Role of TGF-beta 1 in the development of pancreatic fibrosis in Otsuka Long-Evans Tokushima Fatty rats

Hiroyuki Yoshikawa, Yasuyuki Kihara, Masashi Taguchi, Taizo Yamaguchi, Hayato Nakamura, and Makoto Otsuki

Third Department of Internal Medicine, University of Occupational and Environmental Health, Japan, School of Medicine, Kitakyushu, 807 - 8555 Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recently established Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of naturally occurring obesity diabetes, exhibit progressive accumulation of connective tissue in the pancreas. The present study was designed to determine the pathogenic role of transforming growth factor-beta 1 (TGF-beta 1) in the development of pancreatic fibrosis in OLETF rats by investigating the serial changes in the expression of TGF-beta 1 and extracellular matrix (ECM) in the pancreas. Progressive proliferation of connective tissue arose from the interstitial region surrounding islets at 20 wk of age and extended to the exocrine pancreas adjacent to the islets. TGF-beta 1 mRNA levels in the pancreas increased at 20 wk of age and reached a peak value at 30 wk of age. Fibronectin (FN) and procollagen types I and III mRNAs peaked at 20 wk of age and remained at higher levels than those in the nondiabetic counterparts Long-Evans Tokushima Otsuka rats until 50 wk of age. Immunoreactivities for TGF-beta 1 and FN were found in islets of OLETF rats at 20 wk of age and were seen in acinar and interstitial cells at 50 wk of age. Moreover, alpha -smooth muscle actin was located at interstitial region surrounding the islets. Proliferation of the connective tissue in the pancreas of OLETF rats closely correlated with expression of TGF-beta 1 and ECM. Our results suggest that the development of pancreatic fibrosis in OLETF rats extends from endocrine to exocrine pancreas and that TGF-beta 1 is involved in pancreatic fibrosis of OLETF rats.

extracellular matrix; myofibroblast; Type 2 diabetes; pancreatic stellate cell; transforming growth factor-beta 1


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PANCREATIC FIBROSIS IS A CHARACTERISTIC pathological feature of human pancreatic diseases such as chronic pancreatitis (28) and pancreatic cancer. (20). Development of pancreatic fibrosis is associated with accumulation and deposition of extracellular matrix (ECM). However, mechanisms of this fibrotic process in the pancreas are not fully understood. Although the appearance of pancreatic fibrosis in the existing animal models is mostly transient and reversible, it is difficult to investigate the pathophysiology of the development of fibrosis. Research of pancreatic fibrogenesis has been hampered by the lack of suitable in vivo models. The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an established animal model having some characteristics of human Type 2 diabetes (27, 54). OLETF rats exhibits a late onset of chronic and slowly progressive hyperglycemia. Insulin resistance appears at 12-24 wk of age, and obvious diabetes develops at 20-30 wk of age. After 40 wk of age, OLETF rats shows impairment of insulin secretion (25, 27, 54). Histologically, OLETF rats show progressive fibrosis in the pancreas. After 20 wk of age, the fibrosis and enlargement of the islets become prominent, and the islets are separated into clusters by connective tissues (27). After 40 wk of age, the islets are replaced by connective tissues. Finally, extensive atrophy and connective tissue proliferation of the pancreas are observed (27).

Transforming growth factor-beta 1 (TGF-beta 1) is a family of cytokines involved in various pathophysiological processes, including cell proliferation, differentiation, development, angiogenesis, wound healing, and fibrosis (37). Although TGF-beta 1 stimulates the synthesis of ECM components and inhibits matrix degradation in autocrine and paracrine manners (7), it is considered to be a key mediator of fibrosis, including pulmonary fibrosis (9), heart disease (32), liver cirrhosis (11), and glomerulonephritis (55). In fact, TGF-beta 1 mRNA and protein are overexpressed in the pancreas of human chronic pancreatitis (10, 51). Moreover, in the transgenic mice overexpressing TGF-beta 1, disorganization of the islets and fibrosis of both endocrine and exocrine pancreata have been observed (31, 45). Furthermore, upregulation of procollagen type I and TGF-beta 1 mRNA in parallel with the development of pancreatic fibrosis are observed in dibutyltin dichloride-induced pancreatitis (49). In cerulein-induced pancreatitis (40), neutralization with anti-TGF-beta 1 antibody markedly reduces production of ECM components. Pancreatic stellate cells (PSCs), which are characterized by staining for anti-alpha -smooth muscle actin (alpha -SMA) (35), are isolated and cultured from rat and human pancreas (4, 6). PSCs produce ECM components and respond to TGF-beta 1 by increasing collagen synthesis in vitro (5). These studies indicate that TGF-beta 1 plays an important role in pancreatic fibrosis seen after pancreatitis.

