Effects of alpha-lipoic acid on endothelial function in aged diabetic and high-fat fed rats

Abstract

Background and purpose: This study was conducted to investigate the effects of alpha-lipoic acid (alpha-LA) on endothelial function in diabetic and high-fat fed animal models and elucidate the potential mechanism underlying the benefits of alpha-LA.

Experimental approach: Plasma metabolites reflecting glucose and lipid metabolism, endothelial function, urinary albumin excretion (UAE), plasma and aortic malondialdehyde (MDA) and urinary 8-hydroxydeoxyguanosine (8-OHdG) were assessed in non-diabetic controls (Wistar rats), untreated Goto-Kakizaki (GK) diabetic and high-fat fed GK rats (fed with atherogenic diet only, treated with alpha-LA and treated with vehicle, for 3 months). Vascular eNOS, nitrotyrosine, carbonyl groups and superoxide anion were also assessed in the different groups.

Key results: alpha-LA and soybean oil significantly reduced both total and non-HDL serum cholesterol and triglycerides induced by atherogenic diet. MDA, carbonyl groups, vascular superoxide and 8-OHdG levels were higher in GK and high-fat fed GK groups and fully reversed with alpha-LA treatment. High-fat fed GK diabetic rats showed significantly reduced endothelial function and increased UAE, effects ameliorated with alpha-LA. This endothelial dysfunction was associated with decreased NO production, decreased expression of eNOS and increased vascular superoxide production and nitrotyrosine expression.

Conclusions and implications: alpha-LA restores endothelial function and significantly improves systemic and local oxidative stress in high-fat fed GK diabetic rats. Improved endothelial function due to alpha-LA was at least partially attributed to recoupling of eNOS and increased NO bioavailability and represents a pharmacological approach to prevent major complications associated with type 2 diabetes.

Figure 1

Effects of α-lipoic acid (α-LA) treatment on plasma lipid levels. Cholesterol (a), non-HDL cholesterol (b), triglycerides (c) and phospholipids (d) of Wistar and diabetic Goto–Kakizaki (GK) rats fed with standard chow (GK) or atherogenic diet (AD) with or without vehicle (soybean oil, SO) or α-LA. Data are expressed as mean±s.e. (n=10 animals in each group). In (a–d), ^***P<0.001 vs Wistar group; ^§§§P<0.001 vs GK group; ^φφP<0.01, ^φφφP<0.001 vs GK+AD group.

Figure 2

Effects of ageing and α-lipoic acid (α-LA) treatment on vasodilatory responses to acetylcholine (ACh) (a and b) and sodium nitroprusside (SNP) (c and d) in Wistar and Goto–Kakizaki (GK) rats after phenylephrine preconstriction of aortic segments. Vasorelaxation was measured using an isometric force displacement transducer. Data are expressed as mean±s.e. (n=7, 21 vascular ring preparations in 7 animals per group). Rats are 18 months old. In (a–d), ^***P<0.001 vs Wistar 18 month group; ^§§§P<0.001 vs GK group; ^$$$P<0.001 vs Wistar 6 month group; ^φP<0.05, ^φφP<0.01, ^φφφP<0.001 vs GK+AD group.

Figure 3

Reduction of oxidative stress parameters by α-lipoic acid (α-LA). Plasma malondialdehyde (MDA) (a) and urinary 8-hydroxydeoxyguanosine (b) levels in Wistar, Goto–Kakizaki (GK) control and GK rats fed with high-fat diet with or without vehicle (soybean oil, SO) or α-LA with 18 months old. Results are mean±s.e. (n=10 animals in each group). In (a) and (b), ^**P<0.01, ^***P<0.001 vs Wistar group; ^§P<0.05 vs GK group; ^φφP<0.01, ^φφφP<0.001 vs GK+AD group; ^###P<0.001 vs GK+SO.

Figure 4

Effects of α-lipoic acid (α-LA) on aortic NO bioavailability and endothelial nitric oxide synthase (eNOS) expression in aortic tissue. (a) NO metabolites were assessed in aortic homogenates using the Griess reaction. In each group, left and right bars represent basal and acetylcholine (ACh)-stimulated NO synthesis, respectively. (b) Representative western blot analysis of eNOS protein expression in aortas of Wistar, Goto–Kakizaki (GK) control and GK rats fed with high-fat diet with or without vehicle (soybean oil, SO) or LA; 30 μg of protein from aortic lysates was resolved by SDS–polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose membrane and probed with anti-eNOS antibody as described in Materials and methods. (c) Averaged densitometry data for diabetic group expressed as a percentage of elevation over the control value established as 100% previously normalized with β-actin values. Data are expressed as mean±s.e. (n=7 animals per group). In (a–c), ^*P<0.05, ^***P<0.001 vs Wistar group; ^§P<0.05 vs GK group; ^φP<0.05, ^φφP<0.01 vs GK+AD group; ^#P<0.05 vs GK+SO group.

Figure 5

In situ detection of superoxide in rat aorta. Representative dihydroethidium (DHE)-stained aorta artery sections reflect O₂⁻ production with the different treatments. Arrows point to the endothelium. At identical laser and photomultiplier settings, fluorescence in diabetic Goto–Kakizaki (GK) and GK atherosclerotic vessel (b and c, respectively) was markedly increased compared with normal vessel (Wistar, a). Note the increased fluorescence reflecting O₂⁻ levels in the endothelium, intima and media (M) of GK aorta. DHE fluorescence decreased to basal levels in the GK+α-LA-treated group (d). Panel e contains quantification of the fluorescent ethidium signal in the different groups of arteries. Results are mean±s.e. (n=10 animals in each group). ^*P<0.05, ^***P<0.001 vs Wistar group; ^§P<0.05 vs GK group; ^φφφP<0.001 vs GK+AD group; ^###P<0.05 vs GK+SO group.

Figure 6

Effects of α-lipoic acid (α-LA) on aortic contents of malondialdehyde (MDA) (a) and protein-bound carbonyls (b and c) during diabetes. Markers of oxidative stress including MDA and protein-bound carbonyls were measured in aortic homogenates according to the procedures described in Materials and methods. Results are mean±s.e. (n=10 animals in each group). In (a–c), ^*P<0.05, ^**P<0.01, ^***P<0.001 vs Wistar group; ^§P<0.05, ^§§§P<0.001 vs GK group; ^φφφP<0.001 vs GK+AD group; ^###P<0.001 vs GK+SO group.

Figure 7

Effects of α-lipoic acid (α-LA) on aortic content of immunoreactive 3-nitrotyrosine during diabetes. (a) Representative western blot analyses of 3-nitrotyrosine protein expression in aortas of Wistar and Goto–Kakizaki (GK) rats; 30 μg of protein from aortic lysates was resolved by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose and probed with 3-nitrotyrosine antibody as described in Materials and methods. (b) Averaged densitometric data for diabetic group expressed as a percentage of elevation over the control value established as 100%. Data are presented as mean±s.e.; of at least seven animals per group. In panel b, ^*P<0.05, ^***P<0.001 vs Wistar group; ^§P<0.05 vs GK group; ^φφφP<0.001 vs GK+AD group; ^#P<0.05 vs GK+SO group.

Figure 8

Representative histological staining of aorta from Wistar rats (a), Goto–Kakizaki (GK) rats (b) and GK rats treated with atherogenic diet (AD) with (d) or without (c) α-lipoic acid (α-LA). Samples were stained with Oil Red O as described in Materials and methods. GK+AD rats showed marked accumulation, and treatment with α-LA significantly reduced lipid droplets in the vascular wall. The endothelium is facing up in all layers.