Pioglitazone prevents hyperglycemia induced decrease of AdipoR1 and AdipoR2 in coronary arteries and coronary VSMCs

Molecular and Cellular Endocrinology 363 (2012) 27–35

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Pioglitazone prevents hyperglycemia induced decrease of AdipoR1 and AdipoR2 in coronary arteries and coronary VSMCs Xuhua Shen 1, Hongwei Li ⇑, Weiping Li, Xing Wu, Xiaosong Ding Department of Cardiology, Beijing Friendship Hospital Affiliated to the Capital Medical University, China

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Article history: Received 29 December 2011 Received in revised form 9 July 2012 Accepted 10 July 2012 Available online 17 July 2012 Keywords: Pioglitazone Vascular smooth muscle cells Adiponectin receptor Diabetes mellitus

a b s t r a c t Background: Adiponectin receptors play an important role in inflammatory diseases like diabetes and atherosclerosis. Former studies revealed that the regulation of adiponectin receptors expression differs in the receptor responses to pioglitazone. However, expression of AdipoRs has not been investigated in the coronary arteries or the coronary vascular smooth muscle cells (VSMCs). In the present study we investigated the effect of pioglitazone on the adiponectin receptors both in vitro and in vivo. Methods: Male Sprague–Dawley rats were randomly divided in three groups. One of them fed with regular chow (the Control group) and two of them fed with high-fat diet and then received low-dose Streptozotocin once by intraperitoneal injection (the DM groups). Rats in one of the DM groups were further treated with pioglitazone (the PIO group). Blood pressure, serum adiponectin, fasting blood glucose, fasting serum insulin, cholesterol, triglyceride, AdipoR1 and AdipoR2 expression, and TNF-a expression in coronary arteries of these groups were investigated. For the in vitro study, the rat coronary VSMCs maintained under defined in vitro conditions were treated with either PIO or the PIO+ GW9662 (PPAR-c antagonist), and then stimulated with high glucose. AdipoR1 and AdipoR2 expression, TNF-a expression and PPAR-c expression were investigated. Results: Compared to the DM group, treatment with PIO in vivo significantly attenuated cholesterol level, triglyceride level, fasting serum insulin and TNF-a overexpression (p < 0.05). PIO also increased AdipoR1 and AdipoR2 expression in coronary arteries, which were reduced notably in the DM group (p < 0.05). Consistently, in the study with rat coronary VSMCs, PIO prominently downregulated TNF-a expression and induced PPAR-c expression, as well as prevented hyperglycemia induced decrease of AdipoR1 and AdipoR2 expression (p < 0.05). And pretreatment of PIO + GW9662 did not manifest the prevention effect. Conclusion: In this study, we showed that treatment with PIO could ameliorate coronary insulin resistant and upregulate the expression of AdipoR1/R2. PIO showed an anti-atherogenic property via the activation of PPAR-c, suppression of TNF-a overexpression in coronary and coronary VSMCs. Crown Copyright Ó 2012 Published by Elsevier Ireland Ltd. All rights reserved.

1. Introduction Diabetes mellitus is an important risk factor for the development of atherosclerosis. The risk for cardiovascular disease in patients with diabetes is two to sixfold higher than in people without diabetes. The clustering of traditional risk factors such as Abbreviations: PIO, pioglitazone; VSMCs, vascular smooth muscle cells; SD, Sprague–Dawley; STZ, Streptozotocin; DM, diabetic mellitus; APN, adiponectin; FSI, fasting serum insulin; AdipoR, adiponectin receptor; PPAR-c, peroxisome proliferator-activated receptor; AMPK, 50 -AMP-activated protein kinase. ⇑ Corresponding author. Address: Department of Cardiology, Beijing Friendship Hospital Affiliated to the Capital University of Medical Sciences, No. 59 Yong An Road, Xuanwu District, Beijing 100050, China. Tel./fax: +86 10 63139780. E-mail addresses: [email protected] (X. Shen), [email protected] (H. Li). 1 Department of Cardiology, Beijing Friendship Hospital Affiliated to the Capital University of Medical Sciences, No 59 Yong An Road, Xuanwu District, Beijing 100050, China. Tel.: +86 10 63138344; fax: +86 10 63138706.

hypertension and hypercholesterolemia cannot explain the excessive cardiovascular burden of patients with diabetes. The UK prospective diabetes study (UKPDS) showed that while intensive control of blood glucose level could not reduce the cardiovascular risk, multifactorial intervention was effective in improving cardiovascular mortality and morbidity in patients with diabetes (UK Prospective Diabetes Study Group, 1998). Thiazolidinediones (TZDs) are pharmacologic agents that improve glucose homeostasis in type 2 diabetes by increasing insulin sensitivity (Olefsky, 2000). In addition, the pioglitazone (PIO), one of the TZDs, significantly reduces the incidence of major adverse cardiovascular events, strokes, and all-cause mortality in high-risk patients with type 2 diabetes (Dormandy et al., 2005). The PIO is believed to be mediated by their interaction with the nuclear receptor peroxisome proliferator–activated receptor (PPAR-c). PPAR-c is a member of the nuclear hormone receptor superfamily of ligand-activated transcriptional factors. Recently, it has been