In OLETF rats, TGF-beta 1 is known to produce ECM components in the glomeruli, resulting in glomerulosclerosis (2, 53). However, whether TGF-beta 1 is involved in the fibrosis of endocrine and exocrine pancreas in OLETF rats has not yet been determined. In the present study, we investigated the expressions of TGF-beta 1 and ECM genes and determined the correlation between TGF-beta 1 and the development of pancreatic fibrosis in OLETF rats.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Complementary DNA (cDNA) probes were obtained from the following: the rat fibronectin (FN) cDNA probe was kindly provided by Dr. R. O. Hynes (Massachusetts Institute of Technology, Cambridge, MA); the mouse procollagen types I and III cDNA probes from Dr. B. de Crombrugghe (National Cancer Institute, Bethesda, MD); the rat TGF-beta 1 cDNA probe from Dr. S. W. Qian (National Cancer Institute); and the mouse 7S cDNA from Dr. M. Korc (University of California, Irvine, CA). Anti-human alpha -SMA and anti-human FN were purchased from DAKO (Carpinteria, CA), and anti-human TGF-beta 1 was from Santa Cruz Biotechnology (Santa Cruz, CA).

Animals and experimental design. A spontaneously diabetic strain of rats with polyuria and slight obesity was discovered in an outbred colony of Long-Evans rats (Charles River, St. Constant, Canada) and maintained at the Tokushima Research Institute, Otsuka Pharmaceuticals (Tokushima, Japan). After 20 generations of crossbreeding of rats represented hyperglycemia, a diabetic strain of rats, OLETF, was established in the early 1990s (27). OLETF rats exhibit a late-onset of chronic and slowly progressive hyperglycemia (27, 54). Insulin resistance appears at 12-24 wk of age, and overt diabetes develops at 20-30 wk of age. After 40 wk of age, OLETF rats show impairment of insulin secretion (25, 27, 54). On the other hand, the Long-Evans Tokushima Otsuka (LETO) rats were established by different original matings from those of OLETF rats, but both strains originated from the same colony of Long-Evans rats (27). LETO rats represent no diabetic phenotypes such as obesity, hyperglycemia, insulin resistance and an impairment of insulin secretion (27) and are well accepted as control animals of OLETF rats (2, 25, 27, 53). Therefore, LETO rats were used as nondiabetic controls of OLETF rats in the present study.

Male OLETF and LETO rats at 4 wk of age were kindly supplied by the Tokushima Research Institute and maintained in a temperature (23 ± 3°C)- and humidity (55 ± 5%)-controlled room with a 12:12-h light-dark cycle. Animals were provided a standard rat chow (Oriental Yeast, Tokyo) and water ad libitum. Rats were maintained according to the ethical guidelines of our institution, and the experimental protocol was approved by the Animal Welfare Committee. OLETF and LETO rats were killed at 10, 20, 30, 40, and 50 wk of age (OLETF rats n = 5, LETO rats n = 5, at each time point). After body weight was measured, laparotomy was performed under pentobarbital sodium anesthesia and the pancreas was removed. Part of the pancreas was stored at -80°C for Northern blot and immunoblot analyses, and the other part was fixed in 4% paraformaldehyde and then embedded in paraffin.

Histological examination. A portion of the pancreas was cut into 5-µm thin slices for Azan-Mallory staining and light-microscopic examination. All histological samples were examined in a single-blinded fashion.

