Pharmacological activation of PPAR gamma ameliorates vascular endothelial insulin resistance via a non-canonical PPAR gamma-dependent nuclear factor-kappa B trans-repression pathway

European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Endocrine pharmacology

Pharmacological activation of PPAR gamma ameliorates vascular endothelial insulin resistance via a non-canonical PPAR gamma-dependent nuclear factor-kappa B trans-repression pathway Ying Zhang a,b,1, Ri-Xin Zhan a,b,1, Jun-Qun Chen a,b,1, Yan Gao a,b,1, Li Chen a,b, Ying Kong a,b, Xiao-Juan Zhong a,b, Mei-Qi Liu a,b, Jia-Jia Chu a,b, Guo-Qiang Yan a,b, Teng Li a,b, Ming He a,b, Qi-Ren Huang a,b,n,2 a b

Jiangxi Provincial Key Laboratory of Basic Pharmacology, Nanchang University, Nanchang 330006, PR China Department of Pharmacology, Pharmaceutical Science College, Nanchang University, Nanchang 330006, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 12 January 2015 Received in revised form 31 January 2015 Accepted 3 February 2015

Vascular endothelial insulin resistance (IR) is a critically initial factor in cardiocerebrovascular events resulted from diabetes and is becoming a worldwide public health issue. Thiazolidinediones (TZDs) are clinical insulin-sensitizers acting through a canonical peroxisome proliferator-activated receptor gamma (PPARγ)-dependent insulin trans-activation pathway. However, it remains elusive whether there are other mechanisms. In current study, we investigated whether TZDs improve endothelial IR induced by high glucose concentration or hyperglycemia via a non-canonical PPARγ-dependent nuclear factor-kappa B (NF-κB) trans-repression pathway. Our results showed that pre-treatment with TZDs dramatically decrease the susceptibility of endothelial cell to IR, while post-treatment notably improve the endothelial IR both in vitro and in vivo. Moreover, TZDs substantially increase the levels of endothelial nitric oxide synthase (eNOS) and inhibitory κB alpha (IκBα), whereas decrease those of the phosphorylated inhibitory κB kinase alpha/beta (phosphor-IKKα/β) and the cytokines including tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cellular adhesion molecule-1 (sVCAM-1), suggesting that TZDs act indeed through a PPARγdependent NF-κB trans-repression pathway. These findings highlighted a non-canonical mechanism for TZDs to ameliorate endothelial IR which might provide a potential strategy to prevent and treat the diabetic vascular complications clinically. & 2015 Published by Elsevier B.V.

Keywords: Insulin resistance Thiazolidinedione Peroxisome proliferator-activated receptor gamma Endothelium Diabetes Inflammation

1. Introduction Abbreviations: Ang II, angiotensin II; BMI, body mass index; CH, total cholesterol; DMEM, Dulbecco's modified Eagle's medium; eNOS, endothelial nitric oxide synthase; ET-1, endothelin-1; FBS, fetal bovine serum; FINS, fasting serum insulin; FPG, fasting plasma glucose; GL, glucose; HFD, high fat diet; HOMA-IR, homeostatic model assay of IR; HUVEC, human umbilical vein endothelial cells; IκBα, inhibitory κB alpha; IKKα/β, inhibitory κB kinase alpha/beta; IL-6, interleukin-6; INS, insulin; IR, insulin resistance; LBD, ligand binding domain; MNT, mannitol; NF-κB, nuclear factor-kappa B; NO, nitric oxide; PBS, phosphate buffered saline; PE, phenylephrine; PG, pioglitazone; PPARγ, peroxisome proliferator-activated receptor gamma; PPARγLow-HUVEC, HUVEC low-PPARγ-expressing; PPRE, PPARγ response element; RG, rosiglitazone; RXR, retinoid X receptor; sICAM-1, soluble intercellular adhesion molecule-1; SNP, sodium nitroprusside; STZ, streptozotocin; sVCAM-1, soluble vascular cellular adhesion molecule-1; TNFα, tumor necrosis factor alpha; TG, triglycerides; TZDs, thiazolidinediones; WT-HUVEC, wild type human umbilical vein endothelial cells n Corresponding author at: Jiangxi Provincial Key Laboratory of Basic Pharmacology, Nanchang University, Nanchang 330006, PR China. Tel./fax: þ 86 791 86361839. E-mail address: [email protected] (Q.-R. Huang). 1 Equal contributions to this article. 2 Present address: 461 Ba-Yi Street, Nanchang 330006, PR China.

Insulin resistance (IR) is a pathological condition in which cells fail to respond to the normal actions of the hormone insulin. In other words, the cells targeted by insulin (hepatocytes, adipocytes, skeletal muscle cells and vessel cells, etc.) resist to the insulinmediated bioactivities, such as the disposition and metabolism of glucose and lipids. IR is becoming an increasingly pressing worldwide public health issue due to the metabolism-related diseases resulted from IR, such as obesity, diabetes, hypertension and atherosclerosis, which are collectively known as ‘metabolism syndrome’, ‘IR syndrome’ or ‘X syndrome’ (Reaven, 1993; Ford, 2005). As a result, it is of tremendous implications to elucidate the molecular mechanisms, seek for the novel molecular targets and develop new drugs for IR, which are conducive of diminishing the incidence of cardiocerebral vessel events and ameliorating the survival quality of the individuals with diabetes (Jeong et al., 2006).

http://dx.doi.org/10.1016/j.ejphar.2015.02.004 0014-2999/& 2015 Published by Elsevier B.V.

