Arthur Shulkes

Expression, processing, and secretion of gastrin in patients with colorectal carcinoma

BACKGROUND & AIMS The relationship between gastrin and the development of colorectal carcinoma (CRC) remains controversial. Problems with previous studies include failure to measure all forms of gastrin, lack of comparison between stored and secreted gastrin, and not controlling for Helicobacter pylori infection (a known cause of hypergastrinemia). The aim of this study was to quantify progastrin and progastrin-derived peptides in the resected tumor and plasma of patients with CRC and in the antrum and plasma of normal subjects. METHODS Four region-specific gastrin antisera were used to measure progastrin, glycine-extended gastrin, amidated gastrin, and total gastrin. RESULTS Progastrin, amidated gastrin, total gastrin, and glycine-extended gastrin were detected in 100%, 69%, 56%, and 44% of tumors, respectively (n = 32). When allowing for H. pylori infection, circulating amidated gastrin levels were not significantly elevated in patients with CRC. However, compared with control H. pylori-positive and H. pylori-negative subjects, fasting plasma total gastrin levels were increased in H. pylori-positive (5.2-fold) and H. pylori-negative (2.3-fold) patients with CRC. CONCLUSIONS Gastrin or its processing intermediates are present in a high proportion of CRCs. Nonamidated gastrin levels are elevated in the circulation of patients with CRC regardless of H. pylori status. We conclude that gastrin should continue to be assessed as a circulating or autocrine growth factor in the development of CRC.

Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS): a new autosomal dominant syndrome

Objective The purpose of this study was the clinical and pathological characterisation of a new autosomal dominant gastric polyposis syndrome, gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS). Methods Case series were examined, documenting GAPPS in three families from Australia, the USA and Canada. The affected families were identified through referral to centralised clinical genetics centres. Results The report identifies the clinical and pathological features of this syndrome, including the predominant dysplastic fundic gland polyp histology, the exclusive involvement of the gastric body and fundus, the apparent inverse association with current Helicobacter pylori infection and the autosomal dominant mode of inheritance. Conclusions GAPPS is a unique gastric polyposis syndrome with a significant risk of gastric adenocarcinoma. It is characterised by the autosomal dominant transmission of fundic gland polyposis, including areas of dysplasia or intestinal-type gastric adenocarcinoma, restricted to the proximal stomach, and with no evidence of colorectal or duodenal polyposis or other heritable gastrointestinal cancer syndromes.

Inactivating cholecystokinin-2 receptor inhibits progastrin-dependent colonic crypt fission, proliferation, and colorectal cancer in mice

Hyperproliferation of the colonic epithelium, leading to expansion of colonic crypt progenitors, is a recognized risk factor for colorectal cancer. Overexpression of progastrin, a nonamidated and incompletely processed product of the gastrin gene, has been shown to induce colonic hyperproliferation and promote colorectal cancer in mice, but the mechanism of pathogenesis has not been defined. Cholecystokinin-2 receptor (CCK2R) is the primary receptor for cholecystokinin (CCK) and amidated gastrin. Here, we show that Cck2r was expressed in murine colonic crypts and upregulated in the transgenic mice that overexpress human progastrin. Murine deletion of Cck2r abrogated progastrin-dependent increases in colonic proliferation, mucosal thickness, and beta-catenin and CD44 expression in the colon tumor. In addition, either deletion or antagonism of Cck2r resulted in the inhibition of progastrin-dependent increases in progenitors expressing doublecortin and CaM kinase-like-1 (DCAMKL1), stem cells expressing leucine rich repeat-containing G protein-coupled receptor 5 (LgR5), and colonic crypt fission. Furthermore, in the azoxymethane mouse model of colorectal carcinogenesis, Cck2r deletion in human progastrin-overexpressing mice resulted in markedly decreased aberrant crypt foci formation and substantially reduced tumor size and multiplicity. Taken together, these observations indicate that progastrin induces proliferative effects, primarily in colonic progenitor cells, through a CCK2R-dependent pathway. Moreover, our data suggest that CCK2R may be a potential target in the treatment or prevention of colorectal cancer.

