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Oxidative stress in skeletal muscle impairs mitochondrial function in alloxan induced diabetic rats: Role of alpha lipoic acid
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Original article
Oxidative stress in skeletal muscle impairs mitochondrial function in alloxan induced diabetic rats: Role of alpha lipoic acid Ramasamy Saraswathi , Sivasithamparam Niranjali Devaraj ∗ Department of Biochemistry, University of Madras, Guindy campus, Sardar Patel Road, Chennai-600 025, India
a r t i c l e
i n f o
Article history: Received 10 May 2012 Accepted 12 August 2012 Keywords: Diabetes Skeletal muscle Mitochondria Oxidative stress Alpha lipoic acid
a b s t r a c t The role increased free radical production associated with mitochondrial dysfunction is well established in the pathophysiology of diabetes mellitus. A male Wistar rat model was utilized in which diabetes was induced by alloxan at a dose of 140 mg/kg body weight, intraperitoneally for 8 weeks. In alloxan induced diabetes on the activities of Krebs cycle enzymes, oxidative as well as enzymes involved in oxidative metabolism were altered. Oxidative damage to DNA, protein and lipid were observed, which provide insight into the association of elevated free radicals and mitochondrial dysfunction leading to diabetes mellitus. Lipoic acid treatment (100 mg/kg body weight for 30 days orally) restored mitochondrial functioning showing normal activity of TCA cycle enzyme with decreased free radical production and oxidative damage. Thus, our results propose that lipoic acid restores mitochondrial function in experimentally induced diabetic rats through decreasing levels of free radicals and promoting activation of Krebs cycle enzymes. © 2013 Published by Elsevier Masson SAS.
1. Introduction Diabetes mellitus is a chronic metabolic disorder resulting from insulin dyshomeostasis, and it is considered as the major cause of morbidity and mortality affecting ∼5% of the population [1]. Much of the evidence concerning the role of oxidative stress in the induction of diabetes comes from studies using the diabetogenic drug, alloxan [2]. It has the ability to produce free radicals that damages -cells of pancreas [3]. Alloxan induced hyperglycemia aggravates the oxidative stress by auto-oxidation of glucose and their derivatives [4]. Enhanced oxidative stress induced by hyperglycemia may constitute the key and common events in the pathogenesis of different diabetic complications [5]. Mitochondria are central to energy production and are therefore fully integrated into the rest of the cell’s physiological responses to stress. Besides ATP production, mitochondria are also the major endogenous source of superoxide, peroxynitrite, and hydroxyl radicals [6]. Mitochondrial superoxide production is considered as a single unifying mechanism for diabetic complications [7,8]. The resultant mitochondrial decay may ultimately cause mitochondrial dysfunction and/or loss of cellular homeostasis. Lipoic acid is a dithiol compound that exists naturally in mitochondria as a coenzyme for pyruvate dehydrogenase and ␣-ketoglutarate dehydrogenase. It has been used as a therapy
∗ Corresponding author. Tel.: +91 44 22351269; fax: +91 44 22352870. E-mail address: [email protected] (S.N. Devaraj).
for many ailments associated with impaired energy metabolism, including type II diabetes [9] and diabetic polyneuropathies [10]. Dietary supplementation increased the unbound lipoic acid, which can act as a potent antioxidant and ameliorate oxidative stress both in vitro and in vivo [11,12]. Lipoic acid has an ability to improve energy metabolism [13] and lower oxidative stress [14,15] in many pathological conditions and disease states. The present study demonstrates the efficacy of lipoic acid in protection of mitochondrial dysfunction in skeletal muscle of alloxan induced diabetic rats. 2. Materials and methods 2.1. Chemicals dl-alpha-lipoic acid, dichlorofluorescinhydrate-diacetate (DCFH-DA), fattyacid–free bovine, serum albumin (BSA), reduced glutathione (GSH), oxidized glutathione (GSSG), 5,5 -dithiobis2nitrobenzoic acid (DTNB) and alloxan were purchased from Sigma (St. Louis, MO). All other chemicals used were of analytical grade and were obtained from SISCO Research Laboratories Pvt Ltd (Mumbai, India). 2.2. Animals Experiments were conducted in accordance with guidelines approved by the institutional animal ethical committee. Male albino rats weighing between 160 to 180 g were used throughout
2210-5239/$ – see front matter © 2013 Published by Elsevier Masson SAS. http://dx.doi.org/10.1016/j.bionut.2012.08.006
Please cite this article in press as: Saraswathi R, Devaraj SN. Oxidative stress in skeletal muscle impairs mitochondrial function in alloxan induced diabetic rats: Role of alpha lipoic acid. Biomed Prev Nutr (2013), http://dx.doi.org/10.1016/j.bionut.2012.08.006
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the study. The rats were barrier housed two per cage at a temperature of 24 ± 2 ◦ C in a light controlled environment of 12 h light-dark cycle, and provided with free access to food and water. Diabetes was induced by a single subcutaneous injection of 200 mg alloxan/kg body weight in 0.1 M citrate buffer, pH 4.5 at day 0 of the experiment. Body weight of the animals was recorded every 10 days. Rats were identified as diabetic on the basis of fasting blood glucose levels (higher than 200 mg/dL at least 4 days after alloxan treatment), and divided into subgroups as required by the experiment. Group I control rats, group II diabetic rats, group III control rats administered with lipoic acid and group IV diabetic rats administered with lipoic acid. dl-␣-lipoic acid (100 mg/kg body weight/day) dissolved in physiological saline containing 0.5% KOH was administered orally using intragastric canula up to 30 days. Control animals received physiological saline containing 0.5% KOH. On completion of the experimental period, rats were killed by cervical decapitation. Gastrocnemius muscle was excised, weighed, and immediately used for experimental procedure. 2.3. Estimation of serum glucose and plasma insulin Serum glucose was determined in an auto analyzer using Ecoline Kits (E. Merck, Mumbai, India) and plasma insulin was estimated by ELISA method using Boehringer Mannheim Gmbh kit, Werk Penzberg, Germany. 2.4. Isolation of skeletal muscle mitochondria Skeletal muscle mitochondria were isolated by differential centrifugation [16]. Skeletal muscle homogenization was performed in mitochondrial isolation buffer containing 25 M mannitol, 75 mM sucrose, 1 mM EGTA, 0.1% fattyacid–free BSA and 10 mM Tris-HCl (pH 7.4). The homogenate was centrifuged at 1000 g for 10 min in 4 ◦ C and the supernatant was again centrifuged at 12,000 g, and resuspended in isolation buffer without EGTA. The purity of the obtained fraction was evaluated by enzymatic assay of succinate dehydrodenase for monitoring mitochontria, acid phospotase for lysosomes, glucose-6-phosphatase for endoplasmic reticulum and catalase for peroxisomes [17–20]. Protein content was quantified by the method of Lowry et al. [21]. An aliquot of freshly prepared mitochondria was frozen (–80 ◦ C) for enzyme assays and the rest was used immediately for oxidant generation studies. 2.5. Determination of oxidant generation Oxidant generation was evaluated in isolated skeletal muscle mitochondria by using a redox sensitive fluorescent probe, DCFH-DA [22,23]. Isolated mitochondria were incubated in the assay media (137 mM KCl, 2.5 mM MgCl2 , 2 mM K2HPO4, 10 mM Tris-HCl, 5 mM glutamate and 5 mM malate with pH 7.4) containing 5 M DCFH-DA (dissolved in 1.25 mM methanol) at 37 ◦ C for 10 min. The mitochondrial pellets were resuspended in the assay media without DCFH-DA, and 500 l of the suspension (∼0.4 mg protein) was used for the assay. DCF formation was followed at the excitation wavelength of 488 nm and emission wavelength of 525 nm for 25 min by using Shimadzu RF-1501 fluorescence spectrophotometer. The rate of DCFH conversion to DCF was linear for at least 25 min, which was corrected for auto-oxidation rate of DCFH, without protein. To verify that DCFH concentration was saturating allowing maximal rate of oxidation for at least 25 min, 1 mol of H2 O2 was added into the mixture at the end of the assay. The DCFH oxidation rate was found to increase several fold immediately, indicating that sufficient DCFH was still available.
