Gut Microbiota Modulated by Probiotics And

www.nature.com/scientificreports OPEN Gut microbiota Modulated by Probiotics and Garcinia cambogia Extract Correlate with Weight Gain Received: 18 April 2016 Accepted: 31 August 2016 and Adipocyte Sizes in High Fat-Fed Published: 23 September 2016 Mice Jaeyoung Heo1,*, Minseok Seo1,2,*, Hwanhee Park3, Woon Kyu Lee3, Le Luo Guan4, Joon Yoon2, Kelsey Caetano-Anolles5, Hyeonju Ahn5, Se-Young Kim6, Yoon-Mo Kang6, Seoae Cho1 & Heebal Kim1,2,5 Results of recent studies on gut microbiota have suggested that obesogenic bacteria exacerbate obesity and metabolic dysfunction in the host when fed a high fat diet (HFD). In order to explore obesity-associated bacterial candidates and their response to diet, the composition of faecal bacterial communities was investigated by analyzing 16S rRNA gene sequences in mice. Dietary intervention with probiotics and Garcinia cambogia extract attenuated weight gain and adipocyte size in HFD-fed mice. To identify obesity-causative microbiota, two statistical analyses were performed. Forty-eight bacterial species were found to overlap between the two analyses, indicating the commonly identified species as diet-driven and obesity-associated, which would make them strong candidates for host- microbiome interaction on obesity. Finally, correlation based network analysis between diet, microbe, and host revealed that Clostridium aminophilum, a hyper-ammonia-producing bacterium, was highly correlated with obesity phenotypes and other associated bacteria, and shown to be suppressed by the combination of probiotics and Garcinia cambogia extract. Results of the present study suggest that probiotics and Garcinia cambogia extract alleviate weight gain and adiposity, in part via differentially modulating the composition of gut microbiota in HFD fed mice. Obesity is a rapidly growing global health epidemic which confers great risk for the development of serious health issues such as diabetes, cardiovascular disease, and cancer1. Host-microbe studies unveiled the role of gut microbiota modifying immune responses2, inflammation and insulin resistance in the development of diet-induced obesity in animals3. Recent studies which involved transplanting faecal microbiota from obese humans to germ-free mice revealed that the gut microbiota itself may induce obesity2,3. Recent findings in the proportion of two major bacterial phyla (Firmicutes and Bacteriodetes) and their ratio between obese and lean samples (in humans and animals)4,5 have promoted the investigation of microbial candidates associated with obesity. Although growing evidence suggests positively or negatively obesity-associated microbes at various taxa6, only a few species have been well studied and understood6–9. During the past decade, many researchers have focused on studying Firmicutes and its characteristic of increasing the capacity of energy harvest from indigestible sources in diet5,10,11, mainly due to carbohydrate 1C&K genomics, Seoul National University Research Park, Seoul 151-919, Republic of Korea. 2Interdisciplinary Program in Bioinformatics, Seoul National University, Seoul 151-742, Republic of Korea. 3Laboratory of Developmental Genetics, Department of Biomedical Sciences, Inha University School of Medicine, Incheon 22212, Republic of Korea. 4Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada. 5Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea. 6R&D center, CTCBIO, Inc. 38, Hyundaikia-ro, Paltan-myeon, Hwaseong-si, Gyeonggi-do, 18576, Republic of Korea. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to S.C. (email: [email protected]) or H.K. (email: [email protected]) SCIENTIFIC REPORTS | 6:33566 | DOI: 10.1038/srep33566 1 www.nature.com/scientificreports/ metabolism processes. However, recent studies have observed that a western-style diet increases gut microbial amino acid metabolism in humans and animals8,12. High-fat feeding particularly increases microbial proteins of amino acid metabolism, which leads to the exceeding changes of gut microbial community composition in mice12. Short-term consumption of an animal-based diet compared to a plant-based diet significantly increases the faecal concentration of branched short chain fatty acids such as isovalerate and isobutyrate from amino acid fermentation in humans, indicating a quick shift between carbohydrate and amino acid fermentation in gut microbiota8. Previous studies have indicated that dietary intervention such as probiotics and polyphenol-rich fruit extracts changes the composition of gut microbiota, and reduces visceral adiposity and obesity in animals13–16. Probiotics change the proportions of metabolic syndrome-related phylotypes in