0303-7207/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mce.2012.07.005

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demonstrated that PPAR-c is expressed in endothelium, vascular smooth muscle cells, and monocytes/macrophages, which were considered as important cells for atherosclerosis (Marx et al., 2004; Little et al., 2008). It suggests that PIO may exert direct beneficial effects on the vascular wall. Adiponectin (Scherer et al., 1995) is a hormone secreted by adipocytes, which function as the key antidiabetic and anti-atherogenic adipocytokine (Scherer, 2006). Plasma adiponectin levels are decreased in obesity, insulin resistance, and type 2 diabetes mellitus. Many studies have shown that the high levels of adiponectin are associated with insulin sensitization, whereas low levels are found in insulin resistance (Satoh et al., 2005). Recently, two distinct receptors for adiponectin (AdipoR1 and AdipoR2) have been identified (Yamauchi et al., 2003). AdipoR1 was ubiquitously expressed and most abundantly expressed in skeletal muscle, whereas AdipoR2 was most abundantly expressed in mouse liver (Yamauchi et al., 2003). Adiponectin receptors are key innate sensors of endogenous damage signals and play an important role in inflammatory diseases like diabetes and atherosclerosis. Adiponectin receptors mediate the activation of AMPK, PPARa, and fatty acid oxidation, which increases glucose uptake and improves lipid metabolism (Yamauchi et al., 2003). AdipoR1and AdipoR2-mediated signal transduction has been implicated in steatosis, inflammation, and oxidative stress, all key abnormalities associated with obesity and the metabolic syndrome (Kadowaki, 2006). The regulation of AdipoR1 and AdipoR2 expression differs in the receptor responses to PIO. AdipoR1 expression is upregulated in adipose tissue, but downregulated in skeletal muscle by long-term treatment of PIO (Sun et al., 2006). On the other hand, PIO did not change AdipoR1 and AdipoR2 expression in peritoneal macrophages and subcutaneous fatty tissue (Tsuchida et al., 2005). However, little is known about its effect on coronary arteries or coronary VSMC which are the key tissue and cells in diabetes, hypertension and atherosclerosis. In this study, we examined the effects of PIO on AdipoR1 and AdipoR2 expression in rat coronary arteries and coronary VSMC in vivo and in vitro. What we found? 2. Materials and methods 2.1. Materials PIO was donated by Huadong Medicine Co. (Hangzhou, China). GW9662 was purchased from Sigma Co. (Sigma, USA). DMEM, streptomycin, trypsin, fetal bovine serum (FBS), TRIzol reagent, pCR2.1-TOPO vector, LDS sample buffer, and Sample Reducing Agent were from Invitrogen Life Technologies (Shanghai, CN, USA). Anti-GAPDH antibody was from ProMab Biotechnologies Inc. (ProMab, USA). Anti-AdipoR-1 (AHP1824) and AdipoR-2(AHP1900) antibodies were obtained from AbD serotec Corp (AbD, U.K.). Anti-PPAR-c(B0557) antibody was from Assay bioTech Corp(ABT, USA). Anti-TNF-a and horseradish peroxidase conjugated secondary antibodies were from Santa Cruz Biotechnology (Santa, USA).