Please cite this article as: Zhang, Y., et al., Pharmacological activation of PPAR gamma ameliorates vascular endothelial insulin resistance via a non-canonical PPAR gamma-dependent.... Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.02.004i

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Thiazolidinediones (TZDs) are insulin-sensitizing drugs acting through peroxisome proliferator-activated receptors (PPARs), which are widely used to treat the patients with type 2 diabetes (Pershadsingh and Moorel, 2008; Calkin and Thomas, 2008). PPARγ, one of PPARs, belongs to a super-family member of nuclear transcription factors that regulate gene expression in response to the specific ligands including endogenous and exogenous ligands (Burgermeister and Seger, 2008; Holness et al., 2009). Both experimental and clinical data have clearly shown that TZDs improved the insulin sensitivity and slowed down the atherogenic processes. (Defronzo, 2010; PopBusui et al., 2009; Wyatt et al., 2010). Pioglitazone (PG) and rosiglitazone (RG), two of the powerful synthetic PPARγ agonists, have been extensively used in the treatment of type 2 diabetes (Schöndorf et al., 2011; Tzoulaki et al., 2009). Numerous studies also demonstrate that TZDs decrease the circulating triglycerides as well as fatty acids while increase HDL-cholesterol (Karak et al., 2013; Hulsmans et al., 2013). Despite of these, TZDs function through a canonical PPARγ-dependent insulin trans-activation pathway (Zhang et al., 2013; Rao et al., 2012). Recent data reported that TZDs also exhibit many other properties such as anti-inflammation and anti-atherosclerosis, which contribute to their beneficial effects on the development of late diabetic micro-vascular and macro-vascular complications (Maniati et al., 2011; Bardelli et al., 2012). Unfortunately, little is known about their exact mechanisms underpinning antiinflammation and anti-atherosclerosis. To this end, we proposed and sought to investigate the hypothesis that TZDs ameliorate endothelial IR induced by high glucose concentration or hyperglycemia through a non-canonical PPARγ-dependent NF-κB transrepression pathway.

concentration) for an additional 10 min. Finally, the supernatants were collected and used to determine the levels of nitrite (commonly as an index of NO production) and Ang II. On the other hand, the screening of the optimal treatment time of glucose was conducted. In brief, HUVEC with a 90% confluence were first pretreated with a complete DMEM containing the screened optimal glucose concentration (22 mmol/l) for the indicated time (i.e., 0, 6, 12, 24, 48, and 72 h, respectively). The following treatments were the same as abovementioned. Thus, the optimal concentration and treatment time of glucose were selected for the further studies. 2.3. Experimental protocols in vitro In protocols of pre-treatment with TZDs, both WT-HUVEC and PPARγLow- HUVEC were first pretreated with a complete DMEM containing 25 μmol/l of PG (final concentration) or 5 μmol/l of RG (final concentration) for 24 h, and then treated with a fresh complete DMEM containing 22 mmol/l of glucose for 48 h. While in protocols of post-treatment with TZDs, WT- and PPARγLowHUVEC were first pretreated with a complete DMEM containing 22 mmol/l of glucose for 48 h, and then treated further with a fresh complete DMEM containing 25 μmol/l of PG or 5 μmol/l of RG for an additional 24 h. The following procedures were identical in both pre- and post-treatment protocols. Briefly, the cells were further cultured for 4 h in a fresh PBS and subsequently treated with 5 mIU/l of insulin for 10 min. Finally, the supernatants were taken together and used to detect the levels of nitrite, Ang II and cytokines (TNFα, IL-6, sICAM-1 and sVCAM-1); the cells were used to analyze the expression levels of PPARγ, eNOS, IKKα/β, phosphorIKKα/β and IκBα.

2. Materials and methods

2.4. Establishment of systemic and endothelial IR in vivo

2.1. Reagents

All animal procedures were approved by the Institutional Animal Care and Use Committee of Nanchang University School of Medicine and conducted in accordance with the guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996). The model was established according to previous method with minor modifications (Srinivasan et al., 2005). Briefly, male Sprague– Dawley (SD) rats weighing 150–180 g (provided by Department of Experimental Animals, Nanchang University, CHN) were housed in standard polypropylene cages and maintained under a controlled room temperature (22 72 1C) and humidity (55 75%) with a cycle of 12:12 h light/dark. All of the rats were randomly allocated into two dietary regimens by feeding with either standard chow (control) or high fat diet (HFD, 58% fat, 25% protein and 17% carbohydrate, as a percentage of total kcal), respectively, for initial 2 weeks. After the first 2 weeks of dietary manipulation, the HFD-fed rats were injected intraperitoneally (i.p.) with a low dose of streptozotocin (STZ, 35 mg/kg) while the control rats were given a vehicle citrate buffer (pH 4.4) in a dose volume of 1 ml/kg, i.p., respectively. Eventually, all of the rats were further fed with the corresponding diet for 4 weeks. Physical parameters including body weight, body length, body mass index (BMI) and fat coefficient were measured. Also, metabolic estimations such as fasting plasma glucose (FPG), fasting serum insulin (FINS), TG, CH and the homeostatic model assay of IR (HOMA-IR) were detected. Meanwhile, the parameters reflecting the endothelial IR such as the serum levels of nitrite and Ang II were assayed before modeling (pre-model) and after modeling (post-model).

Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco-BRL (NY, USA). The assay kits of tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cellular adhesion molecule-1 (sVCAM-1) and angiotensin II (Ang II) were purchased from R&D Systems, Inc. (MN, USA). The assay kits of nitrite, glucose (GL), triglycerides (TG), total cholesterol (CH) and insulin (INS) were purchased from Beyotime Institute of Biotech (Shanghai, CHN). Antibodies against PPARγ, endothelial nitric oxide synthase (eNOS), inhibitory κB kinase alpha/beta (IKKα/β) and phosphor-IKKα/β and inhibitory κB alpha (IκBα) were purchased from Cell Signaling Technology, Inc. (MA, USA). Antibody against β-actin was purchased from Santa Cruz (CA, USA). Other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless indicated elsewhere. 2.2. Establishment of endothelial IR in vitro Wild type human umbilical vein endothelial cells (WT-HUVEC) were obtained from the American type culture collection (ATCC, Catalog No: CRL-1730, US). HUVEC low-PPARγ-expressing (PPARγLowHUVEC) were prepared by infecting the adenoviruses with a short hairpin RNA (shRNA) targeted Pparγ gene (AD-PPARγ-shRNA, Genechem Tech Inc., Shanghai, CHN). These cells were cultured as described in our previous report (Huang et al., 2010). On one hand, the optimal glucose concentration was screened. Briefly, HUVEC with a 90% confluence were first pretreated with a complete DMEM containing the indicated glucose concentrations (5.5, 11, 22, 33 and 44 mmol/l, respectively) or 44 mmol/l of mannitol (MNT) for 48 h. After that, the DMEM was replaced by a fresh phosphate buffered saline (PBS) and the HUVEC were further cultured for 4 h. Subsequently, the cells were treated with 5 mIU/l insulin (final

2.5. Determination of the physical parameters and fat coefficient The physical parameters (body weight, body length and BMI) and fat coefficient were measured according to our previous report

Please cite this article as: Zhang, Y., et al., Pharmacological activation of PPAR gamma ameliorates vascular endothelial insulin resistance via a non-canonical PPAR gamma-dependent.... Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.02.004i

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(He et al., 1997). Briefly, the rats were first anaesthetized with halothane, and then the body weight as well as body length (from nose tip to anus) were measured, respectively. BMI was calculated as weight over length squared (kg/m2). At the end of the treatments, all of the rats were fasted for 12 h and anaesthetized with ketamine (70 mg/kg, i.p.). Blood samples were collected from the carotid artery and finally the rats were sacrificed. The perigenital fat was isolated, weighed and used to calculate the fat coefficient (fat weight (g)/body weight (g)  100%).

2.6. Blood sample collection and metabolic parameter assays Before blood samples were collected, the rats were fasted for 12 h. The blood samples from the pre-modeling rats were collected from the retro-orbital plexus under halothane anesthesia using capillary tubes into eppendorf tubes containing heparin (20 ml, 200 IU/ml) and heparin-free, respectively. The blood samples from the post-modeling rats were collected from the carotid artery by anaesthetizing with ketamine (70 mg/kg, i.p.). The plasma separated by a centrifugation (5 min, 4000g) was used for detecting the FPG, and the serum for measuring the FINS, TG and CH with assay kits according to the manufacturer's instruction. HOMA-IR was calculated using the formula HOMA-IR¼FINS (mIU/l)  FPG (mmol/l)/22.5 (Hou et al., 2013).

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2.7. Experimental protocols in vivo In the protocols of pre-treatment with TZDs, 24 male SD rats were randomly divided into four groups, i.e., control (Ctrl), IR, PG, and RG, respectively. The rats in PG or RG group were intragastrically administered with PG (10 mg/kg/d) or RG (5 mg/kg/d) respectively, once daily for a week, while the others were given a vehicle DMSO in a dose volume of 10 ml/kg. After an initial week of drug manipulation, the model of systemic and endothelial IR was prepared as above-described. In the protocols of posttreatment with TZDs, additional 18 systemic and endothelial IR rats were randomly divided into three groups (i.e., IR, PG, and RG, respectively). The rats in PG or RG group were intragastrically administered with PG (10 mg/kg/d) and RG (5 mg/kg/d) respectively, once daily for a week; while the corresponding rats in IR group were given a vehicle DMSO in a dose volume of 10 ml/kg. Additional 6 normal rats were considered as control groups. Ultimately, the rats were fasted for 12 h and anaesthetized with ketamine (70 mg/kg, i.p.). The blood samples were collected from the carotid artery and used for detecting the levels of nitrite, Ang II and other cytokines (TNFα, IL-6, sICAM-1 and sVACM-1). Besides, the aortae were isolated and used for detecting the expression levels of IKKα/β, phosphor-IKKα/β as well as IκBα and the functional assessment.

