AJP - Endo Add DOIs to your references at manuscript stage!
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 293: E1703-E1708, 2007. First published September 25, 2007; doi:10.1152/ajpendo.00462.2007
0193-1849/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/6/E1703    most recent
00462.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, R.
Right arrow Articles by Ma, X. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, R.
Right arrow Articles by Ma, X. L.

Adiponectin improves endothelial function in hyperlipidemic rats by reducing oxidative/nitrative stress and differential regulation of eNOS/iNOS activity

Rong Li,1 Wen-Qing Wang,1 Haifeng Zhang,1 Xinchung Yang,2 Qian Fan,2 Theodore A. Christopher,3 Bernard L. Lopez,3 Ling Tao,3 Barry J. Goldstein,4 Feng Gao,1 and Xin L. Ma3

Department of 1Physiology, The Fourth Military Medical University, Xian; 2Department of Cardiology, Chao-Yang Hospital, Beijing, China; and Departments of 3Emergency Medicine and 4Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania

Submitted 18 July 2007 ; accepted in final form 10 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Plasma adiponectin level is significantly reduced in patients with metabolic syndrome, and vascular dysfunction is an important pathological event in these patients. However, whether adiponectin may protect endothelial cells and attenuate endothelial dysfunction caused by metabolic disorders remains largely unknown. Adult rats were fed with a regular or a high-fat diet for 14 wk. The aorta was isolated, and vascular segments were incubated with vehicle or the globular domain of adiponectin (gAd; 2 µg/ml) for 4 h. The effect of gAd on endothelial function, nitric oxide (NO) and superoxide production, nitrotyrosine formation, gp91phox expression, and endothelial nitric oxide synthase (eNOS)/inducible NOS (iNOS) activity/expression was determined. Severe endothelial dysfunction (maximal vasorelaxation in response to ACh: 70.3 ± 3.3 vs. 95.2 ± 2.5% in control, P < 0.01) was observed in hyperlipidemic aortic segments, and treatment with gAd significantly improved endothelial function (P < 0.01). Paradoxically, total NO production was significantly increased in hyperlipidemic vessels, and treatment with gAd slightly reduced, rather than increased, total NO production in these vessels. Treatment with gAd reduced (–78%, P < 0.01) superoxide production and peroxynitrite formation in hyperlipidemic vascular segments. Moreover, a moderate attenuation (–30%, P < 0.05) in gp91phox and iNOS overexpression in hyperlipidemic vessels was observed after gAd incubation. Treatment with gAd had no effect on eNOS expression but significantly increased eNOS phosphorylation (P < 0.01). Most noticeably, treatment with gAd significantly enhanced eNOS (+83%) but reduced iNOS (–70%, P < 0.01) activity in hyperlipidemic vessels. Collectively, these results demonstrated that adiponectin protects the endothelium against hyperlipidemic injury by multiple mechanisms, including promoting eNOS activity, inhibiting iNOS activity, preserving bioactive NO, and attenuating oxidative/nitrative stress.

metabolic syndrome; endothelial dysfunction; cytokine; nitric oxide; endothelial nitric oxide synthase; inducible nitric oxide synthase

METABOLIC SYNDROME IS CHARACTERIZED by a group of metabolic and hemostatic abnormalities, including impaired glucose tolerance, hyperinsulinemia, hypertension, dyslipidemia, oxidant stress, and endothelial dysfunction (7). This cluster generates an increased risk of macroangiopathy, which is the leading cause of mortality for patients with metabolic syndrome and type 2 diabetes (3). Therefore, the discovery of therapeutic interventions that block or attenuate metabolic disorder-induced macroangiopathy holds great promise in reducing metabolic syndrome-related death.

Endothelial dysfunction is an early pivotal event in the development, progression, and manifestation of macroangiopathy (9). Numerous mechanisms have been proposed to explain this pathological alteration, including deficiencies of arginine supply, alteration of signaling mechanisms, alterations of nitric oxide synthase (NOS) expression or one of the cofactors involved in NOS activation, and increased destruction of nitric oxide (NO) by superoxide (O2bullet–) (4). Theoretically, a signaling system that possesses dual actions, i.e., stimulating NO production and inhibiting O2bullet– production, would provide the most protection against endothelial dysfunction. Unfortunately, most, if not all, cytokines identified to date increase NO and O2bullet– production simultaneously with the resultant formation of peroxynitrite (ONOO–), a strong oxidative/nitrative molecule that further aggravates vascular injury.

