Reduced Ghrelin, Islet Amyloid Polypeptide, and Peptide YY Expression in the Stomach of Gastrin-Cholecystokinin Knockout Mice
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《内分泌学杂志》
Department of Clinical Biochemistry (L.F.-H., J.F.R.), Rigshospitalet, University of Copenhagen, DK-2100 Copenhagen, Denmark
Department of Neuroscience and Physiology (N.W., F.S.), University of Lund, S-22100 Lund, Sweden
Abstract
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 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 fasting-induced 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.
Introduction
THE STOMACH IS a major endocrine organ with at least six different endocrine cell types (for reviews, see Refs.1, 2, 3, 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, 11, 12, 13, 14, 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 yet fully understood, but gastrin plays a decisive role in regulating gastric homeostasis (27, 28, 29, 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 μm 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 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 when purified antigen was not available. The morphometrical analysis was performed by cell counting in transversely cut sections as described (33).
CCK-A and CCK-B receptor autoradiography
Two fundic samples were taken from four mice from each of the three strains and frozen on dry ice. Subsequently CCK-A receptor autoradiography was performed on 10- and 20-μm-thick cryostat sections as described (34, 35). The number of binding sites were arbitrary expressed as counts per minute.
Gastrin and CCK replacement experiments
To examine to what extent the changes in the gastrin KO mice could be reversed by exogenous gastrin, the mice were given a sc infusion of nonsulfated gastrin-17 (10 nmol/kg–1·h–1) using osmotic minipumps (Alzet no. 2001; Alza Corp., Cupertino, CA). The mice were anesthetized with intraperitoneal 2,2,2 tribromoethanol (Sigma-Aldrich Corp., St. Louis, MO), and the pumps were implanted sc on the back of the mice. The mice were killed with the minipumps in place after 6 d, and tissue for RNA analysis and blood was collected for measurement of plasma gastrin concentrations. We also examined whether exogenous CCK could reverse the changes in the gastric ghrelin, IAPP, and PYY expression by infusing sulfated CCK-8 (15 nmol/kg–1·h–1) sc into gastrin-CCK KO mice for 2 or 6 d (eight mice for each time point) using osmotic minipumps. Implantation and the after tissue collection were performed as described above.
Fasting
Ten mice from each group (wild-type, gastrin KO, or gastrin-CCK KO mice) were housed in metabolic chambers and fasted for 2 d with unlimited access to water. Changes in body weight and water consumption of the mice were recorded daily. After 2 d of fasting, the mice were killed, plasma collected, and the fundic and antral parts of the stomach were dissected, frozen on liquid nitrogen, and kept at –80 C until processed for further analysis.
RIA
Plasma ghrelin was measured in EDTA plasma without extraction using RIA no. RK-031–31 (Phoenix Peptides, Belmont, CA), which measures the sum of Ser3-octanoyl and Ser3-des-octanoyl ghrelin peptides. The assay has a detection limit of 20 pmol/liter, an interassay variation of 13%, and an intraassay variation of 5% (36). CCK concentrations were measured using antiserum 92128 that does not bind any gastrin peptides (37). Gastrin was measured using antiserum 2604, which does not bind any CCK peptides (38).