Fig. 1. Model confirmation of endothelial insulin resistance in HUVEC. A and B, concentration-response of high glucose concentration-induced insulin resistance. HUVEC with a 90% confluence were pretreated with a complete DMEM containing the indicated glucose concentrations (5.5, 11, 22, 33 and 44 mmol/l, respectively) or 44 mmol/l of mannitol (MNT) for 48 h. After that, the DMEM was replaced by a fresh PBS and the HUVEC were further cultured for 4 h. Subsequently, the cells were treated with 5 mIU/l insulin (final concentration) for 10 min. Finally, the supernatants were collected and used for the assay of the levels of nitrite and Ang II. C and D, time-course of high glucose concentration-induced insulin resistance. HUVEC with a 90% confluence were pretreated with a complete DMEM containing 22 mmol/l glucose for the indicated time (0, 6, 12, 24, 48, and 72 h, respectively). The following treatments were the same as the above-mentioned. nPo 0.05, nnP o0.01, vs. GLU 5.5 or 0 h. Data are expressed as means 7S. E.M. of 4 independent experiments. GLU 5.5 denotes 5.5 mmol/l of glucose, others denote the respective glucose concentrations and MNT44 represents 44 mmol/l of mannitol. NO: nitric oxygen, Ang II: angiotensin II.

Please cite this article as: Zhang, Y., et al., Pharmacological activation of PPAR gamma ameliorates vascular endothelial insulin resistance via a non-canonical PPAR gamma-dependent.... Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.02.004i

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2.8. Functional assessment of rat aortae The functional assessment of rat aortic endothelium was carried out by our previously described method with minor modifications (Liu et al., 2014). Briefly, all rats were fasted for 12 h and anaesthetized with ketamine (70 mg/kg, i.p.). The body weights were recorded and the blood samples were collected immediately. Then, the rats were sacrificed by a cervical dislocation. The thoracic aorta was isolated carefully and prepared to approximately 3 mm-length aortic rings. These aortic rings were isometrically mounted in 10 ml organ baths filled with prewarmed Kreb–Henseleit (K–H) solution (mmol/l, NaCl, 82.8, KCl, 4.7, KH2PO4, 2.4, MgSO4, 1.2, CaCl2, 2.7, dextrose, 11.1, and NaHCO3, 25, pH 7.4, 37 1C) continuously gassed with 95% O2–5% CO2 in a myograph system (model 620 M, DMT, Denmark). Initial passive tension in aortic ring was set as 9.81 mN (1 g). All preparations were then allowed to equilibrate for at least 60 min before further experimentation. The aortic rings were pre-contracted with 1 μmol/l phenylephrine (PE). Dose–response curve was obtained by cumulative addition of acetylcholine (Ach, 10  7–10  4 mol/l) and sodium nitroprusside (SNP, 10  7–10  4 mol/l). The relaxation

at each concentration was measured and expressed as the percentage of force generated in response to PE. 2.9. Western blot assay Western blot assay was performed by a modification of the technique as described in our previous article (Huang et al., 2010). Briefly, HUVEC were harvested and lysed in lysis buffer (25 mmol/l Tris–HCl, pH 7.8, 100 mmol/l NaCl, 1 mmol/l EDTA, 1 mmol/l EGTA, 1 mmol/l Na3VO4, 25 mmol/l glycerol phosphate, 1 mmol/l dithiothreitol, 1% Nonidet P-40 (w/v), 10 μg/ml leupeptin and 10 μg/ml aprotinin). For aorta tissue, 100 mg of aorta tissue were homogenized with a douncer in 1 ml ice-cold lysing buffer (50 mmol/l Tris, 150 mmol/l NaCl, 5 mmol/l EDTA, 1% (v/v) Triton X-100, 1 mmol/l Na3VO4, 100 μg/ml phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin and 0.02% (w/v) sodium azide). The lysates from both cells and tissues were centrifuged at 12,000g at 4 1C for 15 min, and the protein concentration was determined using a Bio-Rad Protein Assay kit (BioRad Laboratories). 50 mg of total proteins was separated by 8% SDSPAGE. Bands on the gels were blotted onto polyvinylidene fluoride membranes (Bio-Rad Laboratories), and then the membranes were

Fig. 2. Model confirmation of systemic and endothelial insulin resistance in obese diabetic rats. A–D, physical parameters (body weight, body length, BMI and fat coefficient) were assessed. E–I, metabolic parameters (FPG, FINS, TG, CH and value of HOMA-IR) were detected. The model of systemic insulin resistance was confirmed by detecting these physical parameters and metabolic parameters in obese diabetic rats. J and K, the model of vascular endothelial insulin resistance was confirmed by measuring the serum levels of nitrite and Ang II in obese diabetic rats. Data are expressed as means 7 S.E.M. of 30 rats. nP o 0.05, nnPo 0.01, vs. pre-model. Po 0.05, Po 0.01, vs. Ctrl, ns ¼no significance; BMI: body mass index, FPG: fasting plasma glucose, FINS: fasting serum insulin, TG: triglyceride, CH: cholesterol, HOMA-IR: homeostatic model assay of IR; Ctrl: control, HFD: high fat diet.