Quantitative analysis of pancreatic fibrosis. A quantitative evaluation of interstitial fibrosis in the pancreatic specimen was performed using an Axiophot microscope (Carl Zeiss, Eching, Germany) connected to an IBAS image analysis system (Carl Zeiss). Ten nonoverlapping fields per pancreatic specimen of Azan-Mallory staining (n = 5, at each time point) were randomly selected at a ×10 magnification. Area (µm2) of total pancreatic specimen and that of interstitial fibrosis stained blue were determined by an IBAS image analysis system. Rate of pancreatic fibrosis was indicated as a percentage of total pancreatic specimen by the following equation: (area of interstitial fibrosis/total area of specimen) × 100.

RNA isolation and Northern blot analysis. Total RNA was extracted from the frozen pancreatic tissue by the acid guanidium thiocyanate/phenol/chloroform method. For Northern blot analysis, 20 µg of total RNA was size fractionated on a 1.2% agarose-1.8 M formaldehyde gel, and RNAs were transferred onto nylon membrane (Hybond-N, Amersham Pharmacia Biotech, London, UK) followed by cross-linking by ultraviolet irradiation (30). Filters were incubated in prehybridization solution containing 50 mM phosphate buffer (pH 7.4), 0.75 M NaCl, 5 mM EDTA, and 50% formamide (vol/vol), 1% SDS (vol/vol), 10% dextran sulfate (vol/vol), 5× Denhardt's solution, and 0.1 mg/ml salmon sperm DNA, and then hybridized overnight with a labeled cDNA probe (1 × 106 counts/min-1 · ml-1) at 42°C. After being washed with saline sodium citrate solution (SSC) containing 0.2% SDS, images were scanned from the Northern blot filters with the FUJIX Bio-Image analyzing system (BAS 2000, Fuji Film, Tokyo). Relative RNA levels were expressed as the ratio of optical density between each cDNA probe and the corresponding 7S signals. Images were analyzed and quantified by National Institutes of Health image analysis software. Purified cDNA probes were labeled with deoxycytidine 5'-[alpha -32P]-triphosphate (Amersham Pharmacia Biotech) by Random Primer DNA Labeling Kit Ver.2 (Takara, Shiga, Japan).

Immunohistochemistry. Paraffin-embedded pancreatic sections were deparaffinized by the standard procedure, immersed in PBS (pH 7.2) for 10 min and then in PBS containing 3% hydrogen peroxide for 10 min to quench endogenous peroxidases. After further incubation in 0.25% casein solution for 10 min, the sections were incubated overnight with a primary polyclonal antibody against human TGF-beta 1 and alpha -SMA at a 1:100 dilution, human FN at a 1:500 dilution at 4°C. The primary antibody was visualized by the labeled streptavidin-biotin method using a commercially available kit (DAKO), and all procedures were performed as recommended by the manufacturer. The bound primary antibody was labeled with the biotinylated link antibody provided in the kit and peroxidase-labeled streptavidin. This procedure was counterstained with Mayer's hematoxylin. To confirm specificity of the immunostaining reactions, consecutive sections were incubated in preimmune serum of rabbit and mouse.

Immunoblot analysis for FN. Frozen pancreatic tissue was homogenized with Polytron homogenizer in ice-cold lysis buffer (pH 7.4) containing 25 mM Tris · HCl, 25 mM NaCl, 0.5 mM EGTA, 10 mM NaF, 1 mM Na3VO4, 10 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 0.1 mg/ml soybean trypsin inhibitor. Samples were then centrifuged at 15,000 rpm for 10 min at 4°C. Protein concentration was determined by the Bradford method (8) using BSA as a standard. Supernatant was prepared for one-dimensional SDS-PAGE. Proteins (20 µg per each lane) were then separated by 8% SDS-PAGE and transferred onto polyvinylidene difluoride membrane (Hybond-P; Amersham Pharmacia Biotech). Membranes were blocked with 10% fat-free dry milk for 1 h in PBS (pH 7.4) and then incubated with anti-FN at a 1:4,000 dilution in PBS containing 0.05% Triton X-100 (pH 7.4) for 1 h at room temperature. After washing, membranes were incubated with appropriate IgG antibody conjugated with horseradish peroxidase in PBS for 1 h at room temperature. Antibody binding was detected by enhanced chemiluminescence detection system (ECL plus, Amersham Pharmacia Biotech) and exposed to X-ray films (Scientific Bio-Imaging Film, Kodak, NY).

