ARTICLE |
Correspondence to: Eugenio Bertelli, Dept. of Biomedical Sciences, Univ. of Siena, Via Aldo Moro, I-53100 Siena, Italy.
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Summary |
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To elucidate the role of intermediate filament proteins in endocrine cells, we investigated the expression and subcellular distribution of GFAP in mouse islets of Langerhans. For this purpose, combined immunocytochemical and biochemical analysis with a panel of antibodies was carried out to identify GFAP-immunoreactive cells in mouse endocrine pancreas. Cell fractionation into NP-40-soluble and detergent/high salt-insoluble components was performed to assess whether GFAP was located in the cytosolic and/or cytoskeletal compartments of immunoreactive cells. Immunoelectron microscopic analysis was carried out to determine the subcellular distribution of the protein. Peripheral islet cells were stained with anti-GFAP antiserum. These cells were identified as glucagon-secreting cells by immunocytochemical staining of consecutive sections with anti-somatostatin, anti-GFAP, and anti-glucagon antisera. Western blotting analysis of both NP-40-soluble and detergent/high-salt insoluble fractions of isolated islets of Langerhans allowed detection of GFAP in both cytosolic and cytoskeletal compartments. Interestingly, however, the former location was highly predominant. In addition, immunoelectron microscopy localized GFAP associated with the periphery of secretory granules. On the basis of these results, an intriguing role for GFAP in secretory events should be strongly suspected. (J Histochem Cytochem 48:12331242, 2000)
Key Words: pancreas, islets of Langerhans, GFAP, intermediate filaments, secretion, glucagon, mouse
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Introduction |
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The cytoskeleton of higher eukaryotic cells is made up of a network of three main filamentous structures (-helical rod domain formed by multiple heptad repeats flanked by two non-
-helical terminal domains (
-internexin, nestin; Type V IFs, nuclear lamins; and Type VI (lens) IFs, phakinin and filensin.
The first step for IF polymerization is the formation of a dimer by the hydrophobic side-to-side interaction of two monomers whose rod domains intertwine in a coiled-coil fashion (
For a long time, IFs have been considered very stable structures. This view, supported because of their resistance to solubilization by high-salt buffers and non-denaturating detergents (
Cytoplasmic IFs, in general, show a developmentally regulated cell type-specific pattern of expression (
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Materials and Methods |
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Materials
Ficoll 400, Hank's balanced salt solution (HBSS), soybean trypsin inhibitor (STI), diphenilthiocarbazone, 3-3' diaminobenzidine (DAB)-HCl, and poly-L-lysine were from SigmaAldrich (Milan, Italy). Collagenase P, and NBT/BCIP stock solution were from Roche Diagnostic (Monza, Italy). Amplified Opti-4CN substrate kit was from Bio-Rad (Segrate, Italy). Purified GFAP from bovine brain was from Cytoskeleton Inc. (Denver, CO).
Antibodies
A rabbit polyclonal antibody (PAb), anti-GFAP, raised using bovine spinal cord as immunogen, was purchased from Zymed Laboratories (South San Francisco, CA). A monoclonal antibody (MAb), anti-GFAP (clone 6F-2), was a kind gift from Dr. R. Salvestroni and Dr. S. Pluchino (Institute of Neurology, University of Siena, Siena, Italy). A rabbit PAb, anti-GFAP, raised using human brain as immunogen, was from Sigma and was generously offered by Dr. C. Nicoletti (Laboratory of Molecular Recognition, The Babraham Institute, Cambridge, UK). Alkaline phosphatase (AP)-conjugated anti-rabbit and anti-mouse IgG were obtained from Roche Diagnostics and Sigma-Aldrich, respectively. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, HRP-conjugated goat anti-mouse IgG, sheep anti-rabbit IgG, and peroxidaseanti-peroxidase complex were from SigmaAldrich. Rabbit anti-glucagon and anti-somatostatin PAbs were a generous gift from Dr. M. Bendayan (Department of Pathology and Cell Biology, University of Montreal, Montreal, PQ, Canada). Twenty-nm gold-conjugated anti-rabbit IgG was from BioCell and was kindly provided by Dr. N. Volpi (Department of Biomedical Science, University of Siena).