RNA extraction and analysis
The stomachs were dissected into fundus and antrum and immediately placed in liquid nitrogen. RNA was extracted using the method described by Chomcynski and Sacchi (39), and quantitative changes in the specific mRNAs were determined by real-time PCR using the Lightcycler (Roche, Mannheim, Germany) as described (31). Quantitations were performed using one of the following primer sets for each analysis: ghrelin forward primer (FP) 5'-TCT GCA GTT TGC TGC TAC TCA-3' and ghrelin reverse primer (RP) 5'-CCT CTT TGA CCT CTT CCC AGA-3'; PYY FP 5'-GCA GCG GTA TGG AAA AAG AG-3' and PYY RP 5'-TTC ACC ACT GGT CCA AAC CT-3'; chromogranin-A FP 5'-CAC AGC AGC TTT GAG GAT GA-3' and chromogranin-A RP 5'-ATG GGG GAC TCT TGG TTA GG-3'; IAPP FP 5'-CAC AGC AGC TTT GAG GAT GA-3' and IAPP RP 5'-ATG GGG GAC TCT TGG TTA GG-3'; CCK-A receptor FP 5'-AAG AGG ATG CGG ACT GTC AC-3' and CCK-A receptor RP 5'-CAG ACG CGG GAT TGT AGG-3'; leptin FP 5'-CTC ATG CCA GCA CTC AAA AA-3' and leptin RP 5'-AGC ACC ACA AAA CCT GAT CC-3'; or the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) FP 5'-GGT GCT GAG TAT GTC GTG GA-3' and GAPDH RP 5'-GTG GTT CAC ACC CAT CAC AA-3'. Each run consisted of one negative control, one sample in which the Moloney murine leukemia virus reverse transcriptase had been omitted in the reverse transcription (RT) step, a standard curve generated by 3-fold serial dilution of RT reactions, and seven to nine RT reactions from each of the three strains. Expression of a given transcript was normalized to a GAPDH quantification performed on the same RT reaction as previously described (31).
Statistical analysis
Student’s unpaired t test statistics were used and differences with a P < 0.05 were considered significant. Where nothing else is stated, results are given as mean ± SEM.
Results
Ghrelin
The density of fundic and antral ghrelin cells was unaffected by the loss of gastrin or gastrin plus CCK in freely fed mice (Figs. 1 and 2A). However, in both KO strains, fundic ghrelin cells were located closer to the base of the glands than in the wild-type mice (Fig. 1). Ghrelin gene expression and ghrelin plasma concentration were also unaffected in the two groups of freely fed KO mice (Fig. 2, B and C). In contrast, after 48 h of fasting (with free access to water), ghrelin secretion was halved in gastrin-CCK KO mice but normal in the gastrin KO mice (Fig. 2C). Furthermore, fasting-induced ghrelin mRNA expression in the gastrin-CCK KO mice was equally attenuated in antral and fundic mucosa (Fig. 2D).
In wild-type mice, the CCK-A receptors were mainly found on fundic chief cells in accordance with earlier studies (40, 41). However, fundic ghrelin cells also expressed CCK-A receptors (Fig. 3). In contrast, antral ghrelin cells lacked the CCK-A receptors. Continuous sc infusion of CCK for both 2 and 6 d in the gastrin-CCK KO mice (Fig. 4A) increased fundic ghrelin mRNA levels 6- to 7-fold (Fig. 4B). In contrast, antral ghrelin expression was up to 2-fold reduced (Fig. 4C). The level of fundic CCK-A receptor expression was the same in all three groups of mice (Table 2). Estimation of the receptors by ligand binding autoradiography also showed equal receptor numbers in the two KO strains and wild-type mice (Table 2). Thus, both CCK-A receptor density and mRNA expression level are unaffected by the loss of CCK.
IAPP
IAPP cells were found frequently in antral mucosa in wild-type mice but were almost absent in the gastrin KO and gastrin-CCK KO mice (Table 2 and Fig. 5A, upper row). Furthermore, antral expression of IAPP mRNA was greatly reduced in both groups of KO mice (Fig. 5B). In wild-type mice, IAPP was predominantly colocalized with somatostatin and PYY but not with serotonin or gastrin (Table 3). In the gastrin KO mice, the antral IAPP expression increased after infusion of gastrin (Table 4). In contrast, infusion of CCK did not alter the expression of IAPP (Table 5).
