Impaired Production of Gastric Ghrelin in Chronic Gastritis Associated with Helicobacter pylori
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《临床内分泌与代谢杂志》
Abstract
Ghrelin is primarily secreted from the stomach and has been implicated in the coordination of eating behavior and weight regulation. The effects of Helicobacter pylori infection on plasma ghrelin concentration and gastric ghrelin production still have not been well known. We determined plasma ghrelin concentration in a total of 160 consecutive individuals with normal body mass index including 110 H. pylori-infected and 50 H. pylori-negative subjects. The expression levels of ghrelin mRNA and ghrelin-producing cells in the gastric mucosa were quantified with real-time quantitative RT-PCR and immunohistochemistry, respectively. The severity of gastric atrophy was evaluated by serum pepsinogen concentrations. Plasma ghrelin concentration, gastric ghrelin mRNA, and ghrelin-positive cell numbers in gastric mucosa were significantly lower in H. pylori-infected subjects. The decrease in plasma ghrelin concentration in H. pylori-positive subjects was accompanied by an attenuation of ghrelin mRNA expression and a reduction of ghrelin-positive cell numbers in the gastric mucosa. Moreover, lower serum pepsinogen I concentrations and I/II ratio were significantly associated with lower plasma ghrelin concentrations in H. pylori-positive subjects. These findings suggest that impaired gastric ghrelin production in association with atrophic gastritis induced by H. pylori infection accounts for the decrease in plasma ghrelin concentration.
Introduction
GHRELIN, A 28-AMINO acid peptide isolated from rat and human stomach possesses strong GH-releasing activity and plays central as well as peripheral roles in food intake, gastric motility, and acid secretion (1, 2). Ghrelin has been shown to evoke weight gain by actions in the hypothalamus (3). Plasma ghrelin concentrations rise before meals and fall after meals. This peptide also contributes to the regulation of both somatic growth and adipose tissue mass and is therefore a short-term, meal-related orexigen as well as a long-term regulator of body weight (4, 5, 6). Circulating ghrelin concentrations in newborns are not associated with gender, body weight, or hormonal parameter (7). In children and adults, however, plasma ghrelin concentrations are lower in obese subjects, compared with those with normal body weight and lean subjects (8, 9). The decrease of plasma ghrelin concentrations appears to compensate for the positive energy balance in obese individuals (9). The majority of circulating ghrelin is produced in the mammalian gastric mucosa by enteroendocrine cells/oxyntic glands, probably the X/A-like cells (10). Thus, there exists the possibility that chronic persistent damage of the gastric mucosa, such as chronic gastritis, might affect ghrelin production, leading to changes in food intake and body weight. Helicobacter pylori is a Gram-negative bacterium that colonizes the stomach. H. pylori infection is involved in the pathogenesis of gastritis, gastric and duodenal ulcer, gastric carcinoma, and mucosa-associated lymphoid tissue lymphoma (11, 12, 13). More than 50% of the adult population are infected with H. pylori worldwide (14, 15). H. pylori infection first leads to atrophic gastritis and intestinal metaplasia, which may further lead to dysplasia and gastric carcinoma (16). Thus, it is an intriguing question whether H. pylori infection affects gastric ghrelin production and consequently alters plasma ghrelin concentration.
In this respect, Nwokolo et al. (17) reported that plasma ghrelin concentrations increased after the eradication of H. pylori. On the contrary, Gokcel et al. (18) reported that H. pylori infection has no effect on plasma ghrelin levels. Thus, the relationship between H. pylori infection and plasma ghrelin concentrations is still controversial, prompting us to further assess the effects of H. pylori infection on plasma ghrelin concentrations. Because previous studies examined serum ghrelin concentrations without investigating gastric ghrelin production (17, 18), the direct relationship between H. pylori infection and gastric ghrelin production, which could influence plasma ghrelin concentrations, is still to be demonstrated. We thus conducted this study to investigate the association of H. pylori infection with both ghrelin mRNA and protein production in the stomach, concomitantly examining plasma ghrelin concentrations. To this end, we applied real-time quantitative RT-PCR and immunohistochemistry of endoscopic biopsy specimens. Moreover, because body weight is an important factor that determines plasma ghrelin concentrations, only individuals with normal body mass index (BMI) were enrolled in this study. We report here that H. pylori infection is associated with lower gastric ghrelin mRNA and protein as well as serum ghrelin concentrations.
Subjects and Methods
Participants
We enrolled 160 consecutive asymptomatic men with normal BMI undergoing gastric cancer surveillance in Tochigi, Japan. They were divided into two groups (110 H. pylori-positive subjects and 50 H. pylori-negative controls) according to the presence or absence of H. pylori in the gastric mucosa evaluated by the bacterial culture and histological examination. The percentage of the H. pylori-positive subjects was similar to that of the same generation in Japan. H. pylori-positive subjects included 23 patients with chronic gastritis alone, 50 patients with chronic gastritis and gastric ulcer, 27 patients with chronic gastritis and duodenal ulcer, eight patients with chronic gastritis and gastric polyp, and two patients with chronic gastritis and gastric adenoma. Neither atrophic changes nor any other abnormal findings were observed in the 50 controls without H. pylori infection by endoscopic or histological examination. Their characteristics were similar to those of the H. pylori-positive subjects in age, gender, BMI, serum cholesterol, and fasting blood sugar as shown in Table 1. No subjects had evidence of a cachectic state such as advanced cancer, thyroid disease, liver disease, or infection. Subjects with diabetes mellitus or renal dysfunction (serum creatinine 1.5 mg/dl) were excluded. None of the 160 individuals recruited had a history of eradication therapy for H. pylori infection or received any antibiotic treatment during the study. Written informed consents were obtained from all participants in accordance with the Declaration of Helsinki and its later revision. This study was approved by the Ethics Committee of the Jichi Medical School. Subjects with H. pylori infection were classified into three groups according to the fasting levels of plasma ghrelin as shown in Table 2: low ghrelin group (<70 fmol/ml; n = 34), middle ghrelin group (70–150 fmol/ml; n = 36), and high ghrelin group (>150 fmol/ml; n = 40). Age, BMI, serum lipid data, and fasting blood sugar were similar in those three groups.
