Effect of cholecystokinin-A receptor blockade on postprandial insulinaemia and gastric emptying in humans.

Our aim was determine the relationship between cholecystokinin (CCK)-A receptor blockade, glucose levels, insulin secretion and gastric emptying in humans, and to assess the effect of CCK-A blockade on pancreatic polypeptide secretion. After a 12-h fast, six healthy volunteers were given [99mTc]iminodiacetic acid monosodium salt (IDA) intravenously (5 mCi). One hour later they were offered a 577 kcal liquid meal containing [99mTc]diethylenetriaminepentaacetic acid (DTPA) (2 mCi) and glucose (105 g). Scintigraphic gastric and gallbladder activity, and plasma glucose, insulin and pancreatic polypeptide responses were monitored. In a second experiment, a continuous intravenous infusion of loxiglumide (7.5 mg kg h(-1)) was started 60 min before and continued until 120 min after test meal ingestion to block the CCK-A receptors. Gallbladder emptying was blocked by loxiglumide. Loxiglumide accelerated gastric emptying, increased insulin secretion without alteration of glucose profiles, and abolished all phases of the postprandial pancreatic polypeptide response. Blockade of peripheral CCK-A receptors accelerates gastric emptying of liquids with an increase in postprandial insulin levels. The lack of changes in glycaemia suggests that alternative homeostatic mechanisms also control postprandial glucose levels. Inhibition of pancreatic polypeptide release may reflect an independent effect of loxiglumide on vagal control involved in pancreatic polypeptide release.

Gene expression in intestinal epithelial cells, IEC-6, is altered by burn injury-induced circulating factors.

Severe burn injury is commonly associated with significant changes in intestinal epithelial function. These changes include mucosal atrophy and increased permeability. To date, the mechanism by which burn injury alters intestinal epithelium function are not clear. We used an in vitro model of intestinal epithelium, IEC-6 cells, and observed that the integrity of confluent culture is disrupted and cell growth and migration rates are reduced in the presence of serum collected from scald burn injury rats (SRS) (6). To identify gene products involved in mechanisms underlying these effects, we used the cDNA expression microarray analysis and found that genes whose expression was affected by SRS in IEC-6 cells were primarily associated with cell shape, growth and death, stress-response, protein turnover and transport of water and electrolytes. These data demonstrate that a burn-induced circulating factor(s) modulates expression of genes, which may affect intestinal epithelial cell survival and function. Thus, these findings provide clues to the nature of molecular mechanisms potentially involved in multiple-organ malfunction, in particular the atrophy and enhanced permeability of gut mucosa, after burn injury.

Acute pancreatitis signals activation of apoptosis-associated and survival genes in mice.

In experimental models of acute pancreatitis (AP), acinar cell death occurs by both necrosis and programmed cell death or apoptosis. Apoptosis is an active form of cell death associated with a tightly regulated expression of gene products that are either pro- or antiapoptotic. The aim of this study was to characterize pancreatic mRNA levels by Northern blotting analysis of apoptosis-associated genes used during the course of cerulein-induced AP in mice. Histone H3 mRNA levels were also examined as an indicator of cell proliferation. Acinar cell apoptosis was confirmed histologically. The findings show that AP modifies pancreatic mRNA levels of both pro- and antiapoptotic genes simultaneously. Pancreatic bclXL, bax, and p53 mRNA levels increased significantly in a temporal fashion during induction of AP. Pancreatic bcl-2 mRNA levels were unchanged during AP. Pancreatic mRNA levels of insulin-like growth factor-1 (IGF-1), a mitogen and cell survival factor, and its receptor (IGF-1R) also increased in a temporal fashion during induction of AP. In summary, this study indicates that acinar cell death during cerulein-induced AP in mice can occur by the apoptotic pathway. Since factors promoting and antagonistic for cell survival are activated simultaneously, regulation of acinar cell survival appears complex and dynamic during AP.

Age-associated changes in gene expression patterns in the duodenum and colon of rats.

