Insulin sensitivity and secretion in obese Type 2 diabetic women after various bariatric operations

Objective: To compare the effects of biliopancreatic diversion (BPD) and laparoscopic gastric banding (LAGB) on insulin sensitivity and secretion with the effects of laparoscopic gastric plication (P). Methods: A total of 52 obese women (age 30-66 years) suffering from type 2 diabetes mellitus (T2DM) were prospectively recruited into three study groups: 16 BPD; 16 LAGB, and 20 P. Euglycemic clamps and mixed meal tolerance tests were performed before, at 1 month and at 6 months after bariatric surgery. Beta cell function derived from the meal test parameters was evaluated using mathematical modeling. Results: Glucose disposal per kilogram of fat free mass (a marker of peripheral insulin sensitivity) increased significantly in all groups, especially after 1 month. Basal insulin secretion decreased significantly after all three types of operations, with the most marked decrease after BPD compared with P and LAGB. Total insulin secretion decreased significantly only following the BPD. Beta cell glucose sensitivity did not change significantly post-surgery in any of the study groups. Conclusion: We documented similar improvement in insulin sensitivity in obese T2DM women after all three study operations during the 6-month postoperative follow-up. Notably, only BPD led to decreased demand on beta cells (decreased integrated insulin secretion), but without increasing the beta cell glucose sensitivity.

The influence of excipients on the diffusion of ibuprofen and paracetamol in gastric mucus

The aim of this study was to examine the diffusion of commonly administered analgesics, ibuprofen and paracetamol, through gastric mucus. As ibuprofen and paracetamol are often formulated with alkalising excipients, or are commonly co-administered with antacids that have been demonstrated to alter their absorption, diffusion was also studied in the presence of a range of soluble and insoluble antacids or buffering agents. The effect of pH, which has been demonstrated to modify the properties of mucus, was also studied. Mucus was a significant barrier to diffusion for both drugs, compared to an unstirred aqueous layer with diffusion rates significantly lower in the presence of a mucus barrier for both drugs; ibuprofen diffusion also demonstrated a significant increase in the lag time. Paracetamol diffusion was not significantly affected by addition of any antacid, whereas ibuprofen rates were affected and the diffusion lag time for ibuprofen was significantly reduced in all cases. Isolated increases in pH increased the rate and reduced the lag time for ibuprofen diffusion. It was shown that mucus acts as a passive barrier in the case of paracetamol diffusion, and an interactive barrier to ibuprofen diffusion. Changes in mucus viscosity at different pH values may be responsible for the observed changes in ibuprofen diffusion rate. © 2004 Elsevier B.V. All rights reserved.

Investigation of liquid sodium alginates as mucoadhesive bandages coating the oesophageal mucosa and protecting it from gastric reflux

Gastro-oesophageal Reflux Disease (GORD), is generally caused by excess gastric reflux back to the oesophagus where damage to the mucosa results in injury. GORD is a very common disease in western countries, more than a quarter of western people are suffering from this disease and there is a trend that the percentage population in eastern countries who are diagnosed as GORD is increasing. GORD and its complications damage the quality of life and can lead to serious oesophageal diseases including Barrett’s disease and oesophageal carcinoma. Sodium alginate dissolved in water forms a viscous liquid and can coat on oesophageal mucosa for a period of time. In this study the ability of the liquid alginate to adhere to the oesophageal mucosa was investigated and the factors that affect this retention were examined. The potential of this liquid alginate as a drug delivery vehicle to extend the duration of contact with the oesophageal mucosa was confirmed by this study. The capacity of an alginate coating to retard acid and pepsin diffusion, the two main aggressive factors in gastric reflux, was investigated. A significant reduction in acid and pepsin diffusion by alginate gel layer was demonstrated in this project, indicating that alginate has great potential to protect against damage caused by acidic reflux. A novel method was introduced using an independent score system to assess the protection of oesophageal tissue by a coating of liquid alginate using microscopy as a technique. This technique demonstrated that alginate can protect the oesophageal epithelial tissue from the damage caused by gastric acid and pepsin. Many techniques were used in this study. The experimental results suggested that liquid sodium alginate is a very promising candidate in treating local oesophageal diseases through forming a coating on the oesophageal mucosal surface, retarding the diffusion of components of gastric refluxate and thus reducing the contact of these noxious factors with the epithelium and minimising injury.

