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Plasma ghrelin isoforms and gastric ghrelin O-acyltransferase expression are influenced by Helicobacter pylori status
Nutrition 28 (2012) 967–972
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Nutrition journal homepage: www.nutritionjrnl.com
Applied nutritional investigation
Plasma ghrelin isoforms and gastric ghrelin O-acyltransferase expression are influenced by Helicobacter pylori status Takashi Ando M.D., Ph.D. a, Shigeto Mizuno M.D., Ph.D. b, *, Tsukasa Ishida M.D., Ph.D. b, Yasuyuki Kondo M.D., Ph.D. b, Ikuya Miki M.D., Ph.D. b, Masaru Yoshida M.D., Ph.D. c, Takeshi Azuma M.D., Ph.D. c, Takeshi Ishikawa M.D., Ph.D. d, Tomohisa Takagi M.D., Ph.D. d, Nobuaki Yagi M.D., Ph.D. d, Satoshi Kokura M.D., Ph.D. d, Yuji Naito M.D., Ph.D. d, Toshikazu Yoshikawa M.D., Ph.D. d, Akihiro Asakawa M.D., Ph.D. e, Akio Inui M.D., Ph.D. e a
Department of Gastroenterology, Social Insurance Kyoto Hospital, Kyoto, Japan Department of Medical Pharmaceutics, Kobe Pharmaceutical University, Kobe, Japan c Division of Gastroenterology, Graduate School of Medicine, Kobe University, Kobe, Japan d Department of Gastroenterology, Kyoto Prefectural University of Medicine, Kyoto, Japan e Department of Psychosomatic Internal Medicine, Kagoshima University, Kagoshima, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 September 2011 Accepted 21 November 2011
Objective: Helicobacter pylori is known to affect the host’s nutritional status. This study was performed to elucidate the relationship between H. pylori status and the dynamics of the ghrelin system, in the context of ghrelin O-acyltransferase (GOAT) expression. Methods: We conducted a clinical study of 30 subjects focusing on the following points: 1) the effects of H. pylori infection on the concentrations of circulating ghrelin isoforms and on ghrelin and GOAT mRNA expression in the gastric mucosa, and 2) the effects of H. pylori eradication on the same parameters. Results: The plasma acyl-ghrelin and desacyl-ghrelin concentrations of 16 H. pylori positive participants were significantly lower than those of 14 H. pylori negative controls. The acyl-ghrelin/ desacyl-ghrelin ratio was not significantly different between the H. pylori positive and H. pylori negative participants. The levels of ghrelin and GOAT mRNA in the gastric mucosa were significantly lower in the H. pylori positive participants than in the H. pylori negative controls. In 11 subjects in whom H. pylori eradication was successful, their plasma acyl-ghrelin levels tended to increase after H. pylori eradication, but the difference was not significant; however, their plasma desacyl-ghrelin levels were significantly reduced. Although gastric ghrelin mRNA expression increased significantly after H. pylori eradication, gastric GOAT mRNA expression tended to increase but was not significantly altered. Conclusion: H. pylori status might affect the host’s nutritional status through changes in the plasma levels of ghrelin isoforms and the gastric expression levels of ghrelin and GOAT mRNA. Ó 2012 Elsevier Inc. All rights reserved.
Keywords: Helicobacter pylori Eradication Acyl-ghrelin Desacyl-ghrelin Acyl-ghrelin/desacyl-ghrelin ratio Ghrelin O-acyltransferase
Introduction Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), consists of 28 amino acids and is primarily produced in the stomach. The peptide is the only circulating orexigenic hormone in humans and has many This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. T. Ando and S. Mizuno contributed equally to this manuscript. * Corresponding author. Tel.: þ81 78 441 7578; fax: þ81 78 441 7578. E-mail address: [email protected] (S. Mizuno). 0899-9007/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2011.11.023
additional physiological activities such as effects on gastric motility, body weight, glucose homeostasis, bone metabolism, and immune regulation [1–3]. Ghrelin has two isoforms: acyl-ghrelin, the serine3 residue of which is posttranslationally octanoylated, and desacyl-ghrelin, in which the abovementioned octanoylation is absent. Only acyl-ghrelin, which accounts for approximately 10% of circulating ghrelin, binds to the GHS-R and performs physiological functions. In contrast, desacyl-ghrelin abolishes the effects of acyl-ghrelin on appetite promotion, gastric motility, and the regulation of pancreatic hormones [4–6]. Recently, the enzyme that octanoylates the serine3 residue of
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ghrelin has been discovered and named ghrelin O-acyltransferase (GOAT) [7]. The enzyme belongs to the membrane-bound O-acyltransferase superfamily and is expressed in many human tissues including the stomach, gut, and pancreas [8]. GOAT has attracted attention because of its unique biological features and pharmacological potential for nutritional regulation. Helicobacter pylori is a Gram-negative spiral bacterium, which has infected more than one-half of the world’s population. Once present within the gastric mucosa, it can persist for the whole of its host’s life, and H. pylori infection is associated with chronic gastritis, gastric atrophy, peptic ulcers, and gastric cancer. As for the relationship between H. pylori infection and circulating ghrelin levels, Nwokolo et al. reported that the plasma total ghrelin concentration increased after H. pylori eradication [9]. They also concluded that the increase in ghrelin levels was responsible for the weight gain observed after H. pylori eradication, and many researchers have been supportive of this hypothesis. Since then, a number of studies have investigated the effects of H. pylori infection and H. pylori eradication on circulating ghrelin levels and/or ghrelin production in the gastric mucosa [10–16]. However, the results have been inconsistent. Until now, there have been no reports about the relationship between H. pylori infection and the expression of GOAT, the only enzyme involved in the activation of ghrelin, in the gastric mucosa. Elucidating the relationship between H. pylori status and the dynamics of ghrelin expression, acylation, and secretion, in the context of GOAT expression, is vital for understanding the impact of ghrelin function on H. pylori related pathophysiology. Thus, we conducted a clinical study of the following points: 1) the effects of H. pylori infection on the concentrations of circulating ghrelin isoforms and on ghrelin and GOAT mRNA expression in the gastric mucosa, and 2) the effects of H. pylori eradication on the same parameters. Materials and methods Subjects This clinical study was conducted from February 2010 to October 2010. One hundred eighteen consecutive patients who underwent upper gastrointestinal endoscopy for gastrointestinal symptoms were enrolled in this study. To prevent interference with the ghrelin concentration, patients with a history of gastric malignancy or upper gastrointestinal surgery were excluded. Given the biological characteristics of ghrelin and GOAT, we excluded any patients whose body mass index (BMI) was less than 20 or more than 30, or whose age was less than 55 or more than 75, from this study. BMI criteria were adopted to avoid the possible influence of metabolic status, and age criteria were adopted in consideration of both the risk of false-negative H. pylori infection results in elderly subjects and the low H. pylori positivity of subjects of less than 55 years of age. The participants’ H. pylori status was evaluated using H. pylori culture tests. Finally, 14 subjects who were negative for H. pylori and 16 subjects who were positive for H. pylori were assessed in this article. The 16 positive subjects underwent H. pylori eradication therapy and successful eradication was achieved in 11 subjects. Consequently, 30 participants were assessed in terms of H. pylori negative/ positive status and 11 participants were assessed in terms of their condition before/after H. pylori cure. Information including the H. pylori status of the subjects is summarized in Table 1. This study was approved by the ethics committee at Social Insurance Kyoto Hospital. The human samples were used in accordance with the hospital’s guidelines, and written informed consent was obtained from all subjects. All patients gave detailed informed consent in accordance with the Helsinki Declaration. Data collection and H. pylori eradication protocol To estimate the atrophy status of the stomach and the expression levels of ghrelin and GOAT in the stomach mucosa, all participants underwent upper gastrointestinal endoscopy and biopsy at the start of the study. Biopsy specimens were taken from the fundic gland region of the gastric corpus. Plasma samples were collected, and body weight and abdominal circumference were measured in the morning after fasting at the start of the study. All of the