Animals
Fifteen 1-year-old male C57 mice were used for this study. After ether anesthesia, animals were decapitated and the splenic portion of the pancreas, the intestines, and the brains were taken. Five pancreata, intestines, and brains were processed for light (LM) and transmission electron microscopy (TEM). The remaining 10 pancreata and brains were used for biochemical evaluations.
Islet Isolation Procedure
After enzymatic digestion of the pancreas, islets of Langerhans were purified using a discontinuous density gradient of Ficoll solutions as previously reported (
Preparation of IF-enriched Cytoskeletons and Cell Fractionation Procedures
Tissues were fractionated into pelletable (insoluble) components and nonpelletable (soluble) proteins as previously reported (
The supernatant was discarded and the pellet was resuspended in the same volume of high-salt buffer and further incubated for 30 min at 4C with magnetic stirring. The pellet of cytoskeletal material obtained after centrifugation at 10,000 x g for 20 min at 4C was washed twice in 0.01 M PBS, pH 7.4, containing 1 mM 2-mercaptoethanol and was dissolved in 40 µl Laemmli's sample buffer for 6 hr at RT with gentle magnetic stirring. Finally, the sample was centrifuged at 20,000 x g for 30 min at 25C and the supernatant (IF-enriched fraction) was used for immunoblotting analysis.
Small cubes of brain tissue were homogenized in lysis buffer with a Potter glassteflon homogenizer and treated with the above-described procedure.
Western Blotting
IF-enriched fractions and a 1:8 dilution of NP40-soluble fractions of islets and brain tissue were separated by electrophoresis on a 12% polyacrylamide gel according to
LM Immunocytochemistry
Small pancreatic, intestinal, and brain samples, as well as aliquots of purified islets, were fixed in 4% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) for 3 hr at 4C and processed for routine embedding in Epon 812. Four pancreata and brains were fixed for 1216 hr in 10% formalin or Bouin's fluid and treated for standard embedding in paraffin.
Sections (~56 µm thick) from paraffin-embedded samples were mounted on poly-L-lysine-coated slides. After deparaffinization and rehydration, sections were pretreated with 1% BSA in PBS, incubated with anti-GFAP PAb for 4 hr at RT, and thoroughly rinsed with PBS. Then the slides were treated again with 1% BSA in PBS and incubated with AP-conjugated anti-rabbit or anti-mouse PAb. AP reaction was developed with NBT/BCIP stock solution as chromogen diluted in 0.1 M Tris buffer, pH 9.5, 0.05 M MgCl2 and 0.1 M NaCl according to manufacturer's recommendations.
Consecutive semithin sections (~0.7 µm thick) from Epon-embedded samples of pancreas and intestine were mounted on poly-L-lysine-coated slides. Epon was removed from slides according to the technique suggested by
Several immunocytochemical control experiments were carried out to substantiate GFAP PAb specificity: omission of the primary antibody or its replacement with normal rabbit serum, liquid-phase absorption with poly-L-lysine (1 mg/ml), and liquid-phase absorption with purified GFAP (50 µg/ml).
Immunoelectron Microscopy
Small samples of pancreatic and brain tissues were fixed in 4% paraformaldehyde in 0.01 M PBS (pH 7.4) for 3 hr at 4C and processed for routine embedding in Lowicryl resin. Thin sections were mounted on formvar-coated nickel grids. Grids were incubated on 1% BSA in PBS for 30 min at RT and then placed on a drop of 1:10 anti-GFAP PAb in PBS overnight at 4C. Then the grids were rinsed with PBS, incubated with 1% BSA in PBS, and placed on a drop of 1:20 20-nm gold-conjugated anti-rabbit IgG in PBS for 90 min at RT. After several rinsings with PBS and distilled water, grids were dried, stained with uranyl acetate, and observed with a Philips 201 transmission electron microscope.
