models have several advantages [2, 4, 16, 21, 22]. In ... tified using NIH Image software with custom-written ... Quantity One Software (PDI, New York, NY).
Exp. Anim. 58(2), 113–121, 2009
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Aortic ER Stress in Streptozotocin-Induced Diabetes Mellitus in APA Hamsters Masaki KUROKAWA, Makoto HIDESHIMA, Yoshiyuki ISHII, Shigeru KYUWA, and Yasuhiro YOSHIKAWA Department of Biomedical Science, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1–1–1, Bunkyo-ku, Tokyo 113-8657, Japan Abstract: Atherosclerosis , is thought to be associated with endoplasmic reticulum (ER) dysfunction and the accumulation of unfolded proteins. In this study, we examined the relationship between atherosclerosis and ER stress and the effect of sodium 4-phenylbutyrate (4-PBA), a kind of chemical chaperone, on atherosclerosis in streptozotocin-induced diabetic APA hamsters. Male, 8-week-old, APA hamsters were injected with streptozotocin (30 mg/ kg body weight) to induce diabetes mellitus, and ER stress was evaluated immunohistochemically or by semi-quantitative RT-PCR analysis using ER stress markers such as calreticulin and GPR78. Control hamsters were injected with citrate buffer and were similarly analyzed. In the aorta of control animals, a weak ER stress was detected, and 4-PBA treatment decreased the calreticulin- and GRP78-positive areas and also reduced the mRNA levels of calreticulin and GRP78. On the other hand, strong ER stress was detected at the lesser curvature of the aortic arch of streptozotocin-induced diabetic APA hamsters. However, 4-PBA treatment failed to lessen the ER stress in the aorta and had no effect on improvement of the atherosclerotic lesions. These results may provide an explanation for the complex etiology of atherosclerosis accompanied by diabetes mellitus and various other clinical phenotypes of atherosclerosis. Key words: aorta, APA hamster, diabetes mellitus, ER stress, streptozotocin
Introduction Diabetes mellitus (DM) is one of the most important problems for public health in developed countries due to recent marked increases in its prevalence and that of its complications. It is well known that DM strongly affects the vascular condition of sufferers and can cause life-threatening vascular complications, including nephropathy, retinopathy, and macro- and micro-vascular damage [13]. Atherosclerosis is the leading cause of
death among DM patients. DM is often complicated by hyperlipidemia including disorders of lipid metabolism and changes in lipoprotein composition and concentrations [25, 27]. In addition, DM facilitates non-enzymatic glycation of protein via the polyol pathway resulting in the accumulation of irreversible end-stage products, i.e., advanced glycation endproducts (AGEs), in blood and tissues. It is believed that the oxidative stress that arises from hyperlipidemia, AGEs, and inflammatory macrophages (foamy cells) is involved in the develop-
(Received 14 May 2008 / Accepted 31 October 2008) Address corresponding: S. Kyuwa, Department of Biomedical Science, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1–1–1, Bunkyo-ku, Tokyo 113-8657, Japan
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ment and progression of atherosclerosis [3, 11]. Some recent findings have shed light on the accumulation of unfolded proteins and endoplasmic reticulum (ER) dysfunction in the pathogenesis of atherosclerosis [31, 32]. The ER regulates protein folding and the quality control systems of membrane-bound and secreting proteins. When cells are exposed to stress that interferes with ER function, unfolded proteins accumulate in the ER lumen, which culminates in ER-mediated cell death [12]. In this situation, which is termed ER stress, several signaling pathways that are derived from the ER, such as the unfolded protein response (UPR) pathway, are simultaneously activated. Under such conditions, inositol-requiring kinase 1 (IRE1) [26, 28], a serine/ threonine kinase with an endoribonuclease domain in the ER membrane, senses the accumulation of unfolded protein in the ER and promotes the transcription of chaperone genes, such as glucose-regulated proteins (GRPs), including GRP78 and GRP94, so as to re-fold the unfolded proteins [25, 27]. In addition to native chaperone proteins, some chemical compounds such as glycerol, dimethylsulfoxide, and sodium 4-phenylbutyrate (4PBA), have been demonstrated to ameliorate the misfolding and mislocalization of proteins [5, 18–20, 23, 24]. 