subjects in the H. pylori positive group were treated with an antimicrobial eradication protocol involving 750 mg amoxicillin, 10 mg sodium rabeprazole, and 200 mg clarithromycin. All drugs were taken twice a day for 7 d. The criterion for eradication success was defined as a negative result on the urea breath test. Upper gastrointestinal endoscopy and biopsy were performed again 3 mo after the eradication treatment of subjects in the H. pylori cure group. On the same day, the subjects also had their plasma samples collected and their body weight and abdominal circumference measured in the morning after overnight fasting. Hormone assays Blood samples were obtained in the morning after overnight fasting and transferred into collection tubes containing ethylenediaminetetraacetic acid-2Na and aprotinin. The samples were immediately centrifuged at 6000 g for 3 min at 4 C, and the plasma was transferred into a clean tube. Then, 100 mL of 1 N hydrochloric acid was added to 1 mL plasma samples. After being mixed, these samples were immediately stored at 80 C until use. Plasma acyl-ghrelin and desacyl-ghrelin concentrations were measured in duplicate using an enzyme immunoassay kit (Mitsubishi Chemical Medience, Tokyo, Japan) according to the manufacturer’s instructions. Isolation of total RNA and quantitative real-time PCR Total RNA was extracted from the biopsy samples obtained from the gastric corpus using the RNeasy Plus Mini Kit (Qiagen, Alameda, CA). cDNA was synthesized from 1 mg total RNA using the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). Real-time PCR was carried out using SYBR Green (Applied Biosystems, Foster, CA) in the Thermal Cycler Dice Real Time System (Takara, Shiga, Japan) according to the manufacturer’s instructions. The PCR primers used were as follows: ghrelin sense: 50 -GGCAGGCTCCAGCTTCCT-30 ; antisense: 50 -TGGCTTCTTCGACTCCTTTCTC-30 ; GOAT sense: 50 -GAGCTGGCAGACCTTGTGT CA-30 ; antisense: 50 -TCCAGATGCTGCCTTCACTTTC-30 ; ACTB sense: 50 -CCACACT GTGCCCATCTACG-30 ; antisense: 50 -AGGATCTTCATGAGGTAGTCAGTCAG-30 . The expression levels of each target mRNA were normalized to that of ACTB. Statistical analysis Data are presented as the mean SD. The statistical significance of the differences between the H. pylori infection positive and negative groups was analyzed using the Student’s t test, and that of the differences between the H. pylori eradication before and after groups was analyzed using the paired t test. A level of probability of 0.05 was used as the criterion for significance.
Results Effect of H. pylori infection on the concentrations of plasma ghrelin isoforms The plasma acyl-ghrelin and desacyl-ghrelin concentrations of the H. pylori positive subjects were significantly lower than those of the H. pylori negative subjects (Fig. 1A and 1B) (H. pylori positive subjects:H. pylori negative subjects; acyl-ghrelin 6.4 3.9 fmol/mL:13.4 9.4 fmol/mL, P < 0.05; desacyl-ghrelin 86.7 81.0 fmol/mL:163.3 73.8 fmol/mL, P < 0.05). However, the acyl-/desacyl-ghrelin ratio of the H. pylori positive subjects was not significantly different from that of the H. pylori negative subjects (Fig. 1C) (0.093 0.049 versus 0.076 0.033, P ¼ 0.28).
Table 1 Subject characteristics
Age (y) BMI (kg/m2) AC (cm) Gender (M/F)
H. pylori negative (n ¼ 14)
H. pylori positive (n ¼ 16)
P value
64. 7 7.1 24.2 2.3 89.6 7.9 7/7
65.4 5.3 23.2 2.0 86.5 5.7 8/8
NS NS NS
AC, abdominal circumference; BMI, body mass index Data are presented as means SD. P values were calculated according to the Student’s t test.
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Fig. 1. Plasma acyl- and desacyl-ghrelin levels in H. pylori negative and positive subjects and the plasma acyl-/desacyl-ghrelin ratio. (A, B) The plasma acyl- and desacylghrelin levels of the H. pylori positive subjects were significantly lower than those of the H. pylori negative subjects. (C) The acyl-/desacyl-ghrelin ratio of the H. pylori positive subjects was not significantly different from that of the H. pylori negative subjects. The subjects’ plasma acyl- and desacyl-ghrelin levels were measured by ELISA. Data are presented as the mean SD. The asterisks indicate significant differences according to the Student’s t test (P < 0.05).
Effect of H. pylori infection on gastric ghrelin and GOAT mRNA expression The gastric ghrelin and GOAT mRNA expression levels of the H. pylori positive subjects were significantly lower than those of the H. pylori negative subjects, as measured by the quantitative real-time PCR method (Fig. 2A and 2B) (positive subjects:negative subjects; ghrelin mRNA 5.3 7.8:54.7 52.6, P < 0.05; GOAT mRNA 372.7 663.2:2774.8 2511.0, P < 0.05). Effect of H. pylori eradication on the concentrations of plasma ghrelin isoforms After H. pylori eradication, the plasma levels of acyl-ghrelin displayed a rising trend, but were not significantly different from those observed before eradication. On the other hand, the plasma levels of desacyl-ghrelin after H. pylori eradication were significantly decreased compared with those observed before eradication (Fig. 3A and 3B) (before eradication:after eradication; acyl ghrelin 5.5 3.1 fmol/mL:6.3 3.2 fmol/mL, P ¼ 0.22; desacyl-ghrelin 71.6 44.4 fmol/mL:51.7 33.5 fmol/mL,
P < 0.05). Consequently, the acyl-/desacyl-ghrelin ratio was significantly increased after H. pylori eradication (Fig. 3C) (before eradication:after eradication; acyl-/desacyl-ghrelin ratio 0.093 0.055:0.15 0.063, P < 0.05). Effect of H. pylori eradication on gastric ghrelin and GOAT mRNA expression Ghrelin expression was significantly increased after H. pylori eradication. The expression levels of GOAT tended to increase after eradication, but the change was not significant (Fig. 4A and 4B) (before eradication:after eradication; ghrelin mRNA 6.3 9.3:16.5 16.9, P < 0.05; GOAT mRNA 361.9 779.5:654.8 732.6, P ¼ 0.12). Effect of H. pylori eradication on BMI and abdominal circumference After H. pylori eradication, we did not observe any significant change in BMI; however, abdominal circumference increased significantly (Table 2) (before eradication:after eradication; BMI 23.1 1.8 kg/m2:23.2 1.6 kg/m2, P ¼ 0.22; abdominal circumference 86.5 6.1 cm:87.9 5.6 cm; P<0.05). Discussion
Fig. 2. Gastric ghrelin and GOAT expression levels in the stomach mucosa in H. pylori negative and positive subjects. (A, B) The gastric ghrelin and GOAT mRNA expression levels of the H. pylori positive subjects were significantly lower than those of the H. pylori negative subjects. The expression of ghrelin and GOAT mRNA was determined by quantitative real-time PCR. The mRNA expression levels were normalized to those of ACTB as an internal control. Data are presented as the mean SD. The asterisks indicate significant differences according to the Student’s t test (P < 0.05).
