This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 25/12/2488    most recent 01.ATV.0000190667.33224.4cv1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lee, W. J. Articles by Park, J.-Y. Search for Related Content PubMed PubMed Citation Articles by Lee, W. J. Articles by Park, J.-Y. Related Collections Energy metabolism

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:2488.)
© 2005 American Heart Association, Inc.

Vascular Biology

-Lipoic Acid Prevents Endothelial Dysfunction in Obese Rats via Activation of AMP-Activated Protein Kinase

Woo Je Lee; In Kyu Lee; Hyoun Sik Kim; Yun Mi Kim; Eun Hee Koh; Jong Chul Won; Sung Min Han; Min-Seon Kim; Inho Jo; Goo Taeg Oh; In-Sun Park; Jang Hyun Youn; Seong-Wook Park; Ki-Up Lee; Joong-Yeol Park

From the Department of Internal Medicine (W.J.L, E.H.K., J.C.W., S.M.H., M.-S.K., S.W.P., K.-U.L., J.-Y.P.), Asan Institute for Life Sciences (H.S.K., Y.M.K.), University of Ulsan College of Medicine, Seoul, Republic of Korea; the Department of Internal Medicine (I.K.L.), Kyungpook National University School of Medicine, Daegu, Republic of Korea; the Division of Cardiovascular Research (I.J.), Korean National Institute of Health, Seoul, Republic of Korea; the Laboratory of Cardiovascular Genomics (G.T.O.), Division of Molecular Life Sciences, Ewha Woman’s University, Seoul, Republic of Korea; the Department of Anatomy (I.-S.P.), Inha University College of Medicine, Incheon, Republic of Korea; and the Department of Physiology and Biophysics (J.H.Y.), University of Southern California Keck School of Medicine, Los Angeles.

Correspondence to Joong-Yeol Park, Department of Internal Medicine, University of Ulsan College of Medicine, Asan Medical Center, Song-Pa PO Box 145, Seoul 138-600, Republic of Korea. E-mail jypark{at}amc.seoul.kr

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Objective— Lipid accumulation in vascular endothelial cells may play an important role in the pathogenesis of atherosclerosis in obese subjects. We showed previously that -lipoic acid (ALA) activates AMP-activated protein kinase (AMPK) and reduces lipid accumulation in skeletal muscle of obese rats. Here, we investigated whether ALA improves endothelial dysfunction in obese rats by activating AMPK in endothelial cells.

Methods and Results— Endothelium-dependent vascular relaxation was impaired, and the number of apoptotic endothelial cells was higher in the aorta of obese rats compared with control rats. In addition, triglyceride and lipid peroxide levels were higher, and NO synthesis was lower. Administration of ALA improved all of these abnormalities. AMPK activity was lower in aortic endothelium of obese rats, and ALA normalized it. Incubation of human aortic endothelial cells with ALA activated AMPK and protected cells from linoleic acid–induced apoptosis. Dominant-negative AMPK inhibited the antiapoptotic effects of ALA.

Conclusions— Reduced AMPK activation may play an important role in the genesis of endothelial dysfunction in obese rats. ALA improves vascular dysfunction by normalizing lipid metabolism and activating AMPK in endothelial cells.

We tested whether reduced AMP-activated protein kinase (AMPK) activity in vascular endothelial cells is responsible for endothelial dysfunction in obese rats. -Lipoic acid (ALA) improved endothelial dysfunction and normalized AMPK activity in obese rats. This study provides a rationale for the therapeutic use of ALA for vascular dysfunction.

Key Words: -lipoic acid • endothelium • AMPK • oxidative stress • vascular dysfunction

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Central obesity is associated with increased cardiovascular morbidity and mortality.¹ Subjects with central obesity show increased lipid accumulation in nonadipose tissues such as muscle, liver, and pancreatic islets.^2,3 It has been proposed that increased lipid accumulation in vascular tissue and the consequent increase in oxidative stress may be a missing link between obesity and atherosclerosis.⁴

-Lipoic acid (ALA), a naturally occurring short chain fatty acid containing sulfhydryl groups, has potent antioxidant capacities. ALA is an essential cofactor for mitochondrial respiratory enzymes and improves mitochondrial function.^5,6 We showed in rodents recently that chronic ALA treatment significantly reduced body weight gain primarily by decreasing food intake, and that this effect was mediated by the effect of ALA to decrease AMP-activated protein kinase (AMPK) activity in the hypothalamus.⁷ AMPK is a major regulator of cellular energy metabolism.^8,9 When activated, AMPK increases glucose uptake and fatty acid oxidation^8,9 and decreases lipid accumulation in the tissues.¹⁰ In contrast to the effect of ALA to inhibit AMPK in the hypothalamus, ALA activated AMPK in skeletal muscle, resulting in enhanced fatty acid oxidation and reduced lipid accumulation.¹¹ AMPK is expressed in vascular endothelial cells,¹² and AMPK dysregulation has been suggested to contribute to endothelial dysfunction.¹⁰ We hypothesized that ALA may improve vascular function via activation of AMPK in vascular endothelial cells. In the present study, we examined whether endothelial dysfunction in obese Otsuka Long Evans Tokushima Fatty (OLETF) rats is associated with impaired AMPK activation in vascular endothelial cells and whether these defects can be normalized by ALA treatment.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
For details regarding animal and cell experiments, measurement of various metabolic parameters, and markers for apoptosis and reactive oxygen species (ROS), please see the online supplement, available at http://atvb.ahajournals.org.