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Results |
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GFAP Is Expressed in Pancreatic Glucagon-secreting Cells
To minimize the risk of crossreaction with other IFs, immunocytochemical detection of GFAP-immunoreactive cells in mouse pancreas was performed with two commercially available anti-GFAP PAbs raised using different sources of immunogen (human brain and bovine spinal cord). Both antibodies gave essentially the same results: acinar and duct cells as well as blood vessels were completely unstained. In contrast, Schwann cells of major nerve fibers were slightly GFAP-immunoreactive. In addition, both PAbs stained cells preferentially located in the periphery of almost all islets of Langerhans (Fig 1). To identify which type of islet cell was GFAP-immunoreactive, we carried out experiments staining three consecutive semithin sections (0.7 µm thick) with anti-somatostatin, anti-GFAP, and anti-glucagon antisera. Whereas no somatostatin-secreting cells were GFAP-immunoreactive (Fig 2A and Fig 2B), all glucagon-secreting cells stained with the anti-GFAP PAb (Fig 2B and Fig 2C).
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To verify whether the antibodies employed in this study recognized actual GFAP, we purified the islets of Langerhans and carried out Western blotting analysis of their IF-enriched cytoskeletal fraction. The same protocol was followed with mouse brain samples as a positive GFAP-immunoreactive control. The enzymatic digestion of the pancreas and its subsequent centrifugation through a discontinuous density gradient (see Materials and Methods) resulted in efficient isolation of islets of Langerhans, which was further improved by hand-picking the islets. This procedure is presumed to remove nerve fibers and periacinar stellate cells that might contaminate the sample with GFAP. To determine if any contamination had occurred during the preparation, aliquots of purified islets were immunocytochemically tested to detect GFAP-containing cells. All the experiments performed revealed GFAP immunoreactivity exclusively in glucagon-secreting cells (Fig 3). No other cells, among the few elements contaminating the isolated islets, stained with anti-GFAP Abs. Western blotting analysis with anti-GFAP PAbs of IF-enriched cytoskeletal fractions revealed a single band in pancreatic samples co-migrating at 50 kD with immunoreactive GFAP from brain tissue (Fig 4B). As an additional control to confirm that the 50-kD band was GFAP, we performed a new series of experiments by immunoblotting membranes with an anti-GFAP MAb (clone 6F-2). In this case, however, whereas brain samples displayed, as expected, a single band with a relative mobility corresponding to 50 kD (Fig 4C), pancreatic samples exhibited two distinguishable GFAP-immunoreactive bands of similar molecular weights (50 and 51 kD).
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GFAP Is Present Mainly in Its NP40-soluble Form
Taken together, these results are consistent with the expression of GFAP in glucagon-secreting cells. However, the amount of GFAP belonging to the cytoskeletal fraction appeared very low because we had to use an amplified Opti-4CN substrate kit to detect it. Because of this, we decided to verify whether the immunocytochemical signal could also be due to a soluble antigen present in the cytosol of glucagon-secreting cells. For this purpose, Western blotting analysis with anti-GFAP PAbs was conducted on NP40-soluble fractions of islets and brain, revealing a single 50-kD band in both pancreatic and brain samples (Fig 5a). To confirm that the 50-kD band of both samples really corresponded to GFAP, we carried out the same controls performed for the IF-enriched cytoskeletal fractions, incubating membranes with an anti-GFAP MAb (clone 6F-2). Western blotting analysis with the latter Ab revealed only one 50-kD band in pancreatic samples (Fig 5B), whereas no bands were detectable in brain samples. On the basis of these results, we can affirm that GFAP is expressed in glucagon-secreting cells as either polymerized or soluble forms.
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Even though we did not directly quantify these two pools of GFAP, the ratio between polymerized GFAP and its detergent-soluble form can be roughly assessed by calculating the dilution factors of the respective samples loaded onto the gels. The GFAP NP40-soluble pool was diluted about 400 times compared to its polymerized form before being loaded onto the gel. Moreover, despite this greater dilution, the demonstration of GFAP-immunoreactive bands in the NP40-soluble sample did not require any particularly sensitive detection device, as did the IF-enriched cytoskeletal fraction, which needed an amplified detection system (Fig 4 and Fig 5).
Taken together, these findings show that in glucagon-secreting cells the GFAP NP40-soluble pool appears to be a hundred times more highly represented than its polymerized form.