In the present study, we first examined the effects of H. pylori infection on circulating ghrelin levels and ghrelin and GOAT mRNA expression in the gastric mucosa. The plasma acyl-ghrelin and desacyl-ghrelin concentrations of the H. pylori positive participants were significantly lower than those of the H. pylori negative controls. The acyl-ghrelin/desacyl-ghrelin ratio showed no significant difference between the H. pylori positive and negative participants. The levels of ghrelin and GOAT transcripts in the gastric mucosa were significantly lower in the H. pylori positive participants than in the negative controls. Regarding the relationship between plasma ghrelin levels and H. pylori infection, many previous studies have measured plasma total ghrelin concentrations. Gokcel et al. measured plasma total ghrelin levels in 39 age- and BMI-matched H. pylori positive and negative women and found no significant relationship between
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Fig. 3. Plasma acyl- and desacyl-ghrelin levels before and after H. pylori eradication therapy and the plasma acyl-/desacyl-ghrelin ratio. (A) After H. pylori eradication, the plasma levels of acyl-ghrelin displayed a rising trend, but the change was not significant. (B) After eradication, the plasma levels of desacyl-ghrelin were significantly decreased compared to those observed before eradication. (C) The acyl-/desacyl-ghrelin ratio significantly increased after eradication. The subjects’ plasma acyl- and desacylghrelin levels were measured by ELISA. Data are presented as the mean SD. Asterisks indicate significant differences according to the paired t test (P < 0.05).
H. pylori infection and plasma ghrelin levels [10]. Osawa et al. measured the plasma ghrelin levels of 160 participants with normal BMI and reported that the H. pylori positive participants displayed lower plasma total ghrelin concentrations [12]. Nweneka et al. conducted a systematic review and metaanalysis of the effects of H. pylori infection on circulating ghrelin levels and concluded that the H. pylori positive participants displayed lower plasma total ghrelin concentrations than the negative controls [17]. As for the levels of plasma acyl-ghrelin, the active isoform of ghrelin, Kawashima et al. conducted a multivariate analysis that examined variables such as age, BMI, gender, H. pylori infection, and gastric atrophy. They concluded that among these variables only atrophic gastritis had a significant lowering effect on the plasma acyl-ghrelin level [15]. The reduced plasma ghrelin levels observed during H. pylori infection are considered to be caused mainly by gastric mucosal injury caused by long-lasting H. pylori infection, particularly the changes in cellular composition produced by gastric mucosal atrophy. In the present study, the plasma concentration of desacyl-ghrelin, which accounts for a large proportion of circulating ghrelin, was lower in H. pylori positive subjects, and these results were consistent with those of previous reports. Plasma
Fig. 4. Gastric ghrelin and GOAT expression levels in the stomach mucosa before and after H. pylori eradication therapy. (A) The subjects’ ghrelin expression levels were significantly increased after H. pylori eradication. (B) The subjects’ GOAT expression levels tended to increase after eradication, but the change was not significant. The expression of ghrelin and GOAT mRNA was determined by quantitative real-time PCR. The subjects’ mRNA expression levels were normalized to those of ACTB as an internal control. Data are presented as the mean SD. Asterisks indicate significant differences according to the paired t test (P < 0.05).
acyl-ghrelin concentrations were also lower in the H. pylori positive subjects. As all of the H. pylori positive subjects in this study had moderate to severe atrophic gastritis (data not shown), H. pylori infection might reduce acyl- and desacylghrelin levels by inducing atrophic changes in the gastric mucosa. Concerning the relationship between gastric ghrelin production and H. pylori infection, Tatsuguchi et al. reported that the number of ghrelin-positive cells was significantly lower in H. pylori positive subjects than in healthy controls [11]. Osawa et al. measured gastric ghrelin mRNA expression levels and circulating ghrelin levels in H. pylori positive and negative subjects and reported that the H. pylori positive subjects displayed lower ghrelin mRNA expression. In the abovementioned systematic review, Nweneka et al. assessed the effect of H. pylori infection on ghrelin-producing cells in the stomach. They could not conduct a meta-analysis because of the divergent methods used to assess ghrelin production; however, in most reports, H. pylori infection negatively contributed to ghrelin production regardless of the measurement method. In the present study, gastric ghrelin mRNA expression was lower in the H. pylori positive subjects than in the negative controls, and these results were consistent with those of previous studies. Until now, there have been no reports about the relationship between GOAT mRNA expression in the gastric mucosa and H. pylori infection. In the present study, we observed lower levels of gastric GOAT transcripts in the H. pylori positive participants than in the H. pylori negative controls. As GOAT and ghrelin usually colocalize in gastric mucosal cells, H. pylori infection and the subsequent mucosal atrophy might be responsible for the observed reduction in GOAT mRNA expression. Lower expression of gastric GOAT mRNA of the H. pylori infected patients might influence the orientation of lower plasma acyl-ghrelin levels of them. Second, we examined the effect of eradicating H. pylori infection on circulating ghrelin levels and on gastric ghrelin and GOAT mRNA expression in this study. After H. pylori eradication, the participants’ plasma acyl-ghrelin levels demonstrated an upward trend but the increase was not significant; however, their plasma desacyl-ghrelin levels significantly deteriorated after H. pylori eradication. Consequently, the plasma acyl-/ desacyl-ghrelin ratio increased significantly after H. pylori
T. Ando et al. / Nutrition 28 (2012) 967–972 Table 2 Comparison of BMI and AC between before and after eradication therapy
BMI (kg/m2) AC (cm) Gender (M/F)
Before eradication (n ¼ 11)
After eradication (n ¼ 11)
P value
23.1 1.8 86.5 6.1 5/6
23.2 1.6 87.9 5.6 5/6
NS <0.05
AC, abdominal circumference; BMI, body mass index Data are presented as means SD. P values were calculated according to the paired t test.