Please cite this article as: Zhang, Y., et al., Pharmacological activation of PPAR gamma ameliorates vascular endothelial insulin resistance via a non-canonical PPAR gamma-dependent.... Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.02.004i

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incubated with antibodies against various proteins at a 1:1000 dilution. A horseradish peroxidase-conjugate anti-rabbit antibody (Amersham Pharmacia Biotech) was used as a secondary antibody at a 1:2000 dilution. Immunoreactive bands were visualized by the enhanced chemiluminescence (ECL) detection system (Perkin-Elmer Life Science Inc., Boston, MA, USA).

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an assay kit according to the manufacturer's instruction. Briefly, a standard curve was prepared using a series of nitrite concentrations. 100 μl of samples were added onto a 96-well microplate, and then Griess reagents I and II were in turn added and mixed. Next, this mixture was incubated at 37 1C for 60 min. Finally, the optical density (OD) was determined at 550 nm with a spectrophotometer (Bio-Rad Laboratories).

2.10. Quantitative assay of Ang II and cytokines by ELISA 2.12. Statistical analysis Quantitative assay of Ang II and cytokines (TNFα, IL-6, sICAM-1 and sVCAM-1) was performed using the Quantikine ELISA Kits according to the manufacturer's instruction. Briefly, 50 μl diluted serum or cell supernatants was first added to a 96-well polystyrene microplate precoated with the indicated monoclonal antibodies and incubated for 2 h at room temperature on a shaker. Next, after five times of gentle washes with PBS, 100 μl secondary antibodies conjugated to horseradish peroxidase were added and further incubated for 2 h. Then, 100 μl substrate solution was added and treated for 30 min at room temperature. Ultimately, 100 μl stop solution was added, and the optical density (OD) was read at 450 nm in a microplate reader (Bio-Rad Laboratories). 2.11. Measurement of nitrite levels Nitrite level in the supernatant or serum is commonly thought as an index of NO production. The nitrite level was determined by

All data were expressed as the means 7S.E.M. Significance was tested with unpaired t-test, one-way ANOVA and homogeneity test of variance. P value o0.05 was considered statistically significant.

3. Results 3.1. The model confirmation of endothelial IR in HUVEC The extents of endothelial IR are evaluated by the release levels of NO and Ang II stimulated by insulin (Kearney et al., 2008). As shown in Fig. 1, upon incubation with the indicated glucose concentrations for 48 h, followed by stimulation with insulin, HUVEC displayed various IR extents responding to the indicated glucose concentrations, presenting a gradual decrease in the NO

Fig. 3. Improved endothelial IR by pre-or post-treatment with TZDs is PPARγ-dependent in HUVEC. A and B, effects of pre-treatment with TZDs on endothelial cell insulin resistance both in WT- and PPARγLow-HUVEC. WT- and PPARγLow-HUVEC were first pretreated with a complete DMEM containing PG (25 μmol/l, final concentration) or RG (5 μmol/l, final concentration) for 24 h, and then treated with a complete DMEM containing 22 mmol/l glucose for 48 h. After that, the DMEM was substituted by a fresh PBS and the HUVEC were further cultured for 4 h. Subsequently, the cells were treated with insulin (5 mIU/l, final concentration) for 10 min. Finally, the supernatants were collected and used for the assay of the levels of nitrite and Ang II. C and D, effects of post-treatment with TZDs on endothelial cell insulin resistance both in WT- and PPARγLow-HUVEC. WT- and PPARγLow-HUVEC were first pretreated with a complete DMEM containing 22 mmol/l glucose for 48 h, and then treated further with a fresh complete DMEM containing PG (25 μmol/l, final concentration) or RG (5 μmol/l, final concentration) for additional 24 h. The following treatments were the same as the A and B. nPo 0.05, nnP o0.01, vs. Ctrl; Po 0.05, P o0.01, vs. IR; && Po 0.01, vs. WT-HUVEC; ns ¼no significance. Data are expressed as means 7S.E.M. of 4 independent experiments. Ctrl: control, IR: insulin resistance, PG: pioglitazone, RG: rosiglitazone, WT-HUVEC: wild type HUVEC, PPARγLow-HUVEC: HUVEC Low-PPARγ-expressing.

Please cite this article as: Zhang, Y., et al., Pharmacological activation of PPAR gamma ameliorates vascular endothelial insulin resistance via a non-canonical PPAR gamma-dependent.... Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.02.004i

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levels and a progressive increase in the Ang II levels with the increase in glucose concentrations. The most significant change occurred at 22 mmol/l of glucose. Although the levels of NO and Ang II continuously decreased or increased with the glucose concentration further increasing, the cells noticeably died. Therefore, we selected 22 mmol/l of glucose as the optimal stimulation concentration for the further studies. 44 mmol/l of MNT, as an osmolarity control, did not affect above parameters (Fig. 1(A) and (B)). In addition, with the elongation in glucose-stimulating time, the IR extents were gradually severe in a time-dependent manner and peaked at 48 h. Hence, we chose 48 h as the optimal stimulation time for further experiments (Fig. 1(C) and (D)).

detecting the physical parameters such as the body weight, body length, BMI, and fat coefficient and metabolic estimations including FPG, FINS, TG, CH as well as HOMA-IR; while the endothelial IR is evaluated by the serum levels of NO and Ang II (Ingelsson et al., 2007). As predicted, HFD-fed rats exhibited not only higher BMI and fat coefficient than those of the control rats (Fig. 2(A)–(D)), but also hyperglycemia, hyperinsulinemia and dyslipidemia (Fig. 2(E)–(I)), demonstrating that the HFD-fed rats appeared in a phenotype of the systemic IR. Also, these rats displayed a phenotype of the endothelial IR, embodying a marked decrease on the NO levels and a substantial increase on the Ang II levels, highly consistent with the in vitro data (Fig. 2(J) and (K)).