Specimens
Five adjacent biopsy specimens from the greater curvatures at the midcorpus of the stomach as well as five from the antrum were obtained endoscopically from all subjects. One biopsy specimen from the corpus of the stomach and one from the antrum were cultured individually to evaluate for the presence of H. pylori infection. Three biopsy specimens from the corpus and three from the antrum were immediately snap frozen and stored in liquid nitrogen for later use. The remaining corpus and antral specimens were fixed and stained with hematoxylin and eosin, Giemsa, and antighrelin antibody. Histological assessments were performed by a single observer (H.Os.). H. pylori infection was evaluated by the bacterial culture and histological examination.
Hormone assays
Blood was drawn into chilled tubes containing EDTA-2Na (1 mg/ml) and aprotinin (500 U/ml), and plasma was harvested after immediate centrifugation and stored at –30 C until assay. Plasma ghrelin levels were measured by a RIA developed in our laboratory. In brief, antiserum against the C-terminal region of human ghrelin was raised in New Zealand white rabbits that were immunized against synthetic human ghrelin (13–28). Human Tyr0-ghrelin (position13–28) was radioiodinated by the lactoperoxidase method for use in the assay. Inter- and intraassay variation was less than 8 and 6%, respectively. The limit of detection of this assay is 12 fmol per tuve of human ghrelin. We described previously the properties of the antiserum for ghrelin used in this study (9, 10).
Immunohistochemistry
We generated antighrelin antiserum as described previously (10). Briefly, synthetic [Cys12]rat-ghrelin (position 13–28) (4 mg) was conjugated with maleimide-activated mariculture keyhole limpet hemocyanin (6 mg; Pierce Chemical Co., Rockford, IL). The antigenic conjugate solution was administered to a New Zealand White rabbit. The antirat ghrelin antiserum (G107) specifically recognizes ghrelin and has 100% cross-reactivity with human ghrelin in immunohistochemistry (10).
Paraffin-embedded sections of the biopsy samples taken from the greater curvature of the stomach were deparaffinized in xylene, immersed in citrate buffer [10 mM (pH 6.0)], heated at 120 C for 20 min in an autoclave, and left at room temperature for 60 min. After incubation with blocking reagent (Dako Japan, Kyoto, Japan) for 10 min, individual sections were incubated with antiserum for ghrelin (diluted to 1:500) in a moist chamber at 4 C overnight. Normal mouse IgG1 was used for control studies. The slides were then washed five times with PBS and incubated with dextran polymer system/peroxidase (EnVision+; Dako Japan) at 37 C for 60 min. The chromogen was developed by incubating the slides with diaminobenzidine solution for 3 min. The slides were counterstained with hematoxylin.
RNA extraction
Total RNA was isolated from the biopsy specimen with ISOGEN (Nippon Gene, Tokyo, Japan). Two microgram of total RNA from each sample was reverse transcribed by using random nanomers and reverse transcriptase (TOYOBO, Osaka, Japan) according to the manufacturer’s protocol.
Real-time quantitative RT-PCR
The expression level of ghrelin mRNA was evaluated by using a real-time quantitative RT-PCR method with an ABI 7700 sequence detector system (PE Applied Biosystems, Foster City, CA). The sense primer for ghrelin was 5'-GGCAGGCTCCAGCTTCCT-3' and the antisense primer was 5'-TGGCTTCTTCGACTCCTTTCTC-3'. The reaction mixture was prepared according to the manufacturer’s protocol using TaqMan PCR kits (PE Applied Biosystems). The reactions also contained target hybridization ghrelin probe labeled with a reporter fluorescent dye, 6-carboxyfluorescein, at the 5' end (5'-AGCCCTGAACACCAGAGA-3'). The thermal cycling conditions included 50 C for 2 min and 95 C for 10 min, followed by 15 sec of denature at 95 C and 1 min of annealing/extension at 60 C for 40 cycles.
The quantitative amplification and expected sigmoid curve of PCR were obtained. The PCR products were also examined by 2% agarose gel electrophoresis to confirm successful amplification of the expected size of the gene. As a control, the mRNA was also subjected to real-time quantitative RT-PCR for measurement of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using TaqMan GAPDH control reagents (PE Applied Biosystems). For relative quantification of the ghrelin expression, calibration curves were constructed using the mRNA obtained from normal gastric mucosa without H. pylori infection. mRNA for GAPDH was used as an endogenous control. The levels of ghrelin mRNA were calculated from the ratio of ghrelin mRNA level to GAPDH mRNA level and shown as the 1000 times data of the mean ratio of three corpus samples or three antral samples. Ghrelin mRNA expression levels in the gastric mucosa were compared between H. pylori-positive and -negative subjects or among groups with different levels of plasma ghrelin.