PYY
In all groups of mice, oxyntic and antral endocrine cells expressed PYY. Nevertheless, in both KO mouse strains, the density of PYY cells was considerably reduced (Table 2 and Fig. 5A, middle and lower rows). Accordingly, the antral and fundic expression of the PYY mRNA was reduced to three fourths of wild-type levels (Fig. 5, C and D). In wild-type mice, both antral and fundic PYY mainly colocalized with somatostatin. In contrast, only a minor fraction of the PYY-expressing cells also stained for serotonin or gastrin (Table 3). The coexpression pattern was not altered by either lack of gastrin alone or combined lack of both gastrin and CCK (data not shown). Infusion of gastrin in gastrin KO mice did not affect the gastric PYY expression levels (Table 4). After 2 d with infusion of CCK into the gastrin-CCK KO mice, the antral as well as the fundic expression levels of PYY increased (Table 5). However, during continued infusion of CCK, the activation of fundic and antral PYY expression diminished, and after 6 d of CCK infusion, antral but not fundic expression had returned to the baseline gastrin-CCK KO levels (Table 5).
Other responses to fasting
Despite the absence of CCK as a satiety signal, the changes in ghrelin secretion, and the alterations in gastric IAPP and PYY expression, there were no differences in body weight among the three groups of mice at 12 wk of age. Furthermore, gastric leptin expression was unaffected by the loss of gastrin or gastrin and CCK (Table 3). Finally, the three groups of mice responded similarly to fasting as evaluated by water consumption and loss of body weight (Fig. 6).
Discussion
This study shows that even though gastrin and CCK are not necessary for the development of gastric endocrine cells as such, lack of gastrin alone or gastrin and CCK affects the expression of IAPP and PYY as well as the ghrelin response to fasting.
The effect of gastrin and CCK on ghrelin cells
The development and maintenance of ghrelin cells is independent of gastrin and CCK (this study and Refs.36, 42). Hence, neither CCK-A nor CCK-B receptor signaling are required for ghrelin cell development. Nevertheless, ghrelin mRNA expression and ghrelin secretion in response to fasting was reduced in mice lacking both gastrin and CCK. Due to lack of CCK KO mice, we were not able to determine whether loss of CCK alone or the combined loss of CCK and gastrin is responsible for the observed alterations in ghrelin expression and secretion in the gastrin-CCK KO mice. However, neither ghrelin expression nor secretion was affected in rats treated with a CCK-B receptor antagonist, which blocks both CCK and gastrin signaling through the CCK-B receptor (36). Furthermore, ghrelin cell density was not affected in the CCK-B receptor KO mice (43). We therefore surmise that the difference between the gastrin KO and gastrin-CCK KO mice arise mainly from lack of CCK signaling via the CCK-A receptor and that lack of gastrin is without significance. However, it cannot be ruled out that only the combined lack of gastrin and CCK affects ghrelin expression and secretion. Analysis of mice deficient of CCK alone is needed to answer this question.
CCK stimulates ghrelin secretion (44), and as shown in this study, CCK replacement increased fundic but not antral ghrelin gene expression. The ability of CCK to stimulate ghrelin secretion is surprising. CCK is a satiety hormone, the secretion of which is reduced during fasting but increased postprandially. CCK presumably stimulates fundic ghrelin secretion directly via the CCK-A receptors on the ghrelin cells. However, because antral and fundic responses to fasting were equally reduced despite the absence of CCK-A receptors on antral ghrelin cells, there also has to be an indirect pathway, perhaps neuronal, for CCK activation. It could be a vagal pathway because vagal activity inhibits ghrelin secretion. Both CCK and fasting have been shown to reduce vagal efferent activity (45, 46). Moreover, increased vagal activity is believed to be one of the mechanisms driving acid secretion in the double-KO mouse (31).
The differential expression of CCK-A receptors on fundic and antral ghrelin cells and accordingly the different response to CCK infusion suggest a direct mechanism for CCK on fundic ghrelin expression. In contrast, antral ghrelin expression was reduced after CCK infusion despite the absence of CCK-A receptors on antral ghrelin cells. Our findings are the first demonstration of phenotypical differences between antral and fundic ghrelin cells. Moreover, they are also in agreement with recent demonstrations of different developmental programs for fundic and antral endocrine cells (47).
