J Clin Endocrinol Metab 44: 795, 1977
ULTRASTRUCTURAL LOCALIZATION OF PANCREATIC POLYPEPTIDE IN HUMAN PANCREAS B. H. Bergstrom, S. Loo, H. J. Hirsch, D. Schutzengel, and K. H. Gabbay Department of Medicine, Children's Hospital Medical Center, and the Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115 ABSTRACT: Human pancreatic polypeptide (HPP) was localized in surgically obtained human pancreas by immunocytochemical electron microscopy. HPP was localized to the secretory granules of cells distinct from the alpha, beta, or delta cells of endocrine islets. The PP cells in 10 patients surveyed were most frequently located in the periphery of the islets. Immunolocalization distinguishes a fifth endocrine cell type in human islets which is distinct ultrastructurally by its smaller granule diameter and lack of PP immunoreactivity. Pancreatic polypeptide (PP) is a straight chain 36 amino acid peptide, originally isolated from chicken pancreas (1,2) along with insulin and glucagon. It has recently been demonstrated in the islets of many animal species (3) by immunocytochemical methods. In man, PP has been assayed in normal pancreas, insulinomas, vipomas, glucagonomas, and gastrinomas, with higher levels noted in the tumorous tissue (4). Because PP levels are assayable in plasma, it has recently been postulated as a new islet hormone. We report here the ultrastructural localization of PP in the granules of a distinct islet cell by immunocytochemical methods, and confirm the presence of yet another islet cell type distinct from the alpha, beta, delta, and PP cells. The fifth cell type is distinguishable on the basis of its ultrastructural characteristics and lack of PP immunoreactivity. MATERIALS AND METHODS Pancreatic tissue obtained at surgery for therapeutic purposes (insulinomas and nesidioblastosis) were immediately fixed in formaldehydeglutaraldehyde as previously described (5). After embedding in plastic, ultra-thin sections were mounted on uncoated nickel grids and etched in 10% H2O2 and processed by a
Submitted December 21, 1976
modification of the peroxidase-antiperoxidase antibody technique of Sternberger (6). Sequential incubations of the sections for 3 minutes each were performed in the following order: 1) normal goat serum, 1:10; 2) rabbit anti-bovine PP (BPP) serum, 1:20 (gift of Dr. R. Chance, Eli Lilly Co.); 3) goat anti-rabbit gamma globulin, 1:10; 4) peroxidase-antiperoxidase complex (PAP) 1:50 (Cappel Laboratories). The sections were then immersed in a solution of 0.075% diaminobenizidine and 0.001% hydrogen peroxide (Sigma Chemicals) for 10 minutes in the dark, rinsed and then exposed to 1% OSO4. Tris-buffered saline (0.05 M Tris, 0.9% NaCl) pH 7.6 was used as the wash reagent between incubations, and as diluent for all reagents. Control subserial sections were treated with either normal rabbit serum, 1:10, or antiBPP serum absorbed with excess BPP. BPP anti-serum showed no crossreactivity with insulin, glucagon, somatostatin, or growth hormone. RESULTS Sections of human pancreas treated with BPP anti-serum and the PAP labelling technique exhibited a positive staining cell distinct from the alpha, beta and delta cells (Fig. 1 ) . The PP granules were densely packed within the cytoplasm and had closely applied limiting membranes. The granules were round with a uniform
795
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796
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JCE & M • 1977 Vol 44 • No 4
Fig. 1. Portion of a human islet stained for pancreatic polypeptide. PAPpositive granules identify the PP cell. Note lack of staining in alpha (A), beta (B), and delta (D) cells (X 10,850).
Fig. 2A. PAP-positive granules in PP cell. 2B. Note absence of PAP-reaction product in adjacent control section using normal rabbit serum (X 23,500).
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RAPID COMMUNICATIONS density, although the density of individual granules varied. Average granule diameter was 134 t 31 ran (mean ± 1 S.D., n = 224 granules, range 80-210 nm). The granules displayed specific positive PAP-staining, and adjacent subserial sections used as controls showed no labelling (Fig. 2 ) . The PP cells were most frequently found in the islet periphery and occasionally within the exocrine parenchyma. PP cells were more easily detected in the pancreas of very young patients (1 month5 years of age) than in the older patients studied. PP cells were identified in every pancreas studied. In addition to the PP cells and the ultrastructurally well defined alpha, beta and delta cells (Fig. 1 ) , we observed a fifth endocrine cell type. This cell contained granules similar in electron density to the PP granules but smaller in diameter (76 ± 29 nm, mean ± 1 S.D., n = 249 granules, range 40-130 nm). These granules are also densely packed within the cytoplasm but did not stain with BPP anti-serum (Fig. 3 ) .