4-PBA is a low molecular weight fatty acid that has been approved for clinical use as an ammonia scavenger in children with urea cycle disorders [14] and for treatment of sickle cell anemia and thalassemia on the basis of its capacity to activate transcription of b- and g-globin [7, 8]. Various animal models of atherosclerosis have been developed and used to investigate the mechanisms of the disease, but there are extremely few models for atherosclerosis associated with DM complications. Syrian hamsters have been used as a model of diet-induced hyperlipidemia and atherosclerosis because hamster models have several advantages [2, 4, 16, 21, 22]. In particular, cholesterol and cholic acid metabolism are fundamentally similar in hamsters and humans [1]. In addition, Syrian hamsters of the APA strain (APA hamsters) are known to show a marked and continuous diabetic state accompanied by hypercholesterolemia and hypertriglycemia after a single injection of streptozotocin (SZ), which specifically attacks b-cells in pancreatic islets, and then develop atherosclerosis and glom-
erulosclerosis [10]. We previously reported that an analysis of the lipoproteins in cholesterol revealed a significant decrease in high density lipoproteins (HDL) and a significant increase in glycated low density lipoproteins (LDL) and oxidized LDLs [30]. Atheromatous lesions, so called “fatty streaks”, which are characterized by a subendothelial accumulation of many foamy cells, are locally generated in the aortic arch of diabetic APA hamsters even at 6 weeks after SZ injection. Diabetic APA hamsters then develop “fibrous plaques” consisting of a huge number of smooth muscle cells (SMCs) with calcium deposits or cholesterol clefts within 26 weeks after SZ injection. These lesions have histological similarities to those encountered in human patients [30]. Thus, the APA hamster is considered to be an important model for investigating the development of atheromatous lesions in DM patients. In this study, we attempted to investigate the association between atherosclerosis and ER stress, and also examined the effect of 4-PBA, a chemical chaperone, on atherosclerotic lesions in SZ-induced diabetic APA hamsters. Materials and Methods Animals Male APA hamsters were purchased from Japan SLC (Shizuoka, Japan). They were maintained under controlled conditions (temperature: 24 ± 2°C; humidity: 55 ± 5%) in plastic cages with sterilized wood shavings and were fed a commercial diet, CMF (Oriental Yeast, Tokyo, Japan) and tap water ad libitum. At the age of 8 weeks, the animals were injected once i.p. with 30 mg/kg body weight of SZ (Sigma, St Louis, Mo.) dissolved in 0.1 M citrate buffer (CB, pH 4.5) or with the same volume of CB only as a control. The former were named as the SZ group and the latter as the CB group. To induce DM with a relatively high frequency, food was withheld from the hamsters for 6 h before and 18 h after SZ administration. This experiment was conducted according to our institutional guidelines for animal experiments and was approved by the Animal Care and Use Committee of the Graduate School of Agricultural and Life Sciences at The University of Tokyo.
AORTIC ER STRESS IN DIABETIC APA HAMSTERS
Experimental schedule Three days after the injection of SZ or CB, 47 animals from both the SZ and CB groups were assigned to the following four experimental groups: animals from the CB group that were injected with saline (CB/saline, n=8), animals from the CB group that were injected with 4-PBA (CB/PBA, n=8), animals from the SZ group that were injected with saline (SZ/saline, n=15), and animals from the SZ group that were injected with 4-PBA (SZ/ PBA, n=16). 4-PBA was prepared by titrating equimolar amounts of 4-phenylbutyric acid (Wako, Osaka, Japan), and the pH was adjusted with sodium hydroxide to pH 7.4. 4-PBA was administered i.p. daily at a volume of 120 mg/kg for 6 weeks from 72 h after the injection. Sampling Blood sampling was performed from the infraorbital vein at one-week intervals, and the samples were used to measure serum glucose, triglyceride, and cholesterol levels using a commercial test kit (Wako, Osaka, Japan). At the end of the administration of 4-PBA, the animals were weighed and sacrificed by exsanguination under anesthesia. The aorta was perfused with 0.01 M phosphate buffered saline (PBS) before being fixed in 4% paraformaldehyde in PBS and removed from each animal. Evaluation of atherosclerotic lesions and histopathological analysis To evaluate the degree of atherosclerotic lesions, the aortas were opened longitudinally, rinsed in 60% isopropanol, stained with 0.3% oil red O in 60% isopropanol for 12 min, and then rinsed again. Photographs of the specimens were digitized for data analysis. The surface area of the atheromatous lesions in the lumen was quantified using NIH Image software with custom-written macros. After being photographed, the specimens were embedded in paraffin by a routine procedure. Fourmicron thick paraffin sections of longitudinal aorta were stained with hematoxylin and eosin (HE), or subjected to immunohistochemistry for alpha-smooth muscle actin (a-SMA), calreticulin, or GRP78 to assess ER stress.