Adiponectin is a novel cytokine secreted from adipose tissue (2, 5). It contains a stalk with 22 collagen repeats and a highly conserved globular domain (gAd). Adiponectin is normally present in plasma at concentrations up to 30 µg/ml but is markedly downregulated in association with obesity-linked diseases including coronary artery disease and type 2 diabetes (11). Clinical observations have demonstrated that hypoadiponectinemia is closely related to endothelial dysfunction in peripheral arteries (13, 15, 17, 21, 22) and that plasma total adiponectin concentrations are inversely related to the risk of myocardial infarction (13, 19). These results suggest that adiponectin might be a potent endothelial protective molecule. However, whether supplementation of adiponectin may attenuate endothelial dysfunction caused by hyperlipidemia has not been previously investigated.

Therefore, the aims of the present study were to determine whether adiponectin might improve endothelial function in vascular segments isolated from hyperlipidemic animals and, if so, to investigate the mechanisms involved.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. All experimental procedures were in accordance with the National Institutes of Health guidelines and were approved by the local authorities for animal research. Male Sprague-Dawley rats (8 wk old) were randomized to receive a regular chow diet or a high-fat (1% cholesterol) supplemented diet. Food and water were provided ad libitum, and animals were maintained in a temperature-controlled barrier facility with a 12:12-h light-dark cycle. Fourteen weeks later, animals were anesthetized by intraperitoneal administration of 20% urethane. Caval blood was withdrawn, the plasma was immediately separated, and lipid profile, glucose, and insulin levels were determined as described below. The aortic segment from the heart to the iliac bifurcation was excised and placed in ice-cold Krebs buffer consisting of (in mM): NaCl 118, KCl 4.8, CaCl2·2H2O 2.5, MgCl2·6H2O 2.5, NaH2PO4·2H2O 1.2, NaHCO3 8.5, and glucose·H2O 11. The aorta was cleaned of adhering tissues, cut into rings ~2 mm in length, and incubated with vehicle or gAd (Biovision, Mountain View, CA) (1–20 µg/ml in pilot study; 2 µg/ml was selected as the optimal dose and used in the rest of the experiments) in a cell culture incubator. After 4 h of incubation, endothelial function and biochemical assays were performed as described below.

Lipid, glucose, and insulin plasma determinations. Plasma cholesterol and triglyceride levels were determined by a biochemistry analyzer (Cobas Integra 400 Plus, Roche). Fasting blood glucose and insulin levels were measured with the use of a blood glucose meter (SureStep, LifeScan) and an RIA test kit (Peninsula Laboratories), respectively.

Determination of endothelial function. Endothelial function was determined by comparing the vasorelaxation response to acetylcholine (ACh), an endothelium-dependent vasodilator, with that of S-nitroso-N-acetylpenicillamine (SNAP), an endothelium-independent vasodilator, as described previously (24). Briefly, aortic rings were mounted onto hooks, suspended in organ chambers filled with Krebs buffer and aerated with 95% O2 and 5% CO2 at 37°C, and connected to force transducers (WPI, Sarasota, FL) to record changes via a Maclab data acquisition system. After equilibration for 60 min at a preload of 1 g, the rings were precontracted with norepinephrine (NE; 0.1 nM). Once a stable contraction was achieved, the rings were exposed to cumulative concentrations of ACh (10–9 to 10–5 M). After the cumulative response stabilized, the rings were washed and allowed to equilibrate to baseline. The procedure was then repeated with an endothelium-independent vasodilator (SNAP, 10–9 to 10–5 M) to determine smooth muscle function and sensitivity to NO. Endothelial dysfunction was defined as a reduced vasorelaxation in response to ACh with a normal response to SNAP.

Total NO production measurement. Total NO production (NOx) by aortic segments was determined by measuring the concentration of nitrite, a stable metabolite of NO in vitro, with a modified Griess reaction method (8). Briefly, 5 min after the highest concentration of ACh (10–5 M) was added, 100 µl of buffer solution were taken from the vascular chamber and mixed with an equal volume of modified Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, and 2% phosphoric acid). After 10 min of incubation at room temperature, the resultant chromophore was spectrophotometrically determined at 540 nm using a spectrophotometer (SpectraMax 190, Molecular Device). The nitrite concentrations in the samples were calculated from freshly prepared nitrite standard curves made from sodium nitrite with the same Krebs buffer.