The effect of gastrin and CCK and PYY and IAPP cells
The colocalization of PYY and IAPP extends earlier observations in fetal rats (6, 48). Contrary to the findings in adult rat (6), there was no colocalization of IAPP and gastrin in the mouse antrum (this study and Ref.49). Fasting reduces the expression of PYY and IAPP (50), but the present study is the first to address the cellular changes in IAPP and PYY expression related to gastrin and gastric acid secretion. Hence, our data suggest that no direct link exists between PYY and IAPP expression, although PYY and IAPP often colocalize in the same cells. Both PYY and IAPP can inhibit gastric acid secretion (51, 52, 53), but the physiological significance is poorly understood.
CCK is a regulator of intestinal PYY secretion, but the data are conflicting as to whether CCK acts directly on PYY cells (54, 55) or whether the vagal nerve mediates CCK actions (56). The presence of CCK-A receptors on the PYY cells favors the idea of a direct CCK action on PYY cells. In contrast, gastrin does not directly regulate gastric PYY expression.
Ghrelin, CCK, and IAPP either alone or together have been shown to affect the regulation of body weight (44, 57, 58). However, despite widespread abnormalities in gastric endocrinology in both groups of KO mice, they responded normally to food withdrawal in terms of water consumption and loss of body weight. Furthermore, gastric leptin expression was unchanged in gastrin-CCK KO mice, although CCK stimulates gastric leptin secretion and expression in chief cells via the CCK-A receptor (57, 59). This suggests that CCK is not important for maintaining gastric leptin expression, although we cannot rule out that the changes due to lack of CCK are ameliorated by the additional lack of gastrin.
In summary, this study shows that lack of gastrin reduces the gastric expression of PYY and IAPP. In contrast, ghrelin expression and secretion is independent of gastrin, but the response to fasting is reduced in the absence of gastrin and CCK. The differential expression of CCK-A receptors on fundic and antral ghrelin cells is the first suggestion that gastric ghrelin cells are functionally heterogeneous.
Acknowledgments
We thank Professor J. C. Reubi (University of Berne, Berne, Switzerland) for the autoradiographic results. The skillful technical assistance of Bo Lindberg and Doris Persson is greatly appreciated.
Footnotes
This work was supported by grants from the Novo Nordisk Foundation, The Swedish Medical Research Council (project 4479), and The Royal Swedish Physiographical Society.
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.
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Department of Neuroscience and Physiology (N.W., F.S.), University of Lund, S-22100 Lund, Sweden
Abstract
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 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 fasting-induced 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.
Introduction
THE STOMACH IS a major endocrine organ with at least six different endocrine cell types (for reviews, see Refs.1, 2, 3, 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, 11, 12, 13, 14, 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 yet fully understood, but gastrin plays a decisive role in regulating gastric homeostasis (27, 28, 29, 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 μm 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 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 when purified antigen was not available. The morphometrical analysis was performed by cell counting in transversely cut sections as described (33).
CCK-A and CCK-B receptor autoradiography
Two fundic samples were taken from four mice from each of the three strains and frozen on dry ice. Subsequently CCK-A receptor autoradiography was performed on 10- and 20-μm-thick cryostat sections as described (34, 35). The number of binding sites were arbitrary expressed as counts per minute.
Gastrin and CCK replacement experiments
To examine to what extent the changes in the gastrin KO mice could be reversed by exogenous gastrin, the mice were given a sc infusion of nonsulfated gastrin-17 (10 nmol/kg–1·h–1) using osmotic minipumps (Alzet no. 2001; Alza Corp., Cupertino, CA). The mice were anesthetized with intraperitoneal 2,2,2 tribromoethanol (Sigma-Aldrich Corp., St. Louis, MO), and the pumps were implanted sc on the back of the mice. The mice were killed with the minipumps in place after 6 d, and tissue for RNA analysis and blood was collected for measurement of plasma gastrin concentrations. We also examined whether exogenous CCK could reverse the changes in the gastric ghrelin, IAPP, and PYY expression by infusing sulfated CCK-8 (15 nmol/kg–1·h–1) sc into gastrin-CCK KO mice for 2 or 6 d (eight mice for each time point) using osmotic minipumps. Implantation and the after tissue collection were performed as described above.
