Treatment with atorvastatin attenuates progression of insulin resistance and pancreatic fibrosis in the Otsuka Long–Evans Tokushima fatty rats

    Treatment with Atorvastatin Attenuates Progression of Insulin Resistance and Pancreatic Fibrosis in the Otsuka Long-Evans Tokushima Fatty Rats Limin Wei, Mitsuyoshi Yamamoto, Masaru Harada, Makoto Otsuki PII: DOI: Reference:

S0026-0495(15)00304-2 doi: 10.1016/j.metabol.2015.10.012 YMETA 53313

To appear in:

Metabolism

Received date: Revised date: Accepted date:

26 January 2015 20 September 2015 1 October 2015

Please cite this article as: Wei Limin, Yamamoto Mitsuyoshi, Harada Masaru, Otsuki Makoto, Treatment with Atorvastatin Attenuates Progression of Insulin Resistance and Pancreatic Fibrosis in the Otsuka Long-Evans Tokushima Fatty Rats, Metabolism (2015), doi: 10.1016/j.metabol.2015.10.012

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Wei et al.

Treatment with Atorvastatin Attenuates Progression of Insulin Resistance and

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Pancreatic Fibrosis in the Otsuka Long-Evans Tokushima Fatty Rats

The Third Department of Internal Medicine, University of Occupational and

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1)

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Limin Wei 1), Mitsuyoshi Yamamoto 1), Masaru Harada 1) and Makoto Otsuki 1) , 2)

Department of Internal Medicine, Kitasuma Hospital, Kobe, Japan

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2)

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Environmental Health, School of Medicine, Kitakyushu, Japan

Corresponding author: Mitsuyoshi Yamamoto, MD, PhD, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan Phone: +81-93-603-1611, Fax: +81-93-692-0107, E-mail: [email protected]

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Abstract

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Purpose: The effects of statins on insulin resistance (IR) and type 2 diabetes mellitus (T2DM) are still controversial and its effects on pancreatic fibrosis are poorly defined.

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The purpose of this study is to examine the effects of atorvastatin on these issues using

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the Otsuka Long-Evans Tokushima Fatty (OLETF) rat, an animal model of IR, T2DM and pancreatic fibrosis.

Methods: Male OLETF rats were divided into 2 groups at 6 weeks of age. The first

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group received a standard diet until the end of experimental period at age 28 weeks. The

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second group was given a diet containing 0.05 % atorvastatin from 6 weeks of age,

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before the onset of IR and pancreatic fibrosis. The age-matched Long-Evans Tokushima Otsuka rats without presence of IR, T2DM and pancreatic fibrosis, received a standard

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diet and were used as a normal control. Results: Atorvastatin slightly decreased serum fasting glucose and insulin levels, but significantly improved index of IR compared with the untreated OLETF rats. In addition, atorvastatin markedly decreased transforming growth factor-β1 mRNA expression, myeloperoxidase activity and proportion of fibrotic area, and elevated superoxide dismutase activity in the pancreas compared with the untreated OLETF rats. Conclusions: These findings suggest that atorvastatin exerts favorable influence on

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progression of IR and pancreatic inflammation and fibrosis via pleiotropic effect such as

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anti-oxidative property.

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Keywords: atorvastatin, pancreatic fibrosis, insulin resistance, OLETF rat

Abbreviations:

AUC; area under curve, FFA; free fatty acids, H&E; hematoxylin and eosin,

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HMG-CoA; 3-hydroxy-3-methylglutaryl-coenzyme A, HOMA-IR; homeostasis model

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assessment of insulin resistance, IVGTT; intravenous glucose tolerance test, LETO;

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Long-Evans Tokushima Otsuka, MPO; myeloperoxidase, OLETF; Otsuka Long-Evans Tokushima Fatty, PSCs; pancreatic stellate cells, SOD; superoxide dismutase, T-cho;

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total cholesterol, TG; triglyceride, TGF-β1; transforming growth factor-β1, TNF-α; tumor necrosis factor-α, T2DM; Type 2 diabetes mellitus, -SMA; -smooth muscle actin, 4-HNE; 4-hydroxy-2-nonenal, 8-OHdG; 8-hydroxy-2'-deoxyguanosine

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1. Introduction

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The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is a diabetic strain established

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from outbred colony of the Long-Evans rat [ ]. This strain of rats lacks cholecystokinin-1 receptor and thus, exhibits a within-meal feedback defect for satiety,

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resulting in hyperphargia and obesity [2]. The male OLETF rats begin to gain weight faster than normal control Long-Evans Tokushima Otsuka (LETO) rats from 5 weeks of age, and the difference gradually increases with age. The fed plasma glucose level of the

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OLETF rats significantly increases from 18 weeks of age compared with those of the

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LETO rats [ ]. In addition, the OLETF rats appear to develop significant hepatic

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steatosis from 20 weeks of age without inflammation or fibrosis [3]. The glucose intolerance is followed by a gradual increase in plasma glucose and insulin, reaching

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levels that are three to four times higher than normal rats. During the progression of type 2 diabetes mellitus (T2DM), the OLETF rats eventually become hypoinsulinemic and develops type 1 like DM after 70 weeks of age [ ]. Histologically, mild to moderate lymphocyte infiltrates into or around the islets and into the acinus area in the pancreas at 6-20 weeks of age. After 20 weeks of age, fibrosis becomes prominent in the islets and progresses into the acinus area [1]. Therefore, the OLETF rats are used as an animal model of pancreatic fibrosis as well as IR and T2DM [4]. Page - 4 -

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Statins are widely used in the first line management of hyperlipidemia due to