other rat received high-fat diet (HFD) for 4 weeks and then a single intraperitoneal injection of STZ (25 mg/kg, in pH 4.5 citrate buffer). After 7 days, blood samples were collected from caudal vein. Fasting Blood Glucose (FBG) and Serum insulin (SI) were estimated, then Insulin Sensitive Index [ISI, ISI = In (FBG  SI)] was calculated. The HFD and STZ-treated rats with the ISI 6 4.88 were randomized into DM groups and PIO group. DM group received the citrate buffer and the PIO group received PIO at 10 mg/kg per day by gavage. After 4 weeks of treatment, 12 h fasted rat (n = 6–8) was anesthetized with an intraperitoneal injection of pentobarbital sodium (60 mg/kg). The blood samples were collected and the body weight was also measured. The coronary arteries were dissected from the ventricle and placed in a phosphate buffered saline (PBS)-precooled plate. The coronary arteries was dissected from the adherent fat and connective tissue on ice, and then frozen in liquid nitrogen and kept at 80 °C for subsequent analysis. This study was performed in accordance with the guidelines for animal experiments of the Capital University of Medical Sciences. 2.3. Measurement of blood pressure Blood pressure (systolic, mean and diastolic) was recorded at end of treatment in all groups, using tail cuff blood pressure recorder (Gene&I Co., Model No. BP-98A, China). Rats were acclimatized to heating chamber (24–26 °C) for 20 min before recording the blood pressure (between 9 and 11 AM). Three recordings were measured for each rat and the average was calculated. 2.4. Oral glucose tolerance test (OGTT) After 4 weeks of treatment, glucose (2 g/kg) was administered to 12 h fasted rats and blood samples were collected from the caudal vein by means of a small incision at the end of the tail at 0 (immediately after glucose load), 30, 60 and 120 min after glucose administration. Blood glucose lever was estimated by the enzymatic glucose oxidase method using a commercial glucometer (Acku-check, sensor confort, Roche, Germany).The results were expressed as the integrated area under the curve for glucose (AUCglucose), which was calculated by trapezoid rule. 2.5. Cell culture and cell treatment Seven-week-old male SD rats (Vital River; Beijing, China) were anesthetized with pentobarbital sodium (60 mg/kg ip). Rat coronary arteries were dissected from the ventricle and the endothelium in the vessels was denuded with air (Liu et al., 1994). Enzymatic isolation of VSMCs were performed according to published methods (Li et al., 2003). Coronary VSMCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 20% fetal calf serum (Gibco, USA), 100 U/mL penicillin G, and 100 mg/mL streptomycin. DMEM was supplemented with normal glucose (NG, 5.5 mmol/L D-glucose). VSMCs were incubated with PIO (10 lmol/L)+ high glucose (HG, 23 mmol/L Dglucose), or PIO(10 lmol/L)+ GW9662(5 lmol/L)+HG, or 0.1% dimethyl sulfoxide vehicle in DMEM HG or DMEM NG for 24 h at 37 °C before each assay. The cells were maintained in a humidified chamber with 5% CO2 at 37 °C.

2.2. Animals 2.6. Quantitative real-time (RT)–PCR analysis Male Sprague–Dawley(SD) rats (Vital River; Beijing, China) weighing 250 g were housed in an environmentally controlled room with a 12-h light/dark cycle and given standard rodent chow and tap water ad libitum. The rats were acclimated to handling before randomization and then divided into three groups in the beginning of the study. The control group fed with regular chow for 5 weeks and received the citrate buffer alone for 4 weeks. The

Total RNA samples were extracted from rat coronary arteries (n = 6–8 in each group) and VSMCs with Trizol reagent (invitrogen, CA), and total RNA was further purified using the RNeasy kitwith RNase-free DNase I treatment according to the manufacturer’s instructions. Total RNA (1 lg) was reverse-transcribed with iScript cDNA Synthesis Kit according to the manufacturer’s instructions

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Table 1 PCR primers and PCR protocols. Forward AdipoR1 AdipoR2 TNF- a GAPDH

0

Reverse 0

5 -CCTGGGACTTGGCTTGAGT-3 50 -ACGAATGGAAGAGTTTGTTTG-30 50 -GAACAACCCTACGAGCACCT-30 50 -CCTGCCAAGTATGATGACATCAAG-30

(invitrogen, CA). Real time-PCR (Q-PCR) used s specific primer paDMs (Table 1). SYBR Green 2X master mix buffer 10 ll, primer-F final concentration 0.1 lmol/L, primer-R final concentration 0.1 lmol/L, cDNA 20 ng, ddH20 4.5 ll, to give a total volume 20 ll. After initial denaturation at 95 °C for 10 min, PCR was performed for a total of 40 cycles, each at 95 °C for 15 s, 60 °C for 1 min, melting curve 95 °C for 15 s, 60 °C for 30 s, and 95 °C for 15 s (StepOne™ Real Time PCR System, ABI). At the end of each reaction, the cycle threshold (Ct) was manually set up at the level that reflected the best kinetic PCR parameters, and melting curves were acquDMed and analyzed. The starting copy number of the unknown samples was determined relative to the known copy number of the calibrator sample using the following formula: DDCt = [Ct target gene (calibrator sample)  Ct b-actin gene (calibrator sample)]–[Ct target gene (unknown sample)  Ct b-actin gene (unknown sample)]. In this case, the target gene is AdipoR1, AdipoR2 and TNF-a. The relative gene copy number was calculated by the expression 2DDCt. Quantitative PCR data were normalized to an internal Control (GAPDH) and were presented as mean ± SD for three independent experiments done in triplicate.