Counting the ghrelin-producing cells
The slides after the immunostaining were viewed at x100 magnification and digitized with a digital HD microscope (VH-7000, Keyence, Tokyo, Japan). The immunoreactive cells in the gastric mucosa were counted and presented as the number of positive cells per branch of oxyntic gland because they were localized in the lower half of fundic epithelial glands. The numbers of ghrelin-expressing cells in the gastric mucosa were also compared between H. pylori-positive and -negative subjects or among groups with different levels of plasma ghrelin.
Statistical analyses
The level of ghrelin mRNA expression was expressed as the median (first quartile to third quartile). The number of the immunoreactive cells and clinical data were presented as mean (± SE). The Mann-Whitney U test was used to compare ghrelin mRNA levels in gastric mucosa from H. pylori-positive and -negative subjects. Two-tailed unpaired t test was used to compare the numbers of the immunoreactive cells in the gastric mucosa between H. pylori-positive and -negative subjects as well as clinical data between two groups. An ANOVA based on Fisher’s protected least significant difference test was used for the assessment of the relationship between the plasma ghrelin levels and either the serum pepsinogen levels or the number of the immunoreactive cells in gastric mucosa among H. pylori-positive subjects. Differences at P < 0.05 were considered significant.
Results
Plasma ghrelin concentrations are lower in H. pylori-positive subjects
Because the relationship between H. pylori infection and plasma ghrelin concentration remains controversial, we first attempted to assess the effect of H. pylori infection on the plasma ghrelin concentrations. To this end, we compared plasma ghrelin concentrations between H. pylori-positive and -negative subjects. The effect of body weight level was minimized because BMIs were similar in both groups (Table 1). As shown in Fig. 1, plasma ghrelin concentrations were significantly lower in H. pylori-positive subjects than in H. pylori-negative controls. A similar difference was also significantly observed between patients with chronic gastritis alone and H. pylori-negative controls (data not shown). Because the majority of ghrelin is known to be produced in the stomach, we hypothesized that H. pylori infection may attenuate ghrelin production in the stomach and may consequently reduce plasma ghrelin concentrations in H. pylori-positive subjects. To examine this hypothesis, we next examined the specific changes of gastric ghrelin production in association with H. pylori infection.
Ghrelin mRNA in gastric mucosa is lower in H. pylori-positive subjects
In an effort to examine the effects of H. pylori infection on ghrelin production in the gastric mucosa, we compared gastric ghrelin mRNA expression levels between H. pylori-positive and -negative subjects using real-time quantitative RT-PCR using corpus mucosa because gastric ghrelin is predominantly produced in the corpus rather than in the antrum (10). As shown in Fig. 2, gastric ghrelin mRNA levels of corpus mucosa were significantly lower in H. pylori-positive patients than H. pylori-negative controls. A similar difference was also significantly observed between patients with chronic gastritis alone and H. pylori-negative controls (data not shown). These results suggest that the expression of ghrelin mRNA in the gastric mucosa is markedly decreased in association with H. pylori infection. It is important to note that the average of gastric ghrelin mRNA expression levels in H. pylori-positive subjects was less than one 45th of that in H. pylori-negative controls. Moreover, as shown in Fig. 3, plasma ghrelin concentrations were in parallel with the ghrelin mRNA expression levels in H. pylori-positive subjects. Taken together, these results suggest that the attenuation of the ghrelin production in the gastric mucosa accounts for the decrease in the plasma ghrelin concentrations in H. pylori-positive individuals.
Ghrelin-producing cells in the gastric mucosa are lower in H. pylori-positive subjects
As an independent test to examine the effect of H. pylori infection on gastric ghrelin production and its relation with plasma ghrelin concentrations, we next investigated the numbers of ghrelin-producing cells of the corpus mucosa in H. pylori-positive and -negative subjects. For this purpose, biopsy samples taken from gastric mucosa were immunostained using an antighrelin polyclonal antibody. Immunoreactive cells were seen in the lower half of fundic epithelial glands as described previously (10). No immunoreactivity was detected in the tissue when control serum was used for staining (data not shown). Immunoreactivity was concentrated in the basal cytoplasm of the positive cells as shown in Fig. 4, A and B. As shown in Fig. 4C, the number of ghrelin-positive cells in the gastric mucosa of H. pylori-positive individuals was significantly lower than those of H. pylori-negative individuals. Furthermore, the numbers of ghrelin-positive cells in the gastric mucosa fell significantly in accompaniment to the decrease in plasma ghrelin concentrations in H. pylori-positive subjects (Fig. 5). These results reinforce that the attenuation of the gastric ghrelin production caused by H. pylori infection accounts for the decrease in the plasma ghrelin concentrations in H. pylori-positive individuals.
