Histochem Cell Biol DOI 10.1007/s00418-006-0248-4

S H O RT C OM M UN IC A TI O N

Catalase expression in pancreatic alpha cells of diabetic and non-diabetic mice Konstantin Bloch · Elina Shichman · Marina Vorobeychik · Daria Bloch · Pnina Vardi

Accepted: 16 October 2006 © Springer-Verlag 2006

Abstract The pancreatic islet beta cells are very sensitive to oxidative stress, probably due to the extremely low level of anti-oxidant enzymes, particularly catalase. In contrast to beta cells, pancreatic alpha cells are signiWcantly more resistant to diabetogenic toxins. However, whether alpha cells express a diVerent level of catalase is not known. The aim of this study was to evaluate catalase expression in alpha cells of diabetic and non-diabetic mice. Diabetes was induced by a single injection of streptozotocin. After 3 weeks of persistent hyperglycemia, pancreatic tissues were collected. Catalase localization in alpha cells was identiWed by a dual-immunoXuorescence staining with anti-glucagon and anti-catalase antibodies. In intact mice, intensive catalase and glucagon immunostaining was found in the peripheral area of islets. Merged images of glucagon and catalase show their localization in the same cell type, namely, alpha cells. Confocal microscopy indicated that the glucagon and catalase staining was distributed throughout the cytoplasm. Similar coexpression of catalase and glucagon was found in the alpha cells of diabetic animals. The results of this study show the intensive catalase expression in alpha cells of diabetic and non-diabetic mice. This knowledge may be useful to better understand the defense mechanisms of pancreatic alpha cells against oxidative stress.

K. Bloch (&) · E. Shichman · M. Vorobeychik · P. Vardi Diabetes and Obesity Research Laboratory, Felsenstein Medical Research Center, Sackler Faculty of Medicine, Tel-Aviv University, Petah Tikva, 49100, Israel e-mail: [email protected] D. Bloch Department of Plant Sciences, Faculty of Life Science, Tel-Aviv University, Tel-Aviv 69978, Israel

Keywords Catalase · Glucagon · Alpha cells · Pancreatic islets · Diabetes

Introduction Catalase is an important scavenging enzyme against reactive oxygen species (ROS), as it removes hydrogen peroxide (HP) produced during metabolic processes. The enzyme is localized in the cytosol and in peroxisomes of cells. Pancreatic islets consist of four types of secretory endocrine cells, namely, the insulin-containing beta, the glucagon-containing alpha, the somatostatincontaining delta, and the pancreatic polypeptide-producing (PP) cells. In rodent islets, beta cells are the predominant cell type and located in the core of the islet, surrounded by a layer of alpha, delta, and PP cells, while human alpha cells were found scattered throughout the islet (Cabrera et al. 2006). In contrast to many other cells, the pancreatic beta cells have a particularly low level of catalase activity and expression, and this poor antioxidant system is believed to be responsible for their high sensitivity to HP (Lenzen et al. 1996; Tiedge et al. 1997). However, transfection of beta cells with catalase gene was able to induce signiWcant protection against streptozotocin (STZ) and ROS (Tiedge et al. 1999; Xu et al. 1999; Lortz et al. 2000; Chen et al. 2005). It was also shown that repeated exposure of insulinoma cells to HP results in the selection of the beta cell subpopulation with increased expression of catalase and improved defense against oxidative stress (Bloch et al. 2003). Alpha cells are the secondary major cell type in pancreatic islets. The known function of these cells is associated with metabolic regulation, mainly to maintain glucose homeostasis during the fasting state. In contrast

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Histochem Cell Biol

Materials and methods

bodies were used at dilutions of 1:100, 1:100 and 1:200, respectively. DeparaVinized sections were pretreated with 3% hydrogen peroxide in absolute methanol for 25 min and blocked with goat or swine serum before incubation with the primary antibodies, which was performed overnight, at room temperature. Peroxidaseconjugated goat anti-guinea pig or swine anti-rabbit Ig were used at a dilution of 1:500. All dilutions were made with Dako antibodies diluent. Substrate solution was 3,3-diaminobenzidine tetrahydrochloride (DAB). Sections were counterstained with hematoxylin and mounted in Entellan.

Materials

Dual-immunoXuorescence staining

Streptozotocin (STZ) and other chemicals were obtained from Sigma (St. Louis, MO, USA). Anti-catalase antibodies were from Rockland (Gilbertsville, PA, USA). Mouse monoclonal anti-glucagon antibodies were from Sigma. Other antibodies and reagents for immunohistochemistry were purchased from DAKO (Carpinteria, CA, USA).