Statistical analysis. All values were expressed as means ± SE. Samples among the same membranes on Northern blot and immunoblot analysis were used for the statistical analysis. Differences between groups were analyzed for statistical significance by Student's t-test, using the StatView computer program (Abacus, Calabasas, CA). A P value <0.05 denoted a statistically significant difference.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Histological findings. Light microscopic findings of the pancreata of OLETF and LETO rats are shown in Fig. 1, using the same magnification. In 10-wk-old OLETF rats, mild enlargement of the islets was observed, but there was no extensive deposition of connective tissue and infiltration of inflammatory cells (data not shown). In 20-wk-old OLETF rats, the islets were enlarged and connective tissue proliferation surrounding the islets was observed (Fig. 1A). Infiltration of inflammatory cells was also seen in the interstitial area around the degenerated islets. At 30 wk of age, islets were replaced by connective tissue. Furthermore, tubular complexes and fibrosis of the exocrine pancreas were observed around the degenerated islets (Fig. 1B). In the pancreas of 50-wk-old OLETF rats, prominent fibrosis was noted, and the islets were separated into clusters by fibrosis. Finally, the exocrine and endocrine components of the pancreas were replaced by connective tissue and fatty deposition (Fig. 1C). Pancreatic fibrosis in OLETF rats arose from islet and extended to the exocrine pancreas adjacent to the fibrotic islets. On the other hand, no significant histological changes, such as enlargement of islet and connective tissue proliferation, were observed in LETO rats throughout the experimental period (Fig. 1D).


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Fig. 1.   Histological findings in the pancreas of Otsuka Long-Evans Tokushima Fatty (OLETF) and Long-Evans Tokushima Otsuka (LETO) rats. A: pancreas of a representative 20-wk-old OLETF rats showing enlargement of islet and infiltration of inflammatory cells around islets. Note proliferation of connective tissue around the islets. B: pancreas of OLETF rats at 30 wk of age demonstrating tubular complexes in the disrupted exocrine pancreas (arrows). Islet was replaced by connective tissue. C: pancreas of a representative 50-wk-old OLETF rat showing fatty replacement and prominent fibrosis. Note that the islets are separated into clusters by connective tissue. D: pancreas of a representative 50-wk-old LETO rat showing islets of normal size with no enlargement of the islets, fatty replacement, or connective tissue proliferation in the exocrine pancreas. (Azan-Mallory staining; original magnification ×25).

Quantitative analysis of pancreatic fibrosis. We also performed a quantitative analysis of pancreatic fibrosis in OLETF and LETO rats by using an IBAS imaging analysis system (Table 1). At 10 wk of age, there was no significant difference in the degree of pancreatic fibrosis between OLETF and LETO rats. However, pancreatic fibrosis in OLETF rats gradually increased with age and peaked at 50 wk of age. The rate of pancreatic fibrosis in OLETF rats at 20, 30, 40, and 50 wk of age was significantly higher than that in LETO rats at the corresponding age (P < 0.01).

                              
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Table 1.   Quantitative analysis of pancreatic fibrosis in OLETF and LETO rats

Northern blot analysis. We then examined the expression levels of TGF-beta 1, FN, and procollagen types I and III mRNAs in the pancreas by Northern blot analysis. Transcript levels were expressed as the ratio of optical density between each gene and the corresponding 7S signal. As shown in Fig. 2A, simultaneous overexpression of TGF-beta 1, FN, and procollagen types I and III mRNAs was noted in 20-wk-old OLETF rats. Expression of TGF-beta 1 mRNA in OLETF rats increased markedly from 20 wk of age, reaching a peak level at 30 wk of age. TGF-beta 1 mRNA levels at 20, 30, and 50 wk of age in OLETF rats were 2.1-, 3.4-, and 1.5-fold, respectively, higher than those of age-matched control LETO rats (Fig. 2B,a). Expression of FN mRNA in OLETF rats reached a peak level at 20 wk of age. FN mRNA levels were significantly higher in OLETF rats than those of control LETO rats at the corresponding age (Fig. 2B,b). As shown in Fig. 2B,c and 2B,d, the expression of levels of procollagen types I and III mRNAs in the pancreas of OLETF rats were maximum at 20 wk of age; thereafter they gradually decreased to levels lower than those at 10 wk of age. Transcript levels of procollagen types I and III in OLETF rats were significantly higher than those of age-matched control LETO rats at 20, 30, 40, and 50 wk of age.