eradication. Although gastric ghrelin mRNA expression increased significantly after H. pylori eradication, gastric GOAT mRNA expression tended to increase, but not significantly. Regarding the effect of H. pylori eradication on the plasma ghrelin concentration, Nwokolo et al. reported that plasma ghrelin levels increased after H. pylori eradication in 10 subjects and hypothesized that an increase in the plasma ghrelin concentration after H. pylori eradication would lead to body weight gain. However, subsequent studies reported inconsistent results: Osawa et al. reported that plasma ghrelin levels decreased after H. pylori eradication in 134 subjects [14], Isomoto et al. reported that there was no significant change in the plasma ghrelin levels of 43 subjects [13]. In the abovementioned systematic review, Nweneka et al. conducted a metaanalysis of the effects of H. pylori eradication on plasma total ghrelin levels and found that there was no significant change in the plasma total ghrelin level between before and after H. pylori eradication. In the previously cited article, Osawa et al. reported that the changes in the plasma ghrelin level induced by H. pylori eradication were inversely correlated with the initial plasma ghrelin level and body weight change. Lee et al. conducted a randomized controlled trial in which H. pylori infected subjects were randomly assigned to the treatment or control group. The plasma acyl-ghrelin concentrations in the treated group tended to increase after H. pylori eradication but the difference was not significant [16]. Regarding the effect of H. pylori eradication on gastric ghrelin mRNA expression, Osawa et al. reported that gastric ghrelin mRNA expression increased nearly four-fold after H. pylori eradication. Lee et al. also reported an increase in the concentration of gastric ghrelin transcripts after H. pylori eradication, and most studies showed consistent results. However, similar to the results of the present study, although gastric ghrelin mRNA expression increased after H. pylori eradication, plasma total ghrelin levels did not increase significantly. In this study, the participants did not show any body weight gain after H. pylori eradication but their abdominal circumference increased significantly, and this might have been the reason for the abovementioned discrepancy. Anyway, there might be a number of mechanisms that regulate ghrelin transcription in the gastric mucosa and ghrelin secretion into the bloodstream. Additionally, we found that gastric GOAT mRNA expression tended to increase after H. pylori eradication, although the increase was not significant. This increase in GOAT mRNA expression might have contributed to the observed elevation of the plasma acyl-/desacyl-ghrelin ratio. There were disparities between the measured values of the H. pylori negative subjects and those of the H. pylori eradicated subjects. We think that the short follow-up periods experienced by our subjects, e.g., 3 months, might have caused these differences. The improvement of gastric atrophy after H. pylori eradication has been demonstrated in long-term follow-up studies [18], and the values of H. pylori eradicated subjects might
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approach those of H. pylori negative subjects in the long term. As for the disparities between the gastric expression levels of ghrelin-related substances and the plasma levels of ghrelin isoforms after H. pylori eradication, other factors such as changes in metabolic status might have affected these results. In the present study, we examined plasma acyl- and desacylghrelin concentrations and the acyl-/desacyl-ghrelin ratio in the context of gastric ghrelin and GOAT mRNA expression, as well as their associations with H. pylori status, for the first time. There are some limitations to the study, such as the small sample size and short follow-up periods. We did not analyze the gender differences in the ghrelin system because of the small sample size. However, our data indicate that the absolute concentrations of ghrelin isoforms and the acyl-/desacyl-ghrelin ratio are important when considering the relationship between ghrelin dynamics and H. pylori status, which is similar to the findings reported for cancer cachexia [19]. To obtain a better understanding of the various pathophysiologies induced by H. pylori infection, clarifying the dynamics of ghrelin expression, acylation, and secretion is vital [20]. A more precise examination of these characteristics is expected in the future. Conclusion The plasma levels of ghrelin isoforms and the gastric expression of ghrelin and GOAT were lower in H. pylori infected individuals. The subjects’ plasma acyl-ghrelin levels tended to increase after H. pylori eradication; however, their desacylghrelin levels significantly decreased. Although the levels of the gastric ghrelin transcript increased after H. pylori eradication, those of the gastric GOAT transcript tended to increase but not significantly. References [1] Chen CY, Asakawa A, Fujimiya M, Lee SD, Inui A. Ghrelin gene products and the regulation of food intake and gut motility. Pharmacol Rev 2009;61:430–81. [2] Peeters TL. Ghrelin: a new player in the control of gastrointestinal functions. Gut 2005;54:1638–49. [3] Vermeulen MA, Richir MC, Garretsen MK, van Schie A, Ghatei MA, Holst JJ, et al. Gastric emptying, glucose metabolism and gut hormones: Evaluation of a common preoperative carbohydrate beverage. Nutrition 2011;27:897– 903. [4] Asakawa A, Inui A, Fujimiya M, Sakamaki R, Shinfuku N, Ueta Y, et al. Stomach regulates energy balance via acylated ghrelin and desacyl ghrelin. Gut 2005;54:18–24. [5] Inhoff T, Mönnikes H, Noetzel S, Stengel A, Goebel M, Dinh QT, et al. Desacyl ghrelin inhibits the orexigenic effect of peripherally injected ghrelin in rats. Peptides 2008;29:2159–68. [6] Qader S, Sh akanson R, Rehfeld JF, Lundquist I, Salehi A. Proghrelin-derived peptides influence the secretion of insulin, glucagon, pancreatic polypeptide and somatostatin: a study on isolated islets from mouse and rat pancreas. Regul Pept 2008;146:230–7. [7] Yang J, Brown MS, Liang G, Grishin NV, Goldstein JL. Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone. Cell 2008;132:387–96. [8] Lim CT, Kola B, Korbonits M. The ghrelin/GOAT/GHS-R system and energy metabolism. Rev Endocr Metab Disord 2011;12:173–86. [9] Nwokolo CU, Freshwater DA, O’Hare P, Randeva HS. Plasma ghrelin following cure of Helicobacter pylori. Gut 2003;52:637–40. [10] Gokcel A, Gumurdulu Y, Kayaselcuk F, Serin E, Ozer B, Ozsahin AK, et al. Helicobacter pylori has no effect on plasma ghrelin levels. Eur J Endocrinol 2003;148:423–6. [11] Tatsuguchi A, Miyake K, Gudis K, Futagami S, Tsukui T, Wada K, et al. Effect of Helicobacter pylori infection on ghrelin expression in human gastric mucosa. Am J Gastroenterol 2004;99:2121–7. [12] Osawa H, Nakazato M, Date Y, Kita H, Ohnishi H, Ueno H, et al. Impaired production of gastric ghrelin in chronic gastritis associated with Helicobacter pylori. J Clin Endocrinol Metab 2005;90:10–6. [13] Isomoto H, Ueno H, Saenko VA, Mondal MS, Nishi Y, Kawano N, et al. Impact of Helicobacter pylori infection on gastric and plasma ghrelin dynamics in humans. Am J Gastroenterol 2005;100:1711–20.
