ARTICLE |
Correspondence to: In-Sun Park, Dept. of Anatomy, College of Medicine, Inha University, Choong-Gu, Shinheung-Dong, Inchon, 400-103, Korea. E-mail: sunpark@inha.ac.kr
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Summary |
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Polyenoylphosphatidylcholine (PPC), a phosphatidylcholine-rich phospholipid extracted from soybean, has been reported to protect liver cells from alloxan-induced cytotoxicity. The present study aimed to investigate whether PPC protects pancreatic ß-cells from the cytotoxic injury induced by streptozotocin, thus preserving insulin synthesis and secretion. ß-Cells of the PPC-treated rats showed a significant reduction of cell death with lesser destruction of plasma membrane on streptozotocin insult. They demonstrated a rapid recovery of GLUT-2 expression, whereas almost irreversible depletion of membrane-bound GLUT-2 was seen in ß-cells of the rats treated with only streptozotocin. A similar cytoprotective effect of PPC was also monitored in the PPC-pretreated MIN6 cells. These ß-cells retained their ability to synthesize and secrete insulin and no alteration of glucose metabolism was detected. These results strongly suggest that PPC plays important roles not only in protecting ß-cells against cytotoxicity but also in maintaining their insulin synthesis and secretion for normal glucose homeostasis. (J Histochem Cytochem 51:10051015, 2003)
Key Words: polyenoylphosphatidylcholine, pancreas, cytoprotection, ß-cell, insulin, streptozotocin, diabetes
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Introduction |
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IMPAIRED insulin secretion of ß-cells results in abnormal glucose homeostasis, leading to diabetes. Type I diabetes is caused by selective and progressive destruction of pancreatic ß-cells (
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Materials and Methods |
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Animals and PPC Feeding
Male SpragueDawley rats (Daehan Experimental Animal Lab; Seoul, Korea) weighing 150200 g with normal fasting blood glucose levels (70120 mg/dl) were used for PPC feeding and STZ treatment. Fifty-six rats were randomly assigned to the experimental and control groups. PPC (RhônePoulenc; Köln, Germany) was dissolved in drinking water and given to the rats at a daily dose of 200 mg/kg (body weight) for 3 weeks according to previous reports (
Tissue Preparation
For light microscopic examination, the pancreatic tissues were fixed overnight with Bouin's solution or 4% paraformaldehyde and 5-µm-thick paraffin sections were mounted on silanized slides (Fisher Scientific; Pittsburgh, PA). For electron microscopic studies, small fragments of tissue were fixed with 4% paraformaldehyde and embedded in Unicryl resin (BBI International; Cardiff, UK).
Immunocytochemical Labeling for Light Microscopy
Immunostaining for light microscopy was carried out using the avidinbiotinperoxidase complex (
Immunoelectron Microscopic Labeling for Insulin
To locate insulin in the secretory granules of ß-cells at the subcellular level, electron microscopic immunogold labeling was performed on ultrathin sections as described previously by
In Situ Hybridization
The tissues fixed with 4% paraformaldehyde were subjected to in situ hybridization as described previously (
Image Analysis for Evaluation of Immunocytochemistry and In Situ Hybridization
The ß-cell number and islet mass were assessed by morphometry on tissue sections immunostained for insulin or glucagon. The entire area of each tissue section was photographed and digitalized for planimetry. The percent area of islet to pancreatic tissue was assessed using an image analyzing system (Image-Pro Plus; Media Cybernetics, Silver Spring, MD). Populations of ß- and -cells were estimated by manual calculation of insulin or glucagon positive cells in each islet. This morphometric assessment was carried out on all islets present in random sections obtained from at least five different rats of each experimental group. It is possible that the actual numbers of insulin cells are higher than the estimated numbers because only the immunoreactive cells displaying their nuclei were counted as positive cells. On the other hand, insulin content in secretory granules of the ß-cell was determined in terms of the insulinimmunogold labeling index at the electron microscopic level (
I = Ni/Sa
Northern Blotting Analysis for Insulin
Total cellular RNA was isolated from pancreatic tissue samples according to the protocol previously described (
Assessment of Insulin Secretion by RIA
Insulin levels were determined in sera and culture media. Culture media were collected each day. An insulin RIA kit was used according to the manufacturer's protocol (Linco Research; St Charles, MO).