Animals
Four-week-old male OLETF rats and their lean controls, Long Evans Tokushima Otsuka (LETO) rats, were obtained from Tokushima Research Institute, Otsuka Pharmaceutical Co. (Tokushima, Japan). All experimental procedures were approved by the institutional animal care and use committee of the Asan Institute for Life Sciences.

Vascular Function Study
Endothelium-dependent and -independent vasorelaxations were measured using an isometric force displacement transducer (Hugo Sachs Elektronik KG D-7806) as described previously.¹³

Electron Microscopy and TUNEL Staining
Apoptosis in the aortic endothelium was examined by electron microscopy and TUNEL staining.

Cell Culture
Human aortic endothelial cells (HAECs) were obtained from BioWhittaker Inc. and maintained in the endothelial basal medium (BioWhittaker Inc.) supplemented with various growth factors and 2% FBS.

Western Blot Analysis of AMPK Phosphorylation and Isoform-Specific AMPK Activity
Phosphorylation and protein levels of AMPK were assayed by Western blot analysis using the antibodies directed against phosphopeptides based on the amino acid sequence surrounding Thr172 of the -subunit of human AMPK and the antibody against -subunit of human AMPK (Cell Signaling), respectively. For isoform-specific AMPK activity measurement, we immunoprecipitated lysates of aortic endothelium (200 µg protein each) with specific antibodies (Upstate Biotechnology) against the 1- and 2-catalytic subunits of AMPK bound to protein G–Sepharose beads. Kinase activity was measured using synthetic "SAMS" peptide and [-³²P]ATP as described.¹⁴

Quantification of Cell Apoptosis
Apotosis was measured by ELISA assay (Boehringer Mannheim) and caspase 3, 8, and 9 activity assay using an Apo Alert caspase florescence assay kit (Clontech).

Adenoviral Gene Transfer of Dominant-Negative 1 and 2 AMPK
Plasmid encoding c-Myc–tagged forms of dominant-negative 1 and 2 AMPK were a kind gift from Dr J. Ha (Department of Molecular Biology, Kyung Hee University College of Medicine, Seoul, Korea). Adenoviruses containing ß-galactosidase (Ad-ß-gal) or mixture of dominant-negative 1 AMPK and 2 AMPK (Ad-DN-AMPK) were added to subconfluent HAECs at a concentration of 10 plaque-forming units (pfu) per cell for 1 hour at 37°C in DMEM without serum, as described previously.¹⁵

Statistical Analysis
All data are shown as mean±SEM. Comparisons between 2 groups were analyzed using unpaired Student’s t tests, and among multiple groups by ANOVA, followed by a post hoc analysis using the Tukey’s multiple comparison test (SPSS). A P value <0.05 was considered to be statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Metabolic Parameters
Compared with LETO rats, OLETF rats had higher body weight, blood pressure, and fasting plasma levels of triglyceride, free fatty acid (FFA), glucose, and insulin. In addition, plasma levels of oxidative stress markers (ie, 8-hydroxydeoxyguanosine and malondialdehyde) were higher and urinary NO excretion was lower in OLETF rats than in LETO rats (Table I, available online at http://atvb.ahajournals.org). ALA treatment completely normalized all of these changes in OLETF rats except for systolic blood pressure, which was substantially lowered but not to the level of LETO rats. These effects of ALA treatment seemed to be mediated by its effect to reduce food intake⁷ because pair feeding of OLETF rats resulted in exactly the same effects. Plasma adiponectin concentrations in OLETF rats were unaltered by ALA treatment.

Endothelium-Dependent Vascular Relaxation
Compared with LETO rats, OLETF rats showed reduced vasodilation of aortic rings in response to acetylcholine, indicating impaired endothelium-dependent vascular relaxation. The endothelium-dependent vasorelaxation in aortic rings of OLETF rats was substantially improved by ALA treatment (Figure 1A). Vascular relaxation in response to sodium nitroprusside, an NO donor, was not different among groups (Figure 1B), indicating that vascular reactivity to NO is intact in OLETF rats. Pair feeding also significantly improved endothelium-dependent vascular function in OLETF rats, but this improvement was significantly less than the improvement achieved by ALA treatment (Figure 1C).

View larger version (26K): [in this window] [in a new window]   Figure 1. Effects of ALA treatment on vasodilatory responses to acetylcholine (A and C) and sodium nitroprusside (B) after phenylephrine preconstriction of aortic segments. Vasorelaxation was measured using an isometric force displacement transducer. Data are expressed as mean±SEM (n=6 per group). In A, *P<0.01 vs LETO group; **P<0.01 vs OLETF group. In C, *P<0.01 vs OLETF group, **P<0.01 vs pair-fed OLETF group.