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their known efficacy in improving plasma lipid profiles [5]. However, previous studies have suggested that statins exert beneficial pleiotropic effects, such as anti-inflammatory,

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anti-fibrotic and anti-oxidative actions [6, 7] beyond their lipid lowering effect. Then we

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previously reported that treatment with pravastatin prevented the progression of T2DM by reducing IR in the OLETF rats probably via its anti-oxidative action [4]. However, clinical [8, 9] and experimental [10-12] studies have investigated the effect of statin

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therapy on IR or glucose intolerance, their results were still controversial. In addition,

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several experimental studies [13, 14] reported that atorvastatin exerted unfavorable

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influence on IR with pravastatin. On the other hand, pancreatic fibrosis is progressive and irreversible, therefore, chronic pancreatitis is considered to be one of the intractable

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pancreatic diseases. Previously, we reported that pravastatin prevented progression of pancreatic fibrosis via its pleiotropic actions [4, 15] and our results suggest that pravastatin exerts therapeutic effect on chronic pancreatitis. However, only a few studies have examined the effect of statins on pancreatic fibrosis in vivo [4, 15]. In the present study, we therefore examined whether atorvastatin elicits beneficial effects on IR and pancreatic fibrosis in the OLETF rats.

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2. Methods

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2.1. Ethical Approval

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The experimental protocol was approved by our institutional animal welfare committee. 2.2. Animals and drug

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The male OLETF and LETO rats at 5 weeks of age were supplied by Otsuka Pharmaceutical Co. (Tokushima, Japan). LETO rats that are developed from the same colony of the OLETF rats, but without presence of hyperphargia, obesity, IR and

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pancreatic fibrosis were used as a normal control [ ]. These rats were maintained in a

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temperature (23 ± 2 °C) and humidity (55 ± 5 %) controlled room with a 12:12-h

guidelines

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light-dark cycle (lights on at 7:00), and received standard care according to the at

our

institution.

Atorvastatin,

a

competitive

inhibitor

of

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3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA), was provided by Pfizer Inc. (New York, NY, USA). 2.3. Administration of Atorvastatin Standard rat diet consisting of 61% carbohydrate, 26% protein and 13% fat (as a percentage of calories, 3.596 kcal/g diet; Oriental Yeast, Tokyo, Japan) was powdered, and atorvastatin was added and thoroughly mixed to a final concentration of 50 mg/100 g food. The drug-diet powder mixture was reconstituted into pellets with a normal Page - 6 -

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appearance. The OLETF rat of 600 g body weight consumes almost 30 g food/day.

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Therefore, the daily dosage of atorvastatin taken by the OLETF rat was approximately

This atoravastatin

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15 mg/rat, which is equivalent to 25 mg/kg body weight

concentration was selected based on the previous studies [14, 16].

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2.4. Experimental Protocol

The OLETF rats were randomly divided into 2 groups (n=10 per group) at 6 weeks of age. The first group of rats received a standard rat diet until the end of experimental

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period at age 28 weeks (O-C group). The second group received the diet containing

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atorvastatin from 6 weeks, before the onset of IR and pancreatic fibrosis, until the end

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of experiment (O-A group). For the normal control, the third group of rats consisting of 10 LETO rats received a standard rat diet throughout the experimental period (L-C

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group). All groups of rats were allowed free access to food and water. The body weight and food intake were measured every 4 weeks. Blood samples were collected to determine fasting serum glucose and insulin concentrations every 4 weeks. At 28 weeks of age, blood samples were collected to determine fasting serum total cholesterol (T-cho), triglycerides (TG) and free fatty acids (FFA), and then an intravenous glucose tolerance test (IVGTT) was performed after a 12 h-fasting under sodium pentobarbital anesthesia (50 mg/kg body wt., i.p.). A bolus dose of 0.2 g/kg body wt. glucose was Page - 7 -

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injected into the jugular vein, and blood samples were collected before and at 5, 10, 30

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and 60 min after glucose loading to determine serum concentrations of glucose and insulin. Rats were sacrificed and the pancreas, liver and adipose depots were taken

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under pentobarbital anesthesia (50 mg/kg body wt ip) at several days after IVGTT. The

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pancreas and liver were cleared of lymph nodes and fat and then weighed. A duodenal portion of the pancreas and a random portion of the liver were used for histological examination. A splenic portion of the pancreas and a portion of the liver were frozen at -

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80 °C for the subsequent assays. White adipose depots were collected from the

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retroperitoneum, mesentery and epididymis, and then weighed.

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2.5. Assays

For the measurement of pancreatic content of protein and amylase activity, the pancreas

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was homogenized in a 0.15 M sodium chloride solution using a motor-driven, teflon-coated glass homogenizer. The homogenates were filtered through three layers of gauze and then sonicated for 1 min. The aqueous phase was used for assays. Protein concentration was measured using Folin phenol reagent with bovine serum albumin (BSA) as a standard [17]. Amylase activity was determined by a chromogenic method with a Phadebas amylase test (Phadebas, Lund, Sweden). Activity of a primary antioxidant enzyme, total superoxide dismutase (SOD), including mitochondrial and

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cytoplasmic SOD, in the pancreas and liver was determined using water-soluble

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tetrazolium salt (SOD Assay Kit-WST; Dojindo Molecular Technologies, Inc, Kumamoto, Japan). Neutrophil sequestration in the pancreas was quantified by

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measuring tissue myeloperoxidase (MPO) activity [18]. The hydroxyproline assay was

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performed as a marker of collagen synthesis in the pancreas using Chloramine T and Ehrlich’s reagent [19]. Hepatic TG content was determined by using a method of Folch et al. [20]. Serum T-cho, TG and FFA concentrations were analyzed enzymatically using Insulin

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commercially available kits (Wako Pure Chemical, Tokyo, Japan).