Determination of NOS activity. Vascular tissue was minced and homogenized in lysis buffer (Tris 20 mM, NaCl 50 mM, NaF 50 mM, Na4P2O7·10H2O 5 mM, C12H22O11 25 mM, DTT 1 mM, Na3VO4 2 mM, and 1% protease inhibitor cocktail, pH 7.4) with a Heidolph DIA900 tissue homogenizer (Heidolph Instruments, Schwabach, Germany). The homogenate was centrifuged (12,000 g at 4°C for 10 min), the supernatant was decanted, and total NOS activity and inducible NOS (iNOS) activity were determined using an NOS activity assay kit (tNOS, Nanjing Jiancheng Bioengineering Institute), following the manufacturer's instructions. In brief, 100 µl of supernatant were added to the reaction buffer containing L-arginine, NADPH, calcium (not present in iNOS assay buffer), calmodulin, tetrahydrobiopterin, nitroblue tetrazolium (NBT), and phenazine methosulfate (PMS). Fifteen minutes after incubation at 37°C, reaction was stopped by adding a termination buffer. Formazan, the reaction product of NBT/PMS with NADPH in the presence of NO, was quantified spectrophotometrically at 530 nm. One NOS enzymatic unit was defined as 1 nmol·NO–1·min–1·mg protein–1. Endothelial NOS (eNOS) activity was obtained by subtracting iNOS activity from the total NOS activity, and results were normalized against the mean value of control and expressed as fold changes.

Determination of eNOS, iNOS, and gp91phox expression by Western blot. The aortic segments were pulverized in liquid nitrogen and resolubilized in lysis buffer. Equal amounts of protein (80 µg protein/lane) were electrophoresed on a 14% SDS-polyacrylamide gel and electrophoretically transferred to a poly (vinylidene difluoride) membrane (Millipore, Billerica, MA). After blocking with 5% skim milk in Tris-buffered saline at room temperature for 1 h, we incubated the membrane with an antibody against eNOS, phosphorylated eNOS (Cell Signaling Technology, Danvers, MA), iNOS, or gp91phox (BD Bioscience Laboratories, San Jose, CA) overnight at 4°C. The membrane was then washed with PBS and incubated with horseradish peroxidase-conjugated IgG antibody (Cell Signaling) for 1 h at 37°C. The blots were developed with an enhanced chemiluminescence detection kit (Pierce Biotechnology, Rockford, IL). The immunoblotting was visualized with ChemiDocXRS (Bio-Rad Laboratory, Hercules, CA), and the blot densities were analyzed with LabImage software.

Determination of tissue antioxidant capacity. Aortic vessels were rinsed, homogenized in 0.9% NaCl solution (1:10, wt/vol), and centrifuged at 3,000 g for 5 min. The pellet was discarded. Total antioxidant capacity was determined with a spectrophotometric assay kit (Nanjing Jiancheng Bioengineering Institute), following the manufacture's instruction. In brief, 30 µl of supernatant were added to the reaction buffer containing xanthine, xanthine oxidase, and hydroxylamine. After 40 min of incubation at 37°C, accumulation of nitrite was quantified by the Griess reaction. Tissue antioxidant capacity is inversely related to the concentration of nitrate. Results were normalized against the mean value of control and expressed as fold changes.

Quantification of vascular superoxide production. Superoxide production from aortic segments was measured by flow injection chemiluminescence as described previously (26). Superoxide production was expressed as chemiluminescence intensity (CI) per milligram of vessel weight (CI/mg tissue).

Quantitation of tissue nitrotyrosine content. Nitrotyrosine content in the aortic tissue, a footprint of in vivo ONOO– formation and an index of nitrative stress, was determined using a nitrotyrosine ELISA kit (Cell Sciences, Canton, MA) as described in our previous study (23).

Statistical analysis. Values are presented as means ± SE. Data were analyzed with one-way ANOVA (GraphPad Software, San Diego, CA). A probability value of <0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Plasma lipid profile. There was no significant difference in any parameters determined before high-fat diet feeding. Compared with animals fed a normal diet, animals fed a high-fat diet exhibited a significant increase in body weight and marked elevation in plasma cholesterol, triglyceride, fasting blood glucose, and insulin concentrations (Table 1).


View this table:
[in this window]
[in a new window]

  		Table 1. Metabolic profiles

 
Treatment of hyperlipidemic aortic segments in vitro with gAd enhanced ACh-induced vasorelaxation in an NO-dependent fashion. Consistent with previously reported results (14), concentration-dependent vasorelaxation in response to ACh was impaired in vascular segments isolated from animals fed a high-fat diet (Fig. 1A). However, concentration-dependent vasorelaxation in response to SNAP, an endothelium-independent vasodilator, remained unchanged in these vessels (Fig. 1B). These results indicate that high-fat diet-induced hyperlipidemia caused significant endothelial dysfunction. Most interestingly, in vitro treatment with gAd for 4 h improved endothelial function, as evidenced by a significant improvement of the dose-response curve to ACh (Fig. 1A). To further determine whether gAd increased ACh-induced vasorelaxation by enhancing NO production, a portion of gAd-treated aortic segments was treated with N{omega}-nitro-L-arginine methyl ester (L-NAME; 0.5 mM). As illustrated in Fig. 1A, addition of L-NAME completely blocked vasorelaxation in response to ACh in those vessels pretreated with gAd.