Fasting
Ten mice from each group (wild-type, gastrin KO, or gastrin-CCK KO mice) were housed in metabolic chambers and fasted for 2 d with unlimited access to water. Changes in body weight and water consumption of the mice were recorded daily. After 2 d of fasting, the mice were killed, plasma collected, and the fundic and antral parts of the stomach were dissected, frozen on liquid nitrogen, and kept at –80 C until processed for further analysis.
RIA
Plasma ghrelin was measured in EDTA plasma without extraction using RIA no. RK-031–31 (Phoenix Peptides, Belmont, CA), which measures the sum of Ser3-octanoyl and Ser3-des-octanoyl ghrelin peptides. The assay has a detection limit of 20 pmol/liter, an interassay variation of 13%, and an intraassay variation of 5% (36). CCK concentrations were measured using antiserum 92128 that does not bind any gastrin peptides (37). Gastrin was measured using antiserum 2604, which does not bind any CCK peptides (38).
RNA extraction and analysis
The stomachs were dissected into fundus and antrum and immediately placed in liquid nitrogen. RNA was extracted using the method described by Chomcynski and Sacchi (39), and quantitative changes in the specific mRNAs were determined by real-time PCR using the Lightcycler (Roche, Mannheim, Germany) as described (31). Quantitations were performed using one of the following primer sets for each analysis: ghrelin forward primer (FP) 5'-TCT GCA GTT TGC TGC TAC TCA-3' and ghrelin reverse primer (RP) 5'-CCT CTT TGA CCT CTT CCC AGA-3'; PYY FP 5'-GCA GCG GTA TGG AAA AAG AG-3' and PYY RP 5'-TTC ACC ACT GGT CCA AAC CT-3'; chromogranin-A FP 5'-CAC AGC AGC TTT GAG GAT GA-3' and chromogranin-A RP 5'-ATG GGG GAC TCT TGG TTA GG-3'; IAPP FP 5'-CAC AGC AGC TTT GAG GAT GA-3' and IAPP RP 5'-ATG GGG GAC TCT TGG TTA GG-3'; CCK-A receptor FP 5'-AAG AGG ATG CGG ACT GTC AC-3' and CCK-A receptor RP 5'-CAG ACG CGG GAT TGT AGG-3'; leptin FP 5'-CTC ATG CCA GCA CTC AAA AA-3' and leptin RP 5'-AGC ACC ACA AAA CCT GAT CC-3'; or the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) FP 5'-GGT GCT GAG TAT GTC GTG GA-3' and GAPDH RP 5'-GTG GTT CAC ACC CAT CAC AA-3'. Each run consisted of one negative control, one sample in which the Moloney murine leukemia virus reverse transcriptase had been omitted in the reverse transcription (RT) step, a standard curve generated by 3-fold serial dilution of RT reactions, and seven to nine RT reactions from each of the three strains. Expression of a given transcript was normalized to a GAPDH quantification performed on the same RT reaction as previously described (31).
Statistical analysis
Student’s unpaired t test statistics were used and differences with a P < 0.05 were considered significant. Where nothing else is stated, results are given as mean ± SEM.
Results
Ghrelin
The density of fundic and antral ghrelin cells was unaffected by the loss of gastrin or gastrin plus CCK in freely fed mice (Figs. 1 and 2A). However, in both KO strains, fundic ghrelin cells were located closer to the base of the glands than in the wild-type mice (Fig. 1). Ghrelin gene expression and ghrelin plasma concentration were also unaffected in the two groups of freely fed KO mice (Fig. 2, B and C). In contrast, after 48 h of fasting (with free access to water), ghrelin secretion was halved in gastrin-CCK KO mice but normal in the gastrin KO mice (Fig. 2C). Furthermore, fasting-induced ghrelin mRNA expression in the gastrin-CCK KO mice was equally attenuated in antral and fundic mucosa (Fig. 2D).