Modelling the gastric epithelium for testing of new chemical entities

A cell culture model of the gastric epithelial cell surface would prove useful for biopharmaceutical screening of new chemical entities and dosage forms. A successful model should exhibit tight junction formation, maintenance of differentiation and polarity. Conditions for primary culture of guinea-pig gastric mucous epithelial cell monolayers on Tissue Culture Plastic (TCP) and membrane insects (Transwells) were established. Tight junction formation for cells grown on Transwells for three days was assessed by measurement of transepithelial resistance (TEER) and permeability of mannitol and fluorescein. Coating the polycarbonate filter with collagen IV, rather with collagen I, enhanced tight junction formation. TEER for cells grown on Transwells coated with collagen IV was close to that obtained with intact guinea-pig gastric epithelium in vitro. Differentiation was assessed by incorporation of [3H] glucosamine into glycoprotein and by activity of NADPH oxidase, which produces superoxide. Both of these measures were greater for cells grown on filters coated with collagen I than for cells grown on TCP, but no major difference was found between cells grown on collagens I and IV. However, monolayers grown on membranes coated with collagen IV exhibited apically polarized secretion of mucin and superoxide. The proportion of cells, which stained positively for mucin with periodic Schiff reagent, was greater than 95% for all culture conditions. Gastric epithelial monolayers grown on Transwells coated with collagen IV were able to withstand transient (30 min) apical acidification to pH 3, which was associated with a decrease in [3H] mannitol flux and an increase in TEER relative to pH 7.4. The model was used to provide the first direct demonstration that an NSAID (indomethacin) accumulated in gastric epithelial cells exposed to low apical pH. In conclusion, guinea-pig epithelial cells cultured on collagen IV represent a promising model of the gastric surface epithelium suitable for screening procedures.

Intracellar signalling in the HGT-1 gastric cell line and in rat isolated parietal cells

The work presented in this thesis was undertaken to increase understanding of the intracellular mechanisms regulating acid secretion by gastric parietal cells. Investigation of the effects of protein kinase C on secretory activity induced by a variety of agents was a major objective. A further aim was to establish the sites at which epidermal growth factor (EGF) acts to stimulate prostaglandin E2 (PGE2) production and to inhibit acid secretion. These investigations were carried out by using the HGT-1 human gastric cancer cell line and freshly isolated rat parietal cells. In HGT-1 cells, the cyclic AMP response to histamine and to truncated glucagon-like peptide 1 (TGLP-1) was reduced when protein kinase C was activated by 12-0-tetradecanoylphorbol 13-acetate (TPA). Receptor-binding studies and experiments in which cyclic AMP production in HGT-1 cells was stimulated by gastric inhibitory polypeptide, cholera toxin and forskolin suggested that the effect of TPA was mediated by uncoupling of the histamine H2 receptor from the guanine nucleotide regulatory protein Gs, possibly by phosphorylation of the receptor. An involvement of protein kinase C α in this effect was suggested because an antibody to this isoform specifically prevented the inhibitory effects of TPA on histamine-stimulated adenylate cyclase activity in a membrane fraction prepared from HGT-1 cells. Carbachol-stimulated secretory activity in parietal cells was specifically inhibited by Ro 31-8220, a bisindolylmaleimide inhibitor of protein kinase C. Thus protein kinase C may play a role in the activation of the secretory response to carbachol. In parietal cells prelabelled with [3H]-arachidonic acid or [3H]myristic acid, EGF did not affect [3H]-fatty acid or [3H] - diacylglycerol content. No evidence for effects of EGF on phosphatidylinositol glycan-specific phospholipase C, phospholipase A2 or on low Km cyclic AMP phosphodiesterase activities were found.

