Cellular Physiology and Biochemistry

Cell Physiol Biochem 2014;34:658-671 DOI: 10.1159/000363031 Published online: August 13, 2014

© 2014 S. Karger AG, Basel www.karger.com/cpb

Wang et al.: Effect FGF21 on ECs Under High Glucose Condition Accepted: June 23,of 2014 1421-9778/14/0343-0658$39.50/0 This is an Open Access article licensed under the terms of the Creative Commons AttributionNonCommercial 3.0 Unported license (CC BY-NC) (www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only.

Original Paper

Fibroblast Growth Factor 21 Protects Against High Glucose Induced Cellular Damage and Dysfunction of Endothelial Nitric-Oxide Synthase in Endothelial Cells Xiao-Mei Wang Shuang-Shuang Song Hang Xiao Pan Gao Xue-Jun Li Liang-Yi Si Department of Geriatrics, Southwest Hospital, Third Military Medical University, Chongqing, China

Key Words )*)‡(QGRWKHOLDOFHOO‡$03.‡H126‡+LJKJOXFRVH Abstract Aims: Fibroblast growth factor 21 (FGF21) is a powerful endocrine hormone modulating glucose and lipid metabolism and represents a promising drug for type 2 diabetes. The present study was to determine the effect of FGF21 on high glucose-induced damage and dysfunction in endothelial cells. Methods: The protein expression of E-klotho was examined in human umELOLFDOYHLQHQGRWKHOLDOFHOO +89(&V XVLQJLPPXQRÁXRUHVFHQFHDQG:HVWHUQEORWWLQJ+8VECs were cultured in medium with normal glucose (NG), high glucose (HG) and HG + FGF21 (30 nM). Cell viability, migration, reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase, nitric oxide (NO) production, intracellular cyclic guanosine monophosphate (cGMP) and endothelial nitric oxide synthase (eNOS) phosphorylation at Ser-1177/Ser633 sites were measured. Results: E-klotho, the anchor protein of FGF21, is expressed in HUVECs. Administration of FGF21 prevented HG-induced impairment of cell viability, migration, oxidant stress, NO production and intracellular cGMP levels in HUVECs. FGF21 also rescued HG-induced decrease of eNOS phosphorylation at Ser-1177 and Ser-633. HG and FGF21 had no effects on eNOS phosphorylation at Ser-617 and Thr-495. Inhibition of AMP-activated protein kinase (AMPK), but not Akt or Ca2+/calmodulin-dependent protein kinase II, abolished the protective effect of FGF21 on eNOS phosphorylation at Ser-1177. The protective effect of FGF21 on eNOS phosphorylation at Ser-633 was also abolished by inhibition of AMPK but not by Akt or cAMP-dependent protein kinase A. Conclusion: 2XU UHVXOWV SURYLGH WKH ÀUVW evidence that FGF21 protects against high glucose induced cell damage and eNOS dysfunction in an AMPK-dependent manner in HUVECs, and suggest that FGF21 may be a promoting therapeutic agent for vascular complications in diabetes. Copyright © 2014 S. Karger AG, Basel Prof. Liang-Yi Si, MD

Department of Geriatrics, Southwest Hospital, Third Military Medical University, Gaotanyan Road 29, Shapinba District, Chongqing, 630008 (China) Tel.+86 023-68754150, E-Mail [email protected]

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Cellular Physiology and Biochemistry

Cell Physiol Biochem 2014;34:658-671 DOI: 10.1159/000363031 Published online: August 13, 2014