In wild-type mice, the CCK-A receptors were mainly found on fundic chief cells in accordance with earlier studies (40, 41). However, fundic ghrelin cells also expressed CCK-A receptors (Fig. 3). In contrast, antral ghrelin cells lacked the CCK-A receptors. Continuous sc infusion of CCK for both 2 and 6 d in the gastrin-CCK KO mice (Fig. 4A) increased fundic ghrelin mRNA levels 6- to 7-fold (Fig. 4B). In contrast, antral ghrelin expression was up to 2-fold reduced (Fig. 4C). The level of fundic CCK-A receptor expression was the same in all three groups of mice (Table 2). Estimation of the receptors by ligand binding autoradiography also showed equal receptor numbers in the two KO strains and wild-type mice (Table 2). Thus, both CCK-A receptor density and mRNA expression level are unaffected by the loss of CCK.
IAPP
IAPP cells were found frequently in antral mucosa in wild-type mice but were almost absent in the gastrin KO and gastrin-CCK KO mice (Table 2 and Fig. 5A, upper row). Furthermore, antral expression of IAPP mRNA was greatly reduced in both groups of KO mice (Fig. 5B). In wild-type mice, IAPP was predominantly colocalized with somatostatin and PYY but not with serotonin or gastrin (Table 3). In the gastrin KO mice, the antral IAPP expression increased after infusion of gastrin (Table 4). In contrast, infusion of CCK did not alter the expression of IAPP (Table 5).
PYY
In all groups of mice, oxyntic and antral endocrine cells expressed PYY. Nevertheless, in both KO mouse strains, the density of PYY cells was considerably reduced (Table 2 and Fig. 5A, middle and lower rows). Accordingly, the antral and fundic expression of the PYY mRNA was reduced to three fourths of wild-type levels (Fig. 5, C and D). In wild-type mice, both antral and fundic PYY mainly colocalized with somatostatin. In contrast, only a minor fraction of the PYY-expressing cells also stained for serotonin or gastrin (Table 3). The coexpression pattern was not altered by either lack of gastrin alone or combined lack of both gastrin and CCK (data not shown). Infusion of gastrin in gastrin KO mice did not affect the gastric PYY expression levels (Table 4). After 2 d with infusion of CCK into the gastrin-CCK KO mice, the antral as well as the fundic expression levels of PYY increased (Table 5). However, during continued infusion of CCK, the activation of fundic and antral PYY expression diminished, and after 6 d of CCK infusion, antral but not fundic expression had returned to the baseline gastrin-CCK KO levels (Table 5).
Other responses to fasting
Despite the absence of CCK as a satiety signal, the changes in ghrelin secretion, and the alterations in gastric IAPP and PYY expression, there were no differences in body weight among the three groups of mice at 12 wk of age. Furthermore, gastric leptin expression was unaffected by the loss of gastrin or gastrin and CCK (Table 3). Finally, the three groups of mice responded similarly to fasting as evaluated by water consumption and loss of body weight (Fig. 6).
Discussion
This study shows that even though gastrin and CCK are not necessary for the development of gastric endocrine cells as such, lack of gastrin alone or gastrin and CCK affects the expression of IAPP and PYY as well as the ghrelin response to fasting.
The effect of gastrin and CCK on ghrelin cells
The development and maintenance of ghrelin cells is independent of gastrin and CCK (this study and Refs.36, 42). Hence, neither CCK-A nor CCK-B receptor signaling are required for ghrelin cell development. Nevertheless, ghrelin mRNA expression and ghrelin secretion in response to fasting was reduced in mice lacking both gastrin and CCK. Due to lack of CCK KO mice, we were not able to determine whether loss of CCK alone or the combined loss of CCK and gastrin is responsible for the observed alterations in ghrelin expression and secretion in the gastrin-CCK KO mice. However, neither ghrelin expression nor secretion was affected in rats treated with a CCK-B receptor antagonist, which blocks both CCK and gastrin signaling through the CCK-B receptor (36). Furthermore, ghrelin cell density was not affected in the CCK-B receptor KO mice (43). We therefore surmise that the difference between the gastrin KO and gastrin-CCK KO mice arise mainly from lack of CCK signaling via the CCK-A receptor and that lack of gastrin is without significance. However, it cannot be ruled out that only the combined lack of gastrin and CCK affects ghrelin expression and secretion. Analysis of mice deficient of CCK alone is needed to answer this question.