Short term infusion of glycine-extended gastrin17 stimulates both proliferation and formation of aberrant crypt foci in rat colonic mucosa

Evidence is accumulating that gastrin precursors may act as growth factors for the colonic mucosa in vivo and for colorectal carcinoma cell lines in vitro. The effect of short term administration of synthetic gastrins on the colonic mucosa in vivo, however, has not been reported. The aim of our study was to determine whether continuous systemic infusion of glycine‐extended gastrin17 stimulated proliferation and accelerated carcinogenesis in the colorectal mucosa. A significant increase in colonic mucosal proliferation as assessed by metaphase index was seen in the caecum (23%, p < 0.02) and distal colon (27%, p < 0.001), but not the rectum, after treatment of intact rats with glycine‐extended gastrin17 for 1 week using implanted miniosmotic pumps. Defunctioning of the rectum reduced both the proliferative index and crypt height of the rectal mucosa of untreated rats. Treatment of rectally defunctioned animals with glycine‐extended gastrin17 for either 1 or 4 weeks resulted in a significant increase in both the proliferative index (40% and 93%, respectively) and crypt height (11% and 19%, respectively) of the rectal mucosa. The total number of aberrant crypt foci in intact rats treated with the procarcinogen azoxymethane plus glycine‐extended gastrin17 was increased by 48% compared to the value in controls treated with azoxymethane only (p = 0.01). We conclude that short term administration of glycine‐extended gastrin17 to mature rats not only has a proliferative effect upon colonic mucosa, but also increases the number of aberrant crypt foci formed in the colorectal mucosa after treatment with azoxymethane. Glycine‐extended gastrin17 could thus potentially act as a promoter of carcinogenesis.

BRIEF REVIEW BIOLOGY OF GUT CHOLECYSTOKININ AND GASTRIN RECEPTORS

1. The stomach hormone gastrin and the intestinal hormone cholecystokinin (CCK) share a common C‐terminal pentapep‐tide sequence but have different biological roles. Gastrin is the major stimulant of gastric acid secretion and has a growth stimulatory effect on the secretory part of the stomach. The physiological roles of CCK are the stimulation of pancreatic secretion and the contraction of the gall‐bladder.

Effect of gastric inhibitory polypeptide on pentagastrin-stimulated acid secretion in man

Eight male subjects were given pentagastrin by intravenous infusion in doses of 25, 74, 222, 667, and 2000 ng/kg/hr, each dose for 30 min. On another day the same subjects were given the same doses of pentagastrin while gastric inhibitory polypeptide (GIP) was being infused intravenously in a dose of 2 μg/kg/hr. At the 222 ng/kg/hr dose of pentagastrin, acid output was significantly lower with GIP; at all other doses of pentagastrin, acid output did not differ significantly in tests with and without GIP. Pepsin output in the tests with and without GIP did not differ significantly at any dose of pentagastrin. Plasma concentration of GIP, measured by radioimmunoassay, showed a mean±SE plateau level of 7.4±1.4 ng/ml during GIP infusion and 0.4±0.1 ng/ml peak level after a standard meal. We conclude that the increase in blood concentration of GIP produced by feeding is probably inadequate to cause significant inhibition of gastric acid or pepsin secretion in man.

PAK1 interacts with β-catenin and is required for the regulation of the β-catenin signalling pathway by gastrins

Beta-catenin regulates cell-cell adhesion by binding to E-cadherin at the cell membrane and, when translocated into the nucleus, mediates signalling by activation of transcription factors such as TCF4. Mutations of the components of the Wnt/beta-catenin pathway are found in many gastrointestinal cancers. Gastrins, including amidated (Gamide) and glycine-extended (Ggly) gastrin(17), stimulate the proliferation of gastrointestinal cancer cells. Gastrins also regulate beta-catenin signalling through multiple pathways which seem to converge on p21-activated kinase 1 (PAK1). In this study, we have investigated the role of PAK1 in the regulation of beta-catenin signalling by gastrins. Here we report for the first time that PAK1 associated with beta-catenin. Both Gamide and Ggly stimulated the phosphorylation and activation of beta-catenin in a PAK1-dependent manner. A kinase-inactive mutant PAK1(K299A) blocked the gastrin-stimulated dissociation of beta-catenin from E-cadherin, translocation of beta-catenin from the cell membrane to the nucleus, and association of beta-catenin with the transcription factor TCF4. The PAK1(K299A) mutant also inhibited the stimulation of the expression of c-myc and cyclin D1, and of cell proliferation and migration, by gastrins. The results indicate that gastrins regulate beta-catenin signalling through a PAK1-dependent pathway. PAK1 seems to be the point of convergence of multiple signalling pathways activated by gastrins.