Regulation of mucus secretion by cells isolated from the rat gastric mucosa

     This study was undertaken to further understanding of the mechanisms which regulate mucus secretion by rat stomach cells. Particular objectives were: (i) to develop and use a radiochemical assay to estimate the secretion of mucin by a suspension of gastric mucosal cells in vitro, (ii) to develop and use a solid-phase enzyme immunoassay (EIA) to study the regulation of the release of bulk gastric mucin from the isolated cells and (iii) to compare the results obtained with the two procedures.      Cells were isolated by exposure of gastric mucosa to pronase and EDTA. Cell suspensions were preincubated with D-[6-3H]glucosamine. [3H]-labelled material of high molecular mass released into the incubation medium, was purified by Fast Protein Liquid Chromatography, and appeared to be gastric mucin. Some unidentified [3H]-labelled material of lower molecular mass was also found in the medium. Release of [3H]-labelled high molecular mass material was essentially linearly related to time. Secretin, isoprenaline and carbachol stimulated release of [3H]-labelled high molecular mass material. The half-maximally effective concentrations of secretin and isoprenaline were 2.3nM and 34nM respectively. Histamine, gastrin and epidermal growth factor were without effect.      A rabbit polyclonal antibody was raised by using purified 'native' rat gastric mucin as immunogen. The antibody preparation appeared specific for rat gastric mucin and was used to establish a quantitative solid-phase EIA. Release of bulk mucin was essentially linearly related to time. Phorbol-12-myristate-13-acetate (PMA), forskolin and A23187 dose-dependently stimulated bulk mucin release. Synergistic interactions were observed between PMA and forskolin, and PMA and A23187. Secretin and isoprenaline were confirmed as mucin secretogogues.      In conclusion gastric mucin release was investigated for the first time by using a suspension of gastric mucosal cells. Two different assay procedures were developed. Some pathways and agents responsible for controlling mucin secretion were identified.

Production and action of local mediators in the rat gastric mucosa

This study concerns the production and action of the local mediators nitric oxide (NO) and prostaglandin E2 (PGE2) in the rat gastric mucosa. The major objectives were: (i) to determine which mucosal cell type(s) contained NO synthase activity, (ii) to establish the functional role(s) of NO in the gastric mucosa and (iii) to investigate regulation of gastric PGE2 production. Gastric mucosal cells were isolated by pronase digestion coupled with intermittent calcium chelation and were separated by either density-gradient centrifugation or by counterflow elutriation. The distribution of Ca2+ -dependent NO synthase activity, measured via the conversion of [14C]-L-arginine to [14C]-L- citrulline, paralleled the distribution of mucous cells in elutriated fractions. Pre-treatment of rats with lipopolysaccharide caused the induction of Ca2+ -independent NO synthase in the elutriator fractions enriched with mucous cells. Incubation of isolated cells with the NO donor isosorbide dinitrate (ISDN) produced a concentration-dependent increase in the guanosine 3',-5'-cyclic monophosphate (cGMP) content which was accompanied by a concentration-dependent increase in release of immunoreactive mucin. Intragastric administration of ISDN of dibutyryl cGMP in vivo increased the thickness of the mucus layer overlying the gastric mucosa. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) produced a concentration-dependent inhibition (IC50 247 μM) of histamine-stimulated aminopyrine accumulation, a measure of secretory activity, in cell suspensions containing > 80% parietal cells. SNAP increased the cGMP content of the suspension but did not decrease cellular viability, glucose oxidation or adenosine 3',5'-cyclic monophosphate content. The inhibitory effect of SNAP was observed in permeabilised cells stimulated with ATP and was stereospecifically blocked by preincubation with Rp-8-bromoguanosine 3'-5'-monophosphorothioate, which inhibits activation of cGMP-dependent protein kinase. Stimulation of PGE2 release by bradykinin in a low density cell fraction, enriched with parietal cells and devoid of vascular endothelial cells and macrophages, involved a bradykinin B1 receptor. In summary, NO synthase activity is probably present in gastric mucous epithelial cells. NO may promote mucus secretion by elevation of cGMP. NO donors inhibit acid secretion at a specific site and their action may involve cGMP. The bradykinin B1 receptor is involved with PGE2 production in the gastric mucosa.