Determination of Proliferation Activity of Islet Cells
To identify the proliferating activity of islet cells, immunostaining for PCNA was carried out on pancreatic tissues using a specific antibody (Zymed). Proliferation rate is presented as the percentage of the PCNA-labeled cells in each islet or in cell culture.
Cell Culture
We induced in vitro cytotoxic injury on a mouse ß-cell line (MIN6) which was highly susceptible to STZ. The cells were grown in RPMI 1640 medium (Gibco Life Technologies; Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotics. The cells were cultured with PPC (0.5 mM) for 1 day and then incubated with STZ (4 mM) for 2 additional days in the presence of PPC. In a second experiment, pretreatment with PPC was omitted, while both PPC and STZ treatments were excluded for control experiment. Media collected from each culture condition were subjected to RIA for insulin.
Assessment of Glucose-stimulated Insulin Secretion
The cells cultured under the above conditions were washed with PBS and then transferred to serum-free KrebsRinger bicarbonate buffer (KRBB; Sigma) for the test of insulin secretion responding to glucose stimulation. The cells were consequently exposed to 5 mM glucose in KRBB and 20 mM for 2 hr each, and insulin contents in KRBBs were determined by combining immunoprecipitation and western blotting (
Assessment of Cell Death
The cultured cells undergoing apoptosis were determined by TUNEL assay (
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Results |
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Modulation of Diabetes Induction in PPC-Treated Rats
The rats fed with PPC maintained normal blood glucose levels (139.28 ± 5.79 mg/dl) after STZ treatment (Fig 1A) and demonstrated normal growth and regular weight gain. However, the rats not treated with PPC but having STZ showed severe hyperglycemia (427.92 ± 27.40 mg/dl) and growth retardation with diabetic symptoms, including polyuria (Fig 1B).
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Modification of GLUT-2 Expression
We examined modification of GLUT-2 expression in islet cells by immunocytochemistry (Fig 2). Because GLUT-2 is specifically localized in the plasma membrane of ß-cells, it can also demonstrate alteration of membrane integrity of ß-cells. GLUT-2 expression was significantly reduced by STZ intoxication in both PPC-pretreated and non-treated control rats, but the latter were more severely affected. GLUT-2 was seen in all ß-cell membranes, with higher immunoreactivity in normal pancreas, but only some ß-cells showed a weak immunoreaction for GLUT-2 in PPC-pretreated rats (Fig 2A and Fig 2D). In non-pretreated control rats, moreover, no GLUT-2 was detectable in the islet cells 24 hr after STZ treatment (Fig 2B). This may be caused not only by loss of the plasma membrane of dying ß-cells but also by complete impairment of GLUT-2 expression by STZ. Membrane-bound GLUT-2 was remarkably recovered after the cytotoxic insult in the PPC-pretreated rats than in those treated only with STZ (Fig 2B, Fig 2C, Fig 2E and Fig 2F). As shown in Fig 2G, ß-cells showing GLUT-2 expression in the plasma membrane were rapidly increased by 75% of the population of normal ones 3 weeks after STZ insult, while poor retrieval of GLUT-2 expression was seen in the diabetic controls at the corresponding period.
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Modification of Islet Cell Death and Proliferation
ß-Cell death and alteration of islet cell population were prominent in the STZ diabetic rats that were not treated with PPC. ß-Cell necrosis showing chromatin condensation and dissolution of plasma membrane was seen 2448 hr after STZ injection, and some cells demonstrated apoptotic cell death (Fig 3A and Fig 3C). ß-Cell death in the core of islets led to shrinkage in islet mass, particularly at 2448 hr after STZ treatment. However, subsequent -cell proliferation resulted in a certain regaining of islet mass and modification of the ß-cell to
-cell ratio in the islets. In contrast, such loss of ß-cells and proliferation of non-ß-cells were not seen in the islet cells of the PPC-fed rats in spite of the STZ treatment. Moreover, mitotic features were frequently seen in the islet cells of the rats fed PPC (Fig 3B). We examined proliferation activity of the islet cells by immunostaining for PCNA, a specific marker for proliferating cells (Fig 3D and Fig 3E). The PCNA labeling index, the number of labeled cells in each islet (Fig 3F), was higher in PPC-fed rats (2.91 ± 0.33) than in the diabetic (0.07 ± 0.05) or control (1.17 ± 0.22) animals.