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concentrations in serum and pancreatic homogenates were determined by RIA using a

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commercially available kit (Rat insulin RIA kit; LINCO Research, Missouri, USA) with crystalline rat insulin (Novo Industria, Copenhagen, Denmark) as a reference standard.

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Serum glucose concentration was determined by the glucose-oxidase method [21] using a commercially available kit (Glucose-E reagent; International Reagents Co., Kobe, Japan). 2.6. Evaluation of IR and Insulin Secretory Function IR was estimated by the homeostasis model assessment of insulin resistance (HOMA-IR) index [22] and IR index [23]. HOMA-IR was calculated with the following formula: fasting insulin (picomolar) × fasting glucose (millimolar) / 22.5. IR index was

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evaluated from serum insulin and glucose responses after intravenous glucose load, and

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was calculated with the following formula: the area under curve (AUC) of serum glucose response × AUC of insulin response. Lower values of HOMA-IR and IR index

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indicate higher insulin sensitivity. The insulin secretory function was evaluated by the

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insulinogenic index [24]. The insulinogenic index is widely used as an index of early-phase insulin response, calculating with the formula: increment of plasma insulin (0 to 5 min) / increment of plasma glucose (0 to 5 min).

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2.7. Quantitative Real-Time Reverse Transcription (RT)-PCR

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To investigate the mechanisms of anti-inflammatory and anti-fibrotic actions of

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atorvastatin, the expression levels of transforming growth factor (TGF)-β1, tumor necrosis factor (TNF)-α and interleukin (IL)-10 mRNA were determined by quantitative

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TaqMan PCR with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as a reference. Total RNA was extracted from the frozen pancreatic tissue by the acid guanidium thiocyanate/phenol/chloroform method [25], and then real-time RT-PCR of TGF-β1, TNF-α, IL-10 and GAPDH were performed using ABI PRISM 7000 sequence detection system (Applied Biosystemss, Foster City, CA, USA). The amounts of TGF-β1, TNF-α and IL-10 transcripts were normalized to the amount of GAPDH transcripts in the same cDNA (expressed as fold change per GAPDH).

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2.8. Histological Examination

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The pancreatic and hepatic tissues were fixed overnight in 4% buffered neutral paraformaldehyde solution, embedded in paraffin, and deparaffinized by standard

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procedure. Thin sections (5 μm) were stained with hematoxylin and eosin (H&E), and

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Azan-Mallory staining for light microscopic examination.

2.9. Quantitative Analysis for Cross Sectional Area of Islets and Distribution of Fibrosis in the Exocrine Glands and Islets of the Pancreas

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Quantitative evaluation of cross sectional area of islets, proportion of fibrotic area in the

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exocrine glands and islets were performed by using an AxioVert 135 microscope (Carl

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Zeiss, Eching, Germany) connected to the digital image processing software, AxioVision 4.8. (Carl Zeiss). Twenty non-overlapping fields and islets of Azan-Mallory

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staining in the pancreas were randomly selected in each experimental group at a × 100 magnification. The area of total pancreatic specimen or cross section of islet and that of blue-stained fibrosis were determined. The proportion of fibrotic area in the exocrine gland and islet were calculated using the following equation: fibrotic area / total area. 2.10. Immunohistochemistry for Detection of Activated Pancreatic Stellate Cells (PSCs), Oxidatively Stressed Cells and Apoptotic Cells in the Pancreas and Oxidatively Stressed Cells in the Liver

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For detection of activated PSCs, oxidatively stressed cells and apoptotic cells in the oxidatively

immunohistochemistry

for

stressed

cells

-smooth

in muscle

the

liver,

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and

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pancreas

actin

we

conducted

(-SMA)

[26],

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8-hydroxy-2'-deoxyguanosine (8-OHdG) [27] and single-stranded regions in the DNA

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(ss-DNA) [28] in the pancreas and 4-hydroxy-2-nonenal histidine adduct (4-HNE) [29] in the liver. For immunohistochemistry for -SMA, ss-DNA and 4-HNE, tissue sections were immersed in PBS (pH 7.2) for 10 min and then in PBS containing 3% H2O2 for 10

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min to quench endogenous peroxidases. Thereafter, the tissue sections were incubated

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with a specific primary antibody for -SMA (Dako Corporation, Carpinteria, CA, USA)

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diluted at 1:50, a specific antibody for ss-DNA (Dako Corporation) diluted at 1:100, or a specific antibody for 4-HNE (HNEJ-2; Japan Institute for the Control of Aging,

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Shizuoka, Japan) diluted at 1: 300 in PBS for 16 hr at 4 °C. The primary antibodies were visualized by the labeled streptavidin-biotin method using a commercially available kit (Dako Corporation). For detection of immunoreactive product of 8-OHdG, the avidin-biotin complex method was used as previously described [30]. The tissue sections were autoclaved for 10 min at 121 °C in 10 mM citrate buffer (pH 6.0). Then the following were sequentially applied: normal rabbit serum (diluted at 1:75) (Dako Corporation), N45.1, biotin-labeled rabbit anti-mouse immunoglobulin (Ig) G serum

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(diluted at 1:300) (Japan Institute for the Control of Aging) and anvidin-biotin-alkaline

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phosphate complex (diluted at 1:100) (Vector Laboratories, Burlingame, CA, USA). All procedures were performed with protocols as recommended by the manufacturer. Quantitative

Analysis

of

Stress

and

Apoptosis

in

the

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Immunohistochemistry

Oxidative

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2.11.