Plasma ghrelin concentrations are associated with serum pepsinogen concentrations in H. pylori-positive subjects
In the last sets of examinations, we further attempted to demonstrate the association between H. pylori infection and plasma ghrelin concentrations. Because H. pylori infection first induces gastric atrophy in its pathological course, we compared plasma ghrelin concentration with serum pepsinogen concentrations in H. pylori-positive patients. Pepsinogen I and pepsinogen II differ in their location in the stomach. Both are located in the chief and mucous neck cells of the oxyntic gland mucosa in the gastric corpus, but only pepsinogen II is present in the gastric antrum. A pepsinogen I to II ratio less than 3 is considered to be a reliable marker for severe atrophic gastritis (19, 20). Therefore, the plasma ghrelin levels in H. pylori-positive patients were compared with serum pepsinogen concentrations and serum pepsinogen I to II ratios. As shown in Fig. 6, serum levels of pepsinogen I and the ratio of pepsinogen I to II fell significantly as plasma ghrelin concentrations decreased, indicating the positive association between plasma ghrelin and pepsinogen I concentrations as well as pepsinogen I to II ratios in H. pylori-positive patients. Collectively, these results reveal that plasma ghrelin concentrations are associated with the progression of gastric atrophy.
Discussion
Plasma ghrelin levels have been associated with several clinical factors including BMI, food intake, and serum insulin levels (9, 21, 22). Although ghrelin-producing endocrine cells have been found mainly in the oxyntic mucosa of the stomach, ghrelin is also released from other tissues including small and large intestines, lung, kidney, the nucleus of the hypothalamus, and A cells of the pancreatic islet (23, 24). In fact, plasma ghrelin concentrations in gastrectomized patients still remain about one third of those in normal subjects (6). Thus, it is important to clarify which organ primarily influences changes in plasma ghrelin concentrations in each disease. In this study, we have demonstrated that plasma ghrelin concentrations are influenced by H. pylori infection. In particular, we focused on the gastric mucosa to better understand the effects of H. pylori infection on the alteration of ghrelin expression. The expression levels of ghrelin mRNA and the numbers of ghrelin-producing cells in the gastric mucosa were much lower in patients with H. pylori infection. Plasma ghrelin concentrations correlated with the gastric ghrelin mRNA as well as the frequency of ghrelin-immunoreactive cells in the gastric mucosa. Finally, we compared plasma ghrelin concentrations with serum pepsinogen levels, a marker for gastric atrophy. Plasma ghrelin concentrations in H. pylori-positive patients correlated with serum pepsinogen I concentration as well as pepsinogen I to II ratio. In addition, we demonstrated that groups with histologically higher degrees of gastric atrophy in the H. pylori-positive subjects tend to have lower plasma ghrelin concentrations (data not shown). These findings strongly suggest that the reduction of ghrelin-producing cells in the gastric mucosa by H. pylori infection results in the lower plasma ghrelin concentration in H. pylori-positive patients.
Our current data are consistent with the report of Nwokolo’s group (17) that plasma ghrelin concentrations increased after H. pylori eradication. In their study, however, gastric ghrelin before and after H. pylori eradication was not measured. Moreover, effects of a change in BMI before and after H. pylori eradication on plasma ghrelin concentrations could not be excluded. In addition, the number of the enrolled subjects in their study was relatively small (10 subjects). Therefore, our present study expanded their observations by enrolling many more subjects with comparable BMI and showing a direct association between H. pylori infection and lower gastric ghrelin production.
On the other hand, Gokcel’s group compared plasma ghrelin concentrations between H. pylori-positive and -negative subjects, and, opposed to our results, they found no differences. Although their study design was similar to ours, they did not provide any data on the gastric atrophy or gastric ghrelin production in their subjects. It is, therefore, only a speculation, but the discrepancy of the results may be due to the different features of gastric atrophy between Western and Japanese populations including disease frequency and severity. The earlier age of acquiring H. pylori infection in Japan, compared with Western countries, may also explain the high incidence of atrophic gastritis in Japanese adults and lower concentrations of plasma ghrelin concentrations as well.
It would be intriguing to clarify how a persistent decrease in plasma ghrelin concentration influences human growth and body weight. Recently several reports demonstrated that H. pylori-positive children have a high incidence of growth retardation (25, 26). H. pylori is acquired early in life in most of the developing world. Together with our results, the decrease of plasma ghrelin levels accompanied by H. pylori gastritis may have considerable influences on growth retardation in childhood. In our study, the plasma ghrelin levels in H. pylori-positive subjects were lower than H. pylori-negative subjects, even in patients with mild atrophic changes, implying that even mild gastric inflammation by H. pylori infection in children may reduce the production of gastric ghrelin. Further study in children including plasma ghrelin levels, degree of atrophy in the stomach, and presence of H. pylori infection may clarify these relationships.
In conclusion, our study indicates that plasma ghrelin concentrations are influenced by the presence of chronic gastritis in association with H. pylori infection. Decreases in gastric ghrelin production may account for lower concentrations of plasma ghrelin in H. pylori-positive individuals. These observations provide novel insights for understanding the physiological function of ghrelin and its relation to various diseases.
Acknowledgments
We are indebted to Ms. Hiroko Hoshino, Ms. Yukari Igarashi, Ms. Keiko Sasaki, Ms. Eiko Watanabe, and Ms. Kaoru Tokuda for their excellent technical assistance and Dr. Christopher L. Bowlus (Division of Gastroenterology, University of California Davis) for careful review of the manuscript.
Footnotes
First Published Online October 13, 2004
Abbreviations: BMI, Body mass index; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Received July 9, 2004.