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Morphological Alteration of Pancreatic Islets and ß-cells
The islets of the PPC-treated rats retained their conventional features. They showed a normal population of ß-cells centrally located, with -cells at the periphery (Fig 4). However, diabetic rats demonstrated a remarkable increase in
-cells in the central area where only few ß-cells remained, resulting in significant modifications of ß- to
-cell ratio (Fig 4C4F and Fig 4H). At the light microscopic level, the highest insulin immunoreactivity was found in ß-cells of the PPC-fed rats, whereas only few cells displayed immunoreaction with variable intensities in the diabetic animals (Fig 4B and Fig 4C). Immunoreaction for insulin at the electron microscopic level reflects subcellular insulin content in ß-cells (
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Insulin mRNA Expression
Expression of insulin mRNA, as an indication of insulin synthesis, was evaluated by in situ hybridization and Northern blotting analysis. Hybridization signals for insulin mRNA were more intense in ß-cells of the PPC-treated rats than in those of the normal and diabetic animals (Fig 6A6C). Higher expression of insulin mRNA was also detected in the pancreatic tissue of the rats fed with PPC by Northern blotting analysis, showing intense expression of insulin mRNA equivalent to that of the normal animals (Fig 6D and Fig 6E).
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Modulation of Insulin Secretion and Glucose Metabolism
Fasting plasma insulin level in the rats fed with PPC (0.38 ± 0.05 ng/ml) was within the normal range, whereas that of the diabetic animals (0.05 ± 0.01 ng/ml) was significantly lower (Fig 7A). PPC-fed rats showed a normal glucose tolerance value of 75 g IPGTT, whereas the diabetic rats manifested impaired glucose tolerance (Fig 7B).
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Preservation of Cell Viability and Insulin Secretion by PPC in ß-cell Culture
ß-Cell death was examined in MIN6 cell culture by TUNEL assay. A higher rate of apoptotic cell death (7.65 ± 3.04%) was monitored after 48 hr of treatment with STZ, whereas a much lesser number of apoptotic cells was demonstrated in PPC-pretreated cells before the STZ insult (1.47 ± 0.378%) and in normal control cells (0.60 ± 0.20%) (Fig 8A8D). Alteration of insulin secretion and glucose sensitiveness in the MIN6 cells was examined by RIA and immunoprecipitationWestern blotting analysis. Insulin concentration in culture media significantly declined after STZ treatment, whereas the media from PPC-treated cells showed no decrease in insulin concentration in spite of the STZ treatment (Fig 9A). We assessed glucose-stimulated insulin secretion in these cultured cells. As shown in Fig 9B, PPC-pretreated MIN cells produced higher levels of insulin level in response to glucose stimulation, whereas STZ-treated cells without PPC treatment displayed loss of glucose responsiveness.
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Discussion |
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We investigated the effects of PPC as a cytoprotector of ß-cells preventing development of diabetes induced by STZ. Membrane phospholipids are important regulators of cell functions such as growth, viability, homeostasis, and signal transduction (
The ß-cells, along with preservation of their population in PPC-treated rats, did not manifest impaired insulin secretion. They displayed well-preserved insulin immunoreactivity and insulin mRNA expression as well as glucose-stimulated insulin secretion, indicating an almost intact process for insulin synthesis and secretion. In fact, the secreted insulin in bloodstream or in culture media was within the normal range in PPC-treated groups.
We suggest that a sufficient supply of phospholipid with unsaturated fatty acids should be considered not only to improve ß-cell function but also to suppress the diabetogenic process in diabetes-prone subjects.
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Acknowledgments |
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Supported by a grant from the Oriental Medicine R&D Project, Ministry or Health and Welfare, Republic of Korea (HMP-00-CO-06-0006).
We wish to thank Dr V. U. Buko (Institute of Biochemistry, National Academy of Science of Belarus) for kind discussion and suggestions.
Received for publication September 30, 2002; accepted March 26, 2003.
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