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1 Accepted Article Preview: Published ahead of advance online publication Omega-3 fatty acids prevent early-life antibiotic exposureinduced gut microbiota dysbiosis and later-life obesity K Kaliannan, B Wang, X-Y Li, A K Bhan, J X Kang Cite this article as: K Kaliannan, B Wang, X-Y Li, A K Bhan, J X Kang, Omega-3 fatty acids prevent early-life antibiotic exposure-induced gut microbiota dysbiosis and later-life obesity, International Journal of Obesity accepted article preview 15 February 2016; doi: /ijo This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply. Received 31 July 2015; revised 12 December 2015; accepted 13 January 2016; Accepted article preview online 15 February 2016

2 Omega-3 fatty acids prevent early-life antibiotic exposureinduced gut microbiota dysbiosis and later-life obesity Kanakaraju Kaliannan 1, Bin Wang 1, Xiang-Yong Li 1, Atul K. Bhan 2, Jing X. Kang 1 * 1 Laboratory of Lipid Medicine and Technology, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02129, USA 2 Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA *Author to whom all correspondence should be addressed: Jing X. Kang, M.D., Ph.D. Laboratory for Lipid Medicine & Technology Department of Medicine Massachusetts General Hospital th Street Boston, MA Tel: (617) Fax: (617) kang.jing@mgh.harvard.edu Running Title: Omega-3 prevents antibiotic-induced obesity Disclosures: The authors have declared that no conflict of interest exists. Keywords: Antibiotics; omega-3; n-6/n-3 ratio; gut microbiota; obesity; metabolic syndrome 1

3 Abstract Early-life antibiotic exposure can disrupt the founding intestinal microbial community and lead to obesity later in life. Recent studies show that omega-3 fatty acids can reduce body weight gain and chronic inflammation through modulation of the gut microbiota. We hypothesize that increased tissue levels of omega-3 fatty acids may prevent antibiotic-induced alteration of gut microbiota and obesity later in life. Here, we utilize the fat-1 transgenic mouse model, which can endogenously produce omega-3 fatty acids and thereby eliminates confounding factors of diet, to show that elevated tissue levels of omega-3 fatty acids significantly reduce body weight gain and the severity of insulin resistance, fatty liver, and dyslipidemia resulting from early-life exposure to azithromycin (AZT). These effects were associated with a reversal of antibiotic-induced dysbiosis of gut microbiota in fat-1 mice. These results demonstrate the beneficial effects of omega-3 fatty acids on antibiotic-induced gut dysbiosis and obesity, and suggest the potential utility of omega-3 supplementation as a safe and effective means for the prevention of obesity in children who are exposed to antibiotics. 2

4 Introduction Early exposure to antibiotics is now linked with an increased susceptibility to obesity later in life (1, 2). The widespread use of antibiotics during childhood could therefore contribute significantly to the modern prevalence of obesity. Azithromycin (AZT) is among the most common broad-spectrum antibiotics prescribed for children (3), and has been reported to induce obesity in children with cystic fibrosis (4). Thus, identification of safe and effective means for preventing antibiotic-induced obesity is warranted Gut dysbiosis is a critical factor in the development of obesity and metabolic syndrome, possibly due to increased energy harvest, production of toxic bacterial metabolites, and greater intestinal permeability leading to elevated levels of lipopolysaccharides (LPS) in systemic circulation and consequently, a low-grade systemic inflammatory state (5). An increased proportion of Firmicutes to Bacteroidetes is recognized as a marker of obesity (6). As antibiotics can significantly alter gut microbiota composition as well as the host response to specific microbial signals (7), disruption of the gut microbiota appears to be a key mechanism underlying antibiotic-induced obesity Recent studies have demonstrated the beneficial effects of omega-3 polyunsaturated fatty acids (n-3 PUFA) on obesity and the gut microbiota (8, 9). Elevating tissue levels of n-3 PUFA can significantly reduce growth of gram-negative Escherichia coli and increase growth of beneficial bifidobacteria and lactobacilli (8), reducing LPS production and thereby suppressing endotoxemia, chronic low-grade inflammation, and metabolic syndrome (9). Omega-3 PUFA supplementation has also been shown to reduce the Firmicutes/Bacteroidetes ratio and fat 3

