RAPID COMMUNICATION
ZIPGRAM
IMMUNOCYTOCHEMICAL LOCALIZATION OF A GASTRIC INHIBITORY POLYPEPTIDE-LIKE MATERIAL WITHIN A-CELLS OF THE ENDOCRINE PANCREAS (1) PHILLIP H. SMITH, FREDERICK W. MERCHANT, DAVID G. JOHNSON, WILFRED Y. FUJIMOTO AND ROBERT H. WILLIAMS Division of Metabolism, Veterans Administration Hospital, Seattle, Washington 98108; and the Departments of Biological Structure and Medicine, University of Washington School of Medicine, Seattle, Washington 98195 ABSTRACT In rat pancreatic islets, the glucagon-producing A-cells also contain a GIP-like material as revealed by the peroxidaseantiperoxidase immunocytochemical technique. Control studies showed that this dual staining was not due to the cross-reactivity of antiGIP with glucagon. It is concluded either that the A-cells synthesize a GIP-like peptide or that it is taken up from the circulation. Gastric inhibitory polypeptide (GIP), a hormone isolated from the intestinal mucosa (Brown et a1 ., ' 7 5 ) , has been localized previously within specific cells of the gastrointestinal tract (Polak et al. , '73; Solcia et al. , '75). Physiologic studies have shown that GIP potentiates the secretion of insulin and glucagon both in vivo (Dupre et al., '73, '76) and in vitro (Pederson and Brown, '76; Johnson et al. , '77). Because a number of gastrointestinal hormones are known to affect pancreatic islet function, an immunocytochemical study was undertaken to evaluate the interaction of GI hormones with receptors on the plasma membrane of islet cells. During the initial phase of this investigation it was found that pancreatic islet A-cells contained an endogenous GIP-like immunoreactive substance. MATERIAL AND METHODS Pancreatic tissue was obtained from neonatal (1-3 days) and adult Wistar rats.
Specimens were fixed using Bouin's
solution, embedded in paraffin and then sectioned serially at 4 urn. 585
These tissues were stained using the sensitive p e r o x i d a s e - a n t i p e r o x i d a s e immunocytochemical technique (Sternberger, '74)after the application of the following anti-hormone sera: guinea-pig anti-insulin, rabbit antiglucagon (30 K ) , rabbit anti-somatostatin, or rabbit anti-GIP (obtained from Doctor John C. Brown, Vancouver, B . C . ) .
All primary antisera were used at
a dilution of 1:lOO for 24 hours at ' 4 C. Sheep anti-rabbit serum (1:lO) was then applied (10 min at 22' C) followed by rabbit p e r o x i d a s e - a n t i p e r o x i d a s e complex (1:50) for 10 min. Between each step the slides were carefully rinsed using Tris-buffered saline (0.9% NcCl containing 0.05 M Tris-HCL buffer and
1% normal sheep serum, pH 7.6). Sites of peroxidase activity were visualized using a modification of the medium of Graham and Karnovsky ('66). Several control procedures were carried out to verify the specificity of the staining reaction. These included substitution of normal serum in place of immune serum; incubation o f the tissue in peroxidase substrate without the appropriate antiserum; or the application of antiserum previously absorbed with an excess of the specific antigen. Anti-GIP (initial dilution 1:20) was absorbed with either 5 pg/ml of highly purified GIP (fromDoctor J.C. Brown) or 100 pg/ml of purified glucagon (Lilly). RESULTS After incubating tissue sections with anti-insulin serum the cells in the central region of the islets of both neonatal and adult rats were stained positively (fig. 5). These cells comprised a majority of the endocrine pancreas. The administration of anti-glucagon (figs. 1 , 8) or anti-somatostatin (fig. 3 ) to adjacent serial sections demonstrated the presence of two distinct populations of endocrine cells at the islet periphery.
In all instances staining was abolished by the use of normal serum or antigenanti body absorbed control sera. When tissue sections were incubated with anti-GIP (fig. 2) many o f 586
the cells at the islet periphery were stained. Using adjacent sections stained with anti-glucagon (fig. 1) a positive reaction was observed over cells having loci within the islet identical t o those reacting with anti-GIP. No correspondence was found between the GIP-positive islet cells and those
staining with anti-somatostatin (compare figs. 3 and 4). To eliminate the possibility of non-specific staining or cross-reactivity of anti-GIP with glucagon, other sections were stained with the control sera. Addition of GIP to anti-GIP (fig. 6) or the use of normal serum completely abolished the staining of the islet cells. In contrast, positive staining was observed following the use of anti-GIP serum absorbed with glucagon (fig. 7). DISCUSSION It is well established that the A-, B-, and D-cells of the pancreatic islets contain glucagon, insulin and somatostatin, respectively.