Absorption and metabolism of chlorogenic acids in cultured gastric epithelial monolayers

Gastric absorption of feruloylquinic acid and di-O-caffeoylquinic acid analogs has never been investigated despite their potential contribution to the proposed beneficial health effects leading to reduced risk of type 2 diabetes. Using a cultured gastric epithelial model, with an acidic apical pH, the relative permeability coefficients (P(app)) and metabolic fate of a series of chlorogenic acids (CGAs) were investigated. Mechanistic studies were performed in the apical to basal direction and demonstrated differential rates of absorption for different CGA subgroups. For the first time, we show intact absorption of feruloylquinic acids and caffeoylquinic acid lactones across the gastric epithelium (P(app) ~ 0.2 cm/s). Transport seemed to be mainly by passive diffusion, because good linearity was observed over the incubation period and test concentrations, and we speculate that a potential carrier-mediated component may be involved in uptake of certain 4-acyl CGA isomers. In contrast, absorption of intact di-O-caffeoylquinic acids was rapid (P(app) ~ 2-10 cm/s) but nonlinear with respect to time and concentration dependence, which was potentially limited by interaction with an efflux transporter and/or pH gradient dependence. For the first time, methylation is shown in gastric mucosa. Furthermore, isoferulic acid, dimethoxycinnamic acid, and ferulic acid were identified as novel gastric metabolites of CGA biotransformation. We propose that the stomach is the first location for the release of hydroxycinnamic acids, which could explain their early detection after coffee consumption.

The isolation of gastric mucosal cells and a study of their isoenzyme patterns

DUE TO COPYRIGHT RESTRICTIONS ONLY AVAILABLE FOR CONSULTATION AT ASTON UNIVERSITY LIBRARY AND INFORMATION SERVICES WITH PRIOR ARRANGEMENT

A collagen IV matrix is required for guinea pig gastric epithelial cell monolayers to provide an optimal model of the stomach surface for biopharmaceutical screening

The surface epithelial cells of the stomach represent a major component of the gastric barrier. A cell culture model of the gastric epithelial cell surface would prove useful for biopharmaceutical screening of new chemical entities and dosage forms. Primary cultures of guinea pig gastric mucous epithelial cells were grown on filter inserts (Transwells®) for 3 days. Tight-junction formation, assessed by transepithelial electrical resistance (TEER) and permeability of mannitol and fluorescein, was enhanced when collagen IV rather than collagen I was used to coat the polycarbonate filter. TEER for cells grown on collagen IV was close to that obtained with intact guinea pig gastric epithelium in vitro. Differentiation was assessed by incorporation of [ 3H]glucosamine into glycoprotein and by activity of NADPH oxidase, which produces superoxide. Both of these measures were greater for cells grown on filters coated with collagen I than for cells grown on plastic culture plates, but no major difference was found between cells grown on collagens I and IV. The proportion of cells, which stained positively for mucin with periodic acid Schiff reagent, was greater than 95% for all culture conditions. Monolayers grown on membranes coated with collagen IV exhibited apically polarized secretion of mucin and superoxide, and were resistant to acidification of the apical medium to pH 3.0 for 30 min. A screen of nonsteroidal anti-inflammatory drugs revealed a novel effect of diclofenac and niflumic acid in reversibly reducing permeability by the paracellular route. In conclusion, the mucous cell preparation grown on collagen IV represents a good model of the gastric surface epithelium suitable for screening procedures. © 2005 The Society for Biomolecular Screening.

Apoptosis induction in gastric mucous cells in vitro:lesser potency of Helicobacter pylori than Escherichia coli lipopolysaccharide, but positive interaction with ibuprofen

Non-steroidal anti-inflammatory drugs (NSAIDs) cause peptic ulcer disease, but whether they interact with Helicobacter pylori to promote damage is controversial. Moreover, the reported induction of apoptosis in gastric cells by H. pylori lipopolysaccharide (LPS) (10-9 g /ml) contrasts with studies showing low immunological potency of this LPS. Therefore, the effects of LPS from H. pylori NCTC 11637 and Escherichia coli 0111:B4 on apoptosis in a primary culture of guinea-pig gastric mucous cells were investigated in the presence and absence of the NSAID, ibuprofen. Cell loss was estimated by a crystal violet assay, and apoptosis determined from caspase activity and from condensation and fragmentation of nuclei. Exposure to E. coli LPS for 24 h caused cell loss and enhanced apoptotic activity at concentrations ≥ 10-9 g/ml, but similar effects were only obtained with H. pylori LPS at concentrations ≥10-6 g/ml. Although ibuprofen (250 μM) caused cell loss and apoptosis, addition of either E. coli or H. pylori LPSs further enhanced these effects. In conclusion, LPS and ibuprofen interact to enhance gastric cell loss and apoptosis. In such interactions, E. coli LPS is more potent than that of H. pylori. The low potency of H. pylori LPS may contribute to a chronic low-grade gastritis that can be enhanced by the use of NSAIDs. © W. S. Maney & Son Ltd.