2.6. Lipid peroxidation (LPO) LPO was determined by measuring the levels of thiobarbituric acid reactive substances (TBARS) in rat skeletal muscle mitochondria using malondialdehyde (MDA) as the standard [24]. Briefly, 500 l of mitochondria (∼4 mg protein ml−1 ) was added to the test tubes containing 0.2 ml of 8.1% SDS, 1.5 ml of 20% acetic acid, pH 3.5, and 1.5 ml of 0.8% thiobarbituric acid (TBA) solution. The mixture was diluted to 4.0 ml with distilled water and heated at 95 ◦ C for 1 h. After cooling on ice, the samples were extracted with 4.0 ml mixture of n-butanol and pyridine (15:1, v/v). After centrifugation at 3000 rpm for 10 min, the organic phase was collected and the absorbance measured at 532 nm. The result was expressed as nanomoles of MDA formed per mg mitochondrial protein. 2.7. Protein carbonyl content Protein carbonyl content in isolated skeletal muscle mitochondria was determined by using 2,4 dinitrophenyl hydrazine (DNPH) [25]. Briefly, 300 l of mitochondrial fraction containing 2.0 mg protein was added into the tubes, to which 300 l of 10 mM DNPH in 2 N HCl was added. The blank tube contained only 2 N HCl. Samples were then incubated for 1 h at room temperature, stirred every 10 min, precipitated with 10% trichloroacetic acid and centrifuged for 3 min at 16,000 g. The pellet was washed thrice with 1 ml ethanol/ethyl acetate mixture (1:1, v/v) and redissolved in 1 ml of 6 M guanidine in 10 mM phosphate trifluroacetic acid (pH 2.3) and insoluble substances were removed by centrifugation. The difference in absorbance between the DNPH-treated and the HCltreated samples was determined at 366 nm and the results were expressed as nanomoles of carbonyl groups per mg mitochondrial protein. 2.8. Assay of enzymatic and non-enzymatic antioxidants Superoxide dismutase (SOD) activity was determined as the degree of inhibition of auto-oxidation of pyrogallol at an alkaline pH [26]. The activity of catalase (CAT) was determined spectrophotometrically at 240 nm as the amount of H2 O2 consumed/min/mg mitochondrial protein [27]. Glutathione peroxidase (GPx) was assayed by measuring the amount of GSH consumed in the reaction mixture [28]. GPx activity was expressed as as nmol of GSH oxidized/min/mg mitochondrial protein. GSH was estimated by the method of [29]. GSSG was measured by the method of Mize et al. [30] by monitoring oxidation of NADPH at 340 nm. ␣-tocopherol (Vit E) was estimated by the method of Desai et al. [31] and ascorbic acid by the method Omaye et al. [32]. 2.9. Assay of TCA cycle enzymes Isocitrate dehydrogenase (ICDH) was assayed according to the method of King et al. [33]. The activity of alpha-ketoglutarate dehydrogenase (␣-KGDH) was assayed by the method of Reed et al., [34] according to the colorimetric determination of ferrocyanide produced by the decarboxylation of ␣-ketoglutarate with ferricyanide as electron acceptor. The activity of succinate dehydrogenase (SDH) was assayed according to the method of Slater et al. [17], in which the rate of reduction of potassium ferricyanide was measured in the presence of potassium cyanide. The activity of malate dehydrogenase (MDH) was assayed by the method of Ochoa et al. [35]. Aconitase activity was assayed spectrophotometrically in Tris-HCl (pH 7.4) at 20 ◦ C by monitoring the formation of cis-aconitate from citrate (20 mM) at 240 nm, using a molar absorption co-efficient of 3600 M−1 [36]. One unit of activity corresponds to nmol of cisaconitate formed per minute.
Please cite this article in press as: Saraswathi R, Devaraj SN. Oxidative stress in skeletal muscle impairs mitochondrial function in alloxan induced diabetic rats: Role of alpha lipoic acid. Biomed Prev Nutr (2013), http://dx.doi.org/10.1016/j.bionut.2012.08.006
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Table 1 Body weight of experimental animals. Groups
Base line
Group I Group II Group III Group IV
293 297 301.5 292
± ± ± ±
10th day 18.1 10.3a 15.1b 14.1c
297 296.8 305 307.8
± ± ± ±
20th day 16.3 13.4a 10.6b 11.8c , *
304.3 291.3 312.3 315.8
± ± ± ±
30th day 13.1 15.1a 19.6b , * 14.1c , **
310.2 285 319.2 327.2
± ± ± ±
15.1 16.1a 13.6b , * 17c , **
Values represent mean ± SD (n = 6); below are the comparisons between groups. a Group I and II. b Group II and III. c Group I and IV. * P < 0.05 ** P < 0.01 Table 2 Serum glucose and plasma insulin levels of experimental animals. Groups
Serum glucose (mg/dl)
Group I Group II Group III Group IV
91.2 338 101 91.8
± ± ± ±
5.3 26.7a , * 7.6b , * 5.8c , ns
Plasma insulin(U/l) 14.98 9.2 14.3 15.7
± ± ± ±
2.3 1.1a , * 0.7b , * 1.3
Values represent mean ± SD (n = 6); below are the comparisons between groups. a Group I and II. b Group II and III. c Group I and IV; ns: non significant. * P < 0.001.
2.10. Data analysis Data for each variable are expressed as the average ± SD and significance of the differences between mean values were determined by one-way analysis of variance (ANOVA) followed by least significant difference (LSD) test for multiple comparisons. Probability (P) values of less than 0.05 were considered significant.
Fig. 1. Effect of lipoic acid on oxidant production in control and alloxan induced diabetic rat skeletal muscle mitochondria. Data expressed as mean ± SD (n = 6 in each group). Values are expressed as mean ± SD for six rats. *: difference compared with control rats (group I); $: difference compared with alloxan induced diabetic rats (group II) significant at P < 0.05.