In humans, decreased intestinal motility, compromised nutritional status and increased risk of colon cancer are commonly associated with aging. Here, we used the cDNA microarray analysis to detect age-associated changes in duodenal and colonic gene expression in male Fischer 344 rats. The primary finding of this study is that the magnitude and direction of age-associated changes in gene expression differs in the colon and duodenum. In the colon, 56 genes showed altered expression, whereas expression of only 25 genes was altered in the duodenum. The magnitude of change was greater in the colon than in the duodenum. The direction of change also differed; in the aged colon, expression of 51 genes increased and only five genes decreased. In contrast, in the aged duodenum, only seven genes increased, whereas 18 genes decreased in expression. In the duodenum of aged rats, expression of genes involved in ATP-generating pathways is decreased. In contrast, in the colon of aged rats, expression of genes involved in energy generating pathways and in lipid oxidation is increased. In addition, in the aging colon, an increased expression of genes that show an aberrant regulation in colon cancer, including CD44, ras, and maspin is observed. Collectively, these findings provide clues to molecular events that may be related to compromised intestinal function and the high incidence of colon cancer in the aged population.

Effects of aging on expression of genes involved in regulation of proliferation and apoptosis in the colonic epithelium.

The purpose of this study was to characterize the effects of aging on colonic messenger ribonucleic acid (mRNA) and protein levels of genes involved in the regulation of cell proliferation and apoptosis, and epithelial morphology in male Fischer 344 rats. Our study shows that, with aging, colonic expression of insulin-like growth factor-1 (IGF-1) and IGF-binding protein-3 (IGFBP-3) is significantly increased and decreased, respectively. Colonic Bax protein levels are increased significantly with aging. Immunohistochemical localization of Bax protein shows a greatly increased expression in colonic crypts, especially in the upper portion of crypts. p53 expression is unchanged with aging. No significant change in proliferation of colonic crypt cells is observed by bromodeoxyuridine (BrdU) labeling, although the increased colonic expression of IGF-1 and the decreased expression of IGFBP-3 with aging may result in an increased colonic IGF-1 bioactivity. The age-related changes in Bax and IGFBP-3 appear to be independent of p53. The finding of an unchanged colonic epithelium with aging in the face of a greatly increased Bax protein levels may suggest that the elevated Bax protein levels function to render colonic epithelial cells more sensitive to apoptotic stimuli.

Minimal enteral nutrient requirements for intestinal growth in neonatal piglets: how much is enough?

BACKGROUND: Parenterally nourished preterm infants commonly receive minimal enteral feedings, the aim being to enhance intestinal function. Whether this regimen increases intestinal growth has not been established. OBJECTIVE: Our objective was to determine the minimal enteral nutrient intakes necessary to stimulate and to normalize neonatal intestinal growth. METHODS: Intestinal growth and cell proliferation were quantified in neonatal pigs given equal amounts of an elemental nutrient solution for 7 d. Different groups (n = 5-7 per group) received 0%, 10%, 20%, 40%, 60%, 80%, or 100% of total nutrient intake enterally, with the remainder given parenterally. RESULTS: In the jejunum, wet weight, protein mass, and villus height were significantly greater at enteral intakes >40%. Stimulation of ileal protein mass required a higher enteral intake (60%). In both segments, abrupt increases in DNA mass, crypt depth, ornithine decarboxylase activity, and crypt cells in S-phase occurred between enteral intakes of 40% and 60%. Circulating concentrations of glucagon-like peptide-2 and peptide YY, but not gastrin, increased significantly between enteral intakes of 40% and 60% and closely paralleled indexes of cell proliferation. CONCLUSIONS: The minimal enteral nutrient intake necessary to increase mucosal mass was 40% of total nutrient intake, whereas 60% enteral nutrition was necessary to sustain normal mucosal proliferation and growth. Our results imply that providing <40% of the total nutrient intake enterally does not have significant intestinal trophic effects.

Effects of aging and caloric restriction on IGF-I, IGF-I receptor, IGFBP-3 and IGFBP-4 gene expression in the rat stomach and colon.

The purpose of this study was to determine the effects of aging and caloric restriction (CR) on insulin-like growth factor-I (IGF-I), IGF-I receptor (IGF-IR), IGF-binding protein-3 (IGFBP-3) and IGFBP-4 expression in the stomach and colon of male Fischer 344 rats. Stomach and colonic RNA were prepared from ad libitum (AL) fed or long-term CR rats. Stomach IGF-I, IGFBP-3 and IGFBP-4 mRNA levels increased significantly (P

Prohormone convertase-1 is essential for conversion of chromogranin A to pancreastatin.

