Reduced Ghrelin, Islet Amyloid Polypeptide, and Peptide YY Expression in the Stomach of Gastrin-Cholecystokinin Knockout Mice

0013-7227/05/$15.00/0 Printed in U.S.A. Endocrinology 146(10):4464 – 4471 Copyright © 2005 by The Endocrine Society doi: 10.1210/en.2004-0938 Reduced Ghrelin, Islet Amyloid Polypeptide, and Peptide YY Expression in the Stomach of Gastrin-Cholecystokinin Knockout Mice Lennart Friis-Hansen, Nils Wierup, Jens F. Rehfeld, and Frank Sundler Department of Clinical Biochemistry (L.F.-H., J.F.R.), Rigshospitalet, University of Copenhagen, DK-2100 Copenhagen, Denmark; and Department of Neuroscience and Physiology (N.W., F.S.), University of Lund, S-22100 Lund, Sweden The antral hormone gastrin and its intestinal relative, cholecystokinin (CCK), are pivotal in the regulation of gastric functions. Other gastric hormones like ghrelin, peptide YY (PYY), and islet amyloid polypeptide (IAPP), however, also contribute to the regulation of acid secretion, motility, and feeding. Because gastrin and CCK are crucial for gastric homeostasis, we examined how loss of gastrin alone and gastrin plus CCK affected the expression of ghrelin, IAPP, and PYY and ghrelin secretion. The expression of ghrelin, IAPP, and PYY and the CCK-A receptor genes were examined in both gastrin and gastrin-CCK double-knockout (KO) mice using immunocytochemistry and quantitative RT-PCR. Ghrelin concentrations in plasma were measured using RIA. Gastrin and CCK were infused in gastrin-CCK KO mice using osmotic minipumps. The number of ghrelin cells and ghrelin gene expression were T HE STOMACH IS a major endocrine organ with at least six different endocrine cell types (for reviews, see Refs. 1– 4). Gastrin is the master hormone of stomach controlling gastric growth and acid secretion (5). In addition to gastrin, peptide hormones like somatostatin, islet amyloid polypeptide (IAPP/amylin) (6), peptide YY (PYY) (7), and ghrelin (8, 9) are also expressed in the gastric mucosa. IAPP, PYY, and ghrelin modulate acid secretion, gastric homeostasis (10 –15), and gut motility (for review see Refs. 16 and 17). Gastric hormones, however, also play a role in the regulation of feeding. PYY and especially the PYY3–36 fragment inhibit feeding (18, 19). IAPP, predominantly produced by pancreatic ␤-cells (20), is involved in the regulation of glucose metabolism (21) and may be involved in the development of non-insulin-dependent diabetes mellitus (22, 23). In the gastric mucosa, PYY and IAPP are often coexpressed with other peptide hormones and amines (6, 10, 24). Ghrelin is expressed in endocrine cells in both the oxyntic and antral mucosa (8, 9). Fasting induces ghrelin secretion and increases gastric ghrelin expression by mechanisms partly involving vagal pathways (25, 26). The full range of interactions between the hormones is not First Published Online July 7, 2005 Abbreviations: CCK, Cholecystokinin; FP, forward primer; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IAPP, islet amyloid polypeptide; KO, knockout; PYY, peptide YY; RP, reverse primer; RT, reverse transcription. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community. unaffected, albeit the ghrelin cells were located closer to the base of the glands in both KO mouse strains when freely fed. However, lack of both gastrin and CCK attenuated fastinginduced ghrelin expression and secretion. Fundic ghrelin cells expressed the CCK-A receptor, and ghrelin expression increased after CCK infusion. Furthermore, gastric IAPP and PYY expression as well as the number of IAPP- and PYY-containing cells were reduced in both gastrin and gastrin-CCK KO mice. Gastrin infusion increased gastric IAPP but not PYY expression. In conclusion, lack of gastrin plus CCK but not gastrin alone reduced ghrelin secretion in response to fasting through both direct and indirect mechanisms. Both gastrin and combined gastrin-CCK deficiency reduced the gastric IAPP and PYY expression. (Endocrinology 146: 4464 – 4471, 2005) yet fully understood, but gastrin plays a decisive role in regulating gastric homeostasis (27–30). Besides gastrin, cholecystokinin (CCK) is also a full agonist for the gastrin/ CCK-B receptor, and in the absence of gastrin, CCK maintains certain gastric functions (31). Furthermore, CCK also activates the CCK-A receptor, which does not bind gastrin. We therefore examined how the gastric expression of PYY, IAPP, and ghrelin was affected in gastrin or gastrin plus CCK KO mice. Materials and Methods Mice Gastrin knockout (KO) mice and gastrin-CCK KO mice and their normal littermates were used (27, 31). All were on a mixed 129/SvJ, C57BL/6J background, backcrossed at least four times to C57BL/6J. Thus, all mice had black coat color. The mice were 12–16 wk old when analyzed, and all groups were gender matched. The mice were kept under specific pathogen-free conditions and monitored according to the Federation of European Laboratory Animal Science Assocations recommendation (32) with 12 h light, 12-h dark cycles. The study was approved by the Danish Animal Welfare Committee. Histological and immunohistochemical/fluorescence analysis Stomachs from freely fed mice aged 12–16 wk were rinsed in ice-cold PBS, fixed in 4% paraformaldehyde in PBS for 4 – 6 h, and embedded in paraffin. Five-micrometer sections were cut and stained with hematoxylin and eosin. Tissues for immunohistochemistry were fixed in Stefanini fixative [2% formaldehyde and 0.2% picric acid (in a pH 7.2 phosphate buffer)] overnight, washed in Tyrodes solution with 10% sucrose (wt/vol) added, and frozen on dry ice. Sections (10 ␮m thickness) were cut and mounted on chrome-alun-coated slides. Immunohistochemistry was performed using the antisera described in Table 1. Fluorescein isothiocyanate- and Texas Red-conjugated secondary antibodies were 4464 Friis-Hansen et al. • Ghrelin, IAPP, and PYY in Gastrin-CCK KO Mice Endocrinology, October 2005, 146(10):4464 – 4471 4465 TABLE 1. Characteristics for the antisera used for immunocytochemistry Antibody Raised against Raised in Dilution 4562 8818 A110 8415 N-Som C-SP-1 H-031-31 9439/16 Human gastrin 2-17 Gastrin-17 Rat IAPP PYY Porcine somatostatin Chromogranin A Ghrelin CCK-A receptor Rabbit Guinea pig Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit 1:2560 1:1280 1:640 1:640 1:800 1:160 1:1000 1:2560 J. F. Rehfeld (Copenhagen, Denmark) Euro-Diagnostica (Malmö, Sweden) P. Westermark (Linköbing, Sweden) Euro-Diagnostica Incstar Corp. (Stillwater, MN) Incstar Corp. Phoenix Peptides Euro-Diagnostica Supplier 1:100 1:400 Dako (Glostrup, Denmark) Jackson (West Grove, PA) Secondary antibodies with conjugates FITC conjugated swine antirabbit IgG Texas Red conjugated goat antiguinea pig IgG FITC, Fluorescein isothiocyanate. used for immunofluorescence detection (see Table 1 for antibody characteristics). The specificity of the immunostaining was tested by absorbing the primary antibodies with antigen before applying them to the slides or omitting the primary antibody wh (...truncated)