Accepted September 10, 2004.
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Ghrelin is primarily secreted from the stomach and has been implicated in the coordination of eating behavior and weight regulation. The effects of Helicobacter pylori infection on plasma ghrelin concentration and gastric ghrelin production still have not been well known. We determined plasma ghrelin concentration in a total of 160 consecutive individuals with normal body mass index including 110 H. pylori-infected and 50 H. pylori-negative subjects. The expression levels of ghrelin mRNA and ghrelin-producing cells in the gastric mucosa were quantified with real-time quantitative RT-PCR and immunohistochemistry, respectively. The severity of gastric atrophy was evaluated by serum pepsinogen concentrations. Plasma ghrelin concentration, gastric ghrelin mRNA, and ghrelin-positive cell numbers in gastric mucosa were significantly lower in H. pylori-infected subjects. The decrease in plasma ghrelin concentration in H. pylori-positive subjects was accompanied by an attenuation of ghrelin mRNA expression and a reduction of ghrelin-positive cell numbers in the gastric mucosa. Moreover, lower serum pepsinogen I concentrations and I/II ratio were significantly associated with lower plasma ghrelin concentrations in H. pylori-positive subjects. These findings suggest that impaired gastric ghrelin production in association with atrophic gastritis induced by H. pylori infection accounts for the decrease in plasma ghrelin concentration.
Introduction
GHRELIN, A 28-AMINO acid peptide isolated from rat and human stomach possesses strong GH-releasing activity and plays central as well as peripheral roles in food intake, gastric motility, and acid secretion (1, 2). Ghrelin has been shown to evoke weight gain by actions in the hypothalamus (3). Plasma ghrelin concentrations rise before meals and fall after meals. This peptide also contributes to the regulation of both somatic growth and adipose tissue mass and is therefore a short-term, meal-related orexigen as well as a long-term regulator of body weight (4, 5, 6). Circulating ghrelin concentrations in newborns are not associated with gender, body weight, or hormonal parameter (7). In children and adults, however, plasma ghrelin concentrations are lower in obese subjects, compared with those with normal body weight and lean subjects (8, 9). The decrease of plasma ghrelin concentrations appears to compensate for the positive energy balance in obese individuals (9). The majority of circulating ghrelin is produced in the mammalian gastric mucosa by enteroendocrine cells/oxyntic glands, probably the X/A-like cells (10). Thus, there exists the possibility that chronic persistent damage of the gastric mucosa, such as chronic gastritis, might affect ghrelin production, leading to changes in food intake and body weight. Helicobacter pylori is a Gram-negative bacterium that colonizes the stomach. H. pylori infection is involved in the pathogenesis of gastritis, gastric and duodenal ulcer, gastric carcinoma, and mucosa-associated lymphoid tissue lymphoma (11, 12, 13). More than 50% of the adult population are infected with H. pylori worldwide (14, 15). H. pylori infection first leads to atrophic gastritis and intestinal metaplasia, which may further lead to dysplasia and gastric carcinoma (16). Thus, it is an intriguing question whether H. pylori infection affects gastric ghrelin production and consequently alters plasma ghrelin concentration.
In this respect, Nwokolo et al. (17) reported that plasma ghrelin concentrations increased after the eradication of H. pylori. On the contrary, Gokcel et al. (18) reported that H. pylori infection has no effect on plasma ghrelin levels. Thus, the relationship between H. pylori infection and plasma ghrelin concentrations is still controversial, prompting us to further assess the effects of H. pylori infection on plasma ghrelin concentrations. Because previous studies examined serum ghrelin concentrations without investigating gastric ghrelin production (17, 18), the direct relationship between H. pylori infection and gastric ghrelin production, which could influence plasma ghrelin concentrations, is still to be demonstrated. We thus conducted this study to investigate the association of H. pylori infection with both ghrelin mRNA and protein production in the stomach, concomitantly examining plasma ghrelin concentrations. To this end, we applied real-time quantitative RT-PCR and immunohistochemistry of endoscopic biopsy specimens. Moreover, because body weight is an important factor that determines plasma ghrelin concentrations, only individuals with normal body mass index (BMI) were enrolled in this study. We report here that H. pylori infection is associated with lower gastric ghrelin mRNA and protein as well as serum ghrelin concentrations.
Subjects and Methods
Participants
We enrolled 160 consecutive asymptomatic men with normal BMI undergoing gastric cancer surveillance in Tochigi, Japan. They were divided into two groups (110 H. pylori-positive subjects and 50 H. pylori-negative controls) according to the presence or absence of H. pylori in the gastric mucosa evaluated by the bacterial culture and histological examination. The percentage of the H. pylori-positive subjects was similar to that of the same generation in Japan. H. pylori-positive subjects included 23 patients with chronic gastritis alone, 50 patients with chronic gastritis and gastric ulcer, 27 patients with chronic gastritis and duodenal ulcer, eight patients with chronic gastritis and gastric polyp, and two patients with chronic gastritis and gastric adenoma. Neither atrophic changes nor any other abnormal findings were observed in the 50 controls without H. pylori infection by endoscopic or histological examination. Their characteristics were similar to those of the H. pylori-positive subjects in age, gender, BMI, serum cholesterol, and fasting blood sugar as shown in Table 1. No subjects had evidence of a cachectic state such as advanced cancer, thyroid disease, liver disease, or infection. Subjects with diabetes mellitus or renal dysfunction (serum creatinine 1.5 mg/dl) were excluded. None of the 160 individuals recruited had a history of eradication therapy for H. pylori infection or received any antibiotic treatment during the study. Written informed consents were obtained from all participants in accordance with the Declaration of Helsinki and its later revision. This study was approved by the Ethics Committee of the Jichi Medical School. Subjects with H. pylori infection were classified into three groups according to the fasting levels of plasma ghrelin as shown in Table 2: low ghrelin group (<70 fmol/ml; n = 34), middle ghrelin group (70–150 fmol/ml; n = 36), and high ghrelin group (>150 fmol/ml; n = 40). Age, BMI, serum lipid data, and fasting blood sugar were similar in those three groups.