In this regard our results agree with previous reports using an identical immunocytochemical technique (Erlandsen et a1 ., '75, '76). The present findings, however, indicate that the pancreatic islet A-cells of neonatal and adult rats contain a GIP-like immunoreactive substance in addition to glucagon. No staining was observed following the absorption of anti-GIP with purified GIP, but staining persisted when glucagon was added to this antiserum. Characterization of the anti-GIP used in this study by radioimmunoassay shows that this antiserum does not cross-react with a variety of hormones including insulin, glucagon, and somatostatin (Doctor J . C. Brown, personal communication).
Therefore, it does not seem likely
that our immunocytochemical results were due to either non-specific staining or cross-reactivity of the anti-GIP with endogenous glucagon of the A-cells.
A recent study by Erlandsen et al. ('76) has shown that pancreatic islet D-cells of humans contain both gastrin and somatostatin. That finding raised the interesting possibility that the D-cell is capable of producing 587
two different hormones. By analogy, our observation that the glucagon-containing A-cells additionally possess an endogenous GIP-like substance suggests that these cells may also synthesize more than one hormone. Alternatively, the present results could be interpreted as evidence for a mechanism whereby GIP is taken up from the circulation. Further studies are in progress to differentiate between these two possibilities in light of the physiologic role that GIP plays in the regulation of insulin and glucagon secretion. ACKNOWLEDGMENTS The skilled technical assistance of Ms. Jean Leik and Ms. Criss Meligro i s gratefully acknowledged. We thank Doctor Ludwig A. Sternberger for the generous supply of rabbit p e r o x i d a s e - a n t i p e r o x i d a s e . LITERATURE CITED Brown, J. C., J. R. Dryburgh, S. A. Ross and J. Dupre 1975 Identification and actions of gastric inhibitory polypeptide. In: Recent Progress in Hormone Research, R. 0. Greep, ed. Academic Press, New York, 31: 487-532. FIGURE LEGENDS Islet of adult rat stained immunocytochemically for glucagon. Glucagon-positive cells (black) are located at islet periphery. X 490. Adjacent section stained immunocytochemically for GIP. Note correspondence o f GIP-positive cells with glucagon-positive cells (compare cells at arrows in figs. 1 , 2). X 490. Rat islet stained using anti-somatostatin. Immunoreactive cells are located at islet periphery (see arrows). X 490. Adjacent section stained immunocytochemically for GIP. Note lack of correspondence with somatostatin-positive cells (compare cells at arrows in figs. 3, 4). X 490. Islet of adult rat stained using anti-insulin. Cells of islet core are stained positively. X 390. Specificity control. Section stained with anti-GIP previously absorbed with highly purified GIP. Islet shows only background staining. X 390. Cross reactivity control. Section stained with anti-GIP previously absorbed with purified glucagon. Note staining of cells at islet periphery. X 390. Adjacent section stained imrnunocytochemically for glucagon. Note correspondence of GIP-and glucagon-positive cells (see arrows figs. 7, 8). X 390.