NO Synthesis and Lipid Contents in Aortic Endothelium
To evaluate whether the changes in endothelium-dependent vasodilation were attributable to changes in NO synthesis, we measured acetylcholine-stimulated NO synthesis in isolated aortas. NO synthesis was significantly lower in aortas of OLETF rats compared with that of LETO rats (Figure 2A). NO synthesis was substantially increased in aortas of OLETF rats by ALA treatment. Pair feeding also significantly improved NO synthesis, but this effect was smaller than that of ALA treatment.

View larger version (33K): [in this window] [in a new window]   Figure 2. Effects of ALA on NO synthesis in aortic tissue (A), triglyceride (B), lipid peroxide (C), AMPK phosphorylation and protein levels (D and F), and 1 AMPK activity (E) in aortic endothelial cells. In A, open and closed bars represent basal and acetylcholine-stimulated NO synthesis, respectively. In D and F, representative Western blot data are shown, together with the bar graphs for AMPK phosphorylation levels. Data are expressed as mean±SEM (n=6 in each group). In A through E, *P<0.01 vs LETO (ie, control) group; **P<0.01 vs OLETF group; #P<0.01 vs OLETF+ALA group. In F, *P<0.01 vs 0 minutes (basal).

Triglyceride and lipid hydroperoxide levels in aortic endothelial cells of OLETF rats were significantly higher than those in LETO rats (Figure 2B and 2C). These changes were reversed completely by ALA treatment but only partially by pair feeding.

AMPK Phosphorylation and Activities in Aortic Endothelium
AMPK phosphorylation was lower in aortic endothelium of OLETF rats compared with that of LETO rats, and ALA treatment completely normalized it (Figure 2D). 1 and 2 AMPK activities were also lower in aortic endothelium of OLETF rats compared with those of LETO rats. ALA treatment increased 1 (Figure 2E) and 2 (Figure I, available online at http://atvb.ahajournals.org) AMPK activities. Interestingly, pair feeding had no effect on these measurements despite its effects on blood metabolic parameters, which were identical to those of ALA treatment (Table I). These data suggest that the effect of ALA treatment on AMPK phosphorylation and activities might be a direct effect of ALA, independent of the effects of ALA on body weight and metabolic parameters.

Effects of Acute Administration of ALA on AMPK Activation and Endothelial Function
To further demonstrate direct effects ALA on AMPK and vascular function, we administrated ALA to 15-week-old OLETF rats (n=12) by an intraperitoneal injection (75 mg/kg body weight). AMPK phosphorylation in aortic endothelium increased 2.3-fold 30 minutes after the ALA administration and remained elevated thereafter until 240 minutes (Figure 2F). Acute administration of ALA also improved endothelium-dependent vascular relaxation, increased NO production, and decreased lipid hydroperoxide levels in the isolated aorta (Figure II, available online at http://atvb.ahajournals.org).

Endothelial Cell Apoptosis
Many in vitro studies have suggested that endothelial cell apoptosis may be an important early event in the pathogenesis of atherosclerosis.¹⁶ Examination by electron microscopy revealed that lumenal walls of aortas of LETO rats were well covered with endothelial cells (Figure 3A). In contrast, in OLETF rats, multiple foci of endothelial cell loss were noticed on lumenal walls of the aorta, and the subendothelial matrix was exposed to the lumen (Figure 3B). Many of the endothelial cells showed apoptotic profiles with nuclear fragmentation and disconnection from neighboring endothelial cells and from underlying subendothelial matrix (Figure 3C). The ultrastructural characteristics of aortas of ALA-treated OLETF rats were between those of LETO rats and of OLEFT rats. Intercellular gap junctions and interdigitations with surrounding tissue were relatively well preserved (Figure 3D). TUNEL staining (Figure 3E) revealed that the number of apoptotic endothelial cells was significantly higher in aortas of OLETF rats than in those of LETO rats, and that ALA treatment of OLETF rats substantially lowered it (Figure 3F). Pair feeding also lowered the number of apoptotic endothelial cells, but this effect was significantly smaller than the effect of ALA treatment (Figure 3F).

View larger version (114K): [in this window] [in a new window]   Figure 3. Electron microscopic examination of endothelial cells. A, Endothelial cells (en) present on the lumenal wall of thoracic aorta of a LETO rat. B and C, Tunica intima of the aorta of OLETF rats. In C, apoptotic figures of an endothelial cell were indicated by nuclear fragmentation (arrows) and spatial detachment (asterisks) from the underlying subendothelial matrix (sem). D, Lumenal wall of thoracic aorta of an ALA-treated OLETF rat. Iel indicates internal elastic lamina. Bar=5 µm. E, Representative figure of TUNEL staining of aortic endothelial cells. Arrows indicate typical TUNEL-positive cells (magnification x1000). F, The percentage of TUNEL-positive cells was calculated as the number of positive cells among total endothelial cells in whole fields at the same magnification. In F, data are expressed as mean±SEM (n=6 in each group). *P<0.01 vs LETO group; **P<0.01 vs OLETF group; #P<0.01 vs OLETF+ALA group.