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[14] Osawa H, Kita H, Ohnishi H, Nakazato M, Date Y, Bowlus CL, et al. Changes in plasma ghrelin levels, gastric ghrelin production, and body weight after Helicobacter pylori cure. J Gastroenterol 2006;41:954–61. [15] Kawashima J, Ohno S, Sakurada T, Takabayashi H, Kudo M, Ro S, et al. Circulating acylated ghrelin level decreases in accordance with the extent of atrophic gastritis. J Gastroenterol 2009;44:1046–54. [16] Lee ES, Yoon YS, Park CY, Kim HS, Um TH, Baik HW, et al. Eradication of Helicobacter pylori increases ghrelin mRNA expression in the gastric mucosa. J Korean Med Sci 2010;25:265–71. [17] Nweneka CV, Prentice AM. Helicobacter pylori infection and circulating ghrelin levels–-A systematic review. BMC Gastroenterol 2011;11:7.
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Nutrition journal homepage: www.nutritionjrnl.com
Applied nutritional investigation
Plasma ghrelin isoforms and gastric ghrelin O-acyltransferase expression are influenced by Helicobacter pylori status Takashi Ando M.D., Ph.D. a, Shigeto Mizuno M.D., Ph.D. b, *, Tsukasa Ishida M.D., Ph.D. b, Yasuyuki Kondo M.D., Ph.D. b, Ikuya Miki M.D., Ph.D. b, Masaru Yoshida M.D., Ph.D. c, Takeshi Azuma M.D., Ph.D. c, Takeshi Ishikawa M.D., Ph.D. d, Tomohisa Takagi M.D., Ph.D. d, Nobuaki Yagi M.D., Ph.D. d, Satoshi Kokura M.D., Ph.D. d, Yuji Naito M.D., Ph.D. d, Toshikazu Yoshikawa M.D., Ph.D. d, Akihiro Asakawa M.D., Ph.D. e, Akio Inui M.D., Ph.D. e a
Department of Gastroenterology, Social Insurance Kyoto Hospital, Kyoto, Japan Department of Medical Pharmaceutics, Kobe Pharmaceutical University, Kobe, Japan c Division of Gastroenterology, Graduate School of Medicine, Kobe University, Kobe, Japan d Department of Gastroenterology, Kyoto Prefectural University of Medicine, Kyoto, Japan e Department of Psychosomatic Internal Medicine, Kagoshima University, Kagoshima, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 September 2011 Accepted 21 November 2011
Objective: Helicobacter pylori is known to affect the host’s nutritional status. This study was performed to elucidate the relationship between H. pylori status and the dynamics of the ghrelin system, in the context of ghrelin O-acyltransferase (GOAT) expression. Methods: We conducted a clinical study of 30 subjects focusing on the following points: 1) the effects of H. pylori infection on the concentrations of circulating ghrelin isoforms and on ghrelin and GOAT mRNA expression in the gastric mucosa, and 2) the effects of H. pylori eradication on the same parameters. Results: The plasma acyl-ghrelin and desacyl-ghrelin concentrations of 16 H. pylori positive participants were significantly lower than those of 14 H. pylori negative controls. The acyl-ghrelin/ desacyl-ghrelin ratio was not significantly different between the H. pylori positive and H. pylori negative participants. The levels of ghrelin and GOAT mRNA in the gastric mucosa were significantly lower in the H. pylori positive participants than in the H. pylori negative controls. In 11 subjects in whom H. pylori eradication was successful, their plasma acyl-ghrelin levels tended to increase after H. pylori eradication, but the difference was not significant; however, their plasma desacyl-ghrelin levels were significantly reduced. Although gastric ghrelin mRNA expression increased significantly after H. pylori eradication, gastric GOAT mRNA expression tended to increase but was not significantly altered. Conclusion: H. pylori status might affect the host’s nutritional status through changes in the plasma levels of ghrelin isoforms and the gastric expression levels of ghrelin and GOAT mRNA. Ó 2012 Elsevier Inc. All rights reserved.
Keywords: Helicobacter pylori Eradication Acyl-ghrelin Desacyl-ghrelin Acyl-ghrelin/desacyl-ghrelin ratio Ghrelin O-acyltransferase
Introduction Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), consists of 28 amino acids and is primarily produced in the stomach. The peptide is the only circulating orexigenic hormone in humans and has many This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. T. Ando and S. Mizuno contributed equally to this manuscript. * Corresponding author. Tel.: þ81 78 441 7578; fax: þ81 78 441 7578. E-mail address: [email protected] (S. Mizuno). 0899-9007/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2011.11.023
additional physiological activities such as effects on gastric motility, body weight, glucose homeostasis, bone metabolism, and immune regulation [1–3]. Ghrelin has two isoforms: acyl-ghrelin, the serine3 residue of which is posttranslationally octanoylated, and desacyl-ghrelin, in which the abovementioned octanoylation is absent. Only acyl-ghrelin, which accounts for approximately 10% of circulating ghrelin, binds to the GHS-R and performs physiological functions. In contrast, desacyl-ghrelin abolishes the effects of acyl-ghrelin on appetite promotion, gastric motility, and the regulation of pancreatic hormones [4–6]. Recently, the enzyme that octanoylates the serine3 residue of
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ghrelin has been discovered and named ghrelin O-acyltransferase (GOAT) [7]. The enzyme belongs to the membrane-bound O-acyltransferase superfamily and is expressed in many human tissues including the stomach, gut, and pancreas [8]. GOAT has attracted attention because of its unique biological features and pharmacological potential for nutritional regulation. Helicobacter pylori is a Gram-negative spiral bacterium, which has infected more than one-half of the world’s population. Once present within the gastric mucosa, it can persist for the whole of its host’s life, and H. pylori infection is associated with chronic gastritis, gastric atrophy, peptic ulcers, and gastric cancer. As for the relationship between H. pylori infection and circulating ghrelin levels, Nwokolo et al. reported that the plasma total ghrelin concentration increased after H. pylori eradication [9]. They also concluded that the increase in ghrelin levels was responsible for the weight gain observed after H. pylori eradication, and many researchers have been supportive of this hypothesis. Since then, a number of studies have investigated the effects of H. pylori infection and H. pylori eradication on circulating ghrelin levels and/or ghrelin production in the gastric mucosa [10–16]. However, the results have been inconsistent. Until now, there have been no reports about the relationship between H. pylori infection and the expression of GOAT, the only enzyme involved in the activation of ghrelin, in the gastric mucosa. Elucidating the relationship between H. pylori status and the dynamics of ghrelin expression, acylation, and secretion, in the context of GOAT expression, is vital for understanding the impact of ghrelin function on H. pylori