Specimens
Five adjacent biopsy specimens from the greater curvatures at the midcorpus of the stomach as well as five from the antrum were obtained endoscopically from all subjects. One biopsy specimen from the corpus of the stomach and one from the antrum were cultured individually to evaluate for the presence of H. pylori infection. Three biopsy specimens from the corpus and three from the antrum were immediately snap frozen and stored in liquid nitrogen for later use. The remaining corpus and antral specimens were fixed and stained with hematoxylin and eosin, Giemsa, and antighrelin antibody. Histological assessments were performed by a single observer (H.Os.). H. pylori infection was evaluated by the bacterial culture and histological examination.
Hormone assays
Blood was drawn into chilled tubes containing EDTA-2Na (1 mg/ml) and aprotinin (500 U/ml), and plasma was harvested after immediate centrifugation and stored at –30 C until assay. Plasma ghrelin levels were measured by a RIA developed in our laboratory. In brief, antiserum against the C-terminal region of human ghrelin was raised in New Zealand white rabbits that were immunized against synthetic human ghrelin (13–28). Human Tyr0-ghrelin (position13–28) was radioiodinated by the lactoperoxidase method for use in the assay. Inter- and intraassay variation was less than 8 and 6%, respectively. The limit of detection of this assay is 12 fmol per tuve of human ghrelin. We described previously the properties of the antiserum for ghrelin used in this study (9, 10).
Immunohistochemistry
We generated antighrelin antiserum as described previously (10). Briefly, synthetic [Cys12]rat-ghrelin (position 13–28) (4 mg) was conjugated with maleimide-activated mariculture keyhole limpet hemocyanin (6 mg; Pierce Chemical Co., Rockford, IL). The antigenic conjugate solution was administered to a New Zealand White rabbit. The antirat ghrelin antiserum (G107) specifically recognizes ghrelin and has 100% cross-reactivity with human ghrelin in immunohistochemistry (10).
Paraffin-embedded sections of the biopsy samples taken from the greater curvature of the stomach were deparaffinized in xylene, immersed in citrate buffer [10 mM (pH 6.0)], heated at 120 C for 20 min in an autoclave, and left at room temperature for 60 min. After incubation with blocking reagent (Dako Japan, Kyoto, Japan) for 10 min, individual sections were incubated with antiserum for ghrelin (diluted to 1:500) in a moist chamber at 4 C overnight. Normal mouse IgG1 was used for control studies. The slides were then washed five times with PBS and incubated with dextran polymer system/peroxidase (EnVision+; Dako Japan) at 37 C for 60 min. The chromogen was developed by incubating the slides with diaminobenzidine solution for 3 min. The slides were counterstained with hematoxylin.
RNA extraction
Total RNA was isolated from the biopsy specimen with ISOGEN (Nippon Gene, Tokyo, Japan). Two microgram of total RNA from each sample was reverse transcribed by using random nanomers and reverse transcriptase (TOYOBO, Osaka, Japan) according to the manufacturer’s protocol.
Real-time quantitative RT-PCR
The expression level of ghrelin mRNA was evaluated by using a real-time quantitative RT-PCR method with an ABI 7700 sequence detector system (PE Applied Biosystems, Foster City, CA). The sense primer for ghrelin was 5'-GGCAGGCTCCAGCTTCCT-3' and the antisense primer was 5'-TGGCTTCTTCGACTCCTTTCTC-3'. The reaction mixture was prepared according to the manufacturer’s protocol using TaqMan PCR kits (PE Applied Biosystems). The reactions also contained target hybridization ghrelin probe labeled with a reporter fluorescent dye, 6-carboxyfluorescein, at the 5' end (5'-AGCCCTGAACACCAGAGA-3'). The thermal cycling conditions included 50 C for 2 min and 95 C for 10 min, followed by 15 sec of denature at 95 C and 1 min of annealing/extension at 60 C for 40 cycles.
The quantitative amplification and expected sigmoid curve of PCR were obtained. The PCR products were also examined by 2% agarose gel electrophoresis to confirm successful amplification of the expected size of the gene. As a control, the mRNA was also subjected to real-time quantitative RT-PCR for measurement of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using TaqMan GAPDH control reagents (PE Applied Biosystems). For relative quantification of the ghrelin expression, calibration curves were constructed using the mRNA obtained from normal gastric mucosa without H. pylori infection. mRNA for GAPDH was used as an endogenous control. The levels of ghrelin mRNA were calculated from the ratio of ghrelin mRNA level to GAPDH mRNA level and shown as the 1000 times data of the mean ratio of three corpus samples or three antral samples. Ghrelin mRNA expression levels in the gastric mucosa were compared between H. pylori-positive and -negative subjects or among groups with different levels of plasma ghrelin.
