Duodenal lipid-induced symptom generation in gastroesophageal reflux disease: role of apolipoprotein A-IV and cholecystokinin.

BACKGROUND: Duodenal lipid intensifies the perception of esophageal acid perfusion. Recently, we showed that genes implicated in lipid absorption were upregulated in the duodenum of fasting gastro-esophageal reflux disease (GERD) patients. This suggests that chylomicron production and secretion may be enhanced and, consequently, the release of apolipoprotein A-IV (apoA-IV), a chylomicron-derived signaling protein. ApoA-IV may stimulate release of cholecystokinin (CCK), an activator of vagal afferents. This study evaluated putative involvement of abnormal apoA-IV and CCK responses to lipid in GERD. METHODS: Ten GERD patients and 10 healthy volunteers (HV) underwent duodenal perfusion with Intralipid 20%, 2 kcal min(-1) , for 60 min. Symptoms were scored, blood samples collected every 15 min during lipid perfusion and 15 min after discontinuation when duodenal biopsies were taken. Plasma and mucosal concentrations of apoA-IV and CCK and transcript levels of 21 genes implicated in lipid absorption, differentially expressed under fasting conditions, were quantified. KEY RESULTS: Heartburn (P = 0.003), abdominal discomfort (P = 0.037) and nausea (P = 0.008) only increased significantly during lipid infusion in GERD patients. Following lipid infusion mean mucosal apoA-IV concentration was lower in GERD patients compared with HV (P = 0.023), whereas plasma concentration tended to be elevated (P = 0.068). Mean mucosal CCK concentration was also lower in GERD patients (P = 0.009). Two genes, HIBADH and JTB, were upregulated in GERD patients (P = 0.008 and P = 0.038, respectively). CONCLUSIONS & INFERENCES: Our results suggest excessive duodenal lipid-induced release of apoA-IV and CCK in GERD. We postulate that the resulting heightened activation of duodenal vagal afferents may underlie central sensitization, thereby increasing the perception of reflux events.

Role of chemical stimulation of the duodenum in dyspeptic symptom generation.

The response to chemical stimuli such as acid, nutrients, and capsaicin at the level of the duodenum is increasingly recognized as important in the etiology of dyspeptic symptoms. Increased duodenal acid exposure has been reported for patients with dyspeptic symptoms. Duodenal hypersensitivity to acid and the enhancing effect of duodenal acid on gastroduodenal mechanosensitivity may also contribute to dyspeptic symptom generation. Serotonergic signaling pathways may be involved in acid-induced dyspeptic symptoms. As for nutrients, lipid has been unequivocally shown to have a function in the pathogenesis of dyspeptic symptoms. Cholecystokinin (CCK) is an important mediator of the effects of duodenal lipid on gastroduodenal sensorimotor activities. It is unclear whether CCK hypersecretion or hypersensitivity to CCK is responsible for symptoms in dyspeptic patients. The presence of capsaicin in the duodenum evokes symptoms and affects gastric sensorimotor function. In patients with dyspepsia, capsaicin-induced symptoms appeared to occur earlier and to be more severe, however the effects of duodenal infusion and putative consequent gastric sensorimotor abnormalities have not been examined. Capsaicin activates transient receptor potential ion channel of the vanilloid type I, which can also be activated and sensitized by acid. The interaction between the different chemical stimuli is complex and has not yet been studied in patients with dyspeptic symptoms. In conclusion, the mechanisms underlying an enhanced response to duodenal chemical stimulation in patients with dyspeptic symptoms are partially understood. At the level of the duodenum, abnormalities may exist in stimulus intensity, mucosal mRNA expression, biosynthesis, release, or inactivation of mucosal mediators, or receptor expression on afferent nerve endings. Elucidation of the abnormalities involved will provide a basis for rational treatment of dyspeptic symptoms.

In GERD patients, mucosal repair associated genes are upregulated in non-inflamed oesophageal epithelium.