Since immunoreactivity of 8-OHdG and ss-DNA are observed in the nuclear compartment, and immunoreactivity of 4-HNE is detected in the cytoplasmic

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compartment, it is possible to quantify the immunoreactions in each experimental group.

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To determine indices of 8-OHdG, ss-DNA and 4-HNE, 20 non-overlapping fields and

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20 non-overlapping islets were randomly selected from 5 rats in each experimental group at × 200 magnification. At least 300 acinar cells and hepatocytes in each field and

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at least 30 cells in each islet were counted, and the indices of 8-OHdG, 4-HNE and ss-DNA represented the percentage of positive cells. 2.12. Statistical Analysis Each experiment was performed in 10 rats, and results were expressed as mean ± SEM. Multiple comparisons among groups were performed by one-way analysis of variance (ANOVA) with post hoc Tukey test or nonparametric ANOVA followed by Kruskal-Wallis test as appropriate using SPSS version 22 (IBM, Armonk, NY, USA).

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Differences with P < 0.05 were considered as statistically significant.

3. Results

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3.1. Serial Changes of Body Weight and Daily Food Intake

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The daily food intake in the L-C group was approximately 21 g/day and that in the both O-C and O-A groups were approximately 32 g/day. Its value in the O-C group was significantly higher than that in the L-C group, but there was no statistically significant

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difference in the values between the O-C and O-A groups (Figure 1A).

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Body weights in the all experimental groups increased with age. Body weight

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in the O-C group was significantly higher than that in the L-C group during

experimental period. Serial change of body weight in the O-A group was similar to that

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in the O-C group (Figure 1B).

3.2. Serial Changes of Fasting Serum Glucose and Insulin Concentrations, and Serum Glucose and Insulin Responses to Intravenous Glucose Tolerance Test at 28 Weeks of Age Fasting serum glucose (Figure 2A) and insulin (Figure 2B) concentrations in the O-C group were progressively elevated during observation period. In contrast, these levels in the L-C group remained unchanged from initial level. These levels in the O-A group

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were slightly lower than those in the O-C group, but there was no significant difference

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during experimental period.

At 28 weeks of age, all time points of serum glucose and insulin levels before

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and after glucose load in the O-C group were significantly higher than those in the L-C

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group (Figure 3A, B). Although all time points of glycemic and insulin secretory responses in the O-A group were lower than those in the O-C group, significant difference was observed at only one time point of serum glucose level (Figure 3A).

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3.3. Evaluation of IR and Insulin Secretory Function

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Chronological alteration of IR estimated by HOMA-IR in the O-C group

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markedly increased with age (Figure 2C). However, its level in the L-C group maintained initial level during experimental period. HOMA-IR in the O-A group was

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lower than that in the O-C group, and significant differences were detected at 24 and 28 weeks of age (Figure 2C). AUCs of glucose and insulin levels during IVGTT and IR index in the O-C group were significantly higher than those in the L-C group (Table 1). Although there was no significant difference in AUCs of glucose and insulin levels between O-C and O-A groups, IR index in the O-A group was significantly lower than that in the O-C group (Table 1).

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Insulin secretory function evaluated by insulinogenic index in the O-C group

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was significantly lower than that in the L-C group (Table 1). Although its index in the

(Table 1).

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3.4. Weight of White Adipose Depots

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O-A group was higher than that in the O-C groups, the difference was not significant

weights of mesentery, epididymis and retoroperitoneum adipose depots in the o-c group were significantly higher than those in the l-c group (table 2). weights of these adipose

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depots in the o-a group were similar levels to those in the o-c groups (table 2).

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3.5. Fasting Serum T-cho, TG and FFA Concentrations at 28 Weeks of Age

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Fasting serum T-cho, TG and FFA concentrations in the O-C group were higher than those in the L-C group and statistically significant differences were detected in the

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concentrations of T-cho and TG (Table 2). Although concentrations of these lipids in the O-A group were lower than those in the O-C group, these differences were not significant (Table 2). 3.6. Pancreatic Weight and Its Contents of Protein, Amylase Activity, and Insulin At age 28 weeks, the pancreatic weight and its contents of protein and amylase activity in the O-C group significantly decreased compared with those in the L-C group (Table 3). However, these parameters in the O-A group significantly increased compared with

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those in the O-C group. In contrast, insulin content in the O-C group was significantly

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higher than that in the L-C group and was similar level to that in the O-A group (Table 3).

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3.7. Hydroxyproline Level and Activities of Myeloperoxidase and Superoxide Dismutase

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in the Pancreas

In the O-C group, hydroxyproline level and MPO activity significantly increased, whereas SOD activity significantly decreased compared with those in the L-C group

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(Table 3). However, in the O-A group, hydroxyproline level and MPO activity were

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O-C group (Table 3).

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significantly lower, whereas SOD activity was significantly higher than those in the

3.8. Expression levels of transforming growth factor (TGF)-β1, Tumor Necrosis Factor

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(TNF)-α and Interleukin (IL)-10 mRNA in the Pancreas Expression levels of TGF-β1 and TNF-α mRNA markedly up-regulated in the O-C group compared with those in the L-C group (Table 3). However, expression levels of these mRNA in the O-A group down-regulated compared with those in the O-C group and significant difference was obtained in TGF-β1 mRNA expression. In contrast, expression level of IL-10 mRNA in the O-A group was significantly elevated compared with that in the O-C and L-C groups (Table 3).