2.7. Immunoblotting analysis The coronary arteries from rats belonging to the different groups was isolated, cleaned and washed in cold saline solution (0.9% NaCl), and frozen in liquid nitrogen. coronary arteries were rinsed and homogenized (1 mg/ml) in lysis buffer of the following composition (1 mg/mM): 50 TrisHCl pH 7.5, 1 EDTA, 150 NaCl, 1 Na3VO4, 10 NaF and complete protease inhibitor cocktail tablet (Sigma–Aldrich, shanghai, CA, USA). The homogenate was centrifuged (1,000g  10 min at 4 °C) to remove cell debris. Serum-starved coronary VSMCs cells were treated with either PIO or the PPAR-c antagonist GW9662 + PIO, and then stimulated with high glucose at 37 °C for 24 h as indicated in each experiment. The cells were lysed in solubilizing buffer containing 20 mM Tris, 1 mM EDTA, 140 mM NaCl, 1% Nonidet P-40 (NP-40), 50 U of aprotinin/ml, 1 mM Na3VO4, 1 mM PMSF, and 10 mM NaF (pH 7.5) for 30 min at 4 °C. The cell lysates were centrifuged at 15,000 rpm for 30 min at 4 °C, to remove insoluble materials. The resulting supernatant was frozen at 80 °C for later analysis by SDS–PAGE and immunoblotting. The protein concentration of the crude lysate was determined by the BCA protein assay reagent kit (Beyotime, China). Proteins separated on a 4–12% (w/ vol) acrylamide gel (Nu-Page Gel, Invitrogen, China) were transferred to polyvinylidene difluoride membranes and probed with Goat ADIPOR1 polyclonal antibodies (1:1000 dilution; AbD Inc., U.K.), Goat ADIPOR2 polyclonal antibodies (1:1000 dilution; AbD Inc., U.K.), Goat TNF-a polyclonal antibodies (1:500 dilution; Santa, USA), Rabbit PPAR-c polyclonal antibody (1:400 dilution; ABT, USA)or with polyclonal anti-Mouse GAPDH (1:1000 dilution; ProMab, CA, USA) overnight at 4 °C. After extensive washings, a rabbit anti-goat peroxidase conjugated antibody was added (1:2000 dilution; Sigma shanghai, CA, USA) and immunodetected bands were visualized by ECL. Densitometric analysis of autoradiographic bands were referred to GAPDH

50 -GGAATCCGAGCAGCATAAA-30 50 -GGCGAAACATATAAAAGATCC-30 50 -GGGTAGTTTGGCTGGGATAA-30 50 -GTAGCCCAGGATGCCCTTTAGT-30

expression taking into account the size and the area of the band (Scion software Image Corp).

2.8. Immunohistochemistry Dissected coronary arteries were embedded in OCT compound and snap-frozen, and subsequently stored at 80 °C before sectioning. Cross cryosections (3 lm thick) were fixed in 4% phosphatebuffered formaldehyde. Endogenous peroxidase activity was quenched by incubation in methanol containing 0.3% hydrogen peroxide. Sections were incubated with anti-goat ADIPOR1 antibody and anti-goat AdipoR2 antibody overnight at 4 °C. Sections were washed with PBS and incubated with HRP-conjugated IgG secondary antibody (MAIXIN, Jinan, China) for 1 h at room temperature. The signal was developed with streptoavidine (NichDMei, Tokyo, Japan) and revealed with 3,30 -diaminobenzidine (Sigma–Aldrich Co.). Sections were counterstained with hematoxylin and mounted. Negative Controls were incubated with PBS without adding primary antibody. Analysis and determination of the integrated optical intensity of AdipoR1 and AdipoR2 was performed by confocal laser scanning microscopy. The analysis was completed by the assistance of the computer software. Acquired images were standardized by ignoring background pixels using the density slice manipulation. For semiquantitative analysis of AdipoR1 and AdipoR2 expression, the integrated optical intensity of AdipoR1 and AdipoR2 immunopositive plaques were measured in a region (50  50 lm2), randomly selected from different areas. 2.9. Statistics analysis Data are expressed as mean ± S.E.M. Results were analyzed using one way analysis of variance (ANOVA) for multiple comparisons followed by Fisher’s Protected Least Significant Difference. A value of P < 0.05 was considered statistically significant.

3. Results 3.1. Effects of lipid and glucose metabolism of rats studied The changes in body weight, AUC from Oral glucose tolerance test, and plasma parameters are summarized in Table 2. Body weight was significantly (P < 0.05) increased in DM rats and unaffected by PIO. AUC from oral glucose tolerance test, plasma FBG and plasma insulin levels in the DM group markedly increased, as compared to those of the Control group. The administration of PIO did not significantly affect the changes of FBG and AUC. Treatment of PIO significantly (P < 0.05) lowered the plasma total cholesterol, triglycerides and plasma insulin, as compared to those of the DM group.

3.2. Effects of pioglitazone on blood pressure PIO didn’t restore the elevated systolic, mean arterial and diastolic blood pressure in DM rats (Table 3).