Previous studies addressing the effects of acid reflux and PPI therapy on gene expression in oesophageal epithelium concentrated on inflamed tissue. We aimed to determine changes in gene expression in non-inflamed oesophageal epithelium of GERD patients. Therefore, we included 20 GERD patients with pathological total 24-hr acid exposure of 6-12% and SAP > or = 95%. Ten patients discontinued PPI treatment (PPI-), 10 took pantoprazole 40 mg bid (PPI+). Ten age/sex-matched healthy controls were recruited. Biopsies were taken from non-inflamed mucosa 6 cm and 16 cm proximal to the squamocolumnar junction (SCJ). Gene expression profiling of biopsies from 6 cm was performed on Human Genome U133 Plus 2.0 arrays (Affymetrix). Genes exhibiting a fold change >1.4 (t-test P-value < 1(E)- 4) were considered differentially expressed. Results were confirmed by real-time RT-PCR. In PPI- patients, 92 microarray probesets were deregulated. The majority of the corresponding genes were associated with cell-cell contacts, cytoskeletal reorganization and cellular motility, suggesting facilitation of a migratory phenotype. Genes encoding proteins with anti-apoptotic or anti-proliferative functions or stress-protective functions were also deregulated. No probesets were deregulated in PPI+ patients. QPCR analysis of 20 selected genes confirmed most of the deregulations in PPI- patients, and showed several deregulated genes in PPI+ patients as well. In the biopsies taken at 16 cm QPCR revealed no deregulations of the selected genes. We conclude that upon acid exposure, oesophageal epithelial cells activate a process globally known as epithelial restitution: up-regulation of anti-apoptotic, anti-oxidant and migration associated genes. Possibly this process helps maintaining barrier function.

Gastroesophageal reflux disease is associated with the C825T polymorphism in the G-protein beta3 subunit gene (GNB3).

OBJECTIVES: Visceral hypersensitivity is involved in the etiology of reflux symptoms. Familial clustering and twin studies demonstrated a genetic predisposition to gastroesophageal reflux disease (GERD). G-protein-coupled receptors (GPCRs) mediate the response to acid, neurotransmitters and humoral factors modulating esophageal sensory function. Thus, polymorphisms in G-proteins are putative genetic factors contributing to GERD manifestation. A functional polymorphism in the G-protein beta3 subunit gene (GNB3) is associated with functional dyspepsia (FD), in which visceral hypersensitivity is implicated in symptom generation. We evaluated the association of the GNB3 C825T polymorphism with GERD and GERD subgroups classified according to esophageal acid exposure time, symptom-reflux correlation, or coexistence of FD and/or irritable bowel syndrome (IBS) symptoms. METHODS: In total, 363 GERD patients, defined as having esophageal pH < 4 > or = 6% of time and/or symptom index (SI) > or = 50% or symptom association probability (SAP) > or = 95%, participated. In addition, 373 healthy controls free of gastrointestinal symptoms were studied. Genotyping was performed by molecular beacon assay. RESULTS: The CT genotype was more prevalent in GERD patients relative to healthy controls (adjusted odds ratio (OR)=1.43, 95% CI 1.04-1.98). GERD patients sensitive to physiological amounts of reflux displayed a higher OR (1.59), as did GERD patients with a positive symptom association score (1.50). The strongest association was detected in patients without concomitant FD and/or IBS symptoms (OR=1.66). CONCLUSIONS: GERD is associated with GNB3 C825T. The results for GERD subgroups support the hypothesis that enhanced perception of reflux events, as a consequence of the increased signal transduction upon GPCR activation associated with the 825T allele, underlies this association.

Trypsinogen IV, serotonin transporter transcript levels and serotonin content are increased in small intestine of irritable bowel syndrome patients.