588
Dupre, J . , S. A. ROSS, D. Watson and J . C . Brown 1973 Stimulation of i n s u l i n se c re ti on by g a s t r i c i nhibitory polypeptide in man. J . Clin. Endocrinol. Metab., 37: 826-828. Dupre, J . , T . J . McDonald, J . Radzink, Y . Caussignac and S. Van Vliet 1976 Glucagonotropic action o f g a s t r i c inhibitory polypeptide in man. Clin. Res., 24: 680A. Erlandsen, S . L., J . A . Parsons, J . P . Burke, J . A. Redick, D. E . Van Orden and L . S . Van Orden 1975 A modification of the unlabeled antibody enzyme method using heterologous a n t i s e r a f o r t h e l i g h t microscopic and u l t r a s t r u c t u r a l l o c a l i z a t i o n of i n s u l i n , glucagon and g r o w t h hormone. J . Histochem. Cytochem., 23: 666-677. Erlandsen, S . L., 0. D. Hegre, J . A . Parsons, R . C . McEvoy and R. P . Elde 1976 Pancreatic i s l e t hormones: Distribution of c e l l types i n the i s l e t a n d evidence f o r t he presence of somatostatin and g a s t r i n within the D-cell. J . Histochem. Cytochem., 24: 883-897. Graham, B . C . , and M . J . Karnovsky 1966 The e a r l y stages of absorption of i n j e ct e d horseradish peroxidase in the proximal tubules of mouse kidney: u l t r a s t r u c t u r a l cytochemistry by a new technique. J . Histochem. Cytochem., 14: 291-302. Johnson, D. G . , F. W . Merchant, P . H . Smith, W . Y . Fujimoto and R. H . Williams 1977 Binding and actions of g a s t r i c inhibitory polypeptide in the endocrine pancreas of t h e r a t . Fed. Proc., 36: 299 ( a b s t r a c t ) . Pederson, R . A . , and J . C . Brown 1976 The insulinotr opic action of g a s t r i c inhibi tory polypeptide in the perfused i s o l a t e d r a t pancreas. Endocrinology, 99: 780-785. Polak, J . M . , S. R. Bloom, M . Kuzio, J . C. Brown and A . G. E . Pearse 1973 C e ll ula r loc a l iz a t ion of g a s t r i c i n h i b i t o r y polypeptide in the duodenum and jejunum. G u t , 14: 284-288. S o l ci a , E . , J . M. Polak, R . Buffa, C . Capella and A . G . E . Pearse 1975 Endocrine c e l l s of t he i n t e s t i n a l mucosa. I n : Gastroi n t e s t i n a l Hormones, J . C . Thompson, ed. University of Texas Press, Austin, p p . 155-168. Sternberger, L . A . 1974 Immunocytochemistry. Prentice-Hall, Englewood C l i f f s , p. 246. REFERENCES
1
Supported in p a r t by Veterans Administration General Medical Research Funds (MRIS 8007), American Diabetes Association, Washington A f f i l i a t e , I n c . , the KROC Foundation, t h e Diabetes-Endocrinology Research Center (AM17047) and USPHS Grants (AM1 531 2 , AM02456). Doctors Johnson and Fujimoto a r e r e c i p i e n t s of Research Career Development Awards AM70727 and AM47142, r espectively. Reprint requests should be se nt t o: Dr. P . H . Smith, Division of Metabolism (151), VA Hospital, 4435 Beacon Avenue South, S e a t t l e , Washington 981 08.
590
ZIPGRAM
IMMUNOCYTOCHEMICAL LOCALIZATION OF A GASTRIC INHIBITORY POLYPEPTIDE-LIKE MATERIAL WITHIN A-CELLS OF THE ENDOCRINE PANCREAS (1) PHILLIP H. SMITH, FREDERICK W. MERCHANT, DAVID G. JOHNSON, WILFRED Y. FUJIMOTO AND ROBERT H. WILLIAMS Division of Metabolism, Veterans Administration Hospital, Seattle, Washington 98108; and the Departments of Biological Structure and Medicine, University of Washington School of Medicine, Seattle, Washington 98195 ABSTRACT In rat pancreatic islets, the glucagon-producing A-cells also contain a GIP-like material as revealed by the peroxidaseantiperoxidase immunocytochemical technique. Control studies showed that this dual staining was not due to the cross-reactivity of antiGIP with glucagon. It is concluded either that the A-cells synthesize a GIP-like peptide or that it is taken up from the circulation. Gastric inhibitory polypeptide (GIP), a hormone isolated from the intestinal mucosa (Brown et a1 ., ' 7 5 ) , has been localized previously within specific cells of the gastrointestinal tract (Polak et al. , '73; Solcia et al. , '75). Physiologic studies have shown that GIP potentiates the secretion of insulin and glucagon both in vivo (Dupre et al., '73, '76) and in vitro (Pederson and Brown, '76; Johnson et al. , '77). Because a number of gastrointestinal hormones are known to affect pancreatic islet function, an immunocytochemical study was undertaken to evaluate the interaction of GI hormones with receptors on the plasma membrane of islet cells. During the initial phase of this investigation it was found that pancreatic islet A-cells contained an endogenous GIP-like immunoreactive substance. MATERIAL AND METHODS Pancreatic tissue was obtained from neonatal (1-3 days) and adult Wistar rats.
Specimens were fixed using Bouin's
solution, embedded in paraffin and then sectioned serially at 4 urn. 585
These tissues were stained using the sensitive p e r o x i d a s e - a n t i p e r o x i d a s e immunocytochemical technique (Sternberger, '74)after the application of the following anti-hormone sera: guinea-pig anti-insulin, rabbit antiglucagon (30 K ) , rabbit anti-somatostatin, or rabbit anti-GIP (obtained from Doctor John C. Brown, Vancouver, B . C . ) .