3. Results The body weight of rats increased significantly (P < 0.05 and P < 0.01) in all groups except alloxan induced diabetes group II (Table 1). Serum glucose and plasma insulin levels in rats of different experimental groups are shown in Table 2. The glucose level was significantly (P < 0.001) high in group II compared with group I. On the other hand, the level of serum glucose was significantly (P < 0.001) decreased in group III compared with group II. There was no significant difference between group I and group IV. The insulin level was significantly (P < 0.001) low in group II compared with group I. On the other hand the level of insulin level was significantly (P < 0.001) increased in group III compared with group II. There was no significant difference between group I and group IV. The DCFH-DA assay in the present study has been applied to measure most, if not all, intracellular oxidants, including ROS and reactive nitrogen species. The oxidant generation by the mitochondria, using glutamate and malate as respiratory substrate, was found to be high in diabetic rats (Fig. 1). The extent of ROS generation was found to be 40% higher when compared with control rats. Oral supplementation of lipoic acid to diabetic rats significantly decreased mitochondrial oxidant generation (P < 0.05), possibly by enhancing redox status of the mitochondria. Since, mitochondria are especially the lipid membrane components, major intracellular source of oxidants; it is possible that mitochondrial macromolecules, are highly susceptible to oxidative damage. Fig. 2 represents the levels of LPO and protein carbonyls, markers of oxidative stress, in rat skeletal muscle mitochondria of control and experimental rats. The levels of both LPO and protein carbonyls were remarkably increased in diabetic rats when compared with
Fig. 2. Effect of lipoic acid on the rate of H2 O2 release in skeletal muscle mitochondria of control and experimental rats. Values are expressed as mean ± SD for six rats. *: difference compared with control rats (group I); $: difference compared with alloxan induced diabetic rats (group II) significant at P < 0.05.
control rats. 2.0 and 1.6-fold increase were observed for LPO and protein carbonyls, respectively. Oral administration of lipoic acid to diabetic rats significantly reduced both LPO and protein carbonyls towards control rats (P < 0.05), indicating the protective effect of lipoic acid on cellular macromolecules. Oxidative damage to cellular macromolecules occurs when the existing balance between oxidants and antioxidants gets out of control in favor of oxidants. Table 3 depicts the antioxidant status in the skeletal muscle mitochondria of control and experimental rats. The activities of enzymatic antioxidants such as SOD, CAT and GPx were found to be decreased by 21%, 36% and 38%, respectively, in diabetic rats when compared with control rats. In contrast, diabetic rats administered with lipoic acid exhibited significantly increased antioxidant enzyme activities than diabetic rats (P < 0.05).
Please cite this article in press as: Saraswathi R, Devaraj SN. Oxidative stress in skeletal muscle impairs mitochondrial function in alloxan induced diabetic rats: Role of alpha lipoic acid. Biomed Prev Nutr (2013), http://dx.doi.org/10.1016/j.bionut.2012.08.006
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Table 3 Effect of lipoic acid on antioxidant enzyme activities in control and alloxan induced diabetic rat skeletal muscle mitochondria. Data expressed as mean ± SD (n = 6 in each group). Parameters
Control
Diabetic rats
Control + LA
Diabetic rats + LA
SOD (units/min/mg protein)
6.7 + 0.77
5.25 + 0.6a
6.65 + 0.71
6.5 + 0.68b
Catalase (mol of H2 O2 consumed/min/mg protein)
37.24 + 4.26
27.40 + 3.13a
37.80 + 4.1
36.59 + 3.9b
GPx (nmoles of GSH consumed/min/mg protein)
1.8 ± 0.17
1.1 ± 0.10
1.99 ± 0.15
1.9 ± 0.17b
GR (nmol of NADPH oxidized/min/mg protein)
25.31 ± 2.24
12.4 ± 1.19a
26.4 ± 1.48
22.46 ± 2.3b
a
Values are expressed as mean ± SD for six rats. LA: lipoic acid. a Difference compared with young control rats (group I). b Difference compared with alloxan induced diabetic rats (group II) significant at P < 0.05.
Table 4 Effect of lipoic acid on non-enzymatic antioxidants in control and alloxan induced diabetic rat skeletal muscle mitochondria. Data expressed as mean ± SD (n = 6 in each group). Parameters
Control
Diabetic rats
Control + LA
Diabetic rats + LA
Vitamin C (g/mg protein) Vitamin E (g/mg protein)
12.2 + 1.34 2.3 + 0.26
7.9 + 0.87a 1.6 + 0.18a
12.20 + 1.28 2.45 + 0.23
12.10 + 1.46b 2.2 + 0.27b
Values are expressed as mean ± SD for six rats. LA: lipoic acid. a Difference compared with control rats (group I). b Difference compared with alloxan induced diabetic rats (group II) significant at P < 0.05.
Fig. 3. Effect of lipoic acid on lipid peroxidation and protein carbonyl content in control and alloxan induced diabetic rat skeletal muscle mitochondria. Data expressed as mean ± SD (n = 6 in each group). LPO and PCO are expressed as nmoles of MDA released/min/mg protein and nmoles/mg protein; DNA damage is expressed as % DNA damage. Values are expressed as mean ± SD for six rats. *: difference compared with control rats (group I); $: difference compared with alloxan induced diabetic rats (group II) significant at P < 0.05.
Here, the levels of GSH, a co-substrate for GPx reaction, were found to be 1.8-fold decreased in alloxan induced diabetic rats when compared with control rats (Fig. 3). Moreover, reduction in GSH levels was accompanied with an increase in GSSG levels (Fig. 3; P < 0.05) and decrease in the levels of vitamin C and vitamin E (Table 4; P < 0.05) in skeletal muscle mitochondria of diabetic rats when compared to control rats. The decrease (P < 0.05) in GSH levels may be attributed to the decrease in GSH/GSSG redox value (Fig. 4) observed in diabetic rats. Oral administration of lipoic acid significantly improves the GSH/GSSG redox value, vitamin C and vitamin E levels (P < 0.05), possibly by regeneration (GSSG to GSH) and/or denovo synthesis of GSH. Fig. 5 depicts the status of TCA cycle enzymes in skeletal muscle mitochondria of control and experimental rats. Here, alloxan induced diabetic rats showed a significant decrease in the activities of TCA cycle enzymes. Thirty-two per cent, 33%, 33% and 35% decrease were observed for SDH, MDH, ICDH and ␣-KGDH, respectively. Oral administration of lipoic acid to diabetic rats improved the activities of TCA cycle enzymes to near normalcy (Figs. 6–9).
Fig. 4. Effect of lipoic acid on redox status in control and alloxan induced diabetic rat skeletal muscle mitochondria. Data expressed as mean ± SD (n = 6 in each group). GSH and GSSG are expressed as g/mg protein. Values are expressed as mean ± SD for six rats. *: difference compared with control rats (group I); $: difference compared with alloxan induced diabetic rats (group II) significant at P < 0.05.