Figure 1
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 1. Concentration-dependent vasorelaxation of control (Con) and hyperlipidemic (HL) aortic segments in response to ACh (A) and S-nitroso-N-acetylpenicillamine (SNAP; B) and effect of in vitro globular domain of adiponectin (gAd) treatment (HL+gAd) on hyperlipidemia-induced endothelial dysfunction. Values are means ± SE; n = 8–10 vascular segments/group from 5–7 rats. **P < 0.01 vs. control. #P < 0.05 and ##P < 0.01 vs. hyperlipidemic vessels treated with vehicle.

 
Treatment with gAd had no significant effect on hyperlipidemia-induced NO overproduction. Having demonstrated that acute treatment of hyperlipidemic aortic segments with gAd significantly improved endothelial function that can be completely blocked by L-NAME, we attempted to obtain direct evidence that treatment with gAd may increase NO production and thus improve vasorelaxation in response to ACh. Surprisingly, although treatment of hyperlipidemic vessels with gAd significantly increased ACh-induced vasorelaxation, this treatment slightly reduced, rather than increased, total NO production (Fig. 2). This paradoxical result suggests that more complex signaling mechanisms are involved in the protective effect of gAd against hyperlipidemia-induced endothelial dysfunction.


Figure 2
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 2. Total nitric oxide production (NOx) by aortic segments isolated from control animals (Con) or hyperlipidemic animals (HL) treated with vehicle or gAd (HL+gAd). NOx concentration in medium containing vascular segments was determined by Griess reaction. dw, Dry weight. Values are means ± SE; n = 8–10 vascular segments/group from 5–7 rats. **P < 0.01 vs. control. $$P < 0.01 vs. HL+gAd.

 
Treatment with gAd significantly reduced superoxide overproduction in hyperlipidemic vessels. The above-mentioned results demonstrating that endothelial dysfunction occurred despite a significant increase in NO production in hyperlipidemic vessels suggest that increased NO destruction is responsible for hyperlipidemic endothelial dysfunction. In addition, our novel observation that gAd improved endothelial function without increasing NO production indicates that gAd may improve endothelial function by preserving bioactive NO. To obtain direct evidence to support this hypothesis, several additional experiments were performed. As summarized in Fig. 3A, hyperlipidemia-induced reduction in total antioxidant capacity was significantly preserved after gAd treatment. In addition, a 2.7-fold increase in superoxide production was observed in aortic segments isolated from hyperlipidemic animals, and treatment with gAd almost abolished (78% reduction compared with vehicle-treated vessels) the superoxide overproduction observed in hyperlipidemic vessels (Fig. 3B). Moreover, protein expression of gp91phox, a major component of NADPH oxidase, was significantly increased in hyperlipidemic vessels. Pretreatment with gAd caused a moderate (~30%), yet statistically significant, reduction in gp91phox expression (Fig. 3C).


Figure 3
View larger version (25K):
[in this window]
[in a new window]

  		Fig. 3. Effect of gAd on hyperlipidemia-induced reduction in antioxidant capacity (A), superoxide overproduction (B), and gp91phox expression (C). Values are means ± SE; n = 6–8 vascular segments/group from 5–7 rats. *P < 0.05 and **P < 0.01 vs. control. #P < 0.05 and ##P < 0.01 vs. hyperlipidemic segments treated with vehicle.

 
Treatment with gAd attenuated peroxynitrite overproduction in hyperlipidemic vessels. Our experiments presented above demonstrated that gAd improved endothelial function in hyperlipidemic vessels, likely by reducing superoxide production and preventing NO destruction. To obtain more evidence to support this notion, ONOO–, the biradical reaction product of superoxide and NO, was determined by measurement of nitrotyrosine, a "footprint" of in vivo ONOO– production. Consistent with previously published results (16, 24), nitrotyrosine content was significantly increased in hyperlipidemic vessels. Importantly, pretreatment with gAd significantly reduced nitrotyrosine content (Fig. 4).