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Fig. 2.   A: Northern blot analysis of transforming growth factor-beta 1 (TGF-beta 1), fibronectin (FN), and procollagen types I and III mRNAs in the pancreas of OLETF and LETO rats at the indicated age. Total RNA (20 µg) was electrophoretically separated in agarose-formaldehyde gel followed by blotting onto nylon membranes. The Northern blots were rehybridized with 7S cDNA probe to verify equivalent RNA loading. Figure shows representative blots from 5 rats. B: densitometric analysis of expression of TGF-beta 1, FN, and procollagen types I and III mRNAs in the pancreas of OLETF and LETO rats. RNA levels were expressed as the ratio of optical density between each gene and the corresponding 7S expression. Data represent the means ± SE values of 5 rats. *P < 0.05, compared with LETO rats at the corresponding age.

Immunohistochemistry. In the pancreas of the 20-wk-old OLETF rats, immunoreactivity for TGF-beta 1 was detected in the islets and inflammatory cells around the degenerated islets (Fig. 3A). At 50 wk of age (Fig. 3B), marked immunoreactivity for TGF-beta 1 was detected in the islets, acinar cells adjacent to the degenerated islets, and interstitial cells in the exocrine pancreas. In contrast, only a weak immunoreactivity for TGF-beta 1 was present in the pancreas of LETO rats, which was limited to a few cells in the islets (Fig. 3E).


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Fig. 3.   Immunohistochemical study of the pancreas of OLETF and LETO rats. A: TGF-beta 1 protein in a representative 20-wk-old OLETF rats. Note the positive immunoreactivity for TGF-beta 1 in the cytoplasm of islets and inflammatory cells. B: TGF-beta 1 protein in a representative 50-wk-old OLETF rat. Note the intense immunoreactivity for TGF-beta 1 in islets, acinar cells adjacent to fibrotic islets, and interstitial cells. C and D: FN protein in representative 20- and 50-wk-old OLETF rats. Note the localization of FN protein in islets at 20 wk of age (C). At 50 wk of age, intense immunoreactivity for FN is noted in islets and interstitial cells in the fibrotic area of the exocrine tissue (D). E: TGF-beta 1 in a representative 50-wk-old LETO rat. Note the weak immunoreactivity for TGF-beta 1 in the cytoplasm of a few islets. F: alpha -smooth muscle actin (alpha -SMA) protein in a representative 20-wk-old OLETF rat. Note the presence of alpha -SMA-positive cells in the interstitial region surrounding islet and periacinar area adjacent to the islet. (original magnification × 50)

Immunoreactivity for FN in the pancreas of 20-wk-old OLETF rats was detected in islets and the interstitial regions surrounding the degenerated islets (Fig. 3C). Immunoreactivity for FN increased markedly in the islets and acinar cells at 50 wk of age. Moreover, intense signals were also observed in the interstitial cells in fibrotic tissues (Fig. 3D). In contrast, in the pancreas of LETO rats, only a weak FN immunoreactivity was noted throughout the experimental period, and such reactivity was noted only in the cytoplasm of a few islets (data not shown).

Spindle-shaped cells expressing alpha -SMA protein were found in the interstitial region surrounding islets and periacinar areas in 20-wk-old OLETF rats (Fig. 3F). At 50 wk of age, alpha -SMA-positive cells were observed in the interstitial regions around the disrupted acinar cells as well as in the clustered islets (data not shown). Distribution of alpha -SMA-positive cells closely correlated with the area of connective tissue proliferation. On the other hand, in the pancreas of LETO rats at 20 and 50 wk of age, alpha -SMA immunoreactivity was seen only in the vessel walls (data not shown).

In all procedures, immunostaining with preimmune rabbit serum (negative control) showed no signal (data not shown).