© 2014 S. Karger AG, Basel www.karger.com/cpb

Wang et al.: Effect of FGF21 on ECs Under High Glucose Condition

Introduction

Endothelial dysfunction is a well-accepted marker of vascular injury. The endothelial dysfunction is related with apoptosis [1], autophagy [2, 3] and the malfunction of intracellular molecular cascades [4]. Endothelial dysfunction is associated with cardiovascular risk factors and precedes the development of hypertension, atherosclerosis, myocardial infarction and etc. Importantly, recent data suggest that loss of modulatory role of the endothelium could be implicated in the pathogenesis of diabetic vascular complications [5]. A large number of evidence indicates that the endothelial dysfunction is impaired in diabetic animal models and patients [5]. The high levels of blood glucose induces endothelial dysfunction, which is characterized by changes in altered barrier function, increased oxidative stress, in lammatory activation and, most importantly, impairment of endothelium-derived nitric oxide (NO). Fibroblast growth factors are a family of structurally related polypeptides characterized by a high af inity for heparin [ ]. ost F Fs act as paracrine factors diffusing away from their origin tissues and functions on cell growth and differentiation [ , ]. Fibroblast growth factor 21 (F F21) is a member of F Fs super family that is reported to be primarily expressed in metabolic organs/tissues such as liver [8]. Unlike most other F Fs, F F21 is released from liver into blood as a hormone and mediates its biological responses as an extracellular protein by binding to and activating cell surface F F receptor [ ]. In this process, E-klotho, a transmembrane anchor protein, is required for the activation of F F21-induced signaling cascade [ , 1 ]. E-klotho enriches in metabolic tissues such as liver and adipose [8]. Recently, F F21 attracts much attention as a relatively new fasting-responding hormone increasing energy production and improving energy utilization [11, 12]. It acts as an autocrine factor regulating PPARg activity and glucose uptake [13, 14]. oreover, F F21 protects from glucolipotoxicity and cytokine-induced islet apoptosis, increases islet insulin content, and enhances glucose-induced insulin secretion [15]. F F21 also regulates lipolysis in white adipose tissue [1 ]. eanwhile, the biological functions of F F21 in non-metabolic tissues have been gradually discovered. F F21 increases systemic glucocorticoid levels, suppresses physical activity and alters circadian behavior by acting on suprachiasmatic nucleus of the hypothalamus and the dorsal vagal complex of the hindbrain [1 ]. oreover, F F21 modulates female reproduction in response to nutritional challenge by acting on the suprachiasmatic nucleus [18]. Serum F F21 levels correlates signi icantly with blood 4 lymphocyte count and immune recovery in HI infection [1 ]. However, the roles of F F21 in other system are largely unknown yet. F F21 is a circulating protein and its serum concentrations are closely associated with metabolic physiology and diseases. Blood F F21 levels are elevated in impaired glucose tolerance and type 2 diabetes and correlate with muscle and hepatic insulin resistance [2 , 21]. Interestingly, blood F F21 levels also correlates with blood glucose in diabetic patients [2 , 21]. It is well known that high glucose is a perturbation suf icient to alter the characteristics of vascular biology in diabetes. Thus, the potential effect of blood F F21 on vascular biology, especially under high glucose condition, is an interesting question. To answer this, we investigated the in luence of F F21 on the biological functions of endothelial cells under high glucose condition. Our results provide the irst evidence that F F21 protects against high glucose-induced damage and regulates endothelial nitric oxide synthase (eNOS) phosphorylation in endothelial cells. Materials and Methods Reagents ouse F F21 full length protein (catalogue No ab 32 ), compound , nitric oxide (NO) assay were purchased from Abcam ( ambridge, U ). EB -2 medium was purchased from onza ( ong Beach, A, USA). Antibodies against phospho-eNOS-Ser11 (p-eNOS-Ser11 ), phospho-eNOS-Ser 33, total-Enos, phosphoA P , total-A P and tubulin were purchased from illipore ( ilford, A, USA). Two antibodies against

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Cell Physiol Biochem 2014;34:658-671 DOI: 10.1159/000363031 Published online: August 13, 2014