3.2. The model confirmation of systemic and endothelial IR in obese diabetic rats

3.3. Improved endothelial IR by pre- or post-treatment with TZDs is PPARγ- dependent in HUVEC

Much evidence demonstrates that the IR arises along with central obesity, hyperglycemia, hyperinsulinemia and dyslipidemia (Schmieder et al., 2007). Hence, the systemic IR is assessed by

In WT-HUVEC, both pre- and post-treatments with PG or RG dramatically antagonized the decrease of the NO levels and increase of the Ang II levels induced by 22 mmol/l of glucose,

Fig. 4. Effects of pre- or post-treatment with TZDs on endothelial insulin resistance and endothelial-dependent vasodilation in vivo. A and B, effects of pre- or post-treatment with TZDs on endothelial insulin resistance. C and D, effects of pre-treatment with TZDs on endothelial-dependent vasodilation. E and F, effects of post-treatment with TZDs on endothelial-dependent vasodilation. Data are expressed as means 7 S.E.M., n¼ 6 rats. nP o 0.05, nnP o 0.01, vs. Ctrl; Po 0.05, Po 0.01, vs. IR, ns ¼no significance. Ach: acetylcholine, SNP: sodium nitroprusside. In Fig. 4C–F, error bars were in part omitted for the sake of clarity.

Please cite this article as: Zhang, Y., et al., Pharmacological activation of PPAR gamma ameliorates vascular endothelial insulin resistance via a non-canonical PPAR gamma-dependent.... Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.02.004i

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suggesting that both PG and RG may improve the endothelial cell IR induced by high glucose concentration, but there was no significant difference between PG and RG. In PPARγLow-HUVEC, however, both pre- and post-treatments with PG or RG, failed to counteract the changes of the levels NO and Ang II induced by the 22 mmol/l of glucose, demonstrating that the improved effects of PG and RG are abolished. Taken together, these data illustrate that both pre- and post-treatments with TZDs may ameliorate the endothelial cell IR induced by high glucose concentration and their improved effects were PPARγ-dependent (Fig. 3(A)–(D)).

3.4. Effects of pre- or post-treatment with TZDs on endothelial IR and endothelial-dependent vasodilation in vivo The pre-treatment with PG or RG did not significantly improve endothelial IR induced by hyperglycemia, while the posttreatment strikingly ameliorated the endothelial IR, consistent with the results in vitro (Fig. 4(A) and (B)). In addition, in IR rats, the aortic vasodilation elicited by Ach was markedly decreased, whereas that induced by SNP was slightly decreased, indicating that only endothelial other than smooth muscle function was impaired in IR state. The pre-treatment with PG or RG failed to combat markedly the decrease of the aortic vasodilation responsive to Ach (Fig. 4(C)), whereas the post-treatment counteracted

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substantially the decrease of the vasodilation responsive to Ach (Fig. 4(E)). Also, neither the pre-treatment nor the post-treatment obviously affected the vasodilation responsive to SNP (Fig. 4 (D) and (F)). These results suggest that post-treatment with TZDs may improve the endothelial-dependent vasodilation.

3.5. Effects of pre- or post-treatment with TZDs on the expression levels of PPARγ and eNOS in vivo and in vitro As the synthetic ligands of PPARγ, it is well-known that TZDs improve systemic IR by regulating the typical PPARγ-dependent insulin pathway (Dahabreh and Medh, 2012). Therefore, we naturally gave a link between the endothelial IR and the expression of PPARγ as well as eNOS. As anticipated, we found that both the pre- and post-treatments with PG or RG enhanced strikingly the expression levels of eNOS both in vitro and in vivo (Fig. 5(A), (C) and (E)). Surprisingly, neither the pre- nor post-treatment had significant impacts on the expression of PPARγ regardless of in vivo and in vitro (Fig. 5(B), (D) and (F)). The reason of this is likely that both PG and RG bind only to the ligand binding domain (LBD) of PPARγ which subsequently activate the PPARγ-dependent insulin pathway and ultimately induce the expression of eNOS other than PPARγ per se.

Fig. 5. Effects of pre- or post-treatment with TZDs on the levels of PPARγ and eNOS in rat aorta or HUVEC. A and B, effects of pre-treatment with TZDs on the levels of eNOS and PPARγ in HUVEC. C and D, effects of post-treatment with TZDs on the levels of eNOS and PPARγ in HUVEC. E and F, effects of post-treatment with TZDs on the levels of eNOS and PPARγ in rat aorta. Grouping and treatments were the same as Figs. 3 and 4. nPo 0.05, vs. Ctrl; Po 0.05, vs. IR, ns ¼no significance. Data in vitro are expressed as means 7 S.E.M. of 4 independent experiments, and in vivo, 6 independent rats. eNOS: endothelial nitric oxygen synthase, AU: arbitrary unit.

