Histological analysis of glucagon-like peptide-1 receptor expression in chicken pancreas.

Glucagon-like peptide-1 (GLP-1) released from intestinal L cells in response to nutrient ingestion inhibits both gastrointestinal emptying and gastric acid secretion and promotes satiety. The main biological effect of GLP-1 is the stimulation of insulin secretion (thereby fulfilling the criterion for an incretin hormone) in order to reduce blood glucose levels in mammalian species. Chicken GLP-1 receptor (cGLP-1R) has also been identified in various tissues by gene expression analysis. Although certain effects of GLP-1 in mammals and birds are consistent, e.g., inhibition of food intake, whether GLP-1 has the same insulinotropic activity in chickens as in mammals is debated. Moreover, the expression of cGLP-1R in chicken pancreatic B cells has not been reported. The localization of cGLP-1R and its mRNA in pancreatic islets is studied by triple-immunofluorescence microscopy and in situ hybridization. Triple-immunofluorescence microscopy with antisera against cGLP-1R, somatostatin and insulin or glucagon revealed that cGLP-1R protein was exclusively localized in D cells producing somatostatin in chicken pancreatic islets. The D cells were localized in peripheral areas of the pancreatic islets and cGLP-1R mRNA was detected in the same areas, indicating that cGLP-1R mRNA was also expressed in D cells. This is the first report to demonstrate that cGLP-1R is expressed by D cells, not B cells as in mammals. Our study suggests that chicken GLP-1 performs its insulinotropic activity by a different mode of action from that of the mammalian hormone.

Influences of protein ingestion on glucagon-like peptide (GLP)-1-immunoreactive endocrine cells in the chicken ileum.

Influences of a specific dietary nutrient on glucagon-like peptide (GLP)-1-containing cells in the chicken intestine are not yet clear. Significance of dietary protein level on GLP-1-containing cells in the chicken ileum was investigated. Chickens fed control or experimental diets of varying protein levels were examined using immunohistochemical and morphometrical techniques. We show that the protein ingestion had an impact on the activities of GLP-1-immunoreactive cells in the chicken ileum. Weight gains declined with decreasing dietary crude protein (CP) levels, but no significant differences were detected in the daily feed intake and villous height. GLP-1-immunoreactive cells with a round or oval shape were frequently observed in the lower CP level groups (4.5% and 0%). Frequencies of occurrence of GLP-1-immunoreactive cells were 41.1 ± 4.1, 38.5 ± 4, 34.8 ± 3.1 and 34.3 ± 3.7 (cells/mm(2) , mean ± SD) for dietary CP level of 18%, 9%, 4.5% and 0% groups, respectively and significant differences were recognized between the control and lower CP level groups (P<0.05). Multiple regression analysis indicated a significant correlation between the daily protein intake and frequencies of occurrence of GLP-1-immunoreactive cells. The protein ingestion is one of the signals that influence GLP-1-containing cells in the chicken small intestine.

Distribution of glucagon-like peptide (GLP)-2-immunoreactive cells in the chicken small intestine: antigen retrieval immunohistochemistry.

An antigen retrieval method for immunohistochemical staining of glucagon-like peptide (GLP)-2-immunoreactive cells was investigated in the chicken small intestine. GLP-2-immunoreactive cells were observed as open-typed endocrine cells in the villous epithelium and crypts on both antigen retrieval agent-treated and untreated preparations. No obvious differences were detected in morphological features of GLP-2-immunoreactive cells between treated and untreated preparations. The frequencies of occurrence of GLP-2-immunoreactive cells, however, were significantly different in treated and untreated preparations: in the proximal and distal regions of jejunum and ileum obtained from untreated preparations, the frequencies of occurrence were 0.5 ± 0.2, 0.7 ± 0.1, 0.9 ± 0.2 and 1.5 ± 0.3, respectively (cell numbers per mucosal area: cells/mm(2), mean ± SD), whereas those from treated sections were 14.7 ± 2.3, 19.8 ± 2.3, 23.5 ± 4.7 and 34.6 ± 4.9 cells/mm(2), respectively. These data indicate that this antigen retrieval method is able to make immunoreactive GLP-2 available for detection and that GLP-2 may act as one of the common hormones secreted by L cells in the chicken small intestine.

Immunoelectron microscopic observation of chicken glucagon-like peptide (GLP)-1-containing cells in tissues derived from thin section, paraffin block and conventional method.

The purpose of the present study was to investigate the possibility of immunoelectron microscopic observation of endocrine cells in paraffin-embedded tissues. The procedure, which involves reprocessing from sliced tissues and immunohistochemical staining by colloidal-gold immunolabeling of paraffin sections from paraffin blocks, was able to reveal the fine characteristics of secretory granules containing glucagon-like peptide-1. Morphometric analyses of the secretory granules showed no significant differences between the reprocessing procedure and a conventional post-embedding procedure, which was performed as a control. The reprocessing procedure has some advantages besides providing information on secretory granules containing the amino acid peptide. For example, the same cell can be observed under both a light microscope and the electron microscope. In addition, the high-electron densities of silver-enhanced gold particles are easily recognized, and the boundary between the profile of the granules and the immunogold labeling is clearly shown at the electron microscopic level. Furthermore, the procedure, which is inexpensive and does not require special devices, can effectively use precious samples that are already paraffin-embedded and unable to be obtained twice, such as the case for endangered animals and rare pathological tissues. To the best of our knowledge, the present study is the first to report the advantages of the reprocessing method for sliced paraffin sections of gut endocrine cells.

Ultrastructural study on colocalization of glucagon-like peptide (GLP)-1 with GLP-2 in chicken intestinal L-cells.

Colocalization of glucagon-like peptide (GLP)-1 with GLP-2 in L-cells was investigated in the chicken ileum by using double immunofluorescent and immunocytochemical techniques. Ultrastructural features of L-cells were also clarified in this study. L-cells showing immunoreactivity for both GLP-1 and GLP-2 were distributed in the whole ileum. They showed comma-like or flask-like shape and were located in epithelium of crypts and lower part of intestinal villi. L-cells showing GLP-1-immunoreactivity only were found in epithelium of lower and middle parts of intestinal villi. Transmission electron microscopy indicated that L-cells identified by colloidal gold-labeled immunocytochemistry were covered apically with microvilli, open-type and contained many secretory granules in their perikarya. These secretory granules without halo were round to oval in shape and showed moderate electron density. The longest and shortest diameters of secretory granules were 355 ± 62 nm (mean ± SD) and 287 ± 48 nm, respectively. Double labeling immunocytochemistry using two different sizes of particles (6 and 12 nm in diameter) of colloidal gold revealed that GLP-1 colocalized with GLP-2 in the same secretory granules. This study advances new morphological data about the endocrine system of the chicken small intestine.