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3.9. Hepatic Weight and Its Contents of TG and Superoxide Dismutase Activity

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The hepatic weight and its content of TG in the O-C group were significantly higher

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than those in the L-C group (Table 3). However, in the O-A group, the hepatic weight and TG content were significantly lower than those in the O-C group. When the hepatic

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weight was expressed as per 100g body weight, its value in the O-A group was almost same level as that in the L-C group (Table 3). In contrast, SOD activity in the O-C group was significantly lower than that in the L-C group, and its activity in the O-A

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group was significantly higher than that in the O-C group (Table 3).

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3.10. Histological Findings of the Pancreas and Liver

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minimal histological changes were noted in the pancreas in the

L-C group (Figure 4A). However, most of islets were enlarged and fibrotic,

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distributions of inflammatory cells and fibrosis were observed in the inter- and intra-lobular areas of the pancreas in the O-C group Figure 4B Although some islets were enlarged, these histological alterations were attenuated in the O-A group (Figure 4C). Azan-Mallory staining clearly demonstrated that blue-stained fibrotic area was rarely noted in the L-C group (Figure 4D). However, marked fibrosis distributed in the islets and the inter- and intra-lobular areas of the pancreas in the O-C group (Figure 4E). In contrast, progression of fibrosis was strongly attenuated in the O-A group (Figure 4F). Page - 18 -

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In the liver, histological alterations were not noted in the L-C group (Figure 5A).

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However, the vacuoles in the hepatocytes, most likely indicating lipid moieties, were

attenuated in the O-A group (Figure 5C).

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commonly seen in the O-C group (Figure 5B). In contrast, its distribution was markedly

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3.11. Quantitative Analysis for Proportion of Fibrotic Area in the Exocrine Glands and Islets and Cross Sectional Area of the Islets in the Pancreas Quantitative analysis using AxioVision 4.8 demonstrated that proportion of fibrotic area

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in the exocrine glands and islets in the O-C group significantly increased compared with

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those in the L-C group (Table 4). However, these levels in the O-A group were

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significantly lower than those in the O-C group. The cross sectional area of the islets in the O-C group was significantly higher than that in the L-C group. However, its area in

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the O-A group was significantly lower than those in the O-C groups (Table 4). 3.12. Immunohistochemistry for Detection of Activated PSCs, Oxidatively Stressed Cells and Apoptotic Cells in the Pancreas and Oxidatively Stressed Cells in the Liver In the L-C group, activated PSCs which express α-SMA were rarely detected except in the vessel walls in the pancreas (Figure 6A). However, 

positive cells were

markedly distributed in the inter- and intra-lobular areas and islets in the O-C group . In contrast, distribution of these positive cells was markedly attenuated Page - 19 -

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in the O-A group (Figure 6C).

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Oxidatively stressed cells detected with staining for 8-OHdG were rarely noted

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in the pancreas in the L-C group (Figure 6D), but were markedly observed in the atrophic and fibrotic lesions of exocrine glands and islets in the O-C group (Figure 6E).

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However, immunoreactivity of 8-OHdG was slightly detected in the O-A group (Figure 6F).

Apoptotic cells detected with antibody for ss-DNA were rarely noted in the . However, these positive cells were distributed

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pancreas in the L-C group

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in the atrophic or fibrotic lesion of the intralobular areas and islets in the O-C group

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(Figure 6H . In contrast, distribution of these positive cells was markedly attenuated in the O-A group (Figure

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Oxidatively stressed cells detected with staining for 4-HNE were rarely noted

in the liver in the L-C group (Figure 5D), but were markedly observed in the O-C group, especially in the hepatocytes with vacuoles (Figure 5E). In contrast, expression of its immunoreactivity was strongly attenuated in the O-A group (Figure 5F). 3.13.

Quantitative

Analysis

of

Oxidative

Stress

and

Apoptosis

in

the

Immunohistochemistry Indices of 8-OHdG in both of the exocrine glands and islets of the pancreas in Page - 20 -

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the O-C group significantly increased compared with those in the L-C group, and these

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indices in the O-A group were significantly lower than those in the O-C group (Table 4). Indices of ss-DNA in the exocrine glands and islets of the pancreas in the O-C group

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were significantly higher than those in the L-C group (Table 4). In contrast, these

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indices in the exocrine glands and islets in the O-A group were lower than those in the O-C group, and statistically significant difference was obtained in the exocrine glands alone (Table 4). Index of 4-HNE in the liver in the O-C group was significantly elevated

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compared with that in the L-C group (Table 4). However, its index in the O-A group

4. Discussion

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was significantly lower than that in the O-C group.

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The present study clearly demonstrated that atorvastatin treatment exerts beneficial effects on progression of pancreatic inflammation, fibrosis and IR in the OLETF rats. Although several studies demonstrated that statins exert different pleiotropic effects [13, 14, 31], and atorvastatin has an unfavorable impact on IR compared with pravastatin [13, 14], the present study indicated that beneficial pleiotropic effects of atorvastatin are similar to those of pravastatin in our former study [4]. In support of our view, a previous study also demonstrated that the effects of atorvastatin on thrombogenic and

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inflammatory parameters are similar to those of pravastatin and simvastatin [32].