Figure 4
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 4. Effect of gAd on hyperlipidemia-induced overproduction of nitrotyrosine in vascular segments. Values are means ± SE; n = 6–8 vascular segments/group from 5–7 rats. **P < 0.01 vs. control. #P < 0.05 and ##P < 0.01 vs. hyperlipidemic segments treated with vehicle.

 
Treatment with gAd differentially regulated eNOS and iNOS activity in hyperlipidemic vessels. The data presented above indicate that the reduction of superoxide production and prevention of NO destruction contribute to the vasculoprotective effect of gAd in hyperlipidemia. However, although gAd has been shown to stimulate NO production in cultured endothelial cells by phosphorylating eNOS (6, 25), treatment with gAd did not increase but slightly reduced total NO production in hyperlipidemic aortic vessels (Fig. 2). These results suggest that the overall effect of gAd on total NO production may involve a complex regulation of gAd on different forms of NOS under hyperlipidemic conditions. To directly investigate this novel possibility, the effect of gAd on eNOS and iNOS activity was determined. As summarized in Fig. 5, a significant reduction in eNOS activity and a marked increase in iNOS activity were observed in hyperlipidemic vessels. Pretreatment with gAd almost completely normalized eNOS activity and significantly reduced iNOS activity. These results demonstrated that gAd had opposite effects on eNOS and iNOS activity, and this differential regulatory role may explain the paradoxical finding that gAd significantly increased ACh-induced vasorelaxation but slightly reduced total NO production.


Figure 5
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 5. Differential regulation of endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) activity by gAd in hyperlipidemic vessels. Values are means ± SE; n = 6–8 vascular segments/group from 5–7 rats. **P < 0.01 vs. control. ##P < 0.01 vs. hyperlipidemic segments treated with vehicle.

 
Treatment with gAd enhanced eNOS phosphorylation and inhibited iNOS expression in hyperlipidemic vessels. In a final attempt to determine, at a molecular level, how eNOS and iNOS were differentially regulated by gAd, eNOS and iNOS protein expression and eNOS phosphorylation were determined. As illustrated in Fig. 6A, neither hyperlipidemia nor treatment with gAd had a significant effect on eNOS protein expression. However, the level of phosphorylated eNOS was significantly reduced in hyperlipidemic vessels, and this pathological change was almost completely normalized by gAd treatment (Fig. 6B). Moreover, hyperlipidemia caused a >10-fold increase in iNOS expression. Acute treatment with gAd moderately attenuated hyperlipidemia-induced iNOS expression (Fig. 6C).


Figure 6
View larger version (32K):
[in this window]
[in a new window]

  		Fig. 6. Effect of gAd on eNOS phosphorylation and iNOS expression in hyperlipidemic vessels. A: expression of eNOS and phosphorylated eNOS (peNOS; at serine 1177, Western blots) and ratio of peNOS/eNOS density analysis. B: representative Western blots and density analysis for iNOS expression. Values are means ± SE; n = 6–8 vascular segments/group from 5–7 rats. *P < 0.05 and **P < 0.01 vs. control. #P < 0.05 vs. hyperlipidemic segments treated with vehicle.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Several important observations were made in the present study. First, we have observed for the first time that acute treatment with gAd significantly attenuated hyperlipidemia-induced endothelial dysfunction. This result raises the possibility that therapeutic application of gAd may be a useful treatment of metabolic disorders with vascular complication. Second, we have provided direct evidence that inhibiting superoxide production, preserving NO from destruction, and blocking the formation of toxic ONOO– are the major mechanisms by which adiponectin exerts its vasculoprotective effect. Finally, we have demonstrated that adiponectin exerts an opposite effect on eNOS and iNOS activity. This novel result indicates that adiponectin possesses a unique property, i.e., differential regulation of eNOS and iNOS activity, that is not shared by any other cytokines identified to date.

The pathogenic relationships among obesity, the metabolic syndrome, and its cardiovascular complications are well established (7). However, mechanisms by which obesity causes vascular dysfunction are not well understood. Increasing attention has been paid to the direct vascular effects of plasma proteins that originate from adipose tissue, especially adiponectin (10). Decreased plasma adiponectin levels are observed in patients with diabetes, metabolic syndrome, and coronary artery disease (18). Moreover, many studies in animal models and human subjects have demonstrated an association between circulating adiponectin levels and endothelial function (13, 17, 21, 22). All these clinical and experimental studies strongly suggest that adiponectin is a critical vascular protective molecule whose reduction may contribute to vascular injury in metabolic disorder-related diseases. The present study took a different approach and provided the first evidence that acute treatment with gAd significantly attenuates endothelial dysfunction associated with hyperlipidemia. This result not only provides additional evidence that reduced adiponectin in metabolic disorders contributes to the development of endothelial dysfunction, but it also suggests that supplementation of gAd in patients with metabolic disorder may normalize endothelial function and prevent or reduce atherosclerosis.

