0023-6837/03/8301-047$03.00/0 LABORATORY INVESTIGATION Vol. 83, No. 1, p. 47, 2003 Copyright © 2003 by The United States and Canadian Academy of Pathology, Inc. Printed in U.S.A. PYY-Tag Transgenic Mice Displaying Abnormal H؉-K؉)ATPase Activity and Gastric Mucosal) Barrier Impairment Umberto Laforenza, Giulia Gastaldi, Guido Rindi, Andrew B. Leiter, Emanuela Cova, Annalisa Marchetti, Maria Elena Candusso, Michele Autelli, Maria Novella Orsenigo, and Ulderico Ventura Department of Experimental Medicine (UL, GG, EC, UV), Section of Human Physiology, and Department of Human Pathology (MEC), University of Pavia, and IRCCS Policlinico S. Matteo (AM, MA), Pavia, and Department of Pathology and Laboratory Medicine (GR), University of Parma, Parma, and Department of General Physiology and Biochemistry (MNO), University of Milan, Milan, Italy; and Division of Gastroenterology (ABL), New England Medical Center, Boston, Massachusetts SUMMARY: The mechanism by which the gastrointestinal hormones peptide YY and glucagon inhibit gastric acid secretion is largely unknown. PYY-Tag transgenic mice develop endocrine tumors in the colon that are composed mainly of peptide YY/enteroglucagon-producing L type cells. Therefore we studied the functional activity of such tumors and the gastric functions of PYY-Tag mice. Fasting and fed PYY-Tag transgenic mice and CD1 controls were assayed for circulating levels of peptide YY, glucagon, insulin, and gastrin. The gastric pH was determined and gastric samples were examined for (a) histologic appearance; (b) Kϩ-stimulated p-nitrophenylphosphatase activity and [14C]aminopyrine accumulation of apical and tubulovesicle membranes; (c) adherent mucus determination by Alcian blue recovery; and (d) DNA/RNA/protein epithelial content and in vivo incorporation of [3H]thymidine into DNA. Transgenic mice showed high serum levels of peptide YY and glucagon, increased gastric pH, and a high incidence of gastric ulcers after fasting. p-Nitrophenylphosphatase activity, [14C] aminopyrine accumulation, and proton pump redistribution from cytoplasmic tubulovesicles to apical membranes were significantly lower in the gastric mucosa of transgenic mice compared with the controls. In addition, the adherent mucus was thinner, and [3H]thymidine incorporation into the DNA was decreased. The abnormal and unregulated levels of circulating peptide YY and glucagon led to gastric acid inhibition and an impairment of gastric barrier function as a result of a striking reduction in epithelial proliferation. (Lab Invest 2003, 83:47– 54). astric acid secretion depends on the activity of vention of neural, endocrine, and paracrine mecha- G (Hϩ-Kϩ)ATPase (EC.3.6.1.36) in parietal cells. In nisms (Urushidani and Forte, 1997; Zeng et al, 1997). humans, basal acid production accounts for 0 to 5 Peptide YY (PYY) and enteroglucagon are synthe- mEq HCl per hour (resting mucosa), whereas after a sized by L-type endocrine cells in the distal ileum and meal it reaches 6 to 40 mEq HCl per hour (stimulated colon and have been shown to inhibit several gut ϩ ϩ mucosa). At rest (H -K )ATPase functions are limited functions including acid secretion (Adrian et al, 1985; by its intracytoplasmic position in vesicles, also called Guo et al, 1987a; Holst, 1997; Pappas et al, 1986; tubulovesicles, and by the reduced permeability of Sheikh, 1991; Walsh, 1994). Considering the location tubulovesicles to KCl (Urushidani and Forte, 1997). of L cells in the distal gut, PYY and enteroglucagon The activation of parietal cells by secretagogues in- may participate in the modulation of transit times, duces tubulovesicle fusion with the apical plasma gastric and pancreatic secretion, and nutrient absorp- membrane (membrane trafficking) and an increase in tion (Goodlad et al, 1991). Both peptides are released permeability to KCl. The regulation of parietal cell into the circulation in response to meal ingestion, with activity is directly or indirectly achieved by the inter- special reference to fats, and are widely considered as potential enterogastrones. Indeed, at physiologic doses, PYY and enteroglucagon may inhibit gastric DOI: 10.1097/01.LAB.0000048720.34096.D7 acid secretion induced by pentagastrin, meal, and Received October 14, 2002. vagal stimulation (Adrian et al, 1985; Anini et al, 1999; This work was supported by COFIN 99 (9905021792_002 and Guo et al, 1987b; Holst, 1997; Sheikh, 1991; Walsh, 9906218982_002 to GR), by the University of Pavia FAR 2001, and by 1994; Wettergren et al, 1994; Yang, 2002; Zeng et al, the Health Ministry (020 RCR 98/03 to GR). Address reprint requests to: Dr. U. Laforenza, Department of Experimental 1997). Although PYY and enteroglucagon colocalize in Medicine, Section of Human Physiology, University of Pavia, Via Forla- the same endocrine cell type and are usually released nini, 6, I-27100 Pavia, Italy. E-mail: [email protected] simultaneously, under certain conditions, only one Laboratory Investigation • January 2003 • Volume 