordinate analysis (PCoA) wherein we observed significant separation between lean mice and Diet-Induced Obesity (DIO) mice. Interestingly, PCoA performed based on distance matrix demonstrated that microbial community structure was more sensitive to SR141716A treatment than dietary fat intake (Fig. 7b). Relative taxa abundance area plots at the genus taxonomical level for individuals from the five groups was assessed by taking the Operational Taxonomic Units (OUTs) Table at genus level as an input (Supplementary Table 4). Individual mice were represented along the horizontal axis, and related taxa frequency at the genus level was denoted by the vertical axis (Fig. 7c). Our data suggested that Operational Taxonomic Units (OTUs) were differentially enriched within the different groups. To investigate the particular effect of SR141716A on the gut-flora of Diet-Induced Obesity (DIO) mice (HFD + SR), we conducted studies with pair-fed to SR141716A (PFSR) and body-weight matched controls to Diet-Induced Obesity (DIO) mice treated with SR141716A (HFD + SR). Our data indicated significant enrichment of Akkermansia muciniphila OTUs in Diet-Induced Obesity-treated mice with SR141716A (HFD + SR) when compared with both pair-fed obese (PFSR) and body-weight matched (BWM) controls. Interestingly, the significant reduction in families, Lanchnospiraceae and Erysipelotrichaceae with SR141716A treatment was beyond the effect of SR141716A on weight loss and diet intake restriction. Because the disruption in gut mucosal layer was improved in Diet-Induced Obesity (DIO) mice with SR141716A treatment (HFD + SR), we investigated the effect of therapy on residential bacteria of mucosal layer, specifically Akkermansia muciniphila 23,24.

Figure 7 SR141716A alters gut microbiota in diet-induced obesity. Experiments Diet-Induced Obesity (DIO) were set up as described in Fig. 1 legend. Metagenomic analysis was performed on 16S rRNA V3 + V4region data, rarefied to a depth of 10,000 reads per sample. (a) Species richness metric based on Chao1 method was calculated. (b) Beta-diversity of the gut microbiome was evaluated by weighted UniFrac-based principal co-ordinate algorithim. The analysis was performed using the abundance matrix of genus-level Operational Taxonomical Units (OTUs) in different samples, and pairwise community distances were determined with 0.97 similarity using the weighted UniFrac algorithm. (c) Relative taxa abundance area plots for individuals from the five populations, summarized at the genus level. Individuals are represented along the horizontal axis, and relative taxa frequency is denoted by the vertical axis. (d) Lanchnospiraceae (e) Erysipelotrichaceae and (f) A. muciniphila abundance (log 10 of bacteria per g of fecal content) was measured in mice (n = 10). Values with different superscript letters are significantly different, (P < 0.01) according to post hoc ANOVA one-way statistical analysis. Full size image

Numerous studies have shown the inverse correlation between the abundance of A. muciniphila and metabolic syndrome. RT-PCR from the isolated fecal DNA demonstrated significant enrichment in A. muciniphila colonization in SR141716A-treated DIO mice (HFD + SR) when compared to vehicle-treated Diet-Induced Obesity (DIO) (HFD + Vehicle), pair-fed to SR141716A (PFSR), and body weight matched to SR141716A (BWM) (Fig. 7f). Previous studies have demonstrated that Lanchnospiraceae and Erysipelotrichaceae, within Firmicutes phylum are implicated in gaining weight and induction of metabolic syndrome10,25. Our studies confirmed that Lanchnospiraceae and Erysipelotrichaceae were significantly decreased in SR141716A-treated Diet-Induced Obesity (DIO) mice (HFD + SR) when compared to vehicle-treated DIO (HFD + Vehicle) control. Also, the RT-PCR from the fecal content validated the 16s rRNA sequenced data (Fig. 7d,e).

Effect of SR141716A on Short Chain Fatty Acid (SCFA) in Diet-Induced Obesity

To investigate the effect of SR141716 intervention treatment in Diet-Induced Obesity (DIO) mice (HFD + SR) on short-chain fatty acids (SCFA), we quantified the level of short-chain fatty acids (SCFA) in serum, cecal and fecal content of mice. Interestingly, we found a significant increase in the concentration of propionic acid, i-butyric, as well as the n-butyric acid in the cecal and fecal content of SR141716A, treated Diet-Induced Obesity (DIO) (HFD + SR) mice when compared to vehicle-treated Diet-Induced Obesity (DIO) (HFD + Vehicle) mice (Fig. 8a,b). The same trend was observed in the concentration of acetic acid as well as valeric acid, but the changes were not significant. To evaluate the alteration in short-chain fatty acids (SCFA) systemically, we assessed the concentration of short-chain fatty acids (SCFA) in serum. Because the short-chain fatty acids (SCFAs) are mostly abundant in colon and stool, the same trend but at the lower level than short-chain fatty acids (SCFAs) in fecal and cecal content, was observed in short-chain fatty acids (SCFAs) of serum. (Supplementary Figure 5).