AMPK and Apoptosis in Cultured Endothelial Cells
It was suggested that endothelial dysfunction in metabolic syndrome is mediated, at least in part, by elevated circulating FFA levels.¹⁷ Incubation of HAECs with 300 µmol/L linoleic acid led to a 2.5-fold increase in apoptosis (Figure 4A), which was accompanied by increases in caspase-3, caspase-9 (a measure of mitochondrial pathway), and caspase-8 (a measure of death receptor signaling pathway) activities (Figure 4B through 4D). The linoleic acid–induced increases in apoptosis and caspase activities were all inhibited by 0.5 mmol/L ALA. Linoleic acid decreased AMPK phosphorylation in HAECs, and ALA substantially reversed this decrease (Figure 4E). ALA increased AMPK phosphorylation rapidly (ie, within 30 minutes; Figure 4F) in HAECs as observed in vivo (Figure 2F).

View larger version (29K): [in this window] [in a new window]   Figure 4. Effects of ALA on linoleic acid–induced apoptosis and AMPK phosphorylation in cultured HAECs. HAECs were incubated in media containing 0.5% FBS, 300 µmol/L linoleic acid, with or without 0.5 mmol/L ALA. After 16 hours, apoptosis (A) was analyzed by measuring the level of cytosolic histone-bound DNA fragments using a cell death ELISA kit. Caspase-3 (B), -8 (C), and -9 (D) activities were analyzed using a caspase fluorescence assay kit. E and F, Immunoblot analysis for AMPK phosphorylation. The values are means±SEM of 3 independent experiments. In A through E, *P<0.01 vs control; **P<0.01 vs linoleic acid. In F, *P<0.01 vs 0 minutes (basal).

Effects of Dominant-Negative 1 and 2 AMPK
Mixture of dominant-negative 1 and 2 AMPK nearly completely reversed the effects of ALA against the changes in intracellular triglyceride, ROS generation, and apoptosis induced by linoleic acid, indicating that these effects of ALA were mediated by AMPK activation (Figure 5A through 5D).

View larger version (27K): [in this window] [in a new window]   Figure 5. Effects of dominant-negative AMPK on linoleic acid–induced intracellular triglyceride accumulation (A), ROS generation (B), apoptosis (C), and caspase-3 activity (D) in HAECs. HAECs were infected with adenoviruses containing plasmids coding for either mixture of 1 and 2 dominant-negative AMPK (Ad-DN-AMPK) or ß-galactosidase (Ad-ß-gal; control). Two days after infection, cells were incubated for 16 hours in media containing 0.5% FBS and 300 µmol/L linoleic acid, with or without 0.5 mmol/L ALA. *P<0.01 vs Ad-ß-gal; **P<0.01 vs linoleic acid; ***P<0.01 vs ALA.

Effects of ALA on NAD(P)H Oxidase Activity and Mitochondrial Membrane Potential
Major sites of intracellular ROS generation are mitochondria and cell membrane NAD(P)H oxidase.¹⁸ The mitochondrial membrane potential increased rapidly and peaked at 30 minutes after exposure to linoleic acid compared with Ad-ß-gal–treated control cells. The membrane potential then decreased and became lower than that of the control cells. ALA treatment nearly completely prevented these changes in membrane potential. On the other hand, pretreatment with Ad-DN-AMPK substantially reversed the effects of ALA on membrane potential (Figure 6A). Similarly, linoleic acid increased NAD(P)H oxidase activity, and ALA decreased it. The effect of ALA on NAD(P)H oxidase activity was prevented by Ad-DN-AMPK (Figure 6B).

View larger version (21K): [in this window] [in a new window]   Figure 6. Effects of ALA on mitochondrial membrane potential (m; A) and NAD(P)H oxidase activity (B). In A, time course of changes in m for 6 hours is shown. m was assessed by loading HAECs with 50 nmol/L TMRM (tetramethylrhodamine methyl ester) for 20 minutes. The uptake of TMRM increases with increased m. *P<0.01 vs ALA. LA indicates linoleic acid. In B, NAD(P)H oxidase activity was accessed by measuring NAD(P)H-induced production of superoxide in HAECs. Data are expressed as mean±SEM. *P<0.01 vs Ad-ß-gal; **P<0.01 vs linoleic acid; ***P<0.01 vs ALA.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Defective endothelium-dependent vascular relaxation is an early event in the development of atherosclerosis.¹⁹ We confirmed that endothelium-dependent vascular relaxation is impaired in obese OLETF rats^20,21 and found that this impairment is associated with increased lipid accumulation and apoptosis and decreased NO synthesis and AMPK activities in endothelial cells. All of these alterations in endothelial cells and vascular dysfunction were substantially improved by ALA treatment.