The purpose of this study was to test the hypothesis that the endoprotease, prohormone convertase-1 (PC-1), is involved in the processing of the precursor protein chromogranin A (CGA) to a smaller peptide called pancreastatin (PST). A human pancreatic carcinoid cell line (BON) that expresses PC-1, CGA and PST was stably transfected with antisense PC-1 mRNA. BON cells expressing antisense PC-1 mRNA showed nearly complete abolishment of PC-1 protein (approximately 95% reduction) and an 80% reduction in cell content of PST immunoreactivity (PST-IR) as assessed by high-performance liquid chromatography in combination with measurement of PST-IR. These findings indicate that PC-1 is essential for processing CGA to PST.

Characterization of two novel proabsorptive peptide YY analogs, BIM-43073D and BIM-43004C.

Effective clinical therapy to augment intestinal absorption of water and electrolytes does not exist; the gut hormone, peptide YY (PYY), is a potent proabsorptive agent in animal models. The purpose of our study was to evaluate the effects of two novel PYY analogs, BIM-43073D and BIM-43004C, on intestinal absorption. Dogs with ileal Thiry-Vella fistulae (TVF) were treated with either PYY, BIM-43073D, or BIM-43004C. Administration of BIM-43073D significantly increased water and sodium absorption over baseline and maintained this level of increased absorption for a longer duration than an equimolar dose of PYY. Administration of BIM-43004C significantly increased sodium and water absorption over baseline at a level equal to that of PYY. The novel PYY analogs, BIM-43073D and BIM-43004C, are effective proabsorptive agents with BIM-43073D producing more sustained effects than PYY. These compounds may be clinically useful in the treatment of gut malabsorption in conditions such as cholera, Crohn's disease, and the short-bowel syndrome.

Novel expression and regulation of gastrin gene in human ovarian cancer cell line, SW626.

Gastrin-secreting tumors have been identified in ectopic locations including the ovary; the mechanisms regulating gastrin gene expression, its distribution, and signaling pathways in these ectopic tissues are not known. The purpose of our present study was to determine: (1) whether the gastrin gene and peptide could be detected in ovarian cancer cell lines, (2) if functional gastrin releasing peptide receptors (GRP-R) are present, and (3) whether gastrin gene expression is altered by GRP. Five ovarian cancer cell lines (SW626, OVCA 420, OVCA 429, OVCA 432, and OVCA 433) were analyzed. We identified gastrin gene and peptide expression in the SW626 cell line but not in the OVCA lines. SW626 cells express a functional GRP-R that is correctly coupled to the Ca2+ signaling pathway. Treatment of SW626 cells with bombesin, the amphibian equivalent of GRP, inhibited expression of the gastrin gene in a time- and dose-dependent fashion. The SW626 ovarian cancer cell line will provide a useful model to further define regulation and expression of both the gastrin gene and peptide in ectopic (nongastrointestinal) tissues.

Stimulation of prohormone convertase-1 mRNA expression by second messenger signaling systems.

The purpose of this study was to investigate the effects of activation of various second messenger signaling systems on gene expression (i.e. mRNA levels) of a peptide hormone processing enzyme called prohormone convertase-1 (PC-1, also called PC-3) in a human pancreatic carcinoid cell line (BON) that expresses several endocrine peptides (chromogranin A, pancreastatin, neurotensin). Pharmacologic activation of adenylate cyclase-cAMP, protein kinase-C and Ca2+ mobilization pathways increased PC-1 mRNA levels and neurotensin secretion. Elevations in PC-1 mRNA levels were dose and time-related. Secretagogue-induced cellular depletion of neurotensin was followed by a replenishment of cellular neurotensin stores and an upregulation of PC-1 mRNA levels. Together, these data indicate that PC-1 mRNA expression is increased with peptide secretion and coordinated with maintenance of cellular stores of peptides.

Bioactivity of intraduodenally and intravenously infused fragments of luminal cholecystokinin releasing factor (LCRF).