Counting the ghrelin-producing cells
The slides after the immunostaining were viewed at x100 magnification and digitized with a digital HD microscope (VH-7000, Keyence, Tokyo, Japan). The immunoreactive cells in the gastric mucosa were counted and presented as the number of positive cells per branch of oxyntic gland because they were localized in the lower half of fundic epithelial glands. The numbers of ghrelin-expressing cells in the gastric mucosa were also compared between H. pylori-positive and -negative subjects or among groups with different levels of plasma ghrelin.
Statistical analyses
The level of ghrelin mRNA expression was expressed as the median (first quartile to third quartile). The number of the immunoreactive cells and clinical data were presented as mean (± SE). The Mann-Whitney U test was used to compare ghrelin mRNA levels in gastric mucosa from H. pylori-positive and -negative subjects. Two-tailed unpaired t test was used to compare the numbers of the immunoreactive cells in the gastric mucosa between H. pylori-positive and -negative subjects as well as clinical data between two groups. An ANOVA based on Fisher’s protected least significant difference test was used for the assessment of the relationship between the plasma ghrelin levels and either the serum pepsinogen levels or the number of the immunoreactive cells in gastric mucosa among H. pylori-positive subjects. Differences at P < 0.05 were considered significant.
Results
Plasma ghrelin concentrations are lower in H. pylori-positive subjects
Because the relationship between H. pylori infection and plasma ghrelin concentration remains controversial, we first attempted to assess the effect of H. pylori infection on the plasma ghrelin concentrations. To this end, we compared plasma ghrelin concentrations between H. pylori-positive and -negative subjects. The effect of body weight level was minimized because BMIs were similar in both groups (Table 1). As shown in Fig. 1, plasma ghrelin concentrations were significantly lower in H. pylori-positive subjects than in H. pylori-negative controls. A similar difference was also significantly observed between patients with chronic gastritis alone and H. pylori-negative controls (data not shown). Because the majority of ghrelin is known to be produced in the stomach, we hypothesized that H. pylori infection may attenuate ghrelin production in the stomach and may consequently reduce plasma ghrelin concentrations in H. pylori-positive subjects. To examine this hypothesis, we next examined the specific changes of gastric ghrelin production in association with H. pylori infection.
Ghrelin mRNA in gastric mucosa is lower in H. pylori-positive subjects
In an effort to examine the effects of H. pylori infection on ghrelin production in the gastric mucosa, we compared gastric ghrelin mRNA expression levels between H. pylori-positive and -negative subjects using real-time quantitative RT-PCR using corpus mucosa because gastric ghrelin is predominantly produced in the corpus rather than in the antrum (10). As shown in Fig. 2, gastric ghrelin mRNA levels of corpus mucosa were significantly lower in H. pylori-positive patients than H. pylori-negative controls. A similar difference was also significantly observed between patients with chronic gastritis alone and H. pylori-negative controls (data not shown). These results suggest that the expression of ghrelin mRNA in the gastric mucosa is markedly decreased in association with H. pylori infection. It is important to note that the average of gastric ghrelin mRNA expression levels in H. pylori-positive subjects was less than one 45th of that in H. pylori-negative controls. Moreover, as shown in Fig. 3, plasma ghrelin concentrations were in parallel with the ghrelin mRNA expression levels in H. pylori-positive subjects. Taken together, these results suggest that the attenuation of the ghrelin production in the gastric mucosa accounts for the decrease in the plasma ghrelin concentrations in H. pylori-positive individuals.
Ghrelin-producing cells in the gastric mucosa are lower in H. pylori-positive subjects
As an independent test to examine the effect of H. pylori infection on gastric ghrelin production and its relation with plasma ghrelin concentrations, we next investigated the numbers of ghrelin-producing cells of the corpus mucosa in H. pylori-positive and -negative subjects. For this purpose, biopsy samples taken from gastric mucosa were immunostained using an antighrelin polyclonal antibody. Immunoreactive cells were seen in the lower half of fundic epithelial glands as described previously (10). No immunoreactivity was detected in the tissue when control serum was used for staining (data not shown). Immunoreactivity was concentrated in the basal cytoplasm of the positive cells as shown in Fig. 4, A and B. As shown in Fig. 4C, the number of ghrelin-positive cells in the gastric mucosa of H. pylori-positive individuals was significantly lower than those of H. pylori-negative individuals. Furthermore, the numbers of ghrelin-positive cells in the gastric mucosa fell significantly in accompaniment to the decrease in plasma ghrelin concentrations in H. pylori-positive subjects (Fig. 5). These results reinforce that the attenuation of the gastric ghrelin production caused by H. pylori infection accounts for the decrease in the plasma ghrelin concentrations in H. pylori-positive individuals.
