Third Department of Internal Medicine, University of Occupational and Environmental Health, Japan, School of Medicine, Kitakyushu, 807 - 8555 Japan
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ABSTRACT |
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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-1 (TGF-
1) in the development
of pancreatic fibrosis in OLETF rats by investigating the serial changes in the expression of TGF-
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-
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-
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,
-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-
1 and ECM. Our results suggest that the
development of pancreatic fibrosis in OLETF rats extends from endocrine
to exocrine pancreas and that TGF-
1 is involved in pancreatic
fibrosis of OLETF rats.
extracellular matrix; myofibroblast; Type 2 diabetes; pancreatic stellate cell; transforming growth factor-1
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INTRODUCTION |
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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-1 (TGF-
1) is a family of cytokines
involved in various pathophysiological processes, including cell
proliferation, differentiation, development, angiogenesis, wound
healing, and fibrosis (37). Although TGF-
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-
1 mRNA and protein are overexpressed in the pancreas of human
chronic pancreatitis (10, 51). Moreover, in the transgenic
mice overexpressing TGF-
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-
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-
1 antibody markedly reduces production of ECM components. Pancreatic stellate cells (PSCs), which are characterized by staining for anti-
-smooth muscle actin (
-SMA) (35), are isolated and cultured from rat and human
pancreas (4, 6). PSCs produce ECM components and respond
to TGF-
1 by increasing collagen synthesis in vitro (5).
These studies indicate that TGF-
1 plays an important role in
pancreatic fibrosis seen after pancreatitis.
In OLETF rats, TGF-1 is known to produce ECM components in the
glomeruli, resulting in glomerulosclerosis (2, 53).
However, whether TGF-
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-
1 and ECM
genes and determined the correlation between TGF-
1 and the
development of pancreatic fibrosis in OLETF rats.
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MATERIALS AND METHODS |
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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-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
-SMA and anti-human FN were purchased from DAKO
(Carpinteria, CA), and anti-human TGF-
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 atHistological 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/min1 · 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'-[
-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-1 and
-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.
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RESULTS |
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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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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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Northern blot analysis.
We then examined the expression levels of TGF-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-
1, FN, and procollagen types I and III mRNAs
was noted in 20-wk-old OLETF rats. Expression of TGF-
1 mRNA in OLETF
rats increased markedly from 20 wk of age, reaching a peak level at 30 wk of age. TGF-
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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Immunohistochemistry.
In the pancreas of the 20-wk-old OLETF rats, immunoreactivity for
TGF-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-
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-
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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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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DISCUSSION |
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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-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-
1 and ECM mRNAs
paralleled the appearance of pancreatic fibrosis. In fact, TGF-
1
mRNA and protein are overexpressed in the pancreas of patients with
chronic pancreatitis (10, 18, 51) and the level of
TGF-
1 gene expression parallels the degree of pancreatic fibrosis
(18). In dibutyltin dichloride-induced pancreatitis,
TGF-
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-
1 and FN mRNAs increase temporarily and
closely parallel the appearance of fibrosis (50). These findings suggest that overexpression of TGF-
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 -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,
-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
-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
-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-1 and FN protein extended from islets
to acinar and interstitial cells in the exocrine pancreas. Distribution
of TGF-
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-
1 mRNA and protein is
predominantly expressed in acinar cells and duct cells (10, 18,
51). In cerulein-induced pancreatitis, TGF-
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-
1 is found in acinar and interstitial cells (50).
On the other hand, the pancreas of transgenic mice overexpressing TGF-
1 in the pancreatic
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-
1 resemble those noted in our
OLETF rats. In this regard, overexpression of TGF-
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-
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-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-
1 protein production and bioactivity as well as the expression of TGF-
1 mRNA (23). Activation
of TGF-
1 mRNA is mediated by the protein kinase C pathway
(17). In addition, a putative glucose-response element in
the promoter region of TGF-
1 has been identified (23).
Indeed, high glucose concentration is shown to activate the
transcription of TGF-
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-
1
transcript (15). Although there is no direct evidence that
HBP or high glucose concentration increases the expression of TGF-
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-
1 mRNA in the pancreas is activated by a
glucose-sensing mechanism, such as HBP. In support of this view,
control of diabetes with
-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-
1 mRNA in the pancreas is
regulated by glucose or HBP.
TGF-1 mRNA encodes a sequence corresponding to 339 amino acids
TGF-
1 precursor, which is released as a latent form
(41). Latent TGF-
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-
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-
1 mRNA. We performed
immunohistochemistry using an antibody that recognizes the mature form
of TGF-
1 (39). It is conceivable, therefore, that an
intense immunostaining without a corresponding increase in TGF-
1
mRNA is due to an activation of latent form as well as an increase in
mature TGF-
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-1
acts as a negative growth factor in epithelial cells (43).
In pancreatic acinar cells, the autocrine inhibitory effect of TGF-
1
on cell proliferation has been demonstrated in vitro (33).
Transgenic mice overexpressing TGF-
1 in the pancreatic
cells
show inhibition of acinar cell proliferation as evidenced by decreased
bromodeoxyuridine incorporation (31). Therefore, the
presence of persistently high levels of TGF-
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-1 and ECM genes play an important role in pancreatic fibrosis in
OLETF rats. Results of time course studies of TGF-
1 and ECM transcript levels were consistent with those of histological changes. Connective tissue proliferation and TGF-
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-
1 gene expression in
the pancreas, resulting in the improvement of pancreatic fibrosis.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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