Fig. 3. Portion of a PP-positive cell and a fifth cell type (arrow) containing smaller granules not stained by the anti-BPP serum (X 9,900). DISCUSSION The presence of endocrine cell types in the human pancreas that are distinct from the alpha, and beta
797
cells had been previously reported in the ultrastructural studies of several investigators (7,8). Recently the delta cell containing somatostatin was identified by specific immunocytochemical techniques (9). In this report, we identify, by a specific immunocytochemical method, a fourth cell type containing pancreatic polypeptide, a recently isolated peptide with putative hormonal function. In addition, we describe the presence of a fifth cell type in human islets which is distinguishable by granules smaller than PP granules and lacking in PP immunoreactivity. Our electron microscopic localization of PP cells in human pancreas confirms the recent report of Larsson et al (10) who identified a population of PP cells at the light microscopic level in the periphery of the islets of Langerhans in humans. Larsson et al (3) further identified PP cells at the ultrastructural level in cat and chinchilla pancreas by sequential examination of adjacent sections by immunofluorescent staining and electron microscopy. In these 2 species, the PP cell granule characteristics differed significantly from the proposed human PP cell granules in size and electron density (3). The PP cell directly identified in this study is identical in its ultrastructural characteristics to the PP cell proposed by Larsson et al (mean granule diameter 134 nm vs. 125 nm respectively). The fifth cell type described in this report does not appear to be the same as the D± cell of Larsson et al (3), as the granules are smaller (mean granule diameter 76 nm vs. 155 nm respectively). Whether this cell type is identical to the enterochromaffin cell (EC) described by others (11) remains speculative. The identification of new endocrine cell types in human pancreas is indicative of the hitherto inapparent complexity of structure and function of the pancreatic islets. ACKNOWLEDGEMENTS:
Supported by USPHS
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798
RAPID COMMUNICATIONS
grant AM-15019 and a Grant-in-Aid and an Established Investigatorship from the .American Heart Association (KHG) . REFERENCES 1.
2.
3.
4.
5.
Kimmel, J.R., H.G. Pollock, and R.L. Hazelwood: Isolation and characterization of chicken insulin. Endocrinology 83:1323, 1968. Langslow, D.R., J.R. Kimmel, and H.G. Pollock: Studies of the distribution of a new avian pancreatic polypeptide and insulin among birds, reptiles, amphibians and mammals. Endocrinology 93:558, 1973. Larsson, L.-I., F. Sundler, and R. Hakanson: Pancreatic polypeptide - a postulated new hormone: identification of its cellular storage site by light and electron microscopic immunocytochemistry. Diabetologia 12:211, 1976. Polak, J.M., T.E. Adrian, M.G. Bryant, S.R. Bloom, P. Heitz, and A.G.E. Pearse: Pancreatic polypeptide in insulinomas, gastrinomas, vipomas, and glucagonomas. Lancet _1:328, 1976. Gabbay, K.H., J. Korff, and
JCE & M • 1977 Vol 44 • No 4
E.E. Schneeberger: Vesicular binesis: glucose effect on insulin secretory vesicles. Science 187: 177, 1975. Sternberger, L.A., P.H. Hardy, Jr., J.J. Cuculis, and H.G. Meyer: The unlabeled antibody enzyme method of immunohistochemistry. J Histochem Cytochem 1£:315, 1970. Jirasek, K., and L. Kubes: A new endocrine cell type of the human pancreatic islets. Experientia 2£:966, 1972. Misugi, K., N. Misugi, J. Sotos, and B. Smith: The pancreatic islet of infants with severe hypoglycemia. Arch Pathol 89_:208, 1970. Goldsmith, P.G., J.G. Rose, A. Arimura, and W.F. Ganong: Ultrastructural localization of somatostatin in pancreatic islets of the rat. Endocrinology 97_:1061, 1975. 10. Larsson, L.-I., F. Sundler, and R. Hakanson: Immunohistochemical localization of human pancreatic polypeptide (HPP) to a population of islet cells. Cell Tiss Res 156: 167, 1975. 11, Deconinck, J.F., P.R. Potvliege, and W. Gepts: The ultrastructure of the human pancreatic islets. I. The islets of adults. Diabetologia 7:266, 1971.