All primary antisera were used at
a dilution of 1:lOO for 24 hours at ' 4 C. Sheep anti-rabbit serum (1:lO) was then applied (10 min at 22' C) followed by rabbit p e r o x i d a s e - a n t i p e r o x i d a s e complex (1:50) for 10 min. Between each step the slides were carefully rinsed using Tris-buffered saline (0.9% NcCl containing 0.05 M Tris-HCL buffer and
1% normal sheep serum, pH 7.6). Sites of peroxidase activity were visualized using a modification of the medium of Graham and Karnovsky ('66). Several control procedures were carried out to verify the specificity of the staining reaction. These included substitution of normal serum in place of immune serum; incubation o f the tissue in peroxidase substrate without the appropriate antiserum; or the application of antiserum previously absorbed with an excess of the specific antigen. Anti-GIP (initial dilution 1:20) was absorbed with either 5 pg/ml of highly purified GIP (fromDoctor J.C. Brown) or 100 pg/ml of purified glucagon (Lilly). RESULTS After incubating tissue sections with anti-insulin serum the cells in the central region of the islets of both neonatal and adult rats were stained positively (fig. 5). These cells comprised a majority of the endocrine pancreas. The administration of anti-glucagon (figs. 1 , 8) or anti-somatostatin (fig. 3 ) to adjacent serial sections demonstrated the presence of two distinct populations of endocrine cells at the islet periphery.
In all instances staining was abolished by the use of normal serum or antigenanti body absorbed control sera. When tissue sections were incubated with anti-GIP (fig. 2) many o f 586
the cells at the islet periphery were stained. Using adjacent sections stained with anti-glucagon (fig. 1) a positive reaction was observed over cells having loci within the islet identical t o those reacting with anti-GIP. No correspondence was found between the GIP-positive islet cells and those
staining with anti-somatostatin (compare figs. 3 and 4). To eliminate the possibility of non-specific staining or cross-reactivity of anti-GIP with glucagon, other sections were stained with the control sera. Addition of GIP to anti-GIP (fig. 6) or the use of normal serum completely abolished the staining of the islet cells. In contrast, positive staining was observed following the use of anti-GIP serum absorbed with glucagon (fig. 7). DISCUSSION It is well established that the A-, B-, and D-cells of the pancreatic islets contain glucagon, insulin and somatostatin, respectively.
In this regard our results agree with previous reports using an identical immunocytochemical technique (Erlandsen et a1 ., '75, '76). The present findings, however, indicate that the pancreatic islet A-cells of neonatal and adult rats contain a GIP-like immunoreactive substance in addition to glucagon. No staining was observed following the absorption of anti-GIP with purified GIP, but staining persisted when glucagon was added to this antiserum. Characterization of the anti-GIP used in this study by radioimmunoassay shows that this antiserum does not cross-react with a variety of hormones including insulin, glucagon, and somatostatin (Doctor J . C. Brown, personal communication).
Therefore, it does not seem likely
that our immunocytochemical results were due to either non-specific staining or cross-reactivity of the anti-GIP with endogenous glucagon of the A-cells.
A recent study by Erlandsen et al. ('76) has shown that pancreatic islet D-cells of humans contain both gastrin and somatostatin. That finding raised the interesting possibility that the D-cell is capable of producing 587
two different hormones. By analogy, our observation that the glucagon-containing A-cells additionally possess an endogenous GIP-like substance suggests that these cells may also synthesize more than one hormone. Alternatively, the present results could be interpreted as evidence for a mechanism whereby GIP is taken up from the circulation. Further studies are in progress to differentiate between these two possibilities in light of the physiologic role that GIP plays in the regulation of insulin and glucagon secretion. ACKNOWLEDGMENTS The skilled technical assistance of Ms. Jean Leik and Ms. Criss Meligro i s gratefully acknowledged. We thank Doctor Ludwig A. Sternberger for the generous supply of rabbit p e r o x i d a s e - a n t i p e r o x i d a s e . LITERATURE CITED Brown, J. C., J. R. Dryburgh, S. A. Ross and J. Dupre 1975 Identification and actions of gastric inhibitory polypeptide. In: Recent Progress in Hormone Research, R. 0. Greep, ed. Academic Press, New York, 31: 487-532. FIGURE LEGENDS Islet of adult rat stained immunocytochemically for glucagon. Glucagon-positive cells (black) are located at islet periphery. X 490. Adjacent section stained immunocytochemically for GIP. Note correspondence o f GIP-positive cells with glucagon-positive cells (compare cells at arrows in figs. 1 , 2). X 490. Rat islet stained using anti-somatostatin. Immunoreactive cells are located at islet periphery (see arrows). X 490. Adjacent section stained immunocytochemically for GIP. Note lack of correspondence with somatostatin-positive cells (compare cells at arrows in figs. 3, 4). X 490. Islet of adult rat stained using anti-insulin. Cells of islet core are stained positively. X 390. Specificity control. Section stained with anti-GIP previously absorbed with highly purified GIP. Islet shows only background staining. X 390. Cross reactivity control. Section stained with anti-GIP previously absorbed with purified glucagon. Note staining of cells at islet periphery. X 390. Adjacent section stained imrnunocytochemically for glucagon. Note correspondence of GIP-and glucagon-positive cells (see arrows figs. 7, 8). X 390.