Our previous study demonstrated strong anorexic effects of ALA in normal and obese rodents.⁷ In the present study, ALA reduced food intake (data not shown) and normalized body weight in OLETF rats. In addition, all of the metabolic changes (FFA, triglycerides, insulin, etc) observed in OLETF rats were normalized by ALA. The normalization of metabolic parameters seemed to be attributable to the reduction in food intake because it also occurred in pair-fed, untreated OLETF rats; the metabolic parameters were identical between ALA-treated and pair-fed OLETF rats. Pair feeding (or normalization of metabolic parameters) alone also improved vascular function and reduced the alterations (lipid accumulation, apoptosis, NO synthesis, etc) in endothelial cells of OLETF rats. However, these effects were only about a half of the effects of ALA treatment, suggesting that ALA improved vascular dysfunction in OLETF rats in part by reducing food intake but also by exerting additional effects. In regard to this, it is of extreme interest that decreased AMPK activities in endothelial cells of OLETF rats was completely normalized by ALA but little affected by pair feeding. In addition, intraperitoneal injection of ALA increased AMPK phosphorylation in aortic endothelial cells within 30 minutes and improved vascular dysfunction in OLETF rats. These data suggest that ALA directly activates AMPK in endothelial cells and that this effect leads to the improvement of vascular dysfunction in OLETF rats beyond that with pair feeding or metabolic normalization. However, to prove the causal relationship between AMPK activation and improvement of vascular function, it would be necessary to demonstrate the effect of selective inactivation of AMPK in aortic endothelial cells, as was done in in vitro experiment.

In cultured HAECs, linoleic acid decreased AMPK phosphorylation and increased triglyceride accumulation, apoptosis, and ROS generation. ALA prevented the linoleic acid–induced decrease in AMPK phosphorylation, and this effect was associated with normalization of triglyceride level, apoptosis, and ROS generation in the presence of linoleic acid. Furthermore, expression of dominant-negative AMPK in these cells led to inhibition of ALA effects. Together, these data suggest that AMPK activity in endothelial cells is an important regulator of endothelial function.

A key feature of defective endothelium-dependent vasodilation is reduced bioavailability of NO.¹⁹ Consistent with previous studies in other animal models of obesity and diabetes,²² aortas isolated from OLETF rats displayed attenuated vascular responses to acetylcholine but not to the direct-acting exogenous NO donor sodium nitroprusside. Urinary NO excretion and acetylcholine-stimulated NO synthesis in aortic endothelium were significantly reduced in OLETF rats compared with LETO rats. These results suggest that vascular reactivity to NO is intact, but NO bioavailability is reduced in OLETF rats. NO bioavailability is determined as the balance between NO production and removal. Evidence from experimental animals with diabetes suggests that the most likely mechanism underlying reduced NO bioavailability is inactivation of NO by oxygen-derived free radicals.²³ Bakker et al⁴ proposed that increased availability of lipid (ie, triglyceride and long chain fatty acyl coenzyme A [LCAC]) in vascular endothelial cells could cause oxidative stress by affecting the mitochondrial respiratory chain. Supporting this hypothesis, the present study demonstrates that intracellular contents of lipid (ie, triglycerides) and the oxidative stress marker (ie, lipid peroxide) were elevated in aortic endothelium of OLETF rats.

Oxidative stress has been also implicated to play a major role in cellular apoptosis.¹⁶ The present study demonstrates that the number of apoptotic cells was significantly increased in aortic endothelium of OLETF rats. In cultured HAECs, linoleic acid increased intracellular triglyceride, oxidative stress, and apoptosis. Thus, increased circulating levels of FFA and triglycerides in obese animals may increase oxidative stress and apoptosis in vascular endothelial cells. This may further contribute to the decrease in NO bioavailability and vascular dysfunction (Figure III, available online at http://atvb.ahajournals.org).

We are the first to show that AMPK phosphorylation was decreased in aortic endothelium of obese rats and to suggest that it could be causally related to vascular dysfunction in obese animals. The mechanism by which this change occurs is unclear. However, because AMPK functions as a fuel sensor in the cell and is activated when cellular energy is depleted, it is conceivable that surplus cellular energy in association with high plasma FFA or glucose levels is responsible for reduced AMPK activity in aortic endothelium of OLETF rats. Consistent with this idea, we demonstrated that incubation of HAECs with linoleic acid significantly decreased AMPK activation. Reduced AMPK activity would decrease fatty acid oxidation by activating acetyl CoA carboxylase and increasing intracellular malonyl coenzyme A levels.²⁴ Thus, increased FFA flux from the circulation and reduced FFA oxidation in aortic endothelial cells could lead to excessive accumulation of triglyceride and LCAC. This may be an early event that leads to a cascade of increased ROS generation, increased apoptosis, and decreased NO bioavailability in endothelial cells and vascular dysfunction, as discussed above.

ALA is well known as an antioxidant. ALA directly scavenges free radicals, chelates transition metal ion (eg, iron and copper), increases cytosolic glutathione and vitamin C levels, and prevents toxicities associated with their loss.^5,6 In addition, the present study demonstrates that antioxidant action of ALA is, at least in part, mediated by its effect on AMPK. Major sites of intracellular ROS generation are mitochondria and cell membrane NAD(P)H oxidase.¹⁸ The mitochondrial respiratory chain generates ROS when the electrochemical gradient between the mitochondrial inner membrane is high and the rate of electron transport is limited.²⁵ Consistent with this concept, recent studies from our group^26,27 and others²⁸ have shown that high glucose or linoleic acid leads to significant increases in mitochondrial membrane potential (hyperpolarization) and ROS generation. In the present study, ALA treatment nearly completely prevented hyperpolarization induced by linoleic acid, and pretreatment with Ad-DN-AMPK reversed the effect of ALA. As stated above, LCAC is known to impair the flow of electrons through the electron transfer chain,⁴ and the reduction of LCAC levels by AMPK activation would be responsible for improvement in electron transfer and reduction in ROS generation.