related pathophysiology. Thus, we conducted a clinical study of the following points: 1) the effects of H. pylori infection on the concentrations of circulating ghrelin isoforms and on ghrelin and GOAT mRNA expression in the gastric mucosa, and 2) the effects of H. pylori eradication on the same parameters. Materials and methods Subjects This clinical study was conducted from February 2010 to October 2010. One hundred eighteen consecutive patients who underwent upper gastrointestinal endoscopy for gastrointestinal symptoms were enrolled in this study. To prevent interference with the ghrelin concentration, patients with a history of gastric malignancy or upper gastrointestinal surgery were excluded. Given the biological characteristics of ghrelin and GOAT, we excluded any patients whose body mass index (BMI) was less than 20 or more than 30, or whose age was less than 55 or more than 75, from this study. BMI criteria were adopted to avoid the possible influence of metabolic status, and age criteria were adopted in consideration of both the risk of false-negative H. pylori infection results in elderly subjects and the low H. pylori positivity of subjects of less than 55 years of age. The participants’ H. pylori status was evaluated using H. pylori culture tests. Finally, 14 subjects who were negative for H. pylori and 16 subjects who were positive for H. pylori were assessed in this article. The 16 positive subjects underwent H. pylori eradication therapy and successful eradication was achieved in 11 subjects. Consequently, 30 participants were assessed in terms of H. pylori negative/ positive status and 11 participants were assessed in terms of their condition before/after H. pylori cure. Information including the H. pylori status of the subjects is summarized in Table 1. This study was approved by the ethics committee at Social Insurance Kyoto Hospital. The human samples were used in accordance with the hospital’s guidelines, and written informed consent was obtained from all subjects. All patients gave detailed informed consent in accordance with the Helsinki Declaration. Data collection and H. pylori eradication protocol To estimate the atrophy status of the stomach and the expression levels of ghrelin and GOAT in the stomach mucosa, all participants underwent upper gastrointestinal endoscopy and biopsy at the start of the study. Biopsy specimens were taken from the fundic gland region of the gastric corpus. Plasma samples were collected, and body weight and abdominal circumference were measured in the morning after fasting at the start of the study. All of the
subjects in the H. pylori positive group were treated with an antimicrobial eradication protocol involving 750 mg amoxicillin, 10 mg sodium rabeprazole, and 200 mg clarithromycin. All drugs were taken twice a day for 7 d. The criterion for eradication success was defined as a negative result on the urea breath test. Upper gastrointestinal endoscopy and biopsy were performed again 3 mo after the eradication treatment of subjects in the H. pylori cure group. On the same day, the subjects also had their plasma samples collected and their body weight and abdominal circumference measured in the morning after overnight fasting. Hormone assays Blood samples were obtained in the morning after overnight fasting and transferred into collection tubes containing ethylenediaminetetraacetic acid-2Na and aprotinin. The samples were immediately centrifuged at 6000 g for 3 min at 4 C, and the plasma was transferred into a clean tube. Then, 100 mL of 1 N hydrochloric acid was added to 1 mL plasma samples. After being mixed, these samples were immediately stored at 80 C until use. Plasma acyl-ghrelin and desacyl-ghrelin concentrations were measured in duplicate using an enzyme immunoassay kit (Mitsubishi Chemical Medience, Tokyo, Japan) according to the manufacturer’s instructions. Isolation of total RNA and quantitative real-time PCR Total RNA was extracted from the biopsy samples obtained from the gastric corpus using the RNeasy Plus Mini Kit (Qiagen, Alameda, CA). cDNA was synthesized from 1 mg total RNA using the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). Real-time PCR was carried out using SYBR Green (Applied Biosystems, Foster, CA) in the Thermal Cycler Dice Real Time System (Takara, Shiga, Japan) according to the manufacturer’s instructions. The PCR primers used were as follows: ghrelin sense: 50 -GGCAGGCTCCAGCTTCCT-30 ; antisense: 50 -TGGCTTCTTCGACTCCTTTCTC-30 ; GOAT sense: 50 -GAGCTGGCAGACCTTGTGT CA-30 ; antisense: 50 -TCCAGATGCTGCCTTCACTTTC-30 ; ACTB sense: 50 -CCACACT GTGCCCATCTACG-30 ; antisense: 50 -AGGATCTTCATGAGGTAGTCAGTCAG-30 . The expression levels of each target mRNA were normalized to that of ACTB. Statistical analysis Data are presented as the mean SD. The statistical significance of the differences between the H. pylori infection positive and negative groups was analyzed using the Student’s t test, and that of the differences between the H. pylori eradication before and after groups was analyzed using the paired t test. A level of probability of 0.05 was used as the criterion for significance.
Results Effect of H. pylori infection on the concentrations of plasma ghrelin isoforms The plasma acyl-ghrelin and desacyl-ghrelin concentrations of the H. pylori positive subjects were significantly lower than those of the H. pylori negative subjects (Fig. 1A and 1B) (H. pylori positive subjects:H. pylori negative subjects; acyl-ghrelin 6.4 3.9 fmol/mL:13.4 9.4 fmol/mL, P < 0.05; desacyl-ghrelin 86.7 81.0 fmol/mL:163.3 73.8 fmol/mL, P < 0.05). However, the acyl-/desacyl-ghrelin ratio of the H. pylori positive subjects was not significantly different from that of the H. pylori negative subjects (Fig. 1C) (0.093 0.049 versus 0.076 0.033, P ¼ 0.28).
Table 1 Subject characteristics
Age (y) BMI (kg/m2) AC (cm) Gender (M/F)
H. pylori negative (n ¼ 14)
H. pylori positive (n ¼ 16)
P value
64. 7 7.1 24.2 2.3 89.6 7.9 7/7
65.4 5.3 23.2 2.0 86.5 5.7 8/8
NS NS NS
AC, abdominal circumference; BMI, body mass index Data are presented as means SD. P values were calculated according to the Student’s t test.
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Fig. 1. Plasma acyl- and desacyl-ghrelin levels in H. pylori negative and positive subjects and the plasma acyl-/desacyl-ghrelin ratio. (A, B) The plasma acyl- and desacylghrelin levels of the H. pylori positive subjects were significantly lower than those of the H. pylori negative subjects. (C) The acyl-/desacyl-ghrelin ratio of the H. pylori positive subjects was not significantly different from that of the H. pylori negative subjects. The subjects’ plasma acyl- and desacyl-ghrelin levels were measured by ELISA. Data are presented as the mean SD. The asterisks indicate significant differences according to the Student’s t test (P < 0.05).