Immunoblot analysis for FN. Although fibrosis of the pancreas became prominent from 20 wk of age in OLETF rats, immunoblot analysis for FN protein was performed in 20- to 50-wk-old rats of both strains. We were able to detect the predictable specific band, and calculated the density of a molecular mass band on 220 kDa for quantification, using National Institutes of Health image analysis software. Expression of FN protein was clearly increased in 40-wk-old OLETF rats (Fig. 4, A and B). Densitometric analysis showed that FN protein levels in OLETF rats at 40 and 50 wk of age was 2.0- and 2.3-fold, respectively, higher than in control LETO rats (Fig. 4B). Specificity of the immunoblot procedures was confirmed by incubating the membranes that stripped the antibody in the preimmune rabbit serum instead of an antibody against human FN.


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Fig. 4.   Immunoblot analysis of FN protein in representative OLETF and LETO rats at indicated age. A: protein samples from homogenized pancreas tissue were resolved on 8% SDS-polyacrylamide gel, transferred onto polyvinylidene difluoride membrane, and immunoblotted with a specific antibody (at a 1:4,000 dilution). Note the marked increase in protein expression in immunoblots of OLETF rats at 40 and 50 wk of age. Representative results of 5 rats. B: densitometric analysis of FN protein expression. Levels of FN protein were significantly higher in 40- and 50-wk-old OLETF rats compared with the age-matched LETO rats. Data represent means ± SE of 5 rats. *P < 0.05, compared with LETO rats at the corresponding age.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, OLETF rats at 20 wk of age showed proliferation of connective tissue in the interstitial areas around the islet, which extended to exocrine pancreas after 30 wk of age. Simultaneously, the degree of pancreatic fibrosis in OLETF rats was significantly increased with age. TGF-beta 1, FN, and procollagen types I and III transcript levels increased markedly in the pancreas of OLETF rats at 20 wk of age. After 30 wk of age, these transcript levels decreased but were still significantly higher than those in the nondiabetic counterparts LETO rats. Thus the increased expression of TGF-beta 1 and ECM mRNAs paralleled the appearance of pancreatic fibrosis. In fact, TGF-beta 1 mRNA and protein are overexpressed in the pancreas of patients with chronic pancreatitis (10, 18, 51) and the level of TGF-beta 1 gene expression parallels the degree of pancreatic fibrosis (18). In dibutyltin dichloride-induced pancreatitis, TGF-beta 1 and procollagen type I mRNAs are upregulated with the development of fibrosis (49). In the pancreas of WBN/Kob rats, a model of transient and spontaneous chronic pancreatitis, the expression levels of TGF-beta 1 and FN mRNAs increase temporarily and closely parallel the appearance of fibrosis (50). These findings suggest that overexpression of TGF-beta 1 in the pancreas of OLETF rats noted in our study might contribute to the accumulation of ECM, resulting in pancreatic fibrosis.

In human alcohol-induced pancreatic fibrosis, spindle-shaped cells stained for alpha -SMA, a marker for activated PSCs, are found in the fibrotic exocrine pancreas where collagen types I and III, FN, and laminin are also identified (6, 10, 35). In experimental pancreatic fibrosis, alpha -SMA-positive cells are found in periacinar areas colocalized with collagen type I (35). Although cultured PSCs exhibit strong positive staining for collagen types I and III, FN, and laminin (5-6), they might be a major source of ECM components (4). In the present study, intense immunoreactivity for alpha -SMA in OLETF rats was located in the stromal area surrounding islets at 20 wk of age and thereafter extended to include the fibrotic exocrine pancreas. Connective tissue stained by Azan-Mallory staining closely correlated with alpha -SMA-positive cells. These results suggest that PSCs are involved in the fibrosis of endocrine and exocrine pancreas in OLETF rats.