We also found that linoleic acid increases and ALA decreases NADPH oxidase activity. Again, DN-AMPK reversed the effect of ALA, implicating that this effect is AMPK mediated. The mechanism by which AMPK activation decreases NAD(P)H oxidase activity is presently unknown but may be attributable to its effect on the cellular NADH/NAD⁺ redox state.²⁹ It can be assumed that linoleic acid–induced mitochondrial membrane hyperpolarization, which reflects impairment of mitochondrial electron transfer and respiration, would increase NAD(P)H oxidase activity by increasing the cytosolic NADH:NAD⁺ ratio.²⁹ Conversely, normalization of mitochondrial membrane potential by AMPK activation would decrease NAD(P)H oxidase activity by reducing the cytosolic NADH:NAD⁺ ratio.

In conclusion, our results suggest that reduced AMPK activity in endothelial cells may play an important role in the genesis of vascular dysfunction in obese rats, and that ALA may improve vascular dysfunction in obese rats by activating AMPK in endothelial cells. This study provides a rationale for the therapeutic use of ALA for vascular dysfunction in obese subjects.

	Acknowledgments

 
This study was supported by the National Research Laboratory grant from the Ministry of Science and Technology (M1040000000804J000000810) and grants from the Korea Ministry of Health and Welfare (02-PJ1-PG10-20708-0007) and the Asan Institute for Life Sciences, (01-122, 01-279, 05-006) Republic of Korea.

	Footnotes

 
W.J.L. and I.K.L. contributed equally to this work.

Received March 7, 2005; accepted September 7, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Poulter N. Global risk of cardiovascular disease. Heart. 2003; 89 (suppl 2): ii2–ii5.[Abstract/Free Full Text]

2. Kelley DE, Goodpaster BH. Skeletal muscle triglyceride. An aspect of regional adiposity and insulin resistance. Diabetes Care. 2001; 24: 933–941.[Abstract/Free Full Text]

3. Unger RH, Zhou YT Lipotoxicity of beta-cells in obesity and in other causes of fatty acid spillover. Diabetes. 2001; 50 (suppl 1): S118–S121.[Free Full Text]

4. Bakker SJ, RG IJ, Teerlink T, Westerhoff HV, Gans RO, Heine RJ. Cytosolic triglycerides and oxidative stress in central obesity: the missing link between excessive atherosclerosis, endothelial dysfunction, and beta-cell failure? Atherosclerosis. 2000; 148: 17–21.[CrossRef][Medline] [Order article via Infotrieve]

5. Packer L, Kraemer K, Rimbach G. Molecular aspects of lipoic acid in the prevention of diabetes complications. Nutrition. 2001; 17: 888–895.[CrossRef][Medline] [Order article via Infotrieve]

6. Smith AR, Shenvi SV, Widlansky M, Suh JH, Hagen TM. Lipoic acid as a potential therapy for chronic diseases associated with oxidative stress. Curr Med Chem. 2004; 11: 1135–1146.[Medline] [Order article via Infotrieve]

7. Kim MS, Park JY, Namkoong C, Jang PG, Ryu JW, Song HS, Yun JY, Namgoong IS, Ha J, Park IS, Lee IK, Viollet B, Youn JH, Lee HK, Lee KU. Anti-obesity effects of alpha-lipoic acid mediated by suppression of hypothalamic AMP-activated protein kinase. Nat Med. 2004; 10: 727–733.[CrossRef][Medline] [Order article via Infotrieve]

8. Winder WW. Energy-sensing and signaling by AMP-activated protein kinase in skeletal muscle. J Appl Physiol. 2001; 91: 1017–1028.[Abstract/Free Full Text]

9. McGarry JD. Banting lecture 2001: dysregulation of fatty acid metabolism in the etiology of type 2 diabetes. Diabetes. 2002; 51: 7–18.[Free Full Text]

10. Ruderman N, Prentki M. AMP kinase and malonyl-CoA: targets for therapy of the metabolic syndrome. Nat Rev Drug Discov. 2004; 3: 340–351.[CrossRef][Medline] [Order article via Infotrieve]

11. Lee WJ, Song KH, Koh EH, Won JC, Kim HS, Park HS, Kim MS, Kim SW, Lee KU, Park JY. alpha-Lipoic acid increases insulin sensitivity by activating AMPK in skeletal muscle. Biochem Biophys Res Commun. 2005; 332: 885–891.[CrossRef][Medline] [Order article via Infotrieve]

12. Dagher Z, Ruderman N, Tornheim K, Ido Y. The effect of AMP-activated protein kinase and its activator AICAR on the metabolism of human umbilical vein endothelial cells. Biochem Biophys Res Commun. 1999; 265: 112–115.[CrossRef][Medline] [Order article via Infotrieve]