Effect of H. pylori infection on gastric ghrelin and GOAT mRNA expression The gastric ghrelin and GOAT mRNA expression levels of the H. pylori positive subjects were significantly lower than those of the H. pylori negative subjects, as measured by the quantitative real-time PCR method (Fig. 2A and 2B) (positive subjects:negative subjects; ghrelin mRNA 5.3 7.8:54.7 52.6, P < 0.05; GOAT mRNA 372.7 663.2:2774.8 2511.0, P < 0.05). Effect of H. pylori eradication on the concentrations of plasma ghrelin isoforms After H. pylori eradication, the plasma levels of acyl-ghrelin displayed a rising trend, but were not significantly different from those observed before eradication. On the other hand, the plasma levels of desacyl-ghrelin after H. pylori eradication were significantly decreased compared with those observed before eradication (Fig. 3A and 3B) (before eradication:after eradication; acyl ghrelin 5.5 3.1 fmol/mL:6.3 3.2 fmol/mL, P ¼ 0.22; desacyl-ghrelin 71.6 44.4 fmol/mL:51.7 33.5 fmol/mL,
P < 0.05). Consequently, the acyl-/desacyl-ghrelin ratio was significantly increased after H. pylori eradication (Fig. 3C) (before eradication:after eradication; acyl-/desacyl-ghrelin ratio 0.093 0.055:0.15 0.063, P < 0.05). Effect of H. pylori eradication on gastric ghrelin and GOAT mRNA expression Ghrelin expression was significantly increased after H. pylori eradication. The expression levels of GOAT tended to increase after eradication, but the change was not significant (Fig. 4A and 4B) (before eradication:after eradication; ghrelin mRNA 6.3 9.3:16.5 16.9, P < 0.05; GOAT mRNA 361.9 779.5:654.8 732.6, P ¼ 0.12). Effect of H. pylori eradication on BMI and abdominal circumference After H. pylori eradication, we did not observe any significant change in BMI; however, abdominal circumference increased significantly (Table 2) (before eradication:after eradication; BMI 23.1 1.8 kg/m2:23.2 1.6 kg/m2, P ¼ 0.22; abdominal circumference 86.5 6.1 cm:87.9 5.6 cm; P<0.05). Discussion
Fig. 2. Gastric ghrelin and GOAT expression levels in the stomach mucosa in H. pylori negative and positive subjects. (A, B) The gastric ghrelin and GOAT mRNA expression levels of the H. pylori positive subjects were significantly lower than those of the H. pylori negative subjects. The expression of ghrelin and GOAT mRNA was determined by quantitative real-time PCR. The mRNA expression levels were normalized to those of ACTB as an internal control. Data are presented as the mean SD. The asterisks indicate significant differences according to the Student’s t test (P < 0.05).
In the present study, we first examined the effects of H. pylori infection on circulating ghrelin levels and ghrelin and GOAT mRNA expression in the gastric mucosa. The plasma acyl-ghrelin and desacyl-ghrelin concentrations of the H. pylori positive participants were significantly lower than those of the H. pylori negative controls. The acyl-ghrelin/desacyl-ghrelin ratio showed no significant difference between the H. pylori positive and negative participants. The levels of ghrelin and GOAT transcripts in the gastric mucosa were significantly lower in the H. pylori positive participants than in the negative controls. Regarding the relationship between plasma ghrelin levels and H. pylori infection, many previous studies have measured plasma total ghrelin concentrations. Gokcel et al. measured plasma total ghrelin levels in 39 age- and BMI-matched H. pylori positive and negative women and found no significant relationship between
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Fig. 3. Plasma acyl- and desacyl-ghrelin levels before and after H. pylori eradication therapy and the plasma acyl-/desacyl-ghrelin ratio. (A) After H. pylori eradication, the plasma levels of acyl-ghrelin displayed a rising trend, but the change was not significant. (B) After eradication, the plasma levels of desacyl-ghrelin were significantly decreased compared to those observed before eradication. (C) The acyl-/desacyl-ghrelin ratio significantly increased after eradication. The subjects’ plasma acyl- and desacylghrelin levels were measured by ELISA. Data are presented as the mean SD. Asterisks indicate significant differences according to the paired t test (P < 0.05).
H. pylori infection and plasma ghrelin levels [10]. Osawa et al. measured the plasma ghrelin levels of 160 participants with normal BMI and reported that the H. pylori positive participants displayed lower plasma total ghrelin concentrations [12]. Nweneka et al. conducted a systematic review and metaanalysis of the effects of H. pylori infection on circulating ghrelin levels and concluded that the H. pylori positive participants displayed lower plasma total ghrelin concentrations than the negative controls [17]. As for the levels of plasma acyl-ghrelin, the active isoform of ghrelin, Kawashima et al. conducted a multivariate analysis that examined variables such as age, BMI, gender, H. pylori infection, and gastric atrophy. They concluded that among these variables only atrophic gastritis had a significant lowering effect on the plasma acyl-ghrelin level [15]. The reduced plasma ghrelin levels observed during H. pylori infection are considered to be caused mainly by gastric mucosal injury caused by long-lasting H. pylori infection, particularly the changes in cellular composition produced by gastric mucosal atrophy. In the present study, the plasma concentration of desacyl-ghrelin, which accounts for a large proportion of circulating ghrelin, was lower in H. pylori positive subjects, and these results were consistent with those of previous reports. Plasma
Fig. 4. Gastric ghrelin and GOAT expression levels in the stomach mucosa before and after H. pylori eradication therapy. (A) The subjects’ ghrelin expression levels were significantly increased after H. pylori eradication. (B) The subjects’ GOAT expression levels tended to increase after eradication, but the change was not significant. The expression of ghrelin and GOAT mRNA was determined by quantitative real-time PCR. The subjects’ mRNA expression levels were normalized to those of ACTB as an internal control. Data are presented as the mean SD. Asterisks indicate significant differences according to the paired t test (P < 0.05).
acyl-ghrelin concentrations were also lower in the H. pylori positive subjects. As all of the H. pylori positive subjects in this study had moderate to severe atrophic gastritis (data not shown), H. pylori infection might reduce acyl- and desacylghrelin levels by inducing atrophic changes in the gastric mucosa. Concerning the relationship between gastric ghrelin production and H. pylori infection, Tatsuguchi et al. reported that the number of ghrelin-positive cells was significantly lower in H. pylori positive subjects than in healthy controls [11]. Osawa et al. measured gastric ghrelin mRNA expression levels and circulating ghrelin levels in H. pylori positive and negative subjects and reported that the H. pylori positive subjects displayed lower ghrelin mRNA expression. In the abovementioned systematic review, Nweneka et al. assessed the effect of H. pylori infection on ghrelin-producing cells in the stomach. They could not conduct a meta-analysis because of the divergent methods used to assess ghrelin production; however, in most reports, H. pylori infection negatively contributed to ghrelin production regardless of the measurement method. In the present study, gastric ghrelin mRNA expression was lower in the H. pylori positive subjects than in the negative controls, and these results were consistent with those of previous studies. Until now, there have been no reports about the relationship between GOAT mRNA expression in the gastric mucosa and H. pylori infection. In the present study, we observed lower levels of gastric GOAT transcripts in the H. pylori positive participants than in the H. pylori negative controls. As GOAT and ghrelin usually colocalize in gastric mucosal cells, H. pylori infection and the subsequent mucosal atrophy might be responsible for the observed reduction in GOAT mRNA expression. Lower expression of gastric GOAT mRNA of the H. pylori infected patients might influence the orientation of lower plasma acyl-ghrelin levels of them. Second, we examined the effect of eradicating H. pylori infection on circulating ghrelin levels and on gastric ghrelin and GOAT mRNA expression in this study. After H. pylori eradication, the participants’ plasma acyl-ghrelin levels demonstrated an upward trend but the increase was not significant; however, their plasma desacyl-ghrelin levels significantly deteriorated after H. pylori eradication. Consequently, the plasma acyl-/ desacyl-ghrelin ratio increased significantly after H. pylori
T. Ando et al. / Nutrition 28 (2012) 967–972 Table 2 Comparison of BMI and AC between before and after eradication therapy
BMI (kg/m2) AC (cm) Gender (M/F)
Before eradication (n ¼ 11)
After eradication (n ¼ 11)
P value
23.1 1.8 86.5 6.1 5/6
23.2 1.6 87.9 5.6 5/6
NS <0.05
AC, abdominal circumference; BMI, body mass index Data are presented as means SD. P values were calculated according to the paired t test.