83 • Number 1 47 Laforenza et al peptide may be selectively released (Anini et al, 1999; suggests that high levels of circulating PYY and glu- Brubaker, 1991; Plaisancié et al, 1996). cagon do not interfere with normal gastric endocrine Recently, transgenic mice expressing the simian cell differentiation. virus 40 large T antigen under the control of the rat PYY gene promoter (PYY-Tag) (Upchurch et al, 1994, 1996) were generated to examine the ontogeny of Gastric Acid Secretion Is Reduced in PYY-Tag Mice PYY-producing cells in the developing mouse pan- Gastric acid secretion analysis revealed that the creas and colon. PYY-Tag transgenic mice develop fasting gastric pH was significantly higher in PYY-Tag well-differentiated endocrine tumors of the proximal transgenic mice than in the controls (3.85 Ϯ 0.5 vs colon mainly composed of L type cells. 2.53 Ϯ 0.14, respectively [mean Ϯ SEM], n ϭ 5; p Յ Our objective was to investigate the functional activity 0.02, Student’s unpaired t test). Under the fed condi- of such tumors, gastric acid secretion, and the mucosal tion, the gastric pH was also significantly higher in barrier in the gastric mucosa of both fasting and fed PYY-Tag transgenic mice than in controls (4.52 Ϯ 0.37 PYY-Tag mice. A preliminary partial account of these vs 2.53 Ϯ 0.46 respectively [mean Ϯ SEM], n ϭ 5; p Յ results has already been published in an abstract form 0.01, Student’s unpaired t test). (Gastaldi et al, 2000; Laforenza et al, 2000). ϩ When assaying the gastric vesicles used for K - Results stimulated p-nitrophenylphosphatase (pNPPase) ac- tivity and aminopyrine uptake measurements (see PYY-Tag Mice Showed High PYY and Glucagon Serum following paragraphs), electron microscopic examina- Levels tion revealed that P1 was multilamellar and multive- sicular with a mean diameter larger than for P3 (P1, Serum PYY levels were significantly higher in fed and 760 Ϯ 32 nm; P3, 173 Ϯ 7.2 nm [mean Ϯ SEM], n ϭ 50 ف fasting transgenic mice and corresponded to 10- and from three different samples). Their appearance and ف 50-fold, respectively, compared with controls (Table 1). morphometry were similar to those previously de- ف Glucagon levels were also significantly higher ( 2-fold) scribed (Forte et al, 1981; Reenstra and Forte, 1990). in transgenic mice compared with controls. PYY, but not The Kϩ-stimulated pNPPase activity in both PYY-Tag glucagon, levels were increased in fasting transgenic and control mice was measured with increasing concen- mice compared with fed transgenic mice. Gastrin was trations of KCl and showed a hyperbolic course up to a significantly higher in fed transgenic mice compared with concentration of 10 mM KCl (not shown). In PYY-Tag and both controls and fasting mice (Table 1). No change was control mice, pNPPase activity gradually decreased at observed in insulin serum levels among any of the animal concentrations of 10 to 50 mM KCl. Vmax values were groups considered. significantly lower in the homogenate and P1 fractions of fed transgenic mice compared with controls. In fasting Gastric Endocrine Cells Are Unchanged in PYY-Tag Mice transgenic mice, Vmax value was significantly reduced only in the P1 fraction (Fig. 1). Kmax values remained Gastric endocrine cells of PYY-Tag mice were investi- unchanged (not shown). gated by Grimelius’ silver and vesicular monoamine The meal-induced proton pump redistribution from transporter 2 (VMAT2) immunohistochemistry to assess cytoplasmic tubulovesicles to the apical plasma mem- histamine-producing enterochromaffin-like cells and by brane was investigated by analyzing the ratio of the immunohistochemistry with specific antisera to deter- total Kϩ-stimulated pNPPase activity in P1 to the total mine ghrelin, gastrin, somatostatin, and the rare PYY activity in P3 (P1/P3 ratio) according to Reenstra and cells. The qualitative assessment of size, shape, and Forte (1990). The total pNPPase activity in P1 and P3 number of argyrophil cells and of VMAT2-, ghrelin-, was obtained by multiplying the pNPPase specific gastrin-, somatostatin-, and PYY-immunoreactive activity (␮mol/mg protein · hour) per milligram of total cells showed no apparent change as compared with protein recovered in each fraction. The P1/P3 ratio nontransgenic CD1 mice, except in correspondence was similar in fasting mice but significantly lower in fed of mucosal ulceration (not shown). This observation transgenic mice (Fig. 2). The time course of [14C] Table 1. Serum Gastrointestinal Hormone (PYY, Glucagon, Insulin, and Gastrin) Concentrations in Normal (CD1) and PYY-Tag Transgenic Mice Under Fasting and Fed Conditions Fasting CD1 Fasting Transgenic Fed CD1 Fed Transgenic PYY (pg/ml) 42 Ϯ 7 2130a Ϯ 235 250b Ϯ 94 2699a,c Ϯ 285 Glucagon (pg/ml) 1102 Ϯ 219 1870a Ϯ 105 1015 Ϯ 224 2396a Ϯ 254 Insulin (␮UI/ml) 1.87 Ϯ 0.1 1.78 Ϯ 0.04 1.96 Ϯ 0.1 2.07 Ϯ 0.2 Gastrin (pg/ml) 124 Ϯ 30 118 Ϯ 7 111 Ϯ 34 264d Ϯ 40 Values are mean Ϯ SEM of at least eight different blood samples each from a different mouse.