5 accretion in animals fed a high-fat diet (10, 11). In this context, we hypothesize that increased tissue levels of n-3 PUFA can prevent the changes in the gut microbiota and onset of later-life obesity caused by early exposure to antibiotics. To test this hypothesis, we used the Fat-1 transgenic mouse model to determine the effects of n-3 PUFA on antibiotic-induced changes in gut microbiota and metabolic syndrome. The Fat-1 mouse expresses the Caenorhabditis elegans fat-1 gene and endogenously produces n-3 PUFA from n-6 PUFA without the need for n-3 PUFA supplementation (Figure S1), thereby eliminating confounding factors of diet (12) Results and Discussion Five-week-old Fat-1 mice and their wild-type (WT) littermates received three intermittent courses of AZT over five weeks, were allowed to recover for six weeks, and then fed a Western diet (high in carbohydrates and fat) for 14 weeks (Figure S2). Compared to the control group, WT+AZT mice gained significantly more body weight after being fed the Western diet (20.04±1.07 vs ±0.44 g, WT vs. WT+AZT, respectively, p<0.001; Figure 1A), primarily due to increased white adipose tissue weight (Figure 1B). Levels of fasting blood glucose (28% increase, p<0.001), fasting serum insulin (25% increase, p<0.05), and HOMA-IR (61% increase, p<0.001) were also significantly elevated by AZT treatment in WT mice (Figure 1C). In contrast, the Fat-1+AZT group did not exhibit significant body weight gain compared to the control Fat-1 group (Figure 1A, B), and changes in fasting blood glucose, fasting serum insulin, and HOMA-IR were much smaller than those in the WT+AZT group (Figure 1C). Food intake and antibiotic water consumption did not differ among groups (Figure S3). Furthermore, WT+AZT mice exhibited marked development of fatty liver disease compared to the untreated WT mice, as shown by histological staining, liver weight (17% increase, p<0.05), hepatic 4

6 triglyceride (TG) levels (22% increase, p<0.05), and ALT (77% increase, p<0.05) and AST (149% increase, p<0.05) enzymatic activity (Figure 1D). Conversely, these markers of fatty liver disease were not increased in Fat-1+AZT mice (Figure 1D). Serum lipid profile abnormalities in total cholesterol (48% increase, p<0.01), TG (44% increase, p<0.01), and low-density lipoprotein-cholesterol (LDL-C) (8% increase, p<0.01) were also observed in WT+AZT mice compared to their untreated WT counterparts, while Fat-1+AZT mice were largely protected against these changes (Figure 1E) To determine the effects of antibiotics on the gut microbiota and whether increased tissue n-3 PUFA could protect against these effects, fecal qpcr analyses were performed to quantify major gut microbial phyla at various time points (Figure S2). Following antibiotic treatment, significant shifts in the Firmicutes/Bacteroidetes ratio (due to increased Firmicutes and decreased Bacteroidetes levels) were observed in both WT and Fat-1 mice, compared to their untreated counterparts (Figures 2A, B & S4). During recovery, WT+AZT mice exhibited a further increase in Firmicutes proportions compared to the other groups, while Fat-1+AZT mice showed markedly recovered levels of Bacteroidetes (Figure 2A, B) and subsequently, a significantly reduced Firmicutes/Bacteroidetes ratio compared to the WT+AZT mice (0.78±0.15 vs. 1.45±0.08, Fat-1+AZT vs. WT+AZT, respectively, p<0.05; Figure 2B). After a Western diet challenge, Firmicutes levels increased roughly 2-fold in all groups, and both the WT and Fat-1 treatment groups exhibited higher Firmicutes/Bacteroidetes ratios than their untreated counterparts (Figure 2A, B). However, Bacteroidetes levels were significantly less decreased in Fat-1+AZT mice compared to the WT+AZT mice, resulting in a significantly lower Firmicutes/Bacteroidetes ratio (4.53±0.84 vs ±4.81, Fat-1+AZT vs. WT+AZT, 5

7 70 71 respectively, p<0.05; Figure 2A, B). These results indicate that elevated tissue n-3 PUFA can attenuate the antibiotic-induced increase of the Firmicutes/Bacteroidetes ratio later in life Furthermore, greater recovery of Bifidobacterium and reduced abundance of Enterobacteriaceae in Fat-1+AZT mice led to a significantly higher Bifidobacterium/Enterobacteriaceae ratio a well-established marker of colonization resistance to opportunistic pathogens (13) during the recovery period, compared to the WT+AZT mice (2.34±0.20 vs. 0.27±0.04, Fat-1+AZT vs. WT+AZT, respectively, p<0.01; Figure 2C). Fat-1+AZT mice also exhibited dramatically lower levels of Proteobacteria (Figure 2D), including the opportunistic pathogen E.coli (Figure 2E), compared to WT+AZT mice. Accordingly, markers of metabolic endotoxemia (LPS and LPSbinding protein (LBP)) and chronic low-grade inflammation (TNF-α, IL-1β, IL-6, and MCP-1) were significantly reduced in Fat-1+AZT mice (Figures S5 & S6). These findings suggest that elevated tissue n-3 PUFA can reduce the antibiotic-induced increase of pathogenic bacteria and related endotoxemia and inflammation Our recent study has clearly demonstrated the capability for n-3 PUFA in modulating gut microbiota by using germ free mouse model and co-housing experiments (9). A series of experiments, including fecal transfer, gene expression and enzymatic assays, has also revealed that elevated tissue omega-3 fatty acids enhance intestinal expression and secretion of intestinal alkaline phosphatase (IAP), which induces changes in the gut bacteria composition resulting in decreased lipopolysaccharide production and gut permeability, and ultimately, reduces the severity of inflammation and metabolic syndrome (9). These results support the notion that the 6