A luminal cholecystokinin releasing factor (LCRF), has been purified from intestinal secretion and found to have a mass of 8136 daltons. The amino-terminal 41 residues have been sequenced. Previous studies showed that intraduodenal infusion of the synthetic amino-terminal 35 amino acid peptide, LCRF1-35 significantly stimulated pancreatic protein and fluid secretion in conscious rats, but the peptide did not stimulate amylase release from isolated, dispersed pancreatic acini. In the present study, several fragments of LCRF were synthesized and tested for CCK-releasing activity (pancreatic protein secretion) to determine whether shorter fragments of LCRF exhibit the characteristic biological activity of native LCRF and synthetic LCRF1-35. Compounds tested were LCRF1-41, LCRF1-35, LCRF1-65 and LCRF11-25. Of the fragments shorter than LCRF1-35, only LCRF11-25 but not LCRF1-6 had significant CCK releasing activity. LCRF1-41 was equivalent to LCRF1-35 in potency and efficacy. Intravenous and intraduodenal infusion of LCRF1-35 elicited nearly identical dose-response curves.

Distribution and localization of a novel cholecystokinin-releasing factor in the rat gastrointestinal tract.

The purpose of this study was to examine the distribution and localization of an intestinal cholecystokinin (CCK)-releasing factor, called luminal CCK-releasing factor (LCRF), in the gastrointestinal tract and pancreas of the rat. RIA analysis indicates that LCRF immunoreactivity is found throughout the gut including the pancreas, stomach, duodenum, jejunum, ileum, and colon with the highest levels in the small intestine. Immunohistochemistry analysis shows LCRF immunoreactivity staining in intestinal villi, Brunner's glands of the duodenum, the duodenal myenteric plexus, gastric pits, pancreatic ductules, and pancreatic islets. These results indicate potential sources for secretagogue-stimulated release of luminal LCRF and support the hypothesis that LCRF is secreted into the intestinal lumen to stimulate CCK release from mucosal CCK cells.

Comparison of somatostatin and pancreastatin on secretion of gastrin, pancreatic polypeptide, and peptide YY.

The purpose of this study was to compare the effects of pancreastatin (PST) (400 pmol/kg/hr) and somatostatin (SRIF) (400 pmol/kg/hr) on food-induced release of gastrin, pancreatic polypeptide (PP), and peptide YY (PYY) in conscious dogs. The present findings indicate that SRIF is more potent than PST on the inhibition of food-induced release of PP; that SRIF and PST do not influence food-induced release of gastrin; and that PST cannot inhibit food-induced release of PYY, whereas SRIF inhibits PYY release in a potent fashion.

Interaction of nicotine and a H2-receptor antagonist, famotidine, on gastrin and chromogranin A expression.

The purpose of this study is to examine the effect of nicotine on famotidine-induced hypergastrinemia in the rat. In addition, the effects of nicotine on gene expression for gastrin and chromogranin A (CGA) in the stomach were examined. Famotidine treatment alone (20 mg/kg. 2 x/day for 14 days) increased serum gastrin levels significantly (P < 0.05) but not antral levels of gastrin mRNA and peptide. Nicotine treatment (12 mg/kg/d) alone did not affect serum gastrin levels; however, nicotine potentiated the hypergastrinemic action of famotidine. The hypergastrinemic action of nicotine was not mediated by a downregulation of stomach somatostatin (SRIF) since stomach SRIF mRNA levels were unaffected by nicotine treatment. Administration of nicotine and famotidine also upregulated stomach CGA gene expression (i.e., mRNA and protein levels) significantly.

Influence of nicotine on gastrin and peptide YY in the rat.

The objective of this study was to examine the effects of nicotine and high-fat diets on gastrin and peptide YY (PYY) homeostasis in the rat. Antral levels of gastrin mRNA and peptide and ileal and colonic levels of PYY mRNA and peptide were examined. Serum levels of gastrin in response to food were also measured. Control rats were ad-lib fed or pair-fed according to the daily food intake of nicotine-treated rats. The results of this study indicate that nicotine treatment and fat diets can influence gastrin and PYY gene expression in the gastrointestinal tract.

Growth hormone upregulates gastrin and peptide YY gene expression.

