www.nature.com/scientificreports colonization is life-long unless eradicated through medical interven- pancreas, ovary and adrenal cortex. In the brain, ghrelin-producing ion. Although most carriers remain asymptomatic, about 20% of neurons have been identified in the pituitary and the hypothalamus them eventually develop gastro-duodenal diseases(such as chronic glands". In the stomach and small intestine, ghrelin is released into gastritis, peptic ulcer and gastric adenocarcinoma) in the later stages circulation as acyl-ghrelin. Leptin(satiety hormone ) is produced of life. Many factors, including H. Pylori, gut microbiota, the host mainly in the adipocytes of white adipose tissue. Brown adios and the environment, may contribute to the development of H. tissue, syncytiotrophoblasts in the placenta,ovaries, skeletal muscle, stomach(the lower part of the fundic glands), mammary In this study, we aimed to investigate the interplay between H. epithelial cells, bone marrow, pituitary, liver, gastric chief cells and P/ ylori and normal gut microbiota during early stages of life using GF DI cells also produce leptin 7. Both ghrelin and leptin receptors are P and SPF mice models. In order to understand the influences of H. localized on the same brain cells(mainly in the hypothalamus), pylori and normal gut microbiota on the development of metabolic therefore these cells receive competing signals of satiety and nd immune systems, we assessed the changes of gut metabolic hor- hunger. In this study, we showed that fasting plasma samples of nones and cytokines/chemokines in response to H pylori colonia- GF and GFH mice had significantly higher ghrelin level by 16 weeks tion in both gFH and SPFH mice into the experiment than SPF and SPFH mice(p<0.01), whether or not they were H pylori colonized(Figure 2A). Therefore, normal gut Results and discussion microbiota, but not H. Pylori, had significant influence on mices We recovered H pylori from inoculated SPFH and GFH mice at 2-, ghrelin level. On the contrary, GF mice had significantly lower 8-and 16-weeks post-colonization. This showed that H pylori strain leptin level than SPF mice(p <0.05)(Figure 2B). Normal gut 298 successfully colonized the stomach of the mice inoculated with microbiota, independent of H. pylori, was sufficient to up-regulate this organism. On the other hand, none of the SPF and GF mice was leptin production and down-regulate ghrelin secretion for H. pylori In addition, H Pylori colonization caused significantly increased In this study, we measured the weight of the control SPF, H pylori- leptin production in SPFH mice compared to SPF mice(p <0.01), ionized SPFH, control GF and H pylori-colonized GFH mice at 2, which became obvious by 8-week post-colonization(Figure 2B). In and 16-weeks post-colonization(Figure 1). At the end of 16 weeks, contrast, H pylori did not cause higher leptin secretion in GFH mice either SPF nor SPFH mice suffered from any significant weight loss compared to GF mice in the absence of normal gut microbiota. Thus, (p >0.05). However, GFH mice suffered from significant retarded H. pylori and normal gut microbiota resulted in up-regulated leptin weight gain compared to GF mice(p <0.01). This showed that in the but not ghrelin production in SPFH mice absence of normal gut microbiota, H. Pylori colonization caused significant reduced weight gain. GF mice weighed significantly less Normal Gut Microbiota and H pylori induced Insulin Production compared to their SPF counterparts(p 0.01), which is consistent Gastric inhibitory polypeptide(GIP) and glucagon-like peptide-1 with previous reports from other research groups 213. Earlier studies (GLP-1) belong to a class of molecules referred as incretins. L-cells have shown that germ-free mice needed 30% more calories com- in the ileum secrete GLP-1 postprandial while K-cells, which are ed to conventional mice in order to keep a similar body mass". found in the mucosa of the duodenum and the jejunum, synthesize imilarly, another study demonstrated that germ-free mice ate more GiP. The incretins play an important role in stimulating insulin but had considerably less body fat than conventional mice. In the production in response to the presence of nutrients in the intestine same study, germ-free mice that were inoculated with microbiota Pancreatic B-islet cells produce insulin, which is the most essential harvested from the distal intestine(cecum)of conventionally raised hormone controlling glucose and energy homeostasis!. Normal gut mice gained 60% of body fat. Indeed, normal gut microbiota plays an microbiome could contribute nutrients and energy to the host via the important role in determining energy harvest from the diet and fermentation of non-digestible dietary components, thereby directly rgy storage in its host. However, the role of H. pylori and its stimulating insulin production in the host Actions of insulin on cells interaction with