Fig. 5. Redox ratio in skeletal muscle mitochondria of control and experimental animals. Values are expressed as mean ± SD for six rats. *: difference compared with control rats (group I); $: difference compared with alloxan induced diabetic rats (group II) significant at P < 0.05.
Please cite this article in press as: Saraswathi R, Devaraj SN. Oxidative stress in skeletal muscle impairs mitochondrial function in alloxan induced diabetic rats: Role of alpha lipoic acid. Biomed Prev Nutr (2013), http://dx.doi.org/10.1016/j.bionut.2012.08.006
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Fig. 6. Effect of lipoic acid on the TCA cycle enzymes of skeletal muscle mitochondria in control and experimental rats. Values are expressed as mean ± SD for six rats. *: difference compared with control rats (group I); $: difference compared with alloxan induced diabetic rats (group II) significant at P < 0.05.
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Fig. 7. Effect of lipoic acid on aconitase activity in skeletal muscle mitochondria in control and experimental rats. Values are expressed as mean ± SD for six rats. *: difference compared with control rats (group I); $: difference compared with alloxan induced diabetic rats (group II) significant at P < 0.05.
Fig. 8. Electron microscopic view of mitochondria in control and experimental animals before and after supplementation of lipoic acid for a period of 30 days (× 100,000).
Please cite this article in press as: Saraswathi R, Devaraj SN. Oxidative stress in skeletal muscle impairs mitochondrial function in alloxan induced diabetic rats: Role of alpha lipoic acid. Biomed Prev Nutr (2013), http://dx.doi.org/10.1016/j.bionut.2012.08.006
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Fig. 9. Effect of lipoic acid on mitochondrial membrane potential in control and alloxan induced diabetic rat skeletal muscle mitochondria. Data expressed as mean ± SD (n = 6 in each group). Values are expressed as mean ± SD for six rats. *: difference compared with control rats (group I); $: difference compared with alloxan induced diabetic rats (group II) significant at P < 0.05.
4. Discussion Diabetes mellitus is one of the pathological conditions associated with oxidative stress [37]. In the present study, oxidant generation was found to be higher in alloxan induced diabetic rat muscle mitochondria. The primary source of oxidative damage brought about by oxidative stress is LPO, which is attributed to its high propagative nature. LPO is significantly increased in diabetic rats. Furthermore, protein carbonyls, a marker of oxidative damage was also found to be higher in diabetic rats. MDA produced during LPO can be incorporated into proteins by reacting with either Eamino moiety of lysine or the sulfhydryl group of cysteine residue to form carbonyl derivatives [38]. Administration of lipoic acid significantly decreased mitochondrial oxidant generation, LPO and protein carbonyls possibly by enhancing cellular redox status. As an antioxidant, lipoic acid and its reduced form dihydro lipoic acid (DHLA) are capable of quenching ROS and reactive nitrogen species such as hydroxyl radical, peroxyl radical, superoxide, hypochlorous acid and peroxynitrite [39]. Furthermore, lipoic acid has the ability to chelate transition metal ions such as Cd2+ , Fe3+ , Cu2+ and Zn2+ and thereby subsequently inhibits the Fenton’s reaction [40]. Therefore, the antioxidant property of lipoic acid may be attributed to the decrease in oxidative stress observed in experimentally induced diabetic rats. Antioxidant enzymes such as SOD, CAT and GPx comprise the major enzymatic antioxidants in neutralizing the ill effects of free radicals produced during diabetes. SOD a family of enzyme that catalyzes dismutation reaction, protects against ROS by catalyzing the removal of superoxide, which damages membrane and biological structures. A decrease in the activities of SOD during diabetes can be attributed to a decrease in the ability of the mitochondrial protective mechanism against disorganizing effects of free radicals. Similar decrease in enzymatic antioxidants has also been reported earlier in alloxan induced diabetic rats [41]. The increased SOD activity seen on treatment could be because of the scavenging effect afforded by lipoic acid against hydroxyl radical, peroxyl radical, superoxide, hypochlorous acid and peroxynitrite [41]. Both CAT and GPx catalyze the transformation of H2 O2 within the cell to harmless byproducts, thereby curtailing the quantity of cellular destruction inflicted by LPO products [42]. The decreased activity of both CAT and GPX is indicative of increased peroxidation of membrane lipids and increased oxidative stress observed in diabetes. CAT is a hemeprotein that requires NADPH for its regeneration to active form. Lipoic acid is able to increase glucose uptake [15] by the cells which serves as a fuel for both the pentose phosphate pathways and oxidative phosphorylation thus bringing up
cellular levels of NADPH thereby significantly enhancing CAT activity in diabetes. GPx, the seleno-cystenine containing antioxidant enzyme is the main scavenger of H2 O2 . GPx is required to repair LPO initiated by superoxide in the membrane to maintain its integrity. The observed increase in the GPx activity may be due to its GSH sparing action. DHLA may serve as an electron donor to GPx, the activity of which is expected to be upregulated in lipoate-treated diabetic rats. GSH has been shown to be protective against auto-oxidation of dialuric acid and generation of alloxan radicals, and also interferes in the redox cycling between alloxan and dialuric acid [43]. Alloxan induced diabetic rats showed a significant decrease in the levels of GSH with a simultaneous increase in GSSG levels. The observed increase in GSH/GSSG status on treatment with lipoic acid could be ascribed to the ability of lipoic acid to increase cysteine availability [44], the rate-limiting factor in its biosynthesis. Lipoic acid could either mitigate GSH consumption by acting as an alternate scavenger of free radicals or by increasing the levels of GSH by stimulating its biosynthesis and thereby correct deficient thiol status of cells [44]. In addition, the levels of both vitamin C and vitamin E were decreased in diabetic rats. Administration of lipoic acid to diabetic rats improved the levels of both the vitamins. The increase in the levels of vitamin C on lipoic acid treatment could be attributed to the ascorbic acid sparing activity of lipoic acid [45]. Lipoic acid has the ability to increase ascorbic acid recycling by providing electrons directly to dehydroascorbic acid [46]. Both lipoic acid and DHLA act as antioxidants in recycling dehydroascorbic acid and tocopheryl radical to ascorbic acid and tocopherol, respectively [47]. The enzymes of the TCA cycle were seen to be decreased significantly in alloxan induced diabetic rats. This is in agreement with earlier reports that show a similar decrease in the mitochondrial enzymes during oxidative stress [15]. Treatment with lipoic acid has shown to improve the activities of these enzymes in diabetic rats to near normalcy. Lipoic acid improved the activities of pyruvate dehydrogenase and ␣-ketoglutarate dehydrogenase by improving the co-factor availability. Lipoic acid has also been shown to increase glucose uptake in cells [15]. This glucose, through the process of glycolysis, could also increase pyruvate availability for TCA cycle. Lipoic acid has been shown to possess potent antioxidant properties against superoxide, peroxyl, and hydroxyl radicals [40]. Therefore, preventing the free radical-induced enzyme inactivation could be another possible mechanism for the increase in the activities of the TCA cycle enzymes upon treatment. In conclusion, our observations suggest that, the diabetes induced oxidative stress in mitochondria results in the decline of both antioxidant and TCA cycle enzymes. Therefore, maintenance of mitochondrial viability would depend, in part, on the ability mitochondria to sense these changes in redox status and respond in an appropriate manner. Lipoic acid supplementation markedly lowers oxidant production and their associated increase in oxidative damage in diabetes by enhancing antioxidant status. Thus, lipoic acid may ameliorate complications associated with diabetes mellitus by improving mitochondrial redox as well as energy metabolism.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