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For the mechanisms of the beneficial effects of atorvastatin on the pancreatic inflammation and fibrosis, it is conceivable that down-regulation of the expression of

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TNF-α and TGF-β1 mRNA and up-regulation of the expression of IL-10 mRNA in the

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pancreas played important roles. It is well documented that TNF-α and TGF-β1 are inflammatory and fibrogenic cytokines and these are involved in the onset of pancreatic inflammation and fibrosis [33], whereas IL-10 inhibits production of inflammatory

ED

cytokines and suppresses inflammatory response [34]. For another mechanism of the

PT

beneficial effects of atorvastatin, we confirmed that atorvastatin exerted anti-oxidative

CE

action in the pancreas, as previous studies have shown [35]. Because oxidative stress has been implicated in the pathophysiology of chronic pancreatitis [36], atorvastatin

AC

seemed to inhibit progression of inflammation and fibrosis in the pancreas via its anti-oxidative action. Previous studies have demonstrated that PSCs are identified [26] and characterized that they are activated upon exposure to cytokines such as TGF-β1 and TNF-α and have a capacity to produce extracellular matrix proteins [37]. In the current study, since treatment with atorvastatin decreased the production of TGF-β1 and TNF-α mRNA, it is quite possible that atorvastatin prevented activation of PSCs, and thereby

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attenuated progression of pancreatic fibrosis.

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The present study showed that treatment with atorvastatin suppressed apoptosis in the pancreas, because atorvastatin significantly attenuated immunoreactivity of

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ss-DNA compared with untreated OLETF rats. Since atorvastatin treatment prevented

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the decrease in pancreatic weight and contents of protein and amylase activity compared with those in the control group of OLETF rats, it seems likely that atorvastatin exerted anti-apoptotic action and thus prevented pancreatic atrophy. For the mechanisms of this

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action, previous studies showed that oxidative stress [38] and inflammatory mediators

PT

such as TGF-β1 and TNF-α [39] strongly induce apoptosis, we therefore speculated that

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atorvastatin inhibits apoptosis via its anti-oxidative and anti-inflammatory actions. It is well documented that IR is closely associated with obesity, in particular,

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abdominal adiposity [40]. However, since atorvastatin prevented the progression of IR without reducing body weight gain and amount of abdominal adipose depots, present study suggests that atorvastatin attenuated the progression of IR via its pleiotropic effect. Similarly, previous experimental studies also demonstrated that statins [4, 12, 16, 41], including atorvastatin [12, 16], improve IR in obese and diabetic models. In addition, we demonstrated that atorvastatin strongly reduced hepatomegaly and liver steatosis as previous clinical and experimental studies have shown [42, 43]. Accumulating data

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suggest that oxidative stress plays an important role in the pathogenesis of steatosis [44],

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and deposition of fat in the liver results in the development of IR [45]. Therefore, we speculated that atorvastatin prevented steatosis in the liver via its anti-oxidative action,

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and thereby attenuated progression of IR in the OLETF rats. In addition, a previous

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study showed that oxidative stress induces IR [46], thus it is also possible that anti-oxidative action of atorvastatin directly prevented the progression of IR. The present results suggest the possibility that atorvastatin has a role to

ED

maintain insulin secretory function, because atorvastatin treatment tended to increase

PT

insulinogenic index and markedly attenuated progression of fibrosis and oxidative stress

CE

in the islets. Since a previous study showed that antioxidant reduces the susceptibility of pancreatic islets to oxidative stress and preserves insulin secretory capacity [47], it is

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therefore likely that anti-oxidative property of atorvastatin exerted beneficial effects on insulin secretory function. In support of this observation, we previously demonstrated that pravastatin maintains insulin secretory function in the OLETF rats until 80 weeks of age [4]. Influences of statin therapy on the new onset of diabetes have been well documented, but conflicting findings were reported [48-51]. Pravastatin was first reported to reduce diabetes occurrence among initial nondiabetic individuals enrolled in

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the West of Scotland Coronary Prevention Study [48]. However, recent analysis of

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randomized clinical trial showed a slightly higher incidence of diabetes in the treatment arm [49-51]. Recently, several possible mechanisms are proposed [52], but it is also

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suggested that previous retrospective studies on statin-related new onset of diabetes

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have limitations [53]. Although further studies are required, present study provides experimental evidence that atorvastatin might exert beneficial effect on the new onset of diabetes.

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There are at least 4 limitations of study. First, this study is an animal study and

PT

the study findings may not be generalizable to humans. Second, although we

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demonstrated that atorvastatin prevented the progression of IR and pancreatic fibrosis via its anti-oxidative action, we cannot exclude the possibility that atorvastatin induced

AC

these beneficial effects via other mechanisms. Third, regarding beneficial effect of atorvastatin on IR, we did not investigate glucose utilization or uptake in the liver, skeletal muscle or adipose tissue, and its molecular mechanisms. Fourth, we tested single statin alone. Since there are controversies regarding the effect of statins on IR (8, 9, 11, 12), further studies examined different agents may be necessary in the future. On the other hand, several strengths of the present study have also been identified. First, the OLETF rats has been used extensively as a model of obesity-associated IR and T2DM,

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and many characteristics of this animal model have striking resemblance to human

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disease. Second, pathological alterations in the pancreas of this animal model are similar to those of human chronic pancreatitis, and only few studies [4, 15] examined the effect

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of statin on inflammation and fibrosis in the pancreas. Third, although clinical studies

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have uncontrolled confounding factors such as varying dose, type or duration of statin treatment, baseline T2DM risk factors and influence of other medications and how insulin sensitivity was assessed, present experimental study was not influenced by these

ED

factors.

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In conclusion, our experimental study demonstrated that treatment with

CE

atorvastatin attenuated progression of IR, pancreatic inflammation and fibrosis via pleiotropic effects such as anti-oxidative action in the OLETF rats. In addition, these

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beneficial effects of atorvastatin were similar to those of pravastatin in our previous study [4]. If these beneficial effects are demonstrated to occur in humans treated with statins, the pleiotropic effect of satins clinically becomes increasingly important and useful for patients with chronic pancreatitis, IR or T2DM. Hopefully, further studies clarify these beneficial effects of statins in humans.

