February 2014
Milena N. Stanković, Dušan Mladenović, Milica Ninković, Ivana Ðuričić,3 Slađana Šobajić, Bojan Jorgačević, Silvio de Luka, Rada Jesic Vukicevic, and Tatjana S. Radosavljević
Abstract
Development of nonalcoholic fatty liver disease (NAFLD) occurs through initial steatosis and subsequent oxidative stress. The aim of this study was to examine the effects of α-lipoic acid (LA) on methionine–choline deficient (MCD) diet-induced NAFLD in mice. Male C57BL/6 mice (n=21) were divided into three groups (n=7 per group): (1) control fed with standard chow, (2) MCD2 group—fed with MCD diet for 2 weeks, and (3) MCD2+LA group—2 weeks on MCD receiving LA i.p. 100 mg/kg/day.
After the treatment, liver samples were taken for pathohistology, oxidative stress parameters, antioxidative enzymes, and liver free fatty acid (FFA) composition. Mild microvesicular hepatic steatosis was found in MCD2 group, while it was reduced to single fat droplets evident in MCD2+LA group. Lipid peroxidation and nitrosative stress were increased by MCD diet, while LA administration induced a decrease in liver malondialdehyde and nitrates+nitrites level.
Similarly, LA improved liver antioxidative capacity by increasing total superoxide dismutase (tSOD), manganese SOD (MnSOD), and copper/zinc-SOD (Cu/ZnSOD) activity as well as glutathione (GSH) content. Liver FFA profile has shown a significant decrease in saturated acids, arachidonic, and docosahexaenoic acid (DHA), while LA treatment increased their proportions.
It can be concluded that LA ameliorates lipid peroxidation and nitrosative stress in MCD diet-induced hepatic steatosis through an increase in SOD activity and GSH level. In addition, LA increases the proportion of palmitic, stearic, arachidonic, and DHA in the fatty liver. An increase in DHA may be a potential mechanism of anti-inflammatory and antioxidant effects of LA in MCD diet-induced NAFLD.
Key Words: antioxidant, DHA, FFA, lipid peroxidation, lipotoxicity, liver, mice, steatosis
Discussion
It is well known that oxidative stress contributes to liver injury regardless of its etiology. Recently, many studies have pointed to the antioxidant effects of LA.25 In accordance with other studies of NAFLD,26 lipid peroxidation and nitrosative stress were also accompanied with mild steatosis in our study after MCD diet (Fig. 1). NO promotes oxidative stress-induced cell injury by formation of peroxynitrite anion, a potent prooxidant that causes protein nitration and tissue injury.27 Oxidative injury in MCD diet model of NAFLD may be due to GSH depletion, as well as to decline in CAT and SOD activity (Figs. 2–4). Poor activity of CAT in both experimental groups may be potentially due to secondary activated alternative GSH-pathways, so the CAT could be more active in the early phase of the model. The role of GSH depletion in the pathogenesis of NAFLD is supported by many other studies.8,26 A decrease in GSH/GSSG ratio reflects the shift of liver redox balance toward pro-oxidants as well as decreased synthesis of GSH due to methionine deficiency in the food. In addition, increased NO production may also contribute to a decline in intracellular GSH level and aggravation of cellular injury.28
Studies in experimental models and in the human population suggest that FFAs may play an important role in the pathogenesis of NAFLD.29 Recent data suggested that saturated fatty acids have more severe lipotoxic and proapoptotic effects in the liver than do unsaturated fatty acids.30,31
However, changes in liver FFA profile in NAFLD still remain controversial. Some studies on patients with NAFLD and NASH have shown that hepatic FFAs were unchanged across the spectrum of liver injury,32 while plasma FFA levels were significantly increased in NAFLD and they were suggested to be the primary source for triglyceride (TAG) synthesis in hepatocytes.33 On the other hand, a high fat diet was found to induce an increase in liver DHA level in mice.34 Our study has shown a decrease in hepatic saturated (palmitic and stearic acid) and arachidonic acid proportions in the group treated with MCD diet for 2 weeks.29,35
LA was found to have antioxidant effects, as well as to affect lipid metabolism in the liver. It has been shown in cultured rat hepatocytes that LA treatment significantly inhibited FFA oxidation or reduced it even by 82%. In the same study, it was also suggested that LA at therapeutic concentrations increased pyruvate oxidation by activation of the pyruvate dehydrogenase complex and decreased gluconeogenesis.36 In addition, LA treatment of insulin-resistant rats for 14 weeks decreased plasma FFA concentrations and this effect appears to be mediated, in part, by increased hepatic PPAR-α expression, which may beneficially affect insulin resistance.37