Pancreatic stellate cells produce acetylcholine and may play a role in pancreatic exocrine secretion

The pancreatic secretagogue cholecystokinin (CCK) is widely thought to stimulate enzyme secretion by acinar cells indirectly via activation of the vagus nerve. We postulate an alternative pathway for CCK-induced pancreatic secretion. We hypothesize that neurally related pancreatic stellate cells (PSCs; located in close proximity to the basolateral aspect of acinar cells) play a regulatory role in pancreatic secretion by serving as an intermediate target for CCK and secreting the neurotransmitter acetylcholine (ACh), which, in turn, stimulates acinar enzyme secretion. To determine whether PSCs (i) exhibit CCK-dependent ACh secretion and (ii) influence acinar enzyme secretion, primary cultures of human and rat PSCs were used. Immunoblotting and/or immunofluorescence was used to detect choline acetyltransferase (ACh synthesizing enzyme), vesicular ACh transporter (VAChT), synaptophysin, and CCK receptors 1 and 2. Synaptic-like vesicles in PSCs were identified by EM. ACh secretion by PSCs exposed to 20 pM CCK was measured by LC-MS/MS. Amylase secretion by acini [pretreated with and without the muscarinic receptor antagonist atropine (10 μM) and cocultured with PSCs] was measured by colorimetry. PSCs express ACh synthesizing enzyme, VAChT, synaptophysin, and CCK receptors; exhibit CCK-dependent ACh secretion; and stimulate amylase secretion by acini, which is blocked by atropine. In conclusion, PSCs express the essential elements for ACh synthesis and secretion. CCK stimulates ACh secretion by PSCs, which, in turn, induces amylase secretion by acini. Therefore, PSCs may represent a previously unrecognized intrapancreatic pathway regulating CCK-induced pancreatic exocrine secretion.

Ferric ions are essential for the biological activity of the hormone glycine-extended gastrin

Amidated and nonamidated gastrins elicit different biological effects via distinct receptors in different tissues. Amidated gastrin 17 stimulates gastric acid secretion and the development of gastric carcinoids, whereas glycine-extended gastrin 17 stimulates proliferation of the colonic mucosa and the development of colorectal cancers. Because glycine-extended gastrin 17 binds two ferric ions with high affinity (Baldwin, G. S., Curtain, C. C., and Sawyer, W. H. (2001) Biochemistry 40, 10741–10746), we have investigated the identity of the iron ligands and the role of ferric ions in biological activity. Here we report the solution structure of glycine-extended gastrin 17, determined by NMR spectroscopy. The spectral changes observed upon the addition of ferric ions revealed that Glu7 acted as a ligand at the first ferric binding site, and that Glu8 and Glu9acted as ligands at the second ferric ion binding site. Fluorescence quenching experiments confirmed that a GglyE7A mutant bound only one ferric ion. The inability of this mutant to stimulate proliferation or migration in the IMGE-5 cell line and the observation that the iron chelator desferrioxamine selectively blocked the effects of glycine-extended gastrin 17 indicated that binding of a ferric ion to Glu7 was essential for biological activity. This is the first report of an essential role for a metal ion in the action of a hormone.

Ketosis and appetite-mediating nutrients and hormones after weight loss

Background/Objectives:Diet-induced weight loss is accompanied by compensatory changes, which increase appetite and encourage weight regain. There is some evidence that ketogenic diets suppress appetite. The objective is to examine the effect of ketosis on a number of circulating factors involved in appetite regulation, following diet-induced weight loss.Subjects/Methods:Of 50 non-diabetic overweight or obese subjects who began the study, 39 completed an 8-week ketogenic very-low-energy diet (VLED), followed by 2 weeks of reintroduction of foods. Following weight loss, circulating concentrations of glucose, insulin, non-esterified fatty acids (NEFA), β-hydroxybutyrate (BHB), leptin, gastrointestinal hormones and subjective ratings of appetite were compared when subjects were ketotic, and after refeeding.Results:During the ketogenic VLED, subjects lost 13% of initial weight and fasting BHB increased from (mean±s.e.m.) 0.07±0.00 to 0.48±0.07 mmol/l (P<0.001). BHB fell to 0.19±0.03 mmol/l after 2 weeks of refeeding (P<0.001 compared with week 8). When participants were ketotic, the weight loss induced increase in ghrelin was suppressed. Glucose and NEFA were higher, and amylin, leptin and subjective ratings of appetite were lower at week 8 than after refeeding.Conclusions:The circulating concentrations of several hormones and nutrients which influence appetite were altered after weight loss induced by a ketogenic diet, compared with after refeeding. The increase in circulating ghrelin and subjective appetite which accompany dietary weight reduction were mitigated when weight-reduced participants were ketotic.