DOI 10.1007/s00535-008-2231-4. Atrophic gastritis: deficient complex I of the respiratory chain in the mitochondria of corpus mucosal cells. MARJU GRUNO.
J Gastroenterol 2008; 43:780–788 DOI 10.1007/s00535-008-2231-4
Atrophic gastritis: deficient complex I of the respiratory chain in the mitochondria of corpus mucosal cells MARJU GRUNO1,2, NADEZHDA PEET1, ANDRES TEIN2, RIINA SALUPERE3, MEELI SIROTKINA4, JULIO VALLE5, ANTS PEETSALU2, and ENN K. SEPPET1 1
Department of Pathophysiology, Centre of Molecular and Clinical Medicine, Faculty of Medicine, University of Tartu, 19 Ravila Street, 50411 Tartu, Estonia Department of Surgery, Faculty of Medicine, University of Tartu, Tartu, Estonia 3 Department of Internal Medicine, Faculty of Medicine, University of Tartu, Tartu, Estonia 4 Department of Pathology, Tartu University Hospital, Tartu, Estonia 5 Department of Gastroenterology, Hospital Virgen de la Salud, Toledo, Spain 2
Background. Mitochondrial dysfunction is one of the most characteristic properties of the cancer cell. However, it is not known whether oxidative energy metabolism has already become altered in conditions of atrophic gastritis, a precancerous state of gastric disease. The purpose of our study was to comparatively characterize oxidative phosphorylation (OXPHOS) in the atrophic and nonatrophic gastric corpus mucosa. Methods. Mucosal biopsies were taken from 12 patients with corpus dominant atrophic gastritis and from 12 patients with nonatrophic mucosa (controls). One part of the tissue samples was permeabilized with saponin for analysis of the function of the respiratory chain using high-resolution respirometry, and another part was used for histopathological examination. The serum level of pepsinogen I (S-PGI) was determined with a specific enzyme immunoassay (EIA). Results. Compared to the control group, the maximal capacity of OXPHOS in the atrophy group was almost twofold lower, the respiratory chain complex I-dependent respiration, normalized to complex II-dependent respiration, was reduced, and respiratory control by ADP in the presence of succinate was increased in the atrophic corpus mucosa. In the whole cohort of the patients studied, serum S-PGI level correlated positively with complex I-dependent respiration or complex Idependent to complex II-dependent respiration ratio. Conclusions. Corpus dominant atrophic gastritis is characterized by decreased respiratory capacity and relative deficiency of the respiratory complex I of mitochondria in the mucosa, the latter defect probably limiting mitochondrial ATP production and energetic support of the secretory function of the zymogenic mucosal cells.
Received: February 7, 2008 / Accepted: May 29, 2008 Reprint requests to: M. Gruno
Key words: respiratory chain, atrophy, gastritis, gastric mucosa, stomach
Introduction Gastric corpus dominant atrophic gastritis, a consistent finding in pernicious anemia (PA), is a definitive risk factor for gastric cancer.1,2 The prevalence of gastric carcinoma in PA patients is 1%–3%, and 2% of patients with gastric carcinoma have PA.3 The mechanisms mediating the progression of atrophic gastritis and its transition to carcinoma are unclear. It has been assumed that decreased acid secretion caused by a loss of parietal cells in the corpus mucosa predisposes to gastric cancer by several mechanisms, including impaired absorption of vitamin C and the constitution of the intragastric milieu allowing overgrowth of salivary and intestinal bacteria.4 At the cellular level, increasing evidence points to the crucial role of disturbances of energy metabolism.5–8 Otto Warburg was the first to propose that development of cancer is associated with suppression of oxidative phosphorylation (OXPHOS) and activation of glycolysis.9 More recent studies have related the pathophysiological role of mitochondria in cancer cell metabolism to their capability to produce reactive oxygen (ROS) and reactive nitrogen species.10–12 Excess ROS in turn causes defects in the mitochondrial genome, thus leading to impaired OXPHOS, which not only limits ATP generation but also further promotes ROS production.13 Recent data aim at mitochondria as key organelles in regulation of expression of the hypoxia-inducible factor-1α (HIF1α), which is responsible for shifting metabolism from OXPHOS to glycolysis, a characteristic change in the tumor cell. This shift is controlled by multiple means including the effects of ROS (see review14) and mitochondrial succinate metabolism.15,16 Along with these
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changes, as a consequence of upregulation of antiapoptotic and downregulation of proapoptotic proteins, mitochondria are deprived of their efficacy in impelling apoptosis, which favors unlimited proliferation of the cancer cells.17,18 There exist only limited data regarding altered OXPHOS in the mucosal cells in gastric diseases.19–22 The status of OXPHOS in patients with atrophic gastritis, a condition known as a precancerous state,23 has not been investigated as yet. Because in this state morphological studies have revealed a decreased number of swollen and vacuolized mitochondria,24 a decreased capacity of OXPHOS might be expected. It is also conceivable that atrophy of the glandular mucosa imposes qualitative alterations upon the systems of electron transport and ATP synthesis, thereby inducing transition from a normal mucosa to cancer tissue. For example, decreased activities of the respiratory chain complexes enhance mitochondrial production of ROS, which in turn stimulates tumour development.25 Our recent study showed that mitochondrial defects in human gastric mucosal cells can be reliably detected by applying high-resolution respirometry in studies of saponinpermeabilized gastrobiopsy specimens.19 At present, it is also unclear how the bioenergetic changes can be related to the secretory function of the corpus mucosal cells. Based on positive correlation between the extent of gastric acid secretion and serum pepsinogen I (SPG-I) level,26 it is expected that the measurements of the SPG-I provide us with useful information for predicting the functional and morphological status of the gastric corpus mucosa.27–29 In this regard, we propose that the value of these tests might be enhanced if the SPG-I levels would correlate with the indices of mitochondrial function. As the experimental proof for such an assumption is still missing, it was one aim of our study to address the relationships between the OXPHOS and SPG-I levels in patients with gastric disease.