The purpose of these studies was to examine the effects of excess growth hormone (GH) on gastrin and peptide YY (PYY) gene expression. Transgenic mice with the bovine GH gene linked to a mouse metallothionein I promoter were used as a model of chronic GH excess. Antral gastrin mRNA and peptide levels were elevated significantly (P < 0.05) in GH transgenic mice compared with wild type littermates. Ileal PYY mRNA and ileal and colonic PYY levels were significantly elevated in GH transgenic mice compared with wild type littermates. The elevations in gastrin and PYY gene expression in GH transgenic mice were independent of food intake. Serum concentrations of gastrin and PYY were also elevated in GH transgenic mice. Immunohistochemical analysis showed that the density of PYY-containing cells in the colon of GH transgenic mice and wild type littermates did not differ. In addition, the mRNA and protein levels of chromogranin A, a marker of endocrine cells, were not increased in the colon of GH transgenic mice. Together, these data indicate that GH, insulin-like growth factor I, or both can upregulate gastrointestinal gastrin and PYY gene expression directly.

Regulation of peptide YY homeostasis by gastric acid and gastrin.

Peptide YY (PYY) is a gut hormone localized primarily in the distal bowel. Because circulating PYY inhibits gastric acid secretion, we investigated the effects of gastric acid secretion and gastrin on gene expression and secretion of PYY. In conscious dogs, PYY release in response to oral food was inhibited (P < 0.05) by pharmacologic inhibition of gastric acid secretion (omeprazole, famotidine). In rats, omeprazole treatment resulted in a significant elevation in serum gastrin concentrations and a simultaneous decrease in PYY messenger RNA (mRNA) and peptide levels in the colon; administration of a gastrin receptor antagonist (L365, 260) prevented the inhibitory actions of omeprazole on colonic PYY mRNA levels. In athymic-nude mice, implantation of a human gastrinoma resulted in an elevation of serum gastrin concentrations and a concomitant depression of colonic PYY mRNA levels. We conclude that endogenous gastric acid secretion up-regulates PYY release and PYY mRNA expression. Circulating gastrin acts to down-regulate PYY release and PYY mRNA expression. This study provides evidence that foregut functions (i.e., gastric acid secretion and gastrin release) exert control over an antiacid signal (e.g. PYY release) emanating from the hindgut.

Downregulation of prohormone convertase-1 by a phorbol ester.

Acute TPA treatment (1h, 100nM) of a human pancreatic carcinoid cell line (BON) depletes cell contents of chromogranin A (CGA) and pancreastatin (PST), a peptide derived posttranslationally from CGA. Despite removal of TPA, BON cells continue to release CGA in an unregulated fashion whereas PST secretion is reduced substantially. TPA treatment also reduced prohormone convertase-1 (PC-1) protein and increased PC-1 mRNA levels. Together, these findings indicate that the TPA-induced switch from a regulated to unregulated pattern of CGA secretion is accompanied by a decrease in the processing of CGA to PST and a decrease in the active form of a processing enzyme potentially involved in processing CGA to a smaller peptide, PST.

Intestinal peptide YY: ontogeny of gene expression in rat bowel and trophic actions on rat and mouse bowel.

The purpose of this study was twofold: 1) to characterize the profile of colonic peptide YY (PYY) gene expression in rats and 2) to examine for potential trophic effects of PYY on the intestine in rats and mice. Expression of PYY mRNA (analyzed by Northern blotting and in situ hybridization) and PYY (analyzed by high-performance liquid chromatography and radioimmunoassay) was detected initially at day 17 of gestation in colonic extracts of Sprague-Dawley and Fischer rats. Expression of colonic PYY mRNA increased until 7 days of age and remained at its highest level (approximately twofold greater than the adult level) through the end of the nursing period. After weaning (21 days of age), PYY mRNA levels declined quickly to adult levels. Colonic PYY concentrations followed, in a coordinated manner, with some temporal delay after birth, the increase and decrease of its mRNA. Administration of PYY increased the weight and DNA content of the duodenum significantly in nursing rats and adult mice. In mice, PYY treatment also increased weight and DNA content of the ileum and colon. The trophic effects of PYY were dose related, peptide specific, and independent of species and sex. From these findings, we hypothesize that PYY plays an important role in intestinal development and dietary adaptation.