Please cite this article as: Zhang, Y., et al., Pharmacological activation of PPAR gamma ameliorates vascular endothelial insulin resistance via a non-canonical PPAR gamma-dependent.... Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.02.004i

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3.6. Trans-repression of pre- or post-treatment with TZDs on NF-κB signaling in vitro Finally, we explored the action mechanisms of TZDs responsible for the improvement of endothelial IR. As expected, HUVEC in IR state showed fewer levels in IκBα, whereas more levels in phosphor-IKKα/β, TNFα, IL-6, sICAM-1 and sVACM-1 than those of control cells, suggesting that high glucose concentration activates the NF-κB signaling, gives rise to inflammation and leads ultimately to IR. Both pre- and post-treatments with PG or RG, reversed the above changes, indicating that they may repress the NF-κB trans-activation, and dwindle the endothelial cell susceptibility to IR or improve the endothelial cell IR induced by high glucose concentration through a canonical PPARγ-dependent NF-κB trans-repression pathway (Fig. 6(A)–(F)). 3.7. Trans-repression of post-treatment with TZDs on NF-κB signaling in vivo To the end, we tested the effects of post-treatment with TZDs on the NF-κB signaling in vivo. Similar to the data in vitro, IR rats presented fewer levels in IκBα, whereas more levels in phosphorIKKα/β, TNFα, IL-6, sICAM-1 and sVACM-1 than those of control rats. Post-treatment with PG or RG may tremendously restore the above- mentioned changes. Taken together, these data suggest that post-treatment with TZDs may repress the NF-κB

trans-activation, and ameliorate the vascular endothelial IR in a canonical PPARγ-dependent NF-κB trans-repression manner (Fig. 7 (A) –(F)).

4. Discussion Recent findings demonstrate that IR and the successive hyperinsulinemia are the predominant risk factors and the common pathophysiological foundation of cardio- cerebrovascullar diseases (Kozakova et al., 2013; Yin et al., 2013). However, the exact mechanism underlying IR remains unknown. Accordingly, the studies on IR mechanisms have been considered as the most cutting-edge issue for the past decades (Mi et al., 2012; Gallagher and LeRoith, 2013). Recently, the researches in IR mechanisms highlight canonical insulin-targeted cells such as hepatocytes, adipocytes and skeletal muscle cells as well (Jocken et al., 2013; Seldin et al., 2012; Fontana et al., 2012), but those coming from the non-canonical insulin-targeted cells such as vascular endothelial cells and smooth muscle cells are still few nowadays. In this paper, we placed the emphasis on the endothelial IR. It is well-known that vascular endothelium is not only a vessel barrier, but also an important endocrinal organ. Upon stimulation by insulin at a physiologic dose, it secretes and releases many vasoactive substances such as NO, Ang II and endothelin-1 (ET-1) which regulate the vessel integrity and tension. The regulatory

Fig. 6. Trans-repression of pre- or post-treatment with TZDs on the NF-κB pathway in vitro. A and B, effects of pre- or post-treatment with TZDs on the expression levels of IκBα, IKKα/β and its phosphorylated protein in HUVEC. C–F, effects of pre- or post-treatment with TZDs on the expression levels of NF-κB target genes in HUVEC. Grouping and treatments were the same as Fig. 3. nPo 0.05, nnP o 0.01, vs. Ctrl; P o 0.05, Po 0.01, vs. IR. Data are expressed as means 7 S.E.M. of 4 independent experiments.

Please cite this article as: Zhang, Y., et al., Pharmacological activation of PPAR gamma ameliorates vascular endothelial insulin resistance via a non-canonical PPAR gamma-dependent.... Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.02.004i

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action is accomplished by fine-tuning the balance between IRS-1/ PI (3) K/ART/NO pathway and RAF/MAPK/ERK/Ang II pathway (Potenza et al., 2005; Montagnani et al., 2002; Hitomi et al., 2011). If the balance is disrupted due to pathological states, i.e., hyperglycemia, hyperinsulinemia and hyperlipidemia, etc., the endothelial dysfunction and IR might occur, giving rise to in turn the cardiocerebral vessel event cascades (Wong and Marwick, 2007; Talbot et al., 2012). Based on these, numerous investigators often choose the levels of NO and Ang II as the indicators of endothelial IR (Kearney et al., 2008; Ingelsson et al., 2007). In this study, we well-established the cell and animal models using the two indicators and found that the concentration of 22 mmol/l and

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48 h were the optimal stimulating concentration and time of glucose, respectively. Based on the successful models of the endothelial IR, we next visualized effects of TZDs on the endothelial IR in vitro and in vivo. Indeed, we observed that post-treatment with PG or RG, both in vitro and in vivo, ameliorated strikingly the systemic and endothelial IR. Nonetheless, pre-treatment with PG or RG, only in vitro, but not in vivo, notably decreased the endothelial cell susceptibility to IR. As for the reasons, it is likely that the duration of pre-treatment is too short to generate an effect powerful enough, i.e., the pre-treatment lasted for only a week, not throughout the period of model-preparing in vivo. Regarding the

Fig. 7. Trans-repression of post-treatment with TZDs on the NF-κB pathway in vivo. A and B, effects of post-treatment with TZDs on the expression levels of IκBα, IKKα/β and its phosphorylated protein in rat aorta. C–F, effects of post-treatment with TZDs on the expression levels of NF-κB target genes in rat serum. Grouping and treatments were the same as Fig. 4. nPo 0.05, nnPo 0.01, vs. Ctrl; Po 0.05, P o0.01, vs. IR. Data are expressed as means 7 S.E.M. of 6 rats.