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Original article
Oxidative stress in skeletal muscle impairs mitochondrial function in alloxan induced diabetic rats: Role of alpha lipoic acid Ramasamy Saraswathi , Sivasithamparam Niranjali Devaraj ∗ Department of Biochemistry, University of Madras, Guindy campus, Sardar Patel Road, Chennai-600 025, India
a r t i c l e
i n f o
Article history: Received 10 May 2012 Accepted 12 August 2012 Keywords: Diabetes Skeletal muscle Mitochondria Oxidative stress Alpha lipoic acid
a b s t r a c t The role increased free radical production associated with mitochondrial dysfunction is well established in the pathophysiology of diabetes mellitus. A male Wistar rat model was utilized in which diabetes was induced by alloxan at a dose of 140 mg/kg body weight, intraperitoneally for 8 weeks. In alloxan induced diabetes on the activities of Krebs cycle enzymes, oxidative as well as enzymes involved in oxidative metabolism were altered. Oxidative damage to DNA, protein and lipid were observed, which provide insight into the association of elevated free radicals and mitochondrial dysfunction leading to diabetes mellitus. Lipoic acid treatment (100 mg/kg body weight for 30 days orally) restored mitochondrial functioning showing normal activity of TCA cycle enzyme with decreased free radical production and oxidative damage. Thus, our results propose that lipoic acid restores mitochondrial function in experimentally induced diabetic rats through decreasing levels of free radicals and promoting activation of Krebs cycle enzymes. © 2013 Published by Elsevier Masson SAS.
1. Introduction Diabetes mellitus is a chronic metabolic disorder resulting from insulin dyshomeostasis, and it is considered as the major cause of morbidity and mortality affecting ∼5% of the population [1]. Much of the evidence concerning the role of oxidative stress in the induction of diabetes comes from studies using the diabetogenic drug, alloxan [2]. It has the ability to produce free radicals that damages -cells of pancreas [3]. Alloxan induced hyperglycemia aggravates the oxidative stress by auto-oxidation of glucose and their derivatives [4]. Enhanced oxidative stress induced by hyperglycemia may constitute the key and common events in the pathogenesis of different diabetic complications [5]. Mitochondria are central to energy production and are therefore fully integrated into the rest of the cell’s physiological responses to stress. Besides ATP production, mitochondria are also the major endogenous source of superoxide, peroxynitrite, and hydroxyl radicals [6]. Mitochondrial superoxide production is considered as a single unifying mechanism for diabetic complications [7,8]. The resultant mitochondrial decay may ultimately cause mitochondrial dysfunction and/or loss of cellular homeostasis. Lipoic acid is a dithiol compound that exists naturally in mitochondria as a coenzyme for pyruvate dehydrogenase and ␣-ketoglutarate dehydrogenase. It has been used as a therapy
∗ Corresponding author. Tel.: +91 44 22351269; fax: +91 44 22352870. E-mail address: [email protected] (S.N. Devaraj).
for many ailments associated with impaired energy metabolism, including type II diabetes [9] and diabetic polyneuropathies [10]. Dietary supplementation increased the unbound lipoic acid, which can act as a potent antioxidant and ameliorate oxidative stress both in vitro and in vivo [11,12]. Lipoic acid has an ability to improve energy metabolism [13] and lower oxidative stress [14,15] in many pathological conditions and disease states. The present study demonstrates the efficacy of lipoic acid in protection of mitochondrial dysfunction in skeletal muscle of alloxan induced diabetic rats. 2. Materials and methods 2.1. Chemicals dl-alpha-lipoic acid, dichlorofluorescinhydrate-diacetate (DCFH-DA), fattyacid–free bovine, serum albumin (BSA), reduced glutathione (GSH), oxidized glutathione (GSSG), 5,5 -dithiobis2nitrobenzoic acid (DTNB) and alloxan were purchased from Sigma (St. Louis, MO). All other chemicals used were of analytical grade and were obtained from SISCO Research Laboratories Pvt Ltd (Mumbai, India). 2.2. Animals Experiments were conducted in accordance with guidelines approved by the institutional animal ethical committee. Male albino rats weighing between 160 to 180 g were used throughout
2210-5239/$ – see front matter © 2013 Published by Elsevier Masson SAS. http://dx.doi.org/10.1016/j.bionut.2012.08.006
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the study. The rats were barrier housed two per cage at a temperature of 24 ± 2 ◦ C in a light controlled environment of 12 h light-dark cycle, and provided with free access to food and water. Diabetes was induced by a single subcutaneous injection of 200 mg alloxan/kg body weight in 0.1 M citrate buffer, pH 4.5 at day 0 of the experiment. Body weight of the animals was recorded every 10 days. Rats were identified as diabetic on the basis of fasting blood glucose levels (higher than 200 mg/dL at least 4 days after alloxan treatment), and divided into subgroups as required by the experiment. Group I control rats, group II diabetic rats, group III control rats administered with lipoic acid and group IV diabetic rats administered with lipoic acid. dl-␣-lipoic acid (100 mg/kg body weight/day) dissolved in physiological saline containing 0.5% KOH was administered orally using intragastric canula up to 30 days. Control animals received physiological saline containing 0.5% KOH. On completion of the experimental period, rats were killed by cervical decapitation. Gastrocnemius muscle was excised, weighed, and immediately used for experimental procedure. 2.3. Estimation of serum glucose and plasma insulin Serum glucose was determined in an auto analyzer using Ecoline Kits (E. Merck, Mumbai, India) and plasma insulin was estimated by ELISA method using Boehringer Mannheim Gmbh kit, Werk Penzberg, Germany. 2.4. Isolation of skeletal muscle mitochondria Skeletal muscle mitochondria were isolated by differential centrifugation [16]. Skeletal muscle homogenization was performed in mitochondrial isolation buffer containing 25 M mannitol, 75 mM sucrose, 1 mM EGTA, 0.1% fattyacid–free BSA and 10 mM Tris-HCl (pH 7.4). The homogenate was centrifuged at 1000 g for 10 min in 4 ◦ C and the supernatant was again centrifuged at 12,000 g, and resuspended in isolation buffer without EGTA. The purity of the obtained fraction was evaluated by enzymatic assay of succinate dehydrodenase for monitoring mitochontria, acid phospotase for lysosomes, glucose-6-phosphatase for endoplasmic reticulum and catalase for peroxisomes [17–20]. Protein content was quantified by the method of Lowry et al. [21]. An aliquot of freshly prepared mitochondria was frozen (–80 ◦ C) for enzyme assays and the rest was used immediately for oxidant generation studies. 2.5. Determination of oxidant generation Oxidant generation was evaluated in isolated skeletal muscle mitochondria by using a redox sensitive fluorescent probe, DCFH-DA [22,23]. Isolated mitochondria were incubated in the assay media (137 mM KCl, 2.5 mM MgCl2 , 2 mM K2HPO4, 10 mM Tris-HCl, 5 mM glutamate and 5 mM malate with pH 7.4) containing 5 M DCFH-DA (dissolved in 1.25 mM methanol) at 37 ◦ C for 10 min. The mitochondrial pellets were resuspended in the assay media without DCFH-DA, and 500 l of the suspension (∼0.4 mg protein) was used for the assay. DCF formation was followed at the excitation wavelength of 488 nm and emission wavelength of 525 nm for 25 min by using Shimadzu RF-1501 fluorescence spectrophotometer. The rate of DCFH conversion to DCF was linear for at least 25 min, which was corrected for auto-oxidation rate of DCFH, without protein. To verify that DCFH concentration was saturating allowing maximal rate of oxidation for at least 25 min, 1 mol of H2 O2 was added into the mixture at the end of the assay. The DCFH oxidation rate was found to increase several fold immediately, indicating that sufficient DCFH was still available.