We have obtained several lines of evidence indicating that gAd improves endothelial function by its novel antioxidative/antinitrative property. First, hyperlipidemia-induced reduction of total antioxidant capacity was significantly reversed by treatment with gAd. Since total antioxidant capacity can be increased by either increasing expression of antioxidant molecules or reducing production of oxidant molecules, we then determined the effect of gAd treatment on superoxide production and demonstrated that gAd significantly reduced superoxide production in hyperlipidemic vessels. It is well documented that superoxide reacts with NO at a near diffusion-limited rate, which is three times faster than the reaction between superoxide and superoxide dismutase (12). This reaction not only causes the inactivation of NO, a cytoprotective and vasodilatory molecule, but also results in the formation of ONOO–, a highly reactive and cytotoxic molecule (1). Thus the superoxide/NO reaction is a "toxic switch" that plays a critic pathogenic role in the development of endothelial dysfunction. We have provided direct evidence that increased ONOO– formation in hyperlipidemic vessels was significantly attenuated after gAd treatment. Finally, considerable evidence now exists that NADPH oxidase is the most important source for superoxide production in vascular tissues (20). We have demonstrated that treatment with gAd moderately reduced gp91phox (a critical component of NADPH oxidase) expression in hyperlipidemic vessels. However, it should be noted that the potent antioxidant effect of gAd (~78% reduction in superoxide production) cannot be completely attributed to its inhibition of NADPH oxidase expression (~30%), and other signaling pathways that may contribute to the antioxidant effect of gAd should be investigated.

The most significant finding of the present study is that gAd differentially regulates eNOS and iNOS activity in hyperlipidemic vessels. Previous studies in cultured endothelial cells or normal vascular tissues have demonstrated that gAd and full-length adiponectin (fAd) activate eNOS through an AMPK-Akt signaling pathway and increase NO production (6, 25). The present study demonstrates that the level of phosphorylated eNOS was significantly reduced in hyperlipidemic vessels and that treatment with gAd reversed eNOS phosphorylation and significantly increased eNOS activity. This result is consistent with the present understanding that gAd stimulates NO production by eNOS phosphorylation. However, total NO production was slightly reduced, rather than increased, after gAd treatment of hyperlipidemic vessels, suggesting that other forms of NOS are involved. Given that hyperlipidemia causes iNOS expression and increased NO production, we investigated the effect of gAd on iNOS expression and iNOS activity. Treatment with gAd resulted in a moderate reduction in iNOS expression. Surprisingly, this treatment markedly inhibited (~70%) iNOS activity in hyperlipidemic vessels. This novel result indicates that gAd inhibits iNOS activity by multiple mechanisms. Further study to explore these mechanisms is not only scientifically significant, because it may reveal new signaling pathways that regulate iNOS activity, but also clinically important, since it could provide a foundation for the application of gAd in the treatment of metabolic disorders.

Endothelial dysfunction due to reduced eNOS activity and nitrative/oxidative stress due to increased iNOS and NADPH oxidase expression and subsequent production of cytotoxic peroxynitrite are early hallmarks of vascular injury in patients with metabolic syndrome. The present study demonstrated for the first time that adiponectin is a unique cytokine that improves endothelial function by enhancing eNOS activity and attenuates oxidative/nitrative stress by blocking iNOS and NADPH oxidase expression and ONOO– production. Loss of this dual-protective effect of adiponectin because of reduced adiponectin production and/or development of adiponectin resistance in patients with metabolic syndrome may play a critical pathogenic role in atherosclerosis and vascular injury.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by the following: National Heart, Lung, and Blood Institute Grant 2R01-HL-63828, American Diabetes Association Grant 7-05-RA-83, the Commonwealth of Pennsylvania Department of Health (X. L. Ma), American Diabetes Association Grant 7-06-JF-59 (L. Tao), National Science Fund for Outstanding Young Investigator Grant 30625033, and the National Basic Research Program of China (program no. 973, grant no. 2007CB512106) (F. Gao).