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Acknowledgments

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We thank Hitomi Mihara, Masami Oe and Yuka Katsuki for the excellent technical assistance.

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Funding

Research Foundation of Japan. Disclosure statement

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This work was supported by Sasagawa Japan China Medical Association and Pancreas

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Author contributions

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There is none of conflict of interest for all authors.

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Design and conduct of the study: Limin Wei, Mitsuyoshi Yamamoto, Makoto Otsuki Data collection and analysis: Limin Wei, Mitsuyoshi Yamamoto, Makoto Otsuki

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Data interpretation and manuscript writing: Limin Wei, Mitsuyoshi Yamamoto, Makoto Otsuki

Final approval of the version to be submitted: Limin Wei, Mitsuyoshi Yamamoto, Masaru Harada, Makoto Otsuki

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Figure Legends

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Figure 1: Serial changes of daily food intake (A) and body weight (B) in rats treated with or without atorvastatin. △: The L-C group given standard rat diet during entire

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experimental period. ○ : The O-C group given standard rat diet during entire

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experimental period. ■: The O-A group given atorvastatin containing diet from 12 to 28 weeks of age. Data are presented as mean ± SEM of 10 rats. +: Significant difference vs. the value in the L-C group at corresponding age (+ P < 0.05, ++ P < 0.01). ×:

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Significant difference vs. the value in the O-C group at corresponding age (× P < 0.05,

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×× P < 0.01). Multiple comparisons among groups were performed by one-way analysis

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of variance (ANOVA) with post hoc Tukey test or nonparametric ANOVA followed by

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Kruskal-Wallis test as appropriate. Figure 2: Serial changes of fasting serum glucose (A) and insulin (B) concentrations and HOMA-IR (C) in rats treated with or without atoravastatin. Data are presented as mean ± SEM of 10 rats. For key to the groups and statistical analysis, see Figure 1. Figure 3: Serum glucose (A) and insulin (B) responses to an intravenous glucose tolerance test (IVGTT) in rats treated with or without atorvastatin at 28 weeks of age. Data are presented as mean ± SEM of 10 rats. For key to the groups and statistical analysis, see Figure 1.

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Figure 4: Representative light microscopic appearances of the pancreas stained with

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H&E (A)-(C) and with Azan-Mallory staining (D)-(F) in the each experimental group at 28 weeks of age. (A): Minimum histological alterations were noted in the L-C group.

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(B): Islets were enlarged, and inflammatory cells and fibrosis distributed in the islets

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and inter- and intra-lobular area in the O-C group. (C): Although some islets were enlarged, infiltrations of fibrosis and inflammatory cells were attenuated in the O-A group. D: Azan-Mallory staining revealed that fibrosis was rarely noted in the L-C

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group. (E): In contrast, marked fibrosis distributed in the periductal, islets and inter- and

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intra-lobular areas in the O-C group. (F): Distribution of fibrosis was markedly

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attenuated in the O-A group. Original magnification, × 100. Scale bar indicates 100 μm. For key to the groups, see Figure 1.

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Figure 5: Representative light microscopic appearances of the liver stained with H&E (A)-(C) and immunohistochemistry for 4-HNE (D)-(F) in the each experimental group at 28 weeks of age. (A): There were notable histological changes in the L-C group. (B): Elevated numbers of vacuoles, indicating hepatic steatosis, were seen in the O-C group. (C): However, steatosis was markedly reduced in the O-A group. (D): Immunoreactivity for 4-HNE was observed only weak in the L-C group. (E): Particular intense staining was observed in the cytoplasm of the hepatocytes with steatosis. (F): However, only

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faint staining was found in the O-A group. (A)-(C): Original magnification, × 100.

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Scale bar indicates 100 µm. (D)-(F): Original magnification, × 200. Scale bar indicates 50 µm.

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Figure 6: Immunohistochemistries for -SMA (A)-(C), 8-OHdG (D)-(F) and ss-DNA

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(G)-(I) in the pancreas in the each experimental group at 28 weeks of age. (A): Immunoreactivity for -SMA was rarely noted except in the vessel walls in the L-C group. (B): In contrast, -SMA-positive cells were markedly detected in the islets,

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periductal and interlobular areas in the O-C group. (C): Expression of -SMA was

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markedly attenuated in the O-A group. (D): Nuclear staining of 8-OHdG was rarely

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detected in the L-C group. (E): Intense immunoreactivity of 8-OHdG was observed in the pancreas, especially in the islet in the O-C group. (F): Immunoreactivity of 8-OHdG

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was markedly attenuated in the O-A group. (G): A minimal number of apoptotic cells detected with antibody for ss-DNA were observed in the L-C group. (H): Several apoptotic cells were distributed in one intralobular area especially in the atrophic and fibrotic portion of the pancreas in the O-C group. (I): However, expression of ss-DNA-positive cells was markedly attenuated in the O-A group. Original magnification, ×200. Scale bar indicates 50 μm. For key to the groups, see Figure 1.

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Table 1 Effects of atorvastatin on parameters of insulin resistance (IR) and insulin secretory function.