2.6. Lipid peroxidation (LPO) LPO was determined by measuring the levels of thiobarbituric acid reactive substances (TBARS) in rat skeletal muscle mitochondria using malondialdehyde (MDA) as the standard [24]. Briefly, 500 l of mitochondria (∼4 mg protein ml−1 ) was added to the test tubes containing 0.2 ml of 8.1% SDS, 1.5 ml of 20% acetic acid, pH 3.5, and 1.5 ml of 0.8% thiobarbituric acid (TBA) solution. The mixture was diluted to 4.0 ml with distilled water and heated at 95 ◦ C for 1 h. After cooling on ice, the samples were extracted with 4.0 ml mixture of n-butanol and pyridine (15:1, v/v). After centrifugation at 3000 rpm for 10 min, the organic phase was collected and the absorbance measured at 532 nm. The result was expressed as nanomoles of MDA formed per mg mitochondrial protein. 2.7. Protein carbonyl content Protein carbonyl content in isolated skeletal muscle mitochondria was determined by using 2,4 dinitrophenyl hydrazine (DNPH) [25]. Briefly, 300 l of mitochondrial fraction containing 2.0 mg protein was added into the tubes, to which 300 l of 10 mM DNPH in 2 N HCl was added. The blank tube contained only 2 N HCl. Samples were then incubated for 1 h at room temperature, stirred every 10 min, precipitated with 10% trichloroacetic acid and centrifuged for 3 min at 16,000 g. The pellet was washed thrice with 1 ml ethanol/ethyl acetate mixture (1:1, v/v) and redissolved in 1 ml of 6 M guanidine in 10 mM phosphate trifluroacetic acid (pH 2.3) and insoluble substances were removed by centrifugation. The difference in absorbance between the DNPH-treated and the HCltreated samples was determined at 366 nm and the results were expressed as nanomoles of carbonyl groups per mg mitochondrial protein. 2.8. Assay of enzymatic and non-enzymatic antioxidants Superoxide dismutase (SOD) activity was determined as the degree of inhibition of auto-oxidation of pyrogallol at an alkaline pH [26]. The activity of catalase (CAT) was determined spectrophotometrically at 240 nm as the amount of H2 O2 consumed/min/mg mitochondrial protein [27]. Glutathione peroxidase (GPx) was assayed by measuring the amount of GSH consumed in the reaction mixture [28]. GPx activity was expressed as as nmol of GSH oxidized/min/mg mitochondrial protein. GSH was estimated by the method of [29]. GSSG was measured by the method of Mize et al. [30] by monitoring oxidation of NADPH at 340 nm. ␣-tocopherol (Vit E) was estimated by the method of Desai et al. [31] and ascorbic acid by the method Omaye et al. [32]. 2.9. Assay of TCA cycle enzymes Isocitrate dehydrogenase (ICDH) was assayed according to the method of King et al. [33]. The activity of alpha-ketoglutarate dehydrogenase (␣-KGDH) was assayed by the method of Reed et al., [34] according to the colorimetric determination of ferrocyanide produced by the decarboxylation of ␣-ketoglutarate with ferricyanide as electron acceptor. The activity of succinate dehydrogenase (SDH) was assayed according to the method of Slater et al. [17], in which the rate of reduction of potassium ferricyanide was measured in the presence of potassium cyanide. The activity of malate dehydrogenase (MDH) was assayed by the method of Ochoa et al. [35]. Aconitase activity was assayed spectrophotometrically in Tris-HCl (pH 7.4) at 20 ◦ C by monitoring the formation of cis-aconitate from citrate (20 mM) at 240 nm, using a molar absorption co-efficient of 3600 M−1 [36]. One unit of activity corresponds to nmol of cisaconitate formed per minute.
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Table 1 Body weight of experimental animals. Groups
Base line
Group I Group II Group III Group IV
293 297 301.5 292
± ± ± ±
10th day 18.1 10.3a 15.1b 14.1c
297 296.8 305 307.8
± ± ± ±
20th day 16.3 13.4a 10.6b 11.8c , *
304.3 291.3 312.3 315.8
± ± ± ±
30th day 13.1 15.1a 19.6b , * 14.1c , **
310.2 285 319.2 327.2
± ± ± ±
15.1 16.1a 13.6b , * 17c , **
Values represent mean ± SD (n = 6); below are the comparisons between groups. a Group I and II. b Group II and III. c Group I and IV. * P < 0.05 ** P < 0.01 Table 2 Serum glucose and plasma insulin levels of experimental animals. Groups
Serum glucose (mg/dl)
Group I Group II Group III Group IV
91.2 338 101 91.8
± ± ± ±
5.3 26.7a , * 7.6b , * 5.8c , ns
Plasma insulin(U/l) 14.98 9.2 14.3 15.7
± ± ± ±
2.3 1.1a , * 0.7b , * 1.3
Values represent mean ± SD (n = 6); below are the comparisons between groups. a Group I and II. b Group II and III. c Group I and IV; ns: non significant. * P < 0.001.
2.10. Data analysis Data for each variable are expressed as the average ± SD and significance of the differences between mean values were determined by one-way analysis of variance (ANOVA) followed by least significant difference (LSD) test for multiple comparisons. Probability (P) values of less than 0.05 were considered significant.
Fig. 1. Effect of lipoic acid on oxidant production in control and alloxan induced diabetic rat skeletal muscle mitochondria. Data expressed as mean ± SD (n = 6 in each group). Values are expressed as mean ± SD for six rats. *: difference compared with control rats (group I); $: difference compared with alloxan induced diabetic rats (group II) significant at P < 0.05.