Please cite this article as: Zhang, Y., et al., Pharmacological activation of PPAR gamma ameliorates vascular endothelial insulin resistance via a non-canonical PPAR gamma-dependent.... Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.02.004i

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Fig. 8. Schematic presentation for putative mechanisms of TZDs. TZDs bind to a complex consisting of PPARγ, retinoid X receptor (RXR), and co-repressor complex and activate it. Subsequently, a disassociation of PPAR/RXR with the co-repressor complex occurs followed by an association with a co-activator complex to form a new complex which binds to the PPARγ response element (PPRE) of IκBα gene. Ultimately, IκBα is induced which represses the activation of NF-κB. In addition, PPARγ activated by TZDs is capable of inhibiting the activation of IKKα/β and improves endothelial insulin resistance.

action mechanisms of TZDs, a large number of experimental and clinical data reported that TZDs act through a canonical PPARγdependent insulin trans-activation pathway. In other words, liganded PPARγ trans-activates its target genes and induces their expressions. These genes include those of insulin signaling and metabolism of glucose and lipid. (Amato et al., 2012; Chen et al., 2012; Gupta et al., 2010). Of course, this is a prevalent viewpoint at present, but we herein proposed and tested another hypothesis that TZDs improve the endothelial IR via a non-canonical PPARγ-dependent NF-κB trans-repression pathway. In detail, TZDs first bind to a heterodimer of PPARγ and retinoid X receptor (RXR), activate PPARγ, and result in the disassociation of the heterodimer with its co-repressor complex (NCoR, Tab2, and TBL1, etc.). Next, the disassociated heterodimer associates with its co-activator complex (PGC-1, etc.) to form a new transcriptional complex. Ultimately, the new complex binds to the PPARγ response element (PPRE) of IκBα gene, which induces the expression of IκBα. In addition, the new complex is capable of inhibiting IKKα/β, an upstream kinase of IκBα, and fails to phosphorylate IκBα at the sites of serine 32 and 36. Consequently, degradation of IκBα and subsequent nucleartranslocation and activation of NF-κB (P65/P50) fail to occur. Eventually, it results in the inhibition of inflammation genes' expression (TNFα, IL-6, sICAM-1 and sVACM-1). A large number of literatures have confirmed that IR is a chronic inflammation resulted from these inflammatory cytokines (Nguyen et al., 2012; Phielix et al., 2011). Indeed, our present data in vitro and in vivo displayed that the endothelial cells in IR state had fewer expression levels in IκBα, whereas more expression levels in phosphorIKKα/β and other cytokines such as TNFα, IL-6, sICAM-1 as well as sVACM-1 than those in the control cells, suggesting that high glucose concentration or hyperglycemia activates the NF-κB pathway, gives rise to inflammation and leads ultimately to IR. However, both pre- and post-treatments with TZDs reversed the above changes, indicating that they may repress the NF-κB transactivation, and alleviate the endothelial cell susceptibility to IR or improve the endothelial cell IR through a NF-κB trans-repression pathway. Here, a question was raised whether the improved effects of TZDs on the endothelial IR are mediated by PPARγ or a direct

action on the NF-κB pathway. To address this question, we applied to an RNA interference (RNAi) technology to knock-down the expression of PPARγ. As expected, our present data showed that after the PPARγ expresion levels were down-regulated, the improvement effects of TZDs were abolished. Consequently, we concluded that the improvement effects of TZDs are PPARγdependent (Fig. 8). In addition, the endothelial IR and endothelial dysfunction have a close link. Moreover, both of them are the triggering factors for atherosclerosis and hypertension (Wang et al., 2011). In our present study, we also viewed when endothelial cells were in the IR states, their endothelial-dependent vasodilations were elicited by Ach rather than the non-endothelial-dependent vasodilations which were induced by SNP severely destroyed, suggesting that in IR or the early state of diabetes, the dysfunction for endothelial cells instead of vascular smooth muscle cells occurs. Yet, treatment with TZDs strikingly improves endothelial-dependent vasodilations other than non-endothelial- dependent vasodilations, implying that the early combination treatment of oral hypoglycemic agents (metformin or TZDs) and anti-inflammation agents might be required for the therapy of diabetes clinically. Thus, vascular complications of diabetes will be well prevented (Schöndorf et al., 2011).

5. Conclusions The present study demonstrated that TZDs improve endothelial IR via a non-canonical PPARγ-dependent NF-κB trans-repression pathway other than a conventional PPARγ-dependent insulin transactivation pathway. The findings enrich the regulatory mechanisms for TZDs to improve the endothelial IR, and provide a potential strategy to prevent and treat diabetic vascular complications.

Acknowledgments This study was supported by the research Grants from the National Natural Science Foundation of China (81360060, 81070633, 30860111,

Please cite this article as: Zhang, Y., et al., Pharmacological activation of PPAR gamma ameliorates vascular endothelial insulin resistance via a non-canonical PPAR gamma-dependent.... Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.02.004i

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