Development of pancreatic fibrosis in OLETF rats exhibited a unique pattern; proliferation of connective tissue arose from islet at 20 wk of age and extended to the exocrine tissue adjacent to fibrotic islets at 50 wk of age. Moreover, TGF-beta 1 and FN protein extended from islets to acinar and interstitial cells in the exocrine pancreas. Distribution of TGF-beta 1 and FN protein corresponded with the area of fibrotic tissue. In patients with chronic pancreatitis, pancreatic fibrosis is found mainly in the exocrine pancreas, and TGF-beta 1 mRNA and protein is predominantly expressed in acinar cells and duct cells (10, 18, 51). In cerulein-induced pancreatitis, TGF-beta 1 mRNA and protein are mainly detected at acinar cells (21, 44). In the pancreas of WBN/Kob rats as well as of OLETF rats, connective tissue proliferation is observed in both endocrine and exocrine pancreata. In WBN/Kob rats, however, connective tissue arises from the interstitial region in the exocrine pancreas (26), and infiltration of inflammatory cells is observed around the pancreatic duct and blood vessels, whereas islets are intact (42). In WBN/Kob rats, TGF-beta 1 is found in acinar and interstitial cells (50). On the other hand, the pancreas of transgenic mice overexpressing TGF-beta 1 in the pancreatic beta  cells shows fibroblast proliferation and abnormal deposition of ECM around the islets from birth onward and finally replaces almost the entire exocrine pancreas (31, 45). Progressive histological changes of the pancreas in transgenic mice that overexpress TGF-beta 1 resemble those noted in our OLETF rats. In this regard, overexpression of TGF-beta 1 in the islet may initiate the fibrotic process that expands from the endocrine pancreas to the exocrine pancreas. Furthermore, marked infiltration of inflammatory cells around the islets in OLETF rats was observed at 20 wk of age. Activated macrophages release a number of cytokines including TGF-beta 1, which stimulates the synthesis of collagen type I and FN in cultured PSCs (46). Therefore, infiltrating inflammatory cells in the pancreas of OLETF rats might promote fibrogenesis.

Tissue fibrosis results from a relative imbalance between synthesis and degradation of ECM (3). Matrix metalloproteinases (MMPs) are the major factors that degrade ECM and display substrate specificity; MMP-1 and -13 degrade interstitial collagens, whereas MMP-2 and -9 degrade collagen type IV and FN (38). On the other hand, tissue inhibitors of metalloproteinases (TIMPs), which are important regulatory molecules in tissue remodeling and repair, inhibit the activity of MMPs. (16). Therefore, it is considered that progression and regression of tissue fibrosis, in part, depend on the relative magnitude of MMPs and TIMPs expression (24). In the present study, in contrast to decreased expression of ECM mRNAs in OLETF rats, the rate of pancreatic fibrosis and FN protein levels dramatically increased at 40 and 50 wk of age. Our results suggest that decreased degradation of ECM protein might be involved in the development of pancreatic fibrosis in OLETF rats.

Pancreatic fibrosis is often observed in diabetes mellitus, and the weight of the pancreas is reduced in patients with a long history of diabetes (19). A moderate degree of acinar atrophy may be associated with periacinar and perilobular fibrosis. Although atrophy and functional abnormality of the exocrine pancreas are prominent features of type 1 diabetes (14), exocrine fibrosis as well as amyloid deposition and fibrosis in islets is reported in the pancreas of human Type 2 diabetes (12). OLETF rats show insulin resistance at 12-24 wk of age and develop overt diabetes at about 20-30 wk of age, resulting in hyperglycemia (25, 54). After 40 wk of age, OLETF rats suffer from defective insulin secretion (25, 27, 54). In our previous studies (25, 54), we have shown that fasting serum glucose levels in OLETF rats were slightly higher at 12 wk of age than those in LETO rats and increased progressively with age, whereas those in LETO rats remained at low levels. In the present study, pancreatic fibrosis in OLETF rats developed with age. These results also suggest that deterioration of serum glucose concentration correlates with the accumulation of interstitial fibrosis in OLETF rats. It is likely, therefore, that hyperglycemia activates the accumulation of ECM, resulting in progressive fibrosis in the pancreas.