CCK stimulates ghrelin secretion (44), and as shown in this study, CCK replacement increased fundic but not antral ghrelin gene expression. The ability of CCK to stimulate ghrelin secretion is surprising. CCK is a satiety hormone, the secretion of which is reduced during fasting but increased postprandially. CCK presumably stimulates fundic ghrelin secretion directly via the CCK-A receptors on the ghrelin cells. However, because antral and fundic responses to fasting were equally reduced despite the absence of CCK-A receptors on antral ghrelin cells, there also has to be an indirect pathway, perhaps neuronal, for CCK activation. It could be a vagal pathway because vagal activity inhibits ghrelin secretion. Both CCK and fasting have been shown to reduce vagal efferent activity (45, 46). Moreover, increased vagal activity is believed to be one of the mechanisms driving acid secretion in the double-KO mouse (31).
The differential expression of CCK-A receptors on fundic and antral ghrelin cells and accordingly the different response to CCK infusion suggest a direct mechanism for CCK on fundic ghrelin expression. In contrast, antral ghrelin expression was reduced after CCK infusion despite the absence of CCK-A receptors on antral ghrelin cells. Our findings are the first demonstration of phenotypical differences between antral and fundic ghrelin cells. Moreover, they are also in agreement with recent demonstrations of different developmental programs for fundic and antral endocrine cells (47).
The effect of gastrin and CCK and PYY and IAPP cells
The colocalization of PYY and IAPP extends earlier observations in fetal rats (6, 48). Contrary to the findings in adult rat (6), there was no colocalization of IAPP and gastrin in the mouse antrum (this study and Ref.49). Fasting reduces the expression of PYY and IAPP (50), but the present study is the first to address the cellular changes in IAPP and PYY expression related to gastrin and gastric acid secretion. Hence, our data suggest that no direct link exists between PYY and IAPP expression, although PYY and IAPP often colocalize in the same cells. Both PYY and IAPP can inhibit gastric acid secretion (51, 52, 53), but the physiological significance is poorly understood.
CCK is a regulator of intestinal PYY secretion, but the data are conflicting as to whether CCK acts directly on PYY cells (54, 55) or whether the vagal nerve mediates CCK actions (56). The presence of CCK-A receptors on the PYY cells favors the idea of a direct CCK action on PYY cells. In contrast, gastrin does not directly regulate gastric PYY expression.
Ghrelin, CCK, and IAPP either alone or together have been shown to affect the regulation of body weight (44, 57, 58). However, despite widespread abnormalities in gastric endocrinology in both groups of KO mice, they responded normally to food withdrawal in terms of water consumption and loss of body weight. Furthermore, gastric leptin expression was unchanged in gastrin-CCK KO mice, although CCK stimulates gastric leptin secretion and expression in chief cells via the CCK-A receptor (57, 59). This suggests that CCK is not important for maintaining gastric leptin expression, although we cannot rule out that the changes due to lack of CCK are ameliorated by the additional lack of gastrin.
In summary, this study shows that lack of gastrin reduces the gastric expression of PYY and IAPP. In contrast, ghrelin expression and secretion is independent of gastrin, but the response to fasting is reduced in the absence of gastrin and CCK. The differential expression of CCK-A receptors on fundic and antral ghrelin cells is the first suggestion that gastric ghrelin cells are functionally heterogeneous.
Acknowledgments
We thank Professor J. C. Reubi (University of Berne, Berne, Switzerland) for the autoradiographic results. The skillful technical assistance of Bo Lindberg and Doris Persson is greatly appreciated.
Footnotes
This work was supported by grants from the Novo Nordisk Foundation, The Swedish Medical Research Council (project 4479), and The Royal Swedish Physiographical Society.
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.
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