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Table 2 Characteristics of rats in the control group (Control), the diabetic mellitus group (DM) and the pioglitazone group (PIO). Parameter

Control

BW (g) Plasma FBG (mmol/L) AUC from OGTT (mmol/Lmin) Plasma T-Cho (mg/dL) Plasma TG (mg/dL) Plasma insulin (pmol) Plasma APN (lg/ml)

476 ± 41 5.49 ± 0.58 981 ± 81

DM 514 ± 61⁄a 9.78 ± 2.65⁄⁄a 1396 ± 225⁄a

PIO

51.5 ± 3.14 46.3 ± 3.23 140 ± 10.2 12.6 ± 1.26

156 ± 21.29⁄⁄a 207.84 ± 23.84⁄⁄a 179.3 ± 19.5⁄a 9.04 ± 0.87⁄⁄a

hyperglycemia, which was measured by immunohistochemistry, was restored by treatment with PIO (Figs. 3 and 4). 3.5. Expression of mRNA and protein for TNF-a in vivo

526 ± 37⁄a 8.94 ± 1.66⁄⁄a 1281 ± 179⁄a 130 ± 10.53⁄⁄a⁄b 158 ± 25.53⁄⁄a⁄b 160.2 ± 15.2⁄a⁄b 11.5 ± 0.71⁄b

Values are means ± S.E.M. (n = 6–8). BW, body weight; FBG, fasting blood glucose, AUC, area under the curve; OGTT, oral glucose tolerance test; T-Cho, total cholesterol; TG, triglycerides; APN, adiponectin. ⁄P < 0.05; ⁄⁄P < 0.01; a vs. control group; b vs. insulin resistant model group.

Table 3 Effect of chronic in vivo treatment of pioglitazone on SBP, DBP and MAP of control group (Control), diabetic mellitus group (DM) and the pioglitazone group (PIO) rats. Parameter

Control

DM

PIO

SBP (mmHg) MAP (mmHg) DBP (mmHg)

119 ± 7.6 93 ± 9.4 80 ± 11.6

135 ± 1.6⁄a 107 ± 10.0⁄a 93 ± 6.2⁄a

138 ± 9.0⁄a 106 ± 2.56⁄a 90 ± 2.53⁄a

Values are means ± S.E.M. (n = 6–8). ⁄P < 0.05; a vs. control group.

The TNF-a mRNA and protein level was significantly greater in the DM group. The increased expression of TNF-a was prevented by treatment with PIO. The expression of TNF-a mRNA was even lower than that of the Control group (Figs. 5 and 6). 3.6. Effects of pioglitazone on the expression of AdipoR1 and AdipoR2 in coronary VSMCs Because the distribution of AdipoR1 and AdipoR2 expression varies between target tissues and among animal species, we hypothesized that adiponectin receptor distribution and/or upregulation by PIO could play a key role in inflammatory diseases in coronary VSMCs. Next, we investigated whether treatment with PIO could alter the expression of AdipoR1 and/or AdipoR2 in coronary VSMCs. As shown in Figs. 7 and 8, we confirmed that AdipoR1 and AdipoR2 mRNA and protein level in coronary VSMCs was significantly decreased by treatment with HG. And PIO prevented hyperglycemia induced decrease of AdipoR1 and AdipoR2 mRNA and protein in coronary VSMCs. The pretreatment of PIO plus GW9662 induced lower expression level of AdipoR1 and AdipoR2 compared with treatment of only PIO. 3.7. Effects of pioglitazone treatment on TNF-a in coronary VSMCs TNF-a is the key molecules in the inflammatory signaling pathway. To assess the potential cellular mechanisms by which the PIO induces increase in inflammation and oxidative stress, we first determined whether PIO affects the activation of inflammatory signaling key components by measuring TNF-a protein level. As shown in Fig. 9, the HG enhanced the protein expression for TNFa in VSMCs compared with the NG group. PIO decreased the hyperglycemia induced increase of TNF-a in coronary VSMCs. Moreover, TNF-a was higher expressed in PIO plus GW9662 group compared with PIO group. 3.8. Expression of protein for PPAR-c in coronary VSMCs

Fig. 1. Effects of pioglitazone on expression of AdipoR1 and AdipoR2 in coronary arteries. Total RNA extracted from coronary arteries was used for gene expression analysis of AdipoR1 and AdipoR2. Levels of GAPDH were used for normalization of sample loading. Data represent the mean ± S.E.M. (n = 6–8). ⁄⁄P < 0.01 vs. the control group (Control), ##P < 0.01 vs. DM group.

Compared with the NG group, the coronary VSMCs expression of PPAR-c protein in HG group was lower by twofold (Fig. 10). What’s more, coronary VSMCs cells with 10 lM PIO pretreatment for 24 h exhibited increase in PPAR-c protein as compared to cells with only HG (Fig. 9). Pretreatment of PIO + GW9662 didn’t manifest the incremental effect. 4. Discussion

3.3. Effects of pioglitazone on plasma APN Compared with the Control group, the DM group had lower levels of APN. PIO prevented the hyperglycemia induced decrease of plasma APN in DM rats (Table 2).