© 2014 S. Karger AG, Basel www.karger.com/cpb

Wang et al.: Effect of FGF21 on ECs Under High Glucose Condition

E-klotho (R Systems and Santa ruz Biotechnology) were used in this study. y-3 conjugated secondary antibody, N- 3, H-8 and -22 were purchased from Santa ruz Biotechnology (Santa ruz, A, USA). ichloro-dihydro- luorescein diacetate ( FH- A), 4 , -diamidino-2-phenylindole ( API) and 1 culture medium were purchased from Invitrogen ( arslbad, A, USA). ell viability assay ( ell ounting it8) was purchased from ojindo olecular Technologies, Inc. ( umamoto, apan). alondialdehyde ( A), cyclic guanosine monophosphate (c P) and manganese-/copper-zinc- superoxide dismutase ( n-SO and u/ n-SO ) kits were purchased from ell Biolab (San iego, A, USA). Protease inhibitors cocktail and phosphatase inhibitor cocktail were purchased from Pierce hemical o. (Rockford, I , USA). siRNA targeting A P D1/2 was purchased from Santa- ruz Biotechnology (Santa- ruz, A, USA). Cell culture and siRNA transfection Human umbilical vein endothelial cells (HU E s) were purchased from American Type ulture ol- lection (Rockville, ) and cultured as described [2]. Brie ly, cells were grown under 1 medium containing 1 fetal bovine serum and penicillin (1 IU/m ) and streptomycin (1 Pg/m ) at 3 in a humidi ied 5 O2 atmosphere. The concentration of glucose in this medium is 5.5 m (normal glucose). To produce high glucose challenge in HU E s, high gluctose (5 m and 1 m ) was applied. For F F21 treatment, PBS-dissolved F F21 was added into the medium. Transfection of siRNA was performed using ipofactamine T with P US (Invitrogen) as described previously [22, 23]. Brie ly, cells were grown in six-well plate to 3 con luence and washed with serum-free medium. About 8 l of serum-free medium were added per well. 1 n siRNA and 5 l of ipofactamine (Invitrogen) were mixed and added into 2 Pl medium. The mixtures was incubated for 3 min at room temperature and then added into cells for hours. One day after the initial transfection, a second round of transfection was performed in the same way as the previous one. At 2 h after initial transfection, cells were treated and harvested as indicated. —‘ϔŽ—‘”‡•…‡…‡ Immuno luorescence was performed as described previously [24, 25]. HU E s were plated on 13mm glass coverslips and cultured for 24 h before ixation. The cells were ixed with 4 paraformaldehyde at room temperature for 3 min, permeabilized with . 5 Triton -1 for 1 min, and immunostained with an anti-E-klotho antibody. In negative control, normal Ig replaced the anti-E-klotho antibody. Then, cells were stained by secondary antibody that had been conjugated with y3. NA staining with API was performed at .5 Pg/ml [2 ]. All immuno luorescence images were observed on a F 1 onfocol microscopy (Olympus, apan). Western blotting Western blotting was performed as described previously [2 , 28]. Brie ly, cells were lysed in lysis buffer with protease inhibitors cocktail and phosphatase inhibitor cocktail. ell lysates were centrifuged at 1 , g for 1 min. The supernatants were collected and boiled for 1 min. After determination of protein concentration, equal amounts of proteins ( 3 Pg) were separated by 12 S S PA E. Blots were transferred to nitrocellulose membrane and incubated overnight at 4 with antibodies against E-klotho (1 8 ), p-eNOS-ser11 (1 4 ), p-eNOS-ser 33 (1 4 ), t-eNOS (1 1 ) and tubulin (1 1 ) and followed by corresponding secondary antibodies (1 5, ). Signals were detected using enhanced chemoluminescence kit (E , Amersham, USA). Cell viability assay ell viability was evaluated by a non-radioactive cell counting kit-8 ( -8) assay as described previously [2 ]. HU E s were maintained in cultured medium with normal glucose, high glucose and high glucose + F F21 (3 n ) in -well plates. edium was changed daily. At 24 and 48 hours post treatment, medium was discarded and cells were incubated in -8 solution for 2 h at the same incubator conditions after which the absorbance was read at 45 nm with a reference wavelength of 5 nm. Transwell migration assay HU E s (1 1 5) were placed in the upper chamber of a 24-well transwell migration insert (pore size 5 Pm) with 1 medium in the presence normal glucose, high glucose and high glucose + F F21 (3 n ).

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Cellular Physiology and Biochemistry

Cell Physiol Biochem 2014;34:658-671 DOI: 10.1159/000363031 Published online: August 13, 2014

© 2014 S. Karger AG, Basel www.karger.com/cpb

Wang et al.: Effect of FGF21 on ECs Under High Glucose Condition

The lower chamber contained migration medium (EB -2 medium, .5 BSA, 2 FBS). After migration for 12 and 24 hours, nonmigrating cells on the upper side of the membrane were removed with a cotton swab [3 ]. ells that had migrated and were attached to the lower side were ixed with 1 formalin and stained with API for 2 min and counted in ive different random ields using a luorescent microscopy [31]. Activity of caspase-3/7 Activity of caspase-3/ was measured using a luorimetric assay from Promega ( adison, WI) as described previously [32, 33]. The luorimetric signal was read in a microplate reader and calculated for activities of caspase-3/ . Measurement of intracellular reactive oxygen species (ROS) levels Intracellular ROS production was evaluated using membrane-permeable probe FH- A assay as described previously [34]. HU E s were cultured in medium of normal glucose, high glucose and high glucose + F F21 (3 n ) for 48 hours, and then washed with PBS followed by incubation in medium containing 2 Pmol/ FH- A at 3 for 2 hours. Subsequently, the cell medium containing FH- A was removed and washed with PBS for three times. Then, the luorescence was revealed by luorescence microscopy. The FH- A luorescence was calculated using Image Pro Plus software ( edia ybernetics, Silver Spring, USA). MDA levels and activities of Mn-SOD and Cu/Zn-SOD Intracellular lipid peroxidation was evaluated by measurement of A levels as described previously [35]. Anti-oxidant activity was evaluated by measurement of n-SO and u/ n-SO activities as described previously [3 ]. Brie ly, the HU E s incubated with normal glucose, high glucose and high glucose + F F21 (3 n ) for 24 and 48 hours were lysed by RIPA buffer, and then the n-SO and u/ n-SO activities and intracellular A concentrations of each sample were detected according to the manufacturer s instruction using microplate reader respectively. NO production measurement The concentration of NO in HU E s was determined as described previously [3 ]. The concentration of nitrite, the stable product of NO was detected to re lect the NO production. HU E s were incubated with medium of normal glucose, high glucose and high glucose + F F21 (3 n ) for 24 and 48 hours. The medium was collected and nitrite content in the medium was measured using a microplate method based on the riess reaction. The optical densities at 5 nm wavelength were obtained using a icroplate Reader (Thermo, USA) and concentrations of NO were calculated according to the calibration curve. Intracellular cGMP measurement HU E s were treated with normal glucose, high glucose and high glucose + F F21 (3 n ) for 12 hours and 24 hours. Then, cells were lysed by RIPA buffer with same volume. Protein concentration was determined by B A assay. Protein concentration of all samples was adjusted to be equal. The c P level was measured using a commercial c P E ISA kit according to the manufacturer s instruction. Statistical analysis Statistical analysis was performed using the raphPad Prism 5 software program. The data were expressed as the mean SE and compared using ANO A with Tukey correction. Statistical signi icance was set at P . 5.

Results

E-klotho, the essential anchor protein for FGF21 function, is expressed in HUVECs As shown in Fig. 1A, immuno luorescence staining with primary antibody against E-klotho showed that E-klotho was indeed expressed in HU E s. In contrast, there was no detectable immuno luorescence signal in negative control experiments with nonspeci ic normal Ig . We also performed Western Blotting analysis using two primary

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Cellular Physiology and Biochemistry

Cell Physiol Biochem 2014;34:658-671 DOI: 10.1159/000363031 Published online: August 13, 2014

© 2014 S. Karger AG, Basel www.karger.com/cpb

Wang et al.: Effect of FGF21 on ECs Under High Glucose Condition

Fig. 1. E-klotho is expressed in HU E s. (A) Immuno luorescence staining of E-klotho in HU E s. ell were ixed by 4 paraformalin and stained by anti-E-klotho antibody or negative control and followed by y3conjugated secondary antibody. The samples of negative control were treated in the same way but incubated with normal Ig in stead of anti-E-klotho. API was used to stain nucleus. The images were captured suing F 1 confocal microscopy. (B) Effects of three concentrations of F F21 (1 , 3 and 1 n ) on E-klotho protein expression was determined by Western Blotting using two primary antibodies (Antibody 1 ab1 4 from Abcam Antibody 2 sc- 4343 from Santa ruz Biotechnology). Tubulin was used as a loading control. *P . 5 vs without F F21, N 4. ( ) Effects of high glucose (5 m and 1 m ) on protein expressions of F F21 and E-klotho. *P . 5 vs control, N 5 per group. ( ) Effects of high glucose (5 m ) incubation for 12, 24 and 48 hours on protein expressions of F F21 and E-klotho. *P . 5 vs control, N 5 per group.

antibodies (Antibody 1 AF588 from R Systems Antibody 2 sc- 4343 from Santa ruz Biotechnology). Both of these two antibodies were able to detect E-klotho protein in HU E s (Fig. 1B). oreover, F F21 treatment (1 , 3 and 1 n ) enhanced E-klotho protein expression in HU E s in a dose-dependent manner (Fig. 1B). Two concentrations of high glucose (5 m and 1 m ) upregulated protein expression of F F21 and E-klotho (Fig. 1 ). There was no signi icant difference between 5 m and 1 m glucose (Fig. 1 ). So we used 5 m glucose in the following experiments. Treatment of high glucose (5 m ) upregulated protein expression of F F21 and E-klotho in a time-dependent manner (Fig. 1 ). These results demonstrate that E-klotho, the essential anchor protein of F F21 biological function, is expressed in HU E s. In the following experiments, we used 3 n F F21 to treat cells because the 3 n F F21 was suf icient to induce E- lotho expression. FGF21 protects against high glucose-induced damage in HUVECs Next, we determined whether F F21 had biological functions on HU E s under high glucose condition stresscondition. High glucose challenge for 24 and 48 hours decreased cell viability by about 2 in HU E s (Fig. 2A). F F21 administration (3 n ) signi icantly attenuated this damage (Fig. 2A). Similarly, high glucose decreased cell migration of HU E s,

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Cellular Physiology and Biochemistry

Cell Physiol Biochem 2014;34:658-671 DOI: 10.1159/000363031 Published online: August 13, 2014

© 2014 S. Karger AG, Basel www.karger.com/cpb

Wang et al.: Effect of FGF21 on ECs Under High Glucose Condition

Fig. 2. F F21 protects against high glucose-induced cell damage in HU E s. (A) Effects of high glucose and F F21 on the cell viability of HU E s. ells were treated with medium of normal glucose (N , 5.5 m ), high glucose (H , 5 m ) and H plus F F21 (3 n ) for 24 hours or 48 hours, and then cell viability was measured using -8 kit. *P . 5 vs N , #P . 5 vs H , N . (B) Effects of high glucose and F F21 on the migration of HU E s. HU E s (1 1 5) were placed in the upper chamber of a 24-well transwell migration insert with medium of N , H and H + F F21 (3 n ). The lower chamber contained migration medium to induce HU E s migration for 12 or 24 hours. *P . 5 vs N , #P . 5 vs H , N 8. ( ) Effects of high glucose and F F21 on the caspas-3 activity of HU E s. *P . 5 vs N , #P . 5 vs H , N 5.

Fig. 3. F F21 decreases high glucose-induced oxidant stress in HU E s. (A) Representative images and quantitative analysis of FH- A luorescence in HU E s treated with normal glucose (N , 5.5 m ), high . 5 vs H , N 8. (B- ) glucose (H , 5 m ) and H + F F21 (3 n ) for 24 hours. *P . 5 vs N , #P Intracellular A levels (B), n-SO activities ( ) and u/ n-SO activities ( ) in HU E s treated with N , H and H + F F21 (3 n ) for 24 or 48 hours. *P . 5 vs N , #P . 5 vs H , N .

which was partly abolished by F F21 administration (Fig. 2B). High glucose also increased activities of caspase-3/ , two key pro-apoptotic factors. F F21 signi icantly decreased the

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Cellular Physiology and Biochemistry

Cell Physiol Biochem 2014;34:658-671 DOI: 10.1159/000363031 Published online: August 13, 2014

© 2014 S. Karger AG, Basel www.karger.com/cpb

Wang et al.: Effect of FGF21 on ECs Under High Glucose Condition

Fig. 4. F F21 prevents high glucose-induced inhibition of NO production and c P levels in HU E s. (A) NO production in the culture medium of HU E s treated with normal glucose (N , 5.5 m ), high glucose (H , 5 m ) and H + F F21 (3 n ) for 24 or 48 hours. *P . 5 vs N , #P . 5 vs H , N . (B) Intracellular c P levels in HU E s treated with N , H and H + F F21 (3 n ) for 24 or 48 hours. *P . 5 vs N , #P . 5 vs H , N .

Fig. 5. F F21 rescues high glucose-induced dysfunction of eNOS phosphorylation in HU E s. (A) Representative images for Western blotting analysis of eNOS phosphorylation at Ser-11 , Ser- 33, Ser- 1 and Thr-4 5 sites in HU E s treated with normal glucose (N , 5.5 m ), high glucose (H , 5 m ) and H + F F21 (3 n ) for 12 or 24 hours. (B) uantitative analysis of eNOS phosphorylation at Ser-11 , Ser- 33, Ser- 1 and Thr-4 5 sites in HU E s. *P . 5 vs N , #P . 5 vs H , N .

upregulation of caspase-3/ activities (Fig. 2 ). These results demonstrate the cytoprotective effect of F F21 against high glucose-induced damage in endothelial cells. FGF21 decreased high glucose-induced oxidant stress in HUVECs We next studied the effects of F F21 on high glucose-induced damage oxidant stress in HU E s. FH- A assay demonstrated that high glucose treatment triggered luorescence in HU E s, suggesting that ROS level in HU E s was remarkably enhanced in high glucose culture (Fig. 3A). Supplement of F F21 (3 n ) signi icantly lowered the FHA luorescence (Fig. 3A). We also measured A level, which is an index of endogenous lipid peroxidation. High glucose signi icantly elevated A level in HU E s (Fig. 3B), which was also partly inhibited by F F21 treatment (Fig. 3B). In contrast, the activities of n-SO and u/ n-SO , two major scavengers of ROS, were decreased by high glucose (Fig. 3 -3 ).

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Cell Physiol Biochem 2014;34:658-671 DOI: 10.1159/000363031 Published online: August 13, 2014

© 2014 S. Karger AG, Basel www.karger.com/cpb

Wang et al.: Effect of FGF21 on ECs Under High Glucose Condition

Fig. 6. The protection of F F21 on eNOS phosphorylation in HU E s is A P -dependent. (A-B) Representative images (A) and quantitative analysis (B) of eNOS phosphorylation at Ser-11 site in HU E s treated with normal glucose (N , 5.5 m ), high glucose (H , 5 m ) and H + F F21 (3 n ) for 12 hours under 22 (2 n ), N- 3 (5 P ) and compound (2 P ). -22 is a speci ic inhibitor of Akt pathway. N- 3 is a speci ic inhibitor of a II pathway. ompound is a speci ic inhibitor of A P pathway. *P . 5 vs N . NS, no signi icance. N . ( - ) Representative images ( ) and quantitative analysis ( ) of eNOS phosphorylation at Ser- 33 site in HU E s treated with normal glucose (N , 5.5 m ), high glucose (H , 5 m ) and H + F F21 (3 n ) for 12 hours under -22 (2 n ), H-8 (2 P ) and compound (2 P ). -22 is a speci ic inhibitor of Akt pathway. H-8 is a speci ic inhibitor of P A pathway. ompound is a speci ic inhibitor of A P pathway. *P . 5 vs N . NS, no signi icance. N .

F F21 treatment partly attenuated these effects (Fig. 3 -3 ). All these results suggest that F F21 is able to decrease high glucose-induced oxidant stress in HU E s. FGF21 rescues high glucose-impaired NO production in HUVECs NO is the key endothelium-derived relaxing factor that plays a pivotal role in the maintenance of vascular tone and reactivity, which is one of the most important functions of endothelial cells [38, 3 ]. We studied the effects of F F21 on NO production in HU E s upon high glucose stress. As shown in Fig. 4A, high glucose treatment for 24 and 48 hours signi icantly reduced the NO production of HU E s. Interestingly, F F21 treatment (3 n ) partly reversed this reduction (Fig. 4A). We also assayed the levels of intracellular c P, which is produced by soluble guanylyl cyclases in responding to NO and thus represents an important index for NO production. We found that c P levels in HU E s was signi icantly decreased by high glucose, which was also partly prevented by F F21 (Fig. 4B). These results indicate that F F21 partly rescues high glucose-induced impairment on NO production in HU E s. FGF21 prevents high glucose-induced inhibition of eNOS phosphorylation eNOS, the rate-limiting enzyme for endothelial NO production, is critical for biological activity of endothelial cells, especially under high glucose stress [4 ]. We measured the in luence of F F21 on eNOS protein expression. High glucose did not alter total eNOS protein expression (Fig. 5A). However, high glucose signi icantly decreased eNOS phosphorylation at Ser-11 (Fig. 5A and 5B) and Ser- 33 (Fig. 5A and 5B), two positive regulatory sites for eNOS enzymatic activity. F F21 treatment signi icantly prevented the high glucoseinduced decrease of eNOS phosphorylation at Ser-11 and Ser- 33 sites partly in HU E s

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Cell Physiol Biochem 2014;34:658-671 DOI: 10.1159/000363031 Published online: August 13, 2014

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Wang et al.: Effect of FGF21 on ECs Under High Glucose Condition

Fig. 7. F F21 activates A P signaling in HU E s. Representative image and quantitative analysis of A P signaling activation in F F21-treated HU E s. *P . 5 vs TR . N 4.

(Fig. 5A-5B). By contrast, high glucose and F F21 had no effects on Ser- 1 and Thr-4 5 sites of eNOS phosphorylation (Fig. 5A-5B). AMPK signaling pathway mediates the regulatory effects of FGF21 on phosphorylation of eNOS in HUVECs Phosphorylation of eNOS on multiple-sites was regulated by complex signaling pathways [41]. We used speci ic chemical inhibitors to explore which molecular signaling pathway is involved in the increase of eNOS phosphorylation by F F21 treatment. As shown in Fig. A and B, F F21 (lane 3) rescued the Ser-11 phosphorylation of eNOS under high glucose stress. Treatment of a2+/calmodulin-dependent protein kinase II ( a II) pathway inhibitor N- 3 or Akt pathway -22 had no in luence on this protective effect of F F21 in HU E s. However, treatment of compound , an inhibitor of A P-activated protein kinase (A P ) pathway, partly impaired this effect of F F21. Similarly, the rescued Ser- 33 phosphorylation of eNOS by F F21 (lane 3) under high glucose stress was also abolished by compound (A P pathway inhibitor) rather than P A pathway inhibiotr H-8 or Akt pathway inhibitor -22 (Fig. - ). Indeed, F F21 activated A P signaling in HU E s (Fig. ). nockdown of A P D1/2 isoforms by siRNA (Fig. 8A) partly inhibited the protective effect of F F21 on eNOS phosphorylation of Ser-11 (Fig. 8B and 8 ) and Ser- 33 sites (Fig. 8B and 8 ). All these results suggest that A P signaling pathway is required for the regulatory effects of F F21 on phosphorylation of eNOS in HU E s. Discussion

In the present study, we demonstrated that E-klotho is expressed in endothelial cells, suggesting F F21 may act on endothelial functions. We found that administration of F F21 potently protects against high glucose-induced decrease of cell viability and migration, decreased high glucose-induced oxidant stress, prevented high glucose-induced decrease of eNOS phosphorylation in HU E s. Furthermore, inhibition of A P signaling pathway effectively abolished the protective effect of F F21 on eNOS phosphorylation in HU E s. These data suggests that F F21 may be an important contributor in endothelial fucntions. The irst important inding of our study is that we found E-klotho is expressed in endothelial cells. For a long time, F F21 was thought to be a hormone whose biological effects was restricted in metabolism system [ ] due to the restricted expression of E-klotho [8]. Whereas the receptors of F F21 are broadly expressed, E-klotho, the essential anchor protein for F F21, is expressed in a more select set of tissues that include liver, white adipose tissue, pancreas, and testes [8, 1 ]. In adult mice, E-klotho mRNA was predominantly expressed in the liver and pancreas, with lower levels in skin, and weakly in the stomach, skeletal muscle, small intestine and lung [8]. According to our knowledge, there was no available literature about E-klotho expression in blood vessel. In this study, we for the irst time reported that E-klotho is expressed in endothelial cells, which suggests that F F21 may directly act on endothelial cells. Blood F F21 levels were elevated in diabetic patients and correlates with insulin resistance [2 , 21]. In addition, a previous study showed that state of obesity and insulin

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Cell Physiol Biochem 2014;34:658-671 DOI: 10.1159/000363031 Published online: August 13, 2014

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Wang et al.: Effect of FGF21 on ECs Under High Glucose Condition

Fig. 8. nockdown of A P D1/2 isoforms partly inhibits the protective effect of F F21 on eNOS phosphorylation. (A) siRNA-mediated knockdown of A P D1/2. *P . 5 vs siRNA-control. N 4. (B) Representative images of eNOS phosphorylation at Ser11 and Ser- 33 sites in HUE with siRNA-A P D1/2. ( - ) uantitative analysis of eNOS phosphorylation at Ser11 ( ) and Ser- 33 ( ) sites. *P . 5 vs without F F21. #P . 5 vs siRNA-control. N 4.

Fig. 9. A working model of the F F21-induced protection on endothelial cells against high-glucose stress.

resistance has increased endogenous levels of F F21 and respond poorly to exogenous F F21 [42], whereas two other groups suggest a compensatory response [43, 44]. These reports indicate that F F21 is linked to metabolic and vascular risk factors. However, the cause of the increment of serum F F21 and its impacts on cardiovascular tissues remain to be determined. It is well known that endothelial dysfunction plays a crucial role in the pathogenesis of diabetic vascular complications [34, 4 , 45-4 ]. High glucose condition increases NA PH oxidase activity in endothelial microparticles that promote vascular in lammation [5 ]. F F21 increased -R expression and mediated bene icial effect on cholesterol uptake, which was additive by statin [51]. Because statin has potent endothelial protection [52, 53], the association between F F21 and endothelial function is a rather interesting question. We determined the biological functions of F F21 on endothelial cells. High glucose induced apoptosis and autophagy, which is associated with cell death [54, 55]. Upon high glucose stress, F F21 displayed cytoprotection, evidence by results from cell viability and migration. F F21 also decreased the high glucose-induced oxidative stress. Finally, and may be most importantly, F F21 rescued the reduction of phosphorylation of eNOS and NO production, which is a central controller of vascular functions and biology. Our data indicate that F F21 may be a promising target in the treatment of vascular complications of hyperglycaemia or diabetes mellitus. We found that F F21 reversed the phosphorylation on Ser-11 and Ser- 33 sites of eNOS decreased by high glucose. No effects on Ser- 1 and Thr-4 5 were observed.

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Cell Physiol Biochem 2014;34:658-671 DOI: 10.1159/000363031 Published online: August 13, 2014

© 2014 S. Karger AG, Basel www.karger.com/cpb

Wang et al.: Effect of FGF21 on ECs Under High Glucose Condition

The roles of eNOS in endothelial functions are extensively studied. Ser-11 and Ser- 33 sites are two positive regulatory sites for eNOS enzymatic activity [41, 5 ]. Ser-11 site of eNOS is phosphorylated by Akt [5 ], a II [58], and A P [5 ]. Ser- 33 of eNOS is phosphorylated by Akt [5 ], cA P-dependent protein kinase A (P A) [ ] and A P [ 1]. The phosphorylation of eNOS also plays important roles in other pathophysiological process such as myocardial infarction and vascular insulin sensitivity [ 2- 4]. To explore how F F21 regulates these two sites phosphorylation, we used chemical inhibitors to selectively block these signaling pathways. Only inhibitor of A P partly blocked the protective effect of F F21 on Ser-11 and Ser- 33 sites of eNOS phosphorylation. Previously, F F21 was reported to increase A P Thr-1 2 phosphorylation in vitro and in vivo, resulting in enhanced mitochondrial oxidative function in adipocytes [ 5]. A P is a energy sensor that maintains energy homeostasis [ ] and is involved in survival and autophagy under diabetic condition [ , 8]. F F21 activated A P and induced cardiac protection on cardiac function in rodent hearts [ ]. F F21 is also correlated with A P activation and underlies the glucagon and lipid interactions in liver [ ]. Furthermore, A P was indispensable for the induction of F F21 by metformin in hepatocytes [ 1]. Our results are in line with these reports, suggesting that A P is critical for the eNOS regulation of F F21 in endothelial cells. In conclusion, our results provide the irst evidence that F F21 can act on endothelial cells. Under high glucose challenge, F F21 protects against cellular damage and eNOS dysfunction in endothelial cells. A P signaling pathway is required for the regulatory effects of F F21 on eNOS phosphorylation. Thus, our results suggest the potential therapeutic effect of F F21 in diabetes-related vascular complications. Acknowledgment

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This work was supported by National Natural Science Foundation of hina (Number 44 ). Disclosure Statement

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Cell Physiol Biochem 2014;34:658-671 DOI: 10.1159/000363031 Published online: August 13, 2014

© 2014 S. Karger AG, Basel www.karger.com/cpb

Wang et al.: Effect of FGF21 on ECs Under High Glucose Condition

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Cell Physiol Biochem 2014;34:658-671 DOI: 10.1159/000363031 Published online: August 13, 2014

© 2014 S. Karger AG, Basel www.karger.com/cpb

Wang et al.: Effect of FGF21 on ECs Under High Glucose Condition

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Wang et al.: Effect of FGF21 on ECs Under High Glucose Condition

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Fibroblast growth factor 21 protects against high glucose induced cellular damage and dysfunction of endothelial nitric-oxide synthase in endothelial cells.

Fibroblast growth factor 21 (FGF21) is a powerful endocrine hormone modulating glucose and lipid metabolism and represents a promising drug for type 2...
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