normal gut microbiota have not been studied muscle cells in the form of glycogen,( 2) prrage of glucose in liver and Gut Microbiota and H pylori influenced Leptin and Ghrelin blood lipids and converting to triglycerides,(3)prompting adipose Levels. Leptin and ghrelin, along with many other hormones, tissue to synthesize fats (i.e, triglycerides)from fatty acid esters,(4) participate in the complex process of energy homeostasis. Ghrelin prompting cells to absorb circulating amino acids, and (5)decreasing ("hunger hormone,")was originally isolated from the stomach, but protein breakdown?". Insulin has a stimulatory effect on leptin, ghrelin has also been identified in other peripheral tissues, such as the which is produced by fat cells". In turn, leptin acts on receptors in here it inhibits h by (1)counteracting Body Weight the effects of neuropeptide Y,(2)counteracting the effects of anandamide, and (3)promoting the synthesis of a-melanocyte simulating hormone(a-MSH). Leptin also interacts with amylin to reduce gastric emptying and creating a feeling of fullness". They work together to maintain energy homeostasis ·SPF We showed that GLP-1 was significantly lower in SPF, SPFH and SPFH GFH mice in comparison to GF mice(p <0.05)when measured at 8 o--GF and 16-weeks post-colonization(Figure 2C). A similar trend was also GFH observed for another member of the incretin family, GIP. However the increase of GIP in GF mice was not statistically significant Figure 2D). This suggested that microbial colonization(H. pylori or normal gut microbiota alone or together)down-regulated the expression of host GLP-I or accelerated the degradation of incretins. Figure 1 Mean body mass of mice. GF mice weighed significantly less However, insulin level in our study did not correlate with levels of the compared to their SPF counterparts(p <0.01). At the end of 16 weeks, incretins. Fasting plasma insulin was low in GF mice despite higher neither SPF nor SPFH mice suffered from any significant weight loss levels of incretins(Figure 2E). Fasting insulin level was significantly (p>0.05). However, GFH mice suffered from significant weight loss higher in 16-week SPF mice than in GF mice(p <0.01), suggesting compared to GF mice(p 0.01) that normal gut microbiota could influence circulating insulin level. SCIENTIFIC REPORTS I 5: 8731 I DO1: 10.1038/ srep08731colonization is life-long unless eradicated through medical interven￾tion9 . Although most carriers remain asymptomatic, about 20% of them eventually develop gastro-duodenal diseases (such as chronic gastritis, peptic ulcer and gastric adenocarcinoma) in the later stages of life11. Many factors, including H. pylori, gut microbiota, the host and the environment, may contribute to the development of H. pylori-associated diseases. In this study, we aimed to investigate the interplay between H. pylori and normal gut microbiota during early stages of life using GF and SPF mice models. In order to understand the influences of H. pylori and normal gut microbiota on the development of metabolic and immune systems, we assessed the changes of gut metabolic hor￾mones and cytokines/chemokines in response to H. pylori coloniza￾tion in both GFH and SPFH mice. Results and Discussion We recovered H. pylori from inoculated SPFH and GFH mice at 2-, 8- and 16-weeks post-colonization. This showed that H. pylori strain 298 successfully colonized the stomach of the mice inoculated with this organism. On the other hand, none of the SPF and GF mice was positive for H. pylori. In this study, we measured the weight of the control SPF, H. pylori￾colonized SPFH, control GF and H. pylori-colonized GFH mice at 2-, 8- and 16-weeks post-colonization (Figure 1). At the end of 16 weeks, neither SPF nor SPFH mice suffered from any significant weight loss (p . 0.05). However, GFH mice suffered from significant retarded weight gain compared to GF mice (p , 0.01). This showed that in the absence of normal gut microbiota, H. pylori colonization caused significant reduced weight gain. GF mice weighed significantly less compared to their SPF counterparts (p , 0.01), which is consistent with previous reports from other research groups12,13. Earlier studies have shown that germ-free mice needed 30% more calories com￾pared to conventional mice in order to keep a similar body mass12. Similarly, another study demonstrated that germ-free mice ate more but had considerably less body fat than conventional mice13. In the same study, germ-free mice that were inoculated with microbiota harvested from the distal intestine (cecum) of conventionally raised mice gained 60% of body fat. Indeed, normal gut microbiota plays an important role in determining energy harvest from the diet and energy storage in its host. However, the role of H. pylori and its interaction with normal gut microbiota have not been studied. Gut Microbiota and H. pylori influenced Leptin and Ghrelin Levels. Leptin and ghrelin, along