from southern Estonia who underwent upper gastrointestinal endoscopy for epigastric complaints were included. None of these subjects exhibited corpus mucosal atrophy, and they had received nonsteroidal anti-inflammatory drugs, H+-pump inhibitors, or antibiotics to cure their illness.
Material and methods
Blood samples and laboratory tests
Patients Twelve patients (5 men and 7 women; mean age, 67 ± 4 years) with PA were included in the study as a group of patients with atrophic corpus gastritis. The criteria for the diagnosis of PA were macrolytic anemia, appearance of parietal cell antibodies, and low serum vitamin B12 and folic acid. The diagnosis of corpus atrophic gastritis was based on low serum pepsinogen I (S-PGI) level and histological confirmation of gastric body mucosal atrophy. As the control group, 12 consecutive patients (7 men and 5 women; mean age, 66 ± 3 years)
Endoscopy and biopsy sampling Mucosal biopsies were taken from the anterior and posterior walls of the medial part of the corpus and from the antrum (2 cm above the pylorus from the anterior and posterior walls of the stomach). One part of each biopsy specimen was used to determine the histology of the gastric mucosa and the presence of Helicobacter pylori (H. pylori), for which these specimens were fixed overnight in neutral buffered formalin and embedded in paraffin. Tissue sections were stained for morphological and H. pylori examination by hematoxylin and eosin and modified Giemsa methods. The presence and severity of chronic gastritis, activity of gastritis, atrophy, and intestinal metaplasia were graded according to the Sidney system, from 0 (no changes) through 1 (mild) and 2 (moderate) to 3 (severe changes).30 The amount of H. pylori in the mucosa was estimated semiquantitatively by microscopic counting as described earlier.31 Another part of the corpus mucosa specimens was placed immediately in ice-cold solution A containing (in mM) CaK2EGTA, 2.77, K2EGTA, 7.23, MgCl2, 6.56, DTT, 0.5, K-MES, 50, imidazole, 20, taurine, 20, Na2ATP, 5.3, phosphocreatine, 15, at pH 7.1, and used for studies of mitochondrial function. The gastric biopsies were carried out in accordance with the European Communities Council Directive 86/609/EEC and with the Declaration of Helsinki.32 Written informed consent was obtained from all patients, and the Tartu University Ethics Committee approved the study.
Basal blood samples for measurements of serum pepsinogen I (S-PGI) were drawn after an overnight fast. Samples for S-PGI were collected into serum tubes. The serum tubes were centrifuged at 1500 g for 10 min and the samples were stored at −70°C until analyzed. S-PGI was determined using specific enzyme immunosorbent assay (EIA) tests (Pepsinogen-I EIA Test Kit; Biohit, Helsinki, Finland), and the procedure was performed on a microwell plate in accordance with the manufacturer’s instructions. All technical equipment required for the EIA techniques was provided by Biohit, Finland.
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Preparation of the permeabilized mucosal tissue
Statistical analyses
The permeabilized mucosal tissue was prepared by the technique described previously.19,33 The mucosal tissue biopsy samples were cut into smaller pieces in ice-cold solution A, and the pieces were gently stretched with thin needles to facilitate the diffusion of the medium into the intracellular space. Next, the tissue was incubated at 4°C, at mild stirring for 30 min in solution A containing 50 μg/ml saponin for permeabilization of the cell plasma membrane. The permeabilized mucosal tissue samples were then washed for 10 min in solution B containing (in mM) CaK2EGTA, 2.77, K2EGTA, 7.23, MgCl2, 1.38, DTT, 0.5, K-Mes, 100, imidazole, 20, taurine, 20, K2HPO4, 3, and 5 mg/ml bovine serum albumin, glutamate, 10, and malate, 2, pH 7.1 at 25°C; this procedure of washing was repeated two more times to remove all metabolites from the cells.
Data are expressed as means ± SE. The unpaired Student’s t test was used to analyze differences between the groups. Correlation analysis was performed by Pearson’s test. A P value