3. Results The body weight of rats increased significantly (P < 0.05 and P < 0.01) in all groups except alloxan induced diabetes group II (Table 1). Serum glucose and plasma insulin levels in rats of different experimental groups are shown in Table 2. The glucose level was significantly (P < 0.001) high in group II compared with group I. On the other hand, the level of serum glucose was significantly (P < 0.001) decreased in group III compared with group II. There was no significant difference between group I and group IV. The insulin level was significantly (P < 0.001) low in group II compared with group I. On the other hand the level of insulin level was significantly (P < 0.001) increased in group III compared with group II. There was no significant difference between group I and group IV. The DCFH-DA assay in the present study has been applied to measure most, if not all, intracellular oxidants, including ROS and reactive nitrogen species. The oxidant generation by the mitochondria, using glutamate and malate as respiratory substrate, was found to be high in diabetic rats (Fig. 1). The extent of ROS generation was found to be 40% higher when compared with control rats. Oral supplementation of lipoic acid to diabetic rats significantly decreased mitochondrial oxidant generation (P < 0.05), possibly by enhancing redox status of the mitochondria. Since, mitochondria are especially the lipid membrane components, major intracellular source of oxidants; it is possible that mitochondrial macromolecules, are highly susceptible to oxidative damage. Fig. 2 represents the levels of LPO and protein carbonyls, markers of oxidative stress, in rat skeletal muscle mitochondria of control and experimental rats. The levels of both LPO and protein carbonyls were remarkably increased in diabetic rats when compared with
Fig. 2. Effect of lipoic acid on the rate of H2 O2 release in skeletal muscle mitochondria of control and experimental rats. Values are expressed as mean ± SD for six rats. *: difference compared with control rats (group I); $: difference compared with alloxan induced diabetic rats (group II) significant at P < 0.05.
control rats. 2.0 and 1.6-fold increase were observed for LPO and protein carbonyls, respectively. Oral administration of lipoic acid to diabetic rats significantly reduced both LPO and protein carbonyls towards control rats (P < 0.05), indicating the protective effect of lipoic acid on cellular macromolecules. Oxidative damage to cellular macromolecules occurs when the existing balance between oxidants and antioxidants gets out of control in favor of oxidants. Table 3 depicts the antioxidant status in the skeletal muscle mitochondria of control and experimental rats. The activities of enzymatic antioxidants such as SOD, CAT and GPx were found to be decreased by 21%, 36% and 38%, respectively, in diabetic rats when compared with control rats. In contrast, diabetic rats administered with lipoic acid exhibited significantly increased antioxidant enzyme activities than diabetic rats (P < 0.05).
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Table 3 Effect of lipoic acid on antioxidant enzyme activities in control and alloxan induced diabetic rat skeletal muscle mitochondria. Data expressed as mean ± SD (n = 6 in each group). Parameters
Control
Diabetic rats
Control + LA
Diabetic rats + LA
SOD (units/min/mg protein)
6.7 + 0.77
5.25 + 0.6a
6.65 + 0.71
6.5 + 0.68b
Catalase (mol of H2 O2 consumed/min/mg protein)
37.24 + 4.26
27.40 + 3.13a
37.80 + 4.1
36.59 + 3.9b
GPx (nmoles of GSH consumed/min/mg protein)
1.8 ± 0.17
1.1 ± 0.10
1.99 ± 0.15
1.9 ± 0.17b
GR (nmol of NADPH oxidized/min/mg protein)
25.31 ± 2.24
12.4 ± 1.19a
26.4 ± 1.48
22.46 ± 2.3b
a
Values are expressed as mean ± SD for six rats. LA: lipoic acid. a Difference compared with young control rats (group I). b Difference compared with alloxan induced diabetic rats (group II) significant at P < 0.05.
Table 4 Effect of lipoic acid on non-enzymatic antioxidants in control and alloxan induced diabetic rat skeletal muscle mitochondria. Data expressed as mean ± SD (n = 6 in each group). Parameters
Control
Diabetic rats
Control + LA
Diabetic rats + LA
Vitamin C (g/mg protein) Vitamin E (g/mg protein)
12.2 + 1.34 2.3 + 0.26
7.9 + 0.87a 1.6 + 0.18a
12.20 + 1.28 2.45 + 0.23
12.10 + 1.46b 2.2 + 0.27b
Values are expressed as mean ± SD for six rats. LA: lipoic acid. a Difference compared with control rats (group I). b Difference compared with alloxan induced diabetic rats (group II) significant at P < 0.05.
Fig. 3. Effect of lipoic acid on lipid peroxidation and protein carbonyl content in control and alloxan induced diabetic rat skeletal muscle mitochondria. Data expressed as mean ± SD (n = 6 in each group). LPO and PCO are expressed as nmoles of MDA released/min/mg protein and nmoles/mg protein; DNA damage is expressed as % DNA damage. Values are expressed as mean ± SD for six rats. *: difference compared with control rats (group I); $: difference compared with alloxan induced diabetic rats (group II) significant at P < 0.05.
Here, the levels of GSH, a co-substrate for GPx reaction, were found to be 1.8-fold decreased in alloxan induced diabetic rats when compared with control rats (Fig. 3). Moreover, reduction in GSH levels was accompanied with an increase in GSSG levels (Fig. 3; P < 0.05) and decrease in the levels of vitamin C and vitamin E (Table 4; P < 0.05) in skeletal muscle mitochondria of diabetic rats when compared to control rats. The decrease (P < 0.05) in GSH levels may be attributed to the decrease in GSH/GSSG redox value (Fig. 4) observed in diabetic rats. Oral administration of lipoic acid significantly improves the GSH/GSSG redox value, vitamin C and vitamin E levels (P < 0.05), possibly by regeneration (GSSG to GSH) and/or denovo synthesis of GSH. Fig. 5 depicts the status of TCA cycle enzymes in skeletal muscle mitochondria of control and experimental rats. Here, alloxan induced diabetic rats showed a significant decrease in the activities of TCA cycle enzymes. Thirty-two per cent, 33%, 33% and 35% decrease were observed for SDH, MDH, ICDH and ␣-KGDH, respectively. Oral administration of lipoic acid to diabetic rats improved the activities of TCA cycle enzymes to near normalcy (Figs. 6–9).
Fig. 4. Effect of lipoic acid on redox status in control and alloxan induced diabetic rat skeletal muscle mitochondria. Data expressed as mean ± SD (n = 6 in each group). GSH and GSSG are expressed as g/mg protein. Values are expressed as mean ± SD for six rats. *: difference compared with control rats (group I); $: difference compared with alloxan induced diabetic rats (group II) significant at P < 0.05.
Fig. 5. Redox ratio in skeletal muscle mitochondria of control and experimental animals. Values are expressed as mean ± SD for six rats. *: difference compared with control rats (group I); $: difference compared with alloxan induced diabetic rats (group II) significant at P < 0.05.