In support of this view, TGF-beta 1 mRNA is reported to be activated by glucose and plays a key role in diabetic kidney disease (47). In cultured mesangial cells, high concentration of glucose stimulates total TGF-beta 1 protein production and bioactivity as well as the expression of TGF-beta 1 mRNA (23). Activation of TGF-beta 1 mRNA is mediated by the protein kinase C pathway (17). In addition, a putative glucose-response element in the promoter region of TGF-beta 1 has been identified (23). Indeed, high glucose concentration is shown to activate the transcription of TGF-beta 1 and collagen synthesis in renal proximal tubular cells and renal cortical fibroblast cells (22). Recently, the hexosamine biosynthetic pathway (HBP) has been identified as a key mechanism of the adverse effects of glucose and development of insulin resistance and diabetic vascular complications (29). HBP accounts for approximately 2-3% of glucose metabolism and generates precursors for protein glycosylation (36). Activation of HBP mediates the increase in TGF-beta 1 transcript (15). Although there is no direct evidence that HBP or high glucose concentration increases the expression of TGF-beta 1 mRNA in the pancreas, it is reported that O-linked N-acetylglucosamine transferase, which is involved in HBP, is present in the islets and pancreatic acinar cells (1). It is possible that TGF-beta 1 mRNA in the pancreas is activated by a glucose-sensing mechanism, such as HBP. In support of this view, control of diabetes with alpha -glucosidase inhibitor (54) or troglitazone (25) has been shown to prevent as well as reverse histologic changes in OLETF rats. Further studies are needed to clarify whether the expression of TGF-beta 1 mRNA in the pancreas is regulated by glucose or HBP.

TGF-beta 1 mRNA encodes a sequence corresponding to 339 amino acids TGF-beta 1 precursor, which is released as a latent form (41). Latent TGF-beta 1 is converted by plasmin or thrombospondin-1 to mature form with biological activity (34, 13). In the present study, despite decreased expression of TGF-beta 1 mRNA, intense immunoreactivities were observed in 50-wk-old OLETF rats. These results suggest that the enhanced immunoreactivity is not induced by synthesis of TGF-beta 1 mRNA. We performed immunohistochemistry using an antibody that recognizes the mature form of TGF-beta 1 (39). It is conceivable, therefore, that an intense immunostaining without a corresponding increase in TGF-beta 1 mRNA is due to an activation of latent form as well as an increase in mature TGF-beta 1 protein.

After 30 wk of age, tubular complexes were observed, and fibrosis and fatty replacement gradually developed in OLETF rats. Tubular complexes are shown to represent degenerated acinar cells that have lost their secretory proteins (52). It is well known that TGF-beta 1 acts as a negative growth factor in epithelial cells (43). In pancreatic acinar cells, the autocrine inhibitory effect of TGF-beta 1 on cell proliferation has been demonstrated in vitro (33). Transgenic mice overexpressing TGF-beta 1 in the pancreatic beta  cells show inhibition of acinar cell proliferation as evidenced by decreased bromodeoxyuridine incorporation (31). Therefore, the presence of persistently high levels of TGF-beta 1 may inhibit proliferation of pancreatic acinar cells in OLETF rats, resulting in atrophy of the exocrine pancreas.

In conclusion, we have demonstrated in the present study that TGF-beta 1 and ECM genes play an important role in pancreatic fibrosis in OLETF rats. Results of time course studies of TGF-beta 1 and ECM transcript levels were consistent with those of histological changes. Connective tissue proliferation and TGF-beta 1 protein extended from peri-islets to exocrine pancreas adjacent to degenerative islets. This model may provide key information for understanding the mechanisms of pancreatic fibrosis. Further studies are necessary to examine whether treatment with anti-diabetic agent reduces TGF-beta 1 gene expression in the pancreas, resulting in the improvement of pancreatic fibrosis.


    ACKNOWLEDGEMENTS

We thank Yoko Uesugi and Hitomi Kamio for excellent technical assistance. We also thank Drs. R. O. Hynes, B. de Crombrugghe, S. W. Qian, and M. Korc for generously providing cDNAs.


    FOOTNOTES

Address for reprint requests and other correspondence: M. Otsuki, Third Dept. of Internal Medicine, University of Occupational and Environmental Health, Japan, School of Medicine, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu, 807-8555 Japan (E-mail: mac-otsk{at}med.uoeh-u.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpgi.00323.2001

Received 14 July 2001; accepted in final form 5 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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