with many other hormones, participate in the complex process of energy homeostasis. Ghrelin (‘‘hunger hormone’’) was originally isolated from the stomach, but ghrelin has also been identified in other peripheral tissues, such as the pancreas, ovary and adrenal cortex14. In the brain, ghrelin-producing neurons have been identified in the pituitary and the hypothalamus glands15. In the stomach and small intestine, ghrelin is released into circulation as acyl-ghrelin16. Leptin (‘‘satiety hormone’’) is produced mainly in the adipocytes of white adipose tissue. Brown adipose tissue, syncytiotrophoblasts in the placenta, ovaries, skeletal muscle, stomach (the lower part of the fundic glands), mammary epithelial cells, bone marrow, pituitary, liver, gastric chief cells and P/ D1 cells also produce leptin17. Both ghrelin and leptin receptors are localized on the same brain cells (mainly in the hypothalamus), therefore these cells receive competing signals of satiety and hunger18. In this study, we showed that fasting plasma samples of GF and GFH mice had significantly higher ghrelin level by 16 weeks into the experiment than SPF and SPFH mice (p , 0.01), whether or not they were H. pylori colonized (Figure 2A). Therefore, normal gut microbiota, but not H. pylori, had significant influence on mice’s ghrelin level. On the contrary, GF mice had significantly lower leptin level than SPF mice (p , 0.05) (Figure 2B). Normal gut microbiota, independent of H. pylori, was sufficient to up-regulate leptin production and down-regulate ghrelin secretion. In addition, H. pylori colonization caused significantly increased leptin production in SPFH mice compared to SPF mice (p , 0.01), which became obvious by 8-week post-colonization (Figure 2B). In contrast, H. pylori did not cause higher leptin secretion in GFH mice compared to GF mice in the absence of normal gut microbiota. Thus, H. pylori and normal gut microbiota resulted in up-regulated leptin but not ghrelin production in SPFH mice. Normal Gut Microbiota and H. pylori induced Insulin Production. Gastric inhibitory polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) belong to a class of molecules referred as incretins. L-cells in the ileum secrete GLP-1 postprandial19 while K-cells, which are found in the mucosa of the duodenum and the jejunum, synthesize GIP20. The incretins play an important role in stimulating insulin production in response to the presence of nutrients in the intestine. Pancreatic ß-islet cells produce insulin, which is the most essential hormone controlling glucose and energy homeostasis21. Normal gut microbiome could contribute nutrients and energy to the host via the fermentation of non-digestible dietary components, thereby directly stimulating insulin production in the host. Actions of insulin on cells include: (1) prompting the uptake and storage of glucose in liver and muscle cells in the form of glycogen, (2) prompting fat cells to take in blood lipids and converting to triglycerides, (3) prompting adipose tissue to synthesize fats (i.e., triglycerides) from fatty acid esters, (4) prompting cells to absorb circulating amino acids, and (5) decreasing protein breakdown22. Insulin has a stimulatory effect on leptin, which is produced by fat cells22. In turn, leptin acts on receptors in the hypothalamus, where it inhibits hunger by (1) counteracting the effects of neuropeptide Y, (2) counteracting the effects of anandamide, and (3) promoting the synthesis of a-melanocyte￾stimulating hormone (a-MSH)23. Leptin also interacts with amylin to reduce gastric emptying and creating a feeling of fullness24. They work together to maintain energy homeostasis. We showed that GLP-1 was significantly lower in SPF, SPFH and GFH mice in comparison to GF mice (p , 0.05) when measured at 8- and 16-weeks post-colonization (Figure 2C). A similar trend was also observed for another member of the incretin family, GIP. However, the increase of GIP in GF mice was not statistically significant (Figure 2D). This suggested that microbial colonization (H. pylori or normal gut microbiota alone or together) down-regulated the expression of host GLP-1 or accelerated the degradation of incretins. However, insulin level in our study did not correlate with levels of the incretins. Fasting plasma insulin was low in GF mice despite higher levels of incretins (Figure 2E). Fasting insulin level was significantly higher in 16-week SPF mice than in GF mice (p , 0.01), suggesting that normal gut microbiota could influence circulating insulin level. Figure 1 | Mean body mass of mice. GF mice weighed significantly less compared to their SPF counterparts (p , 0.01). At the end of 16 weeks, neither SPF nor SPFH mice suffered from any significant weight loss (p . 0.05). However, GFH mice suffered from significant weight loss compared to GF mice (p , 0.01). www.nature.com/scientificreports SCIENTIFIC REPORTS | 5 : 8731 | DOI: 10.1038/srep08731 2