In our study, LA diminished MCD diet-induced lipid peroxidation and nitrosative stress, probably due to increased GSH level and SOD activity (Figs. 3 and and4).4). Similar effects of LA were found in heart and kidney tissues,27 but the effects on GSH level was found to be most marked in the liver because of high activity of GSH-synthesizing enzymes in this organ.38,39 However, the antioxidant effects of the LA in NAFLD may be potentially due to its direct inhibition of the free radicals (in the case of peroxinitrites), rather than increased expression of the antioxidant enzymes. The antioxidative effects of LA may be potentially mediated by changes in fatty acid profile. n-3 Polyunsaturated fatty acids may reduce oxidative damage and restore free radical homeostasis by incompletely understood mechanisms.11 It has been shown that linoleic, eicosapentaenoic acid (EPA), and DHA have beneficial effects on oxidative stress in the kidneys by reducing MDA level, and increasing SOD and CAT activity.40,41 However, our study suggests only the role of DHA, and not of linoleic acid as a mediator of antioxidative effects of LA in the liver, as only the proportion of DHA increases after LA administration (Table 1). This possibly indicates that supplementation of DHA, but not all polyunsaturated acids may potentiate the hepatoprotective effects of LA in NAFLD.
Other studies have also shown that supplementation with DHA significantly reduced NO content and increased SOD and CAT activity in the liver and brain. DHA supplementation improves cognitive functions and delays the onset of pentylenetetrazole-induced seizures, at least partly through attenuation of oxidative stress. In addition, DHA positively modulates phosphatidylserine biosynthesis and accumulation in neuronal cells, increases cell membrane fluidity, and inhibits apoptosis in a phosphatidylserine-dependent manner.42 The effects of DHA on membrane lipid composition in the liver has to be further investigated, but these effects may potentially contribute to modulation of oxidative stress in NAFLD after LA treatment found in our study.
Arachidonic acid was, also, found to cause an increase in MnSOD, Cu/ZnSOD, and CAT activities in rat hippocampal slices.43 Since LA prevented MCD diet-induced decline in arachidonic acid level in the liver, this can possibly be an additional indirect mechanism of LA antioxidant effect. However, this effect has to be further investigated by supplementation with arachidonic acid using various models of NAFLD.
Apart from influence on polyunsaturated fatty acids, antioxidant effects of LA may be potentially further mediated by an elevation of stearic acid proportion in the liver (Table 1). This may be surprising, as saturated fatty acids were found to contribute to the apoptosis of hepatocytes and to the progression of NAFLD, but stearic acid treatment has been shown to increase the activity of SOD, especially Cu/ZnSOD izoenzyme, CAT, and GSH peroxidase.44 This can be considered one of the potentially beneficial effects of stearic acid in NAFLD.
The effects of LA on liver fatty acid profile may, also, be responsible for the anti-inflammatory effects of LA in NAFLD found in the present study. n-3 Polyunsaturated fatty acids reduce the inflammation in the liver through stimulation of resolvin and protectin synthesis, as well as through inhibition of phospholipase A2, an initial enzyme in the synthesis of prostaglandins and leukotrienes. In addition, palmitic acid increases the hepatocyte sensitivity to the cytotoxic effects of TNF-α.45 DHA also exerts anti-inflammatory effect in the brain through reduction of microglial secretion of proinflammatory cytokines such as IL-6 and TNF-α.46 A recent study has shown that α-linoleic acid, EPA, and DHA reduce inflammation in both the heart and the liver and prevent cardiac fibrosis.40 The present study suggests that increased liver DHA proportion may be a potential mechanism of anti-inflammatory effects of LA in MCD-induced model of NAFLD (Table 1).
Based on our results, it can be concluded that LA ameliorates lipid peroxidation and nitrosative stress in MCD diet-induced hepatic steatosis through an increase in SOD activity and GSH level. In addition, LA increases the proportion of palmitic, stearic, arachidonic, and DHA in the fatty liver. Increase in DHA may be considered a potential mechanism of anti-inflammatory and antioxidant effects of LA in MCD diet-induced NAFLD.