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ULTRASTRUCTURAL LOCALIZATION OF PANCREATIC POLYPEPTIDE IN HUMAN PANCREAS B. H. Bergstrom, S. Loo, H. J. Hirsch, D. Schutzengel, and K. H. Gabbay Department of Medicine, Children's Hospital Medical Center, and the Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115 ABSTRACT: Human pancreatic polypeptide (HPP) was localized in surgically obtained human pancreas by immunocytochemical electron microscopy. HPP was localized to the secretory granules of cells distinct from the alpha, beta, or delta cells of endocrine islets. The PP cells in 10 patients surveyed were most frequently located in the periphery of the islets. Immunolocalization distinguishes a fifth endocrine cell type in human islets which is distinct ultrastructurally by its smaller granule diameter and lack of PP immunoreactivity. Pancreatic polypeptide (PP) is a straight chain 36 amino acid peptide, originally isolated from chicken pancreas (1,2) along with insulin and glucagon. It has recently been demonstrated in the islets of many animal species (3) by immunocytochemical methods. In man, PP has been assayed in normal pancreas, insulinomas, vipomas, glucagonomas, and gastrinomas, with higher levels noted in the tumorous tissue (4). Because PP levels are assayable in plasma, it has recently been postulated as a new islet hormone. We report here the ultrastructural localization of PP in the granules of a distinct islet cell by immunocytochemical methods, and confirm the presence of yet another islet cell type distinct from the alpha, beta, delta, and PP cells. The fifth cell type is distinguishable on the basis of its ultrastructural characteristics and lack of PP immunoreactivity. MATERIALS AND METHODS Pancreatic tissue obtained at surgery for therapeutic purposes (insulinomas and nesidioblastosis) were immediately fixed in formaldehydeglutaraldehyde as previously described (5). After embedding in plastic, ultra-thin sections were mounted on uncoated nickel grids and etched in 10% H2O2 and processed by a
Submitted December 21, 1976
modification of the peroxidase-antiperoxidase antibody technique of Sternberger (6). Sequential incubations of the sections for 3 minutes each were performed in the following order: 1) normal goat serum, 1:10; 2) rabbit anti-bovine PP (BPP) serum, 1:20 (gift of Dr. R. Chance, Eli Lilly Co.); 3) goat anti-rabbit gamma globulin, 1:10; 4) peroxidase-antiperoxidase complex (PAP) 1:50 (Cappel Laboratories). The sections were then immersed in a solution of 0.075% diaminobenizidine and 0.001% hydrogen peroxide (Sigma Chemicals) for 10 minutes in the dark, rinsed and then exposed to 1% OSO4. Tris-buffered saline (0.05 M Tris, 0.9% NaCl) pH 7.6 was used as the wash reagent between incubations, and as diluent for all reagents. Control subserial sections were treated with either normal rabbit serum, 1:10, or antiBPP serum absorbed with excess BPP. BPP anti-serum showed no crossreactivity with insulin, glucagon, somatostatin, or growth hormone. RESULTS Sections of human pancreas treated with BPP anti-serum and the PAP labelling technique exhibited a positive staining cell distinct from the alpha, beta and delta cells (Fig. 1 ) . The PP granules were densely packed within the cytoplasm and had closely applied limiting membranes. The granules were round with a uniform
795
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 13:12 For personal use only. No other uses without permission. . All rights reserved.
796
RAPID COMMUNICATIONS
JCE & M • 1977 Vol 44 • No 4
Fig. 1. Portion of a human islet stained for pancreatic polypeptide. PAPpositive granules identify the PP cell. Note lack of staining in alpha (A), beta (B), and delta (D) cells (X 10,850).
Fig. 2A. PAP-positive granules in PP cell. 2B. Note absence of PAP-reaction product in adjacent control section using normal rabbit serum (X 23,500).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 13:12 For personal use only. No other uses without permission. . All rights reserved.
RAPID COMMUNICATIONS density, although the density of individual granules varied. Average granule diameter was 134 t 31 ran (mean ± 1 S.D., n = 224 granules, range 80-210 nm). The granules displayed specific positive PAP-staining, and adjacent subserial sections used as controls showed no labelling (Fig. 2 ) . The PP cells were most frequently found in the islet periphery and occasionally within the exocrine parenchyma. PP cells were more easily detected in the pancreas of very young patients (1 month5 years of age) than in the older patients studied. PP cells were identified in every pancreas studied. In addition to the PP cells and the ultrastructurally well defined alpha, beta and delta cells (Fig. 1 ) , we observed a fifth endocrine cell type. This cell contained granules similar in electron density to the PP granules but smaller in diameter (76 ± 29 nm, mean ± 1 S.D., n = 249 granules, range 40-130 nm). These granules are also densely packed within the cytoplasm but did not stain with BPP anti-serum (Fig. 3 ) .
Fig. 3. Portion of a PP-positive cell and a fifth cell type (arrow) containing smaller granules not stained by the anti-BPP serum (X 9,900). DISCUSSION The presence of endocrine cell types in the human pancreas that are distinct from the alpha, and beta
797
cells had been previously reported in the ultrastructural studies of several investigators (7,8). Recently the delta cell containing somatostatin was identified by specific immunocytochemical techniques (9). In this report, we identify, by a specific immunocytochemical method, a fourth cell type containing pancreatic polypeptide, a recently isolated peptide with putative hormonal function. In addition, we describe the presence of a fifth cell type in human islets which is distinguishable by granules smaller than PP granules and lacking in PP immunoreactivity. Our electron microscopic localization of PP cells in human pancreas confirms the recent report of Larsson et al (10) who identified a population of PP cells at the light microscopic level in the periphery of the islets of Langerhans in humans. Larsson et al (3) further identified PP cells at the ultrastructural level in cat and chinchilla pancreas by sequential examination of adjacent sections by immunofluorescent staining and electron microscopy. In these 2 species, the PP cell granule characteristics differed significantly from the proposed human PP cell granules in size and electron density (3). The PP cell directly identified in this study is identical in its ultrastructural characteristics to the PP cell proposed by Larsson et al (mean granule diameter 134 nm vs. 125 nm respectively). The fifth cell type described in this report does not appear to be the same as the D± cell of Larsson et al (3), as the granules are smaller (mean granule diameter 76 nm vs. 155 nm respectively). Whether this cell type is identical to the enterochromaffin cell (EC) described by others (11) remains speculative. The identification of new endocrine cell types in human pancreas is indicative of the hitherto inapparent complexity of structure and function of the pancreatic islets. ACKNOWLEDGEMENTS:
Supported by USPHS
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 13:12 For personal use only. No other uses without permission. . All rights reserved.
798
RAPID COMMUNICATIONS
grant AM-15019 and a Grant-in-Aid and an Established Investigatorship from the .American Heart Association (KHG) . REFERENCES 1.
2.
3.
4.
5.
Kimmel, J.R., H.G. Pollock, and R.L. Hazelwood: Isolation and characterization of chicken insulin. Endocrinology 83:1323, 1968. Langslow, D.R., J.R. Kimmel, and H.G. Pollock: Studies of the distribution of a new avian pancreatic polypeptide and insulin among birds, reptiles, amphibians and mammals. Endocrinology 93:558, 1973. Larsson, L.-I., F. Sundler, and R. Hakanson: Pancreatic polypeptide - a postulated new hormone: identification of its cellular storage site by light and electron microscopic immunocytochemistry. Diabetologia 12:211, 1976. Polak, J.M., T.E. Adrian, M.G. Bryant, S.R. Bloom, P. Heitz, and A.G.E. Pearse: Pancreatic polypeptide in insulinomas, gastrinomas, vipomas, and glucagonomas. Lancet _1:328, 1976. Gabbay, K.H., J. Korff, and
JCE & M • 1977 Vol 44 • No 4
E.E. Schneeberger: Vesicular binesis: glucose effect on insulin secretory vesicles. Science 187: 177, 1975. Sternberger, L.A., P.H. Hardy, Jr., J.J. Cuculis, and H.G. Meyer: The unlabeled antibody enzyme method of immunohistochemistry. J Histochem Cytochem 1£:315, 1970. Jirasek, K., and L. Kubes: A new endocrine cell type of the human pancreatic islets. Experientia 2£:966, 1972. Misugi, K., N. Misugi, J. Sotos, and B. Smith: The pancreatic islet of infants with severe hypoglycemia. Arch Pathol 89_:208, 1970. Goldsmith, P.G., J.G. Rose, A. Arimura, and W.F. Ganong: Ultrastructural localization of somatostatin in pancreatic islets of the rat. Endocrinology 97_:1061, 1975. 10. Larsson, L.-I., F. Sundler, and R. Hakanson: Immunohistochemical localization of human pancreatic polypeptide (HPP) to a population of islet cells. Cell Tiss Res 156: 167, 1975. 11, Deconinck, J.F., P.R. Potvliege, and W. Gepts: The ultrastructure of the human pancreatic islets. I. The islets of adults. Diabetologia 7:266, 1971.
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