high fat fed mice, which differentially influences gut microbiota14. Garcinia cambogia extract containing hydroxycitric acid, known as an inhib- itor of ATP-citrate-lyase in fatty acid biosynthesis, is polyphenol-rich and shows antibacterial and antioxidant properties. In this study, we aimed to investigate which gut microbes respond to dietary intervention such as high fat, probiotics, and Garcinia cambogia extract, as well as whether they are associated with obesity phenotypes in diet-induced obese mice. After 9 weeks of dietary intervention, three types of metagenome analysis on faecal microbial communities were performed to detect obesity-causative microbes responding to diet: 1) identification of differentially abundant microbiota (DAM); 2) identification of obesity trait-associated microbiota (TAM); and 3) network analysis in order to consider comprehensive information underlying gut microbiome features of diet-microbe interaction, host-microbe interaction, microbe-microbe interaction, and finally all combined interactions. Results Adipocyte sizes sensitively respond to a high-fat diet. To evaluate obesity traits responding to diet, male C57BL/6J mice were fed a low-fat diet (LFD), HFD, HFD with probiotics (HFD + P) and HFD with a combination of probiotics and Garcinia cambogia extract (HFD + P + G) for 9 weeks, respectively (diet information provided in Table S1, n = 7 for each treatment). Body weight was significantly lower in LFD mice after 4 weeks and in HFD + P + G mice after 6 weeks compared to HFD mice at 5% significance level (Fig. 1a and Table S2). Body weight gain was 58%, 13%, 31% lower in LFD mice, HFD + P mice, and HFD + P + G mice, respectively, compared to HFD mice (Table S3). All fat pad mass proportions to body weight in LFD were lower compared to HFD mice (Table S3). The mean sizes of adipocytes from perirenal, epididymal, and mesenteric adipose tissues (PAT, EAT, and MAT, respectively) were lower in HFD + P mice than those from HFD mice (Fig. 1b), except for subcutaneous (SAT) adipocyte. The density plots of adipocyte sizes showed a similar pattern, with mean values arranged in order from LFD to HFD groups (Fig. 1c). Adipocyte sizes of each mouse showed individual variabil- ity responding to diet which we determine, in part, as a result of gut microbiota in this study (Fig. S1a). Moreover, the box-plot patterns were very similar between weight gain and adipocyte mean sizes (Fig. 1b and Fig. S1b), which was concordant with representative tissue-staining images, presenting four kinds of adipocyte sizes and hepatic steatosis gradually increased from LFD to HFD (Fig. 1d). Change of serum biochemical parameters for protein metabolism in response to diet. The serum biochemical parameters were further analyzed to investigate host metabolism in response to diet. Blood urea nitrogen (BUN) and creatinine levels in the serum of HFD and HFD + P mice were found to be significantly lower than those in LFD and HDP + P + G mice (P < 0.001 and P < 0.01, respectively, Fig. S1c). Spearman correlation coefficients among diet, obesity traits and protein metabolism showed that diet order ascending from HFD to LFD had a negative correlation with obesity traits, particularly EAT adipocyte mean size (Correlation coefficient and P-value are − 0.89 and 3.65E-10, respectively), and a positive correlation with serum BUN (0.65 and 1.99E-4) and creatinine (0.76 and 2.74E-06) (Fig. S1d). BUN and creatinine were negatively correlated with obesity traits, particularly with EAT adipocyte mean size (− 0.66 [1.35E-04] and − 0.65 [1.82E-04], respectively). Through correlation analysis, we observed strong linear relationships among diet, obesity traits and protein metabolism. Characterizing changes in microbial abundance in response to diet. Differentially abundant microbiota (DAM) analysis of 494 annotated operational taxonomic units (OTUs) at the species level was performed to characterize the changes of microbial species in response to diet (Dataset S1). Four types of comparisons were performed: a multi-group test (H1: at least one group ≠ 0 in the four dietary ingredients), HFD vs HFD + P, HFD vs HFD + P + G, and HFD vs LFD (descripted in Supplementary Methods). Of these statistical tests, we focused primarily on the result of the multi-group test given our primary goal of detecting causative bacterial species for obesity-related traits in response to diet. As a result, 266 significant differentially abundant species were identified with false discovery rate (FDR) adjusted P-value < 0.05. The distribution of significantly detected species at the phylum level revealed that most of the annotated

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