Colorectal and small intestinal visceral hypersensitivity has been demonstrated in irritable bowel syndrome (IBS). Serine protease signalling via protease-activated receptor (PAR)-2 promotes hyperalgesia to mechanical distension. Furthermore, serotonergic pathways are involved in gastrointestinal visceral sensitivity. Abnormalities of serine protease and serotonergic signalling components have been identified in IBS colorectal mucosal biopsies. We determined the role of altered mucosal serine protease and serotonergic signalling in small intestine of IBS patients. Duodenal mucosal biopsies of 34 IBS patients (10 constipation-,11 diarrhoea-predominant and 13 alternating) and 20 healthy subjects (HS) were collected. Gene transcripts of PAR-2, trypsinogen IV, TPH-1, SERT (serotonin transport protein) and serotonin (5-HT(3)) subunits were quantified using real-time PCR and 5-HT content was measured by ELISA. Irritable bowel syndrome patients showed 1.5-fold higher trypsinogen IV mRNA level compared to HS (P = 0.016). SERT expression was 1.8-fold higher in IBS compared to HS (P = 0.007). Mucosal 5-HT content was 1.7-fold higher in IBS compared to HS (P = 0.015). The increase was 2.1-fold in IBS-C relative to HS (P = 0.018). Transcript levels of PAR-2, TPH-1 and 5-HT(3) receptor subunits did not differ between IBS and HS. In conclusion enhanced trypsinogen IV expression in IBS may cause increased PAR-2 activation. Increased SERT expression and mucosal 5-HT content in IBS suggest higher 5-HT availability. Both may contribute to small intestinal visceral hypersensitivity in IBS patients.

Potential of genetic translational research in gastroenterology.

The concept that genetic variation underlies inter-individual differences in drug response and contributes to the risk of developing common, complex disorders is expanding rapidly. Consequently the interest in genetic translational research has increased. Polymorphic DNA markers, either microsatellites or single nucleotide polymorphisms (SNPs), are used to assess genetic identities and track genetic differences between individuals. Given their abundance and stability, SNPs hold great promise as markers for mapping disease susceptibility loci for common, complex disorders by association studies. For this purpose the development of inexpensive, accurate, high-throughput methods for scoring large numbers of SNPs from hundreds of patients and controls is critical. Furthermore, gene expression profiling using DNA microarrays is likely to become a useful diagnostic tool enabling classification of disease phenotype based on molecular basis of disease pathogenesis, revealing information that cannot be obtained by histological assessment. Moreover, identification of differentially expressed genes in affected versus control tissue or over time in affected tissue will lead to better understanding of the mechanisms underlying disease and ultimately to the development of more effective drug therapies. To illustrate the potential of genetic translational research, several examples in the field of gastroenterology are described.

Transcriptional regulation of YML083c under aerobic and anaerobic conditions.

YML083c and DAN1 were among the Saccharomyces cerevisiae ORFs that displayed the strongest increase in transcript abundance during anaerobic growth compared to aerobic growth, as determined by oligonucleotide microarrays. We here report that transcription of YML083c is regulated by at least three different factors. First, repression under aerobic conditions depends on the presence of heme. Second, deletion analysis of the 5'-flanking region of YML083c and DAN1 revealed two regions responsible for anaerobic induction. Each of these regions conferred anoxia-regulated expression to the heterologous, minimal, CYC1-lacZ reporter. Mutations in the AAACGA subelement, common to the positive acting regions of YML083c and DAN1, almost completely abolished the ability to drive anaerobic expression of the reporter gene. This subelement is similar to the AR1 site, which is involved in anaerobic induction of the DAN/TIR genes. Activation through the AR1 site depends on Upc2. Indeed, transcription from the YML083c promoter was decreased in an upc2 null mutant. Third, expression of Sut1 under aerobic conditions enhanced transcription of YML083c, suggesting that aerobic repression of YML083c is promoted by the general Tup1-Ssn6 co-repressor complex. However, despite the presence of a sequence that matches the consensus for binding of Rox1, YML083c is not controlled by Rox1, since deletion or replacement of the putative binding site did not cause aerobic derepression. Moreover, YML083c expression was undetectable in aerobically grown cells of a rox1 null mutant.