   FOOTNOTES
 

Address for reprint requests and other correspondence: X. L. Ma, Dept. of Emergency Medicine, 1020 Sansom St., Thompson Bldg., Rm. 239, Philadelphia, PA 19107 (e-mail: Xin.Ma{at}Jefferson.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and the ugly. Am J Physiol Cell Physiol 271: C1424–C1437, 1996.[Abstract/Free Full Text]
  2. Berg AH, Combs TP, Scherer PE. ACRP30/adiponectin: an adipokine regulating glucose and lipid metabolism. Trends Endocrinol Metab 13: 84–89, 2002.[CrossRef][Web of Science][Medline]
  3. Buse JB, Ginsberg HN, Bakris GL, Clark NG, Costa F, Eckel R, Fonseca V, Gerstein HC, Grundy S, Nesto RW, Pignone MP, Plutzky J, Porte D, Redberg R, Stitzel KF, Stone NJ. Primary prevention of cardiovascular diseases in people with diabetes mellitus. A scientific statement from the American Heart Association and the American Diabetes Association. Circulation 115: 114–126, 2006.[CrossRef][Web of Science][Medline]
  4. Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res 87: 840–844, 2000.[Abstract/Free Full Text]
  5. Chandran M, Phillips SA, Ciaraldi T, Henry RR. Adiponectin: more than just another fat cell hormone? Diabetes Care 26: 2442–2450, 2003.[Free Full Text]
  6. Chen H, Montagnani M, Funahashi T, Shimomura I, Quon MJ. Adiponectin stimulates production of nitric oxide in vascular endothelial cells. J Biol Chem 278: 45021–45026, 2003.[Abstract/Free Full Text]
  7. Cooper-Dehoff RM, Pepine CJ. Metabolic syndrome and cardiovascular disease: challenges and opportunities. Clin Cardiol. In press.
  8. Cosentino F, Hishikawa K, Katusic ZS, Luscher TF. High glucose increases nitric oxide synthase expression and superoxide anion generation in human aortic endothelial cells. Circulation 96: 25–28, 1997.[Abstract/Free Full Text]
  9. Diamant M, Tushuizen ME. The metabolic syndrome and endothelial dysfunction: common highway to type 2 diabetes and CVD. Curr Diab Rep 6: 279–286, 2006.[Medline]
  10. Goldstein BJ, Scalia R. Adiponectin: a novel adipokine linking adipocytes and vascular function. J Clin Endocrinol Metab 89: 2563–2568, 2004.[Abstract/Free Full Text]
  11. Hotta K, Funahashi T, Arita Y, Takahashi M, Matsuda M, Okamoto Y, Iwahashi H, Kuriyama H, Ouchi N, Maeda K, Nishida M, Kihara S, Sakai N, Nakajima T, Hasegawa K, Muraguchi M, Ohmoto Y, Nakamura T, Yamashita S, Hanafusa T, Matsuzawa Y. Plasma concentrations of a novel, adipose-specific protein, adiponectin, in type 2 diabetic patients. Arterioscler Thromb Vasc Biol 20: 1595–1599, 2000.[Abstract/Free Full Text]
  12. Huie RE, Padmaja S. The reaction of NO with superoxide. Free Radic Res Commun 18: 195–199, 1993.[Web of Science][Medline]
  13. Kumada M, Kihara S, Sumitsuji S, Kawamoto T, Matsumoto S, Ouchi N, Arita Y, Okamoto Y, Shimomura I, Hiraoka H, Nakamura T, Funahashi T, Matsuzawa Y. Association of hypoadiponectinemia with coronary artery disease in men. Arterioscler Thromb Vasc Biol 23: 85–89, 2003.[Abstract/Free Full Text]
  14. Ma XL, Lopez BL, Liu GL, Christopher TA, Gao F, Guo YP, Feuerstein GZ, Ruffolo RR Jr, Barone FC, Yue TL. Hypercholesterolemia impairs a detoxification mechanism against peroxynitrite and renders the vascular tissue more susceptible to oxidative injury. Circ Res 80: 894–901, 1997.[Abstract/Free Full Text]
  15. MacMurray AJ, Moralejo DH, Kwitek AE, Rutledge EA, Van Yserloo B, Gohlke P, Speros SJ, Snyder B, Schaefer J, Bieg S, Jiang J, Ettinger RA, Fuller J, Daniels TL, Pettersson A, Orlebeke K, Birren B, Jacob HJ, Lander ES, Lernmark A. Lymphopenia in the BB rat model of type 1 diabetes is due to a mutation in a novel immune-associated nucleotide (ian)-related gene. Genome Res 12: 1029–1039, 2002.[Abstract/Free Full Text]