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Semi-quantitative RT-PCR Total RNA was isolated from the aortic arch of each animal using TRIzol Reagent (Invitrogen, Carlsbad, CA), and 50 ng of each RNA sample in 20 µl of reaction mixture were reverse-transcribed with oligo (dT) primer and Superscript (Invitrogen, Carlsbad, CA). PCR amplification was carried out with Thermo-Start DNA polymerase (Abgene, Surrey, UK) using the following primers: calreticulin forward primer 5’-aggctccttggaggatgatt-3’, reverse primer 5’-gcttttctgaagccttggtg-3’; GRP78 forward primer 5’-cagcgggttatggaacactt-3’, reverse primer 5’-gacagcagcaccgtatgcta-3’; and b-actin forward primer 5’-tgtcaccaactgggacgata-3’, reverse primer 5’-tctccagggaggaagaggat-3’. PCR was performed via activation of Taq polymerase at 94°C for 2 min, 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 60 s, and final extension at 72°C for 3 min. The PCR products were electrophoresed on 1.5% agarose gels and stained with ethidium bromide, before visualization and densitometry using Quantity One Software (PDI, New York, NY). Relative values were calculated by standardization with the values of b-actin. Immunohistochemistry and imaging analysis Four-micron thick sections were deparaffinized and treated with 0.5% periodic acid for 30 min. In the case of immunohistochemistry for a-SMA, sections were autoclaved at 121°C for 10 min as pretreatment. Subsequently, nonspecific reactions of the sections for anticalreticulin, anti-GRP78, and anti-a-SMA were blocked by 5% skimmed milk for 30 min and Block Ace (Dainippon Pharmaceutical, Osaka, Japan) for 60 min, sequentially, at room temperature. The primary antibodies used were anti-human a-SMA mouse monoclonal antibody (1:200; DAKO A/S, Glostrup, Denmark), rabbit antiGRP78 IgG (1:1000 dilution; Stressgen Biotechnologies, Victoria, Canada), and rabbit anti-calreticulin IgG (1:500; Stressgen Biotechnologies, Victoria, Canada). The secondary antibodies used were anti-mouse IgG (1:500; Vector Laboratories, Burlingame, CA) for aSMA, and anti-rabbit IgG (1:300; Vector Laboratories) for GRP78 and calreticulin. The sections were incubated using the free-floating technique at 4°C overnight with primary antibodies and at room temperature for 1
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h with secondary antibodies. The immunoreactions were visualized with peroxidase-labeled streptavidin (1:300; DAKO A/S, Glostrup, Denmark) and 3, 3’-diaminobenzidine (DAB) (Wako). Photographs of the specimens were digitized for data analysis. The areas of the positive regions were quantified using NIH Image software with custom-written macros. Statistical analysis The 5% level was considered as being statistically significant. When the data of the two groups showed a regular distribution, the F-test was used to check their equality of variance, and Student’s t-test (equal variance) or Welch’ t-test (unequal variance) was used to judge the significance of the differences between their average values. All values are presented as means ± SE. Results Body weight Body weights in the CB groups (CB/saline and CB/ PBA) increased with age. In addition, there was no difference in body weights between CB/saline and CB/PBA (Fig. 1A). On the other hand, body weights in the SZ groups (SZ/saline and SZ/PBA) decreased one week after SZ treatment and subsequently increased with age. Consequently, body weights in the SZ groups were significantly lower than those in the CB groups from one week after 4-PBA treatment till the end of the experiment (Fig. 1A). Although the body weights of the SZ/saline animals were slightly lighter than those of the SZ/PBA animals, the differences were not statistically significant. Serum glucose, total cholesterol, and triglyceride levels After the 4-PBA injection, the animals of the CB/saline and CB/PBA groups maintained standard levels of serum glucose (Fig. 1B), total cholesterol (Fig. 1C), and triglycerides (Fig. 1D), suggesting that there were neither adverse effects of 4-PBA nor age-related changes in these serum biochemical markers in APA hamsters. On the other hand, the SZ group animals (SZ/saline and SZ/ PBA) maintained significantly different hyperglycemia and hyperlipidemia levels throughout the experiment,
Fig. 1. Body weights (A) and serum concentrations of glucose (B), triglycerides (C), and cholesterol (D) in SZ-induced diabetic APA hamsters. CB/saline ( ), CB/PBA ( ) SZ/saline ( ), and SZ/PBA ( ). Mean ± SE. Asterisks (*) and sharps (#) indicate values of SZ/saline and SZ/PBA were significantly different from those of CB/saline and CB/PBA, respectively (P