13. Shinozaki K, Nishio Y, Okamura T, Yoshida Y, Maegawa H, Kojima H, Masada M, Toda N, Kikkawa R, Kashiwagi A. Oral administration of tetrahydrobiopterin prevents endothelial dysfunction and vascular oxidative stress in the aortas of insulin-resistant rats. Circ Res. 2000; 87: 566–573.[Abstract/Free Full Text]

14. Hayashi T, Hirshman MF, Fujii N, Habinowski SA, Witters LA, Goodyear LJ. Metabolic stress and altered glucose transport: activation of AMP-activated protein kinase as a unifying coupling mechanism. Diabetes. 2000; 49: 527–531.[Abstract]

15. Park JY, Takahara N, Gabriele A, Chou E, Naruse K, Suzuma K, Yamauchi T, Ha SW, Meier M, Rhodes CJ, King GL. Induction of endothelin-1 expression by glucose: an effect of protein kinase C activation. Diabetes. 2000; 49: 1239–1248.[Abstract]

16. Choy JC, Granville DJ, Hunt DW, McManus BM. Endothelial cell apoptosis: biochemical characteristics and potential implications for atherosclerosis. J Mol Cell Cardiol. 2001; 33: 1673–1690.[CrossRef][Medline] [Order article via Infotrieve]

17. Steinberg HO, Tarshoby M, Monestel R, Hook G, Cronin J, Johnson A, Bayazeed B. Baron AD. Elevated circulating free fatty acid levels impair endothelium-dependent vasodilation. J Clin Invest. 1997; 100: 1230–1239.[Medline] [Order article via Infotrieve]

18. Schafer M, Schafer C, Ewald N, Piper HM, Noll T. Role of redox signaling in the autonomous proliferative response of endothelial cells to hypoxia. Circ Res. 2003; 92: 1010–1015.[Abstract/Free Full Text]

19. Ross R. Atherosclerosis–an inflammatory disease. N Engl J Med. 1999; 340: 115–126.[Free Full Text]

20. Saito Y, Nakamura T, Ohyama Y, Suzuki T, Iida A, Shiraki-Iida T, Kuro-o M, Nabeshima Y, Kurabayashi M, Nagai R. In vivo klotho gene delivery protects against endothelial dysfunction in multiple risk factor syndrome. Biochem Biophys Res Commun. 2000; 276: 767–772.[CrossRef][Medline] [Order article via Infotrieve]

21. Sakamoto S, Minami K, Niwa Y, Ohnaka M, Nakaya Y, Mizuno A, Kuwajima M, Shima K. Effect of exercise training and food restriction on endothelium-dependent relaxation in the Otsuka Long-Evans Tokushima Fatty rat, a model of spontaneous NIDDM. Diabetes. 1998; 47: 82–86.[Abstract]

22. Durante W, Sen AK, Sunahara FA. Impairment of endothelium-dependent relaxation in aortae from spontaneously diabetic rats. Br J Pharmacol. 1988; 94: 463–468.[Medline] [Order article via Infotrieve]

23. Gryglewski RJ, Palmer RM, Moncada S. Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature. 1986; 320: 454–456.[CrossRef][Medline] [Order article via Infotrieve]

24. Ruderman NB, Saha AK, Vavvas D, Witters LA. Malonyl-CoA, fuel sensing, and insulin resistance. Am J Physiol. 1999; 276: E1–E18.

25. Arsenijevic D, Onuma H, Pecqueur C, Raimbault S, Manning BS, Miroux B, Couplan E, Alves-Guerra MC, Goubern M, Surwit R, Bouillaud F, Richard D, Collins S, Ricquier D. Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production. Nat Genet. 2000; 26: 435–439.[CrossRef][Medline] [Order article via Infotrieve]

26. Park JY, Park KG, Kim HJ, Kang HG, Ahn JD, Kim HS, Kim YM, Son SM, Kim IJ, Kim YK, Kim CD, Lee KU, Lee IK. The effects of the overexpression of recombinant uncoupling protein 2 on proliferation, migration and plasminogen activator inhibitor 1 expression in human vascular smooth muscle cells. Diabetologia. 2005; 48: 1022–1028.[CrossRef][Medline] [Order article via Infotrieve]

27. Lee KU, Lee IK, Han J, Song DK, Kim YM, Song HS, Kim HS, Lee WJ, Koh EH, Song KH, Han SM, Kim MS, Park IS, Park JY. Effects of recombinant adenovirus-mediated uncoupling protein 2 overexpression on endothelial function and apoptosis. Cir Res. 2005; 96: 1200–1207.[Abstract/Free Full Text]

28. Russell JW, Golovoy D, Vincent AM, Mahendru P, Olzmann JA, Mentzer A, Feldman EL. High glucose-induced oxidative stress and mitochondrial dysfunction in neurons. FASEB J. 2002; 16: 1738–1748.[Abstract/Free Full Text]