8 92 93 observed changes in the gut microbiota profile were a result of elevating tissue levels of omega-3 fatty acids A higher Firmicutes/Bacteroidetes ratio (FIR: BAC) reflects greater energy extraction from the diet, and interventions that reduce this ratio have been shown to prevent obesity in animals and humans (14, 15). Increased Enterobacteriaceae and decreased Bifidobacterium levels are known to contribute to metabolic endotoxemia and chronic low-grade inflammation (16), which are important factors for the development of obesity. Although 16s rrna pyrosequencing can provide substantial information on gut microbiota, the key markers of gut microbiota associated with obesity development can be adequately measured by qpcr. Our findings that elevating tissue n-3 PUFA reduces the antibiotic-induced increase in the Firmicutes/Bacteroidetes ratio, improves the Bifidobacterium/Enterobacteriaceae ratio, and prevents obesity, indicate the capability of n-3 PUFA in the management of antibiotic-related obesity The present study underscores the importance of sufficient n-3 PUFA intake during the perinatal period, especially given the frequency of antibiotic use in infants and children. The results from the fat-1 transgenic mouse model demonstrate that the beneficial effects are derived from elevated tissue levels of n-3 PUFA, rather than other dietary components. We have previously shown that elevated tissue n-3 PUFA status and the beneficial effects observed in the fat-1 mice could also be achieved through dietary intervention (9). WT mice supplemented with n-3 PUFA for 2 months exhibited a tissue essential fatty acid profile, gut bacterial profile, and inflammatory status comparable to those of the fat-1 transgenic mice (9). These findings suggest that dietary supplementation with n-3 PUFA can also confer similar protection against antibiotic-related 7

9 obesity as observed in the fat-1 mice, and warrant further investigation. In this context, there may be great clinical utility for n-3 PUFA supplementation to protect individuals from the adverse metabolic effects associated with early antibiotic exposure In summary, we have demonstrated that early antibiotic exposure can lead to gut dysbiosis and obesity later in life, and that elevating tissue n-3 PUFA can largely prevent these problems. Our findings provide insight into the potential utility of n-3 PUFA in preventing antibiotic-related obesity. 8

10 Author Contributions: J.X.K. and K.K designed the study; K.K., B.W., X-Y.L., and A.K.B. performed experiments; K.K. and J.X.K. analyzed data and wrote the paper. Acknowledgments: This study was supported by the generous funding from Sansun Life Sciences and the Fortune Education Foundation. The authors are also grateful to Marina Kang for her editorial assistance and Amy Goodale for her experimental assistance. 9

11 Figure Legends Figure 1. Omega-3 fatty acids prevent antibiotic-induced obesity and metabolic changes. Male wild-type (WT) and Fat-1 mice were treated with AZT over five weeks (Antibiotics), allowed to recover for six weeks (Recovery), and then fed a Western diet for 14 weeks (Western diet). Body weight and metabolic parameters were monitored throughout. Data are expressed as mean ± SE. Data with different superscript letters are significantly different (P < 0.05) according to ANOVA followed by Bonferroni's multiple comparisons test. n = 5 for WT and Fat-1; n=6 for WT+AZT and Fat-1+AZT. Figure 2. Omega-3 fatty acids protect against antibiotic-induced changes in gut microbiota. Stool samples collected from the four animal groups at different time points were tested for 16s rrna copy numbers of various bacteria using qpcr. Data are expressed as mean ± SE. For twogroup comparisons, ***P < Data with different superscript letters are significantly different (P < 0.05) according to ANOVA followed by Bonferroni's multiple comparisons test. n = 5 for WT and Fat-1; n=6 for WT+AZT and Fat-1+AZT. 10

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