Mechanisms and consequences of peroxisome proliferator-induced hyperfunction of the rat gastrinproducing cell
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Peroxisome proliferators (PPs) comprise the largest group of non-genotoxic carcinogens and include both synthetic and physiological ligands like hypoliidemic fibrates, anti-diabetic thiazolidinediones (TZD), non-steroidal anti-intlammatory drugs (NSAIDs), herbicedes, phthalate ester plasticizers, industrail solvents, polyunsaturated fatty acids and arachidonic acid metabolites. These structurally diverse compunds induce predictable responses in the rodent liver, including peroxisome proliferation, hepatocyte hyperplasia and hypertrophy and increased transcription particulary of genes involved in fatty acid meabolism. Long-term exposure results in carcinomas, PPs activate the nuclear receptors named peroxisome proliferator-activated receptors (PPARSs). They exist in three distinct isoforms; PPARα, PParγ and PPARδ, show divergent patterns of tissue expression and are thought to perform different physiological functions. In this thesis, PPs is referred to as all compounds activating either one of the three PPAR isoforms. In the late seventies the hypolipidemic PP ciprofibrate was shown to induce hypergastrinemia and secondary hyperplasia of the ECL cell in rat oxyntic mucosa after 2 months dosing, and formation of malignant carcinoid tumors after life-time (2 years) dosing. The results were explained by inhibition of gastric acid secretion. More recent studies rather suggest that the hypergastrinemia is induced by a mechanism indepentant of acid inhibition. Daily assessments reveal no increase in stomach pH, and co-administration with the proton pump inhibitor (PPI) omeprazole produces a potentiating hypergastrinemic effect. Significant hypergastrinemia is reached after at least 14 days of regular dosing, and further stimulation induces marked increase in antral gastrin mRNA abundance and modest growth of the G-cells. Ciprofibrate on the antral open-type G and D-cells has been suggested. Gastrin has a fundamental function as the main physiological stimulator of gsstric acid secretion and a potent growth factor as the main physiological stimulator of gastric acid secretion and a potent growth factor in the oxyntic mucosa. The specific effect on ECL cell mucosal cells, and particularly the cellular localization of the gastrin receptor (CCK-2), is still debated. In paper I, we used elutriation centrifugation of dispersed oxyntic mucosal cells from hypergastrinemic rats to examine long-term (6 weeks) trophic effects of gastrin, particularly on ECL cells and the parietal cell lineage in comparison to the whole oxyntic mucosa. In two groups of rats with different levels of PPI-evoked hypergastrinemia, total mucosal cell number, percentage of ECL-and parietal cells, and histamine content were determined before elutration fractions. The size and abundance of ECL cells were progressively increased after exposure to elevated levels of gastrin, as assessed by marked shifts in distrubution between the different elutriation fractions. Contrary, independent of the level of hypergastrinemia, the percentage of parietal cells and the distribution in different concentration-dependent specific trophic effect on ECL cells and a general proliferative effect on oxyntic mucosa, including the parietal cell lineage. In paper II, the localization of CCk-2 receptor-binding sites in corpus mucosa was demonstrated by arterial infusion of a fluorescein-labeled CCK-8 peptide (Fluo-CCK-8) to totally isolated, vascularly perfused rat stomachs. Fluo-CCK-8 was added in a concentration physiological for gastrin (130 pmol L-1) and the stomachs were stimulated for 1 minute before biopsies were taken. Using immunohistochemistry, bound Fluo-CCK-8 was found in the basal part of oxyntic mucosa, co-localized with histidine decarboxylase(HDC) immunoreactive ECL cells. No binding was found in the midglandular region were parietal cells dominate. Biological activity of Fluo-CCK-8 was confirmed by an immediate and marked increase of histamine in the venous effluent, and the specificity of the binding was proved by an abolished Fluo-CCK-8 binding in stomachs pre-perfused with excess amounts of native CCK-8. In conclusion, a physiological concentration (for gastrin) of the biologically active Fluo-CCK-8 was used to simultaneously demonstrate a functional response (histamine release) and CCK-2 receptor-binding sites localized to ECL cells in the basal part of corpus, and not to the midglandular region dominated by parietal cells. In paper III, laser microbeam microdissection (LMM) and laser pressure catapulting (LPC) were used to isolate single parietal and ECL cells from cell smears after dispersion of rat oxyntic mucosa and enrichment by eluriation. The mRNA from each single cell was isolated and subjected to one-step multiplex or conventional reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR. For each individual cells, primers specific for the CCK-2 receptor were used in combination with primers for both the parietal cell marker gene H+/K+-ATPase and the ECL cell marker gene HDC. CCK-2 receptor mRNA was demonstrated in 33% of the parietal cells and 40% of the ECL cells. Representative CCK-2 receptor PCR products from both cell types were identical and >99% homologous to the published sequence for rat CCK-2 receptor mRNA, as verified by restriction enzyme cleavage and DNA sequencing. In conclusion, laser-assisted capture of dispersed cells allows sensitive and cell-specific analysis of several mRNA sequences in one individual cell. The results show that at least some of the parietal cells express the CCK-2 receptor mRNA. In paper IV, the effects on rat antral and oxyntic endocrine cells after long-term (8 weeks) oral dosing with the PP ciprofibrate were examined by looking at changes in morphology and mRNA expression. Also, the ability of the somatostatin analogue actreotide LAR to attenuate these effects was examined. Ciprofibrate increased gastrin mRNA abundance, the number and area of G-cells, and induced hypergastinemia. Concomitantly, there was a lack of reduction in antral D-cell parameters, which normally parallels an increase in G-cell function. Profound ECL cell hyperplasia was confirmed by increased HDC and chromogranin A (CgA) mRNA levels, number and area og ECL cells and plasma histamine level. Octreotide LAR did not affect the ciprofibrate-induced increase in G-cell parameters, but all parameters of both normal ECL cells and ECL cell hyperplasia, were ruduced. In conclusion, the PP ciprofibrate stimulates the G-cell activity by a mechanism unaffected by octreotide, but octreotide does inhibit both basal and gastrin-stimulated ECL function and growth. In paper V, implication of the PPARα receptor in the G-cell stimulating mechanism of ciprofibrate was elucidated by investigating whether another PPARα receptor in the G-cell stimulating mechanism of ciprofibrate was elucidated by investigating whether another PPARα-specific ligand (WY-14643) and a PPARγ-specific ligand (troglitazsone) could mimic the effects. Troglitazone did not induce hypergastrinemia, but to a lesser extent than ciprofibrate. Increases in ECL cell parameters were consistant with the level of hypergastrinemia. The oral dosing of both ciprofibrate and WY-15643 increased mRNA abundance of the peroxisomal enzyme acyl-CoA oxidase (ACO), which is a widely used marker of PPARα activation, and reduced mRNA abundance of the PPARα receptor. Both these changes were restricted to the antral mucosa and no effects were seen in oxyntic mucosa. By immunohistochemistry, the PPARα receptor was found in antrum and in co-localization with at least some of the G-cells. Maximal plasma gastrin concentration during the study and the increase in liver weight (in % of total body weight) were signiificantly correlated. In conclusion, these results support the hypotheses that PPARα-specific ligands could stimulate G-cells by acting locally from the stomach lumen through antral PPARα.
UtgiverDet medisinske fakultet
SerieDoktoravhandlinger ved NTNU, 1503-8181
Dissertations at the Faculty of Medicine; 210