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Fig. 6. Effect of lipoic acid on the TCA cycle enzymes of skeletal muscle mitochondria in control and experimental rats. Values are expressed as mean ± SD for six rats. *: difference compared with control rats (group I); $: difference compared with alloxan induced diabetic rats (group II) significant at P < 0.05.
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Fig. 7. Effect of lipoic acid on aconitase activity in skeletal muscle mitochondria in control and experimental rats. Values are expressed as mean ± SD for six rats. *: difference compared with control rats (group I); $: difference compared with alloxan induced diabetic rats (group II) significant at P < 0.05.
Fig. 8. Electron microscopic view of mitochondria in control and experimental animals before and after supplementation of lipoic acid for a period of 30 days (× 100,000).
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Fig. 9. Effect of lipoic acid on mitochondrial membrane potential in control and alloxan induced diabetic rat skeletal muscle mitochondria. Data expressed as mean ± SD (n = 6 in each group). Values are expressed as mean ± SD for six rats. *: difference compared with control rats (group I); $: difference compared with alloxan induced diabetic rats (group II) significant at P < 0.05.
4. Discussion Diabetes mellitus is one of the pathological conditions associated with oxidative stress [37]. In the present study, oxidant generation was found to be higher in alloxan induced diabetic rat muscle mitochondria. The primary source of oxidative damage brought about by oxidative stress is LPO, which is attributed to its high propagative nature. LPO is significantly increased in diabetic rats. Furthermore, protein carbonyls, a marker of oxidative damage was also found to be higher in diabetic rats. MDA produced during LPO can be incorporated into proteins by reacting with either Eamino moiety of lysine or the sulfhydryl group of cysteine residue to form carbonyl derivatives [38]. Administration of lipoic acid significantly decreased mitochondrial oxidant generation, LPO and protein carbonyls possibly by enhancing cellular redox status. As an antioxidant, lipoic acid and its reduced form dihydro lipoic acid (DHLA) are capable of quenching ROS and reactive nitrogen species such as hydroxyl radical, peroxyl radical, superoxide, hypochlorous acid and peroxynitrite [39]. Furthermore, lipoic acid has the ability to chelate transition metal ions such as Cd2+ , Fe3+ , Cu2+ and Zn2+ and thereby subsequently inhibits the Fenton’s reaction [40]. Therefore, the antioxidant property of lipoic acid may be attributed to the decrease in oxidative stress observed in experimentally induced diabetic rats. Antioxidant enzymes such as SOD, CAT and GPx comprise the major enzymatic antioxidants in neutralizing the ill effects of free radicals produced during diabetes. SOD a family of enzyme that catalyzes dismutation reaction, protects against ROS by catalyzing the removal of superoxide, which damages membrane and biological structures. A decrease in the activities of SOD during diabetes can be attributed to a decrease in the ability of the mitochondrial protective mechanism against disorganizing effects of free radicals. Similar decrease in enzymatic antioxidants has also been reported earlier in alloxan induced diabetic rats [41]. The increased SOD activity seen on treatment could be because of the scavenging effect afforded by lipoic acid against hydroxyl radical, peroxyl radical, superoxide, hypochlorous acid and peroxynitrite [41]. Both CAT and GPx catalyze the transformation of H2 O2 within the cell to harmless byproducts, thereby curtailing the quantity of cellular destruction inflicted by LPO products [42]. The decreased activity of both CAT and GPX is indicative of increased peroxidation of membrane lipids and increased oxidative stress observed in diabetes. CAT is a hemeprotein that requires NADPH for its regeneration to active form. Lipoic acid is able to increase glucose uptake [15] by the cells which serves as a fuel for both the pentose phosphate pathways and oxidative phosphorylation thus bringing up
cellular levels of NADPH thereby significantly enhancing CAT activity in diabetes. GPx, the seleno-cystenine containing antioxidant enzyme is the main scavenger of H2 O2 . GPx is required to repair LPO initiated by superoxide in the membrane to maintain its integrity. The observed increase in the GPx activity may be due to its GSH sparing action. DHLA may serve as an electron donor to GPx, the activity of which is expected to be upregulated in lipoate-treated diabetic rats. GSH has been shown to be protective against auto-oxidation of dialuric acid and generation of alloxan radicals, and also interferes in the redox cycling between alloxan and dialuric acid [43]. Alloxan induced diabetic rats showed a significant decrease in the levels of GSH with a simultaneous increase in GSSG levels. The observed increase in GSH/GSSG status on treatment with lipoic acid could be ascribed to the ability of lipoic acid to increase cysteine availability [44], the rate-limiting factor in its biosynthesis. Lipoic acid could either mitigate GSH consumption by acting as an alternate scavenger of free radicals or by increasing the levels of GSH by stimulating its biosynthesis and thereby correct deficient thiol status of cells [44]. In addition, the levels of both vitamin C and vitamin E were decreased in diabetic rats. Administration of lipoic acid to diabetic rats improved the levels of both the vitamins. The increase in the levels of vitamin C on lipoic acid treatment could be attributed to the ascorbic acid sparing activity of lipoic acid [45]. Lipoic acid has the ability to increase ascorbic acid recycling by providing electrons directly to dehydroascorbic acid [46]. Both lipoic acid and DHLA act as antioxidants in recycling dehydroascorbic acid and tocopheryl radical to ascorbic acid and tocopherol, respectively [47]. The enzymes of the TCA cycle were seen to be decreased significantly in alloxan induced diabetic rats. This is in agreement with earlier reports that show a similar decrease in the mitochondrial enzymes during oxidative stress [15]. Treatment with lipoic acid has shown to improve the activities of these enzymes in diabetic rats to near normalcy. Lipoic acid improved the activities of pyruvate dehydrogenase and ␣-ketoglutarate dehydrogenase by improving the co-factor availability. Lipoic acid has also been shown to increase glucose uptake in cells [15]. This glucose, through the process of glycolysis, could also increase pyruvate availability for TCA cycle. Lipoic acid has been shown to possess potent antioxidant properties against superoxide, peroxyl, and hydroxyl radicals [40]. Therefore, preventing the free radical-induced enzyme inactivation could be another possible mechanism for the increase in the activities of the TCA cycle enzymes upon treatment. In conclusion, our observations suggest that, the diabetes induced oxidative stress in mitochondria results in the decline of both antioxidant and TCA cycle enzymes. Therefore, maintenance of mitochondrial viability would depend, in part, on the ability mitochondria to sense these changes in redox status and respond in an appropriate manner. Lipoic acid supplementation markedly lowers oxidant production and their associated increase in oxidative damage in diabetes by enhancing antioxidant status. Thus, lipoic acid may ameliorate complications associated with diabetes mellitus by improving mitochondrial redox as well as energy metabolism.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
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Please cite this article in press as: Saraswathi R, Devaraj SN. Oxidative stress in skeletal muscle impairs mitochondrial function in alloxan induced diabetic rats: Role of alpha lipoic acid. Biomed Prev Nutr (2013), http://dx.doi.org/10.1016/j.bionut.2012.08.006