GFAP Is Associated with Secretory Granules
To localize GFAP in glucagon-secreting cells, we carried out an immunoelectron microscopic analysis of GFAP subcellular distribution. As a positive control, sections from mouse brain were also handled with the same immunocytochemical protocol. Accordingly, in the latter case, gold particles selectively labeled bundles of IFs located in astrocyte processes (Fig 6). On pancreatic sections, confirming the LM findings, the reaction was restricted to glucagon-secreting cells (Fig 7A). In this case, consistent with biochemical analysis that referred most of the GFAP immunoreactivity to the NP40-soluble cell fraction, gold particles did not label filamentous structures. Surprisingly, however, gold particles were not scattered throughout the cytoplasm as would be expected but were associated with the periphery of secretory granules (Fig 7B).
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Immunocytochemical Controls
Light microscopic control experiments consisting of the omission of anti-GFAP PAb or its replacement with normal rabbit serum abolished specific staining on both brain and pancreatic sections (data not shown).
Because unspecific binding of immunoglobulins to basic peptides has been reported to occur in glucagon-secreting cells as well (
Anti-GFAP PAb liquid-phase absorption with purified GFAP abolished specific staining on both brain and pancreatic sections (Fig 8A and Fig 8B). Positive control experiments were carried out on brain and intestine sections, resulting in staining of scattered brain astrocytes and satellite cells of intestinal myoenteric and submucosal plexi (Fig 8C and Fig 8D).
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Discussion |
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In this study we describe the expression of GFAP in glucagon-secreting cells of adult mouse pancreas. This finding confirms previous observations of GFAP-like immunoreactivity in glucagon-secreting cells of rat pancreas (
More important than the simple finding of its presence in glucagon-secreting cells is the demonstration that GFAP is mostly expressed in its non-polymerized form (i.e., NP40-soluble fraction). This raises basic questions about the role of IFps. Even though additional functions for IFps have been strongly suspected owing to their dynamic nature (
At present it is not clear which cellular roles such large amounts of soluble GFAP could play. However, our immunoelectron microscopic analysis of subcellular GFAP distribution in glucagon-secreting cells, sheds some light that enables us to formulate concrete hypotheses. Instead of labeling filamentous structures as occurs in brain astrocytes, in glucagon-secreting cells gold particles localize to the periphery of secretory granules, corroborating previous conjectures about an involvement of GFAP in secretion (
The association of GFAP with the periphery of secretory granules could be due to direct connections with the limiting membrane of granules as the result of IFp interactions with membrane lipids, as previously suggested (
Despite these observations, GFAP interaction with secretory granules may be more likely mediated by associated proteins. Many proteins have been recently described as being associated or as being capable of associating with IFs or with their subunits, thus potentially modulating their role. For example, 14-3-3 protein, in addition to associating with CK8/18 tetramers, has been described to be involved in priming regulated exocytosis in permeabilized chromaffin cells (
However, other proteins described as associating with IFs are also involved in secretion, as in the case of kinesin. Kinesin, a plus-end microtubule-directed motor protein, in addition to binding microtubules, associates with IFs, thus interacting directly or indirectly with IFps (
Whatever the role of GFAP is, its selective expression in glucagon-secreting cells raises a basic question for islet cell biology that will require additional investigation. Is the location of soluble IFps in relation to secretory granules a unique feature that distinguishes glucagon-secreting cells from the other endocrine cell types? In this case, we should assume that GFAP modulates an exclusive function most likely related to specific secretory products of glucagon-secreting cells. Otherwise, if GFAP plays a role in glucagon-secreting cells that is common to all islet cells, it is reasonable to predict the expression of other molecules (maybe still IFps) replacing GFAP in the other pancreatic endocrine cell types.
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Acknowledgments |
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Supported by local funds from the University of Siena (GL and AB).
We wish to thank Mr P. Salvatici and Ms D. Orazioli for excellent technical assistance. We are grateful to Dr A. Gugliucci for critical revision of the manuscript and to Dr M. Bendayan for fruitful discussions during the preparation of the manuscript. We are indebted to Dr L. M. Keith for improving the English text.
Received for publication January 20, 2000; accepted April 6, 2000.
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