588
Dupre, J . , S. A. ROSS, D. Watson and J . C . Brown 1973 Stimulation of i n s u l i n se c re ti on by g a s t r i c i nhibitory polypeptide in man. J . Clin. Endocrinol. Metab., 37: 826-828. Dupre, J . , T . J . McDonald, J . Radzink, Y . Caussignac and S. Van Vliet 1976 Glucagonotropic action o f g a s t r i c inhibitory polypeptide in man. Clin. Res., 24: 680A. Erlandsen, S . L., J . A . Parsons, J . P . Burke, J . A. Redick, D. E . Van Orden and L . S . Van Orden 1975 A modification of the unlabeled antibody enzyme method using heterologous a n t i s e r a f o r t h e l i g h t microscopic and u l t r a s t r u c t u r a l l o c a l i z a t i o n of i n s u l i n , glucagon and g r o w t h hormone. J . Histochem. Cytochem., 23: 666-677. Erlandsen, S . L., 0. D. Hegre, J . A . Parsons, R . C . McEvoy and R. P . Elde 1976 Pancreatic i s l e t hormones: Distribution of c e l l types i n the i s l e t a n d evidence f o r t he presence of somatostatin and g a s t r i n within the D-cell. J . Histochem. Cytochem., 24: 883-897. Graham, B . C . , and M . J . Karnovsky 1966 The e a r l y stages of absorption of i n j e ct e d horseradish peroxidase in the proximal tubules of mouse kidney: u l t r a s t r u c t u r a l cytochemistry by a new technique. J . Histochem. Cytochem., 14: 291-302. Johnson, D. G . , F. W . Merchant, P . H . Smith, W . Y . Fujimoto and R. H . Williams 1977 Binding and actions of g a s t r i c inhibitory polypeptide in the endocrine pancreas of t h e r a t . Fed. Proc., 36: 299 ( a b s t r a c t ) . Pederson, R . A . , and J . C . Brown 1976 The insulinotr opic action of g a s t r i c inhibi tory polypeptide in the perfused i s o l a t e d r a t pancreas. Endocrinology, 99: 780-785. Polak, J . M . , S. R. Bloom, M . Kuzio, J . C. Brown and A . G. E . Pearse 1973 C e ll ula r loc a l iz a t ion of g a s t r i c i n h i b i t o r y polypeptide in the duodenum and jejunum. G u t , 14: 284-288. S o l ci a , E . , J . M. Polak, R . Buffa, C . Capella and A . G . E . Pearse 1975 Endocrine c e l l s of t he i n t e s t i n a l mucosa. I n : Gastroi n t e s t i n a l Hormones, J . C . Thompson, ed. University of Texas Press, Austin, p p . 155-168. Sternberger, L . A . 1974 Immunocytochemistry. Prentice-Hall, Englewood C l i f f s , p. 246. REFERENCES
1
Supported in p a r t by Veterans Administration General Medical Research Funds (MRIS 8007), American Diabetes Association, Washington A f f i l i a t e , I n c . , the KROC Foundation, t h e Diabetes-Endocrinology Research Center (AM17047) and USPHS Grants (AM1 531 2 , AM02456). Doctors Johnson and Fujimoto a r e r e c i p i e n t s of Research Career Development Awards AM70727 and AM47142, r espectively. Reprint requests should be se nt t o: Dr. P . H . Smith, Division of Metabolism (151), VA Hospital, 4435 Beacon Avenue South, S e a t t l e , Washington 981 08.
590