AUC glucose AUC insulin (×103)

13.1 ± 1.4 19.9 ± 2.6

61.1 ± 7.4 ++ 9.4 ± 1.5 ++

40.6 ± 4.7 ++, × 12.7 ± 1.9

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IR index (×106) Insulinogenic index (×103)

O-C O-A0 ++ 895.1 ± 64.2 728.5 ± 49.8 ++ 67.3 ± 5.3 ++ 55.1 ± 3.8 ++

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L-C 461.1 ± 12.9 28.3 ± 2.7

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Values were mean ± SEM of 10 rats. +: Significant difference vs. the value of the L-C group (+ P < 0.05, ++ P < 0.01). ×: Significant difference vs. the value of the O-C group (× P < 0.05, ×× P < 0.01). Multiple comparisons among groups were performed by one-way analysis of variance (ANOVA) with post hoc Tukey test or nonparametric ANOVA followed by Kruskal-Wallis test as appropriate. The O-C group consisting of the OLETF rats received a standard rat diet until the end of experiment at age 28 weeks.

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The O-A group consisting of the OLETF rats received the diet containing atorvastatin from 6 weeks of age until the end of experiment. The L-C group consisting of the LETO rats received a standard rat diet throughout the experimental period.

Table 1. Wei et al.

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O-C

5.3 ± 0.1

15.2 ± 0.9 ++

13.4 ± 0.8 ++

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Epididymis (g) Retroperitoneum (g) Total (g) Serum lipid levels T-cho (mmol/l) TG (mmol/l) FFA (mmol/l)

L-C

13.4 ± 0.9 ++ 26.3 ± 2.2 ++ 54.8 ± 3.3 ++

14.1 ± 0.8 ++ 24.1 ± 1.0 ++ 51.6 ± 1.5 ++

3.1 ± 0.1 ++ 2.6 ± 0.4 ++ 0.7 ± 0.1

2.8 ± 0.2 1.8 ± 0.3 ++ 0.6 ± 0.1

6.4 ± 0.5 6.2 ± 0.4 17.9 ± 0.9

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Weight of fat depots Mesentery (g)

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Table 2 Effects of atorvastatin on distribution of fat depots and fasting serum lipid concentrations.

2.5 ± 0.1 0.5 ± 0.1 0.5 ± 0.1

O-A

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Values were mean ± SEM of 10 rats. +: Significant difference vs. the value of the L-C

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group (+ P < 0.05, ++ P < 0.01). ×: Significant difference vs. the value of the O-C group (× P < 0.05, ×× P < 0.01). For key to the groups and statistical analysis, see Table 1.

Table 2. Wei et al.

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L-C

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Table 3 Effects of atorvastatin on the parameters of inflammation, fibrosis and anti-oxidative action in the pancreas, and steatosis and anti-oxidative action in the liver. O-C

O-A

Pancreas 1.55 ± 0.02

1.40 ± 0.04

(mg/g 202.0 ± 12.0

Amylase activity (×103 IU/g pancreas)

± 185.7 13.6 ×

±

1.3 ± 0.2 + 2.0 ± 0.3

8.9 ± 1.4

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0.6 ± 0.1

(mg/g

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level pancreas)

0.06 ×

1.9 ± 0.2

Insulin (mmol/g pancreas) Hydroxyproline

147.5 5.7 ++

1.57 ±

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Protein pancreas)

+

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Weight (g)

± 20.0 1.9 ++

++

2.9 ± 0.6 +

1.2 ± 0.3 ×

SOD (×104

activity 1.9 ± 0.3 U/g

1.0 ± 0.1 +

1.8 ± 0.4 ×

TGF-β1 1.0 ± 0.2 (TGF-β1/GAPDH: fold increase over L-C)

4.5 ± 1.1 +

1.9 ± 0.4 ×

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17.8 2.2 ++

1.3 ± 0.2 0.9 ± 0.1 ×

MPO activity 1.0 ± 0.3 (U/g pancreas: fold increase over L-C)

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×

pancreas)

TNF-α 1.0 ± 0.4 (TNF-α/GAPDH: fold increase over L-C)

3.7 ± 1.0 + 2.2 ± 0.7

IL-10 1.0 ± 0.4 (IL-10/GAPDH: fold increase

4.5 ± 1.5 + 13.7 ± ++, ×× 3.1

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over L-C) Weight (g)

13.3 ± 0.3

21.2 ± 0.8 18.4 ± 0.5 ++, ××

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Weight/100g BW 3.00 ± 0.04 (g)

3.42 ± 3.08 ± ++ ×× 0.11 0.10

TG (nmol/g liver)

167.5 12.9

± 115.7

±

7.6

++, ×

2.1 ± 0.1 2.4 ± 0.1 ++

+, ××

MA NU

SOD activity 2.6 ± 0.1 4 (×10 U/g liver)

++

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49.9 ± 2.0

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Liver

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Table 4 Effects of atorvastatin on the parameters of fibrosis, size of islets, oxidative stress and apoptosis in the pancreas, and oxidative stress in the liver. L-C

O-C

MA NU

1.0 ± 0.2 1.2 ± 0.2

PT

Islets Liver 4-HNE (%)

7.2 ± 1.2 ++ 14.8 ± 2.5 ++

2.6 ± 0.5 +, × 6.0 ± 0.7 +, ×

16.0 ± 2.6

57.5 ± 11.6 ++

27.0 ± 5.1 ×

0.12 ± 0.06 0.32 ± 0.14

0.45 ± 0.10 ++ 5.46 ± 1.02 ++

0.18 ± 0.07 × 0.86 ± 0.22 +, ××

0.05 ± 0.03

0.35 ± 0.10 +

0.08 ± 0.03 ×

0.03 ± 0.03

0.25 ± 0.11 +

0.07 ± 0.05

1.5 ± 0.4

13.2 ± 1.0 ++

2.5 ± 0.6××

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Exocrine glands Islets Cross-sectional area (×103 μm2) Islets 8-OHdG (%) Exocrine glands Islets ss-DNA (%) Exocrine glands

SC

Pancreas Fibrosis (%)

O-A

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