29. Bassenge E, Sommer O, Schwemmer M, Bunger R. Antioxidant pyruvate inhibits cardiac formation of reactive oxygen species through changes in redox state. Am J Physiol Heart Circ Physiol. 2000; 279: H2431–H2438.[Abstract/Free Full Text]

This article has been cited by other articles:

				S. Wang, J. Xu, P. Song, B. Viollet, and M.-H. Zou In Vivo Activation of AMP-Activated Protein Kinase Attenuates Diabetes-Enhanced Degradation of GTP Cyclohydrolase I Diabetes, August 1, 2009; 58(8): 1893 - 1901. [Abstract] [Full Text] [PDF]

				G. R. Steinberg and B. E. Kemp AMPK in Health and Disease Physiol Rev, July 1, 2009; 89(3): 1025 - 1078. [Abstract] [Full Text] [PDF]

				Y. Wang, Y. Huang, K. S.L. Lam, Y. Li, W. T. Wong, H. Ye, C.-W. Lau, P. M. Vanhoutte, and A. Xu Berberine prevents hyperglycemia-induced endothelial injury and enhances vasodilatation via adenosine monophosphate-activated protein kinase and endothelial nitric oxide synthase Cardiovasc Res, June 1, 2009; 82(3): 484 - 492. [Abstract] [Full Text] [PDF]

				M.-S. Gauthier, H. Miyoshi, S. C. Souza, J. M. Cacicedo, A. K. Saha, A. S. Greenberg, and N. B. Ruderman AMP-activated Protein Kinase Is Activated as a Consequence of Lipolysis in the Adipocyte: POTENTIAL MECHANISM AND PHYSIOLOGICAL RELEVANCE J. Biol. Chem., June 13, 2008; 283(24): 16514 - 16524. [Abstract] [Full Text] [PDF]

				M. C. Cave, R. T. Hurt, T. H. Frazier, P. J. Matheson, R. N. Garrison, C. J. McClain, and S. A. McClave Obesity, Inflammation, and the Potential Application of Pharmaconutrition Nutr Clin Pract, February 1, 2008; 23(1): 16 - 34. [Abstract] [Full Text] [PDF]

				W.-J. Zhang, K. E. Bird, T. S. McMillen, R. C. LeBoeuf, T. M. Hagen, and B. Frei Dietary {alpha}-Lipoic Acid Supplementation Inhibits Atherosclerotic Lesion Development in Apolipoprotein E Deficient and Apolipoprotein E/Low-Density Lipoprotein Receptor Deficient Mice Circulation, January 22, 2008; 117(3): 421 - 428. [Abstract] [Full Text] [PDF]

				G. Ceolotto, A. Gallo, I. Papparella, L. Franco, E. Murphy, E. Iori, E. Pagnin, G. P. Fadini, M. Albiero, A. Semplicini, et al. Rosiglitazone Reduces Glucose-Induced Oxidative Stress Mediated by NAD(P)H Oxidase via AMPK-Dependent Mechanism Arterioscler Thromb Vasc Biol, December 1, 2007; 27(12): 2627 - 2633. [Abstract] [Full Text] [PDF]

				Q. W. Shen, M. J. Zhu, J. Tong, J. Ren, and M. Du Ca2+/calmodulin-dependent protein kinase kinase is involved in AMP-activated protein kinase activation by {alpha}-lipoic acid in C2C12 myotubes Am J Physiol Cell Physiol, October 1, 2007; 293(4): C1395 - C1403. [Abstract] [Full Text] [PDF]

				M. Artwohl, K. Muth, K. Kosulin, R. de Martin, T. Holzenbein, G. Rainer, A. Freudenthaler, N. Huttary, L. Schmetterer, W. K. Waldhausl, et al. R-(+)-{alpha}-lipoic acid inhibits endothelial cell apoptosis and proliferation: involvement of Akt and retinoblastoma protein/E2F-1 Am J Physiol Endocrinol Metab, September 1, 2007; 293(3): E681 - E689. [Abstract] [Full Text] [PDF]

				Y.-W. Kim, S.-Y. Park, J.-Y. Kim, J.-Y. Huh, W.-S. Jeon, C.-J. Yoon, S.-S. Yun, and K.-H. Moon Metformin Restores the Penile Expression of Nitric Oxide Synthase in High-Fat-Fed Obese Rats J Androl, July 1, 2007; 28(4): 555 - 560. [Abstract] [Full Text] [PDF]

				Y. Wu, P. Song, J. Xu, M. Zhang, and M.-H. Zou Activation of Protein Phosphatase 2A by Palmitate Inhibits AMP-activated Protein Kinase J. Biol. Chem., March 30, 2007; 282(13): 9777 - 9788. [Abstract] [Full Text] [PDF]

				M. Arad, C. E. Seidman, and J.G. Seidman AMP-Activated Protein Kinase in the Heart: Role During Health and Disease Circ. Res., March 2, 2007; 100(4): 474 - 488. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 25/12/2488    most recent 01.ATV.0000190667.33224.4cv1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lee, W. J. Articles by Park, J.-Y. Search for Related Content PubMed PubMed Citation Articles by Lee, W. J. Articles by Park, J.-Y. Related Collections Energy metabolism