High permeability of the anionic form restricts accumulation of indomethacin by cultured gastric surface epithelial cells exposed to low apical pH

The 'ion-trapping' hypothesis suggests that the intracellular concentration of acidic non-steroidal anti-inflammatory drugs in gastric epithelial cells could be much higher than in the gastric lumen, and that such accumulation could contribute to their gastrotoxicity. Our aim was to examine the effect of the pH of the apical medium on the apical to basal transfer of the acidic drug indomethacin (pK a 4.5) across a gastric mucous epithelial cell monolayer, and to determine whether indomethacin accumulated in cells exposed to a low apical pH. Guinea-pig gastric mucous epithelial cells were grown on porous membrane culture inserts (Transwells®) for 72 h. Transfer and accumulation of [ 14C] indomethacin were assessed by scintillation counting. Transfer of [ 3H]mannitol and measurement of trans-epithelial electrical resistance were used to assess integrity of the monolayer. Distribution of [ 14C] urea was used to estimate the intracellular volume of the monolayer. The monolayer was not disrupted by exposure of the apical face to media of pH ≥ 3, or by indomethacin. Transfer of indomethacin (12 μM) to the basal medium increased with decreasing apical medium pH. The apparent permeability of the undissociated acid was estimated to be five times that of the anion. The intracellular concentration of indomethacin was respectively 5.3, 4.1 and 4.3 times that in the apical medium at pH 5.5, 4.5 and 3.0. In conclusion, this study represents the first direct demonstration that indomethacin accumulates in gastric epithelial cells exposed to low apical pH. However, accumulation of indomethacin was moderate and the predictions of the ion-trapping hypothesis were not met, probably due to the substantial permeability of anionic indomethacin across membranes. © 2006 Elsevier B.V. All rights reserved.

Disparate effects of non-steroidal anti-inflammatory drugs on apoptosis in guinea-pig gastric mucous cells: inhibition of basal apoptosis by diclofenac

Non-steroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in gastrointestinal cancer cell lines. Similar actions on normal gastric epithelial cells could contribute to NSAID gastropathy. The present work therefore compared the actions of diclofenac, ibuprofen, indomethacin, and the cyclo-oxygenase-2 selective inhibitor, NS-398, on a primary culture of guinea-pig gastric mucous epithelial cells. Cell number was assessed by staining with crystal violet. Apoptotic activity was determined by condensation and fragmentation of nuclei and by assay of caspase-3-like activity. Necrosis was evaluated from release of cellular enzymes. Ibuprofen (250 μM for 24 h) promoted cell loss, and apoptosis, under both basal conditions and when apoptosis was increased by 25 μM N-Hexanoyl-D-sphingosine (C6-ceramide). Diclofenac (250 μM for 24 h) reduced the proportion of apoptotic nuclei from 5.2 to 2.1%, and caused inhibition of caspase-3-like activity, without causing necrosis under basal conditions. No such reduction in apoptotic activity was evident in the presence of 25 μM C6-ceramide. The inhibitory effect of diclofenac on basal caspase-3-like activity was also exhibited by the structurally similar mefenamic and flufenamic acids (1–250 μM), but not by niflumic acid. Inhibition of superoxide production by the cells increased caspase-3-like activity, but the inhibitory action of diclofenac on caspase activity remained. Diclofenac did not affect superoxide production. Diclofenac inhibited caspase-3-like activity in cell homogenates and also inhibited human recombinant caspase-3. In conclusion, NSAIDs vary in their effect on apoptotic activity in a primary culture of guinea-pig gastric mucous epithelial cells, and the inhibitory effect of diclofenac on basal apoptosis could involve an action on caspase activity.