3.4. Expression of mRNA and protein for AdipoR1 and AdipoR2 in vivo As shown in Figs. 1 and 2, the level of AdipoR1 and AdipoR2 mRNA and protein in the coronary arteries from the DM group was lower than that of the Control. The hyperglycemia induced decrease in the AdipoR1 and AdipoR2 mRNA and protein level was prevented by treatment with PIO (Figs. 1 and 2). Likewise, the decreased expression of AdipoR1 and AdipoR2 protein induced by

The plasma glucose tolerance and FPG were not significantly affected by the treatment with PIO. The reason for this could be that the main action of pioglitazone, as with other TZDs, is the reconstitution of fatty tissue, its effects on glycemia occur slowly (Yamanouchi, 2010). A number of trials have reported patients require nearly 6 months of therapy before the maximum glucose-lowering effect is obtained (Yamanouchi et al., 2005; Tan et al., 2004). Furthermore, about 50% of patients receiving PIO monotherapy are ‘‘non-responders’’, defined as a reduction in glycosolated hemoglobin (HbA1c) < 0.7%. In our study, the decreasing trend of glucose was observed after 4 weeks of PIO treatment (8.94 ± 1.66 vs. 9.78 ± 2.65), but the effect was not significant. Elevated triglyceride and/or cholesterol levels were reported to be associated with an increased risk of mortality in coronary

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Fig. 2. Western blotting assay of the expression of AdipoR1 and AdipoR2 protein in the coronary arteries from each type of treated rat. (A) Representative expression of AdipoR1 and AdipoR2 protein, as determined by Western blot analysis. (B) Quantitative analysis of the expression of the protein for AdipoR1 and AdipoR2, analyzed by scanning densitometry. Values are means ± S.E.M. (n = 6–8, AdipoR1/GAPDH, AdipoR2/GAPDH). ⁄⁄P < 0.01 vs. the control group (Control), ##P < 0.01 vs. DM group, #P < 0.05 vs. DM group.

Fig. 3. Immunohistochemical analysis of AdipoR1 and adipoR2 protein levels in the coronary arteries from the control group (Control; A), the diabetic mellitus group (DM; B), DM + pioglitazone (PIO; C). The bar indicates 10 lm.

heart disease (Goldenberg et al., 2009). PIO significantly improves the atherogenic lipid profile that characterizes type 2 diabetes, with effects on small dense low-density and high-density lipoprotein (LDL and HDL) cholesterols and triglyceride-rich lipoprotein particles (Betteridge, 2007). In recent years, much has been learned about the mechanisms underlying the anti-atherogenic properties of triglyceride and cholesterol. PIO treatment is associated with a well-established 15–20% reduction in fasting triglyc-

eride levels, due to a significant decrease in the amount of triglyceride present in very low-density lipoprotein (VLDL), which may relate to a reduction in large VLDL particle concentration and a decrease in mean VLDL particle size (Deeg et al., 2007). In our study, both plasma triglyceride level and plasma total cholesterol level were decreased by the chronic administration of PIO. This suggests that PIO might have beneficial effects of lipid metabolic state.

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Fig. 4. AdipoR1/R2 expression in rat coronary artery measured by Immunohistochemistry. ⁄P < 0.05, compared with the control group; #P < 0.05, compared with group DM.

Fig. 6. Effects of pioglitazone on expression of TNF-a protein in coronary arteries. (A) Representative expression of TNF-a protein, as determined by Western blot analysis. (B) Quantitative analysis of the expression of the protein for TNF-a, analyzed by scanning densitometry. Values are means ± S.E.M. (n = 6–8, TNF-a/ GAPDH). ⁄P < 0.05 vs. the control group (Control), #P < 0.05 vs. DM group.

Fig. 5. RT-PCR assay of the expression of the mRNA for TNF-a in the coronary arteries from each type of treated rat. Values are means ± S.E.M. (n = 6–8, TNF-a/ GAPDH). ⁄⁄P < 0.01 vs. the control group (Control), ⁄P < 0.05 vs. the control group (Control), ##P < 0.01 vs. DM group.

PIO is an antidiabetic insulin-sensitizing agent that improves insulin action in a variety of animal states of insulin resistance and diabetes (Olefsky, 2000). They are thought to exert all these effects by acting as selective ligands of the PPAR-c receptors (Berger et al., 2005). The activated receptors work in a number of ways to achieve these effects. They alter the expression of genes involved in lipid metabolism and promote fatty acid uptake and storage in adipose tissue. PPAR-c activation also increases adiponectin production from adipose tissue which may be due to a direct effect of PPAR-c on adiponectin transcription (Iwaki et al., 2003). Animal and in vitro studies indicate that adiponectin may also protect against atherosclerosis by decreasing adhesion molecule expression on endothelial cells to inhibit foam cell formation and vascular smooth muscle cell proliferation. It inhibits TNF-a-induced adhesion molecule expression on endothelial cells, including vascular cell adhesion molecule-1, intracellular adhesion molecule-1 and E-selectin (Blaschke et al., 2006). We showed that PIO decreased insulin levels and increased plasma APN in DM rats which suggests it act as insulin sensitisers. In addition, numerous experimental studies have demonstrated that PIO not only ameliorate insulin

