Cellular Carbonyl Stress Enhances the Expression of Plasminogen Activator Inhibitor-1 in Rat White Adipocytes via Reactive Oxygen Species-dependent Pathway
2004; Elsevier BV; Volume: 279; Issue: 6 Linguagem: Inglês
10.1074/jbc.m304222200
ISSN1083-351X
AutoresYoko Uchida, Ken‐ichi Ohba, Toshimasa Yoshioka, Kaoru Irie, Takamura Muraki, Yoshiro Maru,
Tópico(s)Antioxidant Activity and Oxidative Stress
ResumoCarbonyl stress is one of the important mechanisms of tissue damage in vascular complications of diabetes. In the present study, we observed that the plasminogen activator inhibitor-1 (PAI-1) levels in serum and its gene expression in adipose tissue were up-regulated in aged OLETF rats, model animals of obese type 2 diabetes. To study the mechanism of PAI-1 up-regulation, we examined the effect of advanced glycation end products (AGEs) and the product of lipid peroxidation (4-hydroxy-2-nonenal (HNE)), both of which are endogenously generated under carbonyl stress. Stimulation of primary white adipocytes by either AGE or HNE resulted in the elevation of PAI-1 in culture medium and at mRNA levels. The up-regulation of PAI-1 was also observed by incubating the cells in high glucose medium (30 mm, 48 h). The stimulatory effects by AGE or high glucose were inhibited by antioxidant, pyrrolidine dithiocarbamate, and reactive oxygen scavenger, probucol, suggesting a pivotal role of oxidative stress in white adipocytes. We also found that the effect by HNE was inhibited by antioxidant, N-acetylcysteine and that a specific inhibitor of glutathione biosynthesis, l-buthionine-S,R-sulfoximine, augmented the effect of subthreshold effect of HNE. Bioimaging of reactive oxygen species (ROS) by a fluorescent indicator, 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate, revealed ROS production in white adipocytes treated with AGE or HNE. These results suggest that cellular carbonyl stress induced by AGEs or HNE may stimulate PAI-1 synthesis in and release from adipose tissues through ROS formation. Carbonyl stress is one of the important mechanisms of tissue damage in vascular complications of diabetes. In the present study, we observed that the plasminogen activator inhibitor-1 (PAI-1) levels in serum and its gene expression in adipose tissue were up-regulated in aged OLETF rats, model animals of obese type 2 diabetes. To study the mechanism of PAI-1 up-regulation, we examined the effect of advanced glycation end products (AGEs) and the product of lipid peroxidation (4-hydroxy-2-nonenal (HNE)), both of which are endogenously generated under carbonyl stress. Stimulation of primary white adipocytes by either AGE or HNE resulted in the elevation of PAI-1 in culture medium and at mRNA levels. The up-regulation of PAI-1 was also observed by incubating the cells in high glucose medium (30 mm, 48 h). The stimulatory effects by AGE or high glucose were inhibited by antioxidant, pyrrolidine dithiocarbamate, and reactive oxygen scavenger, probucol, suggesting a pivotal role of oxidative stress in white adipocytes. We also found that the effect by HNE was inhibited by antioxidant, N-acetylcysteine and that a specific inhibitor of glutathione biosynthesis, l-buthionine-S,R-sulfoximine, augmented the effect of subthreshold effect of HNE. Bioimaging of reactive oxygen species (ROS) by a fluorescent indicator, 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate, revealed ROS production in white adipocytes treated with AGE or HNE. These results suggest that cellular carbonyl stress induced by AGEs or HNE may stimulate PAI-1 synthesis in and release from adipose tissues through ROS formation. Adipose tissue directly secretes biologically active molecules (adipocytokines) including plasminogen activator