Following the blocking of each section with 20% normal goat serum, they were incubated with mixture of primary mouse monoclonal anti-glucagon antibodies (1:2,000) and rabbit anti-catalase antibodies (1:100) overnight, at room temperature. After washing, sections were incubated with a mixture of secondary antibodies: Cytm2-conjugated aYnipure goat anti-mouse Ig (1:200) and Cytm3-conjugated aYnipure goat anti-rabbit Ig (1:200) for 1 h at room temperature. Sections were then washed and prepared for microscope examination.

to beta cells, pancreatic alpha cells are signiWcantly more resistant to diabetogenic toxins, as well as to the autoimmune attack (Nielsen et al. 1999; Reddy et al. 2005). However, defense mechanisms of pancreatic alpha cell are still poorly understood. In order to evaluate the possible involvement of catalase in cytoprotection of alpha cells, we studied the localization and expression of this anti-oxidant enzyme in pancreatic alpha cells of intact and diabetic animals.

Animals, diabetes induction and tissue collection Three-months-old male ICR mice (Harlan, Jerusalem, Israel) of 30–35 g body mass were used in all experiments. All procedures were approved by the Institutional Animal Care and Use Committee at the Tel Aviv University. Diabetes was induced by a single injection of STZ solution at a dose of 100 mg/kg body mass. STZ was injected intraperitoneally after dissolving in citrate buVer (pH 4.5). The severity of diabetes was estimated by blood glucose determination in non fasting animals. After 3 weeks of persistent hyperglycemia (>400 mg/dl) pancreatic tissues were collected. Pancreases from intact mice were used as a control group. Following animal sacriWce, the entire pancreas was removed and Wxed immediately in 4% (v/v) formalin in phosphate-buVered saline. Histochemistry Pancreatic tissues were embedded in paraYn wax. Serial sections (5
m thick) were cut from each pancreas. After deparaVinization and dehydration, the sections were stained with hematoxylin–eosin for routine examination. Staining using the indirect immunoperoxidase technique Polyclonal rabbit anti-catalase antibodies, guinea pig anti-insulin antibodies and rabbit anti-glucagon anti-

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Immunohistochemical controls Immunostaining of catalase was absent when the primary antibodies were replaced with Tris buVer supplemented with 0.1% BSA (pH–7.5) or normal sera, as well as when incompatible secondary antibodies were used at equivalent dilutions. Microscopy and imaging Stained sections were examined with a light microscope. Confocal images were captured with a digital camera attached to a laser scanning confocal microscope with 20£ and 63£ water immersion objectives. Cytm2 was visualized by excitation with an argon laser at 488 nm. Cytm3 was visualized by excitation with HeNe laser at 543 nm. Estimation of alpha cell area The area occupied by alpha cells within islet and total islet area were estimated using Image-Pro Plus, Version 5.1, Media Cybernatics Inc. The alpha cell area was calculated as a percentage of total islet area. Total number of islets examined for diabetic and non-diabetic animals was 37 and 31, respectively.

Histochem Cell Biol

Fig. 1 Representative light and confocal photomicrographs of pancreatic sections of intact mice. Hematoxylin–eosin staining (a). Staining by the indirect immunoperoxidase technique for insulin (b), glucagon (c) and catalase (d). An islet dual-immunoXuorescence labelled for catalase (e) and glucagon (f), merged images of catalase and glucagon (g). Images of subcellular distri-

bution of catalase (h) glucagon (i) and merged images (j). Note: a–d are light micrographs, e–j are confocal micrographs; enlargement of the region speciWed by the rectangle in e, yellow indicates colocalization of catalase and glucagon in overlay images. Bars 20
m (a–g) and 5
m (h–j)

Statistical analysis

groups were studied: diabetic and non-diabetic mice. Six animals from each group were analyzed. Groups of data were compared using unpaired Student’s t test.

The results are presented as mean § SE of three independent experiments for each group. Two

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Histochem Cell Biol

Fig. 2 Representative light and confocal photomicrographs of pancreatic sections of diabetic mice. Hematoxylin–eosin staining (a). Staining by the indirect immunoperoxidase technique for insulin (b), glucagon (c) and catalase (d). An islet dual-immunoXuorescence labelled for catalase (e) and glucagon (f), merged images of catalase

and glucagon (g). Images of subcellular distribution of catalase (h) glucagon (i) and merged images (j). Note: a–d are light micrographs, e–j are confocal micrographs; enlargement of the region speciWed by the rectangle in e; yellow indicates colocalization of catalase and glucagon in overlay images. Bars 20
m (a–g) and 5
m (h–j)

Results

(Fig. 1b.) revealed the well preserved morphology of islet cells and intensive insulin staining of the majority of the islet cells. Strong staining by the indirect immunoperoxidase technique for glucagon or catalase was observed in the cells localized in the peripheral area of islets (Fig. 1c, d, respectively). Dual-immunoXuorescence