Fig. 7. RT-PCR assay of the expression of the mRNA for AdipoR1 and AdipoR2 in coronary VSMCs. After pretreatment with PIO(10 lM) + HG, or PIO(10 lM)+ GW9662(5 lM) + HG or 0.1% dimethyl sulfoxide vehicle in DMEM high glucose (HG, 23 mmol/L D-glucose) or DMEM normal glucose (NG, 5.5 mmol/L D-glucose) for 24 h, followed by measurement of AdipoR1 and AdipoR2 mRNA in coronary VSMCs. Levels of GAPDH were used for normalization of sample loading. Data represent the mean ± SE of three independent experiments (one experiment performed with six samples). ⁄P < 0.05 vs. the NG group, ##P < 0.01 vs. HG group.

sensitivity, but also have pleiotropic effects of anti-inflammatory, antioxidative, and antiproliferative on vascular wall cells. Although it is known that PIO functions as a ligand for PPAR-c, the molecular mechanisms underlying its anti-inflammatory and anti-athero-

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Fig. 8. Western blotting assay of the expression of AdipoR1 and AdipoR2 protein in coronary VSMCs. After pretreatment with PIO (10 lM) + high glucose (HG, 23 mmol/L D-glucose), or PIO (10 lM) + GW9662 (5 lM) + HG or 0.1% dimethyl sulfoxide vehicle in DMEM HG or DMEM normal glucose (NG, 5.5 mmol/L Dglucose) for 24 h, followed by measurement of AdipoR1 and AdipoR2 protein in coronary VSMCs. (A) Representative expression of AdipoR1 and AdipoR2 protein, as determined by Western blot analysis. (B) Quantitative analysis of the expression of the protein for AdipoR1 and AdipoR2, analyzed by scanning densitometry. ⁄⁄P < 0.01 vs. the NG group, #P < 0.05 vs. HG group.

sclerosis effects are not well understood. We showed that PIO restored hyperglycemia induced decrease of AdipoR1 and AdipoR2 expression in coronary arteries and coronary VSMCs. While in vitro, pretreatment of PIO + GW9662 didn’t manifest the upregulation of AdipoR1 and AdipoR2. These findings raised the possibility that PIO may have increased AdipoR1 and AdipoR2 expression in coronary arteries and coronary VSMCs which are PPAR-c dependent. Both AdipoR1 and AdipoR2 receptors activate signaling molecules such as AMPK, PPARa and p38 MAPK (mitogen-activated protein kinase) in vitro (Yamauchi et al., 2007). Using AdipoR1 and AdipoR2 gene knockout mice, it was clearly demonstrated that both receptors are involved in energy metabolism, although they have opposing effects (Bjursell et al., 2007; Liu et al., 2007). AdipoR1/ mice become obese and glucose-intolerant and have decreased energy expenditure while AdipoR2/ mice are lean and resistant to high-fat-diet induced obesity and show increased energy expenditure (Bjursell et al., 2007; Liu et al., 2007). The regulation of AdipoR1 and AdipoR2 expression differs in the receptor responses in different tissues. The expressions of both AdipoR1 and AdipoR2 were significantly decreased in the muscle and adipose tissue in obese mice (Tsuchida et al., 2004). The disruption of both AdipoR1 and AdipoR2 in the mouse liver abolished adiponectin binding and actions, resulting in increased tissue triglyceride content, inflammation, and oxidative stress, thereby leading

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Fig. 9. Effects of pioglitazone on expression of TNF-aprotein in coronary VSMCs. (A) Representative expression of TNF-aprotein, as determined by Western blot analysis. (B) Quantitative analysis of the expression of the protein for TNF-a, analyzed by scanning densitometry. ⁄⁄P < 0.01 vs. the NG group, ##P < 0.01 vs. HG group.