  16. Molnar J, Yu S, Mzhavia N, Pau C, Chereshnev I, Dansky HM. Diabetes induces endothelial dysfunction but does not increase neointimal formation in high-fat diet fed C57BL/6J mice. Circ Res 96: 1178–1184, 2005.[Abstract/Free Full Text]
  17. Ouchi N, Ohishi M, Kihara S, Funahashi T, Nakamura T, Nagaretani H, Kumada M, Ohashi K, Okamoto Y, Nishizawa H, Kishida K, Maeda N, Nagasawa A, Kobayashi H, Hiraoka H, Komai N, Kaibe M, Rakugi H, Ogihara T, Matsuzawa Y. Association of hypoadiponectinemia with impaired vasoreactivity. Hypertension 42: 231–234, 2003.[Abstract/Free Full Text]
  18. Ouchi N, Shibata R, Walsh K. Cardioprotection by adiponectin. Trends Cardiovasc Med 16: 141–146, 2006.[CrossRef][Web of Science][Medline]
  19. Pischon T, Girman CJ, Hotamisligil GS, Rifai N, Hu FB, Rimm EB. Plasma adiponectin levels and risk of myocardial infarction in men. JAMA 291: 1730–1737, 2004.[Abstract/Free Full Text]
  20. Rueckschloss U, Duerrschmidt N, Morawietz H. NADPH oxidase in endothelial cells: impact on atherosclerosis. Antioxid Redox Signal 5: 171–180, 2003.[CrossRef][Web of Science][Medline]
  21. Shimabukuro M, Higa N, Asahi T, Oshiro Y, Takasu N, Tagawa T, Ueda S, Shimomura I, Funahashi T, Matsuzawa Y. Hypoadiponectinemia is closely linked to endothelial dysfunction in man. J Clin Endocrinol Metab 88: 3236–3240, 2003.[Abstract/Free Full Text]
  22. Tan KC, Xu A, Chow WS, Lam MC, Ai VH, Tam SC, Lam KS. Hypoadiponectinemia is associated with impaired endothelium-dependent vasodilation. J Clin Endocrinol Metab 89: 765–769, 2004.[Abstract/Free Full Text]
  23. Tao L, Gao E, Hu A, Coletti C, Wang Y, Christopher TA, Lopez BL, Koch W, Ma XL. Thioredoxin reduces post-ischemic myocardial apoptosis by reducing oxidative/nitrative stress. Br J Pharmacol 149: 311–318, 2006.[Web of Science][Medline]
  24. Tao L, Liu HR, Gao E, Teng ZP, Lopez BL, Christopher TA, Ma XL, Batinic-Haberle I, Willette RN, Ohlstein EH, Yue TL. Antioxidative, antinitrative, and vasculoprotective effects of a peroxisome proliferator-activated receptor-gamma agonist in hypercholesterolemia. Circulation 108: 2805–2811, 2003.[Abstract/Free Full Text]
  25. Xi W, Satoh H, Kase H, Suzuki K, Hattori Y. Stimulated HSP90 binding to eNOS and activation of the PI3-Akt pathway contribute to globular adiponectin-induced NO production: vasorelaxation in response to globular adiponectin. Biochem Biophys Res Commun 332: 200–205, 2005.[CrossRef][Web of Science][Medline]
  26. Yao D, Vlessidis AG, Gou Y, Zhou X, Zhou Y, Evmiridis NP. Chemiluminescence detection of superoxide anion release and superoxide dismutase activity: modulation effect of Pulsatilla chinensis. Anal Bioanal Chem 379: 171–177, 2004.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
D.-E. Lee, S. Kehlenbrink, H. Lee, M. Hawkins, and J. S. Yudkin
Getting the message across: mechanisms of physiological cross talk by adipose tissue
Am J Physiol Endocrinol Metab, June 1, 2009; 296(6): E1210 - E1229.
[Abstract] [Full Text] [PDF]

Home page
 Eur Heart JHome page
S. L. Prior, D. R. Gable, J. A. Cooper, S. C. Bain, S. J. Hurel, S. E. Humphries, and J. W. Stephens
Association between the adiponectin promoter rs266729 gene variant and oxidative stress in patients with diabetes mellitus
Eur. Heart J., May 2, 2009; 30(10): 1263 - 1269.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
L. Gao and G. E. Mann
Vascular NAD(P)H oxidase activation in diabetes: a double-edged sword in redox signalling
Cardiovasc Res, April 1, 2009; 82(1): 9 - 20.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/6/E1703    most recent
00462.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, R.
Right arrow Articles by Ma, X. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, R.
Right arrow Articles by Ma, X. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.