eradication. Although gastric ghrelin mRNA expression increased significantly after H. pylori eradication, gastric GOAT mRNA expression tended to increase, but not significantly. Regarding the effect of H. pylori eradication on the plasma ghrelin concentration, Nwokolo et al. reported that plasma ghrelin levels increased after H. pylori eradication in 10 subjects and hypothesized that an increase in the plasma ghrelin concentration after H. pylori eradication would lead to body weight gain. However, subsequent studies reported inconsistent results: Osawa et al. reported that plasma ghrelin levels decreased after H. pylori eradication in 134 subjects [14], Isomoto et al. reported that there was no significant change in the plasma ghrelin levels of 43 subjects [13]. In the abovementioned systematic review, Nweneka et al. conducted a metaanalysis of the effects of H. pylori eradication on plasma total ghrelin levels and found that there was no significant change in the plasma total ghrelin level between before and after H. pylori eradication. In the previously cited article, Osawa et al. reported that the changes in the plasma ghrelin level induced by H. pylori eradication were inversely correlated with the initial plasma ghrelin level and body weight change. Lee et al. conducted a randomized controlled trial in which H. pylori infected subjects were randomly assigned to the treatment or control group. The plasma acyl-ghrelin concentrations in the treated group tended to increase after H. pylori eradication but the difference was not significant [16]. Regarding the effect of H. pylori eradication on gastric ghrelin mRNA expression, Osawa et al. reported that gastric ghrelin mRNA expression increased nearly four-fold after H. pylori eradication. Lee et al. also reported an increase in the concentration of gastric ghrelin transcripts after H. pylori eradication, and most studies showed consistent results. However, similar to the results of the present study, although gastric ghrelin mRNA expression increased after H. pylori eradication, plasma total ghrelin levels did not increase significantly. In this study, the participants did not show any body weight gain after H. pylori eradication but their abdominal circumference increased significantly, and this might have been the reason for the abovementioned discrepancy. Anyway, there might be a number of mechanisms that regulate ghrelin transcription in the gastric mucosa and ghrelin secretion into the bloodstream. Additionally, we found that gastric GOAT mRNA expression tended to increase after H. pylori eradication, although the increase was not significant. This increase in GOAT mRNA expression might have contributed to the observed elevation of the plasma acyl-/desacyl-ghrelin ratio. There were disparities between the measured values of the H. pylori negative subjects and those of the H. pylori eradicated subjects. We think that the short follow-up periods experienced by our subjects, e.g., 3 months, might have caused these differences. The improvement of gastric atrophy after H. pylori eradication has been demonstrated in long-term follow-up studies [18], and the values of H. pylori eradicated subjects might
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approach those of H. pylori negative subjects in the long term. As for the disparities between the gastric expression levels of ghrelin-related substances and the plasma levels of ghrelin isoforms after H. pylori eradication, other factors such as changes in metabolic status might have affected these results. In the present study, we examined plasma acyl- and desacylghrelin concentrations and the acyl-/desacyl-ghrelin ratio in the context of gastric ghrelin and GOAT mRNA expression, as well as their associations with H. pylori status, for the first time. There are some limitations to the study, such as the small sample size and short follow-up periods. We did not analyze the gender differences in the ghrelin system because of the small sample size. However, our data indicate that the absolute concentrations of ghrelin isoforms and the acyl-/desacyl-ghrelin ratio are important when considering the relationship between ghrelin dynamics and H. pylori status, which is similar to the findings reported for cancer cachexia [19]. To obtain a better understanding of the various pathophysiologies induced by H. pylori infection, clarifying the dynamics of ghrelin expression, acylation, and secretion is vital [20]. A more precise examination of these characteristics is expected in the future. Conclusion The plasma levels of ghrelin isoforms and the gastric expression of ghrelin and GOAT were lower in H. pylori infected individuals. The subjects’ plasma acyl-ghrelin levels tended to increase after H. pylori eradication; however, their desacylghrelin levels significantly decreased. Although the levels of the gastric ghrelin transcript increased after H. pylori eradication, those of the gastric GOAT transcript tended to increase but not significantly. References [1] Chen CY, Asakawa A, Fujimiya M, Lee SD, Inui A. Ghrelin gene products and the regulation of food intake and gut motility. Pharmacol Rev 2009;61:430–81. [2] Peeters TL. Ghrelin: a new player in the control of gastrointestinal functions. Gut 2005;54:1638–49. [3] Vermeulen MA, Richir MC, Garretsen MK, van Schie A, Ghatei MA, Holst JJ, et al. Gastric emptying, glucose metabolism and gut hormones: Evaluation of a common preoperative carbohydrate beverage. Nutrition 2011;27:897– 903. [4] Asakawa A, Inui A, Fujimiya M, Sakamaki R, Shinfuku N, Ueta Y, et al. Stomach regulates energy balance via acylated ghrelin and desacyl ghrelin. Gut 2005;54:18–24. [5] Inhoff T, Mönnikes H, Noetzel S, Stengel A, Goebel M, Dinh QT, et al. Desacyl ghrelin inhibits the orexigenic effect of peripherally injected ghrelin in rats. Peptides 2008;29:2159–68. [6] Qader S, Sh akanson R, Rehfeld JF, Lundquist I, Salehi A. Proghrelin-derived peptides influence the secretion of insulin, glucagon, pancreatic polypeptide and somatostatin: a study on isolated islets from mouse and rat pancreas. Regul Pept 2008;146:230–7. [7] Yang J, Brown MS, Liang G, Grishin NV, Goldstein JL. Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone. Cell 2008;132:387–96. [8] Lim CT, Kola B, Korbonits M. The ghrelin/GOAT/GHS-R system and energy metabolism. Rev Endocr Metab Disord 2011;12:173–86. [9] Nwokolo CU, Freshwater DA, O’Hare P, Randeva HS. Plasma ghrelin following cure of Helicobacter pylori. Gut 2003;52:637–40. [10] Gokcel A, Gumurdulu Y, Kayaselcuk F, Serin E, Ozer B, Ozsahin AK, et al. Helicobacter pylori has no effect on plasma ghrelin levels. Eur J Endocrinol 2003;148:423–6. [11] Tatsuguchi A, Miyake K, Gudis K, Futagami S, Tsukui T, Wada K, et al. Effect of Helicobacter pylori infection on ghrelin expression in human gastric mucosa. Am J Gastroenterol 2004;99:2121–7. [12] Osawa H, Nakazato M, Date Y, Kita H, Ohnishi H, Ueno H, et al. Impaired production of gastric ghrelin in chronic gastritis associated with Helicobacter pylori. J Clin Endocrinol Metab 2005;90:10–6. [13] Isomoto H, Ueno H, Saenko VA, Mondal MS, Nishi Y, Kawano N, et al. Impact of Helicobacter pylori infection on gastric and plasma ghrelin dynamics in humans. Am J Gastroenterol 2005;100:1711–20.
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