to insulin resistance and marked glucose intolerance (Yamauchi et al., 2007). Conversely, adenovDMus-mediated overexpressions of AdipoR1 or AdipoR2 in the liver of obese mice enhanced the effects of adiponectin in the liver. We showed that AdipoR1 and AdipoR2 expression in coronary VSMCs was significantly decreased by treatment with high glucose. A central finding in our study is that PIO ameliorate the downregulation of AdipoR1 and AdipoR2 in coronary artery, which were reduced notably in the DM rat, and prevent hyperglycemia induced decrease of AdipoR1 and AdipoR2 expression in coronary VSMCs. This observation is consistent with three previous reports that have demonstrated positive effects of pioglitazone on adiponectin receptor expression (Kudoh et al., 2011; Shimizu et al., 2007; Coletta et al., 2009). Thus, Kudoh et al. have reported that pioglitazone increases insulin sensitivity by upregulation of AdipoR2 expression in 3T3-L1 adipocytes, but it did not affect AdipoR1 expression (Kudoh et al., 2011). Shimizu et al. have reported that pioglitazone upregulates AdipoR2 mRNA and protein in a hepatocyte cell line (Shimizu et al., 2007). And Coletta et al. have also reported that pioglitazone upregulates AdipoR1 and AdipoR2 mRNA in the human skeletal muscle (Coletta et al., 2009). In contrast, (Li et al., 2007) had previously showed that both adiponectin receptors are expressed in cellular fractions of human adipocytes, but TZD administration did not affect expression of either AdipoR1 or AdipoR2 by short-term stimulation. TZDs improved insulin sensitivity in a dose- and time-dependent manner (Iwaki et al., 2003) TZDs activated the adiponectin promoter, leading to increased production of adiponectin (Iwaki et al., 2003). It has been reported that PIO induced both AdipoR1 and AdipoR2 expression in condition

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In conclusion, the present study demonstrated that PPAR-c ligand PIO attenuated the overexpression of mRNA associated with inflammation in coronary arteries and coronary VSMCs as well as changing the plasma levels of cholesterol and triglyceride. These ameliorations of insulin sensitivity and anti-inflammatory effects of PPARs ligands were found to be related to the enhancement of the expression of AdipoR1 and AdipoR2. In this study, we showed that treatment with PIO could ameliorate coronary insulin resistant and upregulate the expression of AdipoR1 and AdipoR2. PIO had an anti-atherogenic properties via the activation of PPAR-c, suppression of TNF-a overexpression in coronary and coronary VSMCs. Acknowledgements We thank Drs. Bin LI (Institute of Zoology, Chinese Academy of Sciences, Beijing, China) for animal treatment. We acknowledge Yanping Liu, MD (Departments of Internal Medicine and Cardiovascular Center, Milwaukee, Wisconsin) for isolation of coronary VSMCs. PIO were provided by Huadong Medicine Co. (Hangzhou, China). References

Fig. 10. Western blotting assay of the expression of PPAR-c protein in the coronary VSMCs. (A) After pretreatment with PIO (10 lM) + high glucose (HG, 23 mmol/L Dglucose), or PIO (10 lM) + GW9662 (5 lM) + HG or 0.1% dimethyl sulfoxide vehicle in DMEM HG or DMEM normal glucose (NG, 5.5 mmol/L D-glucose) for 24 h, followed by measurement of PPAR-c protein in coronary VSMCs. Representative expression of PPAR-c protein, as determined by Western blot analysis. (B) Quantitative analysis of the expression of the protein for PPAR-c, analyzed by scanning densitometry. ⁄P < 0.05 vs. the NG group, #P < 0.05 vs. HG group, ##P < 0.01 vs. HG group.

with long-term stimulation (Tsuchida et al., 2004). We evaluated the pioglitazone induced AdipoR1 and AdipoR2 expression in vivo and in vitro. Thus it is possible that, the long term-treatment of PIO may attenuate the HG induced decrease of AdioR1 and AdipoR2 expression. Two forms of PPAR-c (c1and c2) are produced because of alternative promoter usage and splicing (Takano and Komuro, 2009). PPAR-c2 is expressed selectively in fat tissue, whereas c1 is found in many tissues including VSMCs (Takano and Komuro, 2009). The functional difference of these two variants is not yet clear. PIO have been reported to antagonize TNF-a–induced insulin resistance both in vitro and in vivo (Díaz-Delfín et al., 2007; Rizza et al., 2011; Iwata et al., 2001). In humans, PIO treatment can convey direct protection against cytokine (TNF-alpha)-induced dysfunction with an increased cardiovascular risk due to type 2 diabetes (Martens et al., 2006; Shimizu et al., 2006). What’s more, several studies in vivo have confirmed the anti-inflammatory and anti-atherogenic properties of adiponectin. Okamoto et al. (2002) have reported that administration of adenovirus-mediated adiponectin reduces atherosclerotic lesion size in mice, accompanied by reductions in the expression of TNF-a. ApoE/APN doubled efficient mice have accelerated T-lymphocyte accumulation in atheromata, and augmented atherogenesis (Okamoto et al., 2008). More recently, it was reported that PIO improves insulin sensitivity, enhances adiponectin expression, and also suppresses TNF-alpha and IL-6 expression (Mohapatra et al., 2009). We showed that PIO significantly induced PPAR-c expression and reduced TNF-alpha expression through agitating PPAR-c. Thus it is possible that PIO has antiatherogenic properties by suppression of TNF-a overexpression and increase of adiponectin, while upregulation of PPAR-c as probable contributors to the prevention of atherosclerosis.

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