Plasma ghrelin concentrations are associated with serum pepsinogen concentrations in H. pylori-positive subjects
In the last sets of examinations, we further attempted to demonstrate the association between H. pylori infection and plasma ghrelin concentrations. Because H. pylori infection first induces gastric atrophy in its pathological course, we compared plasma ghrelin concentration with serum pepsinogen concentrations in H. pylori-positive patients. Pepsinogen I and pepsinogen II differ in their location in the stomach. Both are located in the chief and mucous neck cells of the oxyntic gland mucosa in the gastric corpus, but only pepsinogen II is present in the gastric antrum. A pepsinogen I to II ratio less than 3 is considered to be a reliable marker for severe atrophic gastritis (19, 20). Therefore, the plasma ghrelin levels in H. pylori-positive patients were compared with serum pepsinogen concentrations and serum pepsinogen I to II ratios. As shown in Fig. 6, serum levels of pepsinogen I and the ratio of pepsinogen I to II fell significantly as plasma ghrelin concentrations decreased, indicating the positive association between plasma ghrelin and pepsinogen I concentrations as well as pepsinogen I to II ratios in H. pylori-positive patients. Collectively, these results reveal that plasma ghrelin concentrations are associated with the progression of gastric atrophy.
Discussion
Plasma ghrelin levels have been associated with several clinical factors including BMI, food intake, and serum insulin levels (9, 21, 22). Although ghrelin-producing endocrine cells have been found mainly in the oxyntic mucosa of the stomach, ghrelin is also released from other tissues including small and large intestines, lung, kidney, the nucleus of the hypothalamus, and A cells of the pancreatic islet (23, 24). In fact, plasma ghrelin concentrations in gastrectomized patients still remain about one third of those in normal subjects (6). Thus, it is important to clarify which organ primarily influences changes in plasma ghrelin concentrations in each disease. In this study, we have demonstrated that plasma ghrelin concentrations are influenced by H. pylori infection. In particular, we focused on the gastric mucosa to better understand the effects of H. pylori infection on the alteration of ghrelin expression. The expression levels of ghrelin mRNA and the numbers of ghrelin-producing cells in the gastric mucosa were much lower in patients with H. pylori infection. Plasma ghrelin concentrations correlated with the gastric ghrelin mRNA as well as the frequency of ghrelin-immunoreactive cells in the gastric mucosa. Finally, we compared plasma ghrelin concentrations with serum pepsinogen levels, a marker for gastric atrophy. Plasma ghrelin concentrations in H. pylori-positive patients correlated with serum pepsinogen I concentration as well as pepsinogen I to II ratio. In addition, we demonstrated that groups with histologically higher degrees of gastric atrophy in the H. pylori-positive subjects tend to have lower plasma ghrelin concentrations (data not shown). These findings strongly suggest that the reduction of ghrelin-producing cells in the gastric mucosa by H. pylori infection results in the lower plasma ghrelin concentration in H. pylori-positive patients.
Our current data are consistent with the report of Nwokolo’s group (17) that plasma ghrelin concentrations increased after H. pylori eradication. In their study, however, gastric ghrelin before and after H. pylori eradication was not measured. Moreover, effects of a change in BMI before and after H. pylori eradication on plasma ghrelin concentrations could not be excluded. In addition, the number of the enrolled subjects in their study was relatively small (10 subjects). Therefore, our present study expanded their observations by enrolling many more subjects with comparable BMI and showing a direct association between H. pylori infection and lower gastric ghrelin production.
On the other hand, Gokcel’s group compared plasma ghrelin concentrations between H. pylori-positive and -negative subjects, and, opposed to our results, they found no differences. Although their study design was similar to ours, they did not provide any data on the gastric atrophy or gastric ghrelin production in their subjects. It is, therefore, only a speculation, but the discrepancy of the results may be due to the different features of gastric atrophy between Western and Japanese populations including disease frequency and severity. The earlier age of acquiring H. pylori infection in Japan, compared with Western countries, may also explain the high incidence of atrophic gastritis in Japanese adults and lower concentrations of plasma ghrelin concentrations as well.
It would be intriguing to clarify how a persistent decrease in plasma ghrelin concentration influences human growth and body weight. Recently several reports demonstrated that H. pylori-positive children have a high incidence of growth retardation (25, 26). H. pylori is acquired early in life in most of the developing world. Together with our results, the decrease of plasma ghrelin levels accompanied by H. pylori gastritis may have considerable influences on growth retardation in childhood. In our study, the plasma ghrelin levels in H. pylori-positive subjects were lower than H. pylori-negative subjects, even in patients with mild atrophic changes, implying that even mild gastric inflammation by H. pylori infection in children may reduce the production of gastric ghrelin. Further study in children including plasma ghrelin levels, degree of atrophy in the stomach, and presence of H. pylori infection may clarify these relationships.
In conclusion, our study indicates that plasma ghrelin concentrations are influenced by the presence of chronic gastritis in association with H. pylori infection. Decreases in gastric ghrelin production may account for lower concentrations of plasma ghrelin in H. pylori-positive individuals. These observations provide novel insights for understanding the physiological function of ghrelin and its relation to various diseases.
Acknowledgments
We are indebted to Ms. Hiroko Hoshino, Ms. Yukari Igarashi, Ms. Keiko Sasaki, Ms. Eiko Watanabe, and Ms. Kaoru Tokuda for their excellent technical assistance and Dr. Christopher L. Bowlus (Division of Gastroenterology, University of California Davis) for careful review of the manuscript.
Footnotes
First Published Online October 13, 2004
Abbreviations: BMI, Body mass index; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Received July 9, 2004.
Accepted September 10, 2004.
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