Catalase localization in pancreatic islets of intact mice Both hematoxylin–eosin staining (Fig. 1a) and insulin labeling by the indirect immunoperoxidase technique

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Histochem Cell Biol

labeling demonstrated that catalase and glucagon were presented in the same cell type (Fig. 1e, f). Merged images of glucagon and catalase show localization of catalase in pancreatic alpha cells (Fig. 1g). Confocal microscopy indicated that glucagon and catalase immunostaining was diVusely distributed throughout cytoplasm (Fig. 1h). Merged confocal images of individual cells demonstrated only partial intra-cytoplasm coexpression of catalase and glucagon (Fig. 1i). Catalase localization in pancreatic islets of diabetic mice Histochemical study of islets from diabetic mice showed the loss of the islet architecture with atrophied islets and degenerative changes in the central part of islets (Fig. 2a). These observations are supported by weak immunostaining of insulin in the islet area (Fig. 2b). However, strong immunolabeling for glucagon and catalase was found in both peripheral and central area of islets (Fig. 2c, d). Similar to islets of intact mice, dual labeling demonstrated localization of catalase and glucagon in the same peripheral cells (Fig. 2e, f). Merged images show localization of both catalase and glucagon in pancreatic alpha cells (Fig. 2g). Both glucagon and catalase were diVusely distributed throughout the cytoplasm, as conWrmed by confocal microscopy (Fig. 2h). Merged confocal images of individual cells demonstrated only partial intra-cytoplasm co-expression of catalase and glucagon in diabetic animals (Fig. 2i), also found in intact islets (Fig. 1i). The eVect of diabetes on the alpha cell area The calculated islet area of diabetic mice was signiWcantly lower compared with intact animals (6,643 § 930 vs. 22,671 § 2,570
m2, P < 0.001). This may be explained by the disappearance of insulinlabeled cells in the central part of the islet (Fig. 2b). However, the islet area occupied by alpha cells was signiWcantly increased in diabetic animals compared to intact ones (65.6 § 2.4 vs. 8.7 § 0.9%, P < 0.001).

Discussion Contrasting with other tissue, mouse pancreatic islets display extremely low catalase gene expression and activity (Grankvist et al. 1981; Tiedge et al. 1997). These studies were performed on whole islets, which are known to contain not only beta, but also alpha, delta and PP cells. In order to evaluate catalase expression in alpha cells, we used dual-immunoXuorescence

staining for glucagon and catalase. The presence of catalase and glucagon in alpha cells was found in both intact and diabetic animals, compared to its absence in other islet cells. However, only partial intra-cytoplasm co-expression of catalase and glucagon was found in intact and diabetic animals. Further immunoelectron microscopy studies should be performed in order to verify the exact intra-cytoplasm localization of catalase in alpha cells. In intact animals, alpha cells were distributed mainly in the peripheral area of the islet. However, this islet architecture completely disappeared in diabetic mice, where alpha cells deviated from their natural periphery to occupy nearly the whole islet area. Similar data have been reported by other investigators showing an equivalent distribution of alpha cells in islets of mice with STZ-induced diabetes (Li et al. 2000) and in NOD mice (Reddy et al. 2005), suggesting that in contrast to beta cells, alpha cells are resistant to diabetogenic injury. We hypothesized that this phenomenon may be explained by the fact that alpha cells display a high level of catalase, the enzyme responsible for inactivation of HP, which is generated by STZ (Takasu et al. 1991). For its toxic action, STZ needs glucose transporter 2 (GLUT-2) for transport into the target cells (Schnedl et al. 1994). Although alpha cell resistance to STZ may be dependent on the lack of GLUT-2 expression in these cells (Tal et al. 1992), the other mechanisms related to the expression of the anti-oxidant enzyme may be involved in cell defense. The importance of catalase in the protection against STZ-induced diabetes has been demonstrated on transgenic mice with increased beta-cell expression of this anti-oxidant enzyme (Xu et al. 1999; Chen et al. 2005). In addition, the controlled release of catalase from polymeric microspheres was shown to signiWcantly improve the viability and function of islet cells cultured in vitro (Giovagnoli et al. 2005). In conclusion, for the Wrst time, the present study reveals catalase expression in alpha cells of intact and diabetic mice. These Wndings oVer information that indicates the possible role of catalase in the protection of pancreatic alpha cells against diabetogenic toxins. Acknowledgments This study was supported by a grant from the Research Foundation of Sackler Faculty of Medicine, Tel Aviv University. We thank Pepy Roizman for excellent assistance in the immunohistochemical study, and Sara Dominitz for editing the manuscript.

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