inhibitor-1 (PAI-1) 1The abbreviations used are: PAI-1plasminogen activator inhibitor-1ROSreactive oxygen speciesRCSreactive carbonyl speciesAGEadvanced glycation end productHNE4-hydroxy-2-nonenal8-OHdG8-hydroxy-2′-deoxyguanosineBSAbovine serum albuminPDTCpyrrolidinedithiocarbamateNACN-acetylcysteineBSOl-buthionine-S,R-sulfoximinePBSphosphate-buffered salineELISAenzyme-linked immunosorbent assayDCF-DA6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate. and actively affects other tissues. This is thought to be one of the risk factors for the development of obesity-linked disorders (1Spiegelman B.M. Choy L. Hotamisligil G.S. Graves R.A. Tontonoz P. J. Biol. Chem. 1993; 268: 6823-6826Abstract Full Text PDF PubMed Google Scholar). High plasma PAI-1 activity, which regulates fibrinolytic as well as thrombotic processes, is a frequent finding in obesity (2Vague P. Juhan-Vague I. Chabert V. Alessi M.C. Atlan C. Metabolism. 1989; 38: 913-915Abstract Full Text PDF PubMed Scopus (140) Google Scholar, 3Landin K. Stigendal L. Eriksson E. Krotkiewski M. Risberg B. Tengborn L. Smith U. Metabolism. 1990; 39: 1044-1048Abstract Full Text PDF PubMed Scopus (301) Google Scholar, 4Eriksson P. Reynisdottir S. Lönnqvist F. Stemme V. Hamsten A. Arner P. Diabetologia. 1998; 41: 65-71Crossref PubMed Scopus (229) Google Scholar) and/or in Type II (non-insulin-dependent) diabetes (5Auwerx J. Bouillon R. Collen D. Geboers J. Arteriosclerosis. 1988; 8: 68-72Crossref PubMed Google Scholar, 6Potter van Loon B.J. Kluft C. Radder J.K. Blankenstein M.A. Meinders A.E. Metabolism. 1993; 42: 945-949Abstract Full Text PDF PubMed Scopus (217) Google Scholar, 7McGill J.B. Schneider D.J. Arfken C.L. Lucore C.L. Sobel B.E. Diabetes. 1994; 43: 104-109Crossref PubMed Scopus (0) Google Scholar). Among the studies about PAI-1 expression in adipose tissue, biological significances of visceral fat during the development of obesity have been highlighted (8Shimomura I. Funahashi T. Takahashi M. Maeda K. Kotani K. Nakamura T. Yamashita S. Miura M. Fukuda Y. Takemura K. Tokunaga K. Matsuzawa Y. Nat. Med. 1996; 2: 800-803Crossref PubMed Scopus (819) Google Scholar). Furthermore, Alessi et al. (9Alessi M.C. Peiretti F. Morange P. Henry H. Nalbone G. JuhanVague I. Diabetes. 1997; 46: 860-867Crossref PubMed Scopus (0) Google Scholar) demonstrated that human adipocytes in culture produce PAI-1 and that omental fat produces more PAI-1 than subcutaneous adipose tissue in obese or non-obese individuals. However, the mechanism leading to up-regulation of PAI-1 has not been clearly elucidated. plasminogen activator inhibitor-1 reactive oxygen species reactive carbonyl species advanced glycation end product 4-hydroxy-2-nonenal 8-hydroxy-2′-deoxyguanosine bovine serum albumin pyrrolidinedithiocarbamate N-acetylcysteine l-buthionine-S,R-sulfoximine phosphate-buffered saline enzyme-linked immunosorbent assay 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate. The pathological conditions of diabetes with obesity have been widely associated with reactive oxygen species (ROS) and reactive carbonyl species (RCS), which may be key intermediates leading to diabetic complications. RCS originates from a multitude of mechanistically related pathways, like glycation (10Thornalley P.J. Langborg A. Minhas H.S. Biochem. J. 1999; 344: 109-116Crossref PubMed Scopus (1011) Google Scholar) and lipid peroxidation (11Fu M-X. Requena J.R. Jenkins A.J. Lyons T.J. Baynes J.W. Thorpe S.R. J. Biol. Chem. 1996; 271: 9982-9986Abstract Full Text Full Text PDF PubMed Scopus (722) Google Scholar). Glycation, a spontaneous amino-carbonyl reaction between reducing sugars and proteins, is a major source of RCS production. The complex reaction sequence is initiated by the reversible formation of a Schiff base, which undergoes an Amadori rearrangement to form a relatively stable ketoamine product during early glycation. A series of further reactions involving sugar fragmentation and formation of α-dicarbonyl compound yield stable advanced glycation end products (AGEs) under chronic hyperglycemia (12Vlassara H. Ann. Med. 1996; 28: 419-426Crossref PubMed Scopus (188) Google Scholar, 13Singh R. Barden A. Mori T. Beilin L. Diabetologia. 2001; 44: 129-146Crossref PubMed Scopus (1970) Google Scholar). Carbonyl stress also leads to the intracellular formation of 4-hydroxy-2-nonenal (HNE), one of the active end products of lipid peroxidation. Interestingly, RCS and AGEs can exert their detrimental cellular effects by increasing ROS production (14Lander H.M. Tauras J M. Ogiste J.S. Hori O. Moss R.A. Schmidt A.M. J. Biol. Chem. 1997; 272: 17810-17814Abstract Full Text Full Text PDF PubMed Scopus (679) Google Scholar), thereby forming a vicious cycle of ROS and RCS production. In the present study, to investigate the mechanisms underlying increased PAI-1 levels in obese diabetic conditions, we performed both in vivo and in vitro studies. For in vivo studies, gene expression and secretion of PAI-1 were measured in visceral adipose tissue using OLETF (Otsuka Long-Evans Tokushima fatty) rats, model animals of obese type II diabetes (15Kawano K. Hirashima T. Mori S. Saitoh Y. Kurosumi M. Natori T. Diabetes. 1992; 41: 1422-1428Crossref PubMed Scopus (0) Google Scholar). In parallel with the progression of diabetes, serum levels of both AGE and malondialdehyde derived from lipid peroxidation were shown to increase (16Nakamura S. Makita Z. Ishikawa S. Yasumura K. Fujii W. Yanagisawa K. Kawata T. Koike T. Diabetes. 1997; 46: 895-899Crossref PubMed Google Scholar, 17Tsuji T. Mizushige K. Noma T Murakami K. Ohmori K. Miyatake A. Kohno M. J. Cardiovasc. Pharmacol. 2001; 38: 868-874Crossref PubMed Scopus (66) Google Scholar). For in vitro studies, we examined the effects of AGE and HNE on the PAI-1 activity and its gene expression in rat white adipocytes differentiated in vitro. We focused on the role of carbonyl stress in the upregulation of PAI-1 expression. Animals—Male OLETF rats, model animals of Type II diabetes mellitus established in 1990 at Tokushima Research Institute (Otsuka Pharmaceutical Co., Ltd.) (15Kawano K. Hirashima T. Mori S. Saitoh Y. Kurosumi M. Natori T. Diabetes. 1992; 41: 1422-1428Crossref PubMed Scopus (0) Google Scholar), were generously supplied at 4 weeks of age. Male LETO (Long-Evans Tokushima Otsuka) rats served as normal controls. These animals were kept in an air-conditioned room (23 ± 2 °C, 55 ± 5% humidity) lighted 14 h a day (06:00 to 20:00) and were maintained on a standard diet and water ad libitum. These rats were sacrificed at 12, 20, 30, and 50 weeks of age, and blood was collected for determination of the plasma PAI-1 and 8-hydroxy-2′-deoxyguanosine (8-OHdG). Immediately after decapitation, visceral fat was dissected out and frozen in liquid nitrogen and stored in a deep freezer (–80 °C) until use. At 29 weeks of age, OLETF and LETO rats were divided into four groups, which were injected with probucol (3, 10, or 30 mg/kg subcutaneously) or vehicle (0.1% Me2SO containing ethanol) for 7 days. Cell Culture—White fat precursor cells were isolated from epididymal fat pad of 4-week-old male Wistar-Imamichi rats. Briefly, the minced tissue was incubated in a HEPES-buffered solution, pH 7.4, containing 0.2% collagenase type II (Sigma) for 20 min at 37 °C with vortexing every 5 min. After incubation, the tissue remnants were filtered through a 250-μm nylon screen into plastic test tubes. The tubes were placed on ice for at least 30 min to allow the mature white fat cells and lipid droplets to float on the surface of the cell suspension. The infranatant (stromal-vascular fraction) was collected and filtered through a 25-μm nylon screen to remove cell aggregates. The stromalvascular fraction containing precursor cells was pelleted by a centrifugation for 10 min at 700 × g and resuspended in the culture medium. The cells were plated in six-well multiplates and cultured in Dulbecco's modified Eagle's medium (containing 5.55 mm glucose) supplemented with 10% fetal bovine serum, 4 nm insulin, 25 mg/ml sodium ascorbate, 10 mm HEPES, pH 7.4, 4 mm glutamine, 50 IU/ml penicillin, and 50 μg/ml streptomycin. The cells were grown at 37 °C under an atmosphere of 5% CO2 in air and characterized morphologically on days 11–13 after plating. At this stage, the cells were recognized to be differentiated to mature white adipocytes. AGE-BSA at the concentrations of 0.01, 0.03, 0.1 and 0.3 mg/ml and nonglycated BSA as a control at the concentrations of 0.1 and 0.3 mg/ml were added to the cells. The cells were exposed to AGE-BSA or BSA for 2, 4, 8, 12, and 16 h. Antioxidants such as 10 μm pyrrolidinedithiocarbamate (PDTC) and 50 μm probucol were added to the culture medium 9 h prior to the harvest of the cells. HNE dissolved with 0.1% Me2SO at the concentrations of 3, 10, 30, and 100 μm were added to the cells. The cells were exposed to HNE for 2, 4, 8, and 16 h. N-Acetylcysteine (NAC), a precursor of glutathione, and l-buthionine-S,R-sulfoximine (BSO), a γ-glutamylcysteine synthetase inhibitor, were added to the cells for 24 h at the concentrations of 1, 5, and 20 mm and 10, 50, and 100 μm, respectively. Preparation of AGE—AGEs were prepared by incubating 50 mg/ml BSA (fraction V, fatty acid-free, low endotoxin; Sigma) with 0.5 m glucose-6-phosphate in phosphate-buffered saline (PBS) (10 mm, pH 7.4) at 37 °C for 4 weeks as described (18Doi T. Vlassara H. Kirstein M. Yamada Y. Striker G.E. Striker L.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2873-2877Crossref PubMed Scopus (325) Google Scholar, 19Esposito C. Cerlach H. Brett J. Stern D. Vlassara H. J. Exp. Med. 1989; 170: 1387-1407Crossref PubMed Scopus (343) Google Scholar) with a slight modification. Unmodified BSA was incubated under the same conditions without glucose-6-phosphate as controls. Unincorporated sugar was removed by dialysis against PBS. The concentrations of glycated BSA and control BSA was measured by the method of Lowry et al. (20Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Measurement of PAI-1 Levels in Serum and Culture Medium—PAI-1 levels were determined by measuring of active PAI-1 in the culture medium as follows. After 9–11 days of culture, the medium was replaced with 1 ml of fresh medium with 0.1% instead of 10% fetal bovine serum. AGE-BSA or HNE was added at concentrations of 0.01, 0.03, 0.1, or 0.3 mg/ml or 3, 10, 30, or 100 μm, respectively. In some cells, antioxidants (10 μm PDTC or 50 μm probucol), a glutathione precursor (1, 5, or 20 mm NAC), or a γ-glutamylcysteine synthetase inhibitor (10, 50, or 100 μm BSO) was added to the culture medium 9 h or 24 h prior to the harvest of cells. For the high glucose experiments, the cells were incubated in 30 mm glucose for 48 h. During the last 24 h of incubation, the medium was replaced with 1 ml of fresh medium containing 30 mm glucose with 0.1% instead of 10% fetal bovine serum. In some experiments, the cells were incubated in 30 mm glucose in combination with antioxidants (10 μm PDTC or 50 μm probucol) for 48 h. After incubation, PAI-1 concentrations of the culture medium were determined using a rat PAI-1 ELISA kit (Molecular Innovations, Royal Oak, MI) and were expressed as nanograms of active PAI-1 released from white adipocytes/mg of cell protein. Protein contents of the cells were determined as follows. The cells were rinsed twice with ice-cold phosphate-buffered saline and scraped from dishes with rubber policemen into 1 ml of phosphate-buffered saline and were sonicated for 15 s. After precipitation with 10% trichloroacetic acid, the proteins were measured by the method of Lowry et al. (20Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). PAI-1 levels in the serum were detected using the same ELISA kit. Because we used ELISA kit plated on t-PA antigen, the amount and activity of PAI-1 are simultaneously monitored. Measurement of 8-OHdG in Serum—Serum samples were centrifuged at 10,000 × g for 15 min, and the supernatant was used for determination of 8-OHdG by a competitive ELISA (8-OHdG Check; Japan Institute for Control of Aging, Fukuroi, Shizuoka, Japan). Detection of PAI-1 mRNA by Northern Blotting—Total RNAs was isolated from visceral adipose tissue or white fat cells by using Isogen (Nippongene, Toyama, Japan) and 10 μg of total RNAs/lane were separated by electrophoresis on 1% agarose gel containing 6% formaldehyde, followed by blotting onto nylon filters (Hybond-N; Amersham Biosciences). The blots were prehybridized in Expresshybri solution (Invitrogen) for 30 min at 68 °C and then hybridized with 32P-labeled rat PAI-1 cDNA probe for 1 h at the same temperature. Ribosomal RNAs of 28 S stained with ethidium bromide were utilized as an internal control for the amounts of total RNAs loaded. PAI-1 mRNA levels and 28 S ribosomal RNAs were quantified on autoradiograms using Fluor-S laser scanning densitometry (Fluor-S MultiImager; Bio-Rad). The levels of PAI-1 mRNA were expressed as fold increases over controls after correcting with 28 S ribosomal RNA levels. Intracellular ROS Fluorescence Imaging—To detect intracellular ROS, 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) (Molecular Probe, Eugene, OR) was used. DCF-DA diffuses into cells and is hydrolyzed by intracellular esterases to polar 6-carboxy-2′,7′-dichlorodihydrofluorescein. This nonfluorescent fluorescein analog is trapped in cells and can be oxidized to highly fluorescent 6-carboxy-2′,7′-dichlorofluorescein by intracellular oxidants. The cells on day 9 of culture were exposed to 0.1 mg/ml AGE-BSA or 30 μm HNE for 8 h. After washing twice with PBS(+), 10 μm of DCF-DA was loaded for cells for 30 min. Some cells were pretreated with 10 μm of PDTC for 9 h or 20 mm of NAC for 24 h. Then cells were examined under an inverted fluorescent microscope (IXE70; Olympus, Tokyo, Japan) equipped with an excitation (470–490 nm) and emission (515 nm) filter for fluorescein. Digital images of the fluorescent microscope images were obtained with a CCD camera (Hamamatsu Photonics, Hamamatsu, Japan) and stored in a Macintosh G3 (Apple Japan, Tokyo, Japan). Detection of IκB-α by Western Blotting—Rat white adipocytes on day 9 were exposed to 0.1 mg/ml of AGE for 15 min or 30 μm of HNE for 15 min and were washed twice with phosphate-buffered saline, pH 7.4, and lysed with lysis buffers (50 mm Tris-HCl, pH 7.5, 2% SDS, 6% β-mercaptoethanol, 1% glycerol). Each whole cell lysate was then treated with sample buffers for 5 min at 100 °C. The samples were run on 10% SDS-PAGE slab gels. The gel was transblotted on a nitrocellulose membrane (Hybond ECL; Amersham Biosciences) and incubated with skim milk (10 mg/ml) for blocking. After washing, the nitrocellulose membranes were immunoblotted with a polyclonal anti-IκB-α (Santa Cruz Biotechnology, Santa Cruz, CA) or monoclonal anti-actin (Chemicon international, Temecula, CA) and then with polyclonal peroxidase-conjugated anti-rabbit antibody (Amersham Biosciences) or anti-mouse antibody (Amersham Biosciences) as a secondary antibody. Bound antibodies were detected by enhanced chemiluminescence using ECL Western blotting detection reagents (Amersham Biosciences) and visualized by exposure of the membrane to autoradiography films. The intensity of each band was quantitatively analyzed using Fluor-S laser scanning densitometry (Flour-S MultiImager; Bio-Rad). Enhanced Expression of PAI-1 in Adipose Tissue from OLETF Rats—We examined the serum levels of PAI and its gene expression in adipose tissue from OLETF rats. The serum PAI-1 levels in OLETF rats began to increase at the age of 20 weeks and reached a maximum at the age of 50 weeks, whereas those in control LETO rats remained unchanged (Fig. 1A). The PAI-1 mRNA levels in visceral fat of OLETF rats, but not in LETO rats, increased in parallel with the progression of serum PAI-1 (Fig. 1B). The serum glucose levels of OLETF rats at the ages of 12, 20, 30, and 50 weeks were 120.2 ± 5.6, 160.7 ± 15.2, 397.4 ± 30.5, and 367.5 ± 66.8 mg/dl, respectively, whereas there were no changes in those of LETO rats (123.2 ± 6.5, 121.3 ± 7.6, 123.3 ± 2.9, and 117.2 ± 10.4, respectively). Thus, we confirmed that OLETF rats developed spontaneous and persistent hyperglycemia after the age of 20 weeks as originally described (15Kawano K. Hirashima T. Mori S. Saitoh Y. Kurosumi M. Natori T. Diabetes. 1992; 41: 1422-1428Crossref PubMed Scopus (0) Google Scholar), which we found correlated well with the increased expression of PAI-1 in visceral fat. Correlation between Serum PAI-1 and Serum 8-OHdG—The relationship between the serum PAI-1 and the serum 8-OHdG (8-hydroxy-2′-deoxyguanosine) levels is represented in Fig. 2A. In OLETF rats, a significant correlation was observed between the serum PAI-1 and the 8-OHdG levels (Fig. 2A; r = 0.65, p < 0.001). In contrast, there was no correlation between those two parameters in control LETO rats (Fig. 2A, r = 0.10, NS). Thus, serum PAI-1 could be associated with oxidative stress during the development of diabetes in OLETF rats. Therefore, we examined the effect of probucol, a potent antioxidant, on PAI-1 activity, its gene expression, and serum 8-OHdG concentration. At the age of 30 weeks, the increased levels of serum PAI-1, 8-OHdG and adipose tissue PAI-1 mRNA in OLETF rats were suppressed by successively injected probucol in a dose-dependent manner. However, in LETO control rats, there was no increase in either serum PAI-1 or 8-OHdG level, which was not affected by probucol. Moreover, enhanced expression of PAI-1 mRNA in visceral fat was never observed in vehicle-injected 30-week-old LETO rats (Fig. 2, B and C). Up-regulation of PAI-1 Expression in White Adipocytes by AGEs—Because up-regulation of PAI-1 was observed in in vivo studies using OLETF rats, we performed in vitro studies by directly examining the effect of AGEs on PAI-1 expression in white fat cells in primary culture. AGE-BSA increased PAI-1 activity in culture medium released from white adipocytes differentiated in culture. As shown in Fig. 3A, white adipocytes on days 9–11 exposed to AGE-BSA for 8 h showed a concentration-dependent increase in PAI-1 with a significant elevation at concentrations of 0.1 and 0.3 mg/ml. Time course studies indicated that cells treated with 0.1 mg/ml AGE-BSA showed a steady increase in PAI-1 accumulation in the media by 8 h, and the rate of PAI-1 accumulation was reduced markedly thereafter (Fig. 3B). The PAI-1 accumulation in AGE-BSA-treated cells during 8 h of incubation was approximately twice as high as that in BSA-treated cells. To clarify whether or not the AGE-BSA-induced stimulation of PAI-1 secretion involves activated transcription, PAI-1 mRNA expression was studied by Northern blotting. AGE-BSA induced a concentration-dependent increase in PAI-1 mRNA in white adipocytes (Fig. 3C). White adipocytes exposed to 0.1 mg/ml of AGE-BSA showed a time-dependent increase in PAI-1 mRNA (Fig. 3D). Inhibition of AGE- and High Glucose-induced Increase in PAI-1 Expression by Antioxidant (PDTC) and Reactive Oxygen Scavenger (Probucol)—We further investigated the involvement of oxidative stress in the up-regulation of PAI-1 expression by AGE. As shown in Fig. 4, AGE enhanced PAI-1 expression at both protein and mRNA levels, which could be inhibited by either PDTC (antioxidant) or probucol (reactive oxygen scavenger). Moreover, to examine whether this effect of AGE can be observed also by hyperglycemia, the cells were incubated in high glucose for 48 h. Thirty mm glucose induced an increase in PAI-1 activity and gene expression compared with the control cells incubated in conventional medium (5.55 mm glucose). This enhancement by high glucose was also inhibited by PDTC or probucol (Fig. 5).Fig. 5Inhibition of hyperglycemia-induced increase in PAI-1 expression by antioxidant (PDTC) and reactive oxygen scavenger (probucol). The cells on days 9–11 were incubated with 30 mm glucose (hyperglycemia) or 5.55 mm glucose (normoglycemia) for 48 h. Ten μm PDTC or 50 μm probucol was simultaneously added to the culture. During the last 24 h of incubation, the medium was replaced with 1 ml of fresh medium containing 30 mm glucose and PDTC or probucol with 0.1% instead of 10% fetal bovine serum. PAI-1 concentrations in culture media were measured by ELISA and total RNAs extracted from cells were subjected to Northern blotting. A, inhibitory effects of PDTC and probucol on hyperglycemia-induced increase in PAI-1 concentrations. The values are the means ± S.E. (bars) obtained from five or six experiments. **, p < 0.01 (Student's t test). B, inhibitory effects of PDTC and probucol on hyperglycemia-induced increase in PAI-1 mRNAs. The values are the means ± S.E. (bars) obtained from three or four experiments. ***, p < 0.001 (Friedman's rank test followed by Mann-Whitney U test).View Large Image Figure ViewerDownload Hi-res image Download (PPT) An Increase in PAI-1 Expression in White Adipocytes by 4-Hydroxy-2-nonenal—Because up-regulation of PAI-1 expression was observed in white adipocytes in response to AGE-BSA, we tested to see whether the most prominent intermediate of lipid peroxidation, HNE can induce up-regulation of PAI-1. The effect of an increasing concentration of HNE on PAI-1 expression was determined (Fig. 6, A and C). HNE at concentrations of 30 and 100 μm increased PAI-1 expression 8 h after addition. Time course studies indicated that Me2SO-treated control cells showed a steady increase in PAI-1 accumulation in the media by 8 h, and the rate of PAI-1 accumulation was reduced markedly thereafter. The addition of HNE (30 μm) significantly increased the accumulation of PAI-1 during 8 h of incubation twice as high as Me2SO-treated cells (Fig. 6B). The PAI-1 mRNA levels began to increase 4 h after the addition of HNE and reached a maximum at 8 h (Fig. 6 D). Inhibition of HNE-induced Increase in PAI-1 Expression by Antioxidant (NAC) and Augmentation of Subthreshold Effect of HNE on PAI-1 Expression by a Glutathione Synthesis Inhibitor (BSO)—As shown in Fig. 7, the increases in PAI-1 activity and PAI-1 mRNA by 30 μm HNE were inhibited dose-dependently by antioxidant (NAC). On the other hand, at concentrations of 10–100 μm, a glutathione synthesis inhibitor, l-buthionine-S,R-sulfoximine, which favors intracellular redox environment to reduced state, augmented the effect of subthreshold concentration of HNE (3 μm) on PAI-1 expression (Fig. 8).Fig. 8Augmentation of the effect of HNE in PAI-1 activity and its gene expression by a glutathione synthesis inhibitor, BSO. The cells on days 9–11 were treated with 30 μm HNE or 0.1% dimethyl sulfoxide for 8 h. BSO at concentrations of 10, 50, and 100 μm was added to the culture for 24 h. PAI-1 concentrations in culture media were measured by ELISA, and the total RNAs extracted from cells were subjected to Northern blotting. A, augmenting effects of BSO on PAI-1 concentrations. The values are the means ± S.E. (bars) obtained from five or six experiments. *, p < 0.05; **, p < 0.01 (Student's t test). B, augmenting effects of BSO on PAI-1 mRNA. The values are the means ± S.E. (bars) obtained from four experiments. **, p < 0.01; ***, p < 0.001 (Friedman's rank test followed by Mann-Whitney U test).View Large Image Figure ViewerDownload Hi-res image Download (PPT) AGE- or HNE-induced Intracellular ROS Generation Results in the Activation of the Transcription Factor NF-κB—ROS production from white adipocytes was studied using a membrane-permeable fluorescent indicator, DCF-DA. The fluorescent microscopic images are shown in Fig. 9 (A and B). Cells stimulated with 0.1 mg/ml AGE-BSA for 8 h showed substantial fluorescence 30 min after the addition of 10 μm DCF-DA, whereas minimum fluorescence was observed in unglycated BSA-treated cells. The increase in the fluorescence of white adipocytes stimulated by AGE-BSA was markedly inhibited by 10 μm PDTC (Fig. 9A). Moreover, the stimulatory action of AGE was mimicked by exogenously added HNE. The generation of ROS stimulated by HNE in white adipocytes was blocked by pretreatment with antioxidant, NAC (Fig. 9B). Furthermore, to examine whether the ROS generation under enhanced carbonyl stress could activate the transcription factor NF-κB, we monitored the degradation of IκB-α, an inhibitory subunit of NF-κB, in rat white adipocytes exposed to AGE or HNE. As shown in Fig. 9C, Western blotting revealed that both AGE and HNE markedly reduced IκB-α levels, which were blocked by pretreatment with PDTC or NAC, respectively. A variety of observations suggests that elevated levels of plasma and adipose tissue PAI-1 are frequently found in obese humans (7McGill J.B. Schneider D.J. Arfken C.L. Lucore C.L. Sobel B.E. Diabetes. 1994; 43: 104-109Crossref PubMed Scopus (0) Google Scholar, 9Alessi M.C. Peiretti F. Morange P. Henry H. Nalbone G. JuhanVague I. Diabetes. 1997; 46: 860-867Crossref PubMed Scopus (0) Google Scholar) and rodents (21Samad F. Loskutoff D.J. Mol. Med. 1996; 96: 568-582Crossref Google Scholar, 22Smad F. Yamamoto K. Loskutoff D.J. J. Clin. Invest. 1996; 97: 37-46Crossref PubMed Scopus (287) Google Scholar) and correlate strongly with visceral fat mass (8Shimomura I. Funahashi T. Takahashi M. Maeda K. Kotani K. Nakamura T. Yamashita S. Miura M. Fukuda Y. Takemura K. Tokunaga K. Matsuzawa Y. Nat. Med. 1996; 2: 800-803Crossref PubMed Scopus (819) Google Scholar). Increased plasma PAI-1 levels may contribute to the impaired endogenous fibrinolysis and the increased cardiovascular risk of patients with visceral fat accumulation and/or non-insulin-dependent diabetes mellitus. Despite this well documented link between elevated PAI-1 levels and obesity, little is known about the origin of this plasma inhibitor in obesity or about signals that control its biosynthesis. In the present study, we first showed that circulating PAI-1 was progressively increased during the development of diabetes and hyperglycemia in OLETF rats, and this elevation of PAI-1 accompanied a concomitant increase in PAI-1 mRNA from visceral fat, suggesting that visceral fat tissue can be an important source of elevated PAI-1 in circulation. A considerab
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