001M-7227/91 /1'291 -0139S03.00/0 Endocrinology Copyright ^' 1991 by The Endocrine Society
Vol. 129, No. ]
Printed in U.S.A.
Distribution and Immunocytochemical Colocalization of Peptide YY and Enteroglucagon in Endocrine Cells of the Rabbit Colon* OLA NILSSON, ANTON J. BILCHIK, JAMES R. GOLDENRING, GARTH H. BALLANTYNE, THOMAS E. ADRIAN, AND IRVIN M. MODLIN Gastrointestinal Surgical Research Unit, Yale University Medical School and Veterans Administration Medical Center, West Haven, Connecticut; and Department of Histology (O.N.), University of Gothenburg, Gothenburg, Sweden
population (5%) of D cells. By immunogold labeling serotonin was localized to EC cells, PYY and enteroglucagon to L cells, and somatostatin to the D cell. Double immunogold labeling revealed PYY and enteroglucagon in all L cells examined (93 cells). A majority of the secretory granules (83%) were labeled by both PYY and glucagon antibodies, whereas a significant portion of granules (15%) was labeled by the PYY antibodies alone. The results demonstrate that L cells are the sole source of PYY and enteroglucagon in the rabbit colon and that L cells contain different populations of secretory granules. The existence of different secretory granules in L cells may explain the selective release of PYY and enteroglucagon observed in the rabbit colon. (Endocrinology 129: 139-148, 1991)
ABSTRACT. Peptide YY (PYY) is 36 amino acid peptide hormone present in high concentrations in the colon where it is colocalized with enteroglucagon in L cells. A selective release of PYY and enteroglucagon from the rabbit colon has been described, raising the question of the exact localization of the two hormones in the rabbit colon. We have therefore examined the distribution of PYY and enteroglucagon as well as somatostatin in the rabbit colon using RIA and electron microscopic immunocytochemistry. PYY and enteroglucagon were present in high concentrations in the colorectal mucosa with peak concentrations in the left colon (PYY 544 ± 87 pmol/g, enteroglucagon 152 ± 10 pmol/g). Electron microscopic examination of the colonic mucosa demonstrated a large population (65%) of EC cells, a moderate population (30%) of L cells, and a small
P
ing cholecystokinin (23) have been shown to release PYY from colonic L cells. In a previous study (24) we have been able to demonstrate a selective release of PYY or enteroglucagon from the isolated perfused rabbit colon upon stimulation with bile salts or fatty acids, respectively. Such a differential release of PYY and enteroglucagon from colonic L cells could either be explained by the existence of different populations of L cells {i.e. L cells containing only PYY or only enteroglucagon) or by the existence of different secretory pathways for PYY and enteroglucagon in L cells. Therefore, the aim of this study was to characterize PYY and enteroglucagon containing cell populations of the rabbit colon using both RIA and immunocytochemistry. In particular we have studied the fine structure of colonic L cells and the intracellular localization of PYY and enteroglucagon. The colocalization of PYY and enteroglucagon in secretory granules was investigated by use of double immunogold labeling.
EPTIDE YY (PYY) is a novel gastrointestinal hormone first isolated from the porcine small intestine (1). PYY is a 36 amino acid straight chain peptide which is structurally related to pancreatic polypeptide and neuropeptide Y (2). PYY has been shown to inhibit several proximal gut functions including gastric acid secretion (3, 4), pancreatic secretion (1, 5-8), blood flow (6, 9), and motility (9-11). The major source of PYY is the distal small intestine and the large bowel where high tissue concentrations of PYY have been reported in several species (9, 12-15). PYY has been localized to gut endocrine cells (L cells) by immunocytochemistry (9, 16, 17). It appears to be costored with enteroglucagon (18-21). Various stimuli, including ingestion of food (8, 12), luminal instillation of fatty acids (7, 8, 22, 23), or circulatReceived November 26,1990. Address all correspondence and requests for reprints to: Dr. Irvin M. Modlin, Yale University School of Medicine, Department of Surgery, 333 Cedar Street, P.O. Box 3333, New Haven, Connecticut 065108062. * This work was supported by grants from the VA Research Section and the Yale University Department of Surgery Research Fund. O.N. was supported by Fogarty International Center, NIH Grant 1-FO5TWO4145-01, Swedish Medical Research Council Grants B89-14128617 and B89-14F-8616-01, the Swedish Society of Medicine, and the Tore Nilsson Foundation.
Materials and Methods Animals Male and female New Zealand White Rabbits (CAMM Research Laboratory Animals, Wayne, NJ) were kept on a 12-h
139 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 08:58 For personal use only. No other uses without permission. . All rights reserved.
COLOCALIZATION OF PYY AND ENTEROGLUCAGON
140
day/night schedule with free access to food and water. Rabbits were killed in the morning with an overdose of pentobarbital. Various parts of the colon were resected and used for RIA or microscopy. RIA Tissue extraction. Tissues from right and left colon and from rectum of 10 rabbits were separated into muscle and mucosal layers by blunt dissection. After weighing, tissues were immediately extracted in boiling 0.5 M acetic acid [10 ml/g tissue] for 15 min. Extracts were stored at —20 C until assay. Assay procedure. PYY, enteroglucagon, and somatostatin (SRIF) were measured using RIAs previously described (12, 25). The validity of the PYY assay for rabbit colonic tissue has been established (24). Enteroglucagon was measured using two assays. One using an N- to mid-terminal antibody which fully cross-reacts with glucagon-like immunoreactivity from gut extracts and also with purified glicentin and synthetic oxyntomodulin. A second assay uses a C-terminally directed antibody and is specific for pancreatic glucagon. Enteroglucagon values were calculated by subtracting pancreatic glucagon values from total glucagon values. The specificity of the antisera used is provided in Table 1. [125I]PYY (NEN Research Products, Boston, MA), [125I]glucagon (Amersham Corporation, Arlington Heights, IL) and [125I]SRIF (Amersham) were used as tracers. Aliquots of tissue extracts (100 n\) were assayed in duplicate in a total vol of 0.8 ml phosphate buffer. Incubation was carried out at 4 C for 5-6 days, and free tracer was separated from bound using dextran-coated charcoal. The sensitivity of the assays allowed detection of changes of 0.3-0.5 fmol between adjacent assay tubes with 95% confidence limits. The concentrations of peptide hormones at different levels in the colon were compared by two-sided sign test. Electron microscopy
infiltrated in 10% sucrose, snap frozen in liquid nitrogen and sectioned in a cryostat. Sections (10 /xm) were placed on polyL-lysine coated glass slides and incubated using indirect immunofluorescence techniques (26). After incubation with primary antibodies to glucagon, SRIF, or serotonin (Table 2) sections were incubated with biotin conjugated horse-antimouse immunoglobulin G (IgG) (Vector Laboratories Inc., Burlingame, CA) or sheep-antirat IgG (Amersham International, Amersham, UK) followed by streptavidin-FITC (Amersham) (Table 3, protocols 1,2,3). Sections were examined and photographed using an Olympus BH-2 fluorescence microscope. Immunogold labeling Tissue processing. Tissues from colon and rectum of four rabbits were fixed by immersion in either 3% glutaraldehyde in 0.1 M cacodylate buffer or in a mixture of 4% formaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.4 for 4 h. Specimens were rapidly dehydrated in ethanol and embedded in Lowicryl K4M (Polysciences Inc., Warrington, PA) at low temperature. Thin sections were placed on formvar coated nickel grids. Incubation procedures. Single and double immunogold labeling was performed on thin sections (28). In brief, sections were incubated by floating grids with attached sections on drops of antibody solutions. After preincubation with 1% BSA in PBS for 5 min, sections were incubated with diluted primary antisera for 3 h. Sections were rinsed in PBS and incubated for 0.5 h with secondary antibodies conjugated to colloidal gold particles (Janssen Life Sciences Products, Piscataway, NJ). A complete description of antibodies and incubation protocols used is provided in Tables 2 and 3. All sections were contrasted with 3% aqueous uranyl acetate. Some sections were also contrasted with lead citrate before examination and photography. TABLE
Tissues from colon and rectum were immersed in 3% glutaraldehyde in 0.1 M cacodylate buffer pH 7.2 for 3-12 h. After postfixation in 1% OsO4 for 1 h tissues were dehydrated and embedded in Epox 812 (Ernest F. Fullam Inc., Latham, NJ). Thin sections were placed on copper grids and contrasted with uranyl acetate and lead citrate. Sections were viewed and photographed using a Philips 300 EM.
Endo • 1991 Vol 129 • No 1
2. Characteristics of antisera used for immunocytochemistry
Hormone
Code no.
Species
PYY
B52a
Rabbit
Glucagon
GL77"
Rabbit
Glucagon
GLU-001c
Mouse
Somatostatin
SOM-18d
Mouse
Serotonin
YC 5/45'
Rat
Immunofluorescence Tissues from colon and rectum of four rabbits were fixed by immersion in a mixture of 4% formaldehyde and 0.5% picric acid in phosphate buffer pH 7.4 for 4 h. Specimens were TABLE
1. Characteristics of antisera used for RIA Peptide
PYY Enteroglucagon Pancreatic glucagon Somatostatin
Code no. Y21 GL77 RCS5 K2
Specificity 0
N-terminal N-terminal" C-terminalc Whole cyclic region
Ref. (12) (25) (25) (25)
° No cross-reactivity with NPY or pancreatic polypeptide. * Cross-reacts fully with enteroglucagon and pancreatic glucagon. c No cross-reactivity with enteroglucagon (
Vol. 129, No. ]
Printed in U.S.A.
Distribution and Immunocytochemical Colocalization of Peptide YY and Enteroglucagon in Endocrine Cells of the Rabbit Colon* OLA NILSSON, ANTON J. BILCHIK, JAMES R. GOLDENRING, GARTH H. BALLANTYNE, THOMAS E. ADRIAN, AND IRVIN M. MODLIN Gastrointestinal Surgical Research Unit, Yale University Medical School and Veterans Administration Medical Center, West Haven, Connecticut; and Department of Histology (O.N.), University of Gothenburg, Gothenburg, Sweden
population (5%) of D cells. By immunogold labeling serotonin was localized to EC cells, PYY and enteroglucagon to L cells, and somatostatin to the D cell. Double immunogold labeling revealed PYY and enteroglucagon in all L cells examined (93 cells). A majority of the secretory granules (83%) were labeled by both PYY and glucagon antibodies, whereas a significant portion of granules (15%) was labeled by the PYY antibodies alone. The results demonstrate that L cells are the sole source of PYY and enteroglucagon in the rabbit colon and that L cells contain different populations of secretory granules. The existence of different secretory granules in L cells may explain the selective release of PYY and enteroglucagon observed in the rabbit colon. (Endocrinology 129: 139-148, 1991)
ABSTRACT. Peptide YY (PYY) is 36 amino acid peptide hormone present in high concentrations in the colon where it is colocalized with enteroglucagon in L cells. A selective release of PYY and enteroglucagon from the rabbit colon has been described, raising the question of the exact localization of the two hormones in the rabbit colon. We have therefore examined the distribution of PYY and enteroglucagon as well as somatostatin in the rabbit colon using RIA and electron microscopic immunocytochemistry. PYY and enteroglucagon were present in high concentrations in the colorectal mucosa with peak concentrations in the left colon (PYY 544 ± 87 pmol/g, enteroglucagon 152 ± 10 pmol/g). Electron microscopic examination of the colonic mucosa demonstrated a large population (65%) of EC cells, a moderate population (30%) of L cells, and a small
P
ing cholecystokinin (23) have been shown to release PYY from colonic L cells. In a previous study (24) we have been able to demonstrate a selective release of PYY or enteroglucagon from the isolated perfused rabbit colon upon stimulation with bile salts or fatty acids, respectively. Such a differential release of PYY and enteroglucagon from colonic L cells could either be explained by the existence of different populations of L cells {i.e. L cells containing only PYY or only enteroglucagon) or by the existence of different secretory pathways for PYY and enteroglucagon in L cells. Therefore, the aim of this study was to characterize PYY and enteroglucagon containing cell populations of the rabbit colon using both RIA and immunocytochemistry. In particular we have studied the fine structure of colonic L cells and the intracellular localization of PYY and enteroglucagon. The colocalization of PYY and enteroglucagon in secretory granules was investigated by use of double immunogold labeling.
EPTIDE YY (PYY) is a novel gastrointestinal hormone first isolated from the porcine small intestine (1). PYY is a 36 amino acid straight chain peptide which is structurally related to pancreatic polypeptide and neuropeptide Y (2). PYY has been shown to inhibit several proximal gut functions including gastric acid secretion (3, 4), pancreatic secretion (1, 5-8), blood flow (6, 9), and motility (9-11). The major source of PYY is the distal small intestine and the large bowel where high tissue concentrations of PYY have been reported in several species (9, 12-15). PYY has been localized to gut endocrine cells (L cells) by immunocytochemistry (9, 16, 17). It appears to be costored with enteroglucagon (18-21). Various stimuli, including ingestion of food (8, 12), luminal instillation of fatty acids (7, 8, 22, 23), or circulatReceived November 26,1990. Address all correspondence and requests for reprints to: Dr. Irvin M. Modlin, Yale University School of Medicine, Department of Surgery, 333 Cedar Street, P.O. Box 3333, New Haven, Connecticut 065108062. * This work was supported by grants from the VA Research Section and the Yale University Department of Surgery Research Fund. O.N. was supported by Fogarty International Center, NIH Grant 1-FO5TWO4145-01, Swedish Medical Research Council Grants B89-14128617 and B89-14F-8616-01, the Swedish Society of Medicine, and the Tore Nilsson Foundation.
Materials and Methods Animals Male and female New Zealand White Rabbits (CAMM Research Laboratory Animals, Wayne, NJ) were kept on a 12-h
139 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 08:58 For personal use only. No other uses without permission. . All rights reserved.
COLOCALIZATION OF PYY AND ENTEROGLUCAGON
140
day/night schedule with free access to food and water. Rabbits were killed in the morning with an overdose of pentobarbital. Various parts of the colon were resected and used for RIA or microscopy. RIA Tissue extraction. Tissues from right and left colon and from rectum of 10 rabbits were separated into muscle and mucosal layers by blunt dissection. After weighing, tissues were immediately extracted in boiling 0.5 M acetic acid [10 ml/g tissue] for 15 min. Extracts were stored at —20 C until assay. Assay procedure. PYY, enteroglucagon, and somatostatin (SRIF) were measured using RIAs previously described (12, 25). The validity of the PYY assay for rabbit colonic tissue has been established (24). Enteroglucagon was measured using two assays. One using an N- to mid-terminal antibody which fully cross-reacts with glucagon-like immunoreactivity from gut extracts and also with purified glicentin and synthetic oxyntomodulin. A second assay uses a C-terminally directed antibody and is specific for pancreatic glucagon. Enteroglucagon values were calculated by subtracting pancreatic glucagon values from total glucagon values. The specificity of the antisera used is provided in Table 1. [125I]PYY (NEN Research Products, Boston, MA), [125I]glucagon (Amersham Corporation, Arlington Heights, IL) and [125I]SRIF (Amersham) were used as tracers. Aliquots of tissue extracts (100 n\) were assayed in duplicate in a total vol of 0.8 ml phosphate buffer. Incubation was carried out at 4 C for 5-6 days, and free tracer was separated from bound using dextran-coated charcoal. The sensitivity of the assays allowed detection of changes of 0.3-0.5 fmol between adjacent assay tubes with 95% confidence limits. The concentrations of peptide hormones at different levels in the colon were compared by two-sided sign test. Electron microscopy
infiltrated in 10% sucrose, snap frozen in liquid nitrogen and sectioned in a cryostat. Sections (10 /xm) were placed on polyL-lysine coated glass slides and incubated using indirect immunofluorescence techniques (26). After incubation with primary antibodies to glucagon, SRIF, or serotonin (Table 2) sections were incubated with biotin conjugated horse-antimouse immunoglobulin G (IgG) (Vector Laboratories Inc., Burlingame, CA) or sheep-antirat IgG (Amersham International, Amersham, UK) followed by streptavidin-FITC (Amersham) (Table 3, protocols 1,2,3). Sections were examined and photographed using an Olympus BH-2 fluorescence microscope. Immunogold labeling Tissue processing. Tissues from colon and rectum of four rabbits were fixed by immersion in either 3% glutaraldehyde in 0.1 M cacodylate buffer or in a mixture of 4% formaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.4 for 4 h. Specimens were rapidly dehydrated in ethanol and embedded in Lowicryl K4M (Polysciences Inc., Warrington, PA) at low temperature. Thin sections were placed on formvar coated nickel grids. Incubation procedures. Single and double immunogold labeling was performed on thin sections (28). In brief, sections were incubated by floating grids with attached sections on drops of antibody solutions. After preincubation with 1% BSA in PBS for 5 min, sections were incubated with diluted primary antisera for 3 h. Sections were rinsed in PBS and incubated for 0.5 h with secondary antibodies conjugated to colloidal gold particles (Janssen Life Sciences Products, Piscataway, NJ). A complete description of antibodies and incubation protocols used is provided in Tables 2 and 3. All sections were contrasted with 3% aqueous uranyl acetate. Some sections were also contrasted with lead citrate before examination and photography. TABLE
Tissues from colon and rectum were immersed in 3% glutaraldehyde in 0.1 M cacodylate buffer pH 7.2 for 3-12 h. After postfixation in 1% OsO4 for 1 h tissues were dehydrated and embedded in Epox 812 (Ernest F. Fullam Inc., Latham, NJ). Thin sections were placed on copper grids and contrasted with uranyl acetate and lead citrate. Sections were viewed and photographed using a Philips 300 EM.
Endo • 1991 Vol 129 • No 1
2. Characteristics of antisera used for immunocytochemistry
Hormone
Code no.
Species
PYY
B52a
Rabbit
Glucagon
GL77"
Rabbit
Glucagon
GLU-001c
Mouse
Somatostatin
SOM-18d
Mouse
Serotonin
YC 5/45'
Rat
Immunofluorescence Tissues from colon and rectum of four rabbits were fixed by immersion in a mixture of 4% formaldehyde and 0.5% picric acid in phosphate buffer pH 7.4 for 4 h. Specimens were TABLE
1. Characteristics of antisera used for RIA Peptide
PYY Enteroglucagon Pancreatic glucagon Somatostatin
Code no. Y21 GL77 RCS5 K2
Specificity 0
N-terminal N-terminal" C-terminalc Whole cyclic region
Ref. (12) (25) (25) (25)
° No cross-reactivity with NPY or pancreatic polypeptide. * Cross-reacts fully with enteroglucagon and pancreatic glucagon. c No cross-reactivity with enteroglucagon (
