www.nature.com/scientificreports Table 1 I Parametric Pearson correlation between levels of hormones related to energy homeostasis/ chemokines and body mass accom- panied by two-tailed p-values 8 Weeks 16 Weeks insulin -0.344 0.016 0724* 0.020 0.586 0.732* 0.025 0787* 0.012 0.431 0.286 . Correlation is significant at the 0.01 level [2-tailed) Furthermore, SPFH mice showed increased insulin production com- SPFH mice had significantly higher fasting leptin, insulin andPYY pared to SPF mice(p <0.05). Interestingly, leptin and insulin levels than SPF mice. Despite the differences in hormonal levels, SPFH in plasma of GFH mice were not altered mice had normal body mass. We postulate that H. pylori-associated up-regulation of PYY stimulated the host to produce more insulin PYY induced by H Pylori but suppressed by Normal Gut Microbiota. and leptin in response, thus sustaining normal body mass. However, eptide YY(PYY) is found in L cells in the mucosa of gastrointestinal the long-term implications of higher fasting PYY, insulin and lepti tract, especially in the ileum and colon. In addition, a small amount of remain to be determined On the other hand, GF and GFH mice had PYY, about 1-10%, is found in the esophagus, stomach, duodenum and higher ghrelin, lower leptin and lower insulin than SPF and SPFH ke leptin, PYY exerts its action through NPY receptors to mice. In addition, 16-week GFh mice had higher PYY level than GF low the gastric emptying that increases the efficiency of digestion and mice. All these hormonal differences between GF and GFH mice in nutrient ab after a meal. Interestingly, the presence of normal comparison to SPF and SPFH mice might be linked to their body gut microbiota in SPF mice appeared to suppressed fasting PYY level masses. Interestingly, the effect of H pylori on body mass was most significantly by 8 weeks and onwards into the experiment(p < 0.01) obvious in GFH mice in the absence of normal gut microbiota ( Figure 2F). On the other hand, H Pylori appeared to up-regulate PYY The relationship between body mass and different gut metabolic production, such that fasting level was significantly higher in SPEH and hormones/ eotaxin-l was determined(Table 1). Leptin and PYY GFH mice compared to SPF(p 005)and GF(p <0.01) levels were shown to be significantly related(p <0.05 )to body mass Fasting PYY level was highest among GFH mice followed by those in both 8-week and 16-week mice in direct and inverse relationship consumption of protein and was observed in experimenta with the respectively. However, ghrelin and insulin levels were not related to to reduce hunger and promote weight loss2?. This would help to directly correlated respectively in 16-week mice (p 0.05 explain the weight-loss experienced with high-protein diets. Surprisingly, eotaxin-1 levels correlated significantly with body mass Therefore, it was not surprising that in this study, we found an initially at 8 weeks(p <0.05)but not at the later 16 weeks(p 20.05) inverse correlation between PYY level and the degree of weight loss. These results suggest that both leptin and PYY influenced body mass Since high level PYY was not linked to high protein diets, it might at an early stage and continue to do so at later stage. On the other suggest increased endogenous protein degradation and therefore a hand, ghrelin and insulin, which became related to body mass loss of body mass. Another factor that might be indirectly linked to a later stage, might be in responds to the initial changes in lept the retarded weight gain observed in GF mice is ghrelin, which PYY levels. Eotaxin-1 might be an indirectly respond to the presents the host's response by decreasing energy expenditure. change in body mass but still have an important influence on the Individuals with anorexia nervosa have high plasma level of ghrelin early development in these young mice compared to individuals who are constitutionally thin as well as In our study, H pylori infection in GFH mice was associated with normal-weight controls. Together, high ghrelin and high PYY high plasma PYY level. This may signify high energy expenditure and could account for the retarded weight gain in GF and GFH mice high rate of metabolism in these mice, which translate to rapid loss of and the animals that might be in a state of malnutrition body mass. However, the presence of normal gut microbiota in SPFH The impact of H pylori on the composition and function of the mice might provide buffering against the direct adverse impact of H. normal gut microbiome has not been fully elucidated. Nevertheless, pylori on body mass. On the other hand, high level of leptin in SPFH in this study, our preliminary results demonstrated that H Pylori mice might suppress food intake and thereby preventing the mice may alter the energy homeostasis of its host, and that the normal from becoming obese over long term. Results on SPFH mice from ut microbiome could make a difference on the effect of H. pylori on this study is consistent with the observation in human subjects that H. pylori eradication resulted in increased BMI. Thus, H. pylori regulating metabolic hormones may play a role in keeping obesity Gut Metabolic Hormones and Body Mass. A meta-analysis at bay reporting on H. pylori and obesity prevalence rates in random human subjects and Possible Impacts of Gut Microbiota and H. pylori on Early Develop iscovered there was an inverse correlation between prevalence of ment. Heijtz et al reported that the same GF mice used in this study the bacteria and rate of overweight/obesity in developed countries". demonstrated increased motor activity and reduced anxiety comp Earlier, circulating meal-associated leptin and ghrelin levels were to SPF mice. This correlated with altered expression of genes with an associated increase in body mass intex( BMI)providing report also demonstrated that GF mice exposed to m he same lown to change after H. pylori eradication in human volunteers regulating motor control and anxiety-like behavior. TI evidence that H. pylori colonization might regulate ghrelin and microbiota early in life had similar characteristics to thos leptin levels, which affects body morphometry o. In this study, H. mice. This observation might be related to impaired nu olonization in young GF mice was shown to result in reduced absorption in the GF mice in the absence of normal gut microbiota, n body mass. which led to malnutrition and weight loss. It is well known that the SCIENTIFIC REPORTS I 5: 8731 I DO1: 10.1038/ srep08731Furthermore, SPFH mice showed increased insulin production com￾pared to SPF mice (p , 0.05). Interestingly, leptin and insulin levels in plasma of GFH mice were not altered. PYY induced by H. pylori but suppressed by Normal Gut Microbiota. Peptide YY (PYY) is found in L cells in the mucosa of gastrointestinal tract, especially in the ileum and colon. In addition, a small amount of PYY, about 1–10%, is found in the esophagus, stomach, duodenum and jejunum25. Like leptin, PYY exerts its action through NPY receptors to slow the gastric emptying that increases the efficiency of digestion and nutrient absorption after a meal26. Interestingly, the presence of normal gut microbiota in SPF mice appeared to suppressed fasting PYY level significantly by 8 weeks and onwards into the experiment (p , 0.01) (Figure 2F). On the other hand, H. pylori appeared to up-regulate PYY production, such that fasting level was significantly higher in SPFH and GFH mice compared to SPF (p , 0.05) and GF (p , 0.01) mice. Fasting PYY level was highest among GFH mice followed by those of GF mice (Figure 2F). High PYY is usually associated with the consumption of protein and was observed in experimental subjects to reduce hunger and promote weight loss27. This would help to explain the weight-loss experienced with high-protein diets. Therefore, it was not surprising that in this study, we found an inverse correlation between PYY level and the degree of weight loss. Since high level PYY was not linked to high protein diets, it might suggest increased endogenous protein degradation and therefore a loss of body mass. Another factor that might be indirectly linked to the retarded weight gain observed in GF mice is ghrelin, which represents the host’s response by decreasing energy expenditure. Individuals with anorexia nervosa have high plasma level of ghrelin compared to individuals who are constitutionally thin as well as normal-weight controls28. Together, high ghrelin and high PYY could account for the retarded weight gain in GF and GFH mice and the animals that might be in a state of malnutrition. The impact of H. pylori on the composition and function of the normal gut microbiome has not been fully elucidated. Nevertheless, in this study, our preliminary results demonstrated that H. pylori may alter the energy homeostasis of its host, and that the normal gut microbiome could make a difference on the effect of H. pylori on its host. Gut Metabolic Hormones and Body Mass. A meta-analysis reporting on H. pylori and obesity prevalence rates in random population samples of more than 100 human subjects and discovered there was an inverse correlation between prevalence of the bacteria and rate of overweight/obesity in developed countries29. Earlier, circulating meal-associated leptin and ghrelin levels were shown to change after H. pylori eradication in human volunteers with an associated increase in body mass intex (BMI) providing evidence that H. pylori colonization might regulate ghrelin and leptin levels, which affects body morphometry30. In this study, H. pylori colonization in young GF mice was shown to result in reduced gain in body mass. SPFH mice had significantly higher fasting leptin, insulin and PYY than SPF mice. Despite the differences in hormonal levels, SPFH mice had normal body mass. We postulate that H. pylori-associated up-regulation of PYY stimulated the host to produce more insulin and leptin in response, thus sustaining normal body mass. However, the long-term implications of higher fasting PYY, insulin and leptin remain to be determined. On the other hand, GF and GFH mice had higher ghrelin, lower leptin and lower insulin than SPF and SPFH mice. In addition, 16-week GFH mice had higher PYY level than GF mice. All these hormonal differences between GF and GFH mice in comparison to SPF and SPFH mice might be linked to their body masses. Interestingly, the effect of H. pylori on body mass was most obvious in GFH mice in the absence of normal gut microbiota. The relationship between body mass and different gut metabolic hormones/eotaxin-1 was determined (Table 1). Leptin and PYY levels were shown to be significantly related (p , 0.05) to body mass in both 8-week and 16-week mice in direct and inverse relationships respectively. However, ghrelin and insulin levels were not related to body mass at 8 weeks (p $ 0.05) but were significantly inversely and directly correlated respectively in 16-week mice (p , 0.05). Surprisingly, eotaxin-1 levels correlated significantly with body mass initially at 8 weeks (p , 0.05) but not at the later 16 weeks (p $ 0.05). These results suggest that both leptin and PYY influenced body mass at an early stage and continue to do so at later stage. On the other hand, ghrelin and insulin, which became related to body mass only at a later stage, might be in responds to the initial changes in leptin and PYY levels. Eotaxin-1 might be an indirectly respond to the initial change in body mass but still have an important influence on the early development in these young mice. In our study, H. pylori infection in GFH mice was associated with high plasma PYY level. This may signify high energy expenditure and high rate of metabolism in these mice, which translate to rapid loss of body mass. However, the presence of normal gut microbiota in SPFH mice might provide buffering against the direct adverse impact of H. pylori on body mass. On the other hand, high level of leptin in SPFH mice might suppress food intake and thereby preventing the mice from becoming obese over long term. Results on SPFH mice from this study is consistent with the observation in human subjects that H. pylori eradication resulted in increased BMI30. Thus, H. pylori regulating metabolic hormones may play a role in keeping obesity at bay. Possible Impacts of Gut Microbiota and H. pylori on Early Develop￾ment. Heijtz et al. reported that the same GF mice used in this study demonstrated increased motor activity and reduced anxiety compared to SPF mice8 . This correlated with altered expression of genes regulating motor control and anxiety-like behavior. The same report also demonstrated that GF mice exposed to normal gut microbiota early in life had similar characteristics to those of SPF mice. This observation might be related to impaired nutritional absorption in the GF mice in the absence of normal gut microbiota, which led to malnutrition and weight loss. It is well known that the Table 1 | Parametric Pearson correlation between levels of hormones related to energy homeostasis/chemokines and body mass accom￾panied by two-tailed p-values 8 Weeks 16 Weeks Correlation Coefficient Significance (2-tailed) Correlation Coefficient Significance (2-tailed) Ghrelin 20.344 0.364 20.803* 0.016 Leptin 0.724* 0.027 0.788* 0.020 Insulin 0.586 0.098 0.838** 0.009 PYY 20.732* 0.025 20.873** 0.005 Eotaxin 0.787* 0.012 0.431 0.286 *. Correlation is significant at the 0.05 level (2-tailed). **. Correlation is significant at the 0.01 level (2-tailed). www.nature.com/scientificreports SCIENTIFIC